U.S. patent application number 10/007344 was filed with the patent office on 2002-10-17 for implantable biocompatible immunoisolatory vehicle for delivery of selected therapeutic products.
Invention is credited to Aebischer, Patrick, Christenson, Lisa, Dionne, Keith E., Emerich, Dwaine F., Gentile, Frank T., Hegre, Orion D., Hoffman, Diane, Lacy, Paul E., Lysaght, Michael J., Sanberg, Paul R., Scharp, David W., Vasconcellos, Alfred V..
Application Number | 20020150603 10/007344 |
Document ID | / |
Family ID | 24780436 |
Filed Date | 2002-10-17 |
United States Patent
Application |
20020150603 |
Kind Code |
A1 |
Dionne, Keith E. ; et
al. |
October 17, 2002 |
Implantable biocompatible immunoisolatory vehicle for delivery of
selected therapeutic products
Abstract
An immunoisolatory vehicle for the implantation into an
individual of cells which produce a needed product or provide a
needed metabolic function. The vehicle is comprised of a core
region containing isolated cells and materials sufficient to
maintain the cells, and a permselective, biocompatible, peripheral
region free of the isolated cells, which immunoisolates the core
yet provides for the delivery of the secreted product or metabolic
function to the individual. The vehicle is particularly well-suited
to delivery of insulin from immunoisolated islets of Langerhans,
and can also be used advantageously for delivery of high molecular
weight products, such as products larger than IgG. A method of
making a biocompatible, immunoisolatory implantable vehicle,
consisting in a first embodiment of a coextrusion process, and in a
second embodiment of a stepwise process. A method for isolating
cells within a biocompatible, immunoisolatory implantable vehicle,
which protects the isolated cells from attack by the immune system
of an individual in whom the vehicle is implanted. A method of
providing a needed biological product or metabolic function to an
individual, comprising implanting into the individual an
immunoisolatory vehicle containing isolated cells which produce the
product or provide the metabolic function.
Inventors: |
Dionne, Keith E.; (Rehoboth,
MA) ; Emerich, Dwaine F.; (Providence, RI) ;
Hoffman, Diane; (Cambridge, MA) ; Sanberg, Paul
R.; (Spring Hill, FL) ; Christenson, Lisa;
(New Haven, CT) ; Hegre, Orion D.; (Green Valley,
AZ) ; Scharp, David W.; (St. Louis, MO) ;
Lacy, Paul E.; (Webster Grove, MO) ; Aebischer,
Patrick; (Lutry, CH) ; Vasconcellos, Alfred V.;
(Cranston, RI) ; Lysaght, Michael J.; (E.
Greenwich, RI) ; Gentile, Frank T.; (Warwich,
RI) |
Correspondence
Address: |
MINTZ LEVIN
One Financial Center
Boston
MA
02111
US
|
Family ID: |
24780436 |
Appl. No.: |
10/007344 |
Filed: |
October 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10007344 |
Oct 25, 2001 |
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09563248 |
May 2, 2000 |
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6322804 |
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09563248 |
May 2, 2000 |
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09148671 |
Sep 4, 1998 |
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6083523 |
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09148671 |
Sep 4, 1998 |
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08449837 |
May 24, 1995 |
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5874099 |
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08449837 |
May 24, 1995 |
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08179151 |
Jan 10, 1994 |
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5800828 |
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08179151 |
Jan 10, 1994 |
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PCT/US92/03327 |
Apr 22, 1992 |
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PCT/US92/03327 |
Apr 22, 1992 |
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07692403 |
Apr 25, 1991 |
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Current U.S.
Class: |
424/424 ;
424/93.7 |
Current CPC
Class: |
A61P 37/06 20180101;
C12N 2510/02 20130101; A61K 48/00 20130101; C12N 5/0677 20130101;
Y10S 514/866 20130101; A61K 9/0092 20130101; A61K 35/30 20130101;
A61P 3/10 20180101; C12N 5/0062 20130101; A61P 37/02 20180101; A61P
15/00 20180101; A61P 31/18 20180101; A61K 2035/126 20130101; C12N
5/0614 20130101; A61K 9/0024 20130101; Y10S 514/814 20130101; A61P
43/00 20180101; A61P 25/28 20180101; A61K 35/12 20130101; C12N
5/0012 20130101; A61K 35/39 20130101; A61K 38/185 20130101; C12N
5/0656 20130101; A61K 9/4816 20130101; A61P 25/14 20180101; A61P
25/00 20180101; A61P 5/24 20180101; A61K 2035/128 20130101 |
Class at
Publication: |
424/424 ;
424/93.7 |
International
Class: |
A61K 045/00 |
Claims
What is claimed is:
1. An implantable immunoisolatory vehicle for providing a
biologically active product or function to an individual, the
immunoisolatory vehicle comprising: (a) a core comprising living
cells dispersed in a biocompatible matrix formed of a hydrogen,
said cells being capable of secreting a selected biologically
active product or of providing a selected biological function to an
individual; and (b) an external diffusional surface jacket
surrounding said core formed of a biocompatible hydrogel material
free of said cells projecting externally thereof, said jacket
having a molecular weight cutoff below the molecular weight of
substances essential for immunological rejection of the cells but
permitting passage of substances between the individual and core
through said jacket required to provide said biological product or
function, said core and external jacket forming an interface
substantially free of direct ionic bonding and of an intermediate
linking layer.
Description
RELATED APPLICATIONS
[0001] This is a continuation-in-part application of PCT
application Ser. No. US 92/03327, filed Apr. 22, 1992, which is a
continuation-in-part of application Ser. No. 07/692,403, filed Apr.
25, 1991.
BACKGROUND
[0002] Many clinical conditions, deficiencies, and disease states
can be remedied or alleviated by supplying to the patient a one or
more biologically active moieties produced by living cells or
removing from the patient deleterious factors which are metabolized
by living cells. In many cases, these moieties can restore or
compensate for the impairment or loss of organ or tissue function.
Examples of disease or deficiency states whose etiologies include
loss of secretory organ or tissue function include (a) diabetes,
wherein the production of insulin by pancreatic islets of
Langerhans is impaired or lost; (b) hypoparathyroidism, wherein the
loss of production of parathyroid hormone causes serum calcium
levels to drop, resulting in severe muscular tetany; (c)
Parkinsonism, wherein dopamine production is diminished; and (d)
anemia, which is characterized by the loss of production of red
blood cells secondary to a deficiency in erythropoietin. The
impairment or loss of organ or tissue function may result in the
loss of additional metabolic functions. For example, in fulminant
hepatic failure, liver tissue is rendered incapable of removing
toxins, excreting the products of cell metabolism, and secreting
essential products, such as albumin and Factor VIII. Bontempo, F.
A., et al., Blood, 69, pp. 1721-1724 (1987).
[0003] In other cases, these biologically active moieties are
biological response modifiers, such as lymphokines or cytokines,
which enhance the patient's immune system or act as
anti-inflammatory agents. These can be particularly useful in
individuals with a chronic parasitic or infectious disease, and may
also be useful for the treatment of certain cancers. It may also be
desirable to supply trophic factors to a patient, such as nerve
growth factor or insulin-like growth factor-one or -two (IGF1 or
IGF2).
[0004] In still other cases, the biologically active moiety can be
a secretory substance, such as a neurotransmitter, neuromodulator,
hormone, trophic factor, or growth factor, or a neuroactive
substance for the reduction of pain sensitivity. Such neuroactive
substances include catecholamines, enkephalins, and opioid
peptides.
[0005] In many disease or deficiency states, the affected organ or
tissue is one which normally functions in a manner responsive to
fluctuations in the levels of specific metabolites, thereby
maintaining homeostasis. For example, the parathyroid gland
normally modulates production of parathyroid hormone (PTH) in
response to fluctuations in serum calcium. Similarly, .beta. cells
in the pancreatic islets of Langerhans normally modulate production
of insulin in response to fluctuations in serum glucose.
Traditional therapeutic approaches to the treatment of such
diseases cannot compensate for the responsiveness of the normal
tissue to these fluctuations. For example, an accepted treatment
for diabetes includes daily injections of insulin. This regimen
cannot compensate for the rapid, transient fluctuations in serum
glucose levels produced by, for example, strenuous exercise.
Failure to provide such compensation may lead to complications of
the disease state; this is particularly true in diabetes. Jarret,
R. J. and Keen J., (1976) Lancet(2):1009-1012.
[0006] Many other diseases are, likewise, characterized by a
deficiency in a biologically active moiety that cannot easily be
supplemented by injections or longer-term, controlled release
therapies. Still other diseases, while not characterized by
substance deficiencies, can be treated with biologically active
moieties normally made and secreted by cells. Thus, trophic and
growth factors may be used to prevent neurodegenerative conditions,
such as Huntington's and Alzheimer's diseases, and adrenal
chromaffin cells which secrete catecholamines and enkephalins, may
be used to treat pain.
[0007] It is also fairly well established that the activation of
noradrenergic or opioid receptors in the spinal cord by direct
intrathecal injection of .alpha.-adrenergic or opioid agonists
produces antinociception, and that the co-administration of
subeffective doses of these agents can produce potent analgesia.
The presence of enkephalin-secreting neurons and opiate receptors
in high densities in the substantia gelatinosa of the spinal cord
and the resultant analgesia observed following local injection of
opiates into the spinal cord have suggested a role for opioid
peptides in modulating the central transmission of nociceptive
information. In addition, catecholamines also appear to be
important in modulating pain sensitivity in the spinal cord since
injection of noradrenergic agonists into the subarachnoidal space
of the spinal cord produces analgesia, while the injection of
noradrenergic antagonists produces increased sensitivity to noxious
stimuli.
[0008] Many drugs have been administered intraspinally in the
clinical setting, and numerous methods are available to deliver
intraspinal medications. For instance, the most common method of
intraspinal drug delivery, particularly anesthetics, is continuous
infusion by way of spinal catheters. However, the use of these
catheters, particularly small-bore catheters, has been implicated
in such complications as cauda equina syndrome, a neurological
syndrome characterized by loss of sensation or mobility of the
lower limbs. In fact, the FDA was prompted to issue a safety alert
in May, 1992, alerting Anesthesia Care Providers to the serious
hazard associated with continuous spinal anesthesia by small-bore
catheters and has taken action to remove all small-bore catheters
from the market.
[0009] Accordingly, many investigators have attempted to
reconstitute organ or tissue function by transplanting whole
organs, organ tissue, or cells which provide secreted products or
affect metabolic functions. Moreover, transplantation can provide
dramatic benefits but is limited in its application by the
relatively small number of organs suitable and available for
grafting. In general, the patient must be immunosuppressed in order
to avert immunological rejection of the transplant, which results
in loss of transplant function and eventual necrosis of the
transplanted tissue or cells. In many cases, the transplant must
remain functional for a long period of time, even for the remainder
of the patient's lifetime. It is both undesirable and expensive to
maintain a patient in an immunosuppressed state for a substantial
period of time.
[0010] A desirable alternative to such transplantation procedures
is the implantation of cells or tissues within a physical barrier
which will allow diffusion of nutrients, waste materials, and
secreted products, but block the cellular and molecular effectors
of immunological rejection. A variety of devices which protect
tissues or cells producing a selected product from the immune
system have been explored. These include extravascular diffusion
chambers, intravascular diffusion chambers, intravascular
ultrafiltration chambers, and implantation of microencapsulated
cells. Scharp, D. W., et al., World J. Surg., 8, pp. 221-9 (1984)2.
These devices would alleviate the need to maintain the patient in
an immunosuppressed state. However, none of these approaches have
been satisfactory for providing long-term transplant function. A
method of delivering appropriate quantities of needed substances,
such as enzymes, hormones, or other factors or, providing other
needed metabolic functions, for an extended period of time is still
unavailable and would be very advantageous to those in need of
long-term treatment.
SUMMARY OF THE INVENTION
[0011] This invention relates to a biocompatible, immunoisolatory,
implantable vehicle. The instant vehicle is suitable for isolating
biologically active cells or substances from the body's protective
mechanisms following implantation into an individual. The instant
vehicle is comprised of (a) a core which contains isolated cells,
either suspended in a liquid medium or immobilized within a
hydrogel matrix, and (b) a surrounding or peripheral region
("jacket") of permselective matrix or membrane which does not
contain isolated cells, which is biocompatible, and which is
sufficient to protect the isolated cells in the core from
immunological attack.
[0012] The immunoisolatory vehicle is useful (a) to deliver a wide
range of biologically active moieties, including high molecular
weight products, to an individual in need of them, and/or (b) to
provide needed metabolic functions to an individual, such as the
removal of harmful substances. The instant vehicle contains a
multiplicity of cells, such that implantation of a few or a single
vehicle is sufficient to provide an effective amount of the needed
substance or function to an individual. A further advantage offered
by the instant vehicle is practicality of retrieval.
[0013] In one embodiment of the invention, which is particularly
useful with islets of Langerhans, both the core and the surrounding
or peripheral region of the instant vehicle are hydrogels, which
can be the same composition hydrogel or different composition
hydrogels.
[0014] This invention also relates to a method of delivering a
biologically active moiety or altering a metabolic or immunologic
function in an individual in need of the moiety or altered
metabolic function. An immunoisolatory vehicle of the present
invention is implanted into the individual (referred to as the
recipient), using known techniques or methods and selected for the
particular immunoisolatory vehicle and site of implantation. Once
implanted, cells isolated within the biocompatible immunoisolatory
vehicle produce the desired moieties or perform the desired
function(s). If moieties are released by the isolated cells, they
pass through the surrounding or peripheral permselective membrane
or hydrogel matrix into the recipient's body. If metabolic
functions are provided by the isolated cells, the substances to be
metabolized (e.g., degraded or inactivated) enter the vehicle from
the recipient's body and are removed from the recipient's
bloodstream.
[0015] Thus, this invention relates to a method of isolating cells
within a biocompatible, immunoisolatory implantable vehicle,
thereby protecting the cells within the vehicle from immunological
attack after being implanted into an individual. Although some low
molecular weight mediators of the immune responses (e.g. cytokines)
may be permeable to the membrane, in most cases local or
circulating levels of these substances are not high enough to have
detrimental effects. The isolated cells are protected from attack
by the recipient's immune system and from potentially deleterious
inflammatory responses from the tissues which surround the
implanted vehicle. In the core of the vehicle, the isolated cells
are maintained in a suitable local environment. In this manner,
needed substances or metabolic functions can be delivered to the
recipient even for extended periods of time, and without the need
to treat the recipient with dangerous immunosuppressive drugs.
[0016] This invention relates further to a method of making a
biocompatible immunoisolatory vehicle. In a first embodiment, the
vehicle is formed by coextruding from a nested-bore extrusion
nozzle materials which form the core and surrounding or peripheral
regions, under conditions sufficient to gel, harden, or cast the
matrix or membrane precursor(s) of the surrounding or peripheral
region (and of the core region). A particular advantage of this
coextrusion embodiment is that the cells in the core are isolated
from the moment of formation of the vehicle, ensuring that the core
materials do not become contaminated or adulterated during handling
of the vehicle prior to implantation. A further advantage of the
coextrusion process is that it ensures that the surrounding or
peripheral region is free of cells and other core materials. The
permeability and biocompatibility characteristics of the
surrounding or peripheral region are determined by both the matrix
or membrane precursor materials used, and the conditions under
which the matrix or membrane is formed.
[0017] In another embodiment of the present method, the
immunoisolatory vehicle is formed stepwise. For example, if the
immunoisolatory vehicle being made includes a hydrogel core
containing the isolated cells, the core can be formed initially,
and the surrounding or peripheral matrix or membrane can be
assembled or applied subsequently. Conversely, the surrounding or
peripheral matrix or membrane can be preformed, and then filled
with the preformed isolated-cell containing core material or with
materials which will form the core (i.e., core precursor
materials). The vehicle is sealed in such a manner that the core
materials are completely enclosed. If a core precursor material is
used, the vehicle is then exposed to conditions which result in
formation of the core.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 is a graphic representation of the differences in the
permeability of alginate matrices formed under different conditions
to test solutes of various molecular sizes.
[0019] FIG. 2 is a graphic representation of the results of a
perfusion test of the functionality of immunoisolated versus
unprotected islets maintained in vitro for four weeks. FIG. 2A
depicts the results obtained with an immunoisolatory vehicle having
a hydrogel core matrix and a peripheral jacket made of a
permselective thermoplastic membrane. FIG. 2B depicts the results
obtained with an immunoisolatory vehicle having a hydrogel core
matrix and a peripheral hydrogel jacket.
[0020] FIG. 3 is a graphic representation showing the total amount
of insulin released, and the amounts released during the first and
second phase responses in the perfusion test also shown in FIG. 2.
FIG. 3A depicts the results obtained with the dual-matrix
immunoisolatory vehicle and FIG. 3B depicts the results obtained
with the core matrix-permselective membrane immunoisolatory
vehicle.
[0021] FIG. 4 is a graphic representation of the decrease in plasma
glucose levels observed when immunoisolated xenogeneic islets are
implanted into streptozotocin-induced diabetic mice for a period of
60 days. The immunoisolatory vehicle used was of the configuration
described in Example 5.
[0022] FIG. 5 is a graphic representation of insulin release in a
perfusion experiment using an immunoisolatory vehicle containing
rat islets, of the configuration described in Example 5, recovered
after a period of residence In vivo and challenged with glucose,
with and without theophylline stimulation.
[0023] FIG. 6 is a graphic representation of the decrease in plasma
glucose levels observed when immunoisolated xenogeneic islets are
implanted into streptozotocin-induced diabetic mice for a period of
100 days. The immunoisolatory vehicle used was of the configuration
described in Example 4.
[0024] FIG. 7 is a graphic representation of the permeability of an
alginate matrix to various test solutes. Permeabilities were tested
after storage in Hank's solution after 16 hours and 160 hours. The
change in permeability is due to leaching of Ca.sup.++ from the
matrix.
[0025] FIG. 8 is a graphic comparison of the response to glucose
challenge of rat islets isolated within dual matrix immunoisolatory
vehicles with either thermoplastic or alginate jackets, following a
period of residence in vivo in discordant xenogeneic recipients
(guinea pigs).
[0026] FIG. 9 is a graphic representation of the partial
restoration of normal motor behavior to rodents with experimentally
induced Parkinson-like behavior, following implantation of an
immunoisolatory vehicle containing adrenal chromaffin cells in a
core matrix, with a surrounding jacket of permselective
thermoplastic membrane.
[0027] FIG. 10 is a graphic representation of the mean body weight
changes seen in quinolinic acid lesioned rats. Rats receiving
immunoisolatory capsules containing bovine adrenal chromaffin cells
maintained body weight significantly better than the other lesioned
rats.
[0028] FIG. 11 is a graphic representation of the nonfasting plasma
glucose concentrations of diabetic mice after implantation of type
2 acrylic copolymer hollow fibers containing either 1000 rat islets
(A) or 500 rat islets (B) implanted either intraperitoneally
(circles) or subcutaneously (squares).
[0029] FIG. 12 is a graphic representation of the effects on blood
glucose in diabetic rats implanted. with rat islets encapsulated in
flat sheet devices.
DETAILED DESCRIPTION OF THE INVENTION
[0030] This invention relates to a biocompatible immunoisolatory
vehicle suitable for long-term implantation into individuals. More
particularly, the biocompatible immunoisolatory vehicle of the
instant invention comprises (a) a core which contains a
biologically active moiety, either suspended in a liquid medium or
immobilized within a hydrogel or extracellular matrix, and (b) a
surrounding or peripheral region of permselective matrix or
membrane (jacket) which does not contain isolated cells, which is
biocompatible, and which is sufficient to protect isolated cells if
present in the core from immunological attack.
[0031] The term "individual" refers to a human or an animal
subject.
[0032] A "biologically active moiety" is a tissue, cell, or other
substance, which is capable of exerting a biologically useful
effect upon the body of an individual in whom a vehicle of the
present invention containing a biologically active moiety is
implanted. Thus, the term "biologically active moiety" encompasses
cells or tissues which secrete or release a biologically active
molecule, product or solute; cells or tissues which provide a
metabolic capability or function, such as the removal of specific
solutes from the bloodstream; or a biologically active molecule or
substance such as an enzyme, trophic factor, hormone, or biological
response modifier.
[0033] When the biologically active moiety within the core of the
biocompatible immunoisolatory vehicle comprises cells, the core is
constructed to provide a suitable local environment for the
continued viability and function of the cells isolated therein. The
instant vehicle can be used to immunoisolate a wide variety of
cells or tissues, spanning the range from fully-differentiated,
anchorage-dependent cells or primary tissues, through
incompletely-differentiated fetal or neonatal tissues, to
anchorage-independent transformed cells or cell lines.
[0034] Many transformed cells or cell lines are most advantageously
isolated within a vehicle having a liquid core. For example, PC12
cells (which secrete dopamine and are herein shown to be useful for
the treatment of Parkinsonism) can be isolated within a vehicle
whose core comprises a nutrient medium, optionally containing a
liquid source of additional factors to sustain cell viability and
function, such as fetal bovine or equine serum.
[0035] Unless otherwise specified, the term "cells" means cells in
any form, including but not limited to cells retained in tissue,
cell clusters, and individually isolated cells.
[0036] Implants of the vehicle and contents thereof retain
functionality for greater than three months In vivo and in many
cases for longer than a year. In addition, the vehicle of the
current invention may be prepared of sufficient size to deliver an
entire therapeutic dose of a substance from a single or just a few
(less than 10) implanted and easily retrievable vehicles.
[0037] The core of the immunoisolatory vehicle is constructed to
provide a suitable local environment for the particular cells
isolated therein. In some embodiments, the core comprises a liquid
medium sufficient to maintain the cells. Liquid cores are
particularly suitable for maintaining transformed cells, such as
PC12 cells. In other embodiments, the core comprises a gel matrix
which immobilizes and distributes the cells, thereby reducing the
formation of dense cellular agglomerations. The gel matrix may be
composed of hydrogel or extracellular matrix components.
[0038] Suitably, the core may be composed of a matrix formed by a
hydrogel which stabilizes the position of the cells in cell clumps.
The term "hydrogel" herein refers to a three dimensional network of
cross-linked hydrophilic polymers. The network is in the form of a
gel substantially composed of water, preferably but not limited to
gels being greater than 90% water. Cross-linked hydrogels can also
be considered solids because they do not flow or deform without
appreciable applied shear stress.
[0039] Compositions which form hydrogels fall into three classes
for the purposes of this application. The first class carries a net
negative charge and is typified by alginate. The second class
carries a net positive charge and is typified by extracellular
matrix components such as collagen and laminin. Examples of
commercially available extracellular matrix components include
Matriqel.TM. and Vitrogen.TM.. The third class is net neutral in
charge. An example of a net neutral hydrogel is highly crosslinked
polyethylene oxide, or polyvinyalcohol.
[0040] Cores made of a hydrogel matrix are particularly suitable
for maintaining cells or tissues which tend to form agglomerates or
aggregates, such as the cells in islets of Langerhans, or adrenal
chromaffin cells. The matrix should be of sufficient viscosity to
maintain cell dispersion within the matrix. Optionally, the core of
the instant vehicle can contain substances which support or promote
the function of the isolated cells. These substances include
natural or synthetic nutrient sources, extracellular matrix (ECM)
components, growth factors or growth regulatory substances, or a
population of feeder or accessory cells or O.sub.2 carriers such as
hemoglobins and fluorocarbons.
[0041] Herein, the term "aggregating" refers to a process of
promoting cell clustering. The cells which form clusters may be
obtained from naturally occurring agglomerates, such as pancreatic
islets which are dispersed into single or small-clump suspension
and subsequently reaggregated according to the methods described.
Alternatively, the cells may be obtained originally as single cells
or small cell clumps, and then aggregated to form the desired
cluster size. Such cell clusters will generally contain about 3-400
cells, depending upon cell size and aggregation characteristics.
Typically, the cluster contains about 10 to about 50 cells. The use
of reaggregated pancreatic islet cells is advantageous for insuring
proper diffusion characteristics within the core and maintaining
islet viability.
[0042] Reaggregated islets also allow the use of smaller capsules.
For instance, 500 non-reaggregated islets would generally require a
capsule of approximately 14 cm in length (2% density). In contrast,
capsules containing islets reaggregated to a size smaller than an
intact islet may be as small as 1 to 2 cm in length because of more
efficient packing. More efficient packing allows a lower pO.sub.2
outside the fiber to be tolerated without resultant necrotic cores.
Built in tolerance for lower outside pO.sub.2 has at least two
advantages. Firstly, a smaller capsule size may be used to contain
the same number of cells, i.e. increased tissue density within the
implant is better tolerated. Secondly, implantation sites with
known reduced pO.sub.2, such as subcutaneous locations, may be used
successfully. The presence of the alginate matrix further insures
that the aggregates will not reassociate to large cell masses where
internal cells would be deprived of nutrients and/or oxygen.
[0043] In one advantageous application of the invention, the cells
are formed by reaggregating natural cell clusters into a form
adapted for increased packing per unit volume as described
supra.
[0044] Such reaggregated clusters are preferably characterized by
improved diffusion of critical solutes to and from the cells within
the cluster in comparison to natural cell clusters.
[0045] Cells which do not require an anchorage substrate are those
which are able to form clumps or agglomerates and thus provide
anchorage for each other. Exemplary clumping cell types are
pancreatic islets, pancreatic beta-cell lines, Chinese hamster
ovary (CHO) cells, and adrenal chromaffin cells. These cells are
suitably enclosed in a negatively charged matrix such as
alginate.
[0046] Fibroblasts generally survive well in a positively charged
matrix and are thus suitably enclosed in extracellular-matrix type
hydrogels. Certain cell types tend to multiply rapidly and could
overgrow the space available within the core if they do not exhibit
growth arrest. If the isolated cells do not exhibit growth arrest
upon confluence, substances which induce quiescence can be included
in the interior of the vehicle. In some instances, a hydrogel core
may suffice to limit continued proliferation. For example, a
hydrogel matrix precursor solution can be included but not exposed
to polymerizing conditions. In the case of sodium alginate, a
hydrogel will form slowly after implantation as calcium ions are
scavenged from the surrounding tissues. Alternatively, growth
inhibitory factors, or stimulators of differentiation can be
incorporated into microbeads of a slowly degraded polymer such as
polycarbonate, and cosuspended with the product-secreting cells.
For instance, NGF or FGF can be used to stimulate PC-12 cell
differentiation and terminate cell division.
[0047] Other cells, particularly primary cells or tissues, tend to
adhere to each other and form dense agglomerations which develop
central necrotic regions due to the relative inaccessibility of
nutrients and oxygen to cells embedded within the agglomerated
masses. These dense cellular masses can form slowly, as a result of
cell growth into dense colonies, or rapidly, upon the reassociation
of freshly-dispersed cells or tissue mediated by cell-surface
adhesion proteins. Cells or tissues which are highly metabolically
active are particularly susceptible to the effects of oxygen or
nutrient deprivation, and die shortly after becoming embedded in
the center of an agglomerate. Many endocrine tissues, which
normally are sustained by dense capillary beds, exhibit this
behavior; islets of Langerhans and adrenal chromaffin cells are
particularly sensitive. Cells or tissues which exhibit this
behavior perform most satisfactorily in vehicles comprising a
hydrogel matrix core, sufficient to immobilize the cells or tissue,
thereby preserving the access of nutrients and oxygen to the
majority of them.
[0048] In other circumstances, the immobilizing hydrogel matrix
further performs the additional function of producing or preserving
functional units of a size and/or shape appropriate for maintaining
desirable characteristics of the isolated cells. Moreover, the
presence of the core matrix allows maintenance of a uniform
distribution of cells or clusters of cells within the vehicle
(i.e., the core matrix prevents settling and decreases mobility of
the included cells).
[0049] One particularly advantageous use of hydrogel cores pertains
to the encapsulation of actively dividing cells. Alginate or other
hydrogels may be included in suspensions of actively dividing cells
to be encapsulated. Following encapsulation and generation of the
gel, the encapsulated cells are somewhat immobilized within the gel
and new cells produced during cell division stay localized near the
parent cell. In this manner clusters of cells are produced within
the core. Such a growth method is advantageous in the case of cells
such as the beta cell derived NIT cell line. In the absence of a
core matrix these cells tend to grow attached along the inner walls
of the encapsulation device, with very few cells growing freely
within the cavity of the device. Growth only on the walls of the
capsule leads to a cell population size that is restricted by the
surface area of the inner capsule wall as opposed to a population
that grows to fill the vehicle cavity. When alginate is included in
the core, cell growth is no longer limited to the inner capsule
surface. Rather, discrete spheres of NIT cells are produced
throughout the core, resulting in a significantly larger total cell
population than that which occurs in the absence of alginate.
[0050] Even in the presence of a core matrix, the size of tissue
fragments which can be loaded into a given vehicle volume is
limited by the appearance of central necrosis within the individual
fragments. In one aspect of the instant invention, the useful
amount of tissue fragments or cell clusters that can be placed
within the immunoisolatory vehicle is increased by preparing the
cells in a form with improved diffusional characteristics.
Generally this means preparation of the tissue fragments to a size
less than 75 .mu.m diameter and most optimally to about 35 .mu.m
diameter or less for vehicles to be implanted peritoneally.
Aggregates or clusters of cells in improved diffusional form are
prepared for spontaneously reaggregating cells (e.g. pancreatic
islets or adrenal chromaffin cells) by enzymatically dispersing the
tissue, to single cell and small cell aggregate, suspensions,
followed by controlled reaggregation to the improved diffusional
form.
[0051] Pancreatic islet cells still retain functionality and
secrete insulin in response to glucose in near normal fashion
following enzymatic dispersion and reaggregation. Cells from
dispersed islets are reaggregated to the desired cluster size prior
to loading into the vehicle. Reaggregation can be accomplished by
the methods described by Britt, Diabetes, 34, pp. 898-903, or by
similar methods. The optimal aggregate size for islets is the
smallest size cluster which still maintains the desired
physiological characteristics. The matrix or matrix forming
materials may then be added to the cells, and the combination may
be coextruded into or loaded into biocompatible immunoisolatory
vehicles. If necessary, matrix formation may then be induced. In
preferred embodiments, cells are reaggregated overnight at
37.degree. C., without agitation. The development of aggregates is
monitored by light microscopy until aggregates achieve a size of 25
to 75 .mu.m preferably 35 .mu.m diameter. Liquid uncrosslinked
alginate is then added to the cells to a concentration of from 0.5
to 2%, the cells are incorporated into vehicles, the vehicles are
sealed if necessary, and polymerization is induced by immersion of
the vehicle in an aqueous solution containing CaCl.sub.2.
[0052] Primary cells or tissues may be useful with the vehicle of
the instant invention for various medical applications. For
regulatory reasons and reasons of patient safety, it may sometimes
be useful to employ as sources for primary cultures, animals of
carefully controlled hereditary and developmental background. The
presence of unwanted virus, bacteria and other pathogens may be
limited through the use of specific pathogen free or gnotobiotic
source animals. References and methods for the establishment, care
and use of specific pathogen free and gnotobiotic herds are
provided by Maniatis, O.P., et al. Can. J. Med., 42, p. 428 (1978);
Matthews P. J., et al., Recent Advances in Germ Free Research, pp.
61-64, Tokai Univ. Press (1981), and in the National Accreditation
Standards publication of the National SPF swine Accrediting Agency,
Inc., Conrad, Iowa.
[0053] Optionally, a matrix core can also contain materials which
support or promote the function of the isolated cells. For example,
extracellular matrix (ECM) components can be included to promote
specific attachment or adhesion of the isolated cells. A
combination of ECM components which is particularly suitable for
fostering the growth of certain types of cells is taught in
Kleinman et al., U.S. Pat. No. 4,829,000. The core matrix can
provide a reservoir for soluble or releasable substances, such as
growth factors or growth regulatory substances, or for natural or
synthetic substances which enhance or improve the supply of
nutrients or oxygen to the isolated cells. Thus, it can function in
a manner similar to the bone marrow ECM, which has been reported to
behave as a slow-release reservoir for myeloid lineage-specific
growth factors such as granulocyte-macrophage colony stimulating
factor (gmcsf). Gordon, M. Y., et al., Nature, 326, pp. 403-405
(1987). Thus, the core matrix can function as a reservoir for
growth factors (e.g., prolactin, or insulin-like growth factor 2),
growth regulatory substances such as transforming growth factor
.beta. (TGF.beta.) or the retinoblastoma gene protein or
nutrient-transport enhancers (e.g., perfluorocarbons, which can
enhance the concentration of dissolved oxygen in the core). Certain
of these substances are also appropriate for inclusion in liquid
media.
[0054] Additionally, a population of feeder or accessory cells can
be coisolated within the vehicle. For example, hepatocytes can be
coisolated with endothelial accessory cells, Cai, Z., et al.,
Artificial Organs, 12, pp. 388-393 (1988), or mixed with islet
cells, Ricordi C., et al., Transplantation 45, pp. 1148-1151
(1987), or adrenal chromaffin cells can be coisolated with
accessory cells which provide nerve growth factor (NGF), a
substance needed by the chromaffin cells. In the latter case,
fibroblasts which have been transfected with an expression vector
for NGF can be used as accessory cells.
[0055] The instant vehicle can also be used as a reservoir for the
controlled delivery of needed drugs or biotherapeutics. In such
cases, the core, rather than containing cells or tissues, contains
a high concentration of the selected drug or biotherapeutic. In
addition, satellite vehicles containing substances which prepare or
create a hospitable environment in the area of the body in which a
biocompatible immunoisolatory vehicle containing isolated cells is
implanted can also be implanted into a recipient. In such
instances, the vehicle containing immunoisolated cells is implanted
in the region along with satellite vehicles releasing controlled
amounts of, for example, a substance which down-modulates or
inhibits an inflammatory response from the recipient (e.g.,
anti-inflammatory steroids), or a substance which stimulates the
ingrowth of capillary beds (i.e., an angiogenic factor).
[0056] The surrounding or peripheral region (jacket) of the instant
vehicle is permselective, biocompatible, and immunoisolatory. It is
produced in such a manner that it is free of isolated cells, and
completely surrounds (i.e., isolates) the core, thereby preventing
contact between any cells in the core and the recipient's body.
[0057] To be permselective, the jacket is formed in such a manner
that it has a MWCO range appropriate both to the type and extent of
immunological reaction it is anticipated will be encountered after
the vehicle is implanted and to the molecular size of the largest
substance whose passage into and out of the vehicle is desirable.
The type and extent of immunological attacks which may be mounted
by the recipient following implantation of the vehicle depend in
part upon the type(s) of moiety isolated within it and in part upon
the identity of the recipient (i.e., how closely the recipient is
genetically related to the source of the biologically active
moiety). When the implanted tissue is allogeneic to the recipient,
immunological rejection may proceed largely through cell-mediated
attack by the recipient's immune cells against the implanted cells.
When the tissue is xenogeneic to the recipient, molecular attack
through assembly of the recipient's cytolytic complement attack
complex may predominate, as well as the antibody interaction with
complement.
[0058] In reference to the permselectivity of the membranes and
gels of the instant invention, the phrase "molecular weight cutoff"
(MWCO) is used. It is recognized that there are many methods
available for the determination of the molecular weight cutoff for
a permselective membrane. Depending upon the method used, somewhat
different MWCO estimates may be achieved for the same membrane. In
the context of the current invention, MWCO refers to the results of
the empirical determinations described herein, using the specific
markers described under the specific conditions of the
determination. Other methods of MWCO determination, to apply to the
current invention, will need to be calibrated against the protocol
of the instant invention according to methods known to
practitioners in the art.
[0059] The jacket allows passage of substances up to a
predetermined size, but prevents the passage of larger substances.
More specifically, the surrounding or peripheral region is produced
in such a manner that it has pores or voids of a predetermined
range of sizes; as a result, the vehicle is permselective. The
molecular weight cutoff (MWCO) selected for a particular vehicle
will be determined in part by the type and extent of immunological
rejection it is anticipated will be encountered after the vehicle
is implanted and in part by the molecular size of the largest
substance to be allowed to pass into and/or out of the vehicle. For
example, materials can be used to form permselective membranes or
hydrogel matrices which allow passage of molecules up to about the
size of Clq, a component of complement (about 400 kD), a protein
required for the assembly of the cytolytic complement attack
complex. In this instance, substances smaller than Clq can pass
freely. It is also possible to form permselective matrices or
membranes which allow passage of molecules up to about the size of
immunoglobulin G (about 150 kD) and exclude larger molecules.
Further, membranes or hydrogels which allow passage of molecules up
to about the size of immunoglobulin M (about 1,000 kD) can be used;
only very large substances, such as cells, will be excluded in this
embodiment.
[0060] The MWCO of the surrounding or peripheral region must
therefore be sufficiently low to prevent access of the substances
required to carry out these attacks to the core, yet sufficiently
high to allow delivery of the needed product to the recipient's
body. It will therefore be apparent that the MWCO need not be
strictly restricted to a range which excludes immunoglobulin G from
the core. In fact, there are many cases in which higher MWCOs are
not only permissible, but also advantageous. Indeed, higher MWCOs
allow the delivery of a wide variety of useful products from
immunoisolated cells, as well as the use of such cells to provide
metabolic control of high molecular weight substances.
[0061] Thus, in appropriate cases, the peripheral or surrounding
region can be made of materials which form permselective membranes
or hydrogel matrices allowing the passage of molecules up to about
the size of Clq (about 400 kD), the largest protein required for
the assembly of the complement attack complex. Therefore, any
cellular product or metabolite below about the size of Clq can pass
freely through the vehicle. In other cases, it may still be
desirable to exclude immunoglobulins. In such cases, materials
which form matrices or membranes through which molecules which are
equivalent to or larger than the size of immunoglobulin G (about
150 kD) cannot pass can be used. Cellular products or metabolites
which are smaller than about 150 kD will still pass through the
vehicle. In still other cases, where the patient is
immunosuppressed or where the implanted tissue is syngeneic to the
patient, a vigorous immunological attack is not likely to be
encountered, and passage of a high molecular weight molecule may be
desired. In these latter cases, materials which allow passage of
all molecules up to about the size of immunoglobulin M (about 1,000
kD) can be used. These materials will impede the passage of only
very large substances, such as cells.
[0062] In another aspect of the invention, it has been found that a
molecular weight cutoff for the jacket considerably higher than
that previously contemplated may be employed while maintaining the
viability and function of the encapsulated cells. This permits the
macrocapsules to be used in applications where the cells secrete a
substance of high molecular weight. For this purpose, a
macrocapsule with molecular cutoffs in excess of say 80 to 100 kD
to as high as 200 to 1000 or 2000 kD or more may be employed in
accordance with the present invention.
[0063] As used herein, the term "biocompatible" refers collectively
to both the intact vehicle and its contents. Specifically, it
refers to the capability of the implanted intact vehicle and its
contents to avoid detrimental effects of the body's various
protective systems and remain functional for a significant period
of time. In addition to the avoidance of protective responses from
the immune system, or foreign body fibrotic response,
"biocompatible" also implies that no specific undesirable cytotoxic
or systemic effects are caused by the vehicle and its contents such
as would interfere with the desired functioning of the vehicle or
its contents.
[0064] The jacket is biocompatible. That is, it does not elicit a
detrimental host response sufficient to result in rejection of the
implanted vehicle or to render it inoperable. Neither does the
jacket elicit unfavorable tissue responses such as fibrosis. In
addition, the external surface can be selected or designed in such
a manner that it is particularly suitable for implantation at the
selected site. For example, the external surface can be smooth,
stippled or rough, depending on whether attachment by cells of the
surrounding tissue is desirable. The shape or configuration can
also be selected or designed to be particularly appropriate for the
implantation site chosen.
[0065] The biocompatibility of the surrounding or peripheral region
(jacket) is produced by a combination of factors.
[0066] Important for biocompatibility and continued functionality
are vehicle morphology, hydrophobicity and the absence of
undesirable substances either on the surface of, or leachable from,
the vehicle itself. Thus, brush surfaces, folds, interlayers or
other shapes or structures eliciting a foreign body response are
avoided. The vehicle-forming materials are sufficiently pure that
unwanted substances do not leach out from the vehicle materials
themselves. Additionally, following vehicle preparation, the
treatment of the external surface of the vehicle with fluids or
materials (e.g. serum) which may adhere to or be absorbed by the
vehicle and subsequently impair vehicle biocompatibility are
avoided.
[0067] First, the materials used to form the vehicle are substances
selected based upon their ability to be compatible with, and
accepted by, the tissues of the recipient of the implanted vehicle.
Substances are used which are not harmful to the recipient or to
the isolated biologically active moiety. Preferred substances
include reversibly and irreversibly gellable substances (e.g.,
those which form hydrogels), and water-insoluble thermoplastic
polymers. Particularly preferred thermoplastic polymer substances
are those which are modestly hydrophobic, i.e. those having a
solubility parameter as defined in Brandrup J., et al. Polymer
Handbook 3rd Ed., John Wiley & Sons, NY (1989), between 8 and
15, or more preferably, between 9 and 14 (Joules/m.sup.3).sup.1/2.
The polymer substances are chosen to have a solubility parameter
low enough so that they are soluble in organic solvents and still
high enough so that they will partition to form a proper membrane.
Such polymer substances should be substantially free of labile
nucleophilic moieties and be highly resistant to oxidants and
enzymes even in the absence of stabilizing agents. The period of
residence in vivo which is contemplated for the particular
immunoisolatory vehicle must also be considered: substances must be
chosen which are adequately stable when exposed to physiological
conditions and stresses. There are many hydrogels and
thermoplastics which are sufficiently stable, even for extended
periods of residence in vivo, such as periods in excess of one or
two years. Examples of stable materials include alginate (hydrogel)
and polyacrilonitrile/polyvinylchlo- ride ("PAN/PVC" or
"thermoplastic").
[0068] Second, substances used in preparing the biocompatible
immunoisolatory vehicle are either free of leachable pyrogenic or
otherwise harmful, irritating, or immunogenic substances or are
exhaustively purified to remove such harmful substances.
Thereafter, and throughout the manufacture and maintenance of the
vehicle prior to implantation, great care is taken to prevent the
adulteration or contamination of the vehicle with substances which
would adversely affect its biocompatibility.
[0069] Third, the exterior configuration of the vehicle, including
its texture, is formed in such a manner that it provides an optimal
interface with the tissues of the recipient after implantation.
This parameter will be defined in part by the site of implantation.
For example, if the vehicle will reside in the peritoneal cavity of
the recipient, its surface should be smooth. However, if it will be
embedded in the soft tissues of the recipient, its surface can be
moderately rough or stippled. A determining factor will be whether
it is desirable to allow cells of the recipient to attach to the
external surface of the vehicle or if such attachment must be
avoided. An open-textured or sponge-like surface may promote the
ingrowth of capillary beds, whereas a smooth surface may discourage
excessive overgrowth by fibroblasts. Excessive overgrowth by
fibroblasts is to be avoided, except where capillary undergrowth
has occurred, as it may result in the deposition of a
poorly-permeable basement membrane around the vehicle and walling
off of the isolated cells from contact with the recipient's
body.
[0070] Certain vehicle geometries have also been found to
specifically elicit foreign body fibrotic responses and should be
avoided. Thus vehicles should not contain structures having
interlayers such as brush surfaces or folds. In general, opposing
vehicle surfaces or edges either from the same or adjacent vehicles
should be at least 1 mm apart, preferably greater than 2 mm and
most preferably greater than 5 mm. Preferred embodiments include
cylinders, "U"-shaped cylinders, and flat sheets or sandwiches.
[0071] The surrounding or peripheral region (jacket) of the
biocompatible immunoisolatory vehicle can optionally include
substances which decrease or deter local inflammatory response to
the implanted vehicle, and/or generate or foster a suitable local
environment for the implanted cells or tissues. For example
antibodies to one or more mediators of the immune response could be
included. Available potentially useful antibodies such as
antibodies to the lymphokines tumor necrosis factor (TNF), and to
interferons (IFN) can be included in the matrix precursor solution.
Similarly, an anti-inflammatory steroid can be included.
Christenson, L., et al., J. Biomed. Mat. Res., 23, pp. 705-718
(1989); Christenson, L., Ph.D. thesis, Brown University, 1989,
incorporated by reference.
[0072] Alternatively, a substance which stimulates angiogenesis
(ingrowth of capillary beds) can be included. This may be
particularly desirable where the isolated cells or tissues require
close contact with the recipient's bloodstream to function properly
(e.g., insulin-producing islets of Langerhans). Cell which are
genetically engineered to secrete antibodies may also be included
in the matrix.
[0073] Because of its biocompatibility, the vehicle is suitable for
long-term isolation of biologically useful cells and/or substances
from the various protective systems of the body. As used herein,
the term "protective systems" refers to the types of immunological
attack which can be mounted by the immune system of an individual
in whom the instant vehicle is implanted, and to other rejection
mechanisms, such as the fibrotic response, foreign body response
and other types of inflammatory response which can be induced by
the presence of a foreign object in the individuals' body.
[0074] The jacket of the present vehicle is immunoisolatory. That
is, it protects cells in the core of the vehicle from the immune
system of the individual in whom the vehicle is implanted. It does
so (1) by preventing harmful substances of the individual's body
from entering the core of the vehicle, (2) by minimizing contact
between the individual and inflammatory, antigenic, or otherwise
harmful materials which may be present in the core and (3) by
providing a spatial and physical barrier sufficient to prevent
immunological contact between the isolated moiety and detrimental
portions of the individual's immune system.
[0075] The thickness of this physical barrier can vary, but it will
always be sufficiently thick to prevent direct contact between the
cells and/or substances on either side of the barrier. The
thickness of this region generally ranges between 5 and 200
microns; thicknesses of 10 to 100 microns are preferred, and
thickness of 20 to 50 microns are particularly preferred. Types of
immunological attack which can be prevented or minimized by the use
of the instant vehicle include attack by macrophages, neutrophils,
cellular immune responses (e.g. natural killer cells.and
antibody-dependent T cell-mediated cytoloysis [ADCC]), and humoral
response (e.g. antibody-dependent complement mediated
cytolysis).
[0076] The type and extent of immunological response by the
recipient to the implanted vehicle will be influenced by the
relationship of the recipient to the isolated biologically active
moiety. For example, if the isolated materials comprise syngeneic
cells, these will not cause a vigorous immunological reaction,
unless the recipient suffers from an autoimmunity with respect to
the particular cell or tissue type within the vehicle. There are
several disease or deficiency states which have recently been
determined to have an autoimmune etiology, most notably Type I,
insulin-dependent Diabetes mellitus, wherein the insulin secreting
pancreatic islet .beta. cells are destroyed by the individual's
immune system. Fan, M.-Y. et al., Diabetes, 39, pp. 519-522
(1990).
[0077] Syngeneic cells or tissue are rarely available. In many
cases, allogeneic or xenogeneic cells or tissue (i.e., from donors
of the same species as, or from a different species than, the
prospective recipient) may be available. The immunoisolatory
vehicle allows the implantation of such cells or tissue, without a
concomitant need to immunosuppress the recipient. Therefore, the
instant vehicle makes it possible to treat many more individuals
than can be treated by conventional transplantation techniques. For
example, far more patients suffer from Type 1 diabetes than can be
transplanted with human donor islets (in 1990, fewer than about
4,000 suitable cadaver organ donors became available in the U.S.
for all organ transplants). The supply of donor porcine or bovine
islets is far greater; if these xenoislets are appropriately
immunoisolated according to the instant invention, the diabetic
condition of a far greater number of patients can be remedied.
[0078] The type and vigor of an immune response to xenografted
tissue is expected to differ from the response encountered when
syngeneic or allogeneic tissue is implanted into a recipient. This
rejection may proceed primarily by cell-mediated, or by
complement-mediated attack; the determining parameters in a
particular case may be poorly understood. However, as noted
previously, the exclusion of IgG from the core of the vehicle is
not the touchstone of immunoprotection, because IgG alone is
insufficient to produce cytolysis of the target cells or
tissues.
[0079] Using the macrocapsules of the present invention, preferably
with allogeneic tissue, but even with xenografts, it is possible to
deliver needed high molecular weight products or to provide
metabolic functions pertaining to high molecular weight substances,
provided that critical substances necessary to the mediation of
immunological attack are excluded from the immunoisolatory vehicle.
These substances may comprise the complement attack complex
component Clq, or they may comprise phagocytic or cytotoxic cells;
the instant immunoisolatory vehicle provides a protective barrier
between these harmful substances and the isolated cells. Thus, the
present immunoisolatory vehicle can be used for the delivery even
from allogeneic or xenogeneic cells or tissue, products having a
wide range of molecular sizes, such as insulin, parathyroid
hormone, interleukin 3, erythropoietin, albumin, transferrin, and
Factor VIII.
[0080] The jacket of the instant vehicle is made of a material
which may be the same as that of the core or may be different. In
either case, the material used results in a surrounding or
peripheral region which is permselective, biocompatible and
immunoisolatory. The jacket may be formed freely around the core
without chemical bonding or, alternatively, the jacket may be
directly cross-linked to the core matrix. In either case, formation
of the vehicle of the present invention does not require polymers
of opposite charge to the core being present in an interfacial
layer or in the jacket.
[0081] The surrounding or peripheral region (jacket) can be made of
a hydrogel matrix or of a different material, such as a
thermoplastic membrane. It can also be made of a matrix-membrane
composite, such that a permselective thermoplastic membrane having
matrix-filled pores, is formed.
[0082] Suitably, the external jacket may be formed of a
thermoplastic material known to be biocompatible, such as the ones
described herein. In addition, other jackets which have been used
in the microcapsule field may also be used herein, such as
alginate, suitably cross-linked with a multivalent ion such as
calcium.
[0083] Preferably, the core and external jacket form an interface
free of "ionic bonding" between oppositely charged polymers and
free of an intermediate layer of the type used in prior art
microcapsules. Ionic bonding refers to an ionic interaction of a
core of one charge (positive or negative) and the jacket (or an
intermediate layer) of opposite charge.
[0084] In previously existing devices, the core and jacket were
linked via ionic bonds between oppositely charged polymers in one
of two ways. (1) The devices of Rha (U.S. Pat. No; 4,744,933) were
constructed of a charged inner core material and an outer jacket
material of the opposite charge. (2) The devices of Lim and Sun
(U.S. Pat. Nos: 4,352,833 and 4,409,331) included an intermediate
layer of poly-L-lysine (PLL), which is positively charged, to link
the negatively charged core with the negatively charged jacket
material. The elimination of a PLL layer is advantageous in that
PLL is believed to be fibrogenic in the host. PLL may also have
unwanted growth effects for some cells. Also, the jacket of the
device of the invention can be controlled for permselectivity
better than those made with PLL.
[0085] The vehicle of the present invention is distinguished from
the microcapsules of Lim and Sun (Lim, F., Sun, A. M., Science 210,
pp. 908-910 (1980); Sun, A. M., Methods in Enzymology 137, pp.
575-579 (1988) by its outer jacket which ensures that cells cannot
project outside of the core. The capsules of Lim and Sun suffered
from the disadvantage that portions of encapsulated cells could
potentially project from the core through the poly-L-lysine layer
and thereby be more likely to elicit inflammatory responses from
the host's immune system. That microcapsule technology relies on
the presence of potentially bioactive ionic bonds to form the
microcapsule. By virtue of their ionic nature, those microcapsules
are susceptible to deterioration following implantation due to
competition for the ionic bonds that take place in the body of the
host after capsule implantation. This problem is minimized by the
relatively non-ionic macrocapsules of the present invention. A
further advantage of the macrocapsules of the present invention
lies in their capacity to contain more cells in a single device
than is possible in microcapsule technology.
[0086] The term "dual matrix vehicles" refers to vehicles with a
cell-containing core and an external jacket free of cells. In one
embodiment, the matrix core is, formed of a hydrogel which is
cross-linked to a hydrogel jacket, suitably in the form of a rod or
other shape. The hydrogel jacket may be formed independently as a
sheath around the matrix without cross-linking. The hydrogel care
is not necessarily linked to the outer jacket by means of opposite
ionic charges. In another embodiment, the external jacket is formed
of a thermoplastic material which is not linked to the core matrix
by chemical bonding.
[0087] If a dual matrix immunoisolatory vehicle is to be formed,
the surrounding or peripheral region can be made of a hydrogel
selected from the above-listed matrix precursors. If the
surrounding or peripheral region of the vehicle is to comprise a
permselective membrane, other precursor materials can be used. For
example, the surrounding or peripheral region can be made from
water-insoluble, biocompatible thermoplastic polymers or
copolymers. Several of the polymers or copolymers taught by
Michaels, U.S. Pat. No. 3,615,024, which is hereby incorporated by
reference, fulfill these criteria.
[0088] A preferred membrane casting solution comprises a
polyacrylonitrile-polyvinylchloride (PAN/PVC) copolymer dissolved
in the water-miscible solvent dimethylsulfoxide (DMSO). This
casting solution can optionally comprise hydrophilic or hydrophobic
additives which affect the permeability characteristics of the
finished-membrane. A preferred hydrophilic additive for the PAN/PVC
copolymer is polyvinylpyrrolidone (PVP). Other suitable polymers
comprise polyacrylonitrile (PAN), polymethyl-methacrylate (PMMA),
polyvinyldifluoride (PVDF), polyethylene oxide, polyolefins (e.g.,
polyisobutylene or polypropylene), polysulfones, and cellulose
derivatives (e.g., cellulose acetate or cellulose butyrate).
Compatible water-miscible solvents for these and other suitable
polymers and copolymers are found in the teachings of U.S. Pat. No.
3,615,024.
[0089] In a preferred embodiment, the core is surrounded by a
biocompatible hydrogel matrix free of cells projecting externally
from the outer layer. The macrocapsules of the present invention
are distinguished from the microcapsules of Rha, Lim, and Sun (Rha,
C. K. et al., U.S. Pat. 4,744,933; Sun, A. W., supra) by (1) the
complete exclusion of cells from the outer layer of the
macrocapsule, and (2) the thickness of the outer layer of the
macrocapsule. Both qualities contribute to the immunoisolation of
encapsulated cells in the present invention. The microcapsules of
Rha were formed by ionic interaction of an ionic core solution with
an ionic polymer of opposite charge. The microcapsules of Lim and
Sun were formed by linking an external hydrogel jacket to the core
through an intermediate layer of poly-L-lysine (PLL).
[0090] In the microcapsules of Lim and Sun, the intermediate PLL
layer was not sufficiently thick to guarantee that portions of the
encapsulated cells would not penetrate through and beyond the
layer. Cells penetrating the PLL layer are potential targets for an
immune response. All these capsules, including those disclosed by
Rha, also suffer the following additional limitations: (a) they are
round, and (b) the formation of the outer layer is dependent upon
direct ionic bonding or polyamide linkage with an inner layer or
core substance. The disadvantages of round shape and direct ionic
bonding between polymers are described supra.
[0091] In the capsules of Rha, Lim, and Sun, since the chemical
identity of the inner substance is either dictated by choice of
outer layer, or PLL, the ability to vary growth conditions on the
inside of these capsules is greatly limited. Since there are often
specific growth conditions which need to be met in order to
successfully encapsulate specific cell types, these capsules
generally have a limited utility or require considerable
experimentation to establish appropriate outer layers for a given
internal substance. In contrast, in the instant invention, the
identity of the core material does not place strict limitations on
the identity of the outer jacket material or vice versa. This
allows the material of the inner hydrogel to be selected according
to criteria important for cell viability and growth, and the outer
jacket material to be selected on the basis of immunoisolatory
properties, biocompatibility, and/or manufacturing
considerations.
[0092] The microcapsules of Rha, Lim, and Sun have a greater
potential for bioincompatibility, fibrogenesis, and vehicle
deterioration than do the macrocapsules of the present invention. A
variety of biological systems are known to interact with and break
down the ionic bonds required for the integrity of microcapsules.
PLL evokes unfavorable tissue reactions to the capsule. Most
notably, this is a fibrotic response. Thus, if there is any break
in the external layer, if it is not of sufficient thickness, if the
PLL layer begins to degrade, or if encapsulated cells are entrapped
within the external layer sufficiently close to its outer surface,
the microcapsule can trigger a fibrotic response. The term
"fibrogenic" is used herein in reference to capsules or materials
which elicit a fibrotic response in the implantation site. As set
forth herein, the external jacket of the immunoisolatory,
non-fibrogenic macrocapsule of the present invention may be formed
in a number of ways.
[0093] In one embodiment, the core is preformed by cross-linking a
hydrogel matrix with a cross-linking agent, preferably a
multivalent cation such as calcium. However, other known hydrogel
cross-linking agents may also be employed. After cross-linking, the
core is dipped into a solution of hydrogel to form a second layer
free of cells in the core which, simultaneously or thereafter, is
cross-linked suitably in the same manner. In the instant
embodiment, cross-linking of the core material with the jacket
material is accomplished via the cross-linking agent. For instance,
when the core and jacket materials are both negatively charged
hydrogels, the core and the jacket are cross-linked with each other
via their mutual attraction to the positive charges on the
cross-linking agent, preferably calcium. The core and jacket may be
formed of the same or different type of hydrogel, provided that
both have the same charge. Notably, the instant vehicle is not
formed through direct ionic bonding between anionic and cationic
polymers as described in Rha, C. K., U.S. Pat. No. 4,744,933.
[0094] Herein, the term "direct ionic bonding" refers to the type
of chemical bonding in which two oppositely charged polymers are
attracted to one another because of their oppositely charged
moieties. The instant embodiment is distinguished from that of Rha
because, in the instant embodiment, both the core material and
jacket material have the same charge, and their association is via
an oppositely charged cross-linking agent. This embodiment may be
in the form of a microcapsule or a macrocapsule but, for reasons
set forth herein, the macrocapsule form is preferred.
[0095] The present immunoisolatory vehicle can be formed in a wide
variety of shapes and combinations of suitable materials. A primary
consideration in selecting a particular configuration for the
vehicle when cells are present is the access of oxygen and
nutrients to the isolated cells or tissues, and passage of waste
metabolites, toxins and the secreted product from the vehicle. The
immunoisolatory vehicle can be any configuration appropriate for
maintaining biological activity and providing access for delivery
of the product or function, including for example, cylindrical,
rectangular, disk-shaped, patch-shaped, ovoid, stellate, or
spherical. Moreover, the vehicle can be coiled or wrapped into a
mesh-like or nested structure. If the vehicle is to be retrieved
after it is implanted, configurations which tend to lead to
migration of the vehicle(s) from the site of implantation, such as
spherical vehicles small enough to travel in the recipient's blood
vessels, are not preferred. Certain shapes, such as rectangles,
patches, disks, cylinders, and flat sheets offer greater structural
integrity and are preferable where retrieval is desired.
[0096] The instant vehicle must provide, in at least one dimension,
sufficiently close proximity of any isolated cells in the core to
the surrounding tissues of the recipient, including the recipient's
bloodstream, in order to maintain the viability and function of the
isolated cells. However, the diffusional limitations of the
materials used to form the vehicle do not in all cases solely
prescribe its configurational limits. Certain additives can be used
which alter or enhance the diffusional properties, or nutrient or
oxygen transport properties, of the basic vehicle. For example, the
internal medium can be supplemented with oxygen-saturated
perfluorocarbons, thus reducing the needs for immediate contact
with blood-borne oxygen. This will allow isolated cells.or tissues
to remain viable while, for instance, a gradient of angiotensin is
released from the vehicle into the surrounding tissues, stimulating
ingrowth of capillaries. References and methods for use of
perfluorocarbons are given by Faithful, N. S. Anaesthesia, 42, pp.
234-242 (1987) and NASA Tech Briefs MSC-21480, U.S. Govt. Printing
Office, Washington, D.C. 20402, incorporated herein by reference.
Alternatively for clonal cell lines such as PC12 cells, genetically
engineered hemoglobin sequences may be introduced into the cell
lines to produce superior oxygen storage. NPO-17517 NASA Tech
Briefs, 15, p. 54.
[0097] In general, in the absence of oxygen carrier additives, when
the cells are present the vehicle will have a maximum
depth-to-surface distance of no more than 2 mm in at least one
dimension, with a maximum depth of 800 microns being preferred. One
or several vehicles may be required to produce the desired effect
in the recipient.
[0098] The thickness of the immunoisolatory vehicle jacket should
be sufficient to prevent an immunoresponse by the patient to the
presence of the vehicles. For that purpose, the vehicles preferably
have a minimum thickness of 1 .mu.m or more free of the cells.
[0099] Additionally, reinforcing structural elements can be
incorporated into the vehicle. These structural elements can be
made in such a fashion that they are impermeable, and are
appropriately configured to allow tethering or suturing of the
vehicle to the tissues of the recipient. In certain circumstances,
these elements can act to securely seal the surrounding or
peripheral region (e.g., at the ends of a cylindrical vehicle, or
at the edges of a disk-shaped vehicle), completing isolation of the
core materials (e.g., a molded thermoplastic clip). For many
configurations, it is desirable that these structural elements
should not occlude a significant area of the permselective
surrounding or peripheral region.
[0100] In one preferred embodiment, the implantable immunoisolatory
vehicle of the present invention is of a sufficient size and
durability for complete retrieval after implantation. To be
contrasted with such microcapsules, which have a typical maximum
practical volume on the order of 1 .mu.l, the preferred
immunoisolatory vehicle of the present invention is termed
"macrocapsule". Such macrocapsules have a core of a preferable
minimum volume of about 1 to 1 .mu.l and depending upon use are
easily fabricated to have a value in excess of 100 .mu.l.
[0101] In terms of retrievability, microspheres are generally less
practical than macro-capsules. In order for tissue encapsulated in
microspheres to provide a therapeutic dose of insulin, for
instance, the number of microspheres must be increased to such a
large extent that significant retrievability becomes impossible.
Additionally, an increase in the volume of tissue placed within a
microsphere requires a corresponding increase in surface area.
Within a sphere, because surface area scales with r.sup.2 where as
volume scales with r.sup.3, as the volume of encapsulated tissue
volume increases, the required capsule size to provide sufficient
surface area for nutrient diffusion to the encapsulated tissue
quickly becomes impractical.
[0102] Macrocapsules in the shapes of cylinders or flat sheets do
not have these limitations because volume increases more
proportionately to surface area such that the diffusional transport
of nutrients and products for increased amounts of tissue can be
accommodated by increasing the surface area without unwieldy
increases in total vehicle size. If, for example, about 10,000
islets are required per kg body weight to restore normoglycemia to
a diabetic patient, from 1,000 to 10,000 microcapsules must be
transplanted per kg body weight (e.g. 1-10 islets per capsule).
This number of microcapsules could not be easily retrieved, if
retrieval were required. In contrast, the macrocapsules of the
instant invention may easily hold greater than 1,000 islets to as
high as 500,000 islets or more per vehicle. The preferred
embodiment would require fewer than 5-10 vehicles per patient,
making macrocapsules more easily retrieved than a large number of
microcapsules.
[0103] The macrocapsules of the present invention are distinguished
from microcapsules (Sun, A. M., supra; Rha, U.S. Pat. No.
4,744,933) by the capacity of macrocapsules to contain over
10.sup.4 cells and maintain them in viable condition. The droplet
methods used in the making of microcapsules, in order to ensure
cell viability, necessarily limit the number of cells per capsule
to fewer than 10.sup.4.
[0104] The instant invention also relates to a method for making an
immunoisolatory vehicle. The vehicles of this invention can be
formed either by coextrusion or stepwise assembly. Techniques for
coextrusion which can be used to form the instant vehicle are
taught in copending U.S. patent application Ser. No. 07/461,999,
filed Jan. 8, 1990, which is herein incorporated by reference. For
example, a coextrusion device similar to that taught by U.S.
application Ser. No. 07/461,999 is used in making the subject
vehicle. The device has a nested-bore extrusion nozzle, the lumen
of each bore (inner and outer) being appropriately connected to
sterile chambers for delivery of the core and surrounding region
materials.
[0105] The nozzle can be of any configuration appropriate to
produce an immunoisolatory vehicle whose shape is appropriate to
the metabolic needs of the cells to be immobilized and the
permeability and strength of the matrix which will surround them.
For example, the nozzle can be circular, elliptical, stellate, or
slot-shaped. optionally, the nested bores can be coaxial. The
widest aperture of the nozzle must be commensurate with the maximum
diffusional depth appropriate to the vehicle being formed,
including the metabolic needs of the isolated cells or tissues, and
the materials of the core and peripheral regions.
[0106] Upon extrusion of the core and peripheral region materials
from the inner and outer chambers through the corresponding bores
of the nozzle under conditions sufficient to gel, harden, or cast
the matrix or membrane precursor(s) of the surrounding or
peripheral region (and of the core region), an elongated vehicle of
the selected shape is continuously formed. The length of the
vehicle, and therefore its volume or capacity, can be controlled to
produce vehicles of sizes appropriate for the particular use
contemplated.
[0107] An immunoisolatory vehicle formed by coextrusion in this
manner is particularly preferred because use of this means ensures
that the cells in the core are isolated from the moment of
formation of the vehicle. Thus, it also ensures that the core
materials do not become contaminated or adulterated during handling
of the vehicle prior to implantation.
[0108] Furthermore, the nature of the coextrusion process is such
that it ensures that the surrounding or peripheral region (jacket)
is free of the materials in the core, including the cells, thus
assuring that these cells will be immunoisolated when the vehicle
is implanted into an individual. The permeability, molecular weight
cutoff, and biocompatibility characteristics of the surrounding or
peripheral region are determined by both the chosen matrix or
membrane precursor materials used, and the conditions under which
the matrix or membrane is formed.
[0109] The co-extruded vehicles may be formed with a hydrogel
matrix core and a thermoplastic or hydrogel jacket. Such
macrocapsules may be formed with a core and jacket of the same or
different hydrogel which may be cross-linked to each other or
not.
[0110] If a dual-matrix immunoisolatory vehicle (e.g., an alginate
matrix) is formed, the permeability of the surrounding matrix can
be determined by adjusting the concentration of matrix precursor
used (e;g., sodium alginate), and/or the concentration of gelling
agent (in the case of alginate, a divalent cation such as
Ca.sup.++) present in an immersion bath into which the materials
are coextruded.
[0111] If an immunoisolatory vehicle with a surrounding or
peripheral region of thermoplastic membrane is desired, the pore
size range and distribution can be determined by varying the solids
content of the solution of precursor material (the casting
solution), the chemical composition of the water-miscible solvent,
or optionally including a hydrophilic or hydrophobic additive to
the casting solution, as taught by U.S. Pat. No. 3,615,024. The
pore size may also be adjusted by varying the hydrophobicity of the
coagulant and/or of the bath.
[0112] Typically, the casting solution will comprise a polar
organic solvent containing a dissolved, water-insoluble polymer or
copolymer. This polymer or copolymer precipitates upon contact with
a solvent-miscible aqueous phase, forming a permselective membrane
at the site of interface. The size of pores in the membrane depends
upon the rate of diffusion of the aqueous phase into the solvent
phase; the hydrophilic or hydrophobic additives affect pore size by
altering this rate of diffusion. As the aqueous phase diffuses
farther into the solvent, the remainder of the polymer or copolymer
is precipitated to form a trabecular support which confers
mechanical strength to the finished vehicle.
[0113] The external surface of the vehicle is similarly determined
by the conditions under which the dissolved polymer or copolymer is
precipitated (i.e., exposed to the air, which generates an open,
trabecular or sponge-like outer skin, immersed in an aqueous
precipitation bath, which results in a smooth permselective
membrane bilayer, or exposed to air saturated with water vapor,
which results in an intermediate structure). Again, it will be
readily appreciated that this method of forming the immunoisolatory
vehicle ensures that the peripheral or surrounding membrane is free
of the cells in the core which are desired to be isolated from the
immune system of the individual in whom the vehicle is to be
implanted.
[0114] The surface texture of the vehicle is dependent in part on
whether the extrusion nozzle is positioned above, or immersed in,
the bath: if the nozzle is placed above the surface of the bath a
roughened outer skin of PAN/PVC will be formed, whereas if the
nozzle is immersed in the bath a smooth external surface is
formed.
[0115] The iumunoisolatory vehicle can also be formed in a stepwise
manner. For vehicles wherein the core comprises, in addition to the
cells desired to be isolated, a hydrogel matrix, the core can be
formed initially, and the surrounding or peripheral matrix or
membrane can be assembled or applied subsequently. The matrix core
can either be formed by extrusion or by molding. In a preferred
embodiment, a patch- or sheet-shaped vehicle is formed by stepwise
extrusion of calendared sheets. In this embodiment, a sheet of core
material is layered onto a sheet of peripheral region material,
then covered by a second sheet of peripheral region material. The
edges of the vehicle are then sealed by crimping, compressing,
heating, sealing with a biocompatible glue, or binding into a
preformed biocompatible impermeable clip or combinations of the
above.
[0116] Conversely, the surrounding or peripheral matrix or membrane
can be preformed, filled with the materials which will form the
core (for instance, using a syringe), and subsequently sealed in
such a manner that the core materials are completely enclosed. The
vehicle can then be exposed to conditions which bring about the
formation of a core matrix if a matrix precursor material is
present in the core. Alternatively, a patch- or sheet-shaped matrix
core can be formed by molding, then sandwiched between sheets of
permselective membrane and sealed or clipped in the manner
described above to complete the isolation of the core
materials.
[0117] It is also possible for a single, continuous hydrogel matrix
to provide both immunoisolation and support or immobilization. For
example, this can be accomplished by including the isolated cells
within the vehicle distributed in a concentric gradient about the
core, such that the peripheral region of the vehicle is free of the
immobilized cells. Immunoisolatory vehicles of this nature can be
made in at least two ways. First, a mixture of cells suspended in a
solution of a hydrogel matrix precursor, which is denser than the
isolated cells, can be extruded from the nozzle of a simple
extrusion device. In this manner, the cells are forced into the
core region of the forming vehicle. Alternatively, the cell-matrix
precursor mixture can be extruded from the core lumen of a
nested-bore nozzle, while simultaneously delivering a stream of a
gelling agent (e.g., for alginate, a solution of calcium chloride)
through the peripheral nozzle, whereby the surface and periphery of
the vehicle polymerize first, thus forcing the suspended cells into
the core.
[0118] As noted previously, the vehicle can provide for the
implantation of diverse cell or tissue types, including
fully-differentiated, anchorage-dependent, fetal or neonatal, or
transformed, anchorage-independent cells or tissue. The cells to be
immunoisolated are prepared either from a donor (i.e., primary
cells or tissues, including adult, neonatal, and fetal cells or
tissues) or from cells which replicate in vitro (i.e., immortalized
cells or cell lines, including genetically modified cells). In all
cases, a sufficient quantity of cells to produce effective levels
of the needed product or to supply an effective level of the needed
metabolic function is prepared, generally under sterile conditions,
and maintained appropriately (e.g. in a balanced salt solution such
as Hank's salts, or in a nutrient medium, such as Ham's F12) prior
to isolation.
[0119] In another aspect of the invention, the macrocapsules are of
a shape which tends to reduce the distance between the center of
the macrocapsule and the nearest portion of the jacket for purposes
of permitting easy access of nutrients from the patient into the
cell or of entry of the patient's proteins into the cell to be
acted upon by the cell to provide a metabolic function, such as
interaction with cholesterol or the like. In that regard, a
non-spherical shape is preferred, such as a long tube or flat
plate, or the like. The optimum shape for this purpose may be
calculated by known techniques as set forth herein.
[0120] Four important factors influencing the number of cells or
amount of tissue to be placed within the core of the biocompatible
immunoisolatory vehicle (i.e., loading density) of the instant
invention are: (1) vehicle size and geometry; (2) mitotic activity
within the vehicle; (3) viscosity requirements for core preparation
and or loading; and (4) pre-implantation assay and qualification
requirements.
[0121] With respect to the first of these factors, (capsule size
and geometry), the diffusion of critical nutrients and metabolic
requirements into the cells as well as diffusion of waste products
away from the cell are critical to the continued viability of the
cells. Since diffusional access to the contents of the vehicle is
limited by vehicle surface area, surface to volume relationships of
various shapes and size vehicles will be critical in determining
how much viable tissue can be maintained within the vehicle.
[0122] Among the metabolic requirements met by diffusion of
substances into the vehicle is the requirement for oxygen. The
oxygen requirements of the specific cells must be determined for
the cell of choice. Methods and references for determination of
oxygen metabolism are given in Wilson D. F. et al., J. Biol. Chem.,
263, pp. 2712-2718, (1988). The oxygen requirement for islet cells
has been applied to coupled diffusion reaction models accounting
for diffusional transport from surrounding tissue through the
vehicle wall and tissue compartment (core), and used to calculate
the expected viability of islet cells in a number of vehicles of
different sizes and configurations, after the method of Dionne, K.
E., Ph.D. Thesis, Massachusetts Institute of Technology (1989). For
intact pancreatic islets, these calculations agree well with
experimental observations.
[0123] For a cylindrical vehicle of 900 microns outer diameter,
implanted into the peritoneal cavity (pO2.apprxeq.45-50 mmHg), the
optimal total cell volume is in the range of up to 20%, preferably
1- 15%, most preferably about 5% of the vehicle volume. If this
capsule were 20 cm in length it would have a volume of 100
mm.sup.3. To provide the same amount of surface area with a single
sphere, e.g. to support comparable amounts of tissue; would require
a volume of 1,047 mm.sup.3.
[0124] For a cylindrical vehicle of 400 microns the optimal cell
volume is between 35-65% total vehicle volume, and is preferably
35%. These calculations also take into account the partial oxygen
pressure at the site of implantation. At implantation sites where
the oxygen pressure is less than the peritoneum (e.g., subcutaneous
pO2.apprxeq.20 mmHg), lower loading densities will be required.
Implantation into arteries (pO2.gtoreq.95 mmHg) and the brain
(pO2>75 mmHg) will allow support of greater tissue volume per
unit vehicle.
[0125] Other vehicle configurations, such as disk-shaped or
spherical, are also-possible and optimal cell volumes may be
similarly calculated for these geometries. Actual loading densities
will consider not only these diffusional considerations but also
the other considerations given below.
[0126] With respect to the second factor (cell division), if the
cells selected are expected to be actively dividing while in the
vehicle, then they will continue to divide until they fill the
available space, or until phenomena such as contact inhibition
limit further division. For replicating cells, the geometry and
size of the vehicle will be chosen so that complete filling of the
vehicle core will,not lead to deprivation of critical nutrients due
to diffusional limitations. In general, vehicles that will be
filled to confluency with cells or tissue will be no more than 250
microns in cross-section, such that cells in the interior will have
less than 15 cells between them and an external diffusional
surface, preferably less than 10 cells and more preferably less
than 5 cells.
[0127] In general, for cells not expected to divide within the
vehicle, such as chromaffin cells, pancreatic islet cells and the
like, the appropriate cell densities will be calculated from the
diffusional considerations listed above.
[0128] With respect to the third factor (viscosity of core
materials) cells in densities occupying up to 70% of the vehicle
volume can be viable, but cell solutions in this concentration
range would have considerable viscosity. Introduction of cells in a
very viscous solution into the vehicle could be prohibitively
difficult. In general, for both two step and coextrusion
strategies, discussed below, cell loading densities of higher than
30% will seldom be useful, and in general optimal loading densities
will be 20% and below. For fragments of tissues, it is important,
in order to preserve the viability of interior cells, to observe
the same general guidelines as above and tissue fragments should
not exceed 250 microns in diameter with the interior cells having
less than 15, preferably less than 10 cells between them and the
nearest diffusional surface.
[0129] Finally, with respect to the fourth factor (preimplantation
and assay requirements), in many cases, a certain amount of time
will be required between vehicle preparation and implantation. For
instance, it may be important to qualify the vehicle in terms of
its biological activity. Thus, in the case of mitotically active
cells, preferred loading density will also consider the number of
cells which must be present in order to perform the qualification
assay.
[0130] In most cases, prior to implantation in vivo it will be
important to use in vitro assays to establish the efficacy of the
biologically active moiety within the vehicle. Vehicles containing
the moiety of interest can be constructed and analyzed using model
systems. In a preferred embodiment of the instant invention, the
diffusion of glucose into the vehicle is used to stimulate insulin
release from pancreatic islet cells. The appearance of insulin
outside the vehicle is monitored through the use of an
appropriately specific radioimmunoassay. Such procedures allow the
determination of the efficacy of the vehicle on a per cell or unit
volume basis.
[0131] Following the above guidelines for vehicle loading and for
determination of vehicle efficacy, the actual vehicle size for
implantation will then be determined by the amount of biological
activity required for the particular application. In the case of
secretory cells releasing therapeutic substances, standard dosage
considerations and criteria known to the art will be used to
determine the amount of secretory substance required. Factors to be
considered include; the size and weight of the recipient; the
productivity or functional level of the cells; and, where
appropriate, the normal productivity or metabolic activity of the
organ or tissue whose function is being replaced or augmented. It
is also important to consider that a fraction of the cells may not
survive the imunoisolation and implantation procedures, as well as
whether the recipient has a preexisting condition which can
interfere with the efficacy of the implant. Vehicles of the instant
invention can easily be manufactured which contain many thousands
of cells. In preferred embodiments, therapeutically useful
immunoisolatory vehicles used to provide insulin to insulin
deficient rats contained on the order of 1,000 islets. Larger
vehicles can also be conveniently prepared by the method of the
current invention.
[0132] Because of the potentially large capacity of the
immunoisolatory vehicles, the treatment of many conditions will
require only one or at most a few (less than 10) implanted vehicles
to supply an appropriate therapeutic dose. The use of only a few
therapeutically effective implantable vehicles containing a large
number of cells provides simple retrievability which, for many
applications, will be preferred over microsphere or other small
configurations requiring a large number of vehicles. The
immunoisolatory macrocapsule of the present invention is capable of
storing 10,000 to 100,000 cells to as high as 500,000 cells or
more, in individual or cluster form, depending on their type.
[0133] This invention also pertains to a method of isolating or
protecting biologically active moieties, such as implanted cells,
tissues, or other materials from immunological attack. The methods
and vehicles of the instant invention are useful to deliver a wide
range of cellular products, including high molecular weight
products, to an individual in need of them, and/or to provide
needed metabolic functions to an individual, such as the removal of
harmful substances.
[0134] Products which can be delivered using the instant vehicle
include a wide variety of factors normally secreted by various
organs or tissues. For example, insulin can be delivered to a
diabetic patient, dopamine to a patient suffering from Parkinson's
disease, or Factor VIII to a Type A hemophiliac.
[0135] Other products which can be delivered through use of the
instant vehicle include trophic factors such as erythropoietin,
growth hormone, Substance P, and neurotensin. This invention is
useful for treating individuals suffering from acute and/or chronic
pain, by delivery of an analgesic or pain reducing substance to the
individual. Such pain reducing substances include enkephalins,
catecholamines and other opioid peptides. Such compounds may be
secreted by, e.g., adrenal chromaffin cells. Another family of
products suited to delivery by the instant vehicle comprises
biological response modifiers, including lymphokines and cytokines.
Antibodies from antibody secreting cells may also be delivered.
Specific antibodies which may be useful include those towards tumor
specific antigens. The release of antibodies may also be useful in
decreasing circulating levels of compounds such as hormones or
growth factors. These products are useful in the treatment of a
wide variety of diseases, inflammatory conditions or disorders, and
cancers.
[0136] The instant vehicle can also be used to restore or augment
vital metabolic functions, such as the removal of toxins or harmful
metabolites (e.g., cholesterol) from the bloodstream by cells such
as hepatocytes. The method and vehicle of the instant invention
make possible the implantation of cells without the concomitant
need to immunosuppress the recipient for the duration of treatment.
Through use of the biocompatible immunoisolatory vehicle,
homeostasis of particular substances can be restored and maintained
for extended periods of time. The instant vehicle may contain a
multiplicity of cells, such that implantation of a single vehicle
can be sufficient to provide an effective amount of the needed
substance or function to an individual.
[0137] In one embodiment of this invention, methods are provided
for the prevention or treatment of neural degeneration. Such neural
degeneration occurs naturally as a result of the aging process,
typically in physically mature individuals, or may occur as a
result of a neurological disorder or disease. Examples of human
diseases or disorders which are thought to be associated with
neural degeneration include Alzheimer's disease, Huntington's
chorea, AIDS-related dementia, and Parkinson's disease. These
disorders, often occur in physically mature individuals. However,
these and other neurological disorders may occur in juveniles.
[0138] As used herein, an "aged" individual is an individual in
whom neural degeneration has occurred or is occurring, either as a
result of the natural aging process, or as a result of a
neurodegenerative disorder. Neural degeneration as a result of the
natural aging process means loss or decline of neural function
compared to a previous state not attributable to a defined clinical
abnormality or neurological disorder, such as Alzheimer's,
Parkinson's or Huntington's.
[0139] Animal models for neurodegenerative conditions are based on
the premise that a specific insult may damage or kill neurons. In
some cases this may even lead to a cascade of neuronal death which
affects trophically interdependent neurons along pathways
responsible for specific brain functions.
[0140] A strategy for treatment of neural degenerative condition
involves the localized administration of growth or trophic factors
in order to (1) inhibit further damage to post-synaptic neurons,
and (2) improve viability of cells subjected to the insult. Factors
known to improve neuronal viability include basic fibroblast growth
factor, ciliary neurotrophic factor, brain-derived neurotrophic
factor, neurotrophin-3, neurotensin, and Substance P.
[0141] In one animal model for neurodegenerative excitotoxicity,
the glutamate analog, quinolinic acid, is injected stereotaxically
into the brain region known as the striatum and/or basal ganglia to
produce neuropathology and symptoms analogous to those of patients
suffering from Huntington's disease. Both the model and actual
Huntington's disease are characterized by damage to neurons
necessary for aspects of motor control.
[0142] Furthermore, one of the early symptoms of Huntington's
disease is loss of body weight (Sanberg, P. R. et al. Med J Aust.
1, pp. 407-409 (1981). Similar effects are also seen in the model
system (Sanberg, P. R. et al. Exp Neurol, 66,-pp. 444-466 (1979).
Quinolinic acid is also found at abnormally high levels in
AIDS-related dementia.
[0143] According to the present invention, trophic factors are
provided to the proper brain region by implanting a
vehicle-containing living cells which secret an appropriate factor.
Suitably, the cells are adrenal chromaffin cells which are known to
secrete at least one factor, basic fibroblast growth factdr. Other
as yet unidentified trophic factors may also be secreted by
chromaffin cells. It is to be noted that this embodiment of the
invention is separate from the use of chromaffin cells to secrete
the neurotransmitter, dopamine, for the amelioration of symptoms of
Parkinson's disease. Nerve growth factor-secreting cells such as
fibroblasts engineered to express NGF represent an alternative
therapy for this quinolinic acid induced neurodegeneration. Schwann
cells prepared from myelin may be encapsulated and implanted in
appropriate brain areas to prevent neural degeneration associated
with Parkinson's disease.
[0144] In another embodiment of the invention, the animal model
involves lesion of the fimbria-fornix. In particular, neurons of
the septohippocampal system are axotomized which leads to
degeneration and cell death. This model is thought to be analogous
to the types of lesions which cause Alzheimer's disease in humans.
Suitably, a growth factor, nerve growth factor (NGF), is provided
by the implantation of a vehicle containing cells which secrete
NGF. Astrocytes immortalized (e.g. by transformation with the Large
T antigen) and genetically engineered to express NGF may be
employed. Preferably, the cells are fibroblasts which have been
genetically engineered to produce recombinant NGF. The fibroblasts
survive best in a core composed of a matrix material which mimics
extracellular matrix, such as collagen or laminin-containing
hydrogels. The core is surrounded by an immunoisolatory jacket
which allows the diffusion of oxygen and nutrients to the cells in
the core, and also allows the secreted NGF to diffuse through the
jacket and into the body of the recipient. The vehicle implant
inhibits the death of cholinergic neurons as assayed by the number
of neurons which contain choline acetyl transferase (ChAT), an
indicator of viable cholinergic neurons.
[0145] Fimbria-fornix lesions also cause behavioral deficits in the
animal subjects of the model, most easily observed in tasks
involving learning and memory. It has been reported that chronic
administration of NGF to rats with fimbria-fornix lesions
accelerates the animals' behavioral recovery (Wills, B. et al.
Behav.Brain Res., 17, pp. 17-24 (1985)). In the present invention,
implantation of the vehicle containing NGF-secreting cells provides
a practical way to deliver NGF continuously to the appropriate
brain region of the lesioned animal. By analogy, the vehicle of the
present invention offers a practical form of regenerative and/or
prophylactic therapy for Alzheimer's victims whose conditions may
be ameliorated by continuous delivery of NGF to specific brain
regions.
[0146] A wide variety of biologically active moieties or cells may
be used in this invention. These include well known, publicly
available immortalized cell lines as well as primary cell cultures.
Examples of publicly available cell lines suitable for the practice
of this invention include, baby hamster kidney (BHK), chinese
hamster ovary (CHO), mouse fibroblast (L-M), NIH Swiss mouse embryo
(NIH/3T3), African green monkey cell lines (including COS-a, COS-7,
BSC-1, BSC-40, BMT-10 and Vero), rat adrenal pheochromocytoma
(PC12) and rat glial tumor (C6). Primary cells that may be used
according to the present invention include, bFGF-responsive neural
progenitor-stem cells derived from the CNS of mammals (Richards et
al., Proc. Natl. Acad. Sci. USA 89, pp. 8591-8595 (1992); Ray et
al., Proc. Natl. Acad. Sci. USA, 90, pp. 3602-3606 (1993)), primary
fibroblasts, Schwann cells, astrocytes, .beta.-TC cells, Hep-G2
cells, AT T20 cells, oligodendrocytes and their precursors,
myoblasts, adrenal chromaffin cells, and the like.
[0147] Schwann cells maybe prepared according to the method of
Bunge (PCT published application WO 92/03536), mixed with a
suitable substratum such as Matrigel.TM., and encapsulated. The
encapsulated cells may be implanted in appropriate areas of the
brain to prevent the degeneration of the dopaminergic neurons of
the nigral striatal pathway associated with Parkinson's disease.
Generally, the preferred implant site will be in or near the
striatum. Encapsulating the cells may enhance secretion of trophic
factors since the cells will not be in proximal contact with
neurons, and myelination will not occur. Other glial cell types may
be encapsulated and implanted for this purpose, including
astrocytes and oligodendrocytes.
[0148] The choice of biologically active moiety or cell depends
upon the intended application. The encapsulated cells may be chosen
for secretion of a neurotransmitter. Such neurotransmitters include
dopamine, gamma aminobutyric acid (GABA), serotoninf acetylcholine,
noradrenaline, epinephrine, glutamic acid, and other peptide
neurotransmitters. Cells can also be employed which synthesize and
secrete agonists, analogs, derivatives or fragments of
neurotransmitters which are active, including, for example, cells
which secrete bromocriptine, a dopamine agonist, and cells which
secrete L-dopa, a dopamine precursor.
[0149] The encapsulated cells can also be chosen for their
secretion of hormones, cytokines, growth factors, trophic factors,
angiogensis factors, antibodies, blood coagulation factors,
lymphokines, enzymes, and other therapeutic agents or agonists,
precursors, active analogs, or active fragments thereof. These
include enkephalins, catecholamines, endorphins, dynorphin,
insulin, factor VIII, erythropoietin, Substance P, nerve growth
factor (NGF), Glial derived Neurotrophic Factor (GNDF),
platelet-derived growth factor (PDGF), epidermal growth factor
(EGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), an array of fibroblast growth factors, and ciliary
neurotrophic factor.
[0150] Alternatively, one or more biologically active molecules may
be delivered into the capsule. For example, the capsule may contain
one or more cells or substances which "scavenge" cholesterol, or
other biological factors, from the host.
[0151] Techniques and procedures for isolating cells or tissues
which produce a selected product are known to those skilled in the
art, or can be adapted from known procedures with no more than
routine experimentation. For example, islets of Langerhans can be
isolated from a large-animal pancreas (e.g., human or porcine)
using a combination of mechanical distention and collagenase
digestion, as described by Scharp, D. W., et al., U.S. Pat. No.
4,868,121. Islets may be isolated from small animals such as rats
by the method of Scharp, et al., Diabetes 29, suppl. 1, pp. 19-30
(1980). Similarly, hepatocytes can be isolated from liver tissue
using collagenase digestion followed by tissue fractionation, as
described by Sun, A. M., et al., Biomat., Art. Cells, Art. Org.,
15, pp. 483-496 (1987). Adrenal Chromaffin cells may be isolated by
the method of Livett, B. G., Physiology Reviews, 64, pp. 1103-1161
(1984).
[0152] Many cellular products which are difficult to provide using
primary donor tissues can be provided using immortalized cells or
cell lines. Immortalized cells are those which are capable of
indefinite replication but which exhibit contact inhibition upon
confluence and are not tumorigenic. An example of an immortalized
cell line is the rat pheochromocytoma cell line PC12. Transformed
cells or cell lines can be used in a similar manner. Transformed
cells are unlike merely immortalized cells in that they do not
exhibit contact inhibition upon confluence, and form tumors when
implanted into an allogeneic host. Immortalization can allow the
use of rare or notoriously fragile cell or tissue types for the
long-term delivery of a chosen product or metabolic function.
Suitable techniques for the immortalization of cells are described
in Land H. et al., Nature 304, pp. 596-602 (1983) and Cepko, C. L.,
Neuron 1, pp. 345-353 (1988). Candidate cell lines include
genetically engineered beta-cell lines which secrete insulin such
as NIT cells (Hamaguchi, K., et al., Diabetes 40, p. 842 (1991)),
RIN cells (Chick, W. L., et al., Proc. Natl. Acad. Sci. USA, 74,
pp. 628-632 (1977)), ATT cells (Hughes, S. D., et al, Proc. Natl.
Acad. Sci. USA, 89, pp. 688-692 (1992)), CHO cells (Matsumoto, M.,
et al, 1990, Proc. Natl. Acad. Sci. USA, 87, pp. 9133-9137 (1990)),
and beta-TC-3 cells (Tal, M., et al, 1992, Mol. Cell Biol., 12, pp.
422-432 (1992)). Additionally, recombinant cells or cell lines can
be engineered to provide novel products or functions and
combinations thereof, using a wide variety of techniques well known
to those of ordinary skill in the art.
[0153] For example, fibroblasts can be transfected with an
expression vector for the chosen product (e.g., nerve growth
factor, erythropoietin, insulin, or Factor VIII). It should be
recognized however, that expression of a recombinant protein in a
cell type which does not normally express the protein may lead to
unregulated expression which may not be desirable for certain
medical applications.
[0154] B-cell hybridomas secreting a selected monoclonal antibody,
or T-cell hybridomas secreting a selected lymphokine, can also be
used. It may be particularly desirable to deliver a monoclonal
antibody or fraction thereof, which neutralizes the biological
activity of a disregulated biological response modifier using the
instant invention. Engineered cells which secrete soluble fragments
of receptors for these biological response modifiers can be used in
a similar fashion. The disregulation or overproduction of
particular biological response modifiers has been implicated in the
etiology of certain cancers.
[0155] The encapsulated material can be tissue or cells able to
secrete such antinociceptive agents, including any one of
catecholamines, enkephalins, opioid peptides or mixtures thereof.
Preferably catecholamines are secreted, most preferably a mixture
of catecholamines and enkephalins. Typically, the encapsulated
material can be tissue of the adrenal medulla, or more
particularly, adrenal medulla chromaffin cells. Additionally,
genetically engineered cell lines or other naturally occurring cell
lines able to secrete at least one pain reducing agent such as a
catecholamine, enkephalin, opioid peptide, or agonist analog
thereof, can be used.
[0156] If the cells to be immunoisolated are replicating cells or
cell lines adapted to growth in vitro, it is particularly
advantageous to generate a cell bank of these cells. A particular
advantage of a cell bank is that it is a source of cells prepared
from the same culture or batch of cells. That is, all cells
originated from the same source of cells and have been exposed to
the same conditions and stresses. Therefore, the vials can be
treated as identical clones. In the transplantation context, this
greatly facilitates the production of identical or replacement
immunoisolatory vehicles. It also allows simplified testing
protocols which assure that implanted cells are free of
retroviruses and the like. It may also allow for parallel
monitoring of vehicles in vivo and in vitro, thus allowing
investigation of effects or factors unique to residence in
vivo.
[0157] In all cases, it is important that the cells or tissue
contained in the vehicle are not contaminated or adulterated. If a
vehicle having a matrix core is desired, the cells are mixed under
sterile conditions, with an appropriate amount of a biocompatible,
gellable hydrogel matrix precursor. There are numerous natural and
synthetic hydrogels which are suitable for use in a biocompatible
immunoisolatory vehicle of the instant invention. Suitable
naturally-derived hydrogels include plant-derived gums, such as the
alkali metal alginates and agarose, and other plant-derived
substances, such as cellulose and its derivatives (e.g.,
methylcellulose). Animal tissue-derived hydrogels such as gelatin
are also useful. Alternatively, the core matrix can be made of
extracellular matrix (ECM) components, as described by Kleinman et
al., U.S. Pat. No. 4,829,000. Suitable synthetic hydrogels include
polyvinyl alcohol, block copolymer of ethylene-vinylalcohol, sodium
polystyrene sulfonate, vinyl-methyl-tribenzyl ammonium chloride and
polyphosphazene (Cohen, S. et al. J. Anal. Chem. Soc., 112, pp.
7832-7833 (1990)).
[0158] The newly-formed immunoisolatory vehicle obtained by any of
the methods described herein can be maintained under sterile
conditions in a non-pyrogenic, serum-free defined nutrient medium
or balanced salt solution, at about 37.degree. C., prior to
implantation. Lower temperatures (20.degree. C.-37.degree. C.) may
be optimal for certain cell types and/or culturing conditions.
Other holding temperatures and medium compositions consistent with
good cell viability may also be used. Alternatively, the vehicle
can be cryopreserved in liquid nitrogen, if a cryoprotective agent
such as glycerin has been incorporated into the matrix (Rajotte, R.
V. et al. Transplantation Proceedings, 21, pp. 2638-2640 (1989)).
In such a case, the vehicle is thawed before use and equilibrated
under sterile conditions as described above.
[0159] Implantation of the biocompatible immunoisolatory vehicle is
also performed under sterile conditions. Generally, the
immunoisolatory vehicle is implanted at a site in the recipient's
body which will allow appropriate delivery of the secreted product
or function to the recipient and of nutrients to the implanted
cells or tissue, and will also allow access to the vehicle for
retrieval and/or replacement. It is considered preferable to verify
that the cells immobilized within the vehicle function properly
both before and after implantation; assays or diagnostic tests well
known in the art can be used for these purposes. For example, an
ELISA (enzyme-linked immunosorbent assay), chromatographic or
enzymatic assay, or bioassay specific for the secreted product can
be used. If desired, secretory function of an implant can be
monitored over time by collecting appropriate samples (e.g., serum)
from the recipient and assaying them.
[0160] The invention will now be further illustrated by the
following examples, which are not to be viewed as limiting in any
way.
EXAMPLE 1
[0161] Preparation of Cells for Immunoisolation
[0162] Cells derived from cell lines or from primary sources were
maintained in vitro prior to immunoisolation. (In some cases, cells
can be stored cryopreserved and then thawed and acclimated in
vitro.) Conditions for incubation will vary for specific cell
types, but will be readily ascertainable to those skilled in the
art following no more than routine experimentation. Islets of
Langerhans were obtained by the methods of Scharp et al.(supra) and
maintained at 37.degree. C. in an atmosphere of 5% CO.sub.2-95% air
in a medium consisting of a nutrient broth (e.g., Ham's F12
(GIBCO)), supplemented with serum (e.g., 25% v/v pooled equine
donor serum). Islets were maintained in culture, using Petri dishes
at 24.degree. C. for predetermined periods of time, according to
the method of Lacy, P. E. et. al., Science, 204, pp. 312-313
(1979). Prior to immunoisolation, the islets were collected,
concentrated by swirling the Petri dish, resuspended in Hank's
balanced salt solution (HBSS). The washed islets were resuspended
in a sufficient volume of HBSS to yield the final islet density
required to form an immunoisolatory vehicle containing the desired
number of islets, of an appropriate size and shape for implantation
and subsequent restoration of normoglycemia to a diabetic
individual. This method of preparing the cells prior to
immunbisolation is thought to remove antigen presenting cells from
the islet tissue thus diminishing immunologic attraction to the
outside of the vehicle which could limit its function and
duration.
EXAMPLE 2
[0163] Formation of Hydrogel Matrices with Different Molecular
Weight Cutoffs
[0164] An alginate thin film made from a solution of 1.0% w/v
sodium alginate in H.sub.2O was cross-linked for 6 minutes using
either (1) a 1.0% (w/v) or (2) a 2.0% (w/v) aqueous CaCl.sub.2
solution. The sheet was made by placing a film of liquid on a glass
plate using a draw down blade with a 0.2 mm clearance, then
immersing in the aqueous CaCl.sub.2 solution. A disk was cut from
the film using a 47 mm cutting die. The disk was placed in an
Amicon stirred filtration cell and used to filter solutions of
several marker solutes under a pressure of 10 psi. The
concentration of the marker solute was measured in the retentate
(C.sub.r=average of initial and final retentate concentration) and
similarly in the bulked permeate (C.sub.p). The rejection
coefficient of each hydrogel was calculated as follows:
R=1-C.sub.p/C.sub.r
[0165] Thus, a solute which is completely rejected would have a
coefficient of 1, and conversely, one which is completely passed
through the hydrogel would have a coefficient of 0. The hydrogel
resulting from (1) was permeable to 2,000 kD Dextran (Poly Sciences
corp.) (rejection coefficient equal to 0.64). The hydrogel
resulting from (2) was nearly impermeable to the same Dextran
solution. FIG. 1 describes permeability of the two hydrogels to the
following additional solutes. Bovine Serum Albumin (BSA; ICN
Biochemicals), Vitamin B12 (ICN Biochemicals), .alpha.-chymotrypsin
(ICN Biochemicals), Apoferritin (Sigma). The approximate molecular
weights are given in parenthesis in FIG. 1.
EXAMPLE 3
[0166] Formation of a Dual-Matrix Immunoisolatory Vehicle
[0167] A 2% solution of sodium alginate in physiological saline
(PS; 150 mm NaCl) was prepared under sterile conditions. A sterile
suspension of pancreatic islets in CRML1066 (GIBCO) culture media
isolated from adult rats was diluted 1:1 with the alginate
solution, for a final concentration of 1% alginate in the islet
suspension. The islet suspension was extruded from a single chamber
extrusion nozzle into a 1% CaCl.sub.2 bath. Once the alginate
polyions were crosslinked (approx. 2 min.) and the core containing
immobilized islets formed, the core was placed in a 2% alginate
solution. The core was then drawn up into tubing with a diameter
approximately 500 microns larger then the core and reextruded into
a second crosslinking bath of 2% alginate, surrounding the core
with a jacket formed of a separate cell-free layer of alginate
matrix cross-linked to the core. The thus formed macrocapsule was
cylindrical with dimensions of 30 mm length, 800 mm core diameter,
and 1 mm diameter core to jacket. The core volume was 15 mm. The
core contained 300 islets constituting a volume fraction of 10.6%
of the total core volume.
EXAMPLE 4
[0168] Formation of a Dual-Matrix Immunoisolatory Vehicle by
Coextrusion
[0169] A 2% solution of sodium alginate in physiological saline
(PS; 150 mm NaCl) was prepared under sterile conditions. A sterile
suspension of pancreatic islets in CRML1066 culture media isolated
from adult rats was diluted 1:1 with the alginate solution, for a
final concentration of 1% alginate in the islet suspension.
[0170] The islet suspension was loaded into the inner chamber of a
nested dual-bore coextrusion device of the configuration described
previously, the inner bore of which has a diameter of 500 microns
and the peripheral bore of which has a diameter of 600 microns. The
outer chamber of this device was loaded with a solution of sterile
1% sodium alginate in PS.
[0171] The tip of the nozzle was immersed in a bath containing a
sterile solution of 1% CaCl.sub.2 in PS, which induces the
hardening or gelling of alginate by crosslinkage of alginate
polyions. The materials loaded into the chambers were coextruded
into this bath, generating a continuously forming alginate cylinder
containing a core region of alginate matrix-immobilized islets and
a surrounding region of alginate matrix free of islets. The outer
diameter of the jacket was 1.2 mm. The inner diameter of the core
was 1.0-1.05 mm. The total islet volume of the core was 0.8
mm.sup.3 (200 islets). The total core volume was 25.98 mm.sup.3.
The volume of the islets was 3% of the total core volume. The MWCO
for 1% alginate was as depicted in FIG. 1. However, the MWCO will
increase with time similarly to FIG. 7 due to continued Ca.sup.++
displacement. The alginate of the core was cross-linked with the
alginate of the jacket.
[0172] The relative thickness of the surrounding region was
modified by adjusting the speeds at which the materials were
extruded from the core and peripheral bores of the nozzle. In
general as flow in the core increased, wall thickness decreased. As
flow in the peripheral bore was increased wall thickness increased,
Ranges of flow rates used were the same for both the core and
periphery (0.3-1.5 ml/min.). The ends of the cylinder were sealed
by dipping the cylinder first in a sterile 2% sodium alginate bath,
then in a sterile 1% CaCl.sub.2 bath. The immunoisolatory vehicle
so formed was maintained in sterile tissue culture medium prior to
implantation.
EXAMPLE 5
[0173] Formation of Core Matrix, Permselective Membrane
Immunoisolatory Vehicle by Coextrusion
[0174] A suspension of rat islets in 1% alginate was prepared as
described in Example 3 and loaded into the inner chamber of the
coextrusion device. The outer chamber was loaded with a membrane
casting solution comprising 12.5% (w/w) PAN/PVC in DMSO (i.e.,
dimethyl sulfoxide). The tip of the nozzle was either positioned at
a fixed distance above, or immersed in, a bath containing a sterile
solution of 1% CaCl.sub.2 in PS, which induced the hardening or
gelling of the alginate core matrix, and simultaneously induced the
hardening of the casting solution into a permselective membrane.
The exterior surface characteristics of the vehicle were determined
by whether the nozzle had been positioned above or below the
surface of the bath. When the nozzle was positioned above the bath
and exposed to low relative humidity (RH) air, an anisotropic
membrane with a rough, coriaceous (i.e., leathery) external surface
was formed, but when the nozzle was immersed in the bath, a
membrane-bilayer with a smooth external surface was formed.
Alternatively, when the nozzle was placed above the bath and the
fiber exposed to high RH air, an intermediate was formed. The
materials loaded into the chambers were coextruded into this bath,
generating a continuously forming cylindrical vehicle, comprising a
core region of alginate matrix-immobilized islets and a surrounding
semipermeable membrane having a MWCO of 50 kD.
[0175] The relative thickness of the membrane was modified by
adjusting the relative velocity of extrusion from the bores, as
described in Example 3. The ends of the cylinder were sealed using
methods similar to those described in the copending U.S. patent
application Ser. No. 07/461,999 filed Jan. 8, 1990, the teachings
of which are herein incorporated by reference. The immunoisolatory
vehicle so formed was maintained in sterile PS, a balanced salt
solution or tissue culture medium prior to implantation.
EXAMPLE 6
[0176] Formation of Core Matrix, Permselective Membrane
Immunoisolatory Vehicle by "Hand Loading"
[0177] In other cases, the islets were suspended in a 1-2% alginate
solution, and "hand loaded" into preformed thermoplastic hollow
fibers using a syringe and the ends of the fibers were sealed by a
combination of heat and polymer glue precipitation as described in
copending U.S. Ser. No. 07/461,999. The MWCO of the thermoplastic
jacket was approximately 50 kD. The hydrogel matrix was formed by
incubating the loaded fibers in a 1% calcium chloride solution for
6 minutes.
EXAMPLE 7
[0178] Assessment of Viability and Function of Immobilized,
Immunoisolated Islets In Vitro
[0179] Adult rat islets immunoisolated within double matrix
vehicles by the methods described in Examples 3 and 6. Matrix
liquid core vehicles with thermoplastic jackets were incubated In
vitro for at least two weeks. The vehicle had a core outer diameter
of 800 .mu.m overlain with a wall thickness of 65 .mu.m.
Alginate/Alginate dual matrix vehicles had an 880 .mu.m core
diameter and 60 .mu.m wall thickness. Incubation conditions were:
immersion in Ham's F12 medium supplemented with 25% equine serum at
37.degree. C. in a 5%CO.sub.2-95% air atmosphere. The medium was
refreshed every three to four days.
[0180] Using propidium iodide, the immunoisolated islet cells were
found to be 95% viable after this incubation period in vitro. They
were shown to remain functional as well. When tested by perfusion
with glucose (Dionne, supra) immunoisolated islets were shown to
have an insulin secretory response similar both in magnitude and
pattern to that of unprotected islets incubated in vitro for a
similar period of time and under similar conditions. Insulin
release was measured by the method of Soeldner, J. S. et al.
Diabetes, 14, p. 771 (1965). The results of a typical perfusion
experiment are shown in FIGS. 2A and 2B. The challenge and baseline
concentrations of glucose used were 300 mg % and 100 mg %,
respectively. No significant delay in either the onset of the first
phase of insulin release following glucose stimulation or return to
baseline secretion was observed with immunoisolated islets. In
addition, a rising second phase comparable to that of unprotected
islets was seen. Expressed on a per-islet basis, the total amounts
of insulin released by immunoisolated islets were similar to that
for unprotected islets. These results are summarized in FIGS. 3A
and 3B.
EXAMPLE 8
[0181] Comparison of In Vivo Performance of Xenografted Islets
Isolated within Vehicles Having a Thermoplastic Membrane with and
without an Internal Hdrogel Matrix
[0182] Rat islets were immunoisolated in either matrix or liquid
core thermoplastic vehicles as described in Example 6. Vehicle
dimensions were 800 .mu.m O.D., 55 .mu.m wall thickness, 2 cm fiber
length. Just under 20% loading density was used. In the case of
liquid core capsules,alginate was not included in the cell
suspension. In the first experiment, islets were immunoisolated
within a matrix. Vehicles were is implanted intraperitoneally into
streptozotocin-induced diabetic mice for a concordant xenograft
(i.e., between closely-related species). Free-floating implants
were inserted into the peritoneal cavity. Eight animals were
implanted with 500-1000 immunoisolated rat islets each. One animal
showed no amelioration of hyperglycemia. The others returned to a
normoglycemic state (i.e., the plasma glucose levels of these
animals returned to a normal range defined as 100-125 mg %) within
five days posttransplantation and remained normoglycemic until day
60 when the grafts were removed and the animals again became
hyperglycemic. The average results of 7 such experiments are
summarized in FIG. 4. The absence of significant fluctuations in
plasma glucose levels in these animals should be noted. The
recovered immunoisolatory vehicles were inspected for evidence of
fibrotic overgrowth, and were assessed for the ability to release
insulin in response to glucose perfusion. None of the vehicles had
become completely encapsulated with fibroblasts, however in some
areas three to five layers of fibroblasts around the exterior of
the vehicle were observed. Recovered immunoisolatory vehicles
released insulin in vitro following perfusion in response to
glucose and theophylline stimulation and histological analysis
revealed viable islets with evidence of insulin staining within the
cells. The results of the perfusion experiment with glucose and
theophylline stimulation are shown in FIG. 5.
[0183] These favorable results contrasted sharply with those
obtained when rat islets were immunoisolated within a PAN/PVC
membrane without an immobilizing matrix. For these immunoisolatory
vehicles, functional responsiveness lasted only 12.+-.3 days post
implantation; five out of five animals tested returned to a
hyperglycemic state thereafter. Histological examination of these
immunoisolatory vehicles revealed agglomeration of the islets. The
islets had condensed into a large mass of tissue which exhibited
severe central necrosis, with only a rim of viable and identifiable
islet cells surviving. Thus, the presence of a matrix to prevent
islet aggregation and resulting cell death, significantly improved
viability resulting in long term efficacy of the implant.
EXAMPLE 9
[0184] Assessment of the Restoration of Normoglycemia to
Streptozotocin-Induced Diabetic Mice by Implantation of
Immunoisolated Concordant Xenograft Islets in Immunoglobulin
Permeable Vehicles
[0185] In vivo performance of the double-matrix immunoisolatory
vehicle was assessed using the rat-to-mouse concordant xenograft
model for restoration of normoglycemia to streptozotocin induced
diabetic mice. The vehicles were prepared as in Example 3 and have
significant permeability to 2,000 kD Dextran (FIG. 1). Therefore,
these vehicles were also readily permeable to IgG (150 kD). The
results of this experiment are summarized in Table 2. The animals
were divided into three groups: group 1 consisted of seven control
animals into whom 300 nonimmunoisolated islets per animal were
implanted at the kidney subcapsular site. Only one of these animals
showed amelioration of hyperglycemia for more than 12 days. The
mean duration of normoglycemia in Group 1 was 14.0.+-.3.1 days.
[0186] Group 2 consisted of thirteen animals each implanted with
300 rat islets immobilized in an alginate matrix lacking a
surrounding cell-free region. Graft function was lost within 24
days in 8/13 of these Group 2 animals, indicating that simple
immobilization conferred no advantage over the Group 1 controls.
The other five animals remained normoglycemic until the graft was
removed. The mean duration of normoglycemia in group 2 was
29.3.+-.5.5 days.
[0187] Group 3 consisted of ten animals, each implanted with 300
rat islets immobilized within a dual-matrix immunoisolatory vehicle
of the configuration described in Example 3, i.e., with a
surrounding layer of cell-free alginate matrix. In only four of
these animals, graft function was lost within 14 days
postimplantation. However, six animals remained normoglycemic
beyond 100 days at which time the experiment was terminated and the
grafts removed, precipitating a return to the diabetic state (FIG.
6). The mean duration of normoglycemia in group 3 was 65.8.+-.15.1
days (n=10). Fibromatous reaction to the recovered alginate vehicle
was minimal. Histological analysis of recovered immunoisolated
islets revealed viable islets with evidence of insulin staining
within the .beta. cells. The immunoisolatory vehicles used in this
experiment also demonstrate the functionality and biocompatibility
of vehicles which are permeable to high molecular weight proteins
such as the immunoglobulin G protein.
1TABLE 1 SURVIVAL OF IMMUNOISOLATED XENOGRAFTS IN DIABETIC MICE
Survival (Days) Group Vehicle Individual Group 1 control
7,12,12,12, 14.0 .+-. 3.1 12,12,29 2 immobilized 8,12,12,14,
>29.3 .+-. 5.5 15,16,18,24, 41*-,53*,54*, 54*-,60*- 3 immuno-
8,8,12,13, >65.8 .+-. 15.1 isolated 102*-,102*-, 102*-,102*-,
104*-,104*- *Nephrectomy for removal of islet graft
[0188] A vehicle of the configuration described in Example 3 was
prepared, it contained several hundred islets and had a membrane
MWCO of less than 50 kD.
[0189] The vehicle was implanted into a diabetic BB rat. This
strain of rat is known to be a rodent model for mimicking human
Type 1 autoimmune diabetes. The vehicle was recovered after a
21-day period of residence in vivo. The immunoisolated islets were
found to be viable and functional, as determined by histological
analysis.
EXAMPLE 10
[0190] Assessment of the Survival of Immunoisolated Islets in a
Discordant Xenogenic Recipient
[0191] Dual matrix immunoisolatory vehicles containing immobilized
rat islets were prepared by the process of coextrusion from a
nested dual-bore nozzle as described in Example 4. The conditions
for the gelling of the matrix were chosen to yield a hydrogel
matrix permeable to 2,000 kD blue Dextran (as in FIG. 7), thus the
vehicle so formed was permeable to immunoglobulin G and to Clq.
[0192] Segments of about 0.5 cm in length were prepared from the
continuous cylindrical vehicle by periodically interrupting the
flow of the core material to form cell free regions, which were
readily visible. The fiber was cut in the cell free region; thus
the cells were completely surrounded by a region of cell-free
alginate matrix. The vehicles were implanted between the leaves of
the mesentery of guinea pigs (n=2), a discordant (i.e., distantly
related) host. After 21 days of residence in vivo, the vehicles
were removed and tested in vitro for glucose-responsive insulin
release. These results are summarized in FIG. 8. Following basal
stimulation, a statistically significant rise in insulin release
from the immunoisolated islets was measured when stimulated with
300 mg/dl glucose. A return to basal insulin levels occurred when
glucose returned to 100 mg/dl. Thermoplastic vehicles with alginate
cores gave similar results.
EXAMPLE 11
[0193] Improved Tissue Viability by Controlled Reaggregation
[0194] Purified canine islets, prepared according to the method of
Scharp and Lacy, U.S. Pat. No. 5,079,160, were dispersed into cell
aggregates containing from one to 50 cells according to the
following protocol. 1000 canine islets were rinsed 3 times with 50
ml of Ca++ and Mg++ free Hanks medium containing 1 mm EDTA. After
the final rinse, islets were centrifuged into a pellet at
100.times.g for 8 min., 10.degree. C. The resulting pellet was
resuspended in 5 ml of the same medium per 1000 islets. This slurry
was agitated for 8 min. at 24.degree. C. using a hand held
micro-pipet. At the end of 8 min., trypsin and DNAse were added to
final concentrations of 25 ug/ml and 2 ug/ml respectively. The
slurry was further agitated by repeated pipetting for approximately
5 min at which time microscopic examination indicated that the
largest aggregates consisted of no more than about 50 cells. The
digestion was quenched by adding 10 ml of cold DMEM with 10% new
born calf serum per 1000 islets. Aggregates were pelleted by
centrifugation at 250.times.g for 6 min. Aggregates were cultured
in Ham's F12 with 25% horse serum overnight during which time
limited reaggregation occurred resulting in a volume average
aggregate size of approximately 35 um.
[0195] Following overnight culture, aggregates were pelleted by
centrifugation at 250.times.g for 6 min. A 2% solution of
non-crosslinked alginate was added to the pellet to make a solution
consisting of 1% alginate and 8% tissue (w/v). The final islet
volume of the core was 0.56 mm.sup.3 in the form of aggregates
evenly dispersed throughout the viscous liquid. The tissue slurry
was aseptically aspirated into a length of Pe 90 tubing the end of
which was previously necked down so as to fit into the lumen of 670
.mu.m inner diameter hollow fiber membranes (outer diameter=800
.mu.m. Ten microliters of tissue containing slurry was injected
into each of several PAN/PVC fibers of 2 cm length. The ends of
each fiber were sealed as described previously and the fibers were
stored overnight in Ham's F12 with 25% horse serum. Prior to
implantation, fibers were placed into fresh Hanks without serum for
1 hour in order to remove serum residue.
[0196] After overnight culture, 1/2 of the vehicles were implanted
into the peritoneal cavity of normoglycemic rats (2 vehicles per
animal) and 1/2 of the vehicles were maintained in in vitro tissue
culture. After 2 weeks, implanted vehicles were removed from the
peritoneal cavity and subjected to an in vitro glucose challenge
along with control vehicles cultured in vitro. Explanted vehicles
exhibited insulin release that was as good as or better than that
seen prior to implantation indicating functional survival not only
of insulin release but of glucose sensitivity. Following glucose
challenge, the alginate core of the vehicles was "blown out" and
the tissue was stained with PI for viability. Although each
aggregate had pulled together to form a tight spheroid of
approximately 35 um in diameter, individual aggregates remained
well distributed throughout the alginate core and had not clustered
together to form necrotic regions. Failure to stain with PI stain
indicated 100% viability.
EXAMPLE 12
[0197] Partial Restoration of Motor Behavior Upon Implantation of
Immunoisolated Adrenal Chromaffin Cells in an Experimental Model of
Parkinsonism
[0198] Bovine adrenal chromaff in cells were recovered from adrenal
glands according to the method of Livett B. G.. Physiol. Rev., 64,
pp. 1103-1161 (1984) by collagenase digestion and maintained in
culture for 10 days to ensure lack of bacterial or fungal
contamination. Clumps of chromaffin cells were washed in serum-free
medium by centrifugation and resuspended in 1% alginate solution.
This cell suspension was used to form a matrix-core, thermoplastic
membrane immunoisolatory vehicle by co-extrusion as described in
Example 4. The aqueous precipitation bath comprised a solution of
1% CaCl.sub.2 mixed 1:2 with tissue culture medium for a final
concentration of 0.5% CaCl.sub.2 in the bath. The fiber was
incubated in the bath for 6 minutes to allow the alginate to gel,
and was then transferred to a petri dish containing Dulbecco's
modified Eagle's medium (DMEM). The fiber was visually inspected
for regions with good wall morphology, then spot-checked for the
presence of chromaffin cells. It was then divided into 4 mm long
sections by sealing the ends of the sections with a combination of
heat, solvent, and pressure.
[0199] Eight Sprague-Dawley rats received injections (10 .mu.g/5
ul) of 6-hydroxydopamine (6-OHDA) into the substantia nigra. They
were tested for apomorphine-induced (0.1 mg/kg) rotational behavior
at weekly intervals. By the method of Ungerstedt V. U., Acton.
Physiol. Scand. Suppl., 367, pp. 69-93 (1971) and Ungerstedt V. U.,
Brain Res., 24, pp. 485-493 (1970). Apomorphine induces the
Parkinson's-like motor response of turning away from the side of
the 6-OHDA-induced lesion. The extent of such rotational motor
behavior upon apomorphine injection can be used to monitor the
extent of the lesion and the degree of amelioration provided by the
immunoisolated chromaffin cells. Six weeks after the induction of
Parkinsonism, the animals received intrastriatal implants of either
control (empty) or adrenal-chromaffin cell-containing vehicles, and
were again tested for rotational behavior at weekly intervals.
These results are summarized in FIG. 9.
[0200] Prior to implantation, all animals exhibited an equivalent
number of rotations following apomorphine challenge. Within two.
weeks postimplantation, however, those animals receiving
immunoisolated adrenal chromaffin cells exhibited a significant
35-40% percent decrease in rotations which remained stable
throughout testing; this indicates that the implants significantly
reversed the effects of 6-OHDA-induced lesions. The animals who
received control vehicles did not show any reduction in the number
of rotations.
EXAMPLE 13
[0201] Implantation of Adrenal Chromaffin Cells for Prevention of
Neural Damage Due to Excitotoxicity (Huntington's Disease
Model)
[0202] This example sets forth a method for prevention of neural
damage due to neural excitotoxicity in a subject by implantation of
a vehicle containing cells which secrete a trophic factor. This
animal model of neuroexcitotoxicity is considered analogous to the
types of neural damage suffered by patients with Huntington's
disease.
[0203] Subjects
[0204] Male Sprague-Dawley rats (N=23) 90-120 days old and weighing
approximately 225-250 grams were used in the following experiments.
All animals were housed groups of 2-3 in a temperature and humidity
controlled colony room maintained on a 12 hour light/dark cycle
with lights on at 0700 hours. Food and water were freely available
during testing.
[0205] Preparation of Adrenal Cell-containing Macrocapsules
[0206] Bovine adrenal chromaffin cells were isolated from adrenal
glands and maintained in culture for 10 days to ensure lack of
bacterial or fungal contamination. Clumps of chromaffin cells were
washed in serum-free medium by centrifugation and resuspended in 1%
alginate solution. This cell suspension was used as the bore
solution for coextrusion in 15% PAN/PVC:DMSO solution. The
coextruded fiber containing chromaffin cells was collected in a
bath of 1% CaCl.sub.2 mixed 1:2 with tissue culture medium. The
fiber was left in that solution for 6 minutes to allow the alginate
to gel, and was then transferred to a petri dish containing medium.
Fiber was visually inspected for regions with good wall morphology,
then spot-checked for the presence of chromaffin cells. Capsules
were made by sealing the ends of 4 mm long sections with a
combination of heat, solvent, and pressure. Capsules were then
implanted stereotaxically into the brains of Sprague Dawley
rats.
[0207] Preparation of Quinolinic Acid
[0208] Quinolinic acid (Sigma Chemical Co) was dissolved in 2N
sodium hydroxide and diluted with phosphate buffer at pH 7.2 to a
final pH of 7.4 and concentration of 225 nmole/1 ul.
[0209] Surgical Procedure
[0210] Rats were anesthetized with sodium pentobarbital (45 mg/kg)
and positioned in a Kopf stereotaxic apparatus. A sagittal incision
was made in the scalp and a hole drilled in the skull for placement
of the macroencapsulated adrenal chromaffin cells. Capsules were
placed in a capillary tube mounted to the stereotaxic device and
lowered to the following coordinates: 0.3 mm anterior to Bregma,
3.5 mm lateral to the sagittal suture and 7.5 mm ventral from the
surface of the brain.
[0211] One week later, the animals were anesthetized and mounted in
the stereotaxic instrument prior to intrastriatal administration of
quinolinic acid or the phosphate buffer vehicle. Solutions were
slowly infused (0.125 ul/minute) using a 10 ul Hamilton syringe)
through a hole drilled 0.8 mm lateral to the site of placement of
the capsule. The injection site for guinolinic acid was: 0.3 mm
anterior to Bregma, 2.7 mm lateral to the sagittal suture, and 5.5
mm ventral from the surface of the brain. A total volume of 1.0 ul
was delivered and the injection cannula was left in place for an
additional 2 minutes to promote the local diffusion of the
perfusate. This procedure resulted in the formation of 3
experimental groups: adrenal capsule/guinolinic acid (N=8), empty
capsule/quinolinic acid (N=8), quinolinic acid only (N=7).
[0212] Body weights were recorded on a daily basis for 15 days
following quinolinic acid administration.
[0213] Histology
[0214] Thirty days following quinolinic acid administration,
animals were transcardially perfused using a peristaltic pump, with
0.9% saline (50 ml/min) followed by 1% paraformaldehyde/1.25%
glutaraldehyde in 0.1 M phosphate buffer (4.degree. C.) (800 ml/30
minutes). The brains were then postfixed for 2 hours in the
paraformaldehyde solution prior to being placed in 20% sucrose for
24 hours. The brains were then frozen and serially cut on a sliding
microtome into 30 um coronal sections. Sections were then processed
for cytochrome oxidase histochemistry and adjacent sections were
stained for Nissl.
[0215] Results
[0216] The presence of cytochrome oxidase was considered indicative
of metabolic activity, and thus of neuronal viability. Nissl
staining was used to visualize the cell processes and to assess the
general structure of the neural architecture.
[0217] In the experimental group that received empty capsules, the
striatum was shrunken 20-40% as compared with the lesioned animals
that had received vehicles containing chromaffin cells. The
striatal neurons of the empty capsule group showed lack of
metabolic activity as demonstrated by the absence of staining for
cytochrome oxidase. Furthermore, all animals showed significant
decrease in body weight (FIG. 10).
[0218] In contrast, the neurons of the group which received
vehicles containing chromaffin cells showed normal staining with
both cytochrome oxidase and Nissl, and no loss of body weight.
[0219] Conclusion
[0220] The neurons of subjects which received chromaffin
cell-containing implants were protected from excitotoxic damage
caused by quinolinic acid.
EXAMPLE 14
[0221] Treatment of Neural Degeneration in Rats Using Encapsulated
Genetically Engineered Fibroblasts
[0222] This example sets forth a method of treatment for animals
with a fimbria-fornix lesion. This type of lesion produces neuron
cell death and degeneration of post-synaptic neurons and behavioral
symptoms indicating deficits in memory and learning. The
degeneration of cholinergic neurons produced in this animal model
are considered analogous to similar effects seen in Alzheimer's
disease in humans.
[0223] Surgical Procedure
[0224] Adult male Sprague-Dawley rats (250-350 g) were anesthetized
by intraperitoneal injection of sodium pentobarbital (55 mg/kg).
Unilateral fimbria-fornix lesions were performed by aspiration of
the fimbria, dorsal fornix, medial part of the parietal cortex,
ventral hippocampal commissure, corpus callosum, and overlying
cingulate cortex. An implant vehicle as described below was then
placed into the ipsilateral lateral ventricle of each animal,
oriented perpendicular to the interaural plane.
[0225] Preparation of PAN/PVC fibers Permselective hollow fibers
were prepared via the dry jet-wet spinning technique. Cabasso,
Hollow Fiber Membranes, vol. 12, Kirk-Othmer Encyclopedia of
Chemical Technology, Wiley, New York, 3rd Ed., pp. 492-517 (1980).
A 15% solution of PAN/PVC copolymer in dimethylsulfoxide (DMSO) was
extruded through an annular spinneret, with solvent DMSO (for a
porous inner surface) or nonsolvent water (for a smooth skinned
inner surface) flowing through the bore. The resulting fiber was
collected into a nonsolvent water bath, glycerinated, and
dried.
[0226] Genetically Engineered Fibroblasts
[0227] R208N.8 and R208F rat fibroblasts were a gift of Dr. Xandra
Breakefield, Harvard University. R208N.8 fibroblasts were
engineered to secrete NGF as follows (Short et al., Dev. Neurosci.,
12, pp. 34-45 (1990)). A retroviral vector was constructed from
Moloney murine leukemia virus. It contained the last 777-basepairs
of the coding region for mouse NGF cDNA under control of the viral
5' long terminal repeat. The vector also included a dominant
selectable marker encoding the neomycin-resistance function of
transposon Tn5 under control of an internal Rous sarcoma virus
promoter. Transmissible retrovirus was produced by transfecting
vector DNA into PA317 amphotropic producer mouse fibroblast cells
and by using medium from these cells to infect .Yen.2 ecotropic
cells. Virus from the .Yen.2 clone producing the highest titer was
used to infect the established rat fibroblast cell line R208F,
transforming it to R208N.8. Individual neomycin-resistant colonies,
selected in medium containing the neomycin analog G418, were
expanded and tested for NGF production and secretion by a two-site
enzyme immunoassay.
[0228] Encapsulation of Fibroblasts
[0229] R208F and R208N.8 fibroblasts were dissociated with
Trypsin-EDTA, and suspended at a density of 1.times.10.sup.6
cells/.mu.l in a laminin containing matrigel resuspended in
Matrigel.TM. or Vitrogen.TM., and aspirated with a 1 cc syringe
into a pre-sterilized PAN/PVC hollow fiber. Fibers were cut to 4 mm
length, and the fiber ends were heat sealed with a sterile surgical
cautery.
[0230] ChAT Immunocytochemistry
[0231] After two weeks, the animals were sacrificed, fixed by
transcardial perfusion with cold heparinized physiologic saline and
4% paraformaldehyde in phosphate buffer. The brains were
immediately dissected and postfixed overnight, followed by
immersion in 15% and 25% buffered sucrose solutions. Frozen
sections were cut at 25 .mu.m from anterior to posterior on a
cryostat, and all coronal sections were collected onto slides or
into phosphate buffer. Representative cbronal sections were
processed for immunocytochemistry using a monoclonal antibody to
rat ChAT (2.5 .mu.g/ml) with the biotin-avidin-DAB method. Sections
were mounted and neuronal cell bodies counterstained with cresyl
violet. All ChAT-positive cell. bodies were counted in the medial
septum and vertical diagonal band region ipsilateral and
contralateral to the lesion, between the genu of the corpus
callosum and the decussation of the anterior commissure. A
significant prevention of ChAT(+) cell reduction was observed in
rats receiving R208N.8 capsules.
EXAMPLE 15
[0232] Use of an Immunoisolatory Vehicle to Deliver a
High-Molecular Weight Product to a Recipient
[0233] Immunoisolatory vehicles were prepared by hand loading
350,000 hybridoma cells producing an antibody (iostype
immunoglobulin G), specific for tumor necrosis factor (TNF) into a
7 mm length of a medical grade, olefinic microporous hollow fiber
of the kind used for plasmapheresis (Plasmaphan; Enka). The
internal diameter of the fiber was 300 microns; its MWCO was about
1,000 kD. The ends of the vehicle were sealed as described in
copending U.S. Ser. No. 07/461,999. The vehicle was implanted under
the renal capsule of a mouse, where it was allowed to reside for 14
days. The vehicle was thereafter recovered and found to contain
many cells, over 50% of which were viable as determined by the
exclusion of indicator dye (pI). The release of TNF-specific
antibody into the serum of the recipient mouse was monitored by
ELISA. The results are summarized below:
2 TABLE 2 Days Post Titer of TNF Specific Implantation Antibody in
vivo 0 none detected 1 10 2 30 6 70 8 100 11 60 15 23
[0234] A control immunoisolatory vehicle maintained in vitro
exhibited similar antibody release.
EXAMPLE 16
[0235] Subcutaneous Implantation of Encapsulated Islets
[0236] Preparation of Islet Containing Capsules
[0237] Two types of acrylic copolymer hollow fibers, designated
Type 1 and Type 2 fibers, were used. Fibers were formed by using a
dry-wet spinning technique with a spinneret as described in
Cabasso, Hollow Fiber Membranes, vol. 12, Xirk-Othmer Encyclopedia
of Chemical Technology, Wiley, New York, 3rd Ed., pp. 492-517
(1980). The acrylic co-polymer used was poly(acrylonitrile-co-vinyl
chloride) ( M.sub.n=100,000, M.sub.w=300,000 as measured by
size-exclusion chromatography; CytoTherapeutics, Inc.) dissolved in
dimethyl sulfoxide (12.5% w/w). The acrylic copolymer solution was
pumped through the outer tube of the spinneret and water was pumped
through the inner tube. Type 1 hollow fibers were extruded into
water through an air gap, resulting in a fenestrated outer wall
typical of fibers made by a dry-wet spinning technique. The type 2
fibers were made in a analogous fashion, except the air gap was
replaced by a humidified atmosphere, resulting in a smooth outer
surface.
[0238] Rat islets were isolated from male Wistar-Furth rats as
described in Example 1 above. The islets were immobilized in
alginate gel and encapsulated into 2-cm type 1 or type 2 fibers,
550 or 1000 islets per fiber, as described in Lacy, P. E., et al.,
Science, 254, p. 1782 (1991).
[0239] Implantation
[0240] The encapsulated rat islets were implanted intraperitoneally
or subcutaneously in mice made diabetic by the injection of
streptozotocin. Non-fasting plasma glucose concentrations were
determined three times weekly; the diabetic recipients had
concentrations greater than 400 mg/dl before transplantation. The
loading density was 70 islets per centimeter for 1000 islets and 35
islets per centimeter for 500 islets. Twenty-six mice received
fibers intraperitoneally, and 26 received fibers subcutaneously. In
each group, 14 mice received fibers containing a total of 1,000
islets and 12 received fibers containing a total of 500 islets.
[0241] Results
[0242] The intraperitoneal type 1 fibers induced and maintained
normoglycemia for greater than 60 days in seven of nine recipients
that received 1000 islets and in all of the recipients receiving
500 islets. None of the recipients of subcutaneous type 1 implants
of 500 islets remained normoglycemic for 60 days; three of eight
recipients of 1000 islets were normoglycemic for greater than 60
days. Removal of the fibers from these three recipients returned
the mice to a diabetic state. Transplants of rat islets in the type
2 fibers produced and maintained normoglycemia in the recipient
mice in greater than 80% of either the intraperitoneal or
subcutaneous sites with either 1000 or 500 islets. The recipients
became hyperglycemic again when the fibers were removed at 60 days.
Histologic examination of the removed type 1 and type 2 fibers
revealed that they were biocompatible.
EXAMPLE 17
[0243] Controlled Reaggregation of Rat Islets
[0244] Rat islets were isolated, dissociated, reaggregated and
encapsulated as described in example 11, with the exception that
sealed fibers were not exposed to serum in vitro and were held for
only 1 hour prior to implantation. Either two 2 cm long capsules or
two 2 cm and one 1 cm capsules were implanted in each rat.
[0245] Islets were dissociated and reaggregated to an approximate
size of 35 um. All the reaggregated cells from approximately 500
islets were loaded into each 4-6 cm length capsule. Implanted
capsules are capable of maintaining normoglycemia in rats for
greater than 60 days.
EXAMPLE 18
[0246] Fabrication of Flatsheet Islet Encapsulation Vehicles
[0247] Aseptic materials and methods were used in all the following
procedures. This included autoclave sterilization, EtOH
sanitization, UV sterilization and/or 0.2 um sterile
filtration.
[0248] Casting solution was prepared using a monoacrylic copolymer
with an average molecular weight of 10.sup.5 daltons which was
dissolved in a water-soluble, organic solvent. The casting solution
was 10.0% w/w polymer in the organic solvent. The polymer was
precipitated once under sterile conditions prior to its use to
remove any residual monomers, oligomers, or any additives placed in
the bulk polymer by the manufacturer. This polymer solution was
then dried and redissolved in 100% DMSO to form a 10% w/w polymer
solution. This solution was passed through a 0.2 um sterile nylon
filter and collected under aseptic conditions.
[0249] Next the casting solution was uniformly spread using a
casting bar over a 1/4" glass substrate at a casting thickness of
125 um. In order to cast the film, the substrate, held at a 30
degree incline, was moved under the stationary casting bar into the
precipitation bath. The level of the precipitation bath was between
1/8-1/4" from the casting bar. The substrate can be any material
which prevents premature lifting of the membrane from the substrate
prior to complete precipitation. Simultaneous with spreading, the
casting solution was plunged into 24.degree. C. water resulting in
precipitation of the polymer forming an anisotropic semipermeable
membrane, with the permselective layer appearing as a thin skin on
the quench side (away from substrate) of the membrane. The film was
left in the bath for four minutes to insure that membrane
properties have been adequately established prior to its
removal.
[0250] The film was then carried through a series of rinses to
remove any residual solvent or toxic residue that may compromise
the compatibility of the final product. These rinse baths were
composed of solutions that caused no marked physical or chemical
modification to the initial membrane. The first post bath consists
of water processed through a Milli-Q purification system and was
left to soak for a minimum of 15 min. The material was then removed
and placed into a 70% v/v punctilious ethyl alcohol and water
solution, which had been 0.2 um filtered, for a minimum of 60 mins
and then removed. The final stage was soaking of the film in two
sequential sterile normal saline baths with volumes of 2
ml/cm.sup.2 fiber for a minimum of 60 min each.
[0251] Results
[0252] The final wet-as-cast membrane thickness was between 30 um
to 75 um with a hydraulic permeability of 0.475 cc/min/cm.sup.2 at
5.0 psig. Rejection coefficient data indicated that the membrane
excluded substances larger than approximately 100,000 daltons.
[0253] Encapsulation and Implantation
[0254] Sterile, cast 10% flatsheet membrane soaking in saline was
prepared as described above, and encapsulated islets were prepared
as follows: A 6 by 8 inch sheet of wet membrane was folded over on
itself with the skin sides facing so as to create a strip of double
membrane about 1" wide. The fold was carefully pressed to form a
crease. Using a #10 scalpel blade, the 1" wide strip of double
membrane was cut off from the rest of the sheet. The double strip
was picked up by one end and cut into 1" squares using scissors.
The squares were caught in saline as they were cut off. The tip and
bottom of each square were connected by the fold comprising one
side.
[0255] Immediately prior to loading, a square was lifted out and
placed on the lid of a 3" diameter polystyrene petri dish
previously wet with 1-2 ml of 1% CaCl.sub.2 solution. The membrane
was unfolded and each side was floated on the CaCl.sub.2 solution
with care taken to assure that the solution did not flow into the
tip of the membrane.
[0256] Previous to this, islets were allowed to settle into a
pellet, and resuspended in 1% ungelled sodium alginate solution
(solution was prepared by making a 2% alginate solution in H.sub.2O
and mixing this 50/50 with medium in which islets were cultured).
Islet/alginate slurry was gently mixed by stirring and aspirating.
Slurry was prepared at the rate of 500 islets in 25 ul alginate per
sq cm useable surface area on a single membrane side. This equates
to approximately 125 ul and 2500 rat islets for a 1" sq membrane
sheet.
[0257] Islet/alginate slurry was aspirated into a 200 ul pipet tip
and then evenly spread across the inside of one membrane leaving
about 1/8 inch gap along all edges of the square sheet. Spreading
was done rapidly as alginate slowly crosslinked due to Ca.sup.++
diffusion through the membrane from the underlying CaCl.sub.2
solution.
[0258] Once the alginate was sufficiently crosslinked to prevent
alginate smearing (approx 1-2 min) the other side of the flat sheet
device was folded over to form a tip to the flatsheet sandwich.
Care was taken to eliminate air bubbles.
[0259] The two sides of the membrane were sealed using an impulse
heat sealer with a 1/8" heating element set on medium heat (temp
reached between 80-160.degree. C.). Each edge, including the folded
one, was individually sealed by activating the heat sealer while
pressing down on the 1/8" strip of non-alginate coated membrane
along each edge.
[0260] After sealing, the device was soaked in 1%. CaCl.sub.2
solution for 4 min so as to further crosslink the alginate. The
device was held in Hank's solution until implantation (within 2
hours).
[0261] Flat sheet was implanted in the peritoneal cavity of a
chemically diabetic recipient Wistar-Furth rat by making a midline
incision through the skin and into the peritoneal cavity of the
anesthetized rat. The flat sheet implant was placed in the cavity
by grasping the sealed edge with smooth forceps and gently laying
the device, free floating on top of the gut pile proximal to the
peritoneal wall. The peritoneal cavity and skin were closed by
suturing.
[0262] Animals were studied for 21 days, at which time the devices
were explanted. Blood glucose levels dropped from 375 mg/dl to 150
mg/dl within 4 days of implant and remained there until explant, at
which tine glucose values rose to 275 mg/dl (n=2).
[0263] Histological examination revealed viable islets immobilized
in the alginate layer with less than a monolayer of cells attached
to the outside of the membrane.
EXAMPLE 19
[0264] Implantation of Encapsulated Bovine Adrenal Chromaffin Cells
in the Lumbar Subarachnoid Space in Sheep
[0265] Adrenal Gland Harvesting
[0266] Bovine adrenal chromaffin cells were obtained from healthy
livestock sources in herds tested for adventitious agents and known
to be free of bovine Spongiform Enchephalitis. Two to three week
old calves weighing 52-72 kg (62.+-.7) were premedicated with
atropine (100 mg/kg) and xylazine chlorhydrate (0.15 mg/kg).
Anesthesia was induced with pentobarbital sodium (8 mg/kg), and
maintained with 0.5-1% halothane delivered through an endotracheal
tube. The aorta and vena cava were isolated through a cruciate
organ harvesting ventral incision. The distal aorta and vena cava,
the coeliac axis, and the superior and inferior mesenteric arteries
were ligated. The proximal vena cava was clamped above the liver,
and the proximal aorta above the coeliac axis. Four to six liters
of cold saline solution and 2 liters of a hospital prepared organ
preservation solution (formulated as the University of Wisconsin
solution minus hydroxyethylstarch and adenosine) were perfused by a
cannula introduced in the aorta. The organ preservation solution
comprises potassium lactobionate 100 mM, KH.sub.2PO.sub.4 25 mM,
MgSO4 SmM, raffinose 30 mM, glutathion 3 mM and allopurinol 1 mM.
The adrenal glands were then harvested with their native vessels
and placed in a sterile container filled with enough organ
preservation solution to cover the gland. The container was placed
on ice and send to the tissue culture laboratory for isolation of
the chromaffin cells. Aseptic surgical techniques were utilized for
all procedures. All harvested glands were of suitable quality for
subsequent chromaffin cell isolation. The total amount of cells
obtained by the isolation technique ranged from 2.0 to
3.0.times.10.sup.7 cells per gland with viability greater than 95%,
as assessed with FDA and PI stain. Cells were typically organized
in clusters of 50 to 200 .mu.m in diameter.
[0267] Chromaffin Cells Isolation and Culture
[0268] A cannula was inserted into the isolated gland through the
suprarenal vein. The glands were then perfused with 10 ml of cold
organ preservation solution via the cannula until clear perfusate
was seen dripping from the gland. Five to seven ml of a 0.2%
collagenase solution (Sigma, Type H) was then injected into each
gland. The vein was clamped and the glands were placed in sterile
beakers containing 100 ml of organ preservation solution and shaken
in a 37.degree. C. waterbath at 1 Hz for 30 minutes. This first
digestion allowed mechanical separation of the cortex from the
medulla by gentle pulling. The medullary tissue was then placed in
a tissue culture dish with 1 ml of organ preservation solution and
chopped into approximately 1-2 mm.sup.2 sections. The chopped
tissue pieces were then poured into a dissociation-filtration
chamber with a 250 .mu.m pore size filtration. grid, filled with 10
ml of 0.2% collagenase solution and agitated at 1 Hz for 10 minutes
at 37.degree. C. Constant temperature in the chamber was maintained
by an external water jacket. Every 10 minutes the chamber was
rinsed with cold organ preservation solution. The isolated cells
and cell clumps which passed through the filtration grid were
collected in a sterile 50 ml conical tube. A total of three
digestions were generally needed to fully digest the medullary
tissue.
[0269] The tubes were then spun at 800 g for 5 minutes and washed
two additional times with the organ preservation solution. An
aliquot of the final wash supernatant was placed in thioglycolate
medium for sterility testing.
[0270] The cells from each tube was plated in separate 100 mm
tissue culture dishes, in 10 ml of PC-1 media, a defined medium
containing protein from human recombinant sources (Hycor Biomedical
Inc.).
[0271] Using-a 10 mL pipet, cells were removed from each petri dish
and were pooled in a 50 ml centrifuge tube. The cell solution was
spun at 800 g at ambient temperature for 5 minutes. The supernatant
was disposed and the pellet resuspended in 10 mL of HEPES buffered
NaCl. Two 50 microliter aliquots of cell suspension were placed in
separate Eppendorf tubes. One mL HEPES buffered NaCl and 1 mL
FDA-PI were added to one tube, and 50 microliters Trypan blue
solutions and 50 microliters Triton x-100 were added to the other
tube. The Triton-Trypan cell suspension was examined on a
hemocytomer and the cells were counted. The number of cells was
found to be approximately 2-3.times.10.sup.6 per mL. The FDA-PI
cell solution was examined under a fluorescent microscope and the
percentage of live cells was found to be greater than 95%.
[0272] The tissue culture dishes were held in a 5% CO.sub.2
incubator at 37.degree. C., until the cells were loaded into
capsules.
[0273] Preparation of Alginate Solution
[0274] A 2% alginate solution was prepared dissolving 1 g of Protan
Ultrapure alginate which had been cold cycle ETO sterilized in 50
mL of HEPES buffered 0.9% NaCl. The cell solution was diluted in
the ratio of two parts alginate solution to one part cell
solution.
[0275] Encapsulation Procedure
[0276] Hollow fibers were spun from a 12.5-13.5% poly(acrylonitrile
vinylchloride) solution by a wet spinning technique. Cabasso,
Hollow Fiber Membranes, vol. 12, Kirk-Othmer Encyclopedia of
Chemical Technology, Wiley, New York, 3rd Ed. pp. 492-517 (1980).
The resulting hollow fiber had an outside diameter (OD) of around
900 .mu.m and a wall thickness of around 150 .mu.m. The fibers had
a hydraulic permeability of 18 ml/min/m.sup.2/mmHg and a rejection
coefficient of more than 90% for bovine serum albumin. Fibers were
impregnated with glycerine for storage purposes.
[0277] In order to make implantable capsules, lengths of fiber were
first cut into 5 cm long segments and the distal extremity of each
segment was sealed with an acrylic glue. Encapsulation hub
assemblies were prepared by providing lengths of the membrane
described above, sealing one end of the fiber with a single drop of
LCM 24 (Light curable acrylate glue, available from ICI), and
curing the glue with blue light, and repeating the step with a
second drop. The opposite end was previously attached to a
frangible necked hub assembly, having a silicone septum through
which the cell solution may be introduced. The fiber was glued to
the hub assembly by applying LCM 22 to the outer diameter of the
hub assembly, and pulling the fiber up over it, and curing with
blue light. The hub/fiber assemblies were placed in sterilization
bags and were ETO sterilized.
[0278] Following sterilization with ethylene oxide and outgassing,
the fibers were deglycerinated by ultrafiltering first 70% EtOH,
and then HEPES buffered saline solution through the walls of the
fiber under vacuum.
[0279] The cell/alginate suspension (approx. 20.times.10.sup.6
cells/100 .mu.l) was placed in a 1 ml syringe. A Hamilton 1800
Series 50 microliter syringe was set for a 15 microliter air
bubble, and was inserted into a 1 ml syringe containing the cell
solution and 30 microliters were drawn up. The cell solution was
injected through the silicone seal of the hub/fiber assembly into
the lumen of a modacrylic hollow fiber membrane with a molecular
weight cutoff of approximately 50,000 daltons. Ultrafiltration
could be observed along the entire length of the fiber. After one
minute, the hub was snapped off the sub-hub, exposing a fresh
surface, unwet by cell solution. A single drop of LCM 24 was
applied and the adhesive was cured with blue light. The device was
placed first in HEPES buffered NaCl solution and then in CaCl.sub.2
solution for five minutes to cross-link the alginate. Each implant
was about 5 cm long, 1 mm in diameter, and contained approximately
2.5 million cells.
[0280] After the devices were filled and sealed a silicone tether
(Speciality Silcone Fabrication, Paso Robles, Calif.) (ID: 0.69,
OD: 1.25) was then placed over the proximal end of the fiber. A
radiopaque titanium plug was inserted in the lumen of the silicone
tether to act as a radiographic marker. The devices were then
placed in 100 mm tissue culture dishes in 1.5 ml PC-1 medium, and
stored at 37.degree. C., in a 5% CO.sub.2 incubator for in vitro
analysis and for storage until implantation.
[0281] Implantation
[0282] Devices were implanted one week following the cell loading
procedure. Sheep weighing 42-90 kg (69.+-.15) were given general,
endotracheal anesthesia (pentobarbital sodium 10 mg/kg iv;
halothane 0.5-2%) and preoperative antibiotics (cefazolin sodium 1
g iv). The animals were positioned in the prone position and the
operating table tilted head up at 30.degree.. A 510 cm parasaggital
lumbar incision was made and a spinal tap performed with a Tuohy
needle between L4 and L5 via an oblique paramedian approach. The
appropriate position of the needle in the subarachnoid space was
confirmed by withdrawal of several mls of CSF. This CSF was
analyzed for cell counts, protein level, and microbiology. A guide
wire was introduced through the lumen of the Tuohy needle until it
extended 4-5 cm cranially from the needle opening. The Tuohy needle
was removed and a 7 French dilator introduced over the guide wire
to the level of the dura and removed, enlarging the wire track
through the fascia, paraspinous muscle and ligamentum flavum. This
allowed a 6 French dilator with a 20 cm long outer cannula sheath
to be advanced into the subarachnoid space until the tip of the
cannula was positioned 7 cm within the space. The guide wire and
the dilator were then removed, leaving the cannula within the
subarachnoid space to act as a protective guide for insertion of
the encapsule.
[0283] The cell-loaded and fully assembled device was delivered
into the operating room in a sterile container, bathed in PC-1
medium. The device was prepared for insertion by mounting the
tether on a stainless steel pusher which served to stiffen the very
flexible tether and allowed the capsule to be manipulated within
the lumen of the cannula. The membrane portion of the device was
then introduced into the cannula, handling the device by the
silicone tether and the handle of the pusher. The device was
advanced until the membrane portion lay entirely within the CSF
containing subarachnoid space. The cannula was then removed while
the device was maintained in position using the pusher. Finally,
the pusher was removed and the silicone tether anchored at its free
end by a non-absorbable suture and completely covered with a 2
layer closure of skin and subcutaneous tissue.
[0284] The animal was recovered, examined for possible neurological
complications, and returned to the farm for boarding on the day of
implantation. All animals were able to return to normal diet and
activity on the day of surgery. All experimental, animal care and
surgical protocols were approved by the canton of Vaud Committee on
animal research.
[0285] Explantation
[0286] Four to eight weeks post-implantation each sheep was
anesthetized as described above. The subcutaneous portion of the
silicone tether was isolated through a small skin issue incision.
The device was then retrieved by gentle traction. The capsule was
placed in PC-1 media for analysis of catecholamine release and then
fixed in 4% paraformaldehyde solution for histology. A spinal tap
was performed for all cell counts, protein level and microbiology
prior to the removal of the device. The animal was allowed to
recover and one week following retrieval of the device, CSF samples
were again taken and the animal was sacrificed by overdosage of
pentobarbitral.
[0287] Neurochemical Assays
[0288] The ability of the capsules to release catecholamines was
determined before and after transplantation. Each capsule was first
placed in 2 ml Hank's balanced buffered saline (HBSS) solution for
30 minutes and basal release samples were collected. Evoked release
was obtained by incubating the capsule in 63 .mu.m nicotine
solution in HBSS for another 30 minutes. Perchloric acid (1N) was
added to the collected samples as an antioxidant. Catecholamines
levels were determined by reverse phase high performance liquid
chromatography (HPLC) with electrochemical detection.
[0289] Histology
[0290] Following fixation in 4% paraformaldehyde, the retrieved
capsules were rinsed with phosphate buffered saline (PBS),
dehydrated in graded alcohol up to 95% and embedded in blycol
methacrylate infiltration solution (Historesine Mounting Medium,
Reichert-Jung). Three micron thick sections were cut on a microtome
(Supercut 2065, Leica), mounted on glass slides and stained with
cresyl violet. For immunohistochemistry the capsules were also
fixed in 4% paraformaldehyde, embedded in 5% agarose and cut on a
cryostat (Cryocut 1800, Leica). The immunostain consisted of a
mouse monoclonal antibody to tyrosine hydroxylase (Boehringer
Mannheim) using the peroxidase-anti-peroxidase (PAP) technique and
diaminobenzidine (DAB) coloration.
[0291] Results
[0292] Neurochemic Analysis
[0293] All capsules released a significant amount of catecholamines
under nicotine stimulation. An increase in the catecholamine
release from the capsules on day--1 (one day before implantation,
seven days post-isolation) was observed with each subsequent
isolation and encapsulation series. The mean evoked release of each
batch ranged from 362.+-.14 to 1464.+-.300 pmol/2 ml/30 min for
norepinephrine and 161.+-.11 to 1350.+-.344 pmol/2 ml/30 min for
epinephrin. As indicated by the standard deviation, there were
small but noticeable variations in catecholamine release between
the various capsules of each batch. Basal release was below 230
pmol/2 ml/30 min for both catecholamines measured.
[0294] Typically, the cohorts maintained in vitro were analyzed at
day-1, +7, +14, +21 and +28 days following transplantation for
evoked release of catecholamines. All capsules continued to respond
to nicotine stimulation for at least one month post-encapsulation.
Some showed an increase in their release over time (batch 1,2),
some remained stable (batch 3), and some demonstrated a progressive
decrease of their release over time (batch 4, 5).
[0295] Histology
[0296] Microscopic examination showed good viability of the
encapsulated cells. The cells were organized in small aggregates
entrapped in the alginate matrix. These cell clusters were all
positive for tyrosine-hydroxylase immunochemistry. There was some
disparity in capsule loading within and between batches.
[0297] Surgery and Behavior
[0298] No infections were observed in the implanted sheep and all
CSF samples collected were sterile. No increase in leucocyte counts
were observed in the CSF between the implantation and explantation
times. The same was true for protein levels with exception of sheep
5 which showed a doubling of CSF protein concentration at the
explantation time. A traumatic spinal tap at the explantation may
explain this increase. Of the six implanted sheep, two showed a
transitory weakness of the hind limbs following the transplantation
procedure. An additional sheep showed a complete paralysis at the
time of recovery and did not show any improvement in the following
hours. This animal was sacrificed on the first day
post-transplantation and was therefore not included in the present
series. At autopsy the device appeared to have perforated the
spinal cord of the animal.
[0299] No further surgical complications were encountered. All
devices were retrieved through a small skin incision by gentle
traction on the tether at 4 or 8 weeks post-implantation. The
silicone tethers remained firmly fixed to the capsules; the
membranes remained integral and attached to the tether.
[0300] Morphologic Analysis
[0301] All the retrieved capsules were intact on gross examination.
The membrane was devoid of host cells by microscopic examination.
Clusters of viable cells dispersed in the alginate matrix were
observed throughout the capsule. The cell aggregates were strongly
positive for tyrosine hydroxylase. Capsule loading varied between
batches, with a general upward trend.
[0302] Release
[0303] After retrieval, explanted devices were tested for
catecholamine release in order to assess chromaffin cell viability
and responsiveness to nicotine stimulation. Basal and stimulated
release levels were measured and compared to levels in in-vitro
cohorts. With the exception of sheep 3, the evoked release of
retrieved capsules was in the same range as their respective in
vitro cohort evoked release. In sheep 4, the release of the
retrieved capsule was higher than that of its in vitro cohort. For
Sheep 6, both explanted capsules and in-vitro controls had low
levels of catecholamine release.
EXAMPLE 20
[0304] Implantation of Encapsulated Cellular Grafts in the Lumbar
Subarachnoid Space in Humans
[0305] Fiber Characteristics
[0306] The semipermeable membrane fibers used in this trial were
double skinned PAN/PVC fibers having the following dimensions: an
inner diameter of 773 microns, an outer diameter of 920 microns,
and a wall thickness of 73.1 microns.
[0307] Preparation and Encapsulation of Calf Adrenal Cells
[0308] Bovine adrenal chromaffin cells were prepared and
encapsulated as outlined in example 19.
[0309] Surgical Procedure
[0310] After establishing IV access and administering prophylactic
antibiotics (cefazolin sodium, 1 gram IV), the patient was
positioned on the operating table, generally in either the lateral
decubitus or genu-pectoral position, with the lumbar spine flexed
anteriorly. The operative field was sterily prepared and draped
exposing the midline dorsal lumbar region from the levels of S-1 to
L-1, and allowing for intraoperative imaging of the lumbar spine
with C-arm fluoroscopy. Local infiltration with 1.0% lidocaine was
used to establish anesthesia of the skin as well as the periosteum
and other deep connective tissue structures down to and including
the ligamentum flavum.
[0311] A 3-5 cm skin incision was made in the parasagital plane 1-2
cm to the right or left of the midline and was continued down to
the lumbodorsal fascia using electrocautery for hemostasis. Using
traditional bony landmarks including the iliac crests and the
lumbar spinous processes, as well as fluoroscopic guidance, and 18
gauge Touhy needle was introduced into the subarachnoid space
between L-3 and L-4 via an oblique paramedian approach. The needle
was directed so that it entered the.space at a shallow, superiorly
directed angle that was no greater than 30-35.degree. with respect
to the spinal cord in either the sagittal or transverse plane.
Appropriate position of the tip of the needle was confirmed by
withdrawal of. several ml of cerebrospinal fluid (CSF) for
preimplantation catecholamine, enkephalin, glucose, and protein
levels and cell counts.
[0312] The Touhy needle hub was reexamined to confirm that the
opening at the tip is oriented superiorly (opening direction is
marked by the indexing notch for the obturator on the needle hub),
and the guide wire was passed down the lumen of the needle until it
extended 4-5 cm into the subarachnoid space (determined by
premeasuring). Care was taken during passage of the wire that there
was not resistance to advancement of the wire out of the needle and
that the patient did not complain of significant neurogenic
symptoms, either of which observations might indicate misdirection
of the guide wire and possible impending nerve root or spinal cord
injury.
[0313] After the guide wire appeared to be appropriately placed in
the subarachnoid space, the Touhy needle was separately withdrawn
and removed from the wire. The position of the wire in the midline
of the spinal canal, anterior to the expected location of the caud
equina, and without kinks or unexplainable bends was then confirmed
with fluoroscopy. After removal of the Touhy needle the guide wire
should be able to be moved freely into and out of the space with
only very slight resistance due to the rough surface of the wire
running through the dense and fibrous ligamentum flavum.
[0314] The 7 French dilator was then placed over the guide wire and
the wire was used to direct the dilator as it was gently but firmly
pushed through the fascia, paraspinous muscle, and ligamentum
flavum, following the track of the wire toward the subarachnoid
space. Advancement of the 7 French dilator was stopped and the
dilator removed from the wire as soon as a loss of resistance was
detected after passing the ligamentum flavum. This was done in
order to avoid advancing and manipulating this relatively rigid
dilator within the subarachnoid space to any significant
degree.
[0315] After the wire track was "overdilated" by the 7 French
dilator, the 6 French dilator and cannula sheath were assembled and
placed over the guide wire. The 6 French dilator and cannula were
advanced carefully into the subarachnoid space until the opening
tip of the cannula was positioned 7 cm within the space. As with
the 7 French dilator, the assembled 6 French dilator and cannula
were directed by the wire within the lumen of the dilator. Position
within the subarachnoid space was determined by premeasuring the
device and was grossly confirmed by fluoroscopy. Great care was
taken with manipulation of the dilators and cannula within the
subarachnoid space to avoid misdirection and possible neurologic
injury.
[0316] When appropriate positioning of the cannula was assured, the
guide wire and the 6 French dilator were gently removed from the
lumen of the cannula in sequence. Depending on the patient's
position on the operating table, CSF flow through the cannula at
this point should be noticeable and may be very brisk, requiring
capping the cannula or very prompt placement of the capsule implant
in order to prevent excessive CSF.
[0317] The encapsulated adrenal chromaffin cell graft
(CytoTherapeutics CereCRIB.TM.) was provided in a sterile, double
envelope container, bathed in transport medium, and fully assembled
including a tubular silicone tether. Prior to implantation through
the cannula and into the subarachnoid space, the capsule was
transferred to the insertion kit tray where it was positioned in a
location that allowed the capsule to be maintained in transport
medium while it was grossly examined for damage or major defects,
and while the silicone tether was trimmed, adjusting its length to
the pusher and removing the hemaclip.TM. that plugs its external
end.
[0318] The tether portion of the CereCRIB.TM. capsule was mounted
onto the stainless steel pusher by inserting the small diameter
wire portion of the pusher as the membrane portion of the device
was carefully introduced into the cannula. The capsule was advanced
until the tip of the membrane reached a point that was 2-10 mm
within the cranial tip of the cannula in the subarachnoid space.
This placement was achieved by premeasuring the cannula and the
capsule-tether-pusher assembly, and it assured that the membrane
portion of the capsule was protected by the cannula for the entire
time that it was being advanced into position.
[0319] After the capsule was positioned within the cannula, the
pusher was used to hold the capsule in position (without advancing
or withdrawing) in the subarachnoid space while the cannula was
completely withdrawn from over the capsule and pusher. The pusher
was then removed from the capsule by sliding its wire portion out
of the silicone tether. Using this method the final placement of
the capsule was such that the 5 cm long membrane portion of the
device lay entirely within the CSF containing subarachnoid space
ventral to the cauda equina. It was anchored at its caudal end by a
roughly 1-2 cm length of silicone tether that ran within the
subarachnoid space before the tether exited through the dura and
ligamentum flavum. The tether continued externally from this level
through the paraspinous muscle and emerged from the lumbodorsal
fascia leaving generally 10-12 cm of free tether material that was
available for securing the device.
[0320] CSF leakage was minimized by injecting fibrin glue
(Tissel.RTM.) into the track occupied by the tether in the
paraspinous muscle, and by firmly closing the superficial fascial
opening of the track with a purse-string suture. The free end of
the tether was then anchored with non-absorbable suture and
completely covered with a 2 layer closure of the,skin and
subcutaneous tissue.
[0321] The patient was then transferred to the neurosurgical
recovery area and kept at strict bed rest, recumbent, for 24 hours
postoperatively. Antibiotic prophylaxis is also continued for 24
hours following the implantation procedure.
[0322] Human Pain Patients
[0323] Three human terminally ill patients suffering from
intractable pain were implanted according to the method outlined
above.
[0324] Devices were released for implantation only after individual
testing for sterility and for release of catecholamines. The
protocol called for a thirty day study that could be extended to a
maximum of 90, days upon the request of the patients. Three
patients were eligible with terminal cancer, pain incompletely
relieved by narcotic therapy, and no evidence of active infection
or tumor in the meningeal space. After informed consent was granted
by the patients and approval was received from the Ethical
committee of the Faculty of Medicine of the University of Lausanne,
Switzerland, the devices were implanted under local anesthesia.
[0325] Postoperative recovery was uneventful though all patients
experienced some loss of CSF fluid and one patient experienced
headaches of several days duration. Two of the three patients
recorded improvement on the McGill questionnaire and a visual
analog scale of pain; the third did not. Significant increases were
observed in the cerebrospinal fluid catecholamine levels of the two
patients with improved pain scores. All three patients reduced
their intake of narcotics and analgesics (Table 3).
[0326] The tethered implants were recovered via simple surgical
excision after 43 days and 55 days in two patients and at autopsy
in patient #3 who died from her primary disease at day 42.
Explanted devices were inspected visually and then examined
histologically and for biochemical activity. There was no visible
difference between the devices as implanted and as retrieved. Upon
microcoscopic examination, external surfaces of all three implants
were free of adherent cells, fibrotic overlayers, and other signs
of acute phase response or foreign body reaction.
[0327] Intracapsular populations of healthy chromaffin cells were
observed by histology in all three explants, with cell viability
estimated at 80 percent. Cells recovered from the capsules were
also positive by immunohistochemistry for tyrosine hydroxylase and
metenkaphalins. Basal release of norepinephrine and epinephrine in
explanted capsules was in the range of 0.2 and 3 nanomoles per 24
hours. Autopsy reports on the spinal cords became available in all
three patients and showed no effect from the implant.
3TABLE 3 MORPHINE INTAKE (mg/day) pre-implant post implant.sup.1
oral Patient 1 60 0 Patient 2 0 0 Patient 3 60 0 epidural Patient 1
75 18 Patient 2 60 32 Patient 3 0 0 .sup.1Mean value from day 10
post implant to explant (or death)
EXAMPLE 21
[0328] Implantation of Encapsulated Cellular Grafts Intracranially,
in the Lateral Ventricle in Humans
[0329] Two human patients suffering from intractable pain were
implanted in the ventricle of the brain with encapsulated adrenal
chromaffin cells. The brain ventricles, including the lateral
ventricles, lie rostral to the lumbar region. The CSF drains or
flows from the brain to the spinal cord. The chromaffin cells and
CereCRIB.TM. capsules were prepared as described in Example 20.
[0330] The surgical procedure for implantation into the lateral
ventricle of the brain is described below.
[0331] Immediately before the implantation procedure, the patient
was fitted with a stereotactic head ring assembly and localizer
ring (or image localization/marker device) suitable for guided
cannula placement within the lateral ventricles using local
anesthesia (local infiltration with generally 1% lidocaine). The
Radionics.RTM. BRW frame was used here, however the Radionics.RTM.
CRW, Leksell.RTM., or functionally similar devices are also
appropriate.
[0332] A computed tomography (CT) scan was then performed and used
to define a target site(s) and stereotactic coordinates for the
implant(s). Implantation cannula trajectory and implant site were
chosen with the following considerations: (1) avoiding the frontal
sinuses; (2) avoiding the choroid plexus; (3) allowing straight,
undistorted positioning of the intended implant within the lateral
ventricle. There are three capsule lengths, 2.5, 3.75, or 5.0 cm,
currently in use. The two patients in this study were implanted
with 2.5 cm CereCRIB.TM. capsules.
[0333] A target site must be selected that will allow a length of
the internal end of the cannula that is at least the length of the
membrane portion of the desired capsule to lie within an
acceptable, CSF filled space within the ventricle. The zero
reference point for determining cannula insertion depth is the
surface of the skin, as seen on the CT scan, and the target site is
defined as the intended target of the internal tip (opening) of the
insertion cannula.
[0334] Two implant devices may be placed in one patient at a single
procedure by placing one implant in each lateral ventricle. Future
implantation sites may target the third ventricle and/or the
aqueduct. The current stereotactic guidance technique uses CT
imaging for reference, however magnetic resonance imaging (MRI),
stereotactic at last coordinates, ultrasound or other guidance
methods may also be appropriate. Following completion of the data
gathering for stereotactic placement of the implant(s), the patient
is transferred to the operating room for the implantation
procedure.
[0335] After establishing IV access and administering prophylactic
antibiotics (currently, cefazolin sodium, 1 gram IV), the patient
was positioned on the operating table in the semi-supine/seated
position with the stereotactic head ring assembly secured to the
table. The operative field was sterily prepared and draped exposing
the intended implantation site(s) (generally located in the
paramedian, frontal region) and allowing for sterile placement and
removal of the stereotactic arc system/manipulator to the frame
base.
[0336] Local infiltration with 1.0% lidocaine was used for
anesthesia of the skin and deeper scalp structures down to the
periosteum, and a 4-8 cm skin incision was made down to the skull
at the calculated entry site(s) for the stereotactically guided
insertion canula (generally in the frontal region, in the
parasagital plane 3 cm to the right or left of the midline) using
electrocautery for hemostasis. A twist drill guided by the
stereotactic arc system was then used to create a burr hole
(generally 4 mm diameter) down to the level of the dura. The dura
was sharply penetrated, and the insertion cannula/obturator
assembly was mounted into the stereotaxic microdrive and directed
into the burr hole. Blood from the wound was excluded from the burr
hole by applying the microdrive guide tube directly against the rim
of the burr hole.
[0337] The insertion cannula/obturator assembly were advanced
manually to the preset depth stop on the microdrive, leaving the
tip of the cannula at the target site. The obturator was then
carefully withdrawn from the insertion-cannula, taking care not to
deflect the cannula with the tip of the obturator. Appropriate
position of the tip of the cannula within the ventricle may be
confirmed by a meniscus of cerebrospinal fluid (CSF) rising up
within the clear insertion cannula after removal of the obturator.
Samples of CSF may be taken for preimplantation catecholamine,
enkephalin, glucose, and protein levels and cell counts.
[0338] The encapsulated adrenal chromaffin cell graft
(CytoTherapeutics CereCRIB.TM.) was prepared and mounted onto the
pusher as described in Example 20.
[0339] The CereCRIB.TM. capsule was handled completely by the
silicone tether and the handle of the pusher as the membrane
portion of the device was carefully introduced into the cannula.
The capsule was advanced until the tip of the membrane reached a
point that was within 1-2 mm of the internal tip of the cannula
positioned in the lateral ventricle (but not extending beyond the
tip of the cannula). This placement was achieved by premeasuring
the cannula and the capsule-tether-pusher assembly, and it assured
that the membrane portion of the capsule was protected by the
cannula for the entire time that it was being advanced into
position. After the capsule was positioned manually within the
cannula, the pusher was locked into position in the microdrive and
used to hold the capsule in position in the ventricle (without
advancing or withdrawing) while the cannula was completely
withdrawn from over the capsule and pusher. The pusher was then
removed from the capsule by sliding its wire portion out of the
silicone tether.
[0340] Using this method the final placement of the capsule was
such that the entire membrane portion of the device lay entirely
within an appropriate, CSF containing region of the ventricle. The
membrane capsule was anchored at its external end by a length of
silicone tether that ran (generally) through a portion of the
frontal lobe before it exited through the dura and the skull,
leaving generally 5-10 cm of free tether material that was
available for securing the device. The free end of the tether was
then anchored to the outer table of the skull adjacent to the burr
hole using a standard, maxillo-facial miniplate and screws and
completely covered with a 2 or 3 layer closure.
[0341] The patients were then transferred to the neurosurgical
recovery area and followed for 12 hours postoperatively for
potential hemorrhagic complications with no special restrictions.
Antibiotic prophylaxis was also continued for 24 hours following
the implantation procedure.
EXAMPLE 22
[0342] Implantation of .beta.-NGF Secreting BHK Cells for
Prevention of Neural Damage Due to Excitotoxicity (Huntington's
Disease Model)
[0343] Subjects
[0344] Adult male Sprague-Dawley rats (Taconic Breeders,
Germantown, N.Y.) approximately 3 months old and weighing 300-350
grams were used. The animals were housed in groups of 3 in a
temperature and humidity-controlled colony room which was
maintained on a 12 hr light/dark cycle with lights on at 0700 hrs.
Food and water were available ad libitum throughout the
experiment.
[0345] BHK-NGF Cell Line Production
[0346] Two human genomic clones (phbeta N8D8, phbeta NSB9) coding
for the 5' and 3' ends of the .beta.-NGF gene were obtained from
the ATCC. A 440 bp 5' Sca1-EcoR1 fragment from phbeta N8D8 was
ligated to a 3' 2.0 kb EcoR1 fragment isolated from phbeta N8B9.
The spliced NGF genomic sequence contained approximately 37 bp of
the 3' end of the first intron, the double. ATG sequence believed
to be the protein translation start for pre-pro-NGF and the
complete coding sequence and entire 3' untranslated region of the
human gene (Hoyle et al., Neuron, 10, pp. 1019-34 (1993)). The
combined 2.51 kb .beta.-NGF fragment was subcloned into the
DHFR-based pNUT expression vector immediately downstream from the
mouse metallothionein-1 promotor (-650 to +7) and the first intron
of the rate insulin II gene (Baetge et al., Proc. Natl. Acad. sci.,
83, pp. 5454-58 (1986). The pNUT-.beta.NGF construct was introduced
into BHK cells using a standard calcium phosphate-mediated
transfection method. Mock-transfected cells served as controls in
these experiments. BHK cells were grown in DMEM, 10% fetal bovine
serum, antibiotic/antimycotic, and L-glutamine (GIBCO) in 5% CO2
and at 37.degree. C. Transfected BHK cells were selected in medium
containing 200 .mu.M methotrexate (Sigma) for 3-4 weeks and
resistant cells were maintained as a polyclonal population either
with or without 200 .mu.M methotrexate.
[0347] Encapsulation Procedure
[0348] Asymmetric hollow fibers were cast from solutions of
12.5-13.5% poly (acrylonitrile vinyl chloride, PAN-PVC) copolymer
in dimethyl sulfoxide (w/w). The fabrication process utilized a
dry-wet (jet) spinning technique according to Cabasso, Hollow Fiber
Membranes, vol. 12, Kirk-Othmer Encyclopedia of Chemical
Technology, Wiley, New York, 3rd Ed., pp. 492-517 (1980). Single
cell suspensions of BHX cells were prepared using calcium- and
magnesium-free Hanks' balanced salts (HBSS) and trypsin/EDTA.
Encapsulation devices were manufactured by mounting a 1-1.1 cm
length of dry hollow fiber onto hub with a septal fixture at the
proximal end which has loading access for cells to be injected into
the lumen of the device. Cells were loaded into the prefabricated
capsules as follows: BHK control cells and BHK/hNGF cells were
loaded at a density of approximately 107 cells/ml. The BHK cell
suspensions, at a density of 2.times.10.sup.7 cells/ml, were mixed
1:1 with physiologic Vitrogen.RTM. (Celtrix, Palo Alto, Calif.),
and infused into the capsules through the septal access port. After
infusing 2.2.5 .mu.l of the cellular suspension, the septum was
cracked off and the access port was sealed using a light-cured
acrylate (Luxtrak.TM. LCM 24, ICI Resins US, Wilmington, Mass.).
The capsules were subsequently "tethered" by placing a 1.5 cm
0.020" silastic-tube over the acrylic hub. The cell-loaded devices
were transferred into sterile 5 ml polypropylene snap cap tubes
containing 4.5 ml of PC-1 medium. The 5 ml snap cap tube was then
placed inside a sterile 50 ml conical centrifuge tube and sealed
for transport.
[0349] BHK cell-loaded capsules were maintained in serum-free
defined PC-1 medium (Hycor Biomedical Inc., Portland, Me.) for 2-5
days prior to implantation. After 3 or 4 days In vitro, cell-loaded
capsules were rinsed in HBSS, placed in 1 ml of fresh PC-1 medium
overnight, and the medium analyzed for hNGF by ELISA.
[0350] NGF ELISA
[0351] The quantification of hNGF released from encapsulated
BHK/hNGF cells was performed as follows. All of the reagents were
obtained from Boehringer-Mannheim Biochemicals unless otherwise
noted. Nunc-Immuno Maxisorp ELISA plates were coated with 150 .mu.l
per well of anti-mouse-.beta. (2.5S) NGF at 1 ng/ml in coating
buffer (1.times.PBS without CaCl.sub.2 and without
MgClsub.sub.2/0.1% sodium azide; pH 9.6). The coated plates were
incubated at 37.degree. C. for at least 2 hours or alternatively at
4.degree. C. overnight.
[0352] The coating solution was withdrawn from the wells and the
wells were washed 3.times. with 300 .mu.l wash buffer (50 mm
Tris-HCl/200 mm NaCl.sub.2/1% Triton X-100/0.1% sodium azide; pH
7.0). The wells were then blocked with 300 .mu.l of coating
solution containing 10 mg/ml of BSA at room temperature for 30 min.
The wells were then washed 3.times. with 300 .mu.l wash buffer.
Conditioned medium samples were diluted 1:1 in 2.times. sample
buffer (the sample buffer is the same as wash buffer, only with 2%
BSA), with 10 .mu.l of the prepared samples loaded. into the wells.
The plates were covered and incubated for at least 2 hours at
37.degree. C. or overnight at 4.degree. C.
[0353] The solutions were removed from the wells by suction and
washed 3.times. with 300 .mu.l of wash buffer. To each well, 100
.mu.l of 4 U/ml of anti-mouse-.beta. (2.55) NGF-.beta.-gal
conjugate was added. The plates were incubated at 37.degree. C. for
at least 1 hour. The solutions were removed from the wells by
suction and washed 3.times. with 300 of wash buffer. Finally, 200
.mu.l of chlorophenol red-.beta.-D-galactopyran- oside substrate
solution (40 mg CPRG in 100 mm Hepes/150 mm NaCl/2 mm
MgCl.sub.2/0.1% sodium azide/1% BSA; pH 7.0) was added to the
wells, incubated at 37.degree. C. for 30 min to one hr or after the
color development was sufficient for photometric determination at
570 nm, with the samples analyzed on a plate reader and measured
against recombinant NGF protein standards.
[0354] Surgery
[0355] Immediately prior to surgery, rats were anesthetized with
sodium pentobarbital (45 mg/kg, i.p.), and positioned in a Kopf
stereotaxic instrument. A sagittal incision was made in the scalp
and two holes drilled for the placement of the cell-loaded capsules
into the lateral ventricle. Rats were unilaterally implanted by
placing the capsule within an 18 gauge Teflon catheter mounted to
the stereotaxic frame as previously described. The stereotaxic
coordinates for implantation were: anterior 0.5 mm anterior to
gregma, 1.5 mm lateral to the sagittal suture and 8.0 mm below the
cortical-surface.
[0356] Three days following implantation of cell-loaded capsules,
animals were anesthetized, placed in the stereotaxic instrument and
injected with 225 nmol of quinolinic acid ("QA") or
phosphate-buffered saline (PBS) into the striatum at the following
coordinates: anterior++1.2 mm, lateral=.+-.2.6 mm and ventral=-5.5
mm from the surface of the brain. QA (Sigma Chemical Co.) was
dissolved in 2N sodium hydroxide and diluted with phosphate buffer
at pH 7.2 to a final pH of 7.4 and concentration of 225 nmol/ul. QA
was infused into the striatum, using a 28-gauge Hamilton syringe,
over five min in a volume of 1 .mu.l. The injection cannula was
left in place for an additional two min to allow for diffusion of
the perfusate. This procedure resulted in the formation of three
experimental groups: 1) quinolinic acid only (QA; N=8), quinolinic
acid+NGF-secreting BHK cells (QA/BHK/hNGF; N=6), and quinolinc
acid+control BHK cells (QA/BHK/CONTROL; N=7).
[0357] Immediately following surgery, animals were injected i.p.
with 10 ml of lactated Ringer's solution. Animals were housed
postoperatively with food mash and water available ad libitum.
[0358] At the conclusion of behavioral testing, animals were
anesthetized and placed into the stereotaxic instrument. A
craniotomy was performed. over the implantation site and the dural
scar surrounding the implant site excised. The cortical surface was
cut to expose the underlying capsule witch was retrieved with a
pair of Dumont (#5) forceps.
[0359] Histology
[0360] A subset of animals (3-4 per group) were anesthetized 29-30
days following surgery and prepared for histological analysis.
Animals were transcardially perfused, using a peristaltic pump,
with 20 ml saline followed by 500 ml of paraformaldehyde. All
solutions were ice cold (4.degree. C.) and prepared in 50 mM PBS
(pH 7.4). Brains were removed following fixation, placed in 25%
buffered sucrose (pH 7.4) and refrigerated for approximately 48
hr.
[0361] Tissue was cut at 40 .mu.m intervals on a cryostat and
mounted onto polylysine coated slides. Alternating sections were
taken throughout the striatum and processed for immunocytochemical
localization of choline acetyltransferase (ChAT) and glial
fibrillary acidic protein (CFAP) according to the following
protocol: 1) overnight incubation in PBS containing 0.8% Triton
X-100+10% normal serum, 2) 48 hr incubation with primary antibody
(goat antiserum to ChAT; Chemicon) at a dilution of 1:1000 or
(rabbit antiserum to GFAP at a dilution of 1:5,000), 3) 6.times.5
min rinses in PBS+0.2% Triton X-100 followed by a 1.5 hour
incubation in biotinylated secondary antibody (lgG), 4) 6.times.5
min rinses in PBS+0.2% Triton X-100, 5) incubation with
Avidin-Biotin, Complex (ABC, Vector Elite) for 1.5 hours, 6)
3.times.5 min rinses in PBS, 7) 5 min rinse in distilled water, 8)
incubation DAB (0.05%)+2% nickel ammonium sulfate (ChAT only)
dissolved in 0.1% Tris buffer for 5 min followed by hydrogen
peroxide (0.01%) for 5 minutes, (9) the reaction was terminated by
3.times.1 min rinses in PBS.
[0362] A separate series of sections throughout the striatum were
stained for NADPH-diaphorase according to the procedure of Vincent
et al. Adjacent sections were stained with hematoxylin and eosin
(H+E). Sections were mounted, dehydrated and coverslipped.
[0363] For analysis of retrieved capsules, capsules were fixed in a
4% paraformaldehyde, 0.5% glutaraldehyde solution, rinsed in PBS
and dehydrated up to 95% ethanol. A 1:1 solution of glycol
methacrylate (Historesin, Reichert-Jung, Cambridge Instruments) was
then added to the capsules for one hr. Pure infiltration solution
replaced the 1:1 mixture and remained for a minimum of 2 hrs. The
capsules were then rinsed with the embedding solution, transferred
to flat molds, and embedded in glycol methacrylate. Sections 5
.mu.m thick were sectioned (Reichert-Jung, Supercut microtome
2065), mounted on glass slides and stained for H+E.
[0364] Behavioral Testing
[0365] Beginning 13-14 days following QA injections, animals were
tested for apomorphine-induced (1.0 mg/kg in normal saline
containing 0.2% ascorbate) rotation behavior in one of six rotation
devices (Rotoscan, omnitech Instruments) which were connected to an
IBM computer for automated data collection. Animals were placed
into the test chamber for a 5 min habituation period and were then
injected with apomorphine. Sensitization of apomorphine-induced
rotation behavior occurs following excitotoxin lesions of the
striatum. Therefore, animals were tested 4 times with each session
separated by a 3-4 day interval. Rotations were defined as complete
360 degree ipsilateral turns and were reported as the net
difference between the two directions.
[0366] Statistical Analysis
[0367] The behavioral results were assessed using a two-way
repeated measures analysis of variance (ANOVA). Appropriate
pair-wise comparisons were performed using Fisher's Least
Significant Difference test. Acceptable statistical significance
was established at p<0.05.
[0368] Results
[0369] Behavioral Testing
[0370] No overt signs of behavioral or neurological toxicity were
observed in any animals following implantation of either BHK/hNGF
or BHK control capsules. During the post-operative recovery period
following QA injections, the QA/BHK/CONTROL and QA alone groups
exhibited whole body barrel rotations which persisted for 2-4
hours. These same animals had a transient period of weight loss,
piloerection and diarrhea which subsided 3-4 days following QA.
Animals which received QA together the BHK/hNGF capsules did not
show whole body rotations but did exhibit a slight motor asymmetry
following QA. This asymmetry was transient and recovery was seen
within several hrs. No additional signs of systemic toxicity were
noted.
[0371] Following QA injections, animals displayed
apomorphine-induced rotations ipsilateral to the lesion with the
extent of rotation behavior increasing with repeated testing. hNGF
treatment significantly decreased the extent of rotation behavior
produced by QA. A two-way repeated ANOVA revealed significant
effects of treatment F(2, 17)=16.063, p<0.0001, as well as
repeated testing F(2, 3)=28.861, p<0.0001, and a treatment by
testing interation F (6, 51)=2.937, p<0.05. Post-hoc analysis
revealed that the QA/hNGF group rotated significantly less than
either the QA alone or QA/BHK control at all test times. No
significant differences were observed between the QA alone and
QA/BHK control groups at any time during testing.
[0372] NGF ELISA
[0373] Prior to implantation and following retrieval (immediately
prior to perfusion), the encapsulated BHK cells were incubated and
the conditioned medium was assayed for HNGF by ELISA. Prior to
implantation, the encapsulated BHK/hNGF cells were releasing 34.13
(.+-.6.9) ng NGF/capsule/24 hr while BHK/CONTROL cells showed hNGF
levels no different than measured in control medium (0.06 ng
NGF/capsule/24 hr). The BHK cell-loaded capsules were easily
retrieved with little or no host tissue adhering to the capsule
wall. Post explant values of hNGF from capsules averaged 16.03
(.+-.6.0) ng hNGF/capsule/24 hr and was not detected in conditioned
medium from BHK/CONTROL capsules.
[0374] Histology
[0375] The cell-loaded devices were easily retrieved and induced
minimal damage to the host tissue. Placement of the capsules was
within the lateral ventricle in all cases. Analysis of sections
throughout the implant site revealed that the devices abutted the
cortex and extended through the corpus callosum to the ventral
aspect of the lateral ventricle. The capsules typically extended
into the host striatum and in some cases medially into the lateral
septum.
[0376] Administration of QA produced a substantial atrophy of the
striatum together with a marked ventricular dilation. In some cases
moderate cell loss was observed in the nucleus accumbens and
cortical regions adjacent to the injection site. The lesion core
was virtually devoid of neurons with a nearly complete loss of ChAT
and NADPH-d positive neurons. In general, the remaining neurons
present within the lesion core were shrunken and dystrophic in
appearance. GFAP staining revealed the presence of reactive
astrocytes throughout the lesion site and extending into the
adjacent host tissue.
[0377] Implantation of BHK control cell-loaded devices into the
lateral ventricles did not effect striatal morphology. Furthermore,
no alterations in the size of the lesion core or extent of cell
loss was observed in these animals. Infiltration of reactive
astrocytes dominated the ventricular wall and the striatum
immediately adjacent to the implantation site. In contrast,
implantation of encapsulated BHK cells which released hNGF exerted
a marked protective effect on striatal morphology following QA. In
these animals, the lesion core was significantly reduced and
frequently consisted of cell necrosis surrounding the injection
tract with minimal extension into the surrounding tissue. Within
the lesion core there was no apparent sparing of neurons. On the
other hand, NADPH-d, and Nissl staining revealed a striking
preservation of neurons removed from the central lesion core. In
general, the sparing of ChAT-positive neurons appeared greater than
that of NADPH-d-positive neurons.
[0378] Capsule morphology and cell survival of the encapsulated BHK
cells were determined. Few adhering host cells were found on the
capsule surface. An abundance of viable BHK cells were evenly
distributed throughout the capsule. Areas of focal cell debris were
occasionally observed within the cores of large viable cell
aggregates. Numerous mitotic figures were observed throughout the
capsules and no differences in cell survival were noted between
BHK/hNGF and BHK/CONTROL cells.
[0379] These data indicate implantation of polymer encapsulated BHK
cells, genetically-modified to secrete hNGF, prior to intrastriatal
injections of QA, results in attenuation of the associated
neurotoxicity. QA alone produced a marked striatal atrophy together
with loss of ChAT and NADPH-d-positive neurons. Implantation of
BHK/hNGF cells decreased the overall extent of the lesion produced
by QA. The attenuation of QA-induced toxicity was associated with a
preservation of ChAT and NADPH-d-positive neurons within the
striatum. Associated with the histological protection produced by
hNGF was a significant attenuation of apomorphine-induced
rotational behavior.
EXAMPLE 23
[0380] Treatment of Neural Degeneration in Non-Human Primates Using
Encapsulated Genetically Modified BHK Cells
[0381] In this example we grafted BHK cells into the lateral
ventricle of fornix-transected nonhuman primates and assessed the
ability of polymer encapsulated NGF-secreting cells to prevent the
degeneration of primate cholinergic basal forebrain neurons which
normally occurs following axotomy.
[0382] BHK-hNGF Cell Line Production.
[0383] The BHK-hNGF cell line was produced as described in Example
22.
[0384] Encapsulation Procedure BHK-hNGF cells were encapsulated as
described in Example 22.
[0385] Surgical Procedures
[0386] Young adult cynomolgus (Macaca fascicularis) monkeys of both
sexes (N=8; 4-6 kg) were used in this study. Animals were
tranquilized with ketamine HCL (10 mg/kg, im) and intravenous lines
were secured for fluid administration. The animals were intubated
via the orotracheal method and anesthesia maintained throughout the
procedure with isoflurane (1.5-2.0%). Animals were placed on a
heating pad to maintain body temperature and electrocardiogram
leads placed to monitor heart rate and rhythm. To facilitate
relaxation of the brain and minimize trauma due to retraction
pressure, mannitol (0.250 mg/kg, iv) was administered immediately
prior to the craniotomy.
[0387] Unilateral transections of the left fornix were performed
using an open microsurgical approach developed by Kordower and
Fiandaca (1990). After securing the animals in a Kopf stereotoxic
headframe, a midline incision was made in the scalp and the skin
retracted laterally. The medial attachment of the temporalis muscle
was mobilized and a surgical drill used to create a parasagittal
bone flap (size=1.5 cm.times.4.0 cm) which exposed the frontal
superior sagittal sinus. The dura was retracted and a
self-retaining retractor used to expose the interhemispheric
fissure. The parasagittal bridging veins were coagulated where
needed to facilitate retraction of the cerebral hemisphere. With
the aid of a surgical microscope, arachnoid adhesions were divided.
When necessary, veins overlying the corpus callosum were
coagulated. The corpus callosum was longitudinally incised exposing
medial subcortical structures from the septum and head of the
caudate rostrally through the foramen of Monro caudally. At the
level of the foramen of Monro, the fornix is easily visualized as a
discrete 2-3 mm wide white fiber bundle. The fornix was initially
transected using a ball dissector then the cut ends of the fornix
were suctioned to ensure completeness of the lesion.
[0388] Following the transection of the fornix, individual BHK
cell-containing capsules were manually placed within the lateral
ventricle with fine forceps between the head of the caudate and the
septal nucleus. A total of 5 capsules were implanted in each animal
oriented in a row in the rostrocaudal direction. The capsules
abutted the caudate and septum, remained upright, and did not
require to be secured further. Four animals received BHK/hNGF
capsules, three received BHK-control cell-loaded capsules and one
monkey received a fornix transection but no transplant. With
hemostasis achieved, the dura was reapproximated, the bone flap was
sutured back in place and the galea and skin was sutured using
routine methods. All animals received antibiotics (Cefotaxime, 50
mg/kg, IM) for 4 days postoperatively.
[0389] Histology
[0390] Twenty-three to twenty-eight days following surgery, animals
were anesthetized as described above. Two-three ml of CSF was
obtained from either the lumbar region (N=1 BHK control; 2
BHK/hNGF) or cistema magna (H=2 BHK control; 2 BHK/hNGF) to assay
hNGF levels. Animals were then placed into the stereotoxic frame,
the previously prepared bone flap was removed, the cerebral
hemisphere retracted and the BHK cell-loaded capsules removed.
Immediately following removal of the capsules, animals were
transcardially perfumed using a peristaltic pump with 1 liter of
phosphate-buffered saline (pH=7.4) containing 1 ml of Heparin
followed fixation with 3.5 lifers of 4% paraformaldehyde. The
brains were blocked in the transverse plane following fixation,
stored in 25% buffered sucrose (pH 7.4) and refrigerated for 5-7
days.
[0391] Frozen sections were cut (30 .mu.m) on a sliding knife
microtome and every seventh section through the septal/diagonal
band complex was processed immunocytochemically for choline
acetyltransferase (CHAT) and the low affinity p75 NGF receptor
(NGFr). Immunocytochemical labeling was conducted according to
previously published protocols and briefly consisted of: 1)
overnight incubation in PBS containing 0.4% Triton+2%-normal serum,
2) 48 hour incubation in the primary CHAT polyclonal antibody
(Chemicon; 1:10,000), or NGFR monoclonal antibody (generously
provided by Dr. Mark Bothwell; 1:20,000), 3) overnight rinse in
PBS+0.2% Triton, 4) 6.times.5 minute rinse in PBS followed by a 1.5
hour incubation in the appropriate biotinylated secondary IgG
antibody (Vector; 1:100), 5) 6.times.5 minute rinses in PBS+Triton,
6) incubation with "Elite" Avidin-Biotin complex (Vector, 1:1000)
for 1.5 hours, 7) 3.times.10 minutes rinses in PBS, 8) incubation
in the chromagen solution containing 0.05% 3,3' diaminobenzidine,
2.5% nickel ammonium sulfate dissolved in 0.1% Tris buffer for 5
minutes followed by hydrogen peroxide (0.01%) for 5 minutes. The
reaction was terminated by 3.times.1 minute rinses in PBS. Sections
were mounted, dehydrated in alcohols and coverslipped. Control
sections were processed in an identical manner except the primary
antibody solvent or an irrelevant IgG was substituted for the
primary antibody. Adjacent sections were stained with hematoxylin
and eosin (H & E) to aid in cytoarchitectonic delineation.
[0392] To verify the completeness of the lesion, sections through
the hippocampus were processed histochemically for the
visualization of acetylcholinesterase (AChE) using the procedure of
Hedreen and coworkers ( ). Sections were incubated for 1 hour in a
solution (pH 5.0) containing 100 mM sodium acetate (65 ml), 50 mg
acetythiocholine iodide, 100 mm sodium citrate (5 ml) 30 mm copper
sulfate (10 ml), 15 ml dH20, 5 mM potassium ferricyanide (4 ml) and
0.001M tetraisopropylpyrophophoramide (iso-OMPA). After 3.times.10
minute washes in sodium acetate buffer, the sections were incubated
for 1 minute in 4% ammonium sulfide. After 5.times.10 minute washes
in sodium nitrite, the section were incubated for 1 minute in a
0.05% silver nitrate solution. After 5.times.10 minute washes in
sodium nitrate, sections were mounted, dehydrated, and coverslipped
as before. For control, sections were processed in an identical
manner except that acetylthiocholine iodide was omitted from the
incubation medium.
[0393] For quantification of cholinergic cell loss, the number of
ChAT and NGFr-positive neurons were manually counted within the
medial septum (MS) and vertical limb of the diagonal band (VLDB) at
a total magnification of 10.times.. ChAT-positive neurons on the
midline were excluded from this analysis. Representative sections
(4 per brain) located approximately 200 .mu.M from each animal were
used for this analysis. For statistical analysis, the numbers of
neurons ipsilateral to the lesion were expressed as percentages of
neurons contralateral to the lesion. Student's t test was used to
determine differences between the BHK-control and BHK-hNGF
groups.
[0394] NGF-induced Neurite Outgrowth
[0395] Conditioned media (CM) from encapsulated BHK-control and
BHK-hNGF cells was passed thru a 0.2 .mu.m filter and added to
PC12A cells grown on standard tissue culture 6 or 24 well plates at
a concentration of 200,000 cells/ml to test for the presence of
hNGF in the CM. All medium conditioning and neurite outgrowth
assays were performed in 5% CO2 and at 37.degree. C. As a positive
control, 2.5S mouse NGF was added to some of the wells to induce
neuritic extensions (50 ng/ml). The PC12A cells were scored for
neurite processes that were .gtoreq.3 times the length of the cell
body diameter after a period of 1-4 days. In addition, the rate of
neurite induction, and the stability of the neurites was observed
and a comparison was made between the culture conditions.
[0396] NGF ELISA
[0397] Quantification of hNGF released from both encapsulated
BHX-HNGF cells was performed as described in Example 22.
[0398] Results
[0399] The BHK cell-loaded devices were retrieved from the lateral
ventricles 23-28 days following implantation with little to no host
tissue adhering to the capsules. The level of hNGF produced by the
capsules prior to implantation was 21.4.+-.2.0 ng/capsule/24 hr and
8.3.+-.1.2 ng/capsule/24 hr in the retrieved capsules. The
BHK-control capsules produced no detectable hNGF.
[0400] The BHK-HNGF cell-loaded devices were left in situ in 1 of
the BHK-control animals for fixation to demonstrate placement of
the devices and observe the host tissue response. All capsules were
placed within the lateral ventricle and abutted both the head of
the caudate and the lateral septum. The host response to these
capsules was excellent, with little evidence of immune cells
surrounding the capsules. A proliferation of small to moderate
sized blood vessels and a mild gliotic response was observed around
the capsules particularly at the interface between adjacent
capsules.
[0401] In retrieved capsules containing BHK-.backslash.hNGF cells,
few adhering host cells were found on the capsule wall and a large
number of viable BHK cells, evenly distributed at high density,
were present within the polymeric device. Numerous mitotic figures
were observed throughout all of the cell-loaded capsules.
Morphologic analysis of H & E-stained acrylate sections
revealed that encapsulated cell survival was equivalent between the
control and BHK-hNGF cell-loaded capsules.
[0402] In all animals, histological examination revealed that the
left fornix was completely transected while the contralateral
fornix remained intact. The completeness of the lesion was verified
by demonstrating that within the hippocampus ipsilateral to the
lesion there was a profound reduction in AChE staining. Some
remaining AChE-positive fibers were observed diffusely distributed
within the lesioned hippocampus and little reduction of staining
was observed within the inner third of the molecular layer of the
dentate gyrus.
[0403] No differences in the extent of the fornix lesion or the
loss of cholinergic neurons were observed between the animal that
received no transplant and those receiving control BHK cells.
Accordingly, the data from these groups were combined. In these
animals, a significant reduction was observed in the number of
cholinergic neurons ipsilateral to the lesion. NGFr-positive
neurons were decreased 54% within the MS and 30% within the VLDB
compared to the intact side. The loss of ChAT-positive neurons
paralleled the loss of NGFr labeled neurons and was 53% within the
MS and 21% within the VLDB. While many surviving cholinergic
neurons ipsilateral to the lesion appeared normal, others appeared
shrunken, pale and dystrophic. In contrast, BHK-NGF transplants
resulted in a significant attenuation of the loss of cholinergic
neurons following fornix transection. Analysis of NGFr-positive
neurons revealed a modest loss neurons within the MS (19%) and VLDB
(7%).
[0404] Similarly, ChAT-immunoreactive neurons in NGF-treated
animals were decreased only 20% in the MS and 7% in the VLDB.
Cholinergic neurons in the NGF-treated animals were generally
larger and appeared to be more intensely labeled than those in the
BHK-control animals. Sections through the septum of the NGF-treated
animals revealed a dense sprouting of cholinergic fibers within the
septum in both the ChAT and NGFr preparations. These fibers
ramified against the ependyinal lining of the ventricle adjacent to
the transplant site and were particularly prominent within the
dorsolateral quadrant of the septum corresponding to the normal
course of the fornix. This sprouting of cholinergic fibers was not
observed in animals receiving BHK-control implants. Despite the
prevention of the loss of cholinergic neurons and induction of
sprouting of these same neurons, hNGF was not detectable (limit of
detection equals 25 pg) within CSF taken from either lumbar and
cistema magna taps.
[0405] These findings support use of polymer-encapsulated cell
therapy in treating neurodegenerative diseases such as Alzheimer's
disease where basal forebrain degeneration is a consistent
pathological feature.
EXAMPLE 24
[0406] Treatment of Neural Degeneration in Aged Non-Human Primates
Using Encapsulated Genetically Modified BHK Cells.
[0407] Many mammalian species, including humans, are known to
undergo neuronal loss as a natural consequence of the aging
process. Aged non-human primates were used in this experiment to
evaluate whether aged neurons would respond to growth factors in a
manner similar to neurons in younger animals. Fimbria fornix
lesions were performed in aged non-human primates according to the
method described in example 23. The encapsulated cells, surgical
procedure and analytical methods were the same as reported in
example 23. The animals used in these studies were 24-29 year old
Rhesus monkeys. Similar protection of cholinergic basal forebrain
neurons to that observed in example 23 was also obtained in these
older animals.
EXAMPLE 25
[0408] Delivery of a Putative Parkinson's Factor (GDNF) into the
Rat CNS Using Encapsulated BHK Cells
[0409] Parkinson's disease is a progressive neurodegererative
disorder of unknown etiology in which midbraid dopaminergic neurons
are gradually lost, leading to movement disorders and eventually
death. A growth factor, glial cell line-derived neurotrophic
factor, (GDNF) has been described that exhibits an apparent trophic
activity for midbrain dopaminergic neurons in vitro (Lin et al.,
Science, 260, p. 1130 (1993)). These experiments evaluated the in
vivo effect on dopaminergic function of delivery of rGDNF using
encapsulated genetically modified BHK cells.
[0410] PCR Cloning of GDNF
[0411] Reverse transcription--PCR was performed on total RNA
extracted from E18 rat brain. The PCR primers that were used were
synthesized based on the published sequence (Lin et al., Science,
260, pp. 1130 (1993)) for cloning into the pNUT expression vector
(Baetge et al. Proc. Natl. Acad. Sci., 83, pp. 5454-58 (1986)).
[0412] The cDNA was subcloned into the pNUT vector and restriction
digests were done to determine insert orientation. A sense and
antisense clone were selected and then prepared for transfection. A
modified calcium phosphate transfection method was used to
introduce the expression vectors into BHK cells. The cells were
then selected in methotrexate for 6-8 weeks to amplify the vector
and the gene expression.
[0413] Cell Culture
[0414] The BHK cells were cultured in standard cell cultured medium
containing fetal bovine serum. conditioned medium was obtained by
adding a defined, serum-free medium to both the sense and antisense
BHK cell lines for 48 hours. The primary mesencephalic tissues were
dissected from E15 rat fetuses and enzymatically dissociated and
plated in 24 well plates (Nunc) in a serum-free, defined medium
(Hycor) and incubated at 37.degree. C. and 5% CO2. To assess the
potential of the GDNF to enhance tyrosine hydrosine hydroxylase
(TH) neuron survival, various amounts of the conditioned medium
from both cell lines was added in separate wells to the
mesencephalic cultures for up to 3 weeks.
[0415] Dopaminergic neuron survival was assayed by staining the
cultures for TH after treatment for 1, 2 or 3 weeks.
Immunocytochemistry was performed using a mouse monoclonal antibody
for TH (IncStar) followed by detection with a Vector Mouse ELITE
kit and visualized using diaminobenzidine. Cell counts of
TH-positive neurons was done using an inverted microscope with
bright field optics.
[0416] In general, the TH+ neurons in the cultures treated with the
rGDNF exhibit an increased arborization of processes, increased TH
immunoreactivity and in general a more robust appearance.
[0417] GDNF mRNA and protein expression was verified in the
BHK-rGDNF(sense) cell line using Northern blot analysis and with a
primary ventral mesencephalic neuronal bioassay for dopaminergic
neuron survival (TH-positive).
[0418] To determine whether rGDNF has any effect on dopaminergic
function in vivo, both cell lines (sense or antisense) or
untransfected BHK cells were encapsulated in immunoisolatory
polymeric devices and implanted unilaterally into the striatum of
normal Lewis rats. In those animals receiving rGDNF, behavioral
alterations including movement asymmetries were detected after a
1-2 mg/kg dose of amphetamine. No such asymmetry was seen in the
control animals.
[0419] A repeated measures analysis of variance was conducted
including cell type, and with amphetamine dose and turning
direction included as repeated measures. Rats were more active with
increasing doses of amphetamine, the main effect of amphetamine
dose was statistically significant, F(2,32)=36.90, p=0.0001. The
main effect of movement direction was also statistically
significant, F(1,6)=19.81, p=0.0004. This asymmetry in movement
direction increased as the drug dose increased, the drug dose by
turning direction interaction was statistically significant,
F(2,32)=8.43, p=0.001, and the movement asymmetry was significantly
larger in the rats receiving encapsulated GDNF-transfected bHK
cells than in the rats receiving the encapsulated non-transfected
bHK cells, the movement direction by cell type interaction was
statistically significant, F(1,16)=24.74, p=0.0001. It should be
noted that the direction of the movement asymmetry in the rats
implanted with encapsulated GDNF-transfected BHK cells was such
that they moved more in the direction contralateral to the implant
than in the direction ipsilateral to the implant.
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