Transplantable artificial tissue

A transplantable artificial tissue matrix structure containing viable cells which is suitable for insertion into the body is made by polymerizing precursors in an aqueous solution to form a shape retaining solid matrix comprising viable cells, matrix polymer and reversible gel polymer. The solution contains a matrix polymer precursor, a reversible gel polymer precursor, and viable cells. The reversible gel polymer is dissolved and removed to yield an insoluble, porous matrix containing viable cells. The conditions and reagents are selected to maintain the viability of the cells. The invention is particularly suitable for artificial transplant matrix tissue containing pancreatic islet cells.

FIELD OF THE INVENTION 
This invention relates to an artificial tissue composition for insertion 
into the body and to the process for preparing it. In particular, this 
invention relates to a matrix composition containing cells which can be 
inserted to introduce the cells into the body and to the process of 
preparing this matrix. In one particular aspect, this invention relates to 
preparation of a matrix containing pancreatic B-cells which, when inserted 
into the body, can function as an artificial pancreas, introducing insulin 
produced by the B-cells cells into the body as needed. 
BACKGROUND OF THE INVENTION 
Insertion of a missing cell type into the body can be accomplished by 
implantation, transplantation or injection of cells. The cells can be in 
the form of tissue fragments, clumps of cells or single cells derived from 
the fragmentation of organs or tissues. Alternatively, the cells can be 
clumps of cells or single cells derived from cell culture, tissue culture 
or organ culture. If the cell insertion is to be successful, however, the 
cells must have the physiological environment after insertion which is 
required for the reorganization, growth or differentiation necessary to 
permit normal functioning in the body. The cells inserted into the body 
must be maintained in a physical relationship which permits adaptation to 
the new environment and promotes the changes which are required before 
normal cell functioning can occur. 
Diabetes mellitis is an example of a disease state associated with an 
insufficiency or absence of certain types of cells in the body. In this 
disease, pancreatic B-cells are missing or deficient. The condition can be 
ameliorated by the successful insertion of the missing pancreatic B-cells. 
Prior to this invention, attempts to introduce such cells into the body 
have not achieved the natural reorganization, growth or differenteration 
needed for optimum cell functioning in the body. 
DESCRIPTION OF THE PRIOR ART 
Prior art processes for inserting cells have been generally unsuccessful, 
primarily because they have not satisfied the cellular requirements of 
transplantation. Attempted matrix approaches have failed to form the 
matrix rapidly and without cellular damage. They have failed to provide 
shape and size control, the cell density required for proliferation, the 
matrix porosities for diffusion of nutrients and macromolecules, or the 
environment effecting cell-to-cell contact while permitting cell movement 
during tissue development. 
U.S. Pat. No. 4,352,883 describes a process for encapsulating biological 
materials including cells in a semipermeable spherical membrane with a 
predetermined porosity. Although some size and shape control is possible 
with a membrane envelope, cell-to-cell contact, if present, is incidental 
and uncontrolled. U.S. Pat. 4,391,909 is directed to the improvement of a 
semipermeable membrane envelope, similar to U.S. Pat. No. 4,352,883, but 
with the addition of collagen within the membrane envelope for cell 
anchorage. The semipermeable membrane in these patents is designed to 
prevent macromolecules and cells outside the envelope from reaching the 
cells enclosed within the membrane envelope. Other related patents include 
U.S. Pat. Nos. 4,251,387, 4,324,683, 4,407,957, and 4,495,288. U.S. Pat. 
No. 4,487,758 refines the process, teaching refinements of the older 
processes, optimizing water content of the hydrogel, viscosity of the 
alginate solution and the use of two polymer layers for forming the 
membrane envelope. Cell-to-cell contact is again not promoted; the matrix 
is dissolved to permit the cells to float freely within the membrane 
enclosure. The primary object of the process and composition of the above 
patents is to enclose cells in a protective enveloping membrane which 
prevents cells and macromolecules having a molecular weight averaging more 
than 200,000 daltons in the external environment from reaching the cells 
in the envelope. 
In contrast, an object of this invention is to provide a matrix in which 
the individual cells are supported, confined, anchored and nurtured. The 
matrix can be manufactured to provide any desired pore size, and in some 
embodiments, the matrix pores can be of such a size as to exclude cells 
and molecules from the external environment. In the preferred embodiments 
of this invention, the matrix is constructed to facilitate host cellular 
processes such as angiogenesis and facilitate transport of macromolecules 
and host cells throughout the matrix. 
U.S. Pat. No. 4,353,888 also describes a process for encapsulating 
mammalian cells within a semipermeable polymeric membrane to prevent 
immune rejection of the cells by antibody or immune cells of the 
recipient. The process involves dispersing an aqueous dispersion of the 
cells in a water-in-oil emulsion and coating the aqueous droplets with the 
polymer. Shape is limited to small spheres, size control is poor, 
cell-to-cell contact is not effected, and no matrix is provided within the 
spheres. 
Bell. E. et al. J.Experimental Zoology. 232:277-285 (1984) describe a 
process for enclosing thyroid cells in a collagen matrix which contracts 
around the cells to produce an artificial thyroid gland for implantation. 
Bell et al do not achieve the cell-to-cell contact required for optimum 
implantation, although some cell-to-cell contact could be achieved if the 
number of cells used was sufficiently high. However, no shape, size or 
porosity control is possible with the Bell et al system. Other 
publications applying this procedure to skin grafting are Bell, E. et al. 
J.Invest.Dermatol. 81:2s-10s (1983); Hull, B. et al. J.Invest.Dermatol. 
81:429-436 (1983); Bell, E. et al. Proc.Natl.Acad.Sci. 76:1274-1278 
(1979); Hull, B. et al. J.Invest.Dermatol. 81:436-438 (1983); and U.S. 
Pat. No. 4,485,096. 
Nilsson, K. et al. Eur.J.Appl.Microbiol.Biotech. 17:319-326 (1983) 
describes a process for immobilizing viable cells in various monomers or 
polymers dispersed as a water-in-oil emulsion during matrix formation. The 
process yields only spherical particles and limited ability to control 
particle size. Polymer forming materials tested by Nilsson et al include 
fibrin, alginates, polyacrylamides and other materials. No size or shape 
control is provided. 
U.S. Pat. No. 4,458,678 describes a wound healing product. Viable cells are 
introduced into a fibrous lattice which has some of the attributes 
required for an insertable matrix. However, no shape or size control is 
provided. 
U.S. Pat. No. 4,349,530 describes incorporating biologically active 
substances into cross-linked albumin particles. This process is not 
suitable for cells, and even if modified to accommodate cells, would not 
provide the cell-to-cell contact or control of size, shape or porosity 
required for cellular insertion. 
SUMMARY OF THE INVENTION 
The cell containing matrix of this invention is prepared by 
(1) effecting polymerization of matrix and reversible gel precursors in an 
aqueous mixture of 
(a) viable cells to be incorporated into a matrix, 
(b) matrix precursor, and 
(c) reversible gel precursor to yield an insoluble matrix; 
(2) separating the insoluble matrix from the polymerization promoting 
solution; 
(3) effecting solubilization of gel polymer in the insoluble matrix; and 
(4) recovering an insoluble, porous matrix containing viable cells, 
wherein the solution ingredients, reagents used to effect the 
polymerization and/or dissolution, and the temperatures are selected to 
maintain viability of the cells. Depending upon the selection of matrix 
precursor and reversible gel precursor, the polymerization can be effected 
by exposing the respective precursor to a polymerization promoting reagent 
and/or a polymerization promoting condition. Likewise, depending upon the 
nature of the reversible gel polymer, the dissolution of the gel polymer 
component of the matrix can be effected by choice of a suitable chemical 
composition or other condition of the solution in which the matrix is 
surrounded. 
The cell containing matrix of this invention comprises viable cells 
incorporated into a porous matrix of a non-toxic, cell compatible polymer, 
the matrix having the porosity required for cell movement and the 
cell-to-cell contact required for growth and reorganization to permit 
normal functioning after insertion into the body. 
The cell containing matrix intermediate of this invention comprises viable 
cells incorporated into a solid matrix of matrix polymer and reversible 
gel polymer, the cells having the cell-to-cell contact required for growth 
after dissolution of the reversible gel polymer.

DETAILED DESCRIPTION OF THE INVENTION 
For a cell implant or other type of viable cell insert to be effective, the 
cells must undergo any reorganization, growth and differentiation which is 
required to permit the cells to achieve normal functioning in the body. 
For a three-dimensional matrix holding the cells in a unitary mass to 
promote this result, it should satisfy all of the following criteria. The 
process used for forming the matrix must be rapid and sufficiently gentle 
to prevent cellular damage. The materials used must permit a rapid 
reaction under these gentle conditions. The process should allow shape and 
size control. For a process to be generally applicable for a variety of 
cells, it should produce shapes having controllable final cell densities 
ranging from particles containing one to a few cells to shapes containing 
cells at a density approaching the cell density in tissue, i.e. 
approximately from 10.sup.9 -10.sup.10 cells/cc. 
The ranges of size of the particles should be selected to promote cell 
viability in a physiological environment. Soon after implantation, 
diffusion of oxygen and nutrients into the central interior of particles 
having excessive diameters would be insufficient, causing death of cells 
in the central interior. However, if the particle diameters are very 
small, the particles (and the cells enclosed in their matrix) may be 
successfully attacked by humoral or cellular components of the host immune 
system. Since with increasing diameters, surface areas of particles 
increase more slowly than enclosed volumes, larger particles would be more 
resistant to such attacks. Thus the particle size must be selected to 
balance these factors. The particle sizes are thus limited to a size of 
from 20 to 5000 microns and are preferably within a range of from 50 to 
1000 microns. The optimum particle size is within the range of from 100 to 
500 microns. 
The final matrix must be nontoxic and biocompatible, and it can be 
biodegradable. The process should provide the ability to vary and control 
the matrix porosity to the level necessary to permit the requisite 
diffusion of nutrients and macromolecules. The process should be able to 
provide matrices which partially immobilize the cells to encourage 
cell-to-cell contact while being sufficiently loose to permit cell 
movement which may be necessary for rearrangement during tissue 
development. The matrix composition should also be susceptable to the 
degradation, removal or alteration in the host environment which is 
required for entry of the host cells into the matrix during the 
vascularization process. These criteria can be satisfied with the process 
and composition of this invention. 
The first step of the process of this invention comprises contacting a 
first aqueous solution containing cells and polymer precursors with a 
second aqueous polymerization promoting solution until a solid matrix of 
matrix polymer and reversible gel polymer containing cells is formed. The 
polymer precursors include a matrix precursor and a reversible gel 
precursor. 
Matrix precursors are water-soluble compounds and compositions which can be 
polymerized to form a solid matrix within which cells can achieve the 
reorganization, growth and/or differenteration required for successful 
adaptation. They must form a matrix structure which is sufficiently stable 
to substantially retain its configuration after reversible gel polymer is 
removed to leave a porous structure. They must be non-toxic, biocompatible 
and can be biodegradable. They must undergo the polymerization to form the 
solid, insoluble structure under conditions which do not interfere with 
polymerization of the reversible gel precursor, and must remain insoluble 
under conditions wherein the reversible gel polymer can be solubilized. 
The preferred matrix precursors yield polymers which undergo spacial 
contraction during polymerization. These preferred matrix precursors are 
capable of bringing cells into closer proximity during polymerization, 
increasing cell-to-cell contact. 
Examples of suitable matrix precursors and the reagents and/or temperature 
conditions effecting polymerization are listed in Table A. 
TABLE A 
______________________________________ 
Matrix Polymer Promoter 
______________________________________ 
Plasma Endogenous Thrombin via 
Ca.sup.+2 Activation.sup.b 
Fibrinogen Thrombin.sup.a 
Casein Renin/pepsin.sup.a 
Fibrin Factor XIII(a).sup.a 
Limulus lysate Endotoxin.sup.a 
Milk protein Renin/pepsin.sup.a 
Collagen, all types 
Elevated temperature at 
(I, II and III) neutral pH.sup.b 
______________________________________ 
.sup.a Promoter is provided in the first solution 
.sup.b Promoter is provided in a second solution 
When the matrix precursor is plasma, the matrix polymerization promoter is 
introduced in the second solution with the gel precursor polymerization 
promoter. When the matrix precursor is collagen, the polymerization is 
effected by elevated temperature of the second solution containing the gel 
precursor polymerization promoter. 
The reversible gel precursors must also be water-soluble, non-toxic and 
biocompatible. Additionally, they should rapidly polymerize or gel to form 
a solid structure under conditions which do not interfere with the 
polymerization of the matrix precursor to form the matrix polymer. 
Furthermore, the gel polymer must be susceptable to depolymerization or 
dissolution under conditions which will not significantly affect the 
structure of the matrix polymer or cell viability. The function of the gel 
polymer in the composition is to form an integral structure with the cells 
and matrix polymer which, when removed, will leave a pore structure in the 
matrix. The pore structure, while leaving the cells partially immobilized, 
should allow sufficient cell movement for rearrangement during tissue 
development, as well as permitting diffusion of nutrients and 
macromolecules to and from the cells. 
The gel polymer precursors are gelled by reagent and/or temperature 
conditions in the second solution. 
Examples of suitable gel precursors, the conditions and/or reagents 
promoting polymerization, and the agents and/or conditions effecting 
dissolution are listed in Table B. 
TABLE B 
______________________________________ 
Gel Polymer Dissolution 
Precursor Promoter.sup.b 
Agent 
______________________________________ 
Alginate Ca.sup.+2 Citrate 
Gums.sup.a Ca.sup.+2 Citrate 
Agarose Low Temp. Temp. elevation 
or agarase 
______________________________________ 
.sup.a Carrageenan, agar, guar gum, gum arabic, pectins, tragacanth gum, 
xanthan gum, etc. 
.sup.b Promoter provided in a second solution. 
The aqueous solution for the matrix precursor and the reversible gel 
precursor must be free from compounds which promote polymerization of 
either before the shape of the product is determined. It must also provide 
a non-toxic, biocompatible environment for the cells. 
In general, the first solution is prepared immediately before being 
introduced into the second solution, and the shape of the product is 
determined by interactions of the reagents and/or temperature of the 
second solution with the reactants in the first solution. The gel polymer 
precursor rapidly polymerizes to form an initial shape, and the matrix 
polymer forms later, retaining this shape. By having the gel polymer 
promoting conditions and/or reagents present in the second solution, shape 
formation occurs immediately upon introduction of the first solution into 
the second solution. 
The first solution should be an isotonic solution with a pH which does not 
significantly impair cell viability during particle formation. Preferably, 
the solution is an isotonic saline solution or tissue culture medium of 
the type which is commonly used in cell and tissue cultures. Suitable 
isotonic saline solutions include Hank's Balanced Salt Solution and 
Earle's Balanced Salt Solution. Suitable tissue media include Dulbecco's 
Minimal Essential Medium or RPMI 1640 medium. These formulations are set 
forth in the following tables. 
TABLE C 
______________________________________ 
Earl's Balanced Salt Solution 
Components Concentration, mg/liter 
______________________________________ 
Inorganic salts 
CaCl.sub.2 (anhydrous) 
200.00 
KCl 400.00 
MgSO.sub.4 (anhydrous) 
97.70 
NaCl 6800.00 
NaHCO.sub.3 2200.00 
NaH.sub.2 PO.sub.4.H.sub.2 O 
140.00 
Other Components 
Glucose 1000.00 
Phenol Red 10.00 
______________________________________ 
TABLE D 
______________________________________ 
Hank's Balanced Salt Solution 
Components Concentration, mg/liter 
______________________________________ 
Inorganic salts 
CaCl.sub.2 (anhydrous) 
140.00 
KCl 400.00 
KH.sub.2 PO.sub.4 
60.00 
MgSO.sub.4 (anhydrous) 
97.70 
NaCl 8000.00 
NaHCO.sub.3 350.00 
Na.sub.2 HPO.sub.4 (anhydrous) 
48.00 
Other Components 
Glucose 1000.00 
Phenol Red 10.00 
______________________________________ 
TABLE E 
______________________________________ 
Dulbecco's Minimal Essential Medium 
Components Concentration, mg/liter 
______________________________________ 
Inorganic salts 
CaCl.sub.2 (anhydrous) 
200.00 
Fe(NO.sub.3).sub.3.9H.sub.2 O 
0.10 
KCl 400.00 
MgSO.sub.4 (anhydrous) 
97.70 
NaCl 6400.00 
NaHCO.sub.3 3700.00 
NaH.sub.2 PO.sub.4.H.sub.2 O 
125.00 
Other Components 
Glucose 1000.00 
Phenol Red 15.00 
Sodium pyruvate 110.00 
Amino Acids 
L-Arginine-HCl 84.00 
L-Cystine-2HCl 62.59 
L-Glutamine 584.00 
Glycine 30.00 
L-Histidine-HCl.H.sub.2 O 
42.00 
L-Isoleucine 104.80 
L-Leucine-HCl 104.80 
L-Lysine-HCl 146.50 
L-Methionine 30.00 
L-Phenylalanine 66.00 
L-Serine 42.00 
L-Threonine 95.20 
L-Tryptophan 16.00 
L-Tyrosine 72.00 
L-Valine 93.60 
Vitamins 
D-Calcium pantothenate 
4.00 
Choline chloride 4.00 
Folic acid 4.00 
i-Inositol 7.00 
Nicotinamide 4.00 
Pyridoxal-HCl 4.00 
Riboflavin 0.40 
Thiamine-HCl 4.00 
______________________________________ 
TABLE F 
______________________________________ 
Modified RPMI 1640 Medium 
Components Concentration, mg/liter 
______________________________________ 
Inorganic salts 
Ca(NO.sub.3).sub.2.4H.sub.2 O 
100.00 
KCl 400.00 
MgSO.sub.4 (anhydrous) 
48.90 
NaCl 6000.00 
NaHCO.sub.3 2000.00 
Other Components 
Glucose 2000.00 
Glutathione (reduced) 
1.00 
Phenol Red 5.00 
Amino Acids 
L-Arginine (free base) 
200.00 
L-Asparagine (anhydrous) 
50.00 
L-Aspartic acid 20.00 
L-Cystine.2HCl 65.20 
L-Glutamic acid 20.00 
L-Glutamine 300.00 
Glycine 10.00 
L-Histidine (free base) 
15.00 
Hydroxy-L-proline 20.00 
L-Isoleucine 50.00 
L-Leucine 50.00 
L-Lysine-HCl 40.00 
L-Methionine 15.00 
L-Phenylalanine 15.00 
L-Proline 20.00 
L-Serine 30.00 
Amino acids 
L-Threonine 20.00 
L-Tryptophan 5.00 
L-Tyrosine 20.00 
L-Valine 20.00 
Vitamins 
p-Aminobenzoic acid 
1.00 
d-Biotin 0.20 
D-Calcium pantothenate 
0.25 
Choline chloride 3.00 
Folic acid 1.00 
i-Inositol 35.00 
Nicotinamide 1.00 
Pyridoxine-HCl 1.00 
Riboflavin 0.20 
Thiamine-HCl 1.00 
Vitamin B.sub.12 0.005 
______________________________________ 
The solution pH can be from 5 to 9 and is preferably from 6 to 8. The 
optimum pH is from 6.8 to 7.6. 
The concentration of the matrix precursor and reversible gel precursor in 
the solution are selected to provide the desired pore structure and 
rigidity, and cell attachment. 
The concentration of matrix precursor for most matrices can be from 1 
microgram/ml to 100 mg/ml, preferably from 50 microgram/ml to 20 mg/ml and 
optimally from 0.1 mg/ml to 6 mg/ml. Other ranges may be preferred and 
optimum for matrices having different matrix strengths, rigidity and 
different cell attachment characteristics. 
The reversible gel precursor concentration determines the degree of 
porosity. It should be selected to yield the porosity required to permit 
adequate flow of nutrients and macromolecules, and when necessary, to 
permit sufficient cell movement for reorganization. It can be from 1 
microgram/ml to 100 mg/ml. For low porosity compositions, it is preferably 
from 1microgram/ml to 1 mg/ml, and for high porosity compositions, it is 
preferably from 1 to 100 mg/ml. 
The terms "cell" and "cells", are defined for the purposes hereof to 
include tissue fragments, cell clumps and single cells. The process and 
product of this invention is suitable for preparing a wide variety of 
artificial tissues or organs prepared from single cells for insertion into 
the body. These cells can be fibroblasts, kidney cells, liver cells, 
thymus cells, thyroid cells, epidermal keratinocytes or cells from other 
tissues and organs. The process of this invention is particularly suitable 
for preparing artificial pancreatic endocrine tissue from pancreatic 
islets or islet cells for implantation into the body. This process can be 
used with any type of cell where implantation of the cells as an 
artificial tissue would be beneficial. 
Insulin-dependent diabetes mellitus is a disorder resulting from lack of 
properly functioning pancreatic islet cells which normally produce and 
secrete insulin in response to glucose. For implanted cells to achieve a 
normal function in the body and secrete insulin in response to glucose, 
they may need to reproduce and reorganize to form a functioning cell 
grouping. The process and matrix of this invention is capable of allowing 
the requisite cell-to-cell contact, diffusion of nutrients and 
macromolecules for cell growth, and cell mobility for cellular 
reorganization to the functioning cell grouping required for formation of 
artificial organs and tissues. The optimal pancreatic islet cells for this 
function are substantially fibroblast-free cell preparations derived from 
culturing of fetal islet cells. For purposes of clarity of explanation and 
not by way of limitation, the invention will be described hereinafter in 
terms of pancreatic islet cells, it being understood that the process and 
matrix product is also suitable for implanting other types cf cells 
including cells from adult tissues and organs, and that these other types 
of cells can be considered as alternates in the invention description. 
The cell concentration in the solution determines, together with the 
concentration of matrix precursor, the cell density in the final matrix 
product. Solution cell densities can be from 10.sup.2 to 5.times.10.sup.8 
cells/ml. For matrices having low cell densities, the cell concentration 
in the solution can be from 10.sup.2 to 5.times.10.sup.5 cells/ml, and for 
matrices having high cell densities, approaching the cell density of 
normal tissue, the cell concentration in the solution can be from 
5.times.10.sup.5 to 5.times.10.sup.8 cells/ml. 
Pancreatic islet cells suitable for incorporation into the matrix of this 
invention for implantation can be derived from pancreatic tissue by 
numerous published procedures or they can be derived from tissue, organ or 
cell cultures. The optimum pancreatic islet cells for implantation are 
believed to be fetal pancreatic proislet cells. These can be derived from 
the recipient species or they can be derived from a donor species which is 
different from the recipient species. When implanted into a suitable 
vascularized site in the body, held together with the matrix product of 
this invention, the cells differentiate to produce islet cells in a 
vascularized environment, an artificial endocrine pancreas, which responds 
to serum glucose by producing and secreting insulin. Procedures for 
preparing pancreas tissue for transplant are described by Lafferty, K. et 
al in Transplantation Proceedings. 14:714 (1984) and Lacy, P. et al in 
Ann.Rev.Immunol. 2:183 (1984). 
The polymerization of the matrix precursor and reversible gel precursor is 
effected by ingredients and/or conditions of a second aqueous 
polymerization promotion solution with which the primary cell, matrix 
precursor, and gel precursor components are contacted. The ingredients of 
this second solution must be non-toxic, biocompatible, and must promote 
rapid polymerization of the matrix and reversible gel precursors in the 
primary solution. 
The term "polymerization", for the purposes of this application, is defined 
to include any reaction by which soluble precursor materials are 
transformed to a shape-retaining, insoluble form, including chain 
formation, increase in chain length, and covalent or non-covalent 
cross-linking. 
Preferred polymerization promoters include multivalent ions in solution 
which can form a salt with the acidic gums. The optimum ion is a 
physiologically compatible ion such as calcium in a concentration which 
provides in the mixture of the first and second solution, a calcium ion 
concentration which promotes rapid polymerization of the gel polymer 
precursor. 
The first aqueous solution is contacted with the second aqueous solution in 
a manner which yields the desired solid matrix shape and size. For the 
production of spheres, the primary solution can be dripped or blown under 
air pressure into the secondary solution, the drop size being selected to 
yield the desired particle size. Alternatively, the two solutions can be 
quickly mixed and the mixture placed in a cavity having the general 
configuration of the desired product. A distribution of particles can be 
obtained with certain gel precursors by subjecting the secondary mixture 
to agitation while the primary solution is rapidly poured into the 
secondary solution. 
The mixture is maintained until solidification of the gel precursor occurs. 
For optimal control of particle size and shape, this solidification should 
occur very rapidly. Gel polymer formation should occur in less than 10 
minutes, preferably in less than one minute and optimally in less than10 
seconds. Such solidification is more or less temperature dependent, the 
degree of dependence varying with the characteristics of the selected gel 
precursor and gel polymerization promoter. The temperature dependence of 
solidification is preferably such that rapid solidification occurs at a 
temperature which, is compatible with cell physiology, i,e., from 0 to 
50.degree. C., preferably from 4.degree. to 45.degree. C. and optimally 
from 6.degree. to 37.degree. C. 
Matrix precursor polymerization is initiated by contact with a matrix 
polymerization promoter (or promoting condition), the promoter being 
present in either the first or second aqueous solution. Matrix 
polymerization should occur at a rate which does not interfere with 
adequate gel polymerization. The polymerization rate is dependent upon the 
relative concentrations of matrix precursor and matrix polymerization 
promoter. The rate can be adjusted by adjusting the concentration of one 
or both concentrations. Matrix precursor polymerization should occur in 
from one minute to 24 hours. The optimal rate is determined by the 
physical and chemical characteristics of the particular reaction involved 
and the effect of the total environment during polymerization on the 
particular cell type being used. Preferably, the polymerization should 
occur in from 2 minutes to 10 hours and optimally in from 5 to 200 
minutes. 
The matrix solids comprising polymerized matrix precursor, gelled 
reversible gel precursor and cells are then removed from the solution 
mixture. In a preferred procedure, the matrix solids are then placed into 
a physiologically compatible solution such as described above. This 
solution can contain sufficient quantities of matrix polymerization 
promoting agents to initiate or continue the matrix polymerization if such 
promoting agents were not present in either the first or second aqueous 
solution. During this phase, contraction of the matrix structure may 
continue, and the cells may be brought into closer proximity. 
After matrix polymerization sufficient to provide a matrix integrity which 
will retain pores after removal of the gel polymer has been achieved, the 
solid matrices are removed from the polymerization solution. They are 
preferably rinsed to remove residual solution containing the 
polymerization promoting agent. A suitable rinse solution is an isotonic 
solution, isotonic saline, or tissue culture medium as described 
hereinabove. 
The solid matrices are then placed into a gel polymer dissolution promoting 
solution. The gel polymer dissolution promoting solution may contain 
nutrients required for maintaining viability of the cells and has suitable 
conditions, composition, or agents to reverse the polymerization reaction 
which produced the gel polymer. The solution should not contain any 
components which impair the integrity of the matrix structure or harm the 
cells. The solution preferably contains nutrients which maintain the 
viability of the cells. For pancreatic cells, the nutrients and nutrient 
media described above can be used. 
For removing gel polymers of the polysaccharide type other than agarose, 
the solution should be devoid of multivalent ions, for when the 
cross-linked gel structures are exposed to a solution containing 
monovalent ions and is relatively deficient in multivalent ions, the 
multivalent ions are displaced in a reversable reaction by the monovalent 
ions. Preferably, removal of the multivalent ions during the 
depolymerization treatment is achieved by the use of sequestering agents 
or ion exchange resins having monovalent ions such as sodium ions in the 
exchange sites. Movement of the solution over the matrix surfaces promotes 
the gel dissolution, but excess agitation which damages the matrix 
structure or the cells should be avoided. 
The solution preferably is maintained at a temperature of from 0.degree. to 
50.degree. C. and preferably from 6.degree. to 37.degree. C. during this 
step. The matrix treatment is continued until the desired degree of 
porosity has been produced, that is, until the corresponding amount of the 
gel polymer has been dissolved and removed from the matrix structure. The 
time is temperature dependent and is generally from 5 to 60 minutes for 
temperatures within the range of from 6 to 37.degree. C. 
Agarose polymerization, being initiated by low solution temperatures, can 
be reversed by elevating the solution temperature. Agarose polymer 
dissolution temperatures can be from 37.degree. to 65.degree. C. and are 
preferably from 45.degree. to 55.degree. C. Alternatively, agarose 
polymers can be dissolved by treatment with an agarase solution. 
The final porosity of the particles after gel polymer dissolution is 
dependent upon the initial concentration of the gel polymer in the 
particle, the degree of gel polymer crosslinking obtained, the degree of 
spatial contraction occurring during matrix polymerization, the amount of 
matrix crosslinking or contraction occurring after gel polymer 
dissolution, and other variables, including processing temperatures. These 
variables can affect average pore diameter, pore size distribution, 
average pore length, and total pore numbers. In the process and 
composition of this invention, pore diameters can be from 
5.times.10.sup..times.3 to 40 microns, preferably from 2.times.10.sup.-2 
to 5 microns and optimally from 0.1 to 1 microns. 
The final matrix is then recovered from the solution and placed in a 
nutrient medium such as those described above for maintaining the 
viability of the cells. The nutrient medium can be further supplemented 
with fetal calf serum or other conventional supplements. The nutrients 
required for maintenance and propogation of various cells are not a part 
of this invention, being well established and fully within the knowledge 
and skill of the person skilled in the art. For example, suitable media 
are described in READINGS IN MAMMALIAN CELL CULTURE. Robert Pollack 
(editor) second edition New York: Cold Spring Harbor Laboratory (1981) and 
other publications. 
Further processing of the matrix to preserve or to expand or multiply the 
cells may be desirable, depending upon the cell type and factors involved 
in the insertion. 
The matrix product of this invention can have a particle size as low as 
0.02 mm in diameter and a size as large as 3 mm. The matrix product 
diameter is preferably from 0.05 to 1 mm and optimally from 0.1 to 0.3 mm. 
The matrix can have any desired cell density. For pancreatic islet cells, 
the cell density can range from 10.sup.3 to 5.times.10.sup.8 cells/cc of 
matrix. 
The matrix is porous and has a pore size sufficiently large to permit 
diffusion of nutrients and macromolecules to and from the cells entrapped 
in the matrix, and movement of cells. 
The matrices of this invention can be implanted in the same manner as 
implantation of the corresponding tissue. For effective vascularization of 
the implanted matrix, the matrix is preferably implanted in a portion of 
the body which is not functionally affected by the implant and which will 
readily vascularize the implant. For example, if the matrix supports 
pancreatic islet cells, the matrix can be implanted in the mesenteric 
omentum, kidney subcapsular space, spleenic pulp, portal vein, and other 
sites appropriate for vascularization and function. 
This invention is further illustrated by the following specific, but 
non-limiting examples. Unless otherwise specified, temperatures are given 
in degrees centigrade and concentrations are given as weight percents. 
EXAMPLE 1 
Monolayers of cultured pancreatic pre-islet cells were harvested by 
trypsinization and washed twice in isotonic, neutral pH buffer. The number 
of cells and percent viability were determined by trypan blue exclusion 
with a hemocytometer. Approximately 4.times.10.sup.6 viable cells were 
recovered. The cells were maintained on ice and centrifuged (150.times.g) 
for 5 min before use. After removal of the supernatant, the cell pellet 
was recentrifuged (200.times.g) for 1 min to facilitate removal of 
residual interstitial liquid. 
The following components (maintained on ice) were added to an isotonic 
saline solution in a separate 3 ml conical vial to give the component 
concentrations noted: 
______________________________________ 
Component Concentration 
______________________________________ 
Sodium alginate 1.0% 
Collagen, Type I 0.05 mg/ml 
Collagen, Type IV 0.05 mg/ml 
Fibronectin 0.025 mg/ml 
Lamanin 0.025 mg/ml 
______________________________________ 
The solution was gently mixed and used to resuspend the cell pellet at 
1.times.10.sup.7 cells/ml. To this suspension were added fibrinogen (0.6 
mg/ml final concentration) and thrombin (0.2 units/ml) to clot the 
fibrinogen. This final reaction mixture was rapidly aspirated into a 
syringe and expressed through the droplet-forming device as described in 
BIOMEDICAL APPLICATIONS OF MICROENCAPSULATION. F. Lim (editor) Boca Raton: 
CRC Press (1983) at a rate of 10 ml/hr. The air flow was set at a 
flowmeter reading of 50 psig. Droplets were delivered into 102 mM isotonic 
CaCl.sub.2 for 2-10 min until the insoluble matrix formed. A series of 
washes in isotonic saline to remove Ca+.sup.2 ions and/or free alginate 
were preformed, and the resulting particles were resuspended in RPMI 1640 
containing 10% (v/v) fetal calf serum.