U.S. patent number RE47,439 [Application Number 15/226,341] was granted by the patent office on 2019-06-18 for method for manufacture of macrobeads.
This patent grant is currently assigned to THE ROGOSIN INSTITUTE. The grantee listed for this patent is THE ROGOSIN INSTITUTE. Invention is credited to Lawrence Gazda, Timothy Hamilton, Melissa Laramore, Barry Smith.
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United States Patent |
RE47,439 |
Gazda , et al. |
June 18, 2019 |
Method for manufacture of macrobeads
Abstract
The invention relates to an improved method for making agarose
coated, agarose beads which contain cells. The method which is
preferably automated, involves placing manufactured beads in a
sample of mineral oil at a temperature gradient, such that the
temperature drops as the bead moves through the oil. Preferably, a
"trumpet tool" and a "straw tool" are employed in the method.
Inventors: |
Gazda; Lawrence (Xenia, OH),
Laramore; Melissa (Xenia, OH), Hamilton; Timothy (Xenia,
OH), Smith; Barry (New York, NY) |
Applicant: |
Name |
City |
State |
Country |
Type |
THE ROGOSIN INSTITUTE |
New York |
NY |
US |
|
|
Assignee: |
THE ROGOSIN INSTITUTE (New
York, NY)
|
Family
ID: |
48903127 |
Appl.
No.: |
15/226,341 |
Filed: |
August 2, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61592949 |
Jan 31, 2012 |
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Reissue of: |
13754076 |
Jan 30, 2013 |
9090866 |
Jul 28, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
5/0012 (20130101); C12N 5/0012 (20130101); B25J
15/0616 (20130101); B25J 15/0616 (20130101) |
Current International
Class: |
C12N
5/00 (20060101); B25J 15/06 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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102010040681 |
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Mar 2012 |
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DE |
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102010040684 |
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Mar 2012 |
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DE |
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1522340 |
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Apr 2005 |
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EP |
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Other References
Sigma. Product information, agarose. Oct. 21, 1996. cited by
examiner .
Nussinovitch, A., Bead Formation, Strengthening, and Modification,
Polymer Macro- and Micro-Gel Beads: Fundamentals and Applications,
2010, vol. XXV, pp. 27-52. cited by examiner .
Gradient definition (physics), Oxford Dictionary (American English)
(US), Accessed at
http://www.oxforddictionaries.com/us/definition/american_english/gradient-
, on Mar. 10, 2015. cited by examiner .
International Search Report from PCT/US2013/023802 dated May 9,
2413 (4 pages). cited by applicant.
|
Primary Examiner: Campell; Bruce R
Attorney, Agent or Firm: Abelman, Frayne & Schwab
Parent Case Text
RELATED APPLICATIONS
This application claims priority from U.S. Provisional Application
No. 61/592,949 filed Jan. 31, 2012, incorporated by reference in
its entirety.
Claims
We claims:
1. A method for producing a composition of matter comprising a
sample of live cells in an agarose containing bead, wherein said
bead is coated with agarose, comprising: (a) mixing a first sample
of agarose with said sample of live cells to form a suspension; (b)
moving said suspension to a first sample of mineral oil, to form a
bead from said suspension; (c) removing said bead from said first
sample of mineral oil; (d) rinsing mineral oil from said bead; (e)
moving said bead with a trumpet tool to a second solution of
agarose; (f) coating said bead with said second solution of
agarose, (g) removing said bead from said second solution of
agarose with a straw tool, and (h) dispensing said coated bead into
a second sample of mineral oil, wherein said sample of mineral oil
is kept at a temperature gradient so that said bead moves along a
path from a location in the mineral oil that is at a higher
temperature of about 20.degree. C. to about 30.degree. C. to a
location in the mineral oil at a lower temperature of about
0.degree. C. to about -8.degree. C.
2. The method of claim 1, wherein said cells are secretory
cells.
3. The method of claim 1, wherein said cells are cancer cells.
4. The method of claim 1, wherein said cells are cancer stem
cells.
5. The method of claim 1, wherein said cells are islet cells.
6. The method of claim 1, wherein said cells are stem cells.
7. The method of claim 6, wherein said cells are embryonic stem
cells.
8. The method of claim 1, wherein said cells are pluripotent
cells.
9. The method of claim 1, further comprising removing said bead
from said first or second sample of mineral oil with a trumpet
tool.
10. The method of claim 1, wherein said higher temperature is from
about 20.degree. C. to about 25.degree. C. and said lower
temperature is from about 0.degree. C. to about -2.degree. C.
.Iadd.11. The method of claim 1, wherein said first sample of
agarose has a gel strength of .gtoreq.1000 g/cm.sup.2 at a 1.5%
gel, and a viscosity of from 5.8 to 8.7 cP, a gelling temperature
of 40-43.degree. C. for a 1.5% solution, an electro endosmosis
value of from 0.0 to 0.12, and a sulphate content
.ltoreq.0.30%..Iaddend.
.Iadd.12. The method of claim 1, wherein said second sample of
agarose has a gel strength of .gtoreq.1000 g/cm.sup.2 at a 1.5%
gel, and a viscosity of from 5.8 to 8.7 cP, a gelling temperature
of 40-43.degree. C. for a 1.5% solution, an electroendosmosis value
of from 0.0 to 0.12, and a sulphate content
.ltoreq.0.30%..Iaddend.
.Iadd.13. The method of claim 1, wherein said first and second
samples of agarose have a gel strength of .gtoreq.1000 g/cm.sup.2
at a 1.5% gel, and a viscosity of from 5.8 to 8.7 cP, a gelling
temperature of 40-43.degree. C. for a 1.5% solution, an
electroendosmosis value of from 0.0 to 0.12, and a sulphate content
.ltoreq.0.30%..Iaddend.
.Iadd.14. The method of claim 1, wherein said live cells are cancer
cells..Iaddend.
.Iadd.15. The method of claim 14, wherein said cancer cells are
renal cancer cells..Iaddend.
.Iadd.16. The method of claim 11, wherein said live cells are
islets..Iaddend.
.Iadd.17. The method of claim 11, wherein said live cells are stem
cells..Iaddend.
Description
FIELD OF THE INVENTION
This invention relates to improved methods for making a composition
of matter which comprises cells, secretory cells in particular,
entrapped in a permeable bead or other structure, which is then
coated with agarose. The bead may comprise or consist of agarose.
Litex agarose is especially preferred for the beads.
BACKGROUND AND PRIOR ART
The manufacture of compositions of matter which contain viable
cells, entrapped in permeable media, has been shown to be important
in various fields, such as diabetes therapy, cancer treatment, and
stem cell maintenance. In this regard, see, e.g., Reissue 40,555;
U.S. Pat. Nos. 7,838,291; 6,818,230; 6,808,705; 6,303,151;
6,224,912; 5,888,497; and 5,643,569, and published patent
application 2007/0071732, all of which are incorporated by
reference in their entirety.
The processes for making these compositions of matter, hereafter
referred to as "encapsulates," has been essentially the same,
regardless of the type of cell used. After isolation or securing of
the cells of interest, these are suspended in an aqueous solution
of an agent such as agarose, collagen, or combinations of such, or
placed on a material such as gelatin sponge. When aqueous solutions
are used, semi-solid beads containing the cells of interest are
formed by placing the suspension in mineral oil. If gelatin sponge
is used, the product, containing the cells, is rolled into a
sphere, after which agarose is poured onto it, to form a bead.
The beads are then contacted to a solution of agarose, in, e.g., a
Teflon spoon. The beads roll in the mixture, to form what are
referred to in the art cited supra as agarose-coated
macrobeads.
The initial teachings as shown by, e.g., RE 40,555 and U.S. Pat.
No. 5,888,497 which teach encapsulation of secretory cells, such
insulin producing islets and cancer cells, have been followed by
improvements, including the encapsulation of stem cells, as shown
by U.S. Pat. No. 7,838,291 and via improvements in materials used,
as seen in, e.g., published patent application 2007/0071732;
however, there is an ongoing need to improve the methodology for
making these useful materials.
One improvement, which is very desirable in this field, is the
ability to automate what is a manual process.
In the course of automating the process of manufacture, the
inventors have developed special tools, which facilitate the
manufacturing process. In addition, they have found that in
finishing the process via placing the macrobeads in a vessel of
mineral oil at a temperature gradient, one can produce macrobeads
with more uniform shape and even texture than was believed
possible.
How this is accomplished will be seen in the disclosure, which
follows.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the automated dispensing of cells and agarose into a
multiwell array which contains mineral oil.
FIGS. 2a-2c present different views of a preferred embodiment of
the "trumpet tool" of the invention.
FIG. 3 shows a second embodiment of the trumpet tool.
FIGS. 4a and 4b show different embodiments of the straw tool
invention.
FIG. 5 depicts a summary of the process of the invention.
FIG. 6 shows a device used to maintain temperature gradient in
operation of the invention.
FIG. 7 depicts levels of insulin production from beads made with
different concentrations and types of agarose.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Reference is made to the U.S. Patents cited supra, all of which
describe detailed manual methodologies for making macrobeads, which
contain different types of cells, such as secretory cells as
defined therein. The invention described herein modifies those
methods by replacing the final step of contacting the agarose
coated, agarose beads with mineral oil in a spoon or on a Teflon
surface, with a vessel containing mineral oil kept at a temperature
gradient, as described infra.
Macrobeads such as are described in these patents may also be made
robotically or via automation. One such approach is described
herein.
In one embodiment, as in depicted FIG. 1, a robotic dispenser, such
as an array of pipettes, dispenses secretory cells into a multiple
well plate, such as a standard 96 well plate. Molten agarose is
then added to the wells. The cells and molten agarose are then
transferred via, e.g., automated means such as a multi-pipette
array, to mineral oil containing means such as single well or
multiwell containers, such as 96 well plates, to form beads.
The beads are then removed from the wells, preferably via the use
of a so-called "trumpet tool," depicted in FIGS. "2a-c and 3." The
trumpet tool, as is elaborated upon infra, is designed so as not to
disrupt the surface of the bead.
In a further embodiment, an exit means is provided at the bottom of
the well, by which the beads escape the well and may be used in the
steps which follow. In a still further embodiment, the beads
resulting from the process discussed supra, may be transferred to
the top of the mineral oil via automated means, such as a moveable
platform, and also removed from the mineral oil containing means,
via automated systems.
Following pick up by the trumpet tool, or passage via the exit
means, and removal of any adherent mineral oil by means of rinsing
with an appropriate solution, such as a balanced salt solution, the
beads are placed in wells containing new, warmed agarose. Following
this, the beads are removed together with the warm agarose,
preferably using a so-called "straw tool," as shown in FIGS. 4a and
4b using mild suction, and are dispensed to vessels containing
mineral oil at a controlled temperature gradient.
The vessel containing the mineral oil may be, e.g., a test tube,
cuvette, beaker, or any other object that is suitable, although
test tubes and cuvettes, of various sizes are preferred. The
material used to make these containers, i.e., the test tubes,
cuvettes, etc., may vary. Glass is especially preferred, although
other materials, such as those coated with a substance to provide a
hydrophilic layer, may be used.
The temperature gradient in the vessel may be maintained by any
method known to the art. Key is that the temperature of the mineral
oil be higher at the top of the vessel than at the bottom thereof.
The temperature and amount of gradient will vary based on, e.g.,
the length of the vessel, the type of cell encapsulated, the type
of agarose used, and so forth. The factors which are important to
setting these values include, e.g., temperatures at which the
encapsulated cells are viable, temperatures at which the agarose
employed will solidify, and so forth. The temperature at the start
of the gradient is preferably from 20.degree. C. to 80.degree. C.,
more preferably from 20.degree. C. to 50.degree. C., and even more
preferably, from 20.degree. C. to 40.degree. C. Ideally, the
starting temperature is from about 20.degree. C. to about
25.degree. C. The temperature at the bottom of the vessel may also
vary, ranging from, e.g., 0.degree. C. to -10.degree. C., more
preferably 0.degree. C. to -8.degree. C., and most preferably from
about 0.degree. C. to about -2.degree. C. The total differential
between the highest and lowest temperatures is preferably from
20.degree. C. to 50.degree. C., more preferably from about
20.degree. C. to about 30.degree. C.
The figures which area part of this application will amplify upon
the preceding description.
FIG. 1 shows how both the cells and the agarose are dispensed into
wells of plates. Following removal of the mineral oil via, e.g.,
washing or aspiration, or via the exit means discussed supra, the
"trumpet tool" referred to supra, and as is shown in FIGS. 2a-2c
and 3 is inserted into the well, so as to remove the beads with
entrapped cells. This trumpet tool has a vacuum source attached at
one end, which does not disrupt the surface of the bead so as to
facilitate its removal.
The bead consisting of one layer of agarose and the cells is
deposited via the so-called "trumpet tool" into a new well, filled
with heated agarose which serves as the second layer, or coating of
agarose. The bead is removed together with the warm agarose,
preferably by using a "straw tool," such as one of those shown in
FIGS. 4a and 4b, preferably with the aid of mild suction, and the
bead is dispensed to a vessel containing the temperature gradient
mineral oil. The temperature gradient may be maintained by, e.g.,
the device shown in FIG. 5, but the skilled artisan will be
familiar with other options. When the macrobead has traversed the
vessel and has reached the bottom an elongated form of the trumpet
tool may be used, with suction, to remove the macrobeads for
washing in medium, or as noted, supra, an exit means for removal of
the bead may be employed. At this point, the macrobeads are ready
for use, although in different situations it may be preferable to
permit the cells to proliferate and/or mature within the beads
until a sufficient number are present or until a specific product
is expressed by the cells. This time period could range from, e.g.,
a week to several months.
Referring now to the figures of the claimed invention, FIGS. 2a,
2b, 2c, and 3 show different views of devices in accordance with
the invention, the so called "trumpet tool."
Referring to FIG. 2a, a trumpet tool 40 is shown. The description
of the tool as a "trumpet tool" comes from the resemblance of
negative conical section 41 to a trumpet. At one end, a negative
conical segment 41 is provided, with a rim 42 attached to the
conical end 41. The conical end 41 connects to a hollow,
longitudinal section 43 which is adapted for passage of a fluid
therethrough, such as air.
As FIG. 2a shows, the hollow longitudinal section 43 runs along the
entirety of the device. One can see collar means 44 positioned
about 2/3 to 3/4 of the way along the length of the trumpet tool,
relative to the negative conical end. The collar means 44, as well
as several other features described herein, are artifacts of the
manufacturing process and do not impact the functioning of the
device.
Collar means 44, however, marks the point at which the longitudinal
means expands its circumference. The portion of the device
represented by 45 corresponds to a coupling end which, in
operation, connects the tool to a source of fluid, e.g., air, such
as a vacuum. Connection aperture 46 has the widest circumference of
any part of the device and acts to complete connection to, e.g., a
vacuum means. Internal structures, such as those exhibited in FIG.
2c, are provided to facilitate attachment of vacuum means. These
structures may vary, based upon the apparatus being used.
One observes a ridge means 47 in the device, as well as a series of
evenly spaced projections 48a, b, and c, all of which are, as with
collar means 44, artifacts of manufacturing; however, these
projections also serve to help hold their associated tools in place
in, e.g., a dispensing rack.
The interior of the device can be seen in both FIGS. 2b and 2c,
where it can be seen that in fact the device is hollow along its
entire length. The view of negative conical segment in FIG. 2c
shows in greater detail how it tapers to a point where the
circumference is slightly smaller than an object it is designed to
engage, e.g., a bead.
In operation, the device 40 is connected via connection end 46 to,
e.g., a vacuum pump means, and is positioned, vertically over the
object to be removed, such as a bead. The vacuum pump means (not
shown), removes any air within the tool, and permits uptake of the
bead, e.g., via controlling the force of the suction created by the
vacuum, the configuration of the negative conical end 41 permits
removal of a bead, such as a semi-soft agarose bead, from one
position, and movement to another, such as a solution of coating
agarose. At that point, the vacuum pressure is changed via, e.g.,
providing air to longitudinal means 45, which acts to expel the
bead into the agarose solution from conical end 41 without any
changes to the configuration of the bead.
FIG. 3 shows another embodiment of the device of FIGS. 2a-2c. This
device, prepared via a different manufacturing means than the
embodiment of FIGS. 2a-2c, exhibits ridge means along the
longitudinal section 43. Also, it will be noted that there are
variations in the collar means, as well as in the ridge means 47
and 48a-c. The negative conical end 41 and its rim, as well as the
coupling end 45 and connecting aperture, however, function in the
same way as the comparable structures of FIGS. 2a-2c.
FIGS. 4a and 4b depict embodiments of a device known hereafter as
the "straw tool." Referring now to FIG. 4a a straw tool 50 is
shown. The tool exhibits essentially cylindrical geometry along a
hollow longitudinal axis 51, a coupling end 52, and a receiving end
53.
The straw tool includes a recessed space 54, which extends along
the longitudinal axis 51, opening at receiving end 53.
The receiving space 54 extends along the longitudinal axis and,
which adjoins a canal section 55, which can have a smaller diameter
than the receiving space. This canal section 55 joins connecting
means 52, and the combination of "52," "54" and "55" form a working
channel which accepts changes in pressure of a fluid, such as air
or another gas. In operation, connecting means 52, is coupled to a
device capable of changing pressure of a fluid in the straw tool,
such as an air pump. The straw tool is then placed over an object,
such as a bead, and upon the change in pressure, the bead is
removed by the straw tool. It should be noted that the diameter of
the opening at receiving end 53 is large enough to pick up the
object, e.g., the bead and to permit the bead to move vertically in
the longitudinal cavity of the tool. Once the bead is moved to the
desired location, a second change of pressure causes the bead to
drop, and permit re-use of the straw tool.
FIGS. 4a and 4b differ in that configurations of the connecting
means 52 are different; however, the difference in configurations
results from manufacturing processes and does not impact the
operation of the device.
In each device, broken lines indicate adaptation of the straw tool
for reception of a vacuum connecting means of choice. A recessed
channel 56 is depicted, which is capable of accepting a connection
means, such as an expandable O-ring. Also a feature of the device
is a means 57, for impeding progress of a pipette. This "pipette
shelf" stops passage of a pipette or other delivery means for the
fluid, e.g., air.
The examples which follow should be taken as exemplary, but not
limitative, of the invention as it is described herein.
Example 1
Powdered agarose (Litex HSB-LV) was dissolved in minimal essential
medium to a concentration of either 0.8% or 4.5% (w/v), and then
autoclaved at 121.degree. C., for 20 minutes. This produced viscous
molten solutions. The agarose solutions were cooled and maintained
at 51-53.degree. C., or 61-63.degree. C., respectively.
A total of 150,000 RENCA (renal cancer) cells were added to a well,
after which 0.25 ml of the 0.8% agarose solution was added to form
a suspension, and this cell/agarose suspension was then dispensed
into either room temperature mineral oil in a plastic bowl, or into
a deep well 96 plastic plate, previously filled with room
temperature mineral oil.
RENCA cell containing agarose macrobeads formed, as round beads
with smooth surfaces, and evenly distributed cells. The mineral oil
was then aspirated away from the macrobeads, and the macrobeads
were washed with RPMI cell culture medium.
The coating for the macrobead used the 4.5% w/v solution. The
agarose for the coating was same type as was used for the beads;
however, it should be noted that they can differ.
To carry out the manual mode for preparing the beads, the beads
prepared supra, were rolled in a plastic spoon which contained 1.0
ml of the 4.5% agarose, which had been maintained at 61-63.degree.
C., after which they were dropped into room temperature mineral
oil.
In the method of the invention, the coating material was maintained
at 61-63.degree. C., and was transferred to a 24 well plate. The
"trumpet tool" shown in FIG. 2a and discussed, supra, was then used
to remove the beads from the room temperature mineral oil and to
place them into the second coat, agarose solution following washing
of the beads to remove the mineral oil. A straw tool, depicted in
FIG. 4a, was used together with gentle suction, to remove the
beads, together with 0.5 ml of agarose to a vessel containing
mineral oil maintained at a temperature gradient.
Macrobeads were dispensed from the straw tool, into the top of the
mineral oil gradient, which was maintained at varying temperatures,
as discussed infra. As the macrobead descended through the mineral
oil, the coating solidified on the bead forming a sphere. The
temperature of the mineral oil decreased from the top to the bottom
of the vessel, to a low temperature at the bottom, also as
described infra. The agarose/agarose macrobeads were then removed
from the vessel using the trumpet tool of FIG. 2a and were then
washed in media. The resulting macrobeads were then ready for
routine tissue culture.
Example 2
A series of different experiments, using the temperature gradient
described, supra were used.
TABLE-US-00001 Experiment Top Temperature Bottom Temperature
Control RT RT (Room Temperature) (Room Temperature) 1 25.degree. C.
-2.degree. C. 2 30.degree. C. -2.degree. C. 3 40.degree. C.
-2.degree. C. 4 RT RT 5 50.degree. C. -1.8.degree. C. 6 60.degree.
C. -1.6.degree. C. 7 RT RT 8 70.degree. C. 0.degree. C. 9
25.degree. C. -2.degree. C.
After beads were recovered and washed, as discussed, supra, they
were tested for metabolic activity. This evaluation was carried out
using a standard MTT assay, which is well known in the art.
Metabolic activity was determined at day =0 for non-encapsulated
cells, and at 7 days post-production for the encapsulates. The
results are summarized below.
TABLE-US-00002 Experiment Control 1 2 3 4 5 6 7 8 9 Free Cells
0.603 0.682 0.682 0.682 0.669 0.723 0.723 0.517 0.686 0.686
Encapsulates 0.507 0.411 0.299 0.279 0.261 0.182 0.173 0.448 0.167
0.415
The results indicate that while the encapsulated cells survived
regardless of the starting temperature when dropped into the
gradient, metabolic activity was inversely proportional to the
temperature at which the gradient began, i.e., the higher the
temperature, the lower the resulting metabolic activity.
Example 3
Given that the optimal starting temperature of the mineral oil
gradient was established to be about 25.degree. C., and the ending
temperature about -2.degree. C., this was used in a set of
experiments to determine metabolic activity, and tumor inhibitory
capacity.
Metabolic activity was determined in the same manner set forth
supra.
Tumor inhibitory activity was determined by seeding RENCA cells
into 6 well plates (15,000 cells/well) in either 4 ml of fresh
culture medium, or the same amount of culture medium taken from
cultures of the encapsulates, following 5 days of culture. The free
RENCA cells were cultured in the medium, for 5 days at 37.degree.
C. and a 5% CO.sub.2 atmosphere. The RENCA cells were then fixed
with methanol, stained with 0.33% (w/v)P neutral red, and
absorbance was determined at 540 nm, using 630 nm as a reference
wavelength. Inhibition was defined as the percent difference in
Abs.sub.540 nm-630 nm between treated, and fresh media.
The results that follow present metabolic activity data. "Free
cell" refers to RENCA cells which were not treated with agarose at
all, while "1.sup.st coat" refers to uncoated beads.
TABLE-US-00003 Age (days) Type Control 1 Control 2 2 Control 3 3 4
Control 4 5 6 Control 5 7 0 Free Cell 0.768 0.530 0.378 0.462 0.481
0.532 0.532 0.384 0.518 0.518 - 0.530 0.553 1 1st coat 0.926 1.209
0.912 1.279 1.056 1.120 1.120 1.129 1.002 1.002 1- .157 1.711 7 1
week 0.247 0.260 0.332 0.143 0.739 0.801 0.710 0.409 0.390 0.227
1.- 330 0.697 21 3 week 0.201 0.351 0.328 0.258 0.555 0.560 0.770
0.462 0.531 0.380 0.- 977 0.555 35 5 week 0.647 n/a 0.856 n/a 1.156
n/a 1.092 1.141 n/a n/a 1.447 n/a 49 7 week 1.080 n/a 1.236 n/a
1.324 n/a 1.066 1.215 n/a n/a 1.926 n/a 84 12 week 1.025 n/a 1.548
n/a 1.623 n/a 1.095 1.725 n/a n/a 1.886 n/a 112 16 week 1.652 n/a
2.440 n/a 2.247 n/a 1.128 1.872 n/a n/a n/a n/a
In the table which follows, the tumor inhibitory capacity was
determined:
TABLE-US-00004 Age (days) Type Control 1 Control 2 Control 3 4
Control 5 Control 6 35 % 29.99% 29.80% 12.78% -11.23% 11.10% 7.54%
17.37% 23.97% 26.20% 33.85% 33.28% 49 % 23.62% 33.53% 26.15% 33.98%
32.70% 29.63% 29.63% 34.54% 40.90% n/a n- /a 84 % 33.10% 45.69%
23.41% 33.93% 43.20% 56.02% 56.09% n/a n/a n/a n/a 112 % 50.19%
55.66% 42.21% 46.03% 55.28% 44.84% n/a n/a n/a n/a n/a
The results indicate that the agarose beads produced in accordance
with the invention are equivalent in all relevant respects to
manually produced beads.
Example 4
Beads were produced in accordance with the invention as described
supra, and the manual method as represented via the cited prior
art. Over a period of 7 months, ten beads were selected, at random
to determine their diameters. The results, presented in the table
which follows, shows that beads produced in accordance with the
invention have a more consistent diameter, and hence a more
consistent coating.
TABLE-US-00005 Automated: Manual: Diameter (in) Diameter (in)
January 0.352 0.350 0.363 0.347 0.364 0.347 0.354 0.353 0.350 0.346
0.347 0.348 0.355 0.351 0.348 0.350 0.353 0.352 0.347 0.350
February 0.350 0.350 0.354 0.328 0.351 0.327 0.354 0.326 0.361
0.324 0.353 0.332 0.355 0.325 0.362 0.328 0.353 0.321 0.348 0.316
March 0.335 0.322 0.354 0.317 0.357 0.308 0.364 0.347 0.356 0.331
0.349 0.313 0.351 0.346 0.334 0.333 0.358 0.329 0.352 0.307 April
0.332 0.344 0.342 0.331 0.345 0.324 0.334 0.334 0.351 0.344 0.375
0.346 0.345 0.339 0.343 0.319 0.368 0.339 0.346 0.331 May 0.368
0.335 0.344 0.332 0.349 0.322 0.333 0.317 0.346 0.315 0.337 0.316
0.341 0.318 0.329 0.347 0.347 0.317 0.329 0.332 June 0.370 0.330
0.318 0.357 0.350 0.346 0.350 0.343 0.351 0.347 0.347 0.345 0.353
0.336 0.347 0.344 0.347 0.318 0.363 0.332 July 0.341 0.347 0.349
0.343 0.350 0.345 0.346 0.326 0.366 0.324 0.361 0.342 0.362 0.357
0.348 0.321 0.351 0.314 0.369 0.335 Parameter Auto Manual Mean
0.350386 0.3339714 Standard Error 0.001267 0.0015674 Median 0.35
0.3325 Mode 0.347 0.347 Standard Deviation 0.010601 0.0131137
Sample Variance 0.000112 0.000172 Kurtosis 0.697026 -1.117773
Skewness -0.30036 -0.151897 Range 0.057 0.05 Minimum 0.318 0.307
Maximum 0.375 0.357 Sum 24.527 23.378 Count 70 70
Also, upon examination, it was found that the process of the
invention resulted in reduced cellular contamination. To elaborate,
it has been found that when beads are prepared in accordance with
the manual method of the prior art, there is an issue with cells
that do not become fully encapsulated. When the cells are cancer
cells, plaques, or tumor colonies form, and the beads in the
culture with them must be discarded due to the contamination.
Over an 8-month period, 56 batches of beads were made manually,
each batch containing a number of Petri dish cultures. At least one
culture was contaminated in each batch. In contrast, 62 batches
were prepared over the same period using the method of the
invention. Only 12 of those 62 batches showed any type of
contamination.
Example 5
The following example details the production of three different
groups of agarose coated, agarose bead containing islets.
Islets were prepared from animals over two years of age and with a
history of multiple parities.
The pancreases of the animals were perfused with a
collagenase/neutral protease (either Collagenase P, at 1.0 g/L, or
Liberase MTF/Thermolysin, at 7.5 U/g pancreas), and 0.01 g/L DNase
I, or 2.5 mg/pancreas pulmozyme solution, prepared in either Hanks
Balanced Salt Solution (GBSS) or Cold Storage Purification Stock
Solution.
After quantification, islets were separated into 2000 IEQ aliquots,
before being resuspended in a 0.5 ml solution of one of 1.5% Seakem
Gold ("SG" hereafter, 0.8% SG, or 0.8% Litex ("Li" hereafter). An
"IEQ" as used herein means an islet having a diameter of 150 .mu.m.
Hence, an islet with a diameter of 300 .mu.m is 2 IEQs, while one
with a diameter of 75 .mu.m is 0.5 IEQ. Litex agarose possesses the
following properties: a gel strength .gtoreq.1000 g/cm.sup.2 at a
1.5% gel .Iadd.and a viscosity .Iaddend.of from 5.8 to 8.7 cP when
a 1.5% solution was used, a gelling temperature of 40-43.degree. C.
for a 1.5% solution, an EEO (electro endosmosis) value of from 0.06
to 0.12, and sulphate content .ltoreq.0.30%. The solutions were
prepared in minimal essential medium plus 25 mM HEPES.
Suspensions were expelled beneath the surface of sterile mineral
oil, to yield four, 0.125 ml spherical beads, approximately 5-6 mm
in diameter, each of which contained 500 IEQ.
These, uncoated agarose beads were cultured at 37.degree. C., in a
humidified, 5% CO.sub.2 atmosphere. After 5-7 days, a second coat
of 5%, SG agarose was applied, yielding agarose coated, islet
containing agarose beads, with a final diameter of 8-9 mm.
These beads were cultured at 37.degree. C. in a humidified 5%
CO.sub.2 atmosphere until used in experiments which follow. They
were placed in a culture medium (11 mM glucose, supplemented with
2.5% heat inactivated porcine serum, and 1%
antibiotic/antismycotic). The culture medium was changed weekly,
and samples of media were analyzed, 24 hours after change, every
week, for assays discussed infra.
Example 6
As noted in the prior example, culture media samples were taken
from the samples, and assayed for insulin content, using a
commercially available porcine insulin ELISA. The results are
depicted in FIG. 6. It will be seen that islets encapsulated in
0.8% Li outperformed both islets encapsulated in 0.8% SG, and 1.5%
SG with respect to insulin production.
Example 7
The in vitro experiments presented supra were extended to in vivo
experiments, as discussed herein.
Adult (8 week old rats) were used in these experiments. Animals
were divided into groups of "insulin only" controls, or bead
recipients. All animals received 65 mg/kg of streptozotocin via
tail vein injection to induce diabetes.
Presence of diabetes was confirmed by assaying blood of subject
animals for non-fasting glucose levels. A level over 400 mg/dL of
glucose for 3 consecutive days was taken to indicate diabetes was
present. Any animals which did not exhibit diabetes within 2 weeks
received a second dose of streptozotocin, after which all animals
became diabetic.
The animals chosen for receiving implants received the beads in
accordance with Gazda, et al., Cell Transplant, 16:609-620 (2007),
which is incorporated by reference in its entirety. To elaborate,
12-13 week old animals were anesthetized, using isoflurane.
Following a midline incision along the peritoneal cavity, beads
were placed therein.
The number of beads placed in each animal varied, depending upon
the highest dose of insulin given to the particular animal over the
3 day period prior to the implant, divided by the average insulin
production per macrobead (based upon the insulin produced by the
beads over the 4 week period prior to implantation). Mice received
the amount of beads necessary to provide the exact insulin dose
they had received prior to implantation (1.times.), or 1.7.times.
that dose. Female and male animals received beads equal to
1.times., but only males received 1.7.times., in part because male
rats weight more than female rats.
There were no significant differences seen in pre-implant insulin
requirements with respect to the different implant types (1.5% SG,
0.8% SG, 0.8% Li); however, more macrobeads were required for 1.5%
SG beads, and the fewest were required for 0.8% Li.
Following implantation, the animals were maintained for 90 days,
with blood glucose and body weight being monitored regularly.
Female mice (all in the 1.times. group) showed an immediate,
sustained normalization of blood glucose levels as compared to mice
receiving insulin controls. Male rats in the 1.times. group,
exhibited an immediate, but temporary, improvement in blood glucose
regulation, which returned to pre-implant levels by about 30 days
post-implant.
Example 8
Studies were carried out on the macrobeads after the recipient
animals were sacrificed and examined for structural integrity and
functional capacity.
Gross examination showed that, only two of 1540 macrobeads
recovered showed any structural damage.
The foregoing examples describe various features of the invention,
which relate to secretory cell-containing agarose macrobeads,
coated with agarose, where the agarose used for the macrobeads had
the properties of Litex agarose, set forth supra.
As set forth herein, the term "macrobead" refers to a structure
that is essentially spherical, with a diameter of from about 4 to
about 10-12 mm in diameter, most preferably from about 6 to about 8
mm in diameter. The second agarose layer is preferably from about
0.05 to about 5 mm in thickness, most preferably from about 0.5 mm
to about 5 mm in thickness, even more preferably, from about 1.0 mm
to about 3 mm, and most preferably, from about 1.0 mm to about 2.0
mm in thickness. The second agarose layer may, but need not be, the
same agarose used to make the bead.
"Macrobeads" is used as a preferred structure; however, any solid,
agarose structure which encapsulates secretory cells, and is
preferably coated with a second, agarose layer, are features of the
invention.
The secretory cells may vary. Any cell or organelle which yields a
desirable, secretory product may be encapsulated. Islets, cancer
cells, and stem cells are exemplary of the types of materials which
can be used. Each bead may contain a varying number of cellular
organelles, for islets, for example, from about 50 to about 5000
islet equivalents ("IEQs" hereafter), more preferably, from about
100 to about 2500 IEQs, even more preferably, from about 250 to
about 1000, and most preferably, from about 475 to about 550 IEQs.
About 500 IEQs is most especially preferred.
Also a feature of the invention is an improved method for making
macrobeads, regardless of the agarose used. Cells are mixed with
agarose, and then formed into a suspension which is in turn used to
forma bead. The bead, e.g., a macrobead, is then coated with
agarose, after which the resulting coated bead is dispensed into a
sample of mineral oil at a temperature gradient, such that the bead
contacts mineral oil at a higher temperature and drops to oil at a
lower temperature. The beads may contain any cell type desired such
as, but not being limited to secretory cells, cancer cells, islets,
stem cells, such as pluripotent stem cells, and so forth
In particularly preferred embodiments, "trumpet" and "straw" tools,
as described herein, are used to remove the uncoated, and the
coated beads, respectively.
The temperature gradient of the mineral oil can vary. Preferably,
the gradient is from 20.degree. C. to 50.degree. C., i.e., the
difference between the highest and lowest temperatures falls within
this range. More preferably, the difference is from 20.degree. C.
to 35.degree. C.
The highest temperature of the oil may vary, but is preferably
between 20.degree. C. and 30.degree. C., more preferably between
20.degree. C. and 35.degree. C. Similarly, the lowest temperature
may vary, and run from 0.degree. C. to -8.degree. C., and more
preferably, from 0.degree. C. to -2.degree. C.
Other aspects of the invention will be clear to the skilled
artisan, and need not be elaborated further.
The terms and expressions which have been employed are used as
terms of description and not of limitation, and there is no
intention in the use of such terms and expression of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
* * * * *
References