FABRICATION OF THIN SHEET BIO-ARTIFICIAL ORGANS

AMELIORATION D'UN PROCEDE DE FABRICATION D'ORGANES BIOARTIFICIELS EN FEUILLE FINE

English Abstract

In one embodiment of the present invention, a method is provided for making a
physiologically active and biocompatible cellular implant for implantation
into a host body. The method includes the steps of: (a) forming first and
second layers of first and second polymer solutions, respectively, each layer
having a first substantially uncross-linked surface and an opposing second
cross-linked surface; (b) forming a sandwich of a cell suspension layer of
physiologically active cells in a substantially uncross-linked third solution
between the first and second, and (c) cross-linking the first and second
polymer solutions in a direction toward the cell suspension layer, thereby
forming a cellular implant. In another embodiment, all polymer solutions
initially are uncross-linked and sequentially spread in layers followed by
cross-linking.

French Abstract

La présente invention concerne selon un premier mode de réalisation un procédé permettant de fabriquer un implant cellulaire physiologiquement actif et biocompatible en vue de son implantation dans une anatomie hôte. A cet effet, on commence par (a) réaliser une première et une deuxième couche, respectivement en une première et une deuxième solution polymère, chaque couche présentant une première face sensiblement non réticulée et une deuxième face opposée réticulée. Il faut ensuite (b) prendre en sandwich entre la première et la deuxième couche une couche de cellules physiologiquement actives en suspension dans une troisième solution. Il ne reste plus qu'à (c) réticuler la première et la seconde solution polymère selon un axe orienté vers la couche de cellules en suspension, formant ainsi un implant cellulaire. Selon un autre mode de réalisation, toutes les solutions de polymère sont initialement non réticulées et séquentiellement étendues en couches puis réticulées.

Claims

WHAT IS CLAIMED IS:
1. A method of making a physiologically active and
biocompatible cellular implant for implantation into a host
body, said method comprising the steps of:
(a) forming first and second layers of first and second
polymers, respectively, each layer having a first substantially
uncross-linked polymer solution surface and an opposing second
cross-linked surface,
(b) forming a sandwich of a cell suspension layer
comprising a cell suspension layer of physiologically active
cells in a substantially uncross-linked third polymer solution
between said first and second layers at their respective first
surfaces so that their respective interfaces are in liquid
form, and
(c) cross-linking said first and second polymer
solutions in a direction from the second surface of one or both
of said first or second layers to the cell suspension layer
until the first and second polymer of said first and second
layers, respectively, and said cell suspension layer are cross-
linked at their respective interfaces, thereby forming a
cellular implant.
2. The method of claim 1 in which the interface cross-
linking of said first, second and third polymer is performed by
gelling.
3. The method of claim 2 in which said first, second and
third polymer solutions comprise polysaccharide.
4. The method of claim 3 in. which said first, second and
third polymer solutions comprise alginates.
5. The method of claim 1 in which said cross-linking is
performed by diffusing cross-linking agent from said second
surface of one or both of said first or second layers to said
cell suspension layer.
43

6. The method of claim 5 in which said cross-linking agent
comprises multivalent cations.
7. The method of claim 5 in which said cross-linking agent
is selected from the group consisting of calcium, barium,
strontium or mixtures thereof.
8. The method of claim 5 in which said cross-linking agent
comprises a calcium salt.
9. The method of claim 6 in which said multivalent cations
are in said salt form and the counterion for the multivalent
cation in the salt is selected from the group consisting of
gluconate, lactate, chloride or mixtures thereof.
10. The method of claim 2 in which the temperature of step
(c) is different from the temperature of step (b) and said
gelling occurs in response to the change of temperature from
step (b) to step (c).
11. The method of claim 10 in which said first, second and
third polymer solutions comprise agarose.
12. The method of claim 1 in which said first, second and
third polymer solutions comprise photoactivatable cross-linking
groups, said cross-linking comprising photoactivation of said
photoactivatable cross-linking groups.
13. The method of claim 12 in which said first, second and
third polymer solutions comprise polyethylene glycol
derivatized with photolabile cross-linking groups.
14. The method of claim 1 in which said cells comprise
secretory cells.
15. The method of claim 14 in which said secretory cells
comprise islets of Langerhans.
44

16. The method of claim 1 in which said active cells in said
third polymer solution are first encapsulated in microcapsules
or hollow fibers.
17. The method of claim 1 in which in step (b) said first and
second layers are deposited directly or indirectly on first and
second plates, respectively, said method further comprising
pressing said plates toward each other to form said cell
suspension layer.
18. The method of claim 17 in which during pressing said
first and second plates are precisely spaced apart from each
other by at least one spacer of predetermined thickness during
step (b).
19. The method of claim 17 in which said first and second
layers are separated from said first and second plates by, first
and second flexible membranes, respectively, to form a
membrane-jacketed implant comprising a multi-layer of first
membrane - first layer - cell suspension layer - second -
membrane.
20. The method of claim 19 in which said first and second
membranes include first and second smooth polymer layers,
respectively, facing and in contact with said first and second
layers.
21. The method of claim 19 in which the membrane is selected
from the group consisting of agarose, polyacrylamide, alginate,
polyethylene glycol or mixtures thereof.
22. The method of claim 19 further comprising the step of
removing said membrane-jacketed implant as a unit from said
first and second plates.
23. The method of claim 22 further comprising contacting said
plate-removed membrane-jacketed implant with a solution of said
cross-linking agent.
45

24. The method of claim 22 in which said contacting is
performed by immersing said plate-removed membrane-jacketed
implant in a solution of said cross-linking agent.
25. The method of claim 1 in which said first, second and
third polymer solutions are identical.
26. A method of making a physiologically active and
biocompatible cellular implant for implanting into a host body,
said method comprising the steps of
(a) forming a first layer of a first substantially
uncross-linked polymer solution of predetermined thickness by
substantially uniformly spreading said first polymer solution
on a solid support surface, said first layer having an exposed
surface,
(b) forming a cell suspension second layer of
predetermined thickness. on said first layer exposed surface,
said cell suspension comprising physiologically active cells in
a second substantially uncross-linked second polymer solution,
by substantially uniformly spreading said cell suspension over
said first layer exposed surface, and
(c) forming a third layer of a third substantially
uncross-linked polymer solution on said cell suspension second
layer by substantially uniformly spreading said third polymer
solution on said cell suspension second layer.
27. The method of claim 26 in which said third polymer has a
high viscosity than said second polymer layer and said second
polymer has a higher viscosity than said first polymer.
28. The method of claim 26 in which forming said first,
second and third layers is performed by depositing said first,
second and third polymer solutions successively with spacers of
first, second and third increasing thicknesses sequentially
placed to extend from said solid support surface; and
sequentially sliding a substantially flat edge across said
first spacer in step (a), across said second spacer in step (b)
and said third spacer in step (c).
46

29. The method of claim 27 further comprising diffusing
cross-linking agent through said first, second and third layers
to form a sandwich-type thin cellular implant and removing said
implant from said support surface.
30. A method of treating a disease in which a cellular
implant made by the method of claims 1 or 26 is implanted into
a vascularized site in a host.
47

Description

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IMPROVED METHODS FOR FABRICATION OF THIN SHEET
BIO-ARTIFICIAL ORGANS
BACKGROUND OF THE INVENTION
Field of the Invention:
The present invention concerns methods for making thin sheet
bio-artificial organs for use in treatment of disease. Thin
sheet bio-artificial organs are devices for surgical
implantation which entrap cells or tissue producing desirable
substances or having desirable properties. Specifically, the
field relates to thin sheets containing cells and which have
dimensions and physicochemical properties allowing maintenance
of tissue viability through rapid diffusion of nutrients and
oxygen, and affording protection from contact of said cells
with cells of the recipient's immune system, said sheets
optionally having the further properties of substantially
excluding factors necessary for humoral immune destruction of
the entrapped cells and having the additional properties of
biocompatibility, mechanical strength and chemical stability
sufficient that the entrapped cells or tissue can function in
vivo for a long time.
Bio-Artificial Organs
The potential utility of bioartificial organ implants to treat
disease has been long recognized. The exemplary bioartificial
organ is the bioartificial pancreas containing insulin
producing islets of Langerhans, for which there is great
clinical need in the treatment of diabetes. Evidence that
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islet transplantation can normalize diabetic blood sugars and
arrest and reverse vascular decay continues to mounts. In
essence, the bioartificial pancreas would make possible the
benefits of islet cell transplantation without the need for
immunosuppression. However, the failure of any bioartificial
pancreas to be commercialized after decades of research
emphasizes the difficulty of the task.
The background of this invention was described in a related
patent, No. 5,855,613, incorporated herein by reference.
Recent advances in the bioartificial pancreas field have been
in making smaller and more biocompatible microcapsules. A
microcapsule is so small that each capsule contains a single
islet. Macrocapsules such as sheets comprise many islets each.
Advances in the art of making sheet bio-artificial organs in
the past four years (and macrocapsules generally) are few and
have characteristics such that the essentials of the prior art
have changed little. Therefore, in the remainder of this
background section we describe only developments since the
prior application.
Bioaompatible Materials
As described in patent No. 5,855,613, the material of choice
for manufacture of bioartificial organs is "bioinvisible," that
is, the material by itself or in combination with living
tissues does not produce foreign body reaction or fibrosis when
implanted in a host organism. One known material for this
purpose is highly purified alginates. For example, Van
Schilfgaarde et al.z observe: "Graft failure cannot be readily
explained by immunological rejection, since success rates in
rats are similar with isogenic and allogenic encapsulated islet
transplantation. Insufficient biocompatibility of the capsules
is usually considered to be the main cause...." Recently
additional methods for purification of alginates have been
reported3, as well as methods for analysis of the
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biocompatibility (and "bioinvisibility" as defined in patent
No. 5,855,613) of alginates4.
Coverage of 100 of Cells
Some devices do not cover the entire islet surface. If even a
small bit of the islet is uncovered, macrophages can infiltrate
and destroy the entire islet. The cellular attack and
destruction sensitize the immune system, leading to a humoral
(antibody) response, which may then destroy even those cells
that are completely covered. Thus, complete coverage of all of
the islets is required to protect the islet cells from both
cellular and humoral immune responses.
Previous devices have failed to ensure complete coverage of all
islets while at the same time meeting the dimensional
constraints imposed by oxygen requirement. This problem is
increasingly recognized; for example, Webber et a1.5 observe:
"We observed that a small percentage (2-5~) of the
microcapsules are obviously defective, being misshapen, oblong
or fractured, immediately after their preparation. In
addition, we have found that islets or islet cells are either
attached to or embedded in the microcapsule's membrane.... We
have postulated that these microcapsule defects may allow islet
exposure and host sensitization to the graft, as well~as
reducing durability of the microcapsule's membrane."
Nutrient Diffusion and Bioartificial Implant Dimensions
A successful bioartificial implant must have dimensions that
permit sufficient diffusive flux of nutrients into the implant
and secretion of bioactive agents out of the implant. Yet the
vast majority of bioartificial implants described in the
literature have dimensions too large to permit sufficient
diffusion. The nutrient limiting cell viability and
functionality in bioartificial organs is usually oxygen. The
oxygen available at the center of the bioartificial organ
(where oxygen is at its lowest concentration) is governed by
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the density of oxygen consuming tissue in the bioartificial
pancreas, the geometry of the device, diffusivity of oxygen
through the bioartificial organ and oxygen tension in the
surrounding tissuess.
Further evidence supporting the importance of geometry and
surrounding oxygen tension to the survival of islets has been
reported. For instance, Benson et al'. reported that cells of
the mouse insulinoma line (3TC3 "proliferate while they are
entrapped in . . . alginate beads. During this process, cell
aggregates develop in the bead periphery, which increase in
number and size with time," supporting the concept that the
higher available oxygen at the surface is crucial for cell
growth. Papas et ale. report "Encapsulated cells may
experience hypoxic conditions postimplantation as a result of
one or more of the following: the design of the construct; the
environment at the implantation site; or the development of
fibrosis around the construct.... Results show that, upon
decreasing the oxygen concentration in the surrounding medium,
the encapsulated cell system reached a new, lower metabolic and
secretory state." Davalli et a1.9 report a dramatic decline in
beta cell mass following islet implantation into nude mice and
say that "anoxia may be a major factor."
Other studies have attempted to improve oxygen available to
cells in the bioartificial organ by electrochemically
generating oxygen in situl°, inducing a vascular bed in the
adjacent tissue with VEGF11, and by increasing the diffusivity
of oxygen by addition of perfluorocarbonl2.
Cell Density
The importance of high cell density in a practical
bioartificial organ is now increasingly recognized. As Suzuki
et a113. commented:
"These results strongly suggest that ways must be
found to improve the packing density of
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macroencapsulation devices . . . [T]he surface area
of ,,the membrane required to support a given mass of
islet tissue is so high as to be impractical for
surgical implantation."
Delaunay et a114. made much the same point:
"The design of a geometry to accommodate the number
of islets necessary to treat diabetes in larger
mammals is certainly one of the major challenges to
be overcome in the development of a bioartificial
pancreas . . . "
Calafiore's groupls also recognized the seriousness of this
problem ("it was, in fact, found that a viable human islet cell
quantity . . . would take, upon microencapsulation [in 700 pm
microcapsules] a final graft volume of approximately 180 mZ,
thereby creating quite serious technical implant problems") and
worked to make much smaller microcapsules.
Cellular Trophies Factors
Islet cell mass, viability and functionality may be improved
and enhanced by adding cells and/or other substances in the
bioartificial implant composition. Recent publications include
various trophic cells and substances in the core of the
bioartificial organ to enhance the viability and functionality
of the cells, including Sertoli Cellsls.
Use of Growing Cell Lines
Islet and other primary cells are characterized by very slow
rates of cell division, usually limited to replacement of cells
that die naturally. Embodiments of the thin sheets described
herein can optionally be made that comprise rapidly dividing
cell lines entrapped in rigid capsules, for example alginate-
poly-lysine. This can be done, for example, by the method of
Cochrum, Dorian and Jemtrudl~.
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Previous Approaches
Numerous bioartificial implants have been described; most of
these were discussed in patent, No. 5,855,613. The following
discussion is limited to passive diffusion type implants each
containing multiple islets, and does not include individually
encapsulated islets or vascular devices.
Zanza and Chickl8 summarized the state of the art:
Despite encouraging results [with diffusion-based
chambers], a number technical and safety
issues... must be addressed....includ[ing] long-term
biocompatibility, membrane breakage, and suitability
for retrieval.
Several publications described further research on devices
discussed in patent No. 5,855,613, including work by Gellerl9,
Ohgawara2°, Suzukil3, Tatarkiewicz2l and Trivediz2.
The Ohgawara paper greatly expanded the information available
on the device compared with the earlier paper cited in patent
No. 5,855,613. The planar chamber is 40 mm diameter and 5 mm
thick, fabricated from two membranes made by Nucleopore Corp.
The tissue density reported was 8X106 cells/1.5 mZ. Although
the paper claims that~there is no fibrosis, the relevant
micrograph (Fig. 7) shows a fibrotic mass about 40-50 um thick.
Suzuki et a123. reported on the Baxter double-membrane design,
but in greater detail than ever before. The Suzuki paper
described a flat device substantially similar to the Scharp
device (from the same supplier, Baxter) discussed in the prior
patent.
Tt should be noted that many of these devices fail because the
polymeric membranes employed significantly reduce oxygen
diffusivity and are not amenable to supporting high densities
of entrapped cells even when thickness is minimal.
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Since the review in patent No. 5,855,613 only four new sheets
or planar diffusion systems have published, from Baetge,
Dionne, Tatarkiewicz and Usala.
Baetge et a1.~4 described a flat sheet sealed double-membrane
with loading port. Each membrane was 100 dun thick with a 200
~.un core, and thus a total sandwich of 400 utn thickness. The
disc was 10mm diameter. Thus, with 3.5 X 106 cells the packing
efficiency in the core is 300, or 15o in the total sheet.
Dionne25 describes imunoisolatory vehicles with a core and
permselective jacket, including flat sheets. The methods
described may be distinguished from the present invention in
that the flat sheet fabrication method of Dionne does not work
with alginate. In addition, in the method of Dionne the
membranes are not laminated to the core allowing changes in
dimensions post implant.
Tatarkiewicz at al.2s describe a polylysine coated alginate
slab, 1mm thick, 0.8 cm2. They report that only 4000 islets
survive when up to 8000 are incorporated. The calculated
tissue density is 80.
Usala2' describes an implant in the form of a plate with
multiple wells, each filled with a cell-collagen matrix, the
entire implant coated with poly-para-xylene.
U.S. Patent No. 5,855,613 discloses making the bioartificial
implant using a series of molds constructed with frit materials
that can be molded or milled, and membranes. This allows
diffusion of chelating agents (e.g., sodium citrate) or
multivalent ration gelling agents {e.g., calcium, barium or
strontium chlorides) to liquify and gel, respectively, the
core, coat and overcoat. The manufacturing process makes use
only of materials known to be compatible with the implanted
cells. The shape of the core, coat and overcoat are molded
while the alginate is liquid. The cross-linked core and coats
(and later the coat and overcoats) are bonded together by
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simply contacting the cross-linked surfaces with a small amount
of chelating agent (e. g., sodium citrate). The chelating agent
diffuses into the gelled layer and partially liquefies it. The
layers are brought into contact with each other. When a
cationic cross-linking agent (e.g., calcium, barium or
strontium chlorides) is subsequently added, a tight bond is
formed between the layers. The outer surface is made very
smooth through the simple step of wetting the mold with cross-
linking agent solution before contacting the mold with the
cross-linked coat or overcoat. This assures that the outer
surface is as smooth as the mold surface, limited only by
machining of the frit.
Notes
1. C. Ricordi, Diabetes Reviews 4, 356-3~9 (1996).
2. Van Schilfgaarde, R. and P. De Vos (1999). Factors influencing
the properties and performance of microcapsules for
immunoprotection of pancreatic islets. Journal of Molecular
Medicine 77: 199-205.
3. T. D. Zekorn, et al., Int J Artif Organs I9, 251-7 (1996).
H. A. Clayton, R. F. L. James, N. J. M. London, U.S. Patent
5,529,913 (1996).
G. Skjak-Braek, T. Espevik, M. Otterlei, O. Smidsrod, P. Soon-
Shiong, U.S. Patent 5,459,054 (1995).
Dorian, Randy, Ed., Islet Sheet Medical Useful Methods,
http://www.isletmedical.com/meth0102 (March 31, 1999).
Dorian, Randy, Ed., Islet Sheet Medical Useful Methods,
http://www.isletmedical.com/meth0202 (March 31, 1999).
Van Schilfgaarde, R. and P. De Vos (1999). Factors influencing
the properties and performance of microcapsules for
immunoprotection of pancreatic islets. Journal of Molecular
Medicine. 77: 199-205.
4. M. Maniyama, et al., Cell Transplantation 8, 176 (1999).
S. Arita, et al., Transplantation Proceedings 29, 2125 (1997).
P. Petruzzo, et al., Transplantation Proceedings 29, 2129-2130
(1997).
8

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5. Weber, C. J., S. Safley, M. Hagler and J. Kapp (1999).
Evaluation of graft-host response for various tissue sources
and animal models. Annals of the New York Academy of Sciences
875: 233-254.
6. E. S. Avgoustiniatos, C. K. Colton, Ann N Y Acad Sci 831, 145
(1997).
"Oxygen diffusion limitations in tissue in vivo are far more
severe than for glucose.... whereas oxygen supply limitation
are always serious and are the focus of this paper.... The
effect of...external mass transfer resistences resulting from
the presence of immunoisolation membranes and surrounding host
tissue... can be substantial."
Goosen, M. F. A. (1999). Physico-chemical and mass transfer
considerations in microencapsulation. Annals of the New York
Academy of Sciences 875: 84-104.
7. J. P. Benson, K. K. Papas, I. Constantinidis, A. Sambanis,
Cell Transplantation 6, 395-402 (1997).
8. Papas, K. K., R. C. Long, Jr., A. Sambanis and I.
Constantinidis (1999). Development of a bioartificial
pancreas: II. Effects of oxygen on long- term entrapped
betaTC3 cell cultures. Biotechnology and Bioengineering 66:
231-237.
~9. A. M. Davalli, et al., Transplantation 59, 817-20 (1995).
10. C. K. Colton, et al., Cell Transplantation 8, 212 (1999).
11. Trivedi, N., G. M. Steil, S. Bonner-Weir and G, C. Weir
(1999). Improved Vascularization of.Planar Diffusion Devices
Following Continuous Infusion of Vascular Endothelial Growth
Factor (VEGF). Cell Transplantation. 8: 175.
Trivedi, N., G. M. Steil, C. K. Colton, S. Bonner-Weir and G.
C. Weir (2000). Improved Vascularization of Planar Membrane
Diffusion Devices Following Continuous Infusion of Vascular
Endothelial Growth Factor. Cell Transplantation. 9: 115-124.
12. Z. Inverardi, C. Fraker, M. Mares-Guia, C. Ricordi, Cell
Transplantation 8, 176 (1999).
13. K. Suzuki, et al., Transplantation 66, 21-28 (1998).
14. C. Delaunay, et al., Artificial Organs 22, 291-299 (1998).
15. R. Calafiore, et al., Transplant Proc 28, 812-3 (1996).
16. H. P. Selawry, U.S. Patent 5,843,430 (1998).
17. K. Cochrum, S. Jemtrud, R. Dorian, Transplant proc 27, 3297-
3301 (1995).
9

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K. C. Cochrum, R. E. Dorian, S. Jemtrud, U.S. Patent
5,578,314 (1995).
R. E. Dorian, R. D. Antanavich, K. C. Cochrum U.S. Patent
5643594 (1997).
R. E. Dorian, K. C. Cochrum U.S. Patent 5,693,514 (1997).
R. E. Dorian, K. C. Cochrum U.S. Patent 5,656,468 (1997).
R. E. Dorian, K. C. Cochrum U~.S. Patent 5,639,467 (1997).
18. R. P. Lanza, W. L. Chick, Ann N Y Acad Sci 831, 323-31 (1997).
19. R. L. Geller, T. Loudovaris, S. Neuenfeldt, R. C. Johnson, J.
H. Brauker, Ann N Y Acad Sci 831 (1997).
20. H. Ohgawara, S. Hirotani, J. I. Miyazaki, S. Teraoka,
Artificial Organs 22, 788-794 (1998).
S. Hirotani, H. 0hgawara, Cell Transplantation 7, 407-410
(1998) .
21. K. Tatarkiewicz, et al., Transplantation 67, 665-671 (1999).
22. N. Trivedi, G. M. Steil, S. Bonner-Weir, G. C. Weir, Cell
Transplantation 8, 175 (1999).
23. K. Suzuki, S. Bonner-Weir, J. Hollister, G. C. Weir, Cell
Transplant 5, 613-25 (1996).
Colton, C. K., H. Wu, E. Avgoustiniatos, L. Swette, S. Bonner-
Weir and G. S. Weir (1999). Enhanced oxygen supply to tissue
in planar immunobarrier devices by in situ electrochemical
oxygen generation. Cell Transplantation. 8: 212.
Trivedi, N., G. M. Steil, S. Bonner-Weir and G. C. Weir
(1999). Improved Vascularization of Planar Diffusion Devices
Following Continuous Infusion of Vascular Endothelial Growth
Factor (VEGF). Cell Transplantation 8: 175.
Trivedi, N., G. M. Steil, ~. K. Colton, S. Bonner-Weir and G.
C. Weir (2000). Improved Vascularization of Planar Membrane
Diffusion Devices Following Continuous Infusion of Vascular
Endothelial Growth Factor. Cell Transplantation. 9: 115-124.
Wu, H., E. S. Avgoustiniatos, L. Swette, S. Bonner-Weir, G. C.
Weir and C. K. Colton (1999). In situ electrochemical oxygen
generation with an immunoisolation device. Annals of the New
York Academy of Sciences 875: 105-125.
24. E. E. Baetge, Cell Transplantation 8, 164 (1999).
25. K. E. Dionne, et al., U.S. Patent 5,798,113 (1998).
K. E. Dionne, et al., U.S. Patent 5,874,099 (1999).

CA 02421036 2003-02-26
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K. E. Dionne, et al., U.S. Patent 5,869,077 (1999).
K. E. Dionne, et al., U.S. Patent 5,834,002 (1998).
K. E. Dionne, et al., U.S. Patent 5,800,829 (1998).
K. E. Dionne, et al., U.S. Patent 5,800,828 (1998).
26. K. Tatarkiewicz, E. Sitarek, M. Sabat, T. Orlowski, Transplant
Proc 28, 831-2 (1996).
K. Tatarkiewicz, et al., Transplantation 67, 665-71 (1999).
K. Tatarkiewicz, et al., Cell Transplantation 8, 212 (1999).
Some data included in the table was presented during the
poster session and was not included in the abstract.
27. A.-Z. Usala, U.S. Patent 5,830,492 (1998).
A.-Z. Usala, U.S. Patent 5,824,331 (1998).
A.-Z. Usala, U.S. Patent 5,834,005 (1998).
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Summary of the Invention and Ob-iects
In one embodiment of the present invention, a method is
provided for making a physiologically active and biocompatible
cellular implant for implantation into a host body. The method
includes the steps of:
(a) forming first and second layers of first and second
polymer solutions, respectively, each layer having a first
substantially uncross-linked surface and an opposing second
cross-linked surface,
(b) forming a sandwich of a cell suspension layer
comprising a cell suspension layer of physiologically active
cells in a substantially uncross-linked third polymer solution
between said first and second layers at their respective first
surfaces so that their respective interfaces are in liquid
Z5 form, and
(c) diffusing cross-linking agent in a direction from
the second surface of one or both of said first or second
layers to the cell suspension layer until the first and second
polymer solutions of said first and second layers,
respectively, and said cell suspension layer are cross-linked
at their respective interfaces, thereby forming a cellular
implant.
In another embodiment, the method includes the steps of
(a) forming a first layer of a first substantially
uncross-linked polymer solution of predetermined thickness by
substantially uniformly spreading said first polymer solution
on a solid support surface, said first layer having an exposed
surface,
(b) forming a cell suspension second layer of
predetexmined thickness on said first layer exposed surface,
said cell suspension comprising physiologically active cells in
a second substantially uncross-linked second polymer solution,
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by substantially uniformly spreading s aid cell suspension over
said first layer exposed surface, and
(c) forming a third layer of a third substantially
uncross-linked polymer solution on said cell suspension second
layer by substantially uniformly spreading said third polymer
solution on said cell suspension second layer.
We have invented rapid and convenient methods for fabrication
of a bioartificial implant of dimensions never before achieved,
with many attendant advantages. For example, objects of the
present invention (shared with the previous invention) include
a bioartificial implant in a thin sheet configuration that:
~ is easily retrievable from the host;
~ permits high tissue densities;
~ permits diffusion to the tissue cells of the amounts of
nutrients, oxygen and other substances required for
cellular health, longevity and effective function after
implantation;
~ comprises viable, physiologically active, tissue cells
for implantation in a device which is physiologically
acceptable to the host and which effectively provides
prolonged protection of the tissue cells, after
implantation, from destruction by the host immune system;
~ may contain a mesh or support polymer to improve physical
properties of the sheet;
~ may contain trophic agents such as nurse cells,
nutrients, hormones or oxygen carriers to support the
cellular health, longevity and effective function of the
implant after implantation.
An object of the present invention is to ease scaling up
methods for fabrication of larger sheets. A single simple
13

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device may be used to make sheets ranging from very small up to
a limit imposed only by the spacing of shims.
Another object of the present invention is to reduce the
processing time for making a bioartificial implant. Prior
methods relied on many steps to liquify gelled alginate, time
for the liquids to diffuse, then addition of chelating agent to
gel the alginate again. The new process does not contain any
chelation step.
Another object of the present invention is to fabricate the
coat and overcoat of a bioartificial implant in a single step.
Optionally, an overcoat may be added in an additional step.
Another object of the present invention is incorporation of
rapidly dividing cells into thin sheets without risk of the
cells bursting out of the sheet.
Another object of the present invention is to control the
thickness of the coat/overcoat by physicochemical means, not
relying on a mold. This is accomplished by controlling the
composition of the first polymer and cross-linking solution so
that the layers are substantially cross-linked to a depth of
only several microns or by sweeping a straightedge over shims.
The methods of the present application allow for excellent
cross-linking between layers, smoother outer surface, easier
scale up and more rapid sheet fabrication.
Cells or tissues are entrapped within a core of gel. The core
is laminated and firmly bonded to thin coating and overcoating
gel layers) which do not contain cells or tissue. Sheets of
several polymers are possible. For reasons of biocompatibility
and convenience of fabrication the hydrogel alginate is
preferred.
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Detailed Description of the Preferred Embodiments
The utility of this invention is not limited to encapsulation
of the islets of Zangerhans in a sheet ("islet sheet"). Any
cell that secretes a substance with therapeutic value may be
used. For example, primary parathyroid cells may be used to
treat parathyroid hormone deficiency, or erythropoietin
secreting cells may be used to treat anemia or cytokine
secreting cells may be used to modulate immunity. Another form
of utility would be encapsulation of cells that transform or
metabolize substances found in the body. For instance, hepatic
cells may detoxify toxic compounds, or cells may be used to
oxidize compounds such as ethanol when they are present in
undesirable amounts. For all such uses cells may be, for
example, primary cells, cultured cells, or genetically
engineered cells. Mammalian and non-mammalian cells including
prokaryotes may be used.
The dimensions of the bioartificial implant are such that cell
viability may be maintained by passive diffusion of nutrients,
and preferably, such that a high cell density can be
maintained. The dimensions of the bioartificial implant are
also such that the bioartificial implant is macroscopic and is
easily retrievable from the host and is large enough to contain
a significant fraction of the tissue required to achieve the
desired therapeutic effect. Such high cell density makes
practical surgical use of bioartificial organs possible.
The permeability of the bioartificial implant is such that
passive diffusion of secreted cell products permits rapid
response to changing physiological conditions. At the same
time, the permeability of the membrane~sufficiently impedes
diffusion of antibody and complement to prevent killing of the
implanted cells, even when the tissue is a xenograft or the
host is presensitized to the implant tissue.
The bioartificial implant is biocompatible, meaning it produces
minimal foreign body reaction. We have found that only

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implants that are neutral (causing neither fibrosis nor
neovascularization) have been shown to, last over a year with
minimal decay of function.
In one preferred embodiment, the dimensions of the present
bioartificial implant, when in a thin sheet configuration, are
such that the surface area of a side of a sheet is at least 30
mmz, preferably at least 2.5 cma and more preferably at least 10
cma, as defined by either (a) the diameter (if the sheet is
circular) or (b) the area determined by the method of
converging polygons. Although the maximum dimensions can be
those which are tolerated by the patient into whom the implant
is placed, for ease of fabrication and economy of implanted
cells, a suitable surface area of a face of the present thin
sheet implant may be 400 cm~, more preferably 300 cm2 and most
preferably 250 cm2 (for a human patient with type 1 diabetes).
(A smaller sheet would be sufficient for a more potent hormone
such as erythropoietin.)
In the bioartificial implant the cell density is that which can
be contained within the entire implant. Preferably, the cell
density is at least 100, more preferably 20o and most
preferably at least 30o by volume.
The bioartificial implant is sometimes described using the
terms "core," "coat" and "overcoat." The core comprises the
living tissue, optional trophic factors or nurse cells,
alginate polymer cross-linked with a multivalent canon such as
calcium, and an optional support polymer such as collagen or
fiber mesh for strength. The coat comprises alginate polymer
(optionally of different chemical composition) cross-linked
with a multivalent cation that partly serves to control
permeability. The optional overcoat comprises alginate polymer
(optionally of different chemical composition) cross-linked
with a multivalent cation that serves to render the
bioartificial implant biocompatible. Use of polymers of
different chemical composition may require other methods for
16

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cross-linking, such as covalent bonding or phase change by
cooling.
Previous approaches have responded to inherent limitations on
diffusion of oxygen by lowering cell densities within the
bioartificial implant. We have found that an effective implant
must have medium to high tissue densities to minimize the
volume of the total bioartificial implant, and that the
thickness of a sheet or slab must be very small to permit
effective oxygen diffusion. The sum of the core, coat and
overcoat thicknesses preferably is less than 500 um, more
preferably 350 um or less, and most preferably no more than 300
um. The coat and optional overcoat thickness preferably is
minimized so that the tissue quantity may be maximized. We
have found that preferable coat and overcoat thicknesses may be
from 5 to 100 um thick, more preferably from 10-80 um, and most
preferably from 10-50 um. The length and width of the
bioartificial implant on the other hand preferably is maximized
to permit the greatest possible volume of living tissue t~ be
included in the bioartificial implant and to permit easy
retrieval but not so large as to be surgically impractical.
The thin sheet bioartificial organ made according to the
present invention typically includes an implant core having a
thin sheet configuration comprising viable, physiologically
active tissue or cells and a cross-linked alginate gel and
optionally, trophic factors and nurse cells, and optionally, a
fiber mesh support, being completely covered by an acellular
biocompatible coat and optional overcoat of alginates. The
alginates are preferably free from fibrogenic concentrations of
impurities. The bioartificial implant may have a coat and
overcoat to control permeability and enhance biocompatibility.
The implant sheet is thin and may be permeable enough to
provide a physiologically acceptable oxygen tension at the
center of the sheet when implanted in a suitable site in a
human or animal subject. The thinness and permeability of the
implant allow diffusion of nutrients, especially oxygen,
metabolic waste products and secreted tissue products. The
17

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implant preferably inhibits diffusion of antibody and
complement.
Tn accordance with the present invention, improved methods are
provided for making thin sheet bioartificial implants without
the necessity of a series of molds constructed with frit
materials or membranes that allow diffusion of chelating agents
to liquify the gelled core, coat and overcoat. The use only of
gelling agents and ions known to be compatible with the
implanted cells has been retained. However, the use of
membranes that allow diffusion of gelling agents to gel the
core, coat and overcoat has been made optional, with diffusion
of gelling agents to gel the core, coat and optional overcoat
now possible from a single solution.
The use of a polymer which can be reversibly gelled is no
longer required. This allows selection from a wider range of
polymers.
Although many variations of the method exist, the essence of
the process is simple. The shapes of the core, coat and
overcoat are molded while the polymer solutions are uncross-
linked and in a flowable liquid form preferably at a viscosity
less than that of a gum but more viscous than water (e. g., from
about 100 to 100,000 centipoise). The coat can be formed by
spreading as by sliding a straightedge over two shims to form a
liquid layer of uniform thickness. The liquid core can be
formed by gently suspending cells into a second liquid polymer
solution and sweeping this suspension over the first.
Preferably the first polymer solution is more viscous than the
second. The second layer is formed with thicker shims defining
its thickness. In one embodiment, the layered sheet can be
formed and the core and coat are simultaneously cross-linked by
simply contacting the two liquid polymer solutions formed in
the desired dimensions using a system of shims and
straightedges, then adding a cross-linking agent to form a
tight bond between the layers.
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The outer surface can be made smooth during the coat formation
step through the simple means of gelling the outer surface of
the coat by first wetting a permeable membrane on which it is
formed with cross-linking agent solution or by leaving the coat
liquid against a smooth impermeable mold until the final cross-
linking step. The thickness of the thin sheet is easily
controlled through the use of an impermeable mold of one or two
surfaces with shim spacers.
An important feature of these new methods is that it is no
longer necessary to apply coat and overcoat in separate steps.
~nle have found that a single step produces a coat that
. completely covers the cells in the core, inhibits diffusion of
complement, and has a biocompatible surface free of fibrogenic
properties.
Another important feature of these new methods is that the
outer surface can be made smoother and thus more biocompatible.
The new methods do not require the manufacture of custom frit
molds for each sheet size and geometry.
Due to their simplicity and the small number of steps the new
methods form finished sheets rapidly with less trauma to cells
and less change in the cellular environment.
Brief Description of Drawings
A more complete appreciation of the invention and many of the
attendant advantages thereof will be readily obtained as the
same~becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
Fig. 1 shows the apparatus for the optical glass casting
method.
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Fig. 2 is a diagram of the optical glass casting method showing
initial diffusion of calcium ions into the layered liquid
alginate and islets.
Fig. 3 is a diagram of the optical glass casting method showing
complete diffusion of calcium ions into the Islet Sheet.
Fig. 4 shows the apparatus of the hydrophilic membrane method
for making sheets at the beginning of a cycle.
Fig. 5 shows the apparatus of the hydrophilic membrane method
for making sheets as alginate for the coat/overcoat layer is
applied.
Fig. 6 shows the apparatus of the hydrophilic membrane method
at the addition of the core, cells suspended in alginate.
Fig. 7 shows the apparatus of the hydrophilic membrane method
closing.
Fig. 8 shows the apparatus of the hydrophilic membrane method
completely closed.
Fig. 9 shows the apparatus of the hydrophilic membrane method
as the finished sheet is withdrawn wrapped in a protective
membrane, about to be transferred to a bath of cross-linking
2 0 agent .
Fig. 10 is a diagram of the hydrophilic membrane method; the
membrane is prepared by soaking in a solution of calcium
gluconate.
Fig. 11 is a diagram of the hydrophilic membrane method: the
membrane is covered with a layer of liquid sodium alginate at
the instant of application.

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Fig. 12 is a diagram of the hydrophilic membrane method;
calcium diffuses from the membrane into the liquid alginate,
gelling the alginate.
Fig. 13 is a diagram of the hydrophilic membrane method; two
membranes layered with partially gelled alginate squeeze a
dollop of liquid alginate containing cells or islets.
Fig. 14 is a diagram of the.hydrophilic membrane method; the
cell/alginate suspension is flattened and the liquid alginate
from the membrane layer is displaced by the advancing liquid
alginate from the cell dollop (arrows).
Fig. 15 is a diagram of the hydrophilic membrane method; at the
completion of the squeezing process the sheet is of uniform
thinness and the liquid portion of the membrane alginate has
been displaced from view.
Fig. 16 is a diagram of the hydrophilic membrane method; the
calcium diffuses in through both membranes and gels all the
liquid alginate into a uniform sheet.
Fig. 17 is a photograph of a thin sheet comprising erythrocytes
encapsulated in alginate. The U.S. quarter dollar coin gives
scale.
Fig. 18 is an electron micrograph of the surface of an islet
containing thin sheet. The surface is smooth. The bumps show
that the surface of the sheet is convex over the islets.
Fig. 19 is an electron micrograph of the surface of a thin
sheet reinforced with a non-woven fabric. The sheet is
fractured to reveal the fabric.
Fig. 20 is a micrograph of an Islet Sheet that has been
retrieved after 2'~ months sutured on to the omentum of a dog.
The islets are stained with dithizone.
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Fig. 21 is a photograph of an Islet Sheet as it is being
sutured on to the omentum of a diabetic dog described in
Example 8.
Fig. 22 is a chart showing the blood sugars of a canine
allograft described in Example 8.
Fig. 23 is a chart showing intravenous glucose tolerance tests
of the canine allograft described in Example 8.
In one embodiment of the present invention, termed "the casting
method," a sheet is fabricated as illustrated in Figs. 1-3.
Suitable apparatus for this method illustrated in F~.g. 1,
included a flat plate (40), preferably with the smoothness of
optical glass, shims (41), a straightedge for sweeping
solutions and suspensions (43), dispensers for solutions and
suspensions (42) and for dispensing calcium solutions (44).
Figs. 2 and 3 show the beginning and final stages of diffusion
of calcium ions into the sheet.
In the first step of the casting method, a first substantially
uncross-linked polymer solution is formed by substantially
uniformly spreading the polymer solution onto a solid support
surface, preferably one that is flat and smooth, e.g., optical
quality glass. A suitable polxmer solution is a substantially
uncross-linked soluble alginate salt (such as sodium alginate
or another monovalent cation salt) in a viscosity (e. g., less
than about 50,000 centipoise) which permits it to be readily
flowable and spreadable in contrast to a hydrogel (which is
cross-linked). The polymer solution (42) is spread as a thin
layer on a smooth surface, such as optical glass (40). A
convenient means of sweeping out this and the subsequent layers
described below is by spreading the alginate across the smooth
surface with a straightedge (43) while controlling the
thickness by means of guide shims (41). These outer layers
should be less than 100 ~.un so that oxygen flux is not unduly
impeded. Optionally, a reinforcing fabric or membrane is
22

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placed on top of the layer (e.g., of the type illustrated in
U.S. Patent No. 5,855,613).
In the second step of the casting method, a cell suspension of
physiologically active cells in a substantially uncross-linked
second polymer solution is substantially uniformly spread over
the exposed layer of the first polymer solution. Suitably, the
suspension of cells (such as islets or other cells) dispersed
in alginate solution is spread across the surface of the layer
(or layer overlayed with fabric) using a straightedge and
thicker spacing shim, typically 100-200 ~.un thicker than the
first shim. In one embodiment, the alginate used for
suspension of particulates is of a lower viscosity (e.g., at
least about 50 less) than the underlying alginate layer to
minimize disturbing the bottom layer while sweeping out the
particulate suspension. Optionally, a reinforcing fabric or
membrane can be placed on top of the spread particulate
suspension.
In the third step of the casting method, another layer of a
third substantially uncross-linked polymer solution (e. g.,
sodium alginate) is spread over the top of the cell-containing
layer (or cell-containing layer and reinforcing fabric) with a
straightedge (43) using a shim, typically less than 100 ~.lm
thicker than the combined thickness of the prior layers. The
over-laying alginate solution can optionally be of a lower
viscosity than that of the particulate suspension to minimize
disturbance of the suspension during sweeping out of the
topmost layer.
The cross-linking solution can be spread across the surface
with a straightedge (43) guided by thick shims or allowed to
pool freely. The entire laminate can be cross-linked by
submerging in a solution of cross-linking ions (e.g., a
multivalent ion such as calcium) (44) to cross-link the polymer
(alginate). Calcium from the overlying liquid quickly diffuses
into the layers (Fig. 2) and gels the entire sheet (Fig. 3).
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After cross-linking, the implant can be removed from the
support surface.
The entire sheet may be strengthened by immersion in a solution
of barium chloride. Barium exchanges with calcium in the
sheet, and the stronger barium-alginate bonds result in a sheet
that is physically stronger.
The cell-containing layer of alginate is preferably constrained
to a smaller area than the under- and over-lying layers so that
the edges of the device will be devoid of cells.
The reinforcing fabric or membrane is preferably constrained to
an area smaller than the under- and over-lying layers and
larger than the cell-containing layer so that it may be used as
a surgical cuff without any danger of exposing cells in the
cell-containing layer during suturing.
As used herein, the term "cells" includes cells or
physiologically active tissue. Such cells are washed in
isotonic buffer solution lacking cross-linking agent and
resuspended in a soluble polymer solution.
Another embodiment of the invention, termed "the partially pre-
cross-linked method" is illustrated in Figs. 4-9.
Suitable apparatus to perform this method includes two flat
plastic plates (51) supporting two smooth flat glass plates,
e.g., f~rmed of optical glass (52). The plastic plates are
hinged (53) so that, when the hinge is closed, the flat plates
(52) are held apart, preferably spaced equidistant across their
facing surfaces by spacers or shims (54).
In the first step of the partially pre-cross-linked method, two
layers of polymer solutions are formed each having one cross-
linked surface adjacent a support surface and an uncross-linked
opposed surface. This can be performed with a pair of smooth,
absorbent membranes (or a single, folded membrane 55) wetted
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with a solution containing cross-linking agent, such as calcium
(see Fig. 10). The membranes may be gel films cast from PEG,
polyacrylamide, polysulfone, agarose or other polymers. The
wet membranes are laid out onto rigid flat surfaces, such as
glass optical windows (52). Excess liquid is blotted or
displaced from the membrane surfaces.
The flat surfaces are fitted with spacers (shims or rails) (61)
which serve as guides to control thickness of a polymer (e. g.,
alginate) layer to be spread over the surfaces of the
membranes. A liquid solution of polymer, such as sodium
alginate, is applied from an impermeable depot resting atop one
end of the membranes (61) and spread across the membranes using
a straightedge knife (62) guided by the shims (61). (In Fig. 5
the depot and the shims are a single rectangular mask 61.)
As the polymer contacts the wet membrane surfaces (see Fig.
11), a smooth skin of cross-linked gel is rapidly formed at the
interface by interaction of the polymer with the cross-linking
solution. Gradually over the course of seconds to minutes, the
cross -linking agent diffuses from the membrane into the polymer
solution, interacting with the latter and producing a front of
gelation (see Fig. 12). The degree of cross-linking of the
polymer layers decreases in a moving or kinetic gradient from
the surface contacting the membranes outward, and is dependent
on the composition of the cross-linking solution. Parameters
which effect the rate of cross-linking include concentration,
counterion, viscosity, and temperature. For example, a dilute
solution of a weakly dissociable salt of cross-linker in a
viscous solution will result in slower diffusion into the
liquid polymer.
By appropriate selection of the membrane thickness and
porosity, the composition of the polymer solution, composition
of the cross-linking solution and the concentration of the
cross-linker in the cross-linking solution, conditions can be
established whereby within a minute or so, the polymer layers
are substantially cross-linked to a depth of only several

CA 02421036 2003-02-26
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microns. For example, using Gelman 0.2 micron cellulose
nitrate filter membranes soaked in a solution of 4.630 sucrose,
3.25 calcium gluconate and 10 mM HEPES, pH 7.0, substantially
complete cross-linking of a neutral 4~ solution of moderate
molecular weight, mannuronate-rich sodium alginate in isotonic
sucrose extends to about 30 or 40 microns from the membrane
surface, regardless of the total depth of the alginate layer.
Under these specific conditions, calcium-binding uronate
residues are in excess of available calcium, and further
progression of the gel front is governed by calcium exchange
between gelled and liquid alginate. This is a relatively slow
process because the gelled calcium alginate does not diffuse
and diffusion of liquid alginate is slow by virtue of its high
molecular weight. Thus, there is a comfortable window of time
during which the layer exists in this particular state.
This process may be adapted to polymers that gel as a result of
a physical process rather than a cross-linking agent, for
example, gelation of a liquid that cools to its gel point.
The next step in the partially pre-cross-linked method is to
form a sandwich of an uncross-linked cell suspension layer
(e.g., of the type previously described) sandwiched between the
exposed uncross-linked surfaces of the two partially pre-cross-
linked polymer layers. In one embodiment, the suspension is
deposited onto one of the two partially cross-linked
overcoating polymer layers (by syringe 71). The other
partially cross-linked layer is lowered on top of the
suspension (81), contacting it, and is pressed down until the
spacer shims which control the distance between the two
membrane surfaces (Fig. 8) collide. As the cell suspension is
flattened between the opposing layers, the soluble polymer
component of said layers is displaced radially (91); see Figs.
13 through 15. The gelled and highly viscous, partially gelled
polymer components of the layers are unperturbed because of
their cross-linked, macromolecular structure, so that the
suspended cells are substantially uniformly separated from the
membranes by a layer of gelled polymer, the thickness of which
26

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is determined by the extent of cross-linking prior to collision
of the shims.
The sandwich consisting of membrane-polymer layer-cell
suspension-polymer layer-membrane may then be slid out from
between the two rigid, flat surfaces (Fig. 9). This operation
can be facilitated by infusing cross-linking solution between
the flat surfaces around the sandwich, for instance by using a
syringe or suitable device for applying the solution to the
space between the surfaces. The presence of a solution around
the membrane sandwich both continues the cross-linking process
and lowers the surface tension between the damp membranes and
the flat surfaces.
The sandwich may then submersed in cross-linking solution (92)
and incubated for sufficient time to ensure that polymer is
cross-linked entirely throughout its thickness. Alternatively
the cross-linking agent can be infused solely between the flat
surfaces, or through semipermeable flat, rigid surfaces such as
glass frits. The membranes are removed from the cell-
containing multilayered polymer sheet and the sheet is
equilibrated in an appropriate buffered salt solution or
nutrient medium, for example, HEPES buffered normal saline with
5 mM CaCl2, to prepare it for tissue culture or surgical
implantation.
The entire sheet may be strengthened by immersion in a solution
of, e.g., barium chloride. Barium exchanges with calcium in
the sheet, and the stronger barium-alginate bonds result in a
sheet that is physically stronger.
Figs. 10-16 illustrate formation of a sheet by the partially
pre-cross-linked method (the hydrophilic membrane method).
Fig. 10 shows a porous membrane (101) prepared by soaking in a
solution of a multivalent canon salt cross-linking agent
(e. g., calcium gluconate). Fig. 11 shows the membrane (101)
covered with a layer of liquid sodium alginate (102) at the
instant of application before any calcium has diffused out of
27

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the membrane. Fig. Z2 shows calcium diffusing from the
membrane (101) into the liquid alginate (102), gelling the
alginate; gelling is complete near the membrane. The amount of
calcium in the membrane is chosen so that the alginate remains
liquid at a distance from the membrane. Fig. 13 shows two
membranes (101) layered with partially gelled alginate (102)
beginning to squeeze a dollop of liquid alginate containing
cells or islets (103). Fig. 14 shows the squeezing continues
as the cell/alginate suspension is flattened (103) and the
liquid alginate from the membrane layer is displaced by the
advancing liquid alginate from the cell dollop (arrows). Fig.
shows the completion of the squeezing process. The sheet is
of uniform thinness and the liquid portion of the membrane
alginate has been displaced from view. The spacing of the
15 membrane is determined by shims (54). Fig. 16 shows calcium
diffusing in through both membranes and gelling all the liquid
alginate into a sheet.
Proliferating cells entrapped in a sheet of polymer may break
out of the sheet during expansion. In order to prevent this
eventuality, such cells may be first entrapped within shells
which have sufficient mechanical strength to contain them.
For example, cells which either are free or entrapped within an
intraluminal gel matrix can be contained within shells in the
form of small, semipermeable hollow fibers, sealed at either
end. See, e.g., Sharp et al., Diabetes 43(9):1167-70 (1994).
These sealed tubes containing cells may be layered onto one of
the two partially cross-linked polymer layers of hydrophilic
membrane method prior to layering with the apposing layer.
Alternatively, the cells may be entrapped within microcapsules,
such as polylysine stabilized alginate microcapsules. In order
to ensure complete entrapment within poly-Iysine/alginate
microcapsules without defects, it is preferable to coat the
cells in such a way as to ensure complete coverage of all cells
with alginate before stabilization of the alginate with
polylysine. This can be done, for example, by the method of
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Cochrum, Dorian and Jemtrud (Note 17). Such cell-containing
microcapsules, after stabilization by polylysine treatment and
optional dissolution of the core gel matrix, can then be
substituted for free cells or tissue in the method for sheet
manufacture in both the hydrophilic membrane method and the
casting method.
The polymer for the cell-containing core may have a different
composition from that used in preparation of the partially
cross-linked coating layers. For example, to provide for
improved mechanical strength without severely limiting
permeability, the core can be composed of alginate with a
higher guluronate content (such as that isolated from Laminaria
hyperborea, FG 0.68) or a higher molecular weight. The
partially cross-linked layers of the hydrophilic membrane
method (or the outer liquid layers of the casting method),
which will ultimately separate the cell-containing core of the
sheet from the external environment, may be composed of a
different alginate composition chosen for desired permeability
and biocompatibility properties. In general, the
characteristics of the polymer solution are described in U.S.
Patent No. 5,855,613, except that there is no need for polymers
that can be reversibly gelled.
Polymers other than alginate can be used. These polymers must
be liquid in one phase (which allows cell viability) and gelled
in another phase (which also allows cell viability), for
instance by cross-linking by addition of a cross-linking agent
(which also allows cell viability). For example, chitosan can
be substituted for alginate and cross-linked by similar
divalent ion diffusion. Acrylamide monomer may be cross-linked
with ammonium persulfate and TEMED (N,N,N',N'-
tetramethylathylenediamine).
Suitable cross-linking agents are multivalent cation salts
(e. g., of calcium, barium, strontium, or mixtures thereof).
Such salts include as counterions gluconate, lactate, and
chloride.
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In the case of casting method, an even greater variety of
polymers may be used. For example, a free-radical polymer may
be made by sweeping the three layers in the dark (or under red
light) then exposing the sheet to light. Similarly,
polyacrylamide may be cross-linked with TEMED mixed with
ammonium persulfate. Agarose, gelled by a change in
temperature can be used.
Essentially any method may be used for cross-linking the
polymer solution in the casting method or partially cross-
linked method so long as it causes a phase transition from
liquid to a gel or solid. In the. foregoing description, cross-
linking takes place by diffusion of cross-linking agent toward
the interface of the cell suspension layer from one or more of
the sandwiching layers.
In another form of cross-linking, a temperature-induced phase
transition may be used. For example, 1o agarose In
physiological solutions undergoes a liquid-to-gel phase
transition at approximately 37°C. Cells for encapsulation can
be suspended in 1% liquid agarose thermostated at temperatures
slightly higher than this, e.g., 40°C. This liquid suspension
is used for the core polymer. After formation of a sandwich
with substantially cell-free polymer solutions, cooling below
37°C would cross-link and thereby gel the suspension,
entrapping the cells, without need for diffusible cross-linking
agents.
Alternatively, the substantially uncross-linked polymer
solution may be cross-linked by photoactivation using solutions
of polymers such as polyethylene glycol derivatized with
photolabile cross-linking groups. For example, the chemistry
described in U.S. Patent 5,573,934 (Hubbell, et al.) can be
implemented in the described methods. In a preferred
embodiment, Hubbell describes the use of polyethylene glycol-
diacrylates as a soluble "macromer" in which the cells to be
entrapped are suspended. To this solution is added ethyl eosin
and triethanolamine to act as a photon-induced free radical

CA 02421036 2003-02-26
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donor. Upon illumination with 415nm light the eosin produces a
free radical which can be transferred via the triethanolamine
to the acrylate group on the macromer, which becomes activated
and cross-links with other acrylate groups forming a larger
polymer which will no longer be soluble, but will form a cross-
linked gel phase entrapping the cells and thereby forming a
cellular implant. Other photoactivated cross-linking systems
are well-known in the art.
Each polymer will have distinct permeability, stability, and
biocompatibility characteristics which can be tailored to
specific applications. For long-term cellular implants, we
have found alginate possesses the best balance of these
properties, especially biocompatibility, but this in no way
limits the choices available for this or other applications of
this invention.
As briefly described above, to improve mechanical properties of
the sheet, a mesh or other fabric (13) can be laid down on the
first over-coating film together with polymer suspended cells
by either the hydrophilic membrane method or the casting
method. The mesh or fabric can optionally be treated in such a
manner as to improve bonding to the polymer. Examples of such
treatment are covalent modification of the fabric material to
allow coupling of polyaminoacids or other polycations and
amination. Wettability of the fabric can also be improved by
chemical modification, corona treatment or pre-wetting with
dilute alginate or other polymer solution and drying.
An alternative means of improving mechanical strength of the
sheet is to incorporate a cuff of mesh or other fabric by
similar means to provide an outer annulus of support, which can
be used for suturing the sheet onto a vascularized surface (see
Fig. 22). The integral cuff can be placed so as to be distant
from the cells within the core and can be imposed in such a way
as to ensure its total encapsulation within the coating polymer
layers to improve biocompatibility. Alternatively, the cuff
31

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t
can be imposed in such a way as to allow presentation to cells
of the implant recipient to encourage engraftment of the sheet
without stimulation of cellular deposition within the immediate
environment of the contained cells.
In another alternative, the polymer solution of the coat or
core can contain microfibrillar collagen, fibrin or other
microfibrillar material, which will enhance mechanical
strength.
Treatment of fully cross-linked thin polymer layers or a fully
cross-linked cell-containing polymer core with multi-functional
reagents such as poly-lysine, poly-asparagine or other poly-
cationic polymers provides another means of improving
mechanical strength, while simultaneously providing another
means of controlling permeability. The presence of unreacted
reactive moieties of polymer-bound multi-functional reagent
provides a means for bonding between the so treated cross-
linked polymer layer and the layer of soluble polymer (coat or
core, interchangeably). Following application of coats to
core, immersion in cross-linker stabilizes the entire sheet.
In the case of multi-functional reagent treatment of the core
(as opposed to the coating layers) an advantage is afforded by
the fact that the multi-functional reagent so applied will be
buried beneath the over-lying polymer layer and thus not
exposed to the recipient, which may react to said multi-
functional reagent.
A number of other optional materials may be co-entrapped with
cells in the sheet. For~example, immobilized enzymes may be
desirable for modification of sloughed or secreted cellular
substances or immobilized hemoglobin or fluorocarbon fluid may
be included to improve oxygen and COZ solubility and transport
through the gel matrix. It may be desirable to add materials
which will be released slowly over time, such as agents to
minimize inflammation in response to the initial trauma of
surgical implantation.
32

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It may also be desirable to co-encapsulate different cell
types, where such cells are capable of interacting in some
desirable fashion. For example, inclusion of a "feeder" cell
may improve viability or survival of a particular cell.
Another example is the use of Sertoli cells to induce tolerance
(U.S. Patent No. 5,849,285, Selawry, Helena P).
Inclusion of materials which promote engraftment and/or
neovascularization may also be desirable. For example,
hyaluronic acid may be included in the coat. For example, a
cell line that secretes known vascularization factors may be
included in the core and/or coat or materials that encourage
vascular growth in adjacent tissues e.g., VEGF.
Insolublized or entrapped enzymes which catalyze desirable
conversion of naturally present substances in the recipient may
also be entrapped alone within a sheet.
In order to minimize abrasion of the device and to maintain it
in intimate contact with a well vascularized site within the
recipient, the implant may be sutured to the omentum or trapped
beneath an omental flap sutured to the surface of a
vascularized organ (Fig. 21). A support cuff can optionally be
sutured into place exterior to the space between the omental
patch and the organ. A sheet can be sutured onto any
vascularized site including the surface of the liver or other
organs or subcutaneously. Optionally, the device may be held
in place during engraftment by fibrin glues, preferably
autologous plasma concentrates. Treatment with serum, plasma,
platelet releasate or other compositions which promote healing
may offer the additional advantage of diminishing foreign body
reaction and scar formation.
33

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Figs. 17-21 show various views of thin sheets. Fig. 17 shows a
sheet with a red cell containing core. Fig. 18 shows an
electron micrograph of the surface of an islet sheet; the
islets inside appear as convex areas of the surface. Fig. 19
shows an electron micrograph of a sheet reinforced with
nonwoven fabric mesh, fractured; the flat surface (11): the
edge of the broken upper half of the sheet (12); the mesh (13)
and the lower half of the sheet (14). Fig. 20 shows a sheet
retrieved after over 2 months in a dog and Fig. 21 shows an
islet sheet made by the method of Example 8 being attached to
the dog's omentum.
Fig. 22 shows blood sugars following implant of a canine Islet
Sheet into a pancreatectomized diabetic dog of Example 8. Fig.
23 shows intravenous glucose tolerance tests (IVGTT) of the
same dog 30 days and 60 days after the implant.
Exam ale 1
Preparation of guluronate-rich alginate
Guluronate-rich alginate was purified by a modification of the
method of Dorian, et al. (U. S. Patent No. 5,643,594;
http://www.isletmedical.com/meth0202.htm (March 31, 1999)).
Briefly, 1 gram Protan MVG alginate was dissolved in 1 liter
0.5 mM EDTA, 10 mM HEPES, pH 7Ø The solution was filtered to
0.45 microns to remove particulates then mixed with 4 grams
bleached, activated charcoal for 30 minutes to adsorb organic
contaminants (bleaching of charcoal comprised stirring 30
minutes in 100 mL 0.1 M sodium perchlorate then washing by
centrifugation 2x times with 100 mL H20, 4 times with 100 mL
EtOH, 4 times with 100 mL H20). The charcoal adsorbed alginate
was filtered sequentially to 0.22 microns then 0.1 microns. To
the filtered solution was added 10.2 mL 10o MgClz~2H20. While
stirring, 3.8 mL of 34~ CaClz~2H20 was added to precipitate
larger, guluronate-rich chains. After 30 minutes of stirring,
the precipitate was pelleted and the supernatant was discarded.
34

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The pellet was dissolved in 150 mZ 0.1 M EDTA disodium salt, 10
mM HEPES, pH 7Ø After dilution to 1 liter with water, the
solution was filtered three times by concentrating ten fold in
a 17 kD hollow fiber cartridge. The retentate was diluted to
250 mZ with water and NaCl was added to a final.concentration
of 100 mM. While stirring vigorously, an equal volume of
anhydrous ethanol was added slowly. The precipitated alginate
was then pelleted by centrifugation, dissolved in 250 mZ 100 mM
NaCl, reprecipitated by addition of an equal volume alcohol as
above and pelleted. After 2 more identical washes with 500
alcohol, the final pellet was resuspended in 40 mL 50 mM NaCl
and combined with 160 mZ ethanol, pelleted and washed 3 times
by centrifugation with 200 mL ethanol to remove water, salts
and residual organic contaminants. After the last wash, the
pellet was pressed to remove excess alcohol, teased and fluffed
with forceps and dried overnight in vacuo at 60 degrees.
Examt~le 2
Preparation of ma.nnuronate-rich alginate
Mannuronate-rich alginate was purified by a modification of the
method of Dorian, et al. (U. S. Patent No. 5,429,821
http://www.isletmedical.com/meth0102.htm (March 31, 1999)).
One gram Kelco HV alginate was dissolved in 1 liter 0.5 mM
EDTA, 10 mM HEPES, pH 7Ø The solution was filtered to 0.45
microns then mixed with 4 grams bleached, activated charcoal
for 30 minutes (charcoal bleached as in example 1). The
charcoal adsorbed alginate was filtered sequentially to 0.22
microns then 0.1 microns. The filtered solution was then
filtered with a hollow-fiber cartridge and alcohol washed as in
example 1. After the last wash, the pellet was pressed to
remove excess alcohol, teased and fluffed with forceps and
dried overnight in vacuo at 60 degrees.

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Examt~le 3
Preparation of Sheets directly on smooth glass surface
Purified canine islets were sedimented by gravity from serum
free medium and washed twice by gentle centrifugation with a
wash buffer comprised of 0.9% NaCl, 3 mM glucose, 0.5 mM sodium
citrate, 10 mM HEPES, pH 7Ø The washed islets were
resuspended in a 3% solution of purified mannuronate-rich
alginate (example 2) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM
HEPES, pH 7Ø
A solution of soluble alginate salt (3% hi-M in normal
saline/10 mM HEPES, pH 7.0) is spread as a thin layer on a
perfectly smooth surface, such as optical glass (Borofloat
Window, Edmund Scientific cat# K45-686. A layer was formed by
dragging the alginate across the smooth surface with a
straightedge (precision stainless steel straightedge, McMaster-
Carr cat# 2215A2) while controlling the thickness by means of
guide shims fabricated from 75 micron polycarbonate film. A
reinforcing fabric (Hollytex, Ahlstrom cat# 3251) was placed on
top of the alginate layer. An additional 187.5 um shim was
stacked atop the 75 um shim. The suspension of islets in
alginate solution was spread with a straightedge across the
surface of the layer..
A coating layer of alginate (2% hi-M in normal saline/10 mM
HEPES, pH 7.0) was spread with a straightedge over the top of
the islet-containing layer. The entire laminate was submerged
in a solution of cross-linking ions (1.7% CaC12~2H~0, 10 mM
HEPES, pH 7.0) to cross-link the alginate.
36

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Examt~le 4
Preparation of non-reinforced sheets
by hydrophilic membrane method
Partially cross-linked layers of alginate (6 cm wide and 20 cm
long) were prepared by spreading to a depth of 125 microns a 5%
solution of 0.45 micron sterile filtered purified mannuronate-
rich alginate (example 2) in 0.9% NaCl, 0.5 mM sodium citrate,
mM HEPES, pH 7.0 onto a 0.2 micron nitrocellulose filter
membrane (Micro Filtration Systems) which had been pre-wetted
10 with 4.63% sucrose, 3.2% calcium gluconate, 10 mM HEPES, pH 7.0
and blotted before alginate application. Backing the filter
membrane at its underside was a hinged pair of plates of
optical glass positioned relative to the membrane in such a way
that the membrane and spread alginate layer spanned the hinged
junction of the 2 plates. Control of the thickness of the
spread alginate layer was achieved by using a straightedge
guided by shim rails to sweep the alginate onto the wet
membrane surface. The shim rails were then removed.
Two hundred microliters of 0.45 micron sterile filtered 5%
solution of purified guluronate-rich alginate (example 1) in
0.9% NaCl, 3 mM glucose, 0.5 mM sodium citrate, 10 mM HEPES, pH
7.0 were dispensed from a syringe onto the layer in the
approximate center of one of the hinged plates. The hinged
plates were then pivoted toward each other so that the alginate
layer on the membrane filter folded into juxtaposition,
squeezing the droplet of guluronate-rich alginate into a disk
entrapped within a sandwich of under- and over-lying alginate.
The thickness of the. sandwich was determined by spacers which
held the two plates at a defined distance from each other. The
plates were then hinged apart, leaving the membrane/alginate
layer/guluronate-rich core/alginate layer/membrane sandwich,
overlaid by an optional thin polycarbonate film lying on one of
the 2 plates. The plastic film provided a dry interface to
break the surface tension which otherwise binds the plates
together, and was peeled off and the overcoated sheet trapped
37

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within the folded membrane was transferred into a dish of 1.7%
CaC12~2H20, 10 mM HEPES, pH 7.0 to complete cross-linking of the
alginate. One fifth volume of 115 mM BaCl~ was added to the
fixative solution and the sheet was incubated for an additional
5 minutes. Finally, the sheet was rinsed with 5 mM CaClz~2H~0,
mM HEPES, pH 7.0 and transferred to serum free DMEM culture
medium with added 3 mM CaC12~2H~0 and 10 mM HEPES, pH 7.0 in
normal saline for maintenance during shipment to the surgical
facility.
10 Example 5
Encapsulation of islets in paper-reinforced sheets by
hydrophilic membrane method
Purified canine islets were sedimented by gravity from serum
free medium and washed twice by gentle centrifugation with a
wash buffer comprised of 0.9% NaCl, 3 mM glucose, 0.5 mM sodium
citrate, 10 mM HEPES, pH 7Ø The washed islets were
resuspended in a 5% solution of purified guluronate-rich
alginate (example 1) in 0.9% NaCl, 0.5 mM sodium citrate, 10 mM
HEPES, pH 7Ø
Partially cross-linked layers of alginate (6 cm wide and 20 cm
long) were prepared by spreading to a depth of 125 microns a 5%
solution of purified mannuronate-rich alginate (example 2) in
0.9% NaCl, 0.5 mM sodium citrate, 10 mM HEPES, pH 7.0 onto a
0.2 micron nitrocellulose filter membrane (Micro Filtration
Systems) which had been pre-wetted with 4.63% sucrose, 3.2%
calcium gluconate, 10 mM HEPES, pH 7.0 and blotted before
alginate application. Backing the filter membrane at its .
underside was a hinged pair of plates of optical glass
positioned relative to the membrane in such a way that the
membrane and spread alginate layer spanned the hinged junction
of the 2 plates. A 25 micron thick sheet of polycarbonate film
had been interposed between the filter membrane and one of the
optical glass plates prior to spreading of the alginate layer.
Control of the thickness of the spread alginate layer was
38

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achieved by using a straightedge guided by shim rails to sweep
the alginate onto the wet membrane surface.
A 5 cm disk of 25 micron thick polyester non-woven scrim
(Ahlstrom) was placed onto the layer in the approximate center
of one of the hinged plates. Two hundred microliters of the
islet suspension in alginate were dispensed from a syringe onto
the polyester disk. The hinged plates were then pivoted toward
each other so that the alginate layer on the membrane filter
folded into juxtaposition, flattening the droplet of suspended
islets into a disk entrapped within a sandwich of overlying
alginate. The thickness of the sandwich was determined by
spacers which held the two plates at a defined distance from
each other. The plates were then hinged apart, leaving the
membrane/alginate layer/islet suspension/alginate
layer/membrane sandwich, overlaid by the thin polycarbonate
film lying on one of the 2 plates. The plastic film was peeled
off and the overcoated sheet trapped within the folded membrane
was transferred into a dish of 1.7% CaC12~2Hz0, 10 mM HEPES, pH
7.0 to complete cross-linking of the alginate. One fifth
volume of 115 mM BaClZ was added to the fixative solution and
the sheet was incubated for an additional 5 minutes. Finally,
the sheet was rinsed with 5 mM CaCl~~2H20, 10 mM HEPES, pH 7.0
and transferred to serum free DMEM with added 3 mM CaCl~~2H~0
and 10 mM HEPES, pH 7.0 for maintenance during shipment to the
surgical facility.
Example 6
Preparation of VEGF-containing sheets
Sheets containing Vascular-Endothelial Growth Factor (VEGF)
complexed to sucralfate, an 0.-D-glucopyranoside, !3-D-
fructofuranosyl-, octakis-(hydrogen sulfate), aluminum complex,
were constructed by the following method. The dry
sucralfate/VEGF complex when added to the alginate suspension
at 400 mg/ml formed a thick paste which could not be swept
easily over the pure alginate layer. The paste was applied to a
39

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polyester scrim in a smear of uniform 500 um thickness. A 75 ~m
thick layer of liquid 3~ alginate was made by sweeping the
alginate suspension with a straightedge and shim. The paste and
embedded scrim were carefully lifted with filter forceps and
gently laid atop the liquid alginate layer. A second layer of
liquid 2°s alginate was swept over the paste/scrim with a
straightedge guided by a 750 um thick shim. This sandwich was
gelled by overlaying the liquid alginate with a solution of
1.7o CaCl~~2H2o. After 5 minutes the gel sandwich was lifted
from the glass substrate and transferred to a solution of 7mM
CaCl2 in normal saline until implanted in rats.
Examt~le 7
Preparation of Agarose Sheets
Agarose 2% (SIGMA Agarose Type I-B Low EEO) was suspended in
normal saline/10 mM HEPES, pH 7.0 and heated in a water bath to
60°C to dissolve it. The agarose was cooled to 45°C (at which
temperature it remains liquid) in a water bath and divided into
two aliquots. Dextran beads (Sephadex G-50) were suspended in
one solution 10o v/v to serve as mock islets and maintained at
45°C.
The solution of agarose without mock islets was spread as a
thin layer on optical glass (Borofloat Window, Edmund
Scientific cat# K45-686). A layer was formed by dragging the
hot agarose across the smooth surface with a straightedge
(precision stainless steel straightedge, McMaster-Carr cat#
2215A2) while controlling the thickness by means of guide shims
fabricated from 75 micron polycarbonate film. The layer was
allowed to cool for a few seconds and solidify. An additional
187.5 um shim was stacked atop the 75 um shim. The 45°C
suspension of dextran beads in agarose solution was spread with
a straightedge across the surface of the layer and allowed to
cool briefly. The two shims were removed and replaced with a
single polycarbonate shim 300 microns thick.

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6
A coating layer of agarose was spread with a straightedge over
the top of the bead-containing layer using the shims as guides.
The entire laminate was allowed to cool to room temperature
then submerged in a solution of saline/10 mM HEPES, pH 7Ø
Examt~le 8
Biooompatibility test of non-reinforced
sheets in mice, rats and dog
Sheets prepared as described in Example 5 were implanted
intraperitoneally in mice, subcutaneously in rats, and on the
omentum of a'dog (Fig. 21). The mice were sacrificed after 3
weeks and the implants removed for histological analysis. The
rats were sacrificed after 3 weeks and the implants removed for
histological analysis. The dog was sacrificed after 13 days
and the implants removed for histological analysis. The
explanted sheets were physically intact and minimal foreign
body reaction was observed.
Example 9
Implantation of islet containing sheets into a
pancreatectomized dog
A total of 200 microliters of islets were obtained from 2
mongrel dogs by standard collagenase treatment and Ficoll
density gradient purification. Islets were divided into 6
aliquots and encapsulated in polyester reinforced sheets
prepared as described above in Example 3. The sheets were
implanted into a pancreatectomized beagle on the dog's omentum
(Fig. 21).
Management of the dog's blood sugars required.a single
injection of 2 units insulin on day 9 (Fig. 22). Fasting blood
sugars remained in the normal range for 10 weeks.
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Eighty-four days after implantation the sheets and adjacent
omentum were removed. Seven days later the dog had become
diabetic with fasting blood sugar over 200 mg/ml. The sheet was
wet mounted and frozen for histology. Examination of the sheets
before fixation showed viable, islet cells (by Trypan Blue
exclusion) containing insulin (Fig. 20) as assessed by
dithizone stain.
At 30 and 60 days an intravenous glucose tolerance test was
performed according to the standard protocol. Briefly, the dog
is injected with 7 ml 50o glucose, and blood sugars are
measured at the times shown in the table below.
Dog Beagle Total Pancreatectomy/Allograft
Body weight 7 kg
IV 50o Dextrose 7 mL 30 days 60 days
Sample # Sample Glucose Glucose (Beckman)
time (Beckman) (mg/dL)
min m dL
-5 87 86
~ 0 94 79
3 1 299 279
4 5 258 236
5 10 219 211
6 20 187 169
7 30 177 144
8 45 145 117
9 60 136 97
10 90 95 84
11 120 93 79
The results of these tests are shown in Fig. 23. The IVGTT was
closer to normal at 60 days compared to 30 days showing an
increase in sheet function.
42

Representative Drawing