Note: Descriptions are shown in the official language in which they were submitted.
~\
117Z5~36
~ckqround of th- ~n~ntlon
ln~-ntlon r-l~t-- to ~tho~ of eult~l ~51
~nch~- d~p-n~-nt c~ of e~- typ~ h
nor~lly ~-t ~ a ~ u~ t- u,a
o~nnot b gra~ 1~ u-~n~
al~vans-- ln ~llulu~ ~lolo~y ~uv- d ~ ~t th-
~ncho~g- d-p-~t ~all~ of vlo~ ~lp~ 11119 ~n ~uc-
~aîl qu~ntltl~- of r ~vl ~ lflc~t ~-ntl~l cr
tr bl- utlllty ~ t~nt of ~lo~-~, o.g., int~r-
10 f-on. ~uc~ o~ l-o u -ful ~o~ r~ h p~--.
In u~l ¢~11 oultur-- ~- 1- ~ ~ pro--nt ~ang-r
of b~ct-rlal o~ r co~t~lnatlon. Al-o, 11~ ~o t l~ n~
qu~ntlt~-~ of th~ tu- of lnt--t ~rotlu~l by ~
cultur-~ vy ~ lnc-, ~llk u~n-$on ~l~lt~--,
~ncho~gc n~t ~ # c~ltur~ to flll ~olu~,
th- 1- b~ d to ~ ~o ~ ntly ~ tln~ Factlc~l tho~ of
growln5~ l rg- J~ of ~11- r-~aulrd f~ ~o~uctlo~ of
lqnlfle~n~ quantltl-~l of o~b~o-- of ~t~-t ~ro~u6~tl by u~
e~
-- 2 --
~L17;~586
; 1 Curr-ntly, u~chorag- a ~-nt Q 11- uch ~
flbrcbla~t- ~Ad o rt~ln kl~ y U-a ~p~ t ~-
cultur~ on ub-tr~ la-tlo ~h- t- for ~ t--
~r- co rcl-lly av~ll bl-. ~ ty~- of uuhoras~ a-nt
o~ 11 r-~o~uo u~tll th~ t 1- oo~-r~ ~th ~ ~OAol~y r
of c-ll- ~ th-n o~-- ultl~lylaS~ - ~111 contlnu to
~ultl~ly to f~ A or ~o~- ~ltloAal lay~- ~ th- ~r~t.
- - ~ - . . , . r . ~
_, ( , . _
~L3 72S86
1 Summary of the Invention
This invention provides a method of culturing anchorage
dependent cell~ in su~pension and thereby enables greater numbers
of cell~ to be grown per unit volume of culture medium as com-
pared with monolayer cultures.
In accordance with the invention, a seed culture of
anchorage dependent cells iB encapsulated within a plural$ty of
microcapsule6 comprising ~emipermeable membranes. Synthesis of
the membranes is controlled 80 that they have a selected upper
limit of permeability, that i~, the membranes define micropores
of dimensions sufficient to allow passage of molecules having a
molecular weight up to, for ex~mple, 2x105 daltons, but substan-
tially preclude passage of materials of higher molscular weight.
The capsule membrane thus defines a microenvironment within which
the cell together with high molecular weight ~erum components
are confined, but which allow the cells free acce~s to lower
molecular weight cell nutrients such a~ amino acid~, and allow
low molecular weight cell metabolites to exit the environment.
The microcApsules containing cell~ are then dispersed
in a conventional culture medium. The interior ~urfaces of the
cap~ule membrane and/or certain high molecular weight water-
disper~ible materials which are includ~d ln the m~crocap~ule ~ct
as a ~ubstrate to which the cells attach. Bec~u~e the ratio of
the aurface area of the c~psulea to the volume of the extracap-
sular medium may be quite large, individu~l cells are afforded
adequ~te acc~s to required nutrients, and the ~rea on which the
cell~ can grow iB increased a~ compared with conYentional mono-
layer cultures. Generally, the ~verage diameter of the microcap-
:1~7~586
1 sules may be varied between a few microns to a millimeteror more. A preferred average size is on the order of
100-500 micrometers in diameter.
If an anchorage substrate such as collagen or the
like is included within the capsules, then fibroblasts also
grow inwardly of the capsule membranes and will display
normal fibroblastic morphology.
It is an object of the invention to provide an
improved method of culturing anchorage dependent cells.
Another object is to improve the yield of anchorage dependent
or contact inhibited cells grown in vitro. Another object
is to provide throughout the volume of a cell growth medium
a large surface area suitable as a substrate for anchorage
dependent cells. Another object is to provide a method
of growing cells which can produce interfereon. These and
other objects and features of the invention will be apparent
from the description which follows and from the drawing
wherein the sole figure is a photomicrograph (200X) showing
anchorage dependent cells grown within a microcapsule in
accordance with the invention and illustrating classical
fibroblastic morphology.
- 5 -
1~72586
1 Descrip-tion
The broad concept of the invention is to provide a
multiplicity of semipermeable membranes about individual
cells or groups of cells to act as a high surface area
substrate to which the cells can anchor and/or to confine a
high molecular weight material within microcapsules to serve
as an anchorage substrate. Cells which grow in suspension
may also be encapsulated as taught herein and in the above-
referenced copending applications. The microcapsules serve
as a microenvironment for the cells together with at least
high molecular weight components of its culture medium,
and separate the cells from an extracapsular aqueous
medium. Bacteria and other relatively large contaminants
cannot penetrate the membranes.
Anchorage dependent cells of mammalian origin, such
as fibroblast, epithelial, or kidney cells require for ongoing
viability the presence of serl~ components, a portion of which
may have a molecular weight in excess of the upper per-
meability limit of the membranes. Such components may be
included with the encapsulated cells and need not be presentin the extracapsular medium. -
It is required also to include within themicrocapsules a high molecular weight material to serve as an
anchoring substrate. Collagen, a natural protein which is a
major constituent of connective tissues, has been used with
success for the purpose. Other compatible high molecular
weight, water dispersible proteins may also be used, e.g.,
polylysine. If the proteins have free amino groups, they may
be rendered water-insoluble by reaction with a water-soluble
gum during membrane formation as disclosed hereinafter. The
~72586
1 use of such materials is believed to result in the creation of
a matrix within the intracapsular volume. The inclusion of
such a material can improve the cell density within the cap-
sules as the cells grow within the intracapsular volume instead
of or in addition to the growth about the interior of the
capsule membrane.
The encapsulated seed culture is then suspended in a
suitable growth medium of the type employed for growth of con-
ventional cultures. Serum proteins which are not injested
by the cells may be omitted from the extracapsular medium.
However, pH, temperature, ionic concentration, and the like
should be the same as in conventional media. Also, oxygen
and C02 transfer may be promoted by the same means as in
conventional cultures, as these dissolved gasses freely
traverse the membrane.
Incubation of the encapsulated cell culture results
in cell mitosis. Fibroblast cell growths display classical
fibroblastic morphology and form arrays of cells on the
interior of the membrane or on the anchoring substrate contained
within the capsules. Fresh growth medium may be supplied as
required either on a continuous or intermittent basis by change
of the extracapsular medium. If the purpose of the culture is
to produce a cell metabolite of interest, the metabolite may
be harvested either from the intracapsular volume or the
extracapsular medium, depending on its molecular weight and the
upper permeability limit of the membranes (see copending
Canadian application Serial No. 398,218. In a preferred
embodiment of the process, the membranes are of a type which
may be selectively disrupted without damage to the cells.
This allows the cells to be released from the capsules as
,. . .
Z586
1 desired (see copending Canadian application Serial
No. 398,210).
One reason for releasing the cells after their growth
period is to stimulate the production of a substance of interest
by the cells. An example is the production of interferon from
human fibroblasts, leukocytes, ox lymphoblastoid cells which
are induced to excrete interferon by treatment with certain
viruses or high molecular weight nuclei acids. In such a
situation, if the upper permeability limit of the membranes is
less than the molecular weight of the inducing factor, the
cells must be sub~ected to interferon induction prior to en-
capsulation, or the capsule membranes, after culture of the
cells, must be selectively disrupted to allow such high
molecular weight materials to come into contact with the
cell.
The process of the invention depends on one's
ability to form semipermeable membranes about cells without
simultaneously adversely affecting their ongoing viability.
One suitable encapsulation process is set forth in detail below.
Cell Encapsulation
The tissue or cells to be encapsulated are suspended
in an aqueous medium preferably suitable for growth of the
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
about a millimeter. However, best results are achieved with
capsules of a size in the range of 100-500 micrometers. Indi-
vidual anchorage dependent cells such as fibroblasts from
human or other animal tissues, kidney cells, and epithelial
cells may be encapsulated as desired. Also another cells such
,~
~172586
1 as leukocytes, lymphoblastoids, pancreatic beta cells, alpha
cells, delta cells, various ratios thereof, or other tissue
units may be encapsulated.
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. Heretofore, 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. 348,524, 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
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 cells 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,
typically at a concentration on the order of less than 1.0
weight percent, to the tissue culture medium which also con-
tains cells of the seed culture, serum components (if required),
and collagen or another high molecular weight, water-dispen-
sible material which acts as an anchoring substrate. The
g _
,~
- (
1172S86
1 concentration of the material employed a8 ~ substrate ~hould be
within the range of about 10 ug/ml to about 1 mg/ml but is pre-
ferably on the order of 100-500 ug/ml.
The solution i~ then formed into droplets containing
tissue together with its medium and iB immediately rendered
water-insoluble and gelled, at least in a surface layer.
Thereafter, the shape-retaining temporary cap~ule~ are provided
with a more permanent membrane which may itself ~ubsequently be
selectively disrupted if it is desired to release the tisaue
without damage. Where the material used to form the temporary
capsules permits, the capsule interior may be reliquified af~er
formation of the permanent membrane. This i8 done by re-
e~tablishing 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 ehould also contains plural, easily
ionized anionic moieties, e.g., carboxyl groups, which can react
by salt formation with polymers containing plural cationic
groups. As will be e~plained below, use of thi6 type of material
enables one to deposit a permanent membrane of a ~elected upper
limit of permeability (generally no greater than 100,000 to
150,000 daltons) without difficulty in ~urface layer~ of the tem-
porary cap~ule.
The presently preferred materials for forming the tem-
porary capsule are acidic, w~ter-~oluble, natural or synthetic
--10--
_
~7~5136
1 polysaccharide gums. Such materials are commercially
available. They are typically extracted from vegetable matter
and are often used as additives to various foods. Sodium algi-
nate 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 permeate the finally formed capsule
membranes. Lower molecular weight alginate, e.g., 50,000-
80,000 daltons, is more easily removed from the intra~
capsular volume by diffusion through a membrane of sufficient
porosity and is therefore preferred. Other useable gums
include acidic fractions of guar gum, carageenan, 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 strontium 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
resolubilized 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 physiologically compatible ions, e.g., calcium,
be employed. This tends to preserve the tissue in the living
state. Other multivalent 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 ~with an anchoring sub-
strate) and a one or two percent solution of gum in physiolo-
-- 11 --
~7i~586
1 gical saline. When employing ~odium algina~e, a 1.0 to 1.5 per-
cent solution has been used with BUCce~B. 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 i8 employed, e.g., polylysine, the cationic groups react
with anionic sites in the water-soluble gum to form a substan-
tially water-insoluble matrix intertwined with the gum.
Preferred concentrations for such materials are on the order of
100-500 ug per ml of suspension (including gum solution).
In the next ~tep of the encapsulation process, the gum
aolution containing the ti~sue i~ formed into droplet~ of a
de6ired size, and the droplets are immediately gelled to form
shape-retaining spherical or spheroidal masses. The drop forma-
tions may be conducted as follows.
A tube containing an aqueous solution of multivalent
cations, e.g., 1.5% CaC12 solution, iB fitted with a stopper
which holds a drop forming apparatus. The appartus consists of a
hou6ing having an upper air intake nozzle and an elongate hollow
body friction fitted into the stopper. A 10 cc eyringe equipped
with a stepping pump is mounted atop the housing with, e.g., a
O.01 inch I.D. Teflon coated needle pa~sing through the length of
the housing. The interior of the housing iB designed such that
the tip of the needle iB subjected to a con~tant laminar air flow
which act~ aB an air knife. In u~e, with the syringe full of
solution containing the material to be encap~ulated, the stepping
pump i8 actuated to incr~mentally force droplets of ~olution from
-12-
- ~ )
72586
1 the tip of the needle. Each drop i~ ~cut off" by the air stream
and falls approximately 2.5 cm into the CaC12 ~olution where it
is immediately gelled by absorption of calcium ions. ~he
distance between the tip of the needle and the surface of the
CaC12 BolUtion iB great enough, in this instance, to allow the
sodium alginate/cell suspension to assume the most physically
favorable shape; a sphere (masimum volume for minimum surface
area). Air within the tube bleeds through an opening in the
stopper. This results in "croas-linking" of the gel and in the
formation of a hi~h viscosity shape-retaining protective tem-
porary capsule containing the suspended tissue and its mediwm.
The capsules collect in the solution as a separate phase and may
be separated by 2spiration.
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 groups reactive with anionic
functionalities in the gel molecules. Polymers containing acid
reactive groups such as free imine or amine groups are preferred.
In this situation, the polysaccharide gum $8 cro~slinked by
interaction (salt bond formation) between the carbosyl groups and
the amine or imine groups. Permeabil$ty can be controlled within
limits by selecting the molecular weight of the cross-linking
polymer u~ed and by regulating the concentration of the polymer
solution and the duration and temperature of esposure. A 801u-
tion of polymer having a low molecular weight, in a given time
period, will penetrate further into the temporary capsules than
' -13-
- (;
1~72586
1 will a high molecular weight polymer. The degree of penetration
of the cross-linker has been correlated with the resulting per-
meability. In general, the higher the molecular weight and the
le6s penetration, the larger the pore size. Broadly, polymers
within the molecular weight range of 3,000 to 100,000 daltons or
greater may be used, depending on the duration of the reaction,
the concentration of the polymer solution, and the degree of per-
meability desired. One ~ucce~sful set of reaction conditions,
using polylysine of average molecular weight of about 35,00~
daltons, involved reaction for two minutes, with stirring, of a
physiological saline solution containing 0.0167 percent polyly-
sine. This results in membranes having an upper permeability
limit 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 guide-
lines. Using this method it i~ possible to set the upper perme-
ability limit of the membranes at a selected level generally
below about 150,000 daltons.
Examples of suitable cross-linking polymers include
proteins and polypeptides, either natural or ~ynthetic, having
free amino or imino groups, polyethyleneamine~, polyethylene-
imines, and polyvinylamines. Polylysine, in both the D and L
forms, has been used with euccess. Proteins such a~ polyargi-
nine, polycitrulline, or polyornithine are also operable.
Polymers in the higher range of positive charge den~ity, e.g.,
polyvinylamine, vigorouely adhere to the anionic groupa of the
gel molecules to form stable membranes, but the membrane~ are
somewhat difficult to di~rupt.
-14-
. . .
~3.72586
1 Treatment with a dilute ~olution of gum or a ~wi~-
terionic buffer will tie up free amino group~ on the surfaces of
the capsules which otherwise may impart to the cap~ule6 a
tendency to clump.
At this point in the encapsulation, c~psule~ may be
collected which compri~e a semipermeable membrane surrounding a
gelled solution of gum, cell-type compatible culture medium,
cells, and ~yri~*rLLy an internal matrix of collagen or another
- anchorage sub~trate. Since mass transfer should be promoted
within the capgules and acros~ the membranes, it i~ preferred to
reliquify the gel to its water-soluble form. This may be done by
re-establishing the condition~ under which the gum i~ a liquid,
e.g., removing the calcium or other multifunctional cation~ from
the interior gel. The medium in the capsule can be resolubilized
simply by immer~ing the capsule~ 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 im~er~ed in the solution
with stirring. 80dium ci~rate solutions may be u~ed for the ~ame
purpose, and serve to ~equester the divalènt ions.
Cell cultures encapsulated as described above may be
suspended in growth medium designed specifically to ~atisfy all
of the requirements of the particular cell type involved and will
continue to undergo normal in vitro metaboli~m and mitosis. If
the culture requires an environment of high molecular weight com,
ponent~ uch a8 sorum components, tho~e may be omitted from the
estrac~p~ular medium. Typically, the component~ normally
ingested by cells are of relatively low molecul~r weight and
. .
~ 72586
1 readily diffuse across the cap~ule membranes into the microen-
vironment of the cells where they permeate the cell me~brane.
Metabolites of the cells which are e~creted into the intracap-
sular medium, if they have a molecular weight below the upper
limit of permeability of tbe capsule membrane, likewige diffuse
thereacros~ and collect in the extracapsular medium.
The encapsulated cells are grown under condition~ of,
e.g., temperature, pH, and ionic environment, identical to con-
ventional cultures. Cell metabolites may be harve~ted from the
extracapsular medium or from the intracapsular volume by conven-
tional techniques. However, the culturing technique disclosed
herein has the following advantagess
1. The cells of the culture are protected from con-
tamination by factors having dimensions in escess of the upper
permeability limit of the membranes. This meAns that ~terility
requirements normally incident to culturing procedures can be
somewhat relaxed, ~ince microorganisms cannot reach the encap-
sulated cells.
2. The cap~ules in effect immbbili~e the cells within
an environment in which enclosed high molecular weight mAterials
are confined, yet lower molecular weight cell nutrient~ and
products are readily romoved and introduc~d. Thi8 allowe the
- nutrient medium to be intermittently or continuously collected
and upplQmontsd a~ desired, without dicturbing the cells.
3. 8ubstance~ of intero~t produced by the cellfi ~re
more ea~ily rocovered. Cell products of molecular dimen~ion~
small enough to permeate the cap~ule me~brane~ collect in the
-16-
1172586
1 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. Cell products of
molecular dimensions in excess of the upper permeability limit
of the membranes collect within the capsules. These may be
recovered in relatively concentrated form by isolating the
capsules and subsequently selectively disrupting the membranes
using, for example, the technique disclosed hereinafter.
4. The intracapsular volume provides an environment
well suited for cell division. Suspension 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 sub-
strate and/or anchor to the high molecular weight materials
set forth above which are disposed within the capsules. This
leads to significant increases in cell density as compared
with conventional cultures. The ongoing viability of such cell
clusters is aided by the fact that the surface area to volume
ratios of the capsules can be quite large, and thus all cells
have access to required nutrients, oxygen, etc.
In certain situations it is advantageous to selectively
disrupt the capsule membranes to release the cells without
damage. One notable example is in the production of interfereon
(IFN). Cells capable of producing IFN must be subjected to cer-
tain viruses or necleic acids in preparation for the IFN produc-
tion stage. Also, in several IFN induction procedures, reagents
.
- 17 -
,
~72586
1 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 IFN in-
duction stage. If the substances used for IFN induction are
of a molecular weight in excess of the upper permeability limit
of the capsule membranes (as will be the case in virus induc-
tions) the induction process cannot be accomplished in the
encapsulated cell culture. Accordingly, IFN 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 process.
Disruption of Membranes
Cells confined in membranes of the type set forth
above may be released by a process involving commercially
available reagents having properties which do not significantly
adversely affect the encapsulated cells. First, the capsules
are separated from their suspending medium, 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 polymer having plural anionic moieties such as
a salt of a polysulfonic or polyphosphoric acid. Heparin,
a natural sulfonated polysaccharide, is preferred for this
step. The anionic charge density of the polymer used should
be equal to or preferably greater than the charge density of
the acidic gum originally employed to form the membranes.
The molecular weight of the polymer should be at least com-
parable 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
- 18 -
- (
:L172586
mixed solution, the calcium ions compete w~th the cationic
polymer chains used to form the membrane for anioDic ~ites on the
water-soluble gum. Simultaneouely, the heparin or ot~er polymer
having plural anionic moieties dissolved in the solution coanpetes
with the ~Inionic gum in the men~rane for cationic sites on the
polymer chains. This result~ in a water-dispersable or pre-
ferably water-soluble complex of e.g., polyly~ine and heparin,
and in association of the monatomic cations with molecules of the
gel.
This step rendere the membrane suseptible to dis~olu-
tion upon subsequent expoEure to a ~equestering`agent which
completes the disruption proces~ by ~aking up monatomic ions from
the gel. Cap~ule membrane debris which remains in the medium, if
any, can be easily separated from the calls.
The currently preferred solution for the first stage of
the ~elective disruption proce~s canprises 1.1% calcium chloride
tw/v) and between 500 to 1,500 units of heparin per milliliter of
solution. A volume of microcapsules i~ added to this solution
sufficient to constitute between about 20~ and 30% of the total
20 volume of suspension. Calcium chloride and heparin are preferred
since both reagents are physiologically compatible with most
cells and therefore minimize the possibility of cell damage.
Mixtures of strontium salts or other multiv&lent cations (but not
Mg++ ion~) may also be used together with the poly~ulfonic or
polyphosphoric acid salts of the type set for~h nbove.
In general, the conc~ntration~ of monatomic ions an
anionic polymer used in this step may vary widely. Optimum con-
--19--
~7ZS86
1 centrations may be readily determined empirically, and depend
on exposure time as well as the particular polymer used toform the membranes.
The currently preferred sequestering agent for per-
forming the selective disruption is sodium citrate, although
other alkali metal citrate salts and alkali metal EDTA salts
may also be used. When sodium citxate is employed, the optimum
concentration is on the order of 50-60 mM. It is preferred to
dissolve 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: Human Fibroblasts
Human fibroblasts obtained by treating conventional
monolayer culture with trypsin and EDTA for 5 minutes at 37 C
in a known manner are suspended in a complete growth medium
(CMLR 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 10 cells~ml.
Next, a 1,5 percent calcium chloride solution is used
to gel droplets formed by using a drop forming apparatus as
described above. Droplets on the order of 50-500 microns in
diameter leave the tip of the needle and immediately gel upon
entering the calcium solution.
After 5 minutes, the supernatant solution is removed by
aspiration. The gelled capsules are then transferred to a beaker
- 20 -
.~
1172S86
1 containing 15 ml of a golution comprising one part of a 2% 2
(cyclohexylamino) ethane sulfonic acid buffer solution in 0.6~
NaCl (isotonic, phe8.2) diluted with 20 parts 1% CaC12. After a
3 minute immersion, the capsule3 are wa3hed twice in 1~ CaC12.
The capsules are then transferred to a 32 ml solution
comprising 0.005% (w/v) polylysine (average MW 43,000 daltons) in
physiological saline. After 3 minutes, the polyly~ine solution
is decanted. The resulting capsules, having "permanent" semiper-
meable membranes, are then washed twice with 1% CaC12, twice with
physiological saline, and mixed with 10 ml of 0.03 percent algi-
nic acid fiolution.
The capsules resist clumping, and all cfin be seen to
contain fibroblasts. Gel on the interior of the cap~ules i8
reliquified by immersing the capsules in a mixture of saline and
citrate buffer (pH-7.4) for 5 minutes. All of the foregoing
procedures are conducted at 22--37-C.
Under the micro~cope, these cap~ules are obaerved to
comprise a very thin membrane which enclose celle. Molecule~
having a molecular weight up to about one-hundred thousand can
traverse ~he membranes.
The resulting capsules are ~uspended in CMLR-1969
supplemented with 10~ fetal calf ~erum. After incubation at 37-C
for 4-5 days, the capsules, if e%amined under the microscope,
will be found to contain fibroblasts which have undergone mito~ia
and display a classical fibrobla~tic morphology within the micro-
capsules.
-21-
,. . . .. .. . . .. ...
11725~36
1 The capsule membranes may be disrupted without
damaging the cells by allowing a 10 ml portion of the micro-
capsule suspension containing about 500-1000 capsules per ml to
settle. After aspiration of the medium, the capsules are
washed twice with saline. The washed capsules are then mixed
with a 3.0 ml aliquot 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 settle, the
supernatant is aspirated off, and the capsules are washed twice
with 3.0 ml of 0.15M NaCl. After aspiration 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 fibroblasts are subjected to an IFN-~ superinduc-
tion technique according to the Vilcek procedure. Under a 5%
C2 atmosphere (95% air~, the cell suspension is incubated at
37C for one hour in the presence of 100 ug/ml Poly I-Poly C,
a double stranded RNA (known IFN-inducer) available from PL
Biochemicals, Milwaukee, Wisconsin and 50 ug/ml cycloheximide
(protein synthesis inhibitor, Calbiochem, La Jolla, California.)
After one hour, the suspended cells are washed in medium ~CMLR-
1969) containing 50 ug/ml cycloheximide and then resuspended in
the same solution for 3 hours at 37C under a 5% CO2 atmosphere.
At the completion of this incubation the washing step is
repeated and the cells 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 atmosphere. The cells are then washed
- 22 -
~,
:1~7Z586
1 twice in medium and suspended in serum-free medium at 37C
for 18-24 hours, during which time the fibroblasts secrete
IFN-~, which has a molecular weight on the order of 21,000
daltons and may be harvested from the extracapsular medium.
In one experiment conducted with Poly I-Poly C(5S)
(sedimentation value, Poly I and Poly C annealed to form
double stranded RNA) 2,500 units of IFN-~ were produced per
105 cells in the culture. An identical yield was obtained in
a second run using Poly I-Poly C (12S) (double stranded as
purchased).
Other embodiments are within the following claims.
- 23 -
