U.S. patent application number 10/808276 was filed with the patent office on 2004-09-16 for methods, compositions and devices for growing hematopoietics cells.
This patent application is currently assigned to The Regents of the University of Michigan. Invention is credited to Clarke, Michael F., Emerson, Stephen G., Palsson, Bernhard O..
Application Number | 20040180432 10/808276 |
Document ID | / |
Family ID | 27379002 |
Filed Date | 2004-09-16 |
United States Patent
Application |
20040180432 |
Kind Code |
A1 |
Emerson, Stephen G. ; et
al. |
September 16, 2004 |
Methods, compositions and devices for growing hematopoietics
cells
Abstract
Methods, compositions and devices are provided for the growth of
hematopoietic cells in culture. Bioreactors are provided in which
diverse cell types are simultaneously cultured in the presence of
appropriate levels of nutrients and growth factors substantially
continuously maintained in the bioreactor while removing
undesirable metabolic products. This simultaneous culture of
multiple cell types is required for the successful reconstruction
of hematopoietic tissue ex vivo. At least one growth factor is
provided through excretion by transfected stromal cells,
particularly heterologous cells. Means are provided for maintaining
the stromal cells and hematopoietic cells separately, to allow for
early removal of the hematopoietic cells.
Inventors: |
Emerson, Stephen G.; (Ann
Arbor, MI) ; Clarke, Michael F.; (Ann Arbor, MI)
; Palsson, Bernhard O.; (Ann Arbor, MI) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
The Regents of the University of
Michigan
Ann Arbor
MI
|
Family ID: |
27379002 |
Appl. No.: |
10/808276 |
Filed: |
March 25, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10808276 |
Mar 25, 2004 |
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09970934 |
Oct 5, 2001 |
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10808276 |
Mar 25, 2004 |
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08787044 |
Jan 28, 1997 |
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08787044 |
Jan 28, 1997 |
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08352196 |
Dec 1, 1994 |
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5605822 |
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08352196 |
Dec 1, 1994 |
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08100337 |
Jul 30, 1993 |
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08100337 |
Jul 30, 1993 |
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07628343 |
Dec 17, 1990 |
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07628343 |
Dec 17, 1990 |
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07366639 |
Jun 15, 1989 |
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Current U.S.
Class: |
435/372 |
Current CPC
Class: |
C07K 14/5403 20130101;
C12N 2740/13043 20130101; C07K 14/535 20130101; C12N 5/0647
20130101; C12M 35/08 20130101; C12N 5/0642 20130101; A61K 48/00
20130101; C12M 29/10 20130101; C12N 2830/002 20130101; C07K 14/705
20130101; C07K 14/5412 20130101; C12N 15/85 20130101; C12N
2502/1394 20130101; C12N 15/86 20130101; C12M 35/04 20130101; C12N
2501/125 20130101; C12N 2502/99 20130101; C12N 2502/13 20130101;
C12N 2501/14 20130101; C12N 2830/85 20130101; C12N 2740/10043
20130101; C12N 5/0641 20130101; C12N 2501/22 20130101; C12N 2501/23
20130101; C12M 25/02 20130101; C12N 2501/39 20130101 |
Class at
Publication: |
435/372 |
International
Class: |
C12N 005/08 |
Claims
What is claimed is:
1. A method of growing human hematopoietic cells in culture, said
method comprising: inoculating a reactor vessel comprising stromal
cells adherent to a protein substrate with human hematopoietic
cells comprising progenitor cells, wherein at least a portion of
said stromal cells are transformed fibroblast cells capable of
adhering to a protein surface and capable of excreting at least one
growth factor which directs the proliferation and/or
differentiation of said progenitor hematopoietic cells;
substantially continuously perfusing said cells in said reactor
with a nutrient medium comprising any additional growth factors
necessary for proliferation and/or differentiation of said
hematopoietic cells, while removing metabolic products and
replenishing depleted nutrients, while maintaining said reactor
under physiologically acceptable conditions; and harvesting
hematopoietic cells from said reactor, with the proviso that when
said human hematopoietic cells inoculated into said reactor vessel
are suspected of comprising neoplastic cells, said perfusing is at
a rate providing a force greater than the affinity of neoplastic
cells to said stromal cells and less than the affinity of normal
hematopoietic cells.
2. A method according to claim 1, wherein said stromal cells
excrete at least one growth factor.
3. A method according to claim 2, wherein at least one growth
factor is human GM-CSF or IL-3.
4. A method according to claim 1, wherein said perfusion rate
resulting in a shear stress at the surface of the hematopoietic
cells greater than about 1.0 dyne/cm.sup.2.
5. A method according to claim 1, wherein said protein substrate is
a protein coated membrane or protein sponge.
6. A method according to claim 5, wherein said protein is collagen
and/or fibronectin.
7. A method according to claim 1, wherein said transformed cells
are physically separated from said normal bone marrow cells by a
physical barrier.
8. A method according to claim 1, wherein said stromal cells are
maintained prior to harvesting substantially at a subconfluent
stage.
9. A method according to claim 1, further comprising recycling
hematopoietic stem cells from said nutrient medium exiting said
reactor.
10. A method according to claim 1, wherein said perfusing is at a
flow rate to maintain production of hematopoietic growth factors at
about the endogenous level produced by said normal bone marrow
stromal cells.
11. A method according to claim 1, wherein said perfusing with said
nutrient medium and said stromal cells supports the division of
human bone marrow stem cells, whereby human bone marrow stem cells
are produced in said vessel, and said method further comprises
transfecting said human bone marrow stem cells with a gene of
interest present in a retroviral vector.
12. A method of growing human hematopoietic cells in culture, said
method comprising: inoculating a reactor vessel comprising
heterologous stromal cells adherent to one side of a protein
substrate with pores in the range of about 1-5 microns with human
hematopoietic cells comprising progenitor cells, said inoculation
being on the opposite side of said membrane from said stromal
cells, wherein at least a portion of said stromal cells are
transformed fibroblast cells capable of adhering to a protein
surface and capable of excreting at least one growth factor which
directs the proliferation and/or differentiation of said progenitor
hematopoietic cells; substantially continuously perfusing said
cells in said reactor with a nutrient medium comprising any
additional growth factors necessary for proliferation and/or
differentiation of said hematopoietic cells, while removing
metabolic products and replenishing depleted nutrients, while
maintaining said reactor under physiologically acceptable
conditions; and harvesting hematopoietic cells from said reactor,
with the proviso that when said human hematopoietic cells
inoculated into said reactor vessel are suspected of comprising
neoplastic cells, said perfusing is at a rate providing a force
greater than the affinity of neoplastic cells to said stromal cells
and less than the affinity of normal hematopoietic cells.
13. A method according to claim 2, wherein said hematopoietic cells
are bone marrow cells.
14. A method according to claim 12, wherein said perfusing provides
a glucose concentration in the range of about 5 to 20 mM and a
glutamine concentration in the range of about 1 to 3 mM, while the
lactate concentration is maintained below about 35 mM and the
ammonia concentration is maintained below about 2.5 mM.
15. A bioreactor comprising: a reactor chamber; means for
introducing and removing a nutrient medium from said reactor
chamber and means for monitoring the effluent from said reactor
chamber; in said reactor chamber, stromal cells adherent to a
protein substrate with human hematopoietic cells comprising
progenitor cells, wherein at least a portion of said stromal cells
are transformed fibroblast cells capable of adhering to a protein
surface and capable of excreting at least one growth factor which
directs the proliferation and/or differentiation of said progenitor
cells.
16. A bioreactor according to claim 15, wherein said protein
substrate is a protein coated membrane with pores of a size in the
range of about 1-5 microns, with said stromal cells adherent to one
side of said membrane and said hematopoietic cells present on the
opposite side.
17. A bioreactor according to claim 15, wherein said protein
substrate is protein sponge.
18. A bioreactor according to claim 15, further comprising means
for maintaining said stromal cells substantially at a subconfluent
stage.
19. A bioreactor according to claim 16, wherein said means for
introducing and removing a nutrient medium comprises: a media
reservoir for storing media; means for transporting fresh media
into said reservoir and removing partially spent media from said
reservoir; means for transporting media from said reservoir to said
bioreactor and from said bioreactor to said reservoir; means for
oxygenating said media prior to introduction into said bioreactor;
and means for monitoring the composition of said media from said
bioreactor.
20. A bioreactor reactor according to claim 16, further comprising
means for isolating hematopoietic stem cells from said exiting
nutrient medium and returning said hematopoietic stem cells to said
reactor chamber.
21. Transformed fibroblast cells comprising a DNA expression
construct capable of expressing at least one human growth factor in
a form capable of excretion which growth factor directs the
proliferation and/or differentiation of progenitor hematopoietic
cells.
22. Transformed fibroblast cells according to claim 21, wherein
said growth factor is a colony stimulating factor or a
interleukin.
23. Transformed fibroblast cells according to claim 22, wherein
said colony stimulating factor is GM-CSF and said interleukin is
IL-3.
24. Transformed fibroblast cells according to claim 21, wherein
said DNA expression construct comprises a promoter inducible in
hematopoietic cells.
25. Transformed fibroblast cells according to claim 21, wherein
said cells are other than primate.
26. A method of separating hematopoietic neoplastic cells from
normal cells comprising: combining a cell population of
hematopoietic cells with stromal cells, wherein said stromal cells
have limited mobility and said hematopoietic cells contact said
stromal cells; and subjecting said hematopoietic to a fluid flow
producing a force at least Sufficient to remove neoplastic cells
from contact with said stromal cells, without significant removal
of normal cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of application
Ser. No. 366,639, filed Jun. 15, 1989, which disclosure is
incorporated herein by reference.
TECHNICAL FIELD
[0002] The field of the invention is the growth of normal mammalian
cells in culture.
BACKGROUND
[0003] There is significant interest in the ability to use cells
for a wide variety of therapeutic purposes. The hematopoietic
system exemplifies the extraordinary range of cells involved in
protection of mammalian hosts from pathogens, toxins, neoplastic
cells, and other diseases. The hematopoietic system is believed to
evolve from a single stem cell, from which all the lineages of the
hematopoietic system derive. The particular manner in which the
stem cell proliferates and differentiates to become determined in
its lineage is not completely understood, nor are the factors
defined. However, once the stem cell has become dedicated to a
particular lineage, there appear to be a number of factors, for
example colony stimulating factors, which allow, and may direct the
stem cell to a particular mature cell lineage.
[0004] There are many uses for blood cells. Platelets find use in
protection against hemorrhaging, as well as a source of platelet
derived growth factor. Red blood cells can find use in transfusions
to support the transport of oxygen. Specific lymphocytes may find
application in the treatment of various diseases, where the
lymphocyte is specifically sensitized to an epitope of an antigen.
Stem cells may be used for genetic therapy as well as for rescue
from high dose cancer chemotherapy. These and many other purposes
may be contemplated.
[0005] In order to provide these cells, it will be necessary to
provide a means, whereby cells can be grown in culture and result
in the desired mature cell, either prior to or after administration
to a mammalian host. The hematopoietic cells are known to grow and
mature to varying degrees in bone, as part of the bone marrow. It
therefore becomes of interest to recreate a system which provides
substantially the same environment as is encountered in the bone
marrow, as well as being able to direct these cells which are grown
in culture to a specific lineage.
[0006] Relevant Literature
[0007] U.S. Pat. No. 4,721,096 describes a 3-dimensional system
involving stromal cells for the growth of hematopoietic cells. See
also references cited therein. Glanville, et al., Nature
292:267-269, (1981), describe the mouse metallothionein-I gene.
Wong et al., Science 228:810-815, (1985), describe human GM-CSF.
Lemischka, et al., Cell 45:917-927, (1986), describe
retrovirus-mediated gene transfer as a marker for hematopoietic
stem cells and the tracking of the fate of these cells after
transplantation. Yang, et al., Cell 47:3-10, (1986), describe human
IL-3. Chen and Okayama, Mol. Cell. Biol. 7:2745-2752, (1987),
describe transformation of mammalian cells by plasmid DNA. Greaves,
et al., Cell 56:979-986, (1989), describe the human CD2 gene. Civin
C., Strauss L. C., Brovall C., Fackler M. J., Schwartz J. R.,
Shaper J. H., J. Immunol. 133:1576-165, (1984), describes the CD34
antigen. Martin F H, Suggs S V, Langley K E, et al., Cell
63:203-211, (1990), describes human S-CSF; Forrester, J V, Lackie J
M, J. Cell Science, 70:93-110, (1984), discusses parallel flow
chamber. Coulombel, L. et al., J. Clin. Invest., Vol. 75:961-
(1986)., describes the loss of CML cells in static cultures.
SUMMARY OF THE INVENTION
[0008] Methods are provided employing reactors and compositions
which allow for the efficient proliferation of hematopoietic cells
in culture, particularly cells at an early stage in maturation,
including stem cells. The methods employ stromal cells, normally
transformed, which provide constitutive or inducible production of
growth factors, which cells are physically separated to allow for
easy separation of hematopoietic cells. By providing for continuous
perfusion, and recycling of cells as appropriate, high densities
and yields of viable hematopoietic cells may be achieved. The
reactor employs a protein surface for the stromal cells and either
the surface or other barrier for maintaining separation of stromal
cells and hematopoietic cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a schematic view of a perfusion chamber;
[0010] FIG. 2 is a schematic representation and flow diagram of the
perfusion medium pathway;
[0011] FIG. 3a is a schematic view of a flow chamber for measuring
shear stress for separation of cells;
[0012] FIG. 3b is a side view of the flow chamber of FIG. 3a;
[0013] FIG. 3c is a graph of a shear stress profile for
hematopoietic cells; and
[0014] FIGS. 4a and 4b are top and side views of a flow chamber for
growing and separating hematopoietic cells.r
[0015] FIGS. 5a and 5b are views of a flow chamber in which
barriers are removed sequentially allowing the continued growth of
stromal cells.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0016] Methods are provided for the growth of hematopoietic cells
in culture, employing fibroblast cells, normally transformed, for
providing growth factors, with proteinaceous components added to
the mixtures of the fibroblast cells and hematopoietic cells, and
substantially continuous perfusion, optionally with recycling, to
maintain an effective growth environment.
[0017] The description of the method therefore may be divided into
descriptions of the perfusion conditions, the reactor and its
internal structure, and the transformed fibroblasts.
[0018] The reactor comprises a vessel which may be of any
convenient shape which allows for the necessary cell distribution,
introduction of nutrients and oxygen, removal of waste metabolic
products, optional recycling of hematopoietic cells, substitution
of stromal cells, and harvesting of hematopoietic cells. The
reactor should provide for conditions which substantially mimic
bone perfusion. In vivo, about 0.08 ml of serum per ml of bone
marrow per minute is perfused. This translates into about 0.3 ml of
serum per 10.sup.6 cells per day. The media will therefore be
changed on the average at least 50%, preferably at least 100%, in
any 24 hour period, so as to maintain a level of metabolic products
which is not growth limiting. The rate of change will generally be
from about 0.5 to 1.0 ml of perfusion medium per 10.sup.6 cells per
day, empirically mimicking in vivo perfusion rates.
[0019] The rate of perfusion in the bioreactor will vary depending
on the cell density in the reactor. For cells cultured at
2-10.times.10.sup.6 cells/ml, this rate is 1 ml/ml reactor volume
per 24-48 hours, where the medium used contains 20% serum, either
10% fetal calf serum and 10% horse serum, or 20% fetal calf serum.
For higher cell densities, the perfusion rate will be increased
proportionately to achieve a constant serum flux per cell per time.
Thus if the cells are cultured at 5.times.10.sup.8 cell/ml the
perfusion rate will be 0.1 ml/ml reactor volume per minute. These
flow rates, matching serum and medium flux rates to cell density,
are essential to stimulating the endogenous production of
hematopoietic growth factors from the normal human bone marrow
stromal cells in the culture. The hematopoietic growth factors
induced by these serum and medium flux rates include GM-CSF, and
may also include S-CSF, IL-6 and G-CSF as well as other
hematopoietic growth factors. These rates will be established in
the bioreactors such that the shear stress from longitudinal flow
experienced by the stem cells and progenitor cells at their stromal
cell attachment sites are between 1.0 and 5.0 dynes/square cm.
[0020] Various media may be employed for the growth of
hematopoietic and stromal cells. Illustrative media include MEM,
IMDM, and RPMI, which may be supplemented by combinations of 5-20%
fetal calf serum, 5-20% calf serum, and 0-15% horse serum, and/or
serum free media supplemented with PDGF, EGF, FGF, HGF or other
growth factors to stimulate stromal cells or stem cells. To
supplement the growth factors provided by the transformed
fibroblasts, additional growth factors may be included in the
perfusion medium, particularly where dedicated cells of a
particular lineage are desired. Among the growth factors which may
be included in the perfusion medium, either by stromal cell
secretion or addition, are GM-CSF, G-CSF, or M-CSF, interleukins
1-7, particularly 1, 3, 6, and 7, TGF-.alpha. or .beta.,
erythropoietin, or the like, particularly human factors. Of
particular interest is the presence of about 0.5-2, preferably 1,
ng/ml G-MCSF, and 0.5-2, preferably 1 ng/ml, as well as a 0.1-2
U/ml/day of final concentration of erythropoietin, from about
100-300 ng/ml/day of G-CSF and about 1-10 ng/ml/day of stem cell
growth factor (S-CSF, also referenced as Mast Cell Growth Factor or
Kit ligand). It is understood that one or more, preferably at least
two of the growth factors will be provided by secretion from
transformed cells, which will be present in an amount sufficient to
maintain the desired level of the growth factors in the perfusion
medium.
[0021] Conveniently, in the reactor, physiologic temperature will
be employed, namely 37.degree. C., although lower temperatures may
also be employed, including 330, usually not being below 25.degree.
C. Humidity will generally be about 100%, where the air will
contain about 5% carbon dioxide. The perfusion medium may be
oxygenated external to the reactor or internal to the reactor,
various means being provided for internal oxygenation. Internal
oxygenation may be achieved with hollow fibers, porous sintered
disks, silicone tubing or other membranes of suitable porosity and
hydrophobicity. The nutrient level and metabolic product level will
normally be maintained in a relatively narrow range. Glucose level
will usually be in the range of about 5 to 20 mm, usually about 10
to 20 mM, lactate concentration will usually be maintained below
about 35 mM and may be allowed to be over 20 mM. Glutamine
concentration will generally be maintained in the range of about 1
to 3 mM, usually 1.5 to 2.5 mM, while ammonia concentration will
usually be maintained below about 2.5 mM, preferably below about
2.0 mM.
[0022] The flow of fluid may be by gravity, by a pump, or other
means, where the flow may be in any direction or a multiplicity of
directions, depending upon the nature of the packing in the
reactor. Desirably, laminar flow may be employed where the flow may
be substantially horizontal across the reactor or vertical flow may
be employed, where the flow is from the bottom to the top of the
reactor or vice-versa.
[0023] Where the source of human hematopoietic cells is suspected
of having neoplastic cells, e.g., leukemic lymphoma or carcinoma,
the perfusion flow can be selected so as to segregate the normal
progenitor cells from the neoplastic hematopoietic cells. It is
found that normal hematopoietic progenitor cells adhere to stroma
and matrix proteins with an affinity able to withstand
approximately 1.5-2.0 dynes/cm.sup.2 stress from longitudinal fluid
flow. By contrast, neoplastic cells and their progenitors have a
substantially weaker affinity for stroma, in the range of about
0.05-1.2 dynes/cm.sup.2. By providing for a perfusion flow rate
which provides sheer stress rates intermediate between that
tolerated by normal and neoplastic progenitor cells, generally
greater than 1 dyne/cm.sup.2, one can provide for separation of the
neoplastic progenitor cells from the normal progenitor cells,
generally maintaining the perfusion for at least about two days,
preferably at least about five days, and more preferably seven days
or more. In this manner, one can expand normal hematopoietic cells
from a human patient, while at the same time using the appropriate
flow rates, separate neoplastic cells. In this manner, one can
provide for autologous hematopoietic cells from a patient suffering
from neoplasia, expand the normal hematopoietic cells during a
period of treatment of the patient by chemotherapy or
X-irradiation, and then restore normal hematopoietic cells to the
patient to restore hematopoiesis and the immune system of the
patient.
[0024] Illustrative of the use of shear stress to separate
hematopoietic tumor cells from normal hematopoietic cells is the
situation of chronic myelogenous leukemia (CML). Shear stress
tolerance for CML cells is in the range of 0.05-1.2 dyne/cm.sup.2.
This difference permits the efficient removal of CML cells with an
individual bone marrow sample. By employing a shear rate of about
1.2-1.5, preferably 1.3, dynes/cm.sup.2, the CML cell may be
efficiently separated.
[0025] The shear stress tolerance within an individual's bone
marrow cells may be determined using a tapered radial flow chamber.
In the radial flow chamber, the shear stress experienced by the
cell decreases with distance "d" from the start of the chamber as a
function of 1/d. Bands may then be analyzed for cell population and
the shear stress set for the desired cell population to be
retained.
[0026] For the removal of leukemic stem cells, progenitor cells and
stem cells from bone marrow samples from patients with leukemia are
first placed into a radial flow chamber. The radial flow chamber
consists of two parallel plates, made of polycarbonate or glass,
which permit the adhesion of bone marrow stromal cells to the lower
plates. The initial measurements can be performed by either 1)
establishing a preformed confluent monolayer of bone marrow stromal
cells prior to hematopoietic cell infusion and then initiating
fluid flow after 12-24 hours, or 2) inoculating the patient's bone
marrow directly into the flow chamber without using a preformed
stromal monolayer, and then waiting 3-4 days before establishing
the fluid flow, usually 0.05-1.0 cc/min. The plates are sealed
together at the edges through a rubber gasket, and held together
with adjustable screws. At the narrow, infusion, end of the chamber
a tube brings fluid into the chamber from a reservoir delivered by
a constant pressure syringe-type pump. At the wide, collection end,
the fluid and removed cells are collected through a separate tube
(see FIGS. 3 and 3b). After the period of perfusion (usually 3-7
days), the nonadherent cells are removed, and the plates are
separated, cells from each of 3-5 regions are separately removed by
aspiration and rubber policeman, and each fraction is analyzed for
the presence of leukemic cells by standard techniques (usually
karyotypic analysis by chromosomal banding). Comparison of the
leukemic analyses of each fraction demonstrates in which fraction
(i.e. at which shear stress), the leukemic cells fail to adhere to
the stroma and are removed. In these chambers, the shear stress
perceived by the cells declines exponentially as a function of the
distance are from the inlet. (See FIG. 3c). Typically, the
nonadherent cells are all or nearly all leukemic, whereas cells
adhering at the in the narrowest 1/2 of the chamber are all or
nearly all normal.
[0027] Based upon the results of these measurements, a series of
parallel, rectangular chambers is established in which the rate of
fluid flow (see FIGS. 4a and 4b over the lower surface creates a
shear stress rate which was found in the tapered chamber to remove
leukemic cells from the stroma without removing all of the normal
cells. In the case of chronic myelogenous leukemia patient bone
marrows, this shear stress is typically 0.01-0.05 dynes/square cm.
The actual flow rate employed will depend on the size and geometry
of the chambers. Bone marrow cells from the patient will be
cultured in these rectangular chambers at a concentration of
5.times.10.sup.6/ml to 50.times.10.sup.6/ml in Iscove's Modified
Dulbecco's Medium with 5-20% (typically 10%) Fetal calf serum plus
0-15% (typically 10%) horse serum, with or without 10.sup.-6M
hydrocortisone. The bone marrow cells will be cultured for 12-24
hours without fluid flow, and then fluid flow will be initiated.
The cells will be cultured for 3-7 days, at which time all of the
nonadherent cells will be discarded. The adherent cells will be
recovered from the rectangular plates by aspiration and mechanical
agitation, and then collected. These cells can then be either
directly returned to the patient, or stored in liquid nitrogen by
standard techniques for later use.
[0028] Cells other than those of the hematopoietic system also may
be separated using differential tolerance to shear stress. Thus,
where there are distinct subpopulations of cells within a complex
mixture of cells the methods described above can be used to
separate out a cell type of interest from within a suspension of
cells derived from, e.g. skin, liver, muscle, nerve, or epithelium.
Of particular interest is the separation of tumor cells from within
a population of normal cells. The population of cells to be
separated will be contacted with a suitable stromal substrate as
described below, such as a purified protein or cellular component
to which the cells of interest adhere. The shear stress tolerance
for each of the adherent subpopulations is determined as described
above. The fluid flow can then be adjusted appropriately so as to
retain the desired subpopulation of cells on the stroma. The
desired cells are then collected as desribed above.
[0029] A variety of packings may be used in the reactor to provide
for adherent growth of the cells, while maintaining some physical
separation between the stromal cells and the hematopoietic cells,
and while allowing for some contact or close juxtaposition between
the stromal cells and the hematopoietic cells. In this way, the
factors secreted by the stromal cells may be readily taken up by
the hematopoietic cells to encourage their proliferation and, as
appropriate, differentiation and maturation.
[0030] The protein matrix to support the cells may take the form of
shredded collagen particles, e.g., sponges or porous collagen
beads, sponges or beads composed of extra-cellular bone matrix
protein from bone marrow, or protein coated membranes, where the
protein may be collagen, fibronectin, hemonectin, RGDS peptide,
mixed bone marrow matrix protein, or the like. Pore sizes of
membranes will generally range from about 1 to 5 .mu. to allow for
interaction between the different cell types, while still retaining
physical separation.
[0031] Membranes may be employed, which will be protein coated.
Various membrane materials may be employed such as polypropylene,
polyethylene, polycarbonate, polysulfonate, etc. Various proteins
may be employed, particularly collagen or the other proteins which
were indicated previously. The membrane should have sufficiently
small pores, that the transformed cells may not pass through the
membranes, but may grow and form a confluent layer on one side of
the membrane and extend portions of the cell membrane into the
pores. Generally the pores will be in the range of about 1 to 5
.mu.. In this manner, the hematopoietic stem cells may grow on the
opposite side of the membrane and interact with the transformed
cells, whereby factors may be transferred directly from the
transformed cells to the hematopoietic progenitor cells. The
progenitor cells, the stem cells, are able to attach to the
intruded cytoplasmic projections which have passed into the pores.
Hematopoietic differentiation from the stem cells occurs on one
side of the membrane and differentiated progeny are unable to
squeeze back through the pores, which are already largely occupied
by cytoplasmic projections from the fibroblasts. As hematopoietic
cells mature and differentiate, they will be released from the
membrane into the nutrient medium.
[0032] The reactor may be packed with the various particles in a
central portion of the reactor to define a central chamber, which
will be separated from an upper chamber and a lower chamber.
Alternatively, one or a plurality of membranes may be introduced,
where two membranes will define a region associated with either the
stromal cells or the hematopoietic cells, where the regions will
alternate between stromal and hematopoietic cells. In this way, one
may provide for differential perfusion rates between the chambers
of the hematopoietic cells and the stromal cells. The medium
exchange rate will generally fall within the ranges indicated
above.
[0033] Instead of or in addition to contact between hematopoietic
cells and stromal cells, one may provide for stromal cells in an
environment where the two types of cells are inhibited from any
contact. Thus, the stromal cells would provide for the various
factors to support the growth of the hematopoietic cells and the
stromal cell conditioned medium used for perfusion of the
hematopoietic cells. For example, one could use two hollow-fiber
cartridges in series, where the first cartridge would contain
stromal cells under conditions which allow for the continued growth
of stromal cells, while a second cartridge would contain the
hematopoietic cells which would be perfused with the conditioned
medium.
[0034] Furthermore, it is desirable to provide for continuous
stromal cell proliferation as a source of growth factors. In order
to maintain continuous stromal cell proliferation, it is desirable
to initially employ stromal cells at a subconfluent stage. Various
techniques may be employed to provide for replacement of the
stromal cell layer when confluence is approached or reached. For
example, one could provide for a plurality of chambers in which
stromal cells may grow and the hematopoietic cells may be moved in
accordance with the chamber which has the stromal cells at a
subconfluent level. Thus, by having a movable barrier between the
chambers, when the stromal cells approach confluence, generally
after about 8-12 weeks, one could open or remove the barrier
between the chambers and allow for the stromal cells to migrate
into the new chamber and allow for the hematopoietic cells to come
in contact with the subconfluent stromal cells, while the
subconfluent stromal cells feed the factors to the chamber
comprising the hematopoietic cells (FIG. 5a and FIG. 5b). The
transfer of the hematopoietic cells can be achieved by appropriate
flow rates or by other convenient means. One can provide for
various wells in the chamber, which are divided by appropriate
walls, after seeding in one well, when the cells become confluent,
cells will then move over into the next well and seed the next well
in a subconfluent manner. Another modification of the system is one
in which, after 8-12 weeks in culture, the hematopoietic cells are
exposed to new, proliferating stromal cells. This is accomplished
in one of several ways. This exposure to proliferating stromal
cells is accomplished in one of several ways. In the first
technique, the culture are exposed to EDTA for 3-5 minutes, which
removes the hematopoietic stem cells from the stromal cells. The
removed cells are then transferred to a new culture vessel, which
may itself contain bone marrow stromal cells seeded 3-7 days prior.
This process is repeated every 8-12 weeks. Another alternative
approach is to add additional surface area by increasing the volume
of the cultures and adding additional collagen beads to the
cultures at 8-12 weeks. Finally, small organic molecules or
proteins, particularly hormones, such as platelet-derived growth
factor (at 100-500 ng/ml), interleukin 1 alpha, tumor necrosis
factor alpha, or basic fibroblast growth factor or other molecules
mitogenic to fibroblasts, can be added to the cultures every 3-7
days. This exposure to stromal mitogenic stimulatory factors
promotes the continued proliferation of bone marrow stromal cells,
and their continued production of hematopoietic growth factors.
Thus, one can provide for the continuous subconfluent stage of the
stromal cells.
[0035] Continuous fluid flow can also be used to selectively
separate normal from cancerous cells within a bone marrow
population. In this approach, a radial flow chamber is first used
to determine the specific stromal adhesive properties of normal
versus cancerous cells, and then a rectangular flow chamber with
flow rates established to achieve a shear stress sufficient to
remove the cancerous cells is used to preoperatively separate the
normal and cancerous cells.
[0036] The subject method and apparatus also provides for the
opportunity to recycle stem cells which are lost by the flow of the
perfusion medium. The surface membrane protein marker CD34
substantially separates immature hematopoietic cells from mature
hematopoietic cells. Thus, by capturing and recycling those cells
which are CD34+, one may avoid the loss of stem cells to the
medium.
[0037] Various techniques may be employed for capturing and
returning the immature fraction of cells to the reactor. For
example, one could label the cells with an antibody specific for
CD34 and then use antibodies to the antibody for collecting the
CD34+cells and recycling them to the reactor. Alternatively to
positive selection, one may use negative selection, whereby one
would remove the mature cells employing antibodies to various
markers associated with mature cells, such as antibodies to
glycophorin A, CD33, MO1, OKT3, OKT4, OKT8, OKT11, OKT16, OKM1,
OKMS Leu7, Leu9, Leu M1, Leu M3, and the like. Various antibodies
are available for markers specific for mature cells of the various
hematopoietic lineages, lymphoid, myeloid and erythroid, and these
antibodies may be used to remove the mature cells from the effluent
from the reactor, followed by harvesting of the remaining cells and
restoring them to the reactor. In this way, one can avoid forced
decline in the cultures due to loss of stem cells and maintain
unlimited stem survival in vitro.
[0038] Separation using antibody markers can be achieved in various
ways, using standard techniques, individually or in combination,
such as panning, fluorescence activated cell sorting, antibodies
bound to various surfaces, e.g. polystyrene surface, metal
microspheres and magnets, and the like. The antibodies are bound to
a surface which allows for separation between adherent and
non-adherent cells or the antibodies are labeled, directly or
indirectly, which permits selection between labeled and unlabeled
cells.
[0039] By following the subject procedures greatly extended periods
of in vitro growth of hematopoietic cells may be achieved,
generally providing ex vivo human hematopoiesis for at least six
months in culture, with granulopoiesis being supported for at least
four months and erythropoiesis for at least three months. In
addition, hematopoietic progenitor cells are continuously generated
throughout the culture resulting in net expansions of progenitor
cells of over 10-fold from input cells.
[0040] In addition, by following the subject procedures greatly
increased rates of stem cell division are supported, permitting the
efficient insertion of retrovirally transfected genetic material.
Genes inserted by the appropriate retroviral vector during an
initial two week infection period can be expressed in up to 10-30%
of all progenitor and precursor cells arising during subsequent
culture for over four months in culture. These subject procedures
thus support the successful transfer of genetic material into a
highly proliferative human hematopoietic stem cell.
[0041] FIG. 1 is a schematic view of a perfusion chamber. Reactor
10 with cover plate 12 and floor plate 14 are joined by bolts 16,
held in position by wing nuts 18. Three bolts are employed, so as
to avoid warping. The chamber 20 has three sections, the middle
section 22 containing the support matrix for the stromal cells, the
bed of stromal cells, and the bone marrow cells. The central
section 22 is separated from the top section 24 and the bottom
section 26 by membranes or mesh 28 and 30 respectively.
Conveniently, polysulfonate membrane may be employed or a stainless
steel mesh, whose mesh size is small enough so that cells are
contained within the central section of the chamber. The separating
interphase may be placed in the chamber using an inner cylinder 27
which is sectioned to provide the separating membrane mechanical
support. The top section 24 and the bottom section 26 need not be
identical and will have tubing or membranes across which liquid
media and gases are exchanged. The gases are exchanged across a
hydrophobic, e.g., silicone, tube whose length (and thereby
gas/liquid contact area) may be varied to allow for sufficient gas
fluxes to support the needs of the cell population that is
metabolizing in the central section. The media can be pumped or
withdrawn directly from the top or bottom sections through port 32
and may be fed through delivery tube 34.
[0042] If desired, the top and bottom sections may be eliminated by
using an external oxygenator. In this situation, the separating
membrane is held in place under the glass cylinder 36 which fits
into cylindrical groove plates 12 and 14 and the area inside of the
cylindrical groove is indented to allow for good flow distribution
across the membrane. This geometry allows the fluid from the finite
number of inlet ports to mix and for radial pressure to
equilibrate, leading to a uniform liquid flow across the separating
membrane. This setup is suitable for chambers which have relatively
few cells, so that oxygenation does not become limiting.
[0043] In FIG. 2 is depicted a schematic representation of the loop
that connects the perfusion chamber to the side media reservoir,
oxygenator, sensor chamber, and sample/injection ports.
[0044] An external fresh media source 50 is pumped by means of pump
52 to a media reservoir through line 56 and spent media is
withdrawn through line 58 from reservoir 54 by means of pump 52 to
the spent media container 60 for further processing. A second pump
62 pumps media from the media reservoir 52 through line 64 through
a hollow fiber oxygenator 66. The media is directed through line 68
to the first chamber of bioreactor 70. As appropriate, a means for
injection of media component 82 is provided, for introducing the
component into line 68 for transport by the media into the first
chamber of bioreactor 70. The component may be test components,
additional factors, or the like. The media from bioreactor 70 is
directed through central chamber 72 into the second chamber 74 of
the bioreactor. From there the media is directed by line 76 to
in-line sensors 78 for detecting the change in composition of the
media.
[0045] For example, it is desirable that the glutamine:glucose
ratio be in the range of about 1:5-8, depending on the cell lines
used; for instance, preferably 1:8 for transfected 3T3 cells.
Furthermore, ammonium concentrations will preferably be below about
2.0 mM and lactate concentrations are preferably less than about 40
mM. By monitoring the effluent from the bioreactor, the media
introduced into the bioreactor may be modified, oxygen partial
pressure may be changed, gas flow rate may be altered, various
components may be augmented, or the rate of perfusion may be slowed
or increased.
[0046] From the sensors 78, the media is directed through line 80
by means of pump 62 to the reservoir 54.
[0047] By means of the flow path described above, the media in the
side reservoir is slowly exchanged using a separate pump. This
organization allows for separate control of the media exchange rate
(the outer pump) and the flow rate through the oxygenator and
perfusion chamber. The former is used to control the longer term
change in the media composition and perfusion, while the latter may
be used to control the dissolved oxygen tension and flow patterns
in the chamber. The use of a small mesh biocompatible membrane
allows for plug (piston) flow in the chamber and thus allows the
precise control of delivery of growth factors and other special
compounds that one may wish to introduce to the hematopoietic cells
and stromal cells in very precise amounts.
[0048] After autoclaving the chamber and components of the loop,
the reactor is assembled in a sterile environment. The media may be
circulated through the side loop and chamber for a few days while
signs of contamination are monitored. If sterile assembly is
accomplished, the central section of the chamber is inoculated with
either the extra-cellular matrix alone or a pre-inoculated
extra-cellular matrix support that contains the stromal cells. The
stromal cells are then either: 1) kept in the chamber for a period
of a few days while their metabolic performance and/or growth
factor responsiveness is monitored and if results are satisfactory,
the bone marrow is inoculated; or 2) immediately seeded with bone
marrow. In either case, the cell layer is kept at the bottom of the
central section of the perfusion chamber. The cells lay down
additional extra-cellular matrix and the cell layer adheres to the
separating membrane. At this time, the chamber may be inverted and
the cell layer may then be located at the ceiling of the central
section. In this configuration, the maturing cells will settle on
the bottom of the central chamber as they lose their adherence to
the stromal layer. This feature is important to prevent the damage
caused by mature cells to the stromal layer and/or the less mature
hematopoietic cells. This feature also makes the continuous removal
of mature cells easier.
[0049] These cells are harvested by withdrawing the cells by
syringe, or by continuously allowing the cells to flow out of the
chamber, by the pressure of the perfused medium, through the exit
tubing.
[0050] The stromal cells will, for the most part, be fibroblasts
transformed with one or more genes providing for desired
hematopoietic growth factors. The same or different cells may be
transfected with the genes, depending upon the particular selection
of host cells, the same or different cells may be used for a
plurality of genes.
[0051] A wide variety of normal cells or stable lines may be
employed. However, it is found that not all cell strains are
permissible, since transformation of some cell lines may result in
the overgrowth of the cells. Desirably, the cells which are
employed will not be neoplastic, but rather require adherence to a
support. The mammalian cells need not be human, nor even primate. A
variety of nontransformed cells may be included in the adherent
cell layer as well, including normal human bone marrow adherent
cells, normal human spleen adherent cells, and normal human thymic
epithelium.
[0052] Methods for transforming mammalian cells, including
fibroblasts, are well known and there is an extensive literature of
which only a few references have been previously given. The
constructs may employ the naturally occurring transcriptional
initiation regulatory region, comprising the promoter and, as
appropriate the enhancer, or a different transcriptional initiation
region may be involved, which may be inducible or constitutive.
[0053] A large number of transcriptional initiation regions are
available which are inducible or constitutive, may be associated
with a naturally occurring enhancer, or an enhancer may be
provided, may be induced only in a particular cell type, or may be
functional in a plurality or all cell types. The transcriptional
initiation region may be derived from a virus, a naturally
occurring gene, may be synthesized, or combinations thereof.
[0054] Promoters which are available and have found use include the
chromosomal promoters, such as the mouse or human metallothionein-I
or II promoters, actin promoter, etc., or viral promoters, such as
SV40 early gene promoters, CMV promoter, adenovirus promoters,
promoters associated with LTRs of retroviruses, etc. These
promoters are available and may be readily inserted into
appropriate vectors which comprise polylinkers for insertion of the
transcriptional initiation region as well as the gene of interest.
In other instances, expression vectors are available which provide
for a polylinker between a transcriptional initiation region and a
transcriptional termination region, also providing for the various
signals associated with the processing of the messenger for
translation, i.e., the cap site and the polyadenylation signal. The
construction of the expression cassette comprising the regulatory
regions and the structural gene may employ one or more of
restriction enzymes, adaptors, polylinkers, in vitro mutagenesis,
primer repair, resection, or the like.
[0055] The expression cassette will usually be part of a vector
which will include a marker and one or more replication systems.
The marker will allow for detection and/or selection of cells into
which the expression cassette and marker have been introduced.
Various markers may be employed, particularly markers which provide
for resistance to a toxin, particularly an antibiotic. Preferably,
gentamicin resistance is employed, which provides resistance to
G418 for a mammalian cell host. The replication systems may
comprise a prokaryotic replication system, which will allow for
cloning during the various stages of bringing together the
individual components of the expression cassette. The other
replication system may be used for maintenance of an episomal
element in the host cell, although for the most part the
replication system will be selected so as to allow for integration
of the expression cassette into a chromosome of the host.
[0056] The introduction of the expression cassette into the host
may employ any of the commonly employed techniques, including
transformation with calcium precipitated DNA, transfection,
infection, electroporation, ballistic particles, or the like. Once
the host cells have been transformed, they may be amplified in an
appropriate nutrient medium having a selective agent, to select for
those cells which comprise the marker. Surviving cells may then be
amplified and used.
[0057] Host cells which may be employed include African green
monkey cell line CV1, mouse cells NIH-3T3, normal human bone marrow
fibroblasts, human spleen fibroblasts, normal mouse bone marrow
fibroblasts, and normal mouse spleen fibroblasts. It should be
noted that in some instances, depending upon the choice of vector
and cell line, the cells may become neoplastic. It is important
that the resulting transformed cells be capable of adherence,
whereby the transformed cells maintain binding to a support, such
as protein sponges, protein coated membranes, or the like.
[0058] Once the vector for expressing the appropriate growth
factors has been constructed, it may be used to transform the cells
by any convenient means. The resulting transformed cells may then
be used to seed the supports, which have already been described.
These supports may be introduced into the reactor or may be present
at the time of seeding in the reactor. The cells will be allowed to
grow for sufficient time to ensure that the cells are viable and
are capable of producing the desired growth factors.
[0059] The reactor may then be seeded as appropriate with the
hematopoietic cells. The hematopoietic cells may include
substantially pure stem cells, a mixture of hematopoietic cells
substantially free of mature hematopoietic cells of one or more
lineages, or a mixture comprising all or substantially all of the
various lineages of the hematopoietic system, at various stages of
their maturation.
[0060] The cells are allowed to grow with substantially continuous
perfusion through the reactor and monitoring of the various
nutrients and factors involved. For the most part, the primary
factors will be provided by the stromal cells, so that a steady
state concentration of growth factors will normally be achieved.
Since conditioned supernatants are found to be effective in the
growth of the hematopoietic cells, one can provide for a ratio of
stromal cells to hematopoietic cells which will maintain the growth
factor at a appropriate concentration level in the reactor.
[0061] Transfected stroma can provide for the introduction of genes
into human stem cells. In mice, retroviral mediated gene transfer
into stem cells is made possible by pretreating mice with 5-FU and
then growing the harvested bone marrow cells in WEHI conditioned
media, which contains IL-3 and GM-CSF (Lemischka Cell 45:917,
1986). The artificial stroma, grown with a retroviral packaging
cell line secreting a retroviral vector of interest, may be used to
efficiently introduce genes into human stem cells. For example,
human T-cells could be made resistant to HIV infection by infecting
stem cells with the retroviral vector containing an HIV antisense
sequence under control of a CDC2 regulatory sequence (Greaves, Cell
56:979-986, 1989) which would allow for tissue specific expression
in T-cells. There would be a factor provided by the retroviral
packaging cell line essential for replication of the retrovirus;
this factor would be absent in the hematopoietic target cells. Once
the virus was transferred to the hematopoietic target cells, it
would no longer be able to replicate.
[0062] In FIGS. 3a and b are depicted radial flow chamber 100
having inlet 102 and outlet 104, and with chamber 106 where the
arrows 108 indicate the direction of flow. Hematopoietic cells 110
are seeded onto a stromal layer 112 in the chamber and grown. The
flow rate will determine which cells are able to adhere, the
non-adherent cells 114 passing out through outlet 104.
[0063] In FIGS. 4a and 4b, growth chamber 120 is provided having
inlet 122 and outlet 124. In FIG. 4b, inlet 122 comprises a
manifold 128 which feeds individual chambers 126 containing cells
110 and stroma 112 in the chamber 126 for growth and
separation.
[0064] In FIGS. 5a and 5b are shown growth chambers in which
barriers 134, 136, 138 are removed schematically during culture:
barriers 134 at about week 0.8-10; barrier 136 at about week 18-20
and barrier 138, at about week 28-32.
[0065] The following examples are offered by way of illustration
and not by way of limitation.
Experimental
[0066] I. Formation of Transformants
[0067] The growth factor human GM-CSF (Wong, Science, 228:810-815,
(1985)) was inserted into a eukaryotic expression vector. The
hGM-CSF cDNA (EcoRI to AhaIII, approximately 700 bp fragment) was
cloned into an EcoRI to PstI fragment of pSP65. (Melton, Nucl.
Acids Res. 2:7035-7056 (1984)). The resulting plasmid was
pSP65GM-CSF. The mouse metallothionein promoter (Glanville, Nature,
292:267-269, (1981)) was digested with EcoRI and BglII and the
approximately 2 kb fragment containing the promoter was inserted
into the EcoRI to BamHI fragment of pSP65 to make p65MT. The
plasmid pMT GM-CSF was then constructed by digesting pSP65GM-CSF
with EcoRI, filling in the overhang with the Klenow fragment of DNA
polymerase I and then digesting the resulting linearized DNA with
HindIII to isolate the 700 bp fragment comprising the coding region
of GM-CSF. This fragment was subcloned into the SalI filled/HindIII
site of p65MT. The 2.7 kb fragment comprising the metallothionein
promoter and the GM-CSF coding region was then isolated and placed
into pSV2neo (Southern and Berg, J. Mol. Appl. Genet 1:327 (1982))
from which the SV-40 promoter was removed. This results in the
SV-40 poly A signal downstream of the GM-CSF coding sequence.
[0068] The neomycin resistant gene, which confers resistance to the
antibiotic gentamicin (G418) was taken from pSV2neo by isolating
the approximately 3 kb PvuII to EcoRI fragment and placing EcoRI
linkers onto the PvuII site. The neo resistance gene with EcoRI
ends was subcloned into the EcoRI site of the GM-CSF expression
plasmid to create the plasmid MTGM-CSFneo.
[0069] The plasmid MTGM-CSFneo alone and as a cotransfection with
the plasmid (Yang, Cell 47:3-10, 1986) encoding the gibbon ape IL-3
gene under the control of the SV-40 promoter and poly A site, were
transfected by electroporation of linearized DNA into the African
green monkey cell line CV1 and the mouse cell line NIH 3T3 cells.
Transformants were selected by selection in media containing 500
mg/ml of G418, isolated, and screened for production of GM-CSF or
IL-3 by bioassay of supernatants using AML-193 cells (Adams, et
al., Leukemia 3:314 (1989)). Several of the positive lines were
then employed as stroma for human bone marrow cells in Dexter
culture.
[0070] In addition, normal mouse bone marrow cells were transfected
with the above plasmids using the calcium/phosphate method of
Okayama (Chen, Mol. Cell. Biol. 7:2745-2752, 1987) and were found
to efficiently express the introduced genes.
[0071] GM-CSF and IL-0.3 secretion by the transfected fibroblasts
was investigated. Serum free 72 hour culture supernatants were
obtained from the NIH-3T3 cells and assayed for hGF secretion by 3H
uptake on target cells inhibitable by neutralizing rabbit
anti--GM-CSF or anti-IL-3 antibodies. Proliferation induced by 20
mg/ml GM-CSF was set as 100 units GM-CSF and that induced by 10
ng/ml IL-3 was set as 100 units IL-3. The co-transfected cells
produced about 35 units/ml of GM-CSF and about 57 units/ml of
IL-3.
[0072] II. Perfusion Chamber
[0073] The perfusion chamber is a glass cylinder with Delrin caps
to allow for autoclaving without deformation and biocompatability.
The caps have cylindrical groves into which the glass cylinder
fits. At the bottom of the grove an O-ring is placed to seal the
lumen of the chamber. The caps have several holes into which Luer
(Luer Lok) fittings are provided into which media and gas delivery
lines are put as well as an extended tube into the central section
of the chamber to sample adherent and/or non-adherent cells. The
caps are attached with three long bolts, spaced 1200, placed
outside the glass cylinder; wing nuts and washers are used to
tighten the assembly.
[0074] The chamber is hooked to a side reservoir. The loop contains
a pump, a chamber of on-line sensors, oxygenator, and sample and
injection ports in addition to the side media reservoir. The media
in the side reservoir is then slowly exchanged using a separate
pump. This configuration allows for separate control of the media
exchange rate and the flow rate through the oxygenator and
perfusion chamber. The former is used to control the longer term
change in the media composition and perfusion, while the latter may
be used to control the dissolved oxygen tension and flow patterns
in the chamber. The use of a small mesh polysulfonate membrane
allows for plug flow in the chamber and the precise control of
delivery of growth factors and other special compounds which one
may wish to introduce into the bioreactor in very precise
amounts.
[0075] The transfected stromal cells are seeded either over a bed
of shredded collagen sponge or the stromal cells are placed on one
side of a 5.mu. porous polycarbonate filter precoated with collagen
and the stromal cells allowed to adhere to the filter over a number
of hours. The cells are allowed to grow in an appropriate nutrient
medium until the cells become confluent on one side while sending
cytoplasmic projections through the pores. Bone marrow cells are
then seeded on the other side of the membrane and the stem cells
attach to the intruded cytoplasmic projections which have-passed
through the pores.
[0076] After autoclaving the chamber and components of the loop,
the reactor is assembled in a sterile environment. The media is
then circulated through the side loop and chamber for a few days
while signs of contamination are monitored. The central section of
the bioreactor is then inoculated with either the extra-cellular
matrix alone or a pre-inoculated extracellular matrix support that
contains the stromal cells. The stromal cells may then be kept in
the chamber for a period of a few days while their metabolic
performance and/or growth factor responsiveness is monitored and if
results are satisfactory, the bone marrow is inoculated or
immediately seeded with bone marrow. In either case, the cell layer
is kept at the bottom of the central section of the perfusion
chamber.
[0077] The cells lay down additional extra-cellular matrix and the
cell layer adheres to the support. Where the membrane is used, the
chamber may be inverted and the cell layer is then located at the
ceiling of the central section. In this configuration, the maturing
cells settle on the bottom of the central chamber as they loose
their adherence to the stromal layer. The non-adherent cells are
then harvested by constant cell flow, driven by the medium
perfusion pressure, into the exit tubing.
[0078] In a typical run, the chamber was inoculated with NIH-3T3
cells on day one on shredded collagen sponge support. For the first
40 days perfusion rates and other operating variables were
adjusted. At day 40 a reasonable steady state was achieved which
was maintained for about 20 days. On day 64 the chamber was seeded
with 33.times.10.sup.6 human bone marrow cells. For the first 10
days the harvested cell count decreased until it settled in a
steady state of about 7-8.times.10.sup.5 cells produced every three
days. Flow cytometric analysis showed that a constant fraction,
about 20% of the harvested cells were HLA-DR positive. On day 90 a
pump failure was experienced and the pH dropped below 6.9
overnight. When the perfusion rate was restored the production of
non-adherent cells recovered and was approaching the previous
steady state production rate when a bacterial contamination
occurred. At this point, the study was terminated.
[0079] The above results demonstrated that a perfusion chamber is
capable of performing ex vivo hematopoiesis, hematopoiesis may be
restored ex vivo after a pH drop, the glucose concentration data
showed that the hematopoietic cells grow primarily aerobically on
glucose, since the glucose concentration drops after inoculation
without increasing the lactate concentration indicating that
oxygenation is limiting. The glucose/lactate (anaerobic) metabolism
appears to be primarily due to the NIH-3T3 stromal bed. Similarly,
the glutamine and ammonia concentrations reach pre-inoculum levels
once the hematopoietic cell number levels off, implying that the
glutamine consumption by the bone marrow cells is much less than
that of the stromal bed.
[0080] III. Monitoring of Metabolic Products
[0081] The consumption and formation rates of glucose and lactate
as well as glutamine and ammonia were determined for transfected
NIH-3T3 cells. (The medium was IMDM plus 20% FCS). Increased
glucose consumption was only observed for daily fed T-flasks,
whereas all less frequently fed cultures follow the same slowly
diminishing glucose uptake rate pattern. Cultures that were
exchanged 50% daily were switched to the 100% daily exchange
schedule on day 18, which resulted in an immediate increase in
glucose consumption following the same trend as that observed for
cultures exchanged 100% daily from day one. Lactate production
rates follow a similar pattern, as the lactate yield on glucose is
essentially a constant (0.9 lactate/glucose; indicating a largely
anaerobic stromal metabolism).
[0082] The glutamine and ammonia concentrations show a pattern
analogous to the glucose/lactate metabolism. Using values corrected
for chemical decomposition of glutamine at 37.degree. C., the
glutamine consumption rate versus the glucose consumption rate
shows relative uptake rates are constant, about 1:8 glutamine:
glucose. The predicted-optimum ratio varies with oxygen uptake rate
--the ratio drops with increasing optimum uptake rate.
[0083] Analogous conclusions were supported by glucose/lactate
metabolic data derived from normal bone marrow stromal fibroblasts.
Under conditions of infrequent medium exchange the cultures were
primarily anaerobic, with high steady state levels of lactate
rapidly achieved and maintained. With more frequent medium
exchange, the cell metabolism became more rapid, with increased
glucose consumption and lactate production. No detectable
consumption of glutamine was observed after correcting the data for
spontaneous chemical decomposition. For both 3T3 cells and normal
human bone marrow cells, the cells continue to divide and crowd
when the serum/media exchange rate was above what appears to be a
critical replacement schedule.
[0084] To further ascertain the relative importance of perfusion
rate of serum versus that of nutrients, the following experiments
were performed: 1) one set of T-flasks with 20% serum containing
media exchanged daily; 2) two sets of T-flasks, one with 20% serum
and the media exchanged every other day and one with 10% serum with
the media exchanged daily; 3) two sets of T-flasks, one with 10%
serum and the media exchanged every other day, one with 5% serum
with the media exchanged daily; 4) two sets of T-flasks, one with
5% serum and the media exchanged every other day and one with 2.5%
serum with the media exchanged daily. The serum exchange rate is
the same within each group while the exchange rate of the nutrient
containing media varies. The results from these experiments show
that it is the exchange rate of the serum that is critical. While
for the experiment 1) glucose consumption increased and by day four
had substantially flattened out to a rate of about 9.5 mmoles/per
day, in all of the other cases, the glucose consumption started
below the original glucose consumption of Group I and dropped off
in a substantially linear manner regardless of whether twice the
amount of serum was used and changed every other day or half the
amount of serum was used and the media changed every day. This
supports the need for a critical perfusion rate of serum or one or
more serum components that influence the metabolic growth behavior
of the stromal cells.
[0085] It is evident from the above results, that one may grow
hematopoietic cells in a bioreactor in an efficient manner. Stromal
cells can be provided from homologous or heterologous sources,
where the stromal cells have been transfected with genes to provide
for the important growth factors. In this manner, serum need not be
added to the media to support the growth of the cells. By providing
for stromal cells which adhere to a support in a manner which
allows for separation of hematopoietic cells from the stromal
cells, the hematopoietic cells may be continuously harvested for
use. By appropriate choice of combinations of growth factors,
specific lineages of hematopoietic cells may be grown. In addition,
if desired, the stromal cells may provide for a reservoir of
transfecting viruses for the introduction of genes into the
hematopoietic cells.
[0086] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0087] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *