Note: Descriptions are shown in the official language in which they were submitted.
1~7Z~61
1 8ackground of the Invention
This invention relates to a method of producing bio-
logical material6 of the type generated by cells.
Advance6 in cellular biology have shown that the cells
of various higher organisms produce 6mall quantitie6 of
substances having significant potential or demon6trable utility
for the treatment or diagno6is of disease. Examples of such
; 6ubstances abound in the literature and include various
biological response modifiers such as hormones, interferons, and
lymphokine6, as well as other sub6tance6 such as antibodies used
in diagnostic testing. Cell cultures of microbial origin have
long been used to produce large quantities of antibiotics.
Especially in cell cultures derived from higher
animal6, there i6 an ever pre6ent danger of bacterial or other
contamination. Al60, in most in6tance6 the quantitie6 of the
substance of interest produced by cell cultures are very small
and collect in the culture medium which contains a complex
mixture of serum proteins and other substances. This make
isolation and purification of the substance of interest
difficult.
~'
--2--
1~7~3ti:1
1 SummarY of the Invention
This invention provide6 a system and process for pro-
ducing substances which are produced by living cells. The
practice of the invention has ~he inherent dual advantages of
providing a protective environment for the cells of the culture
and providing a means of collecting sub6tance6 of interest in a
medium having fewer admixed extraneous component~. The invention
may be used to separate the 6ubstance of interest from higher
molecular weight serum proteins and the like normally required to
support the ongoing viability and metabolism of the producing
cell6. Alternatively, the invention may be used to collect the
substance of interest in a medium containing relatively small
quantitie~ of low molecular weight nutrients or cell metabolic
products.
The process comprises the steps of encapsulating cells
within a membrane which is permeable to the nutrients, ions, and
other relatively low molecular weight material~ needed for normal
metabolism and ongoing viability of the cell~. The membrane may
or may not be permeable to the substance of interest secreted by
the cells, but in any case will have an upper limit of
permeability sufficient to allow traver6e of molecules having a
molecular weight of some selected level generally below about
2.0 x 105 dalton6. The cap6ules 80 produced are suspended in a
conventional aqueous culture medium, and the encapsulated cells
are allowed to undergo normal in vitro metabolism. Sub6tances of
a molecular weight below the upper permeability limit of the
membrane which are secreted by the cells permeate the membrane
and collect in the culture medium. Advantageou61y, high molecu-
lar weight substances ~uch a6 serum protein~ which are required
~L'7;~
1 for health and viability of many types of cell cultures from
higher animals, but which typically are themselves not consumed,
may be included in the microcapsules where they are confined and
prevented from diffusing into the culture medium. Substances
which the cell culture consumes during metabolism having a mole-
cular weight low enough to permit diffusion through the capsule
membranes pa~s therethrough from the culture medium. Metabolic
products of the cells having molecula, dimension 6ufficiently
small to allow pagsage through the membrane diffuse into the
medium external to the capsules. The substances of interest, if
of a molecular weight below the upper limit of permeability, dif-
fuse into the extracapsular medium where they can be harvested
relatively easily because of the absence of contaminating higher
molecular weight materials present in prior art unencapsulated
cell cultures. If the substance of interest has a molecular
weight in excess of the upper limit of permeability of the
membranes, then it collects in the capsules which may sub-
sequently be isolated from the medium and disrupted for recovery
procedures .
The invention is essentially unlimited with respect to
the types of cells which may be included within the capsule
membranes. Specifically, it is contemplated that cultures of
cells from the tissue of all higher animal~ as well as micro-
organisms may be employed. Fused cells, e.g., hybridoma cells,
or genetically modified cells produced, for example, by the
emerging recombinant DNA technology, can likewise be encapsulated
without difficulty. In short, provided there exists a culture
medium operable to maintain in vitro the cell type in question,
that cell type can be utilized in accordance with the techniques
1~ 7~tj~
1 disclosed herein. Non-limiting examples of the types of substan-
ces that may be produced in accordance with the proceæs and by
the system of the invention include insulin, glycogen, prolactin,
60matostatin, thyroxin, steroid hormones, pituitary hormones,
interferons, folliclegtimulating hormones (FSH), PTH, and antibo-
dies .
The system of the invention comprises encapsulated
viable cells suspended in an aqueous culture medium. The encap-
sulated cells comprise membranes characterized by an upper limit
of permeability sufficient to allow traverse of the nutrients
needed for cell metabolism and ongoing viability. The membranes
enclose viable cells dispo6ed in a medium which includes all
components needed to maintain metabolism of the cells and which
are of a size range in excess of the upper permeability limit of
the membrane. The culture medium comprises components needed to
maintain viability of the cells which have a molecular weight
below the upper permeability limit of the membranes.
Accordingly, an object of the invention i0 the provi-
sion of a system and method for producing biological materials of
the type produced by cells. Another object of the invention is
to provide such a system wherein the producing cells are con-
tained within a protective, healthful microenvironment confined
by a semipermeable membrane which serves to separate products of
cell metabolism from high molecular weight materials needed for
viability and maintenance of the cells. Another object is to
provide an improved process for producing biologically active
materials from cell cultures. Yet another object i8 to produce
antibodies and biological response modifiers such as hormones,
interferons, and lymphokines in a serum-free medium.
--5--
.~
1 These and other objects and features of the invention
will be apparent from the following description and from the
drawing wherein Figure 1 is a schematic diagram illustrating the
concept of the invention and Figure 2 is a graph ~howing the
results of the experiment de6cribed in example 5.
~ ~L7;~
1 Descri~tion
The broad concept of the invention is to interpose a
semipermeable membrane about individual cells or groups of cells
so as to provide a microenvironment for the cells complete with
the cell culture medium and separated by the membrane from an
external aqueous medium. Cell6 of mammalian origin typically
require for ongoing health and viability the presence of serum
proteins, a portion of which have a molecular weight in excess of
about 65,000-150,000 daltons. In the prior art technique of un-
encapsulated cell culturing, materials of interest secreted from
the cells are dispersed in the culture medium and mixed with both
high and low molecular weight components. Since the quantities
of cell-produced products are typically rather small, isolation
of the substance of interest becomes an arduous purification
task. Furthermore, mammalian cell cultures are notoriously sen-
sitive to contamination by bacterial or other sources. This
necessitates that culturing be conducted using various techniques
to maintain sterility and often that antibiotics be included in
the medium.
:;
According to the practice of this invention, the
foregoing difficulties are alleviated by encapsulating the cells
of the culture within semipermeable membranes having a selected
limit of permeability generally no greater than about 200,000
daltons, that is, the mernbrane contains pores which allow
substances having a maximum molecular weight at or below the
upper permeability limit to traverse the membrane whereas
substances of molecular weight above the upper permeability limit
are precluded from traversing the mernbrane. This allows one to
encapsulate cell3 together with a culture medium containing all
--7--
1~7;~61
1 componentg needed for ongoing viability, metab~lism, and even
mitosis, and then to suspend the so encapsulated cells in a
culture medium which contains lower molecular weight substances
consumed by the cells but which need not include the required
high molecular weight substances.
Typically, cells from higher organisms do not ingest
high molecular weight serum proteins and the like, but rather
require them in close proximity for ongoing normal biological
responses. Salts, amino acids and other lower molecular weight
factors which are ingested or metabolized by the cells pass
freely through the membrane and may be replenished as needed by
simple change of the culture medium external t~ the capsules.
Secreted products of cell metabolism having a molecular weight
below the upper limit of membrane permeability collect in the
extracapsular medium, where, because of the absence in the medium
of the high molecular weight materials, harvesting and isolation
of the metabolic product~ of interest are simplified. Harvesting
of products of interest having a molecular weight above the upper
permeability limit is also aided in that such products collect
within the capsules and are not dispersed in the extracapsular
volume.
The concept of the invention, as applied to lower
molecular weight cell products, is schematically illustrated in
the drawing. As shown, a cell 10 is disposed within a capsule
membrane 12 having pores 16. High molecular weight factors 18
are enclosed within membrane 12 and are free to circulate within
the confines of the membrane in the medium 14. Components 20
needed by the cell as well as metabolic products 22 including the
substance of interest 22' freely circulate in both the
13~7i~61
1 intracapsular and extracapsular medium and traverse the membrane
through pores 16. As required on a periodic (or continuous)
basis, the extracapsular medium together with all of its
components can be separated by aspiration or the like from the
capsules themselves and replaced with fresh medium. The
collected medium will be substantially free of high molecular
weight component~ 18, thus simplifying the harvesting and
isolation procedures. Furthermore, the cell 10 remains protected
within the intracapsular microenvironment at all times.
In 60me cases, e.g., in order to stimulate production
by encapsulated cells of a particular substance of interest, it
i8 required to subject the cells to high molecular weight com-
ponents having molecular dimensions too large to traverse the
membrane. An example is the production of interferon from human
fibroblasts, leukocytes, or lymphoblastoid cells which are
induced to secrete interferon by treatment with certain viruses
or high molecular weight nucleic acids. In such a case, if the
upper permeablity limit of the membranes is less than the
molecular weight of the inducing factor, the cells must be
subjected to interferon induction prior to encap6ulation, or the
capsule membranes, after culture of the cells, must be
~ selectively disrupted to allow such high molecular weight
~ can~lJ~
tJ~ materials to be ingested by the cell. Copending~application
Serial No. ~ , discloses a method of selectively disrupting
certain types of capsule membranes which may be used for these
and other purposes without damage to the cells.
The process of the invention depends on one's ability
to form semipermeable membranes about cells without simultane-
ously adversely affecting their ongoing viability. One suitable
encapsulation process is set forth in detail below.
1 Cell Encapsulation
The tissue or cells to be encapsulated are suspended
in an aqueous medium suitable for maintenance or for supporting
the ongoing metabolic processes of the particular tissue or
cell type involved. Media suitable for this purpose are
available commercially. The average diameter of the material
to be encapsulated can vary widely between a few micrometers to
several millimeters. However, best results are achieved with
capsules of a size in the range of 300-1000 micrometers.
10 Mammalian islets of Langerhans are typically 50 to 200 micro-
meters in diameter. Individual cells such as fibroblasts,
- leukocytes, lymphblastoids, pancreatic beta cells, alpha cells,
delta cells, various ratios thereof, or other tissue units
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
dependent, inter alia, on the availability of required nutrients,
oxygen transfer, absence of toxic substances in the medium,
and the pH of the medium. ~eretofore, it has not been possible
to maintain such living matter in a physiologically compatible
environment while simultaneously encapsulating. The problem
has been that the conditions required for membrane formation
have been lethal or harmful to the tissue, and prior to the
invention of Canadian application Serial No. 384,524, now
Patent No. 1,145,258, no method of membrane formation which
allowed tissue to survive in a healthy state had been
forthcoming.
However, it has been discovered that certain water-
soluble substances which are physiologically compatible with
.~
-- 10 --
~3L7~$~1
1 living tissue and can be rendered water-insoluble to form a
shape-retaining, coherent mass, can be used to form a "temporary
capsule" or protective barrier layer about individual cells or
groups of cell~ and that this temporary capsule can be treated to
deposit a more permanent semipermeable membrane about the cells
without damage to the cells. Such a substance is added, typi-
cally at a concentration on the order of a few weight percent, to
the tissue culture medium, which also contains cells of the
culture, serum components (if required) and optionally, a cellu-
' 10 lar substr~te such as collagen or another high molecular weight,
water dispersible material which acts as an anchoring substrate.
When using collagen, the concentration should be within the range
of about 10 ug/ml to about 1 mg/ml, but preferably on the order
of 100-500 ~g/ml.
The solution is then formed into droplets containing
tis~ue together with its medium and is immediately rendered
water-insoluble and gelled, at least in a surface layer.
Thereafter, the shape-retaining temporary cap~ules are provided
with a more permanent membrane which may itself ~ubsequently be
6electively disrupted if it i6 desired to release the tissue
without darnage. Where the material used to form the temporary
capsules permits, the capsule interior may be reliquified after
formation of the permanent membrane. This is done by re-
establishing the conditions in the medium at which the material
is soluble.
The material used to form the temporary capsules may be
any non-toxic, water-soluble material which, by a change in ionic
environment or concentration, can be converted to a shape-
retaining mass. The material should also contains plural, easily
~7~
1 ionized anionic moieties, e.g., carboxyl groups, which can react
by salt formation with polymers containing plural cationic
groups. As will be explained below, use of this type of material
enables one to deposit a permanent membrane of a selected upper
limit of permeability without difficulty in surface layers of the
temporary capsule.
The presently preferred materials for forming the tem-
porary capsule are acidic, water-soluble, natural or synthetic
polysaccharide gums. Such materials are commercially available.
They are typically extracted from vegetable matter and are often
used as additives to various foods. Sodium alginate is the
presently preferred water-soluble gum. Alginate in the molecular
weight range of 150,000~ daltons may be used, but because of its
molecular dimensions and viscosity will usually be unable to per-
- meate the finally formed capsule membranes. Lower molecular
weight alginate, e.g., 50,000-80,000 daltons, is more easily
removed from the intracapsular volume by diffusion through a
membrane of sufficient porosity and is therefore preferred.
Other useable gums include acidic fractions of guar gum, cara-
geenan, pectin, tragacanth gum, or xanthan gum.
These materials comprise glycoside-linked saccharide
chains. Their free acid groups are often present in the alkali
metal ion form, e.g., sodium form. If a multivalent ion such as
calcium or aluminum is exchanged for the alkali metal ion, the
water-soluble polysaccharide molecules are "cross-linked" to form
a water-insoluble, shape-retaining gel which can be resolublized
on removal of the ions by ion exchange or via a sequestering
agent. While essentially any multivalent ion which can form a
salt with the acidic gum is operable, it is preferred that phy-
-12-
1~7~61
1 siologically compatible ions, e.g., calcium, be employed This
tends to preserve the tis6ue in the living state. Other multiva-
lent cations can be used. Magnesium ions are ineffective in
gelling sodium alginate.
A typical solution composition comprises equal volumes
of a cell culture in its medium and a one or two percent solution
of gum in physiological saline. When employing sodium alginate,
a 1.2 to 1.~ percent solution has been used with success. If the
cells to be encapsulated are of the type which require attachment
to an anchoring substrate to undergo mitosis, and if the cells
are to be grown within the capsules, then collagen or another
high molecular weight water-dispersible protein or polypeptide,
either natural or synthetic, may be included in the cell culture,
and will be confined within the intracapsular volume of the
finally formed capsules. If a polymer having plural cationic
groups, e.g., polylysine, i~ employed for this purpose, the
cationic groups react with anionic sites on the water-soluble gum
to form a sub6tantially water-insoluble matrix intertwined with
the gum. Preferred concentrations for such materials are on the
order of 100-500 ug/ml of suspension (including gum solution).
In the next step of the encapsulation process, the gum
solution containing the tissue iB formed into droplets of a
desired size. Thereafter, the droplets are immediately gelled to
form shape-retaining masses preferably but not
necessarily in spherical or ~pheroidal form. The drop formation~
may be conducted by known methods. An exemplary procedure
follows .
A tube containing an aqueous solution of multivalent
cation~, e.g., 1.5~ CaC12 solution, is fitted with a stopper
1~7~6~
1 which holds a drop forming apparatus. The appartus consists of a
housing having an upper air intake nozzle and an elongate hollow
body friction fitted into the stopper. A 10 cc syringe equipped
with a stepping pump is~ mounted atop the housing with, e.g., a
-f~-f~ ~3,y
~~~ O.01 inch I.D.-Tcflon coated needle pagsing through the length of
the housing. The interior of the housing is designed such that
the tip of the needle is subjected to a constant laminar air flow
which acts as an air knife. In use, with the syringe full of
solution containing the material to be encapsulated, the stepping
pump is actuated to incrementally force droplets of solution from
the tip of the needle. Each drop is "cut off" by the air stream
and falls approximately 2.5 cm into the CaC12 solution where it
is immediately 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 alginate/cell suspension to assume the most physically
favorable shape; a sphere (maximum volume for minimum surface
area). Air within the tube bleeds through an opening in the
stopper. This results in "cross-linking" of the gel and in the
formation of a high viscosity shape-retaining protective tem-
porary capsule containing the suspended tissue and its medium.
The capsules collect in the solution as a separate phase and may
be separated by aspiration.
In the next step of the process, a semipermeable
membrane is deposited about the surface of the temporary capsules
by "cross-linking" surface layers. This may be effected by
subjecting the gelled temporary capsules to an aqueous solution
of a polymer containing cationic group~ reactive with anionic
functionalities in the gel molecules. Polymers containing acid
/~`c~ f~
-14-
:~'
.
1~7~$t;1
1 reactive groups such as free imine or amine groups are preferred.
In this situation, the polysaccharide gum is crosslinked by
interaction (salt bond formation) between the carboxyl groups and
the arnine or imine groups. Permeability can be controlled within
limits by selecting the molecular weight of the cross-linking
polymer u6ed and by regulating the concentration of the polymer
solution and the duration of exposure. A solution of polymer
having a low molecular weight, in a given time period, will
penetrate further into the temporary capsules than will a high
molecular 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 the pore size. Broadly, polymers within the
molecular weight range of 3,000 to 100,000 dalton6 or greater may
be used, depending on the duration of the reaction, the con-
centration of the polymer solution, and the degree of permeabil-
ity desired. One successful set of reaction conditions, using
polylysine of average molecular weight of about 35,000 daltons,
involved reaction for two minutes, with stirring, of a physio-
logical saline solution containing 0.0167 percent polylysine.
This results in membranes having an upper limit of permeability
of about 100,000 daltons. Optimal reaction conditions suitable
for controlling permeability in a given system can readily be
determined empirically in view of the foregoing guidelines.
Using this method it is possible to set the upper permeability
limit of the membranes at a selected level below about 200,000
daltons.
Examples of suitable cross-linking polymers include
proteins and polypeptides, either natural or synthetic, having
'
'7~
1 free amino or imino groups, polyethyleneamines, polyethylene-
imines, and polyvinyl amines. Polylysine, in both the D and L
forms, has been used with 6uccess. Proteins such as polyarge-
nine, polycitrulline, or polyornithine are also operable.
Polymers in the higher range of positive charge density, e.g.,
polyvinylamine, vigorously adhere to the anionic groups of the
gel molecules to form Qtable mernbranes, but the membranes are
rather difficult to disrupt.
Treatment with a dilute solution of gum will tie up
free amino groups on the surfaces of the capsules which otherwise
may impart to the capsules a tendency to clump.
At this point in the encapsulation, capsules may be
collected which comprise a semipermeable membrane surrounding a
gelled solution of gum, cell-type compatible culture medium,
cells, and optionally an internal matrix of collagen or another
anchorage substrate. Since mass tran6fer should be promoted
within the capsules and across the membranes, it is preferred to
reliquify the gel to its water-soluble form. This may be done by
re-establishing the conditions under which the gum is a liquid,
e.g., rernoving the calcium or other multifunctional cations from
the interiox gel. The medium in the capsule can be resolubilized
simply by immersing the capsules in phosphate buffered saline,
which contains alkali metal ions and hydrogen ions. Monovalent
ions exchange with the calcium or other multifunctional ions
within the gum when the capsules are immersed in the solution
with stirring. Sodium citrate 601utions may be used for the same
purpose, and serve to sequester the divalent ions.
Cell cultures encapsulated as described above may be
suspended in culture media designed specifically to satisfy all
-16-
1~7 '~1
1 of the requirements of the particular cell type involved and will
continue to undergo normal in vitro metabolism. If the culture
requires an environment of high molecular weight components such
as ~erum components, these may be omitted from the extracapsular
medium. Typically, the components normally ingested by cell6 are
of relatively low molecular weight and readily diffuse across the
cap6ule membranes into the microenvironment of the cell6 where
they permeate the cell membrane. Products of metabolism of the
cells which are 6ecreted into the intracapsular medium, if they
have a molecular weight below the upper limit of permeability of
the capsule membrane, likewise diffu6e thereacross and collect in
the extracapsular medium.
The encapsulated cell6 may be cultured under conditions
of, e.g., temperature, pH, and ionic environment, identical to
conventional cultures. Al60, cell-produced products may be har-
vested from the extracap6ular medium or from the cap6ules by con-
ventional techniques. However, the culturing technique di6closed
herein has the following advantages:
1. The cells of the culture are protected from
shearing forces and mechanical damage and from contamination by
factors having dimensions in excess of the upper permeability
limit of the membranes. This means that handling and sterility
re~uirements normally incident to culturing procedures can be
somewhat relaxed, since microorganisms cannot reach the encap-
sulated cells, and viru~ infected cells need not contaminate
other cells.
2. The cap6ules in effect immobilize the cells within
an environment in which enclosed high molecular weight materials
.,
-17-
~7;~
1 are confined, yet lower molecular weight cell nutrients and
products are readily removed and introduced. This allows the
nutrient broth to be intermittently or continuously collected and
supplemented as desired, without disturbing the cells.
3. Substances of interest produced by the cells are
more easily recovered. Secreted cell products of molecular
dimensions small enough to permeate the capsule membranes collect
in the extracapsular medium in admixture with nutrients.
However, high molecular weight serum components and the like are
not released into the extracapsular medium, thus simplifying
recovery of a cell product of interest. Secreted cell products
of molecular dimensions in excess of the upper permeability limit
of the membranes collect within the capsules. Of course, cell
products not secreted through the cell membrane may also be of
intere~t. These may be recovered in relatively concentrated form
by isolating the capsules and subsequently selectively disrupting
the capsule membranes using, for example, the technique disclosed
hereinafter, and if necessary by disrupting the cell membranes.
4. The intracapsular volume provides an environment
well suited for cell division. Su~pen~ion cultures have been
observed to undergo mitosis within the capsule. Anchorage depen-
dent cells which in normal cultures grow in a two-dimensional
monolayer multiply to form an array within the capsule. Such
cells use the interior surfaces of the membrane as a substrate
and/or anchor to the high molecular weight materials set forth
above which are disposed within the capsule. This leads to
significant increases in cell density as compared with conven-
tional cultures. The ongoing viability of such cell clusters is
aided by the fact that the surface area to volume ratios of the
-18-
~7~
capsules can be quite large, and thus all cells have access to
re~uired nutrients, oxygen, etc.
In certain situations it would be advantageous to
selectively disrupt the capsule membranes to release the cells
without damage. One notable example is in the production of
interferon (INF). Cells capable of producing INF must be sub-
jected to certain viruses or nucleic acids in preparation for the
INF production ~tage. Also, in several INF induction procedures,
reagents are added to the culture to inhibit protein synthesis.
Accordingly, the growth stage of the culturing process must be
conducted under conditions quite different from the INF induction
stage. If the substances used for INF induction are of a molecu-
lar weight in excess of the upper permeability limit of the cap-
sule membranes (as will be the case in virus inductions) the
induction process cannot be accompliqhed in the encapsulated cell
culture. Accordingly, INF producing cells, if grown within the
capsule, would have to be released by disruption of the membrane
in order to be subjected to the induction proce~.
Disruption of Membrane~
Cells confined in membranes of the type set forth above
rnay be released by a process involving cornmercially available
reagent~ having propertie6 which do not significantly adversely
affect the encapsulated cell~. First, the capsules are separated
from their suspending mediurn, washed thoroughly to remove any
; contaminants present on the exterior of the microcapsules, and
then dispersed, with agitation, in a mixed solution of monatomic,
multivalent cations such as calcium ions and a stripping polymer
having plural anionic moieties such as a salt of a polysulfonic
or polyphosphoric acid. Heparin, a natural sulfonated poly-
--19--
:1~7~
1 saccharide, is preferred for this step. The anionic charge den-
sity of the stripping polymer used should be equal to or
preferably greater than the charge density of the polyanionic
material originally employed to form the membranes. The molecu-
lar weight of the polymer should be at least comparable to and
preferably greater than the molecular weight of the polymer
having plural cationic groups used in forming the membrane.
Within the suspension of capsules in the mixed solution, the
calcium ions compete with the polycationic polymer chains used to
form the membrane for anionic sites on ~he water-soluble gum.
Simultaneously, the heparin or other polymer having plural
anionic moieties dissolved in the 601ution competes with the gum
in the membrane for cationic sites on the polymer chains. This
result6 in a water-dispersable or preferrably water-soluble com-
plex of e.g., polylysine and heparin, and in association of the
monatomic cations with molecules of the gel.
This step renders the membrane suseptible to dissolu-
tion upon subsequent exposure to a sequestering agent which
completes the disruption process by taking up monatomic ions from
; 20 the gel. Capsule membrane debris which remains in the medium, if
any, can be easily separated from the cells.
The currently preferred solution for the first stage of
the selective disruption process comprises 1.1~ calcium chloride
(w/v) and between 500 to 1,500 unit~ of heparin per milliliter of
solution. A volume of microcapsules is added to this solution
sufficient to constitute between about 20% and 30~ of the total
volume of suspension. Calcium chloride and heparin are preferred
for disrupting membranes of cell-containing capsules since both
reagents are physiologically compatible with most cells and
-20-
1~7 ~6i
1 therefore minimize 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 polyphosphoric
acid 6alts of the type 6et forth above.
In general, the concentrations of monatomic ions and
anionic polymer used in this step may vary widely. Optimum con-
centrations may be readily determined empirically, and depend on
exposure time as well as the particular polymer used to form the
membranes .
The currently preferred sequestering agent for per-
forming the selective disruption is sodium citrate, although
other alkali metal citrate ealts and alkali metal EDTA salts may
also be used. When sodium citrate is employed, the optimum con-
centration is on the order of 55 mM. It i8 preferred to di6solve
the citrate or other sequestering agent in isotonic saline so as
to minimize cell damage.
.
The invention will be further understood from the
following non-limiting examples.
Example 1: Insulin Production
Islets of Langerhans are obtained from human cadaver of
animal pancreas and added to a complete tissue culture (CMRL-1969
Connaught Laboratories, Toronto, Canada) at a concentration of
approximately 103 islets per milliliter. The tissue culture con-
tains all nutrients needed for continued viability of the islets
a~ well as the amino acids employed by the Beta cells for making
insulin. Four-tenths of a milliliter of a 10-~ islet per milli-
- liter suspen~ion is then added to a one-half milliliter volume of
1.2 percent sodium alginate (Sigma Chemical Company) in
physiological saline.
-21-
:~P~ 61
1 Next, a 1.5 percent calcium chloride solution is used
to gel droplets of the solution formed as set forth above.
Droplets on the order of 300-400 microns in diameter emanating
from the tip of the needle immediately gel upon entering the
calcium solution. The gelled capsules are then transferred to a
beaker containing 15 ml of a solution comprising 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% CaC12. After a
3 minute immersion, the capsules are washed twice in 1~ CaC12.
The capsules are then transferred to a 32 ml solution
coraprising 1/80 of one percent polylysine (average MW 35,000
daltons) in physiological saline. After 3 minutes, the poly-
lysine solution is decanted. The capsules are washed with 1%
CaC12, and optionally resuspended for 3 minutes in a solution of
polyethyleneimine (MW 40,000-60,000) produced by diluting a stock
3.3% polyethyleneimine solution in morpholino propane sulfonic
acid buffer (0.2M, ph=6) with sufficient lX CaC12 to result in a
final polymer concentration of 0.12~. The resulting capsules,
having "permanent" semipermeable membranes, are then washed twice
with 1% CaC12, 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 islets of Langerhans. Gel on the interior of the cap-
sules i8 reliquified by immersing the capsules in a mixture of
saline and citrate buffer (pH-7.4) for 5 minutes. Lastly, the
capsules are suspended in CMLR-69 medium.
Vnder the microscope, these capsules are seen to con-
sist of a very thin membrane which encircles an islet within
-22-
1 7;~
1 which individual cells can be seen. Molecules having a molecular
weight up to about one-hundred thousand can traverse the
membranes. This allows oxygen, amino acids, nutrient~, and
plasma components used in culture media (i.e., lower molecular
weight fetal calf plasrna components) to reach the islet an
allows insulin to be 6ecreted.
After repeated washings in physiological saline, micro-
capsules made in accordance with the above procedure containing
approximately 15 iglets are suspended in 3 milliliters of CMRL-
1969. When eight days old, in the presence of 600 mg/dl glucose,
the capsules secreted into the extracapsular medium, in one run,
67 units/ml insulin in 1.5 ho~rs. In a second run, 68 units/ml
insulin were produced in the same amount of time. One week old
capsules, in the same medium, but in the presence of 100 mg/dl
glucose, in a first run, secreted 25 units/ml insulin in 1.2
hours, and in a second run, secreted 10 units/ml.
^ Exam~le 2: INF-~ Production
Human fibroblasts obtained by treating human foreskin
tissue with trypsin and EDTA for 5 minutes at 37C in a known
~ C~R~
manner are suspended in a complete growth medium (~Mb~ 1969,
Connaught Laboratories~ supplemented with 40% (v/v) purified
fetal calf serum, 0.8% sodium alginate (Sigma) and 200 ug/ml
purified calf skin collagen. The density of the cell suspension
is about 1.5 x 107 cells/ml. Temporary alginate capsules are
formed as set forth in Example 1. Semipermeable membranes are
deposited in surface layers of the capsules by suspending them in
a .005~ (w/v) aqueous solution of polylysine, (MW 43,000 daltons)
for 3 minutes.
1~7~
"~ c r: ~ ~
~ The resultinq capsules are suspended in eM~-1969
supplemented with 10~ fetal calf serum. The foregoing steps are
all conducted at 37C. After incubation at the same temperature,
the capsules, if examined under the microscope, will be found to
contain fibroblasts which have undergone mitosis and display
three-dimensional fibroblastic morphology within the microcap-
sules .
After 4-5 days of incubation, the encapsulated
fibroblasts are subjected to an INF-p superinduction techni~ue
according to the Vilcek procedure. Under a 5~ CO2 atmosphere
(95~ air), the capsule suspension is incubated at 37C for one
hour in the presence of lOO ug/ml Poly I-Poly C, a double
stranded RNA (known INF-~ inducer) available from PL
Biochemicals, Milwaukee, Wiscon~in and 50 ~g/ml cycloheximide
(protein synthesis inhibitor, Calbiochem, La Jolla, California.)
After one hour, the suspended capsules are washed in medium
~ RI
(CMLR 1969) containing 50 ,ug/ml cycloheximide and then
resuspended in the same solution for 3 hours at 37C under a 5%
C2 atmosphere. At the completion of this incubation the
washing step is repeated and the capsules are resuspended in
medium containing 50 ug/ml cycloheximide and 5 ug/ml actimomycin
D (a known RNA synthesis inhibitor, Calbiochem) and incubated for
2 hours at 37C under a 5~ CO2 a~nosphere. The capsules are then
washed twice in medium and suspended in serum-free medium at 37~C
for 18-24 hours, during which time the fibroblasts secrete INF-~,
which has a molecular weight on the order of 21,000 daltons and
may be harvested from the extracapsular medium.
Example 3: INF-p Production
The procedure of Example 2 i~ repeated, except that
?c~ k.
-24-
1 ~7~1
1 prior to induction the capsule membranes are selectively
disrupted so that the Poly I-Poly C can more ea~ily gain access
to the fibroblasts. The disruption procedure is conducted as
follows.
10 ml portions of microcapsule suspensions containing
about 500-1000 capsules per ml are allowed to settle and the
suspen6ion medium is aspirated off. The capsules are washed
twice with phosphate buffered saline (PBS, pH=7.4). The washed
- capsules are then mixed with a 3.0 ml aliquot of PBS containing
1000 units/ml heparin and 1.1% (w/v) CaC12. The suspension is
agitated at 37C for 3 minutes, after which the capsules are
allowed to ~ettle, the supernatant is aspirated off, and the cap-
; ~ules 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 0.15M NaCl (pH=7.4). The mixture is hand vortexed
for 1 minute to induce dissolution of the membranes after which
cells are washed twice in medium.
The cells are then suspended in medium, ~ubjected to
the induction procedure set forth in example 2, and then re-
encapsulated as set forth in Example 2. The capsule suspension
is then incubated in serum-free medium for 18-24 hours, during
which time INF-~ is secreted from the cells, permeates the cap-
sule membranes, and collects in the extracapsular medium.
Examples 2 and 3, if conducted with Poly I-Poly C (5S)
(sedimentation value, Poly I and Poly C annealed to form double
ætranded RNA) result in the following INF-~ production levels, in
units of INF-~/105 cells:
-25-
~L7;~
1 Example 2 1. 2.
; 25 25
Example 3 2,500 2,500
Examples 2 and 3, if conducted with Poly I-Poly C (12S)
(sedimentation value, double stranded as purchased) result in the
following INF-P production levels, in units of INF-~ /105 cells:
Example 2 1. 2.
Example 3 2,500 2,500
The one-hundred fold increase in production using the procedure
of Example 3 over that of Example 2 is believed to be due, at
least in part, to the fact that the Poly I-Poly C has better
access to the cells in the Example 3 procedure.
Exam~le 4:
The procedure of Example 2 is repeated except that cap-
sules containing no collagen are employed. The encapsulated
cells were grown in conventional monolayer culture, treated with
trypin, and induced with Poly I-Poly C (5S) and microencapsulated
simultaneously. The extracap6ular medium is found to contain
2,500 units INF-~/105 cells.
ExamPle 5: Monoclonal Antibodies
Hybridoma cells obtained from Herman Eisen at MIT were
culture to a density of 3.0 x 106 cells/ml. These cells had been
fused from mouse pleen cells and mouse myeloma cells in a manner
now well known in the prior art and constituted an immortal cell
line which in culture produced antibodies against dinitrophenyl
-26-
1~72~61
bovine serum albumin. Three ml aliquots of the cell suspension
were made up by adding 2.1 ml of suspension containing 1.4%
sodium alginate to 0.6 ml fetal calf serum and 0.3 ml physiologi-
cal (150mM) saline. Droplets of the 6uspension were immediately
gelled in CaCl~ solution and then treated with a 0.016 weight
percent solution of poly L lysine. The interior of the resulting
capsules was then reliquified by immersion in a solution of one
part llOmM sodium citrate and three parts 150mM saline for 6
minutes. The capsules containing hybridoma cells were then
suspended in a mixture of RPMI-1640 medium (Gibco) containing 2096
heat inactivated fetal calf serum.
Cell counts of encapsulated and unencapsulated hybri-
ds)ma cultures, and the amount of monoclonal antibody produced by
both the encapsulated and unencap6ulated cultures were determined
periodically. The re6ults are set forth in graphical form in
Fig. 2.
Example 6: INF- o~ from Leukocytes
30 ml buffy coats obtained from the American Red Cross
were treated with 3.0 ml of 5% EDTA and repeated 10 minute
exposures to 0.8396 NH4Cl at 4C to lyse the red cells. A five
minute centrifuge (1200 rpm at 4C) between NH4Cl treatments
separated debris from the remaining intact leukocytes. The cells
were next suspended in MEM (minimum essential medium, ~erum free
- Gibco), diluted by a factor of 100, and stained with tryptan
blue for 15 minutes. A cell count conducted on a sample showed
that about 1.3 x 109 leukocytes per 30 ml buffy coat survived.
The cells were then suspended at a density of 1 x 107 cells/ml in
medium supplemented with 2% heat inactivated fetal calf serum.
11 ~7~
l Induction was effected by exposing the cell suspension
to Sendai virug (various concentrations in heamagglutinating
units/ml - Flow Laboratories, Md.) for one hour a~ 37C with
stirring. The virus was then separated from the cell by centri-
fugation at room temperature and the cells were resuspended in
equal volumes of MEM - 4~ heat inactivated fetal calf serum and
1.4~ sodium alginate. Capsules were formed as set forth above
and then regugpended in serum-free and seruM-containing media.
There were no 6ignificant differences in the ~uantities of INF
detected in the extracapsular medium of these test samples. INF
production levels were also identical in unencapsulated control
cultures. The results of these experiments are set forth below
Units Sendai Virus INF Produced
(HA Units/ml) Units 107 Cells
600 10
300 20
150 33
Example 7: INF ~ from Lymphoblastoids
Namalwa cells from the American Type Culture Collection
were grown both in conventional culture and within microcapsules
in RPMI-1640 medium supplemented with 10% heat inactivated fetal
calf serum. Volumes of the cell suspensions were then subjected
to INF induction and production procedures, with one volume
encapsulated and the other unencapsulated. The cultures con-
tained substantially equal numbers of cells. To both the encap-
sulated and unencapsulated cultures wa~ added 25 mg/ml bromo
deoxyuridine in double distilled water to inhibit mitosis. After
incubation for 36 hours at 37C, the cells of both cultures were
wa~hed and then suspended in RPMI1640 medium supplemented with 2%
heat inactivated fetal calf serum.
-28-
61
1 The encap6ulated culture was then treated to selec-
tively disrupt the capsule membranes. The capsules were washed
three times in phsiological saline incubated in 1000 units/ml
heparin solution containing 1.1% CaC12 for 10 minutes at 37C,
and then rewashed in saline. The washed capsules are next incu-
bated for 5 minutes at 37C with dilute sodium citrate solution
in physiological saline. Agitation of the cap6ule suspension at
this point results in dissolution of the membranes and release of
the Namalwa cells. The cell suspension is then centrifuged to
remove debris and washed several times in citrate/saline solu-
tion.
Both cultures were next suspended in fresh RPMI-1640
culture medium supplemented with 2% heat inactivated fetal calf
serum and buffer (pH = 7.4) at a density of 1.0 x 106 cells/ml.
To both the conventional culture and the formerly
encapsulated culture were then added the Bankowski strain of
Newcastle Disease Virus in amniotic fluid. The virus was at a
concentration of 1.0 x 108 pfu/ml and was purchased from Poultry
Health Laboratories, Davis, California. One ml of the virus was
added for each 10 ml of cell suspension. The cultures were incu-
bated for 24 hours at 37C.
The conventional culture was then divided into five
parts ~1-5 below); the formerly encapsulated culture was divided
into 4 partq (6-9 below). Each of the 9 aliquots of culture were
then assayed for INF production following the treatments set
forth below.
1. untreated
2. resuspended in RPMI-1640 medium with 2%
- heat inactivated fetal calf serum
-29-
13~7~
1 3. resuspended in RPMI-1640 medium serum-free
4. encapsulated together with RPMI-1640 medium and
5% heat-inactivated fetal calf serum-capsules
suspended in serum-free medium
5. encapsulated together with RPMI-1640 medium and
5% heat-inactivated fetal calf serum-capsules
suspended in medium with 2% fetal calf serum
6. resuspended in serum-free mediwn
7. resuspended in medium containing 2% heat-
1~ inactivated fetal calf serum
8. reencapsulated together with medium plus 5%heat-inactivated fetal calf serum-capsules
suspended in serurn-free medium
9. reencapsulated together with medium plus 5%
heat-inactivated fetal calf serum-capsules
suspended in medium plus 2~ serw~l
The following table sets forth the quantity of cells required in
each of the cell cultures 1-9 to produce 1 unit of INF ~ :
-30-
~7~6~
1 1 30 6 40
2 45 7 40
3 - 8 1000, 360
4 680 9 200, 100
. 5 2000
Other embodiments are within the following claims.
What i8 claimed is:
