Note: Descriptions are shown in the official language in which they were submitted.
~.4q5~L~
BackgroUnd
This invention relates to a method of encapsulation
which is reversible, that is, a method which may be used to
encapsulate a liquid or a solid material and thereafter to
release the material by selectively disrupting the capsule
membranes~ An important embodimen-t of the invention involves
the microencapsulation of living cells which may subsequently
be released from within the produced capsule membranes without
damage.
Canadian Application Serial No. 348,524, now Patent
No. 1,145,258, entitled "Encapsulation of Viable Tissue
and Tissue Implantation Mlethod", by F. Lim discloses a micro-
encapsulation technique which can be used to encapsulate
esscntially any solid or liquid material within semipermeable
or substant:ially impermeable capsule membranes. An outstan~ing
advantage of the process is that the conditions under which
th~ capsule membranes are formed involve no toxic or denatur-
.ing reagents, ~xtremes of temperature, or other conditions which
damacJe living cells. The process of that application is
~ according:ly well-suited for the production of microencapsulated
l:Lving materials which remain viable and in a healthy state.
Because the process allows a degree of control of the perme-
~bility o~ the membrane, it is now possible to microencapsulate
cell cultures of procaryotic, eukaryotic~ or other origin such
that cells of the culture are protected from contaminating
bacteria, high molecular weight immunoglobulins, and other
potentially deleterious factors, and remain confined within a
microenvironment well-suited for their continuing viability
and ongoing metabolic functions. If the
-- 2
~sj D~
1 microcapsules are suspended in a conventional culture medium suf-
ficient to suppo~t growth of the living cells involved, the
microencapsulated cells are free ~o ingest substances needed for
metabolism which diffuse through the membrane and to excrete
their me~abolic products through the capsule membrane into the
surrounding medium.
5~
1 Summar~ of the Invention
The instant invention is directed to a method of selec-
tively disrupting certain membranes synthesized during the micro-
encapsulation procedure se~ forth in the above-referenced
application without any detectable damage to the encapsulated
core rnaterial, ~ore specifically, the process of this invention
is practiced on membranes comprising a water-insoluble matrix
formed froTn at least two water-soluble componen~s: a polyme_
which includes multiple cationic moieties [polycationic polymer,
e.g. polye-thylene amine); and a polymer having multiple anionic
rnoieties (polyanionic polymer, e.g. sodium alginate gum). The
two components are connected by ~alt bridges between the anionic
and cationic moieties to form the matrix.
The process of the invention comprises the steps of
expo~ing membranes of the type set forth above to a solution of
ca~ions, peferably, monatomic or very low molecular weight multi-
~alent cations/ and a solution of a stripping polymer having
plural anionic rnoieties. Preferably, these solutions are mixed
together. The anionic charge of the polymer ~hould be sufficient
to disrupt the salt bridges and should preEerably be equal to or
yreater than the charge density of the polyanionic polymer of the
membrane. The solution is contacted with the capsules to allow
the cations, e.g., calcium or aluminum, to eornpete against the
polycationic polymer in the capsule m~m~rane for anionic sites on
the polyanionic polymer. Sirnul~aneously, the stripping polymer
having plural anionic moieties competes with the polyanionic
polymer for cationic sites on the polymer chains~ This results
; in "softening" or "unæipping" of the capsule membranes. To
1 complete the disruption, the capsules are wa8hed a~d then exposed
to a sequestering agent to r~move cations associated with the
polyanionic polymer~ The preferred sequestering agents are che-
lating ayents such as citrate ion~ or EDTA ions. If~ as in an
important embodiment, the capsules con~ain viable cells, it is
preferred to mix the sequestering agent with an isotonic saline
solution.
The currently preferred cation is calciumO Examples of
the stripping polymer having plural anionic yroups used in the
mixed solution include polysulfonic acids or (pre~erably) their
salt~, either natural or synthetic. Outstanding results have
been obtained using heparin, a natural polymer containing plural
~ulfonate groups. Polymers containing polyphosphoric or
polyacrylic acid salt moie~ies may also be used. The currently
preferred seques~ering agent is sodium citrate.
The invention also contemplates a method of encap-
sulati.ng viabLe cells within a pro~ective environment and sub-
sequently releasing the cells. Thus, the invention provides what
may be described as a package which maintains living cell
cultures of whatever origin in a sterile, stablizing environment
in which they can undergo normal metabolism and even mitosis and
from which they subsequently can be released.
Accordingly, an object of the invention is to provide a
method for encapsulating living cells within permeable membranes
and subsequently selectively disrupting the membranes to release
the cells. Another object is to provide a process for selec-
tively disrupting membranes. Another object is to disrupt mem-
1 branes without damage to proximate living tissue. Still anotherobject is to provide a method of encapsulatiny and subsequently
releasing finely divided materials J liquids J and solutions.
These and other objects and features of the invention
will be apparent from the following description of some important
embodirnents.
Description
The selective membrane disruption process of the inven-
tion is practiced on membranes consisting of a salt bridge-bonded
]0 matrix of a polycationic polymer and a polyanionic polymer.
U~ually, the membranes will have a spheroidal form defining an
enclo~ed int~rior containing an encap~ulated substanceO However,
~he proce~s may al50 be practiced on membranes o~ this type which
take other than spheroidal form,
Although es~entially any material ~compatible with
aqueous environments) in liquid or solid form can be encapsulated
and subsequently released without damage by the process of this
irlvention, its most notable utility, as prPsently contemplated,
lies in it~ abili~y ko encapsulate and subsequently release
living systems such as cell cultures. Accordingly, the descrip-
tion which follows will be primarily confined to a discussion of
the encapsulation and release of cell~. Those skilled in the art
will be able to adap~ ~he process withou~ difficulty to the
encapsulation of less fragile materials.
lation
__.
The tissue or cells to be encapsulated are suspended in
an aqueous medium ~uitable for maintenance and for supporting the
1 ongoing me~abolic processes of the particular cell type involved.
Media sui~able for this purpose are well known to those sXilled
in the art and often ar~ availahle commercially. The average
diameter of the cell mass or ot~er material to be encapsula~ed
can vary widely between a few microns to a millimeter or more.
Mammalian Islets of Langerhans, for examples, are typically 50 to
200 rnicrons in diameter. Tissue fragments and individual cells
such as fibroblasts, leukocytes, lymphoblastoids, pancreatic
beta, alpha or delta cells, islet of Langerhans, hepatocytes, or
the cells of other tisgue may be encapsulated as desired~ Also,
microorganisms may be encapsulated including those which have
been genetically modified by recombinant DNA or other techniques,
The ongoing viability of such living matter is depen-
dent, inter alia, on the availability o~ required nutrients, oxy-
gen transfer, absence of toxic substances in the medium, and the
pH o~ the medium. Heretofore, it has not been possible to main-
tain suc~l living rnatter in a physiologically compatible environ-
ment while simultaneouæly encapsulating, The problem has been
that the conditions required for membrane formation have been
~0 l~tllal or harmful to the tifisue, and prior to the invention of
the above-referenced application, no method of membrane ormation
which allowed tissue ~o survive in a healthy state had been
forthcoming.
However, it has been discovered that certain water-
~oluble substances which are physiologically compatible with
living tissue and can be rendered water-insoluble to form a
æhape-retainingO coherent mass, can be used to form a "temporary
cap~ule" or protective barrier layer about individual cells,
--7~
l groups of cells, or tissues. Such a subs~ance is added,
typically at a concen~ration on the order of 1-2 weight percent
to the tissue culture medium. The solution is then formed into
droplets containing tissue together with its maintenance or
growth medium and is immediately rendered water-insoluble and
gelled, at least in a surface layer. Thereafter, the
shape-retaining temporary capsules are provided with a more
permanent membrane which, in accordance with this invention, may
be subsequently ~electively disrupted to release the encapsulated
tissue without damage. Where the ma~erial used to form the
temporary capsules permi~s, the capsule interior may be
reliquified ater formation of the permanent membrane. This is
done by re-establishing the conditions in the medium at which the
ma~erial is soluble.
The material used to form the temporary capsules may be
any non-toxic, water-soluble material which, by a change in ~he
~urrounding io~ic environment or concentration, can be converted
to a shape-retaining mass. The material also comprises plural,
easily ionized anionic moieties, e.g., carboxyl grc,ups, which can
react by sal~-bond forrnation with polymers containing plural
cationic groups. As will be explained below, this type of
material enables the d~position of the permanent membrane of a
selected permeability (including substantially non-porous to a
level of several hundred thousand daltons).
The presently preferred polyanionic material for
forming the temporary capsule ars acidic, water-soluble, natural
or syIl~hetic polysaccharide gumsO Many such materials are com-
mercially available~ They are typically extracted from vegetable
1 matter and are often used as additives ~o various foodsO Sodium
alginate i3 the presently preferred anionic polymer. Alginate in
the molecular weigh~ range of 150,000~ daltong may be used, but
because of its molecular dimensions will usually be unable to
permeate the finally formed capsule mernbranes. To make capsules
without trapped liquid alginate, lower molecular weight alginate,
e.g., 40,000--80,000 daltons can be used. In the inished cap-
sule, the alginate can then be more easily removed from the
intracapsular volume by diffusion through a membrane of suf-
ficient porosity. Other useable polyanionic yums include acidic
fractions of guar gum, carageenan, pectin, tragacanth gum, or
x~nthan gurQ.
These materials compriee glycoside-linked saccharide
chain~. Their free acid groups are often present in the alkali
m~tal ion form, e.g., ~odium form. If a multivalent ion such as
calcium, strontium, or aluminum is exchanged for the alkali metal
ion, the liquid, water-soluble polysaccharide rnolecules are
"croas-linked" to forrn a water-in~oluble, shape retaining gel
which can be resolublized on rernoval of the ions by ion exchange
or via a suquestering agent. While essentially any multivalent
ion which can form a salt with the acidic gum is operable, it is
preferred that physiologically compatible ions, e.g., calcium, be
employed. This tends to preserve the tissue in the living state.
Other multivalent cations can be used for less fragile materialO
Magrlesiurn ions are ineffective in gelling sodium alginate.
A typical solution composition comprises equal volumes
o a cell suspensiorl in its medium and a one to two percent solu-
tion of gum in physiological saline. When employing sodium algi-
nate, a 1.2 to 1.6 percent solution has been used with success.
1 In the next s~ep of the encapsulation process, the gum
solution containing the tissue i5 formed into droplets of a
desired size sufficient to envelop the cells ~o be encapsulated.
Thereafter, the droplets are immediately gelled to form
shape-re~aining spherical or spheroidal masges~ The drop
formation may be conducted by known techniques.
A tube containing an aqueous solution of rnultivalent
cations, e.g., 1.5~ CaC12 solution, is fitted with a stopper
which holds a drop forming apparatus. The appartus comprises a
housing having an upper air intake nozzle and an elongate hollow
body friction fitted into the stopper. ~ lO cc syringe equipped
with a stepping pump is mounted atop the housing with, e~g., a
O.Ol inch I.D. Teflon coated needle passing through the length of
the housing. The interior of the housing is designed such that
the tip of the needle is subjec~ed to a constant laminar air flow
which acts as an air knife. In use, with the syringe full of
~olution cont~ining the rnaterial to be encapsulated, the stepping
pump i~ actuated to incrementally force droplets of solution from
the tip o~ the needle~ Each drop is "cut of-f" by the air stream
and fall~ approximately 2.5 cm into the CaC12 solution where it
is immedia~ely gelled by absorption of calcium ions. The
distance between the tip of the needle and the surface of the
CaC12 solution is great enough, in this instance, to allow the
sodium algina~e/cell suspension to assume the most physically
favorable æhape; a æphere (maximurn volume for minimwn surface
area). Air within the tube bleeds through an opening in ~he
stopper. This results in "cross-linking" of the gel and in the
formation of a high viscosity, shape-retaining protective tem-
--10--
1 porary capsule containing the suspended ti~sue and its medium.The capsules collect in the solution as a separate phase and are
separated by aspiration.
In the next step of the proc~ss, a membrane is
deposited about the eurface of the temporary capsules by cxoss-
linkiny surface layers. This is done by subjecting the temporary
cap~ules comprising polyanion to an aqueous solution of a polymer
containing cationic groups reactive with anionic functiorlalities
in the polyanionic polymer. Polymers containing reactive
cationic groups such as free amine groups or combina~ions of
amine and imine groups are preferred. In this situation/ the
polysaccharide gurn is crosslinked by interaction (sal~ bond
~ormati.on) between the carboxyl groups and the amine or imine
groups of the polycationic polymer. Advantageously, permeability
can be controlled within limi~s by selecting the molecular weight
o~ the cro~s-linking polymer used and by varying exposure time
and the concentration of poLymer in solution. A solution of
polymer having a low molecular weight, in a given time period,
will penetra~e further into the temporary capsules than will a
high molecul~r weight polymer. The degree of penetration of the
cross-linker has been correlated with the resulting permeability.
In general, the higher the molecular weight and the less penetra-
tion, the larger ~he pore size. Longer exposures and more con-
centrated polymer solutions tend to decrease the resulting
mernbrane'~ upper limit of permeability However, the average
molecular weight of the polymer is the dominarlt determinant.
Broadl~, polymers within the molecular weight range o~ 10,000 to
100,000 daltons or greater may be used, depending on the duratio
--11--
1 of the reaction, th~ concentration of the polymer solution, and
the degree of permeability desirzd. One successful ~et of reac-
tion conditions, using polylysine of average molecular weight of
about 35,000 daltons, involved reaction for three minutest with
stirring, o a physiological saline solution containing 0~0167
percent polylysine~ This results in membranes having an upper
limit of permeability of about lOOrOOO daltons. Generally,
higher molecular weight materials form membrane which are more
difficult to subsequently disrupt as compared with lower
molecular weight ma~erials. The charge densi~y of the
crosslinking polycationic polymer also affects the pore size and
ea~e of membrane disruption. Generally, higher charge density
materials form less porous membranes which are more di~ficult to
dlsrupt. Optimal reac~ion conditions suitable for controlling
permeability in a given sys~em can readily be determined
emp~rically in view of the foregoing guidelines.
Example~ of suitable cross-linking polymers include
proteins and polypeptides, either natural or synthetic, having
~ree amino or cornbinationa of amino and imino groups, polyethyl-
enearnines, polyethyleneimines, and polyvinylamines Polylysine,in both the D and L forms, has been used with success. Proteins
such as polyarginine, polycitrulline, or polyornithine are also
operableO Polymers in the higher range of positive charge den-
sity (e.g., polyvinylamine) vigorously adhere to the anionic
groups of the polyanionic molecules and are rnore difficult to
disrupt.
At this point in the encapsu]ation, capsules may be
collected w~ich cornprise a "psrmanent" semipermeable membrane
1 surrounding a gelled solution of gum, cell type compatible
culture medium, and the cells. If the obje~t is simply to
preserve the cells in a protective environment, no further steps
need be done. However, if mass transfer is to be promoted within
the capsules and across the membranes, it i6 preferred to
re~lj.quify the gel to its water- oluble form, This may be done by
reestahlishing the conditions under which the gum is a liquid,
e.g., removing the calcium or other multifunctional ca~ions from
the gel. The medium in the capsule can be resolubilized simply
by immersin~ the capsules in phosphate buffered saline, which
contains alkali metal ions and hydrogen ions. Monovalent ions
exchange with the calcium or other multifunc~ional ions within
the gum when the capsules are immersed in the solution with
stirring. Sodiurn citra~e solu~ions may be used for the same
purpose, and serve to sequester the divalent ions~ Gum molecules
having a molecular weight below the upper limit of permeability
o~ th~ rnernbranes may subsequently be removed from the intracap-
sular volurne by diffusion.
La3tly, it may be desirable to -treat the capsules so as
to tie up free amino groups of the like which might otherwise
impart to the cap~ules a tendency to clump~ This can be done,
for example, by immersing ~he capsules in a dilute solution of
sodium alginate.
From the foregoing it will be apparent that no harsh
reagents, extremes of tempera~ure, or other conditions
deleterious to the health and viability of the cells need be used
in the membrane formation process. Thuss even very sensitive
cells such as mammalian hepatocytes, leukocytes, ~ibroblasts,
-13-
1 lymphoblasts, and cells from various endocrine ~issues may be
encapsulated withou~ difficul~y~ Of course, ce~ls of microbial
oriyin such as yeasts, molds, and bacteria which are bet~er
adapted to survive in hostle environtnentsl as well as inert
reagents, solids, or biologically active materials may also be
encapsulated without damage.
Encapsulated cells of the type described above may be
suspended in maintenance medium or growth medium for storage or
culture and will remain free of bacterial infection. If
suspended in growth medium, cells which undergo rni~osis ln vitro
will do so within the capsules Normal in vitro metabolism con-
tinues provided the faetors needed for metabolic processes are of
~u~iciently low molecular weight that ~hey can penetrate the
capsule mellbrane, or are encapsulated together with the cells.
Metaboli.c products of the cells (if of a molecular weight below
the upper limit of permeability) penetrate the membrane and
~ollect in the medium. The cells in encapsulated form may be
stored, shipped, or cultured as desired, and may be released from
their protective environmen~ without damage by means of the
following process of selectively disrupting the membranes.
Disruption oi Membrane
In accordance with the invention the encapsulated
material may be released by a two step process involving commer-
cially available reagents having properties which do not
adversely affect the encapsulated cells.
Eirst, the capsules are separated from their suspending
medium, washed thoroughly to remove any contaminants and then
-14-
s~
1 dispersed, with agitation, in a separate or preferably mixed
solution o~ cations such as calcium ions or ot~er monatomic (low
molecular weigh~ multivalen~ cation) and a stripping polymer
having plural anionic moieties such as polysulfonic acid groups~
Polymers containing polyphosphoric or polyacrylic acid moieties
ma~ also be used. Heparin, a natural sulfonated polysaccharide,
is preferred ~or disrupting membranes containing cells. The
anionic charge of the stripping polymer used must be sufficient
to diRrupt the salt bridges. Thus the anionic charge density may
be equal to or preferably greater than the charge density of th~
interior polyanionic polymer (e.g~, ~he gum) originally employed
to forrn the membranes. The molecular weight of the stripping
polymer should be at least comparable to and preferably greater
than the molecular weight of the interior polycationic polymer
used in forrning the merabrane. Within the suspension, the calcium
ions com~ete with the interior polycationic polymer comprising
the membrane for anionic sites on the polyanionic polymer.
5imultaneously, the stripping polymer dissolved in the solution
competes wi~h the polyanionic gum in the membrane for cationic
~0 site~ on the polycationic polymer. This results in a water-
dispersible or preferPably water-soluble complex of, e.g~, poly-
lysine and the polyanionic polymer, and in association of the
cations with gel molecules.
This step renders the membrane susceptible to
subsequent exposure to a sequestering agent which completes the
disruption process by taking up di or trivalent ions from the
gel. Typically, capsule membrane debris, if any, which remains
in the medium can be separated easily from the cells.
-15-
1 The currently preferred ~olution for the first stage of
the selective disruption process comprises 1.1~ calcium cloride
(w/v) and between 500 to 1,500 units of heparin per milliliter
of solu~ion. A volume of microcapsules is added ~o this solution
sufficient to constitute between about 20~ and 30~ of the total
volume of suspensionO Calcium chloride and heparin are preferred
when disrup~ing membranes of cell-containing capsules since both
reagents are physiologically compatible with most cells and mini-
mize the possibility of cell damage. Mixtures of aluminum salts
or other multivalent cations ~but not Mg+~ ions) may also be used
together with the polysulfonic or other acid salt of the type set
~orth above.
In general, the concentration of the ions ~nd anionic
polymer in the solution used in this step may vary widely.
Optimum concentrations may be readily determined empirically.
The lowe~t operable concentration for a particular batch o~
~ncap~ula~ed cell~ is preferred.
The curren~ly preferred sequestering agent for per-
forming the selective disruption is sodium citrate, although
other allcali metal citrate salts and alkali metal EDTA may also
be used. When sodium citrate is employed, the optimum con~
centration is on the order of 55 mM. Where the cap~ule membranes
being disrupted contain viable tissuel it is preferred that the
citrate be dissolved in isotonic saline so as to minimize cell
darnageO
The inverltion will be further understood from the
following non-limiting examples.
-16-
1 Capsule Formation
Exam~le_l: Encapsulat.ion of Panc eatic Tissue
Islets of Langerhans are obtained rom rat pancreas and
added to a complete tissue culture (C~RL-1969 Connaught
Laboratories, Toronto, Canada) a~ a concentration of approxima-
tely 103 i51et3 per rnilliliter, The tissue culture contains all
nutrients needed for continued viability of the islets as well as
the amino acids employed by the cells for making hormones. Four-
tenths of a milliliter of an islet suspension containing approxi-
mately 103 islets per milliliter is then added to a one-half
milliliter volume of 102 percen~ sodium alginate ~Sigma Chemical
Company) in physiological saline.
Next, a 1.5 percent calcium chloride solution is used
to gel droplets on the order of 300-400 microns in diameter.
After the supernatant solution is removed by aspiration, the
~lled droplets are transferred to a beaker containing 15 ml o a
~olution cornpri~ing one part of a 2% 2 (cyclohexylamino) ethane
sulfonic acid buffer solution in 0~6~ NaCl (isotonic, pH-8,2)
diluted with 20 parts 1% CaCl~. After a 3 minute immersion, the
capsules are washed twice in 1% CaC12.
The capsules are then transferred to a 32 ml solution
comprising 1/80 of one percent polylysine (average MW 35,000 amu~
in physiological saline. After 3 minutes~ the polylysine solu~
tion is decantedO The capsules are washed with 1% CaC12, and
optionally re~uspended for 3 minutes in a solution of polyethyl-
eneimine (MW 40,000-60,000) produced by diluting a stock 303~
polyethyleneimine solution in morpholino propane sulfonic acid
bufer (0.2M, pH=6) with sufficient 1% CaCl~ to result in a final
-17-
1 polymer concentration of 0 D 12~. The re~u~ting capsules, having
"permanent" semipermeablP membranes, are then washed twice with
1% CaCl ~, twice with physiological saline/ and mixed with 10 ml
of 0.12 percent alginic acid solution.
The capsules resist clumping, and many can be seen to
contain islet~ of Langerhans. Gel on the in~erior of the cap-
sules is reliquified by immersing th~ capsules in a mixture of
~aline and citrate buffer (pH-7~4) for 5 minutes~ Lastly, the
capsules are suspended in CMLR-69 medium.
Under the microscope, these capsules are observed to
compri e a thin membrane w~ich encircles an islet within which
individual cells can be seen. Molecules having a molecular
weight up to about one-hundred thousand can traverse the membra-
nes. This allows oxygen, amino acids, nutrients, and plasma com-
pon~nts used in cul~ure media (e.g,, lower molecular weight fetal
cal~ serurn co~nponents) ko reach khe islek and allows insulin to
be excreted.
Exam~le 2. _Encapsulation of Hepatocyte~
The procedure of example 1 is repea~ed except ~ha~ 0.5
ml of a liver cell suspension containing about 105 cells per
milliliter is used in place of the 0.4 ml islet suspension. The
ongoing viability of the liver cells has been demonstrated by the
dye exclusion technique (~rypan blue exclusion) and by their
observed ability to con~inuously produce urea. It is known that
liver tissue, in vitro, can ingest toxins from its environmen~.
Accordingly, toxins of a molecular weight low enough to pass
t~rough khe semipermeable membranes are detoxiied by the cells.
-18-
1 Example 3_ Activated Charcoal Encaps la~ion
The procedure of exarnple l is repeated except that par-
ticulate activated charcoal is suspende~ directly in the sodium
alginate solution, the half milliliter of tissue suspension is
omitted, and polylysine of of an average rnolecular weight of
35,000 is used as a cross-linkerG As long as the charcoal par-
ticles are smaller than the smallest inside diameter of the
capi.llary used to produce the dropl~ts, charcoal of high surface
area surrounded by a semipermeable membrane resultsO These
effectively prohibit the escape of charcoal chips or dust, yet
can be used to absorb materials of any pre-selec~ed molecular
weight ranye from fluid passed through the capsules~
4: Enca~sulation of Human Fibroblasts
Human fibroblasts obtained by treating human foreskin
tiRsue with trypsin and EDTA for 5 minutes at 37C in a known
manner are suspended in a complete growth medium (CML,R 1969,
C~nnaught Laboratories) ~upplemented with 40% (v/v) puriEied
fetal calf ~erurn, 0.8% sodium alginate (Sigma) and 200 mg/ml
puri1ed cal~ skin collagen. The density of the cell suspension
is about 1.5 x 107 cells/ml. Temporary alginate capsules are
formed as set forth abovel Semipermeable membranes are deposited
in surface layers of the capsules by suspending them in a .005~
~w/v) aqueous solution of poly L lysine, (MW 43,000 daltons) for
3 minutes.
Th~ resulting ~apsules are suspended in CMLR-1969
P~upplemented with 10% fetal calf serum. The foregoing steps are
all conducted at 37C. After incubation at the same tempera~ure,
the capsules, if examined under the microscope, will be found to
-lg-
1 contain fibrobla~ts which have undergone mito~is and display
thr~e-dimensional fibroblastic morphology within the microcap-
sules.
Selective Dlsru~tion of the Membranes
Exa~le 5
._
Microcapsules from any of examples 1-4 may be treated
as follows in order to ~electively disrupt the capsule membranes
without damage to the encapsulated core material.
10 ml por~ions of microcapsule suspensions containing
about 500-5000 capsules per ml are allowed ~o se~tle and the
suspension mediurn is aspirated ofq The capsules are washed
twice with saline. The washed capsules are then mixed with a 300
ml aliquot of saline con~aining heparin in various concentrations
a~ set forth below and 1.1% (w/v) CaC12. Capsules having algi-
nate enclosed therewithin, on completion of this step, display a
c3elled, ~hape-retairling interior core. The ~uspension i8 agi-
tated at 37C for 10 minutes, after which the capsules are
allowed to settle, the supernatent is a~pirated off~ and the cap-
sules are washed twice with 3.0 ml of 0.15M NaCl. After aspira-
tion of the second wash solution, the capsules are mixed with 2.0
ml of a mixed solution comprising equal volumes of 110 mM sodium
citrate and 0.15 M NaCl (pH~-7~4).
Capsule membranes which had been treated with 1,000
u~its/ml heparin and vortexed in the NaCl-NaCitrate solution for
1 minute were completely disintegrated~ The same reault is
achieved with capsules treated with 2,000 units/ml hep~rin for 2
rninutes, followed by 15-30 seconds o hand vortexingO Lower
-20-
~f~
1 concentrations of heparin are preferred as the possibility of
cell damage is decreased.
Af~er dissolution of the mer~ranes any membrane debris
may be removed by aspiration and washing. After ~he released
cells are resuspend~d in cul~ure medium, they may be tested by
the ~ryptan hlue dye exclusion technique and will be found to be
in a healthy, viable conditionl with relatively few cells exhi-
biting dye up~ake.
Capsules produced in accordance with example 3 are
treated, ater washing, with a 3.0 ml solution containing 1,000
units/rnl heparin and 1.0~ AlC13 for 6 minutes with agi~ation.
After aspira~ion of the supernatant, the core material is
released by vortexing the capsules wi~h a O.lM solution of sodium
ci~rate for 30-90 seconds.
'rhe procedure of example 6 i 8 repeated except that
O.lOM EDTA (sodiurn form~ at a pH of 7.0 is used in place of the
sodium citrate, resulting in rapid disruption of the capsule
membranes.
Example 8
Cap~ules produced in accordance with example 3 are
treated, after washing, with a 3.0 ml aqueous solution containing
10 mg/ml of polyvinyl ~ulate (mw approximately 50,000 daltons)
and 1~ CaCl~. Post tre~tment with O.lOM sodium citrate results
in essentially cornplete dissolution o the capsules.
~21-
Other embodimentg are within ~he following claims.
What is claimed is:
--22--
