FR2501715A1 - PROCESS FOR CULTURING CELLS REQUIRING FASTENING FOR THE MANUFACTURE OF THERAPEUTIC SUBSTANCES - Google Patents

Abstract

THE INVENTION RELATES TO CULTURE OF CELLS REQUIRING FIXATION. IT RELATES TO A PROCESS WHICH INCLUDES THE SUSPENSION OF THE CELLS IN A CULTURE MEDIUM CONTAINING THE INGREDIENTS NECESSARY FOR THE VIABILITY AND THE CARYOCINESE OF THE CELLS, THESE INGREDIENTS HAVING A HIGH MOLECULAR WEIGHT IN THE SEMI-SEMI-ENCAPS, THE MEMBRAPS -PERMEABLES HAVING AN UPPER LIMIT OF PERMEABILITY SUCH AS PREVENTS THE PASSAGE OF THE ABOVE INGREDIENTS, THE SUSPENSION OF THE CAPSULES FORMED IN A CULTURE MEDIUM CONTAINING INGREDIENTS OF LOW MOLECULAR WEIGHT, CAPABLE OF CARVING AND CARVERSING THE NATURAL MEMBERS, CELLS CONTAINED IN THE INTERNAL VOLUMES OF THE CAPSULES. APPLICATION TO THE PREPARATION OF INTERFERON BY CULTURE OF FIBROBLASTS.

Description

The present invention relates to a method of

  cell culture requiring fixation, that is,

  to say cells of the type that should normally reproduce as one or more layers formed on a substrate and that can not reproduce in suspension. Advances in cell biology have shown that cells requiring binding, belonging to various higher organisms, can produce small amounts of substances with potential or demonstrable utility important for the treatment of certain diseases, including interferons. Such cells are

also useful in research.

  In such cell cultures, there is

  a permanent risk of bacterial or other contamination.

  In addition, in most cases, the quantities of the substance of interest produced by the cell culture

  are very weak. Like cells requiring a

  tion, unlike in suspension cultures, can not

  not be cultivated to fill a volume, it is considered that there is currently no useful method in practice for the cultivation of a large number of cells necessary for the production of significant quantities.

  interesting substances produced by such cells

  the. Cells requiring fixation, such

  normal fibroblasts and certain epithelial cells

  liales and kidney should be commonly grown on a substrate. Plastic sheets intended for

  to form substrates are commercially available.

  Some types of cells requiring fixation will reproduce until the leaf is covered with a

  monolayer of cells, and they then cease to multiply

  to fold. Others continue to multiply by forming a

  or several additional layers on the first.

  The present invention relates to a suspension cell culture method which requires fixation, and exhibits the growth of larger numbers of cells per unit volume of culture medium, by

  compared to monolayer cultures.

  According to the invention, a germ culture of cells

  requiring fixation is embedded in microcapsules

  formed of semi-permeable membranes. The membrane synthesis is adjusted so that they have a selected upper limit of permeability, i.e.

  that the membranes form micropores of sufficient size

  large enough to allow the passage of molecules

  Such molecules have a molecular weight of, for example, 2.105 daltons, but substantially prevent the passage of higher molecular weight material. The membrane of

  the capsule therefore forms a micro-medium in which the cells

  and the components of a serum having a high molecular weight are maintained, but allowing the cells free access to the nutrients of the cells, having a lower molecular weight, for example amino acids, and the low molecular weight metabolites formed by

  the cells leave the micro-medium.

  The microcapsules containing cells are then dispersed in a conventional culture medium. The

  internal surfaces of the capsule membrane and / or

  some high molecular weight materials, which can

  dispersed in water, are incorporated in the micro-

  capsule and act as a substrate to which cells attach themselves. Since the ratio of the surface of the capsules to the volume of the medium outside them can be very high, the individual cells have a convenient access to

  necessary nutrients, and the surface area

  what cells can grow is increased compared to conventional monolayer cultures. The average diameter

  microcapsules may vary in general between

  microns and 1 mm or more. An average size

  The tagger is in the range of 100 to 500 microns in diameter.

  When a fixing substrate, for example collagen or the like, is incorporated in the capsules,

  fibroblasts also grow inward

  branes and exhibit normal fibroblastic morphology.

  The invention thus relates to an improved method

  cell culture requiring fixation. It is

  also the increase in cell output required

  both a fixation or inhibited by contact, grown in vitro. It also relates to the production, in the volume of the cell culture medium, of a large area suitable as a substrate for the growth of cells requiring fixation. It also relates to a method

  cell growth allowing the production of inter-

will.

  Other features and advantages of the invention

  better stand out from the detailed description which

  following, with reference to the drawing on which the

  gure unique is a photomicrograph at a magnification of 200, representing cells requiring fixation, which multiply in a microcapsule, by implementation

  of the invention, and shows fibroblast morphology

than classic.

  The invention generally relates to the formation of a large number of semipermeable membranes around individual cells or groups of cells so that these membranes form a large surface substrate to which

  the cells can cling and / or allow the confi-

  of a high molecular weight material in the microcapsules to form a fixing substrate. Cells that grow in suspension can also be encapsulated according to the invention. The microcapsules form a micro-medium for the cells, with at least the high molecular weight constituents of the culture medium, and they separate the cells from the aqueous medium that bathes the capsules. Bacteria and other relatively large impurity materials can not cross

membranes.

  Cells requiring fixation and having an origin in mammals, for example fibroblasts, epithelial cells and kidney cells, require,

  to have permanent viability, the presence of

  components of the serum, some of which may have a

  higher than the upper limit of membrane permeability. These constituents may be incorporated into the encapsulated cells and need not be present in

the external environment.

  It is advantageous but not essential also that the microcapsules contain a weight material

  high molecular weight intended to form a fixing substrate.

  Collagen has been successfully used for this purpose, which is a natural protein forming one of the constituents

  main connective tissues. Other proteins

  high molecular weight and dispersible

  water can also be used, including poylysine.

  If the proteins have free amino groups, they can be made insoluble in water by reaction with a water-soluble gum during the formation of the

  membrane, as described hereinafter.

  The use of such materials probably comes from

  the creation of a binder in the volume internal to the capsule.

  The incorporation of such a material can increase the density of

  cells in the capsules because the cells grow in the outer volume to the capsules instead of growing around the inside of the membrane or in addition to this

internal growth.

  The encapsulated seed culture is then suspended in a suitable growth medium of the type used for the growth of conventional cultures. The

  serum proteins that are not ingested by the cells

  they can be omitted in the environment external to the capsules.

  - However, pH, temperature, ionic concentration and similar parameters should be the same as

  in classical circles. In addition, the transfer of oxy-

  gene and CO2 can be promoted in the same way that

  in conventional crops, because these dissolved gases pass through

feel the membrane freely.

  Incubation of encapsulated cell culture causes cell karyocytosis. Fibroblast growths have a classical fibroblastic morphology and form cell arrangements on the

  inner side of the membrane or on the substrate of

  contained in the capsules. A new environment of growth

  may be provided, where appropriate, either continuously

  naked, either intermittently, by changing the environment

  external to the capsules. If the purpose of culture is to produce

  of a metabolite of interest to the cells, the metabolite can be harvested either in the internal volume of the capsules

  outside, depending on its molecular weight.

  and the upper limit of membrane permeability.

  In an advantageous embodiment of the method, the

  membranes are of a type that can be selectively fractured

  effectively without damaging the cells. In this way, the cells can be released from the capsules if necessary. A reason for the release of cells after

  their period of growth is the stimulation of

  of a substance of interest by the cells. An example is the production of interferons by human fibroblasts, leukocytes or lymphoblastoid cells whose interferon excretion is induced by treatment with certain viruses or nucleic acids of high molecular weight. In this case, if the upper limit

  membrane permeability is less than the average weight of

  of the inducing factor, the cells must be subjected to induction of interferons before encapsulation or the membranes of the capsules, after culturing the cells,

  should be selectively fractured so that they allow

  to such high molecular weight materials of

come into contact with the cell.

  The process according to the invention is based on the ability to form semipermeable membranes around the cells without simultaneous harmful interference with their permanent viability. The rest of this memoir describes

  such a suitable method of encapsulation.

  We now consider the encapsulation of cells

  the. The tissues or cells to be encapsulated are put in sus-

  pension in an aqueous medium suitable in preference to the growth of the type of cells considered. Suitable media for this purpose are available commercially. The average diameter of the material to be encapsulated can

  much vary between a few microns and 1 mm.

  However, the best results are obtained with

  capsules whose size is between 100 and 500 hm.

  Individual cells requiring fixation, by

  fibroblasts of human tissue or other animal

  sore, kidney cells and epithelial cells, can be encapsulated as appropriate. In addition, other cells such as leukocytes, lymphoblasts, pancreatic beta cells, alpha cells,

  delta cells, various ratios of such cells or

  very cellular elements can be encapsulated.

  The permanent viability of such living matter depends among other things on the availability of materials

  necessary nutrients, the transfer of oxygen, the ab-

  toxic substance in the medium and the pH of that medium

  this. Until now, it has not been possible to maintain such a living material in a physiologically compatible medium with simultaneous encapsulation. The problem that arose was that the conditions required for membrane formation were lethal or tissue-damaging, so that membrane formation was not available.

  allowing the tissues to survive in good health.

  However, it has been found that certain water-soluble substances that are physiologically compatible with living tissues and that can be rendered insoluble in water by forming a coherent mass that conserves

  its configuration, can be used for training-

  of a "temporary capsule" or a protective layer around individual cells or groups of cells and that this temporary capsule can be treated so that a more permanent semi-permeable membrane can

  deposit around the cells without damaging them

  this. Such a substance is added, for example at a concentration of less than about 1.0% by weight, to the tissue culture medium which also contains germ culture cells, serum constituents (if any) and possibly collagen. or another water-dispersible high molecular weight material acting as a fixation substrate. The concentration of the material used as substrate must be between

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  about 10 g / cm and about 1 mg / cm, but is preferably in the range of 100 to 500 g / cm

  The solution is then put in the form of

  containing a tissue with its environment and is immediately

  naturally rendered insoluble in water and gelled, at least

  in a superficial layer. Then the time capsules

  preserving their configuration receive a more permanent membrane which can itself be subsequently fractured

  selectively if the tissue is to be released without deterioration.

  When the material used for the formation of the temporary capsules allows it, the inside of the capsules may be

  reliquefied after the formation of the permanent membrane.

  The operation is performed by recovery in the middle

  conditions in which the material is soluble.

  The material used for the formation of temporary capsules may be any water soluble and non-toxic material which, by changing the concentration or the ionic medium can be converted into a mass retaining its configuration. The material should also contain a plurality of readily ionized anionic units, for example, carboxyl groups that can react with the formation of a salt with polymers containing several cationic groups. As described later in this specification, the use of

  This type of material allows the deposition of a permanent membrane

  having a selected upper limit of permeability (generally not exceeding 100,000 to 150,000 daltons) without difficulties, in the superficial layers

of the temporary capsule.

  The most advantageous materials for the formation of the temporary capsule are gums of natural or synthetic polysaccharides acidic and water-soluble. These materials are commercially available, for example they are extracted from plant materials and are often used as additives in various foods. Sodium alginate

  is a water-soluble gum particularly advantageous.

  Alginate having a molecular weight greater than 150,000 daltons can be used but, because of its molecular size and viscosity, it can not usually pass through the membranes of the finally formed capsules. A lower molecular weight alginate, for example between 50,000 and 80,000 daltons, is more readily removed from the inner volume of the capsules by diffusion through a membrane of sufficient porosity, and is therefore preferable. Other usable gums are acidic fractions of guar gum, carrageenan,

  pectin, gum tragacanth and xanthan gum.

  These materials include glycoside-bonded saccharide chains. Their free acid groups are often present in the form of an alkali metal ion, for example sodium. When a multivalent ion such as calcium or

  strontium replaces the alkaline ion, the hydro-

  The polysaccharide liquids are "crosslinked" and form a water-insoluble and retaining gel that can be dispensed. in solution, after removal of the ions by ion exchange or with the aid of a sequestering agent. Although any multivalent ion substantially capable of forming a salt with the acidic gum can be used, it is preferable that they be physiologically compatible ions, for example

  example calcium. This characteristic tends to preserve

  to worm the tissue in the living state. Other multivalent cations can be used. Magnesium ions do not allow

  not an effective gelation of sodium alginate.

  An exemplary solution composition contains equal volumes of a culture of cells in its medium (with or without a binding substrate) and a 1 to 2% solution of a gum in saline. then

  the use of sodium alginate, a 1.0-

  1.5% gives satisfaction. Collagen or other water dispersible protein or polypeptide having a high molecular weight, either natural or synthetic, can be incorporated into the cell culture and is contained in the volume.

  internal capsules eventually formed. When using

  In one embodiment, the cationic groups react with anionic sites in the water-soluble gum and form a substantially water-insoluble binder embedded in the gum. Advantageous concentrations for these materials are of the order of 100 to 500 g / cm3 of suspension

(including the gum solution).

  In the next step of encapsulation, the gum solution containing the tissue is formed into droplets of the desired size, and these droplets immediately gel to form spherical or spheroidal masses that retain their shape. The formation of the droplets can be carried out as follows. A tube containing an aqueous solution of multivalent cations, for example a 1.5% CaCl 2 solution, is provided with a stopper which supports a drop-forming apparatus. This apparatus comprises a housing having an upper air inlet nozzle and an elongate and hollow body frictionally held in the plug. A 10 cm3 syringe equipped with a stepper pump is mounted on the casing with for example a needle of 0.25 mm internal diameter, coated with "Teflon" passing in the length of the housing. The interior of the latter is made so that the tip of the needle is subjected to a constant laminar flow of air acting as an air curtain. During

  when the syringe is full of

  containing the material to be encapsulated, the step pump is controlled so that it gradually

  droplets of solution at the end of the needle.

  Each drop is "torn off" by the stream of air and falls a distance of about 2.5 cm into the solution of

  CaCl2 in which it gels immediately by ab-

  calcium ion sorption. The distance between the tip of the needle and the surface of the CaCl 2 solution

  is large enough, in this case, for the suspension to

  sodium alginate and the cells take the confi-

  the most favorable treatment from the physical point of view,

  that is, a sphere (the maximum volume for the minimum area). The air in the tube escapes through an orifice formed in the plug. The operation causes a "crosslinking" of the gel and the formation of a temporary protective cap of high viscosity, retaining

  its shape and containing the suspended tissue and its medium.

  Capsules collect in solution in form

  of a separate phase and can be extracted by aspiration.

  In the next step of the process, a semipermeable membrane is deposited around the surface of the capsules

  by "crosslinking" of the superficial layers.

  The operation can be carried out by treating the gelled temporary capsules with an aqueous solution of a polymer containing cationic groups which can

  react with the anionic functions of the gel molecules.

  Polymers containing reactive acid groups, for example

  Examples of free imine or amine groups are advantageous.

  In this case, the polysaccharide gum is crosslinked by interaction (formation of salt bonds) between the carboxyl groups and the amine or imine groups. The permeability can be adjusted between limits by selecting the molecular weight of the polymer used for crosslinking and by adjusting the concentration of the polymer solution.

  as well as the duration and temperature of exposure.

  A solution of low molecular weight polymer, in a determined period, passes more inwardly of the temporary capsules than a high molecular weight polymer. The degree of penetration of the crosslinking agent can be related to the resulting permeability. In general, 1 1 plus the molecular weight is high and the penetration

  is small, the larger the pore size.

  Polymers having a molecular weight of between 3,000 and 100,000 daltons or more can be used in general depending on the duration of the reaction, the concentration

  of the polymer solution and the desired degree of permeability

  you. A satisfactory set of reaction conditions, when using polylysine of average molecular weight equal to about 35,000 daltons, gives a reaction of 2 min with agitation of physiological saline containing 0.0167% polylysine. Formed membranes have an upper permeability limit of about 100,000 daltons. Optimum reaction conditions suitable for adjustment

  permeability in a given system can be easily

  determined in an empirical way, once known

  the preceding guidelines. The implementation of this procedure

  allows the establishment of the upper limit of membrane permeability at a chosen level that is in

  less than about 150,000 daltons.

  Examples of suitable polymers of

  Lation are naturally occurring or synthetic proteins and polypeptides having free amino or imino groups, polyethyleneamines, polyethyleneimines, and polyvinylamines. Polylysine, both in its D and L forms, gives satisfaction. Proteins such as polyarginine, polycitrulline or polyornithine can also be used. Polymers having a high density of positive charges, for example a polyvinylamine, strongly adhere to the anionic groups of the gel molecules during the formation of stable membranes, but the fracture of the

  membranes presents some difficulties.

  Treatment with a dilute solution of a gum or a hermaphrodite buffer causes the binding of the free amino groups to the capsule surfaces which could

  elsewhere tend to agglomerate.

  At this stage of encapsulation, the capsules may be collected in a form that comprises a semipermeable membrane surrounding a gelled gum solution, a culture medium compatible with the cell type,

  cells and possibly an internal binder of collagen

  gene or other attachment substrate. As the trans-

  Massive fertility should be promoted inside the capsules and through the membranes, it is advantageous that the gel

  returned in a liquid and water-soluble form.

  The operation can be carried out by restoring the conditions

  in which the gum is liquid, for example by removal of the calcium or multifunctional cations of another type of the internal gel. The medium defined in the capsule can be brought back into solution by simply immersing the capsules in a phosphate buffered saline solution containing alkali metal ions and hydrogen ions. Monovalent ions replace other calcium or multifunctional ions in the gum when

  the capsules are immersed in the solution with stirring.

  Sodium citrate solutions can be used for this purpose to ensure the sequestration of ions.

bivalent.

  The cell cultures encapsulated as described above can be suspended in a growth medium more specifically designed to meet all the criteria of the particular type of cell considered, and continue to have a metabolism and karyokinesis.

  in vitro normal. If the culture requires a medium containing

  For high molecular weight ingredients such as serum components, these may be omitted from the external capsule medium. For example, the constituents

  normally ingested by the cells are of molecular weight.

  relatively weak and diffuse easily through the membranes of the capsules towards the micro-medium of the cells and thus cross the membrane of the cells. The metabolites of the cells that are excreted in the inner capsule medium, when they have a molecular weight below the upper limit of permeability of the capsule membranes, diffuse similarly across these membranes.

  and gather in the outer medium to the capsules.

  The encapsulated cells grow under conditions

  temperature, pH and ionic medium, for example

  ple, which are identical to those of classical cultures.

  The metabolites of the cells can be collected in the external medium to the capsules or in the internal volume

  capsules, using conventional techniques.

  However, the culture technique described herein has the following advantages: 1. The culture cells are protected against contamination by factors having dimensions that exceed the upper limit of permeability of the membranes. This means that the sterility criteria normally required in culture procedures may be less demanding since microorganisms

  can not reach the encapsulated cells.

  2. The capsules actually immobilize the cells in a medium in which high molecular weight materials are confined, and the nutrient products and ingredients of the cells that have a weight

  lower molecular weight are easily extracted and introduced

  ucts. In this way, the nutrient medium can be col-

  read or renewed intermittently or continuously

  if necessary, without disturbing the cells.

  3. The interesting substances produced by the cells can be very easily recovered. The products of the cells having sufficiently small molecular dimensions to pass through the capsule membranes gather in the external medium to the capsules, in the form of a mixture with the nutritive ingredients. However, high molecular weight serum components and the like are not extracted to the external capsule medium so that recovery of the product of interest from the cells is simplified. The products of the cells having molecular dimensions greater than the upper limit of permeability of the membrane collect in the capsules. These products

250 1715

  can be recovered in a relatively

  by isolation of the capsules and then by selective frac-

  subsequent membranes using, for example,

  technique described later in this memo.

  4. The internal volume of the capsules forms a medium well suited for dividing the cells. It is observed that suspension cultures show karyokinesis inside the capsule. The cells requiring fixation and which, in normal cultures, grow as a two-dimensional monolayer, multiply forming an arrangement in the capsule. These cells use the inner surfaces of the membrane as substrates and / or attach to the high molecular weight materials indicated above placed in the capsule. The density of cells therefore increases significantly compared to conventional cultures. The permanent viability of these groups of cells is accentuated by the fact that the surface / volume ratios of the capsules can be very important if

  although all cells have access to nutrients

  oxygen, etc. which are necessary.

  In some cases, it is advantageous for capsule membranes to be selectively fractured

  so that the cells are released without deterioration.

  An example to note is the production of interferon. Cells capable of producing interferon must be subjected to certain viruses or nucleic acids during

  preparation, in a production step of inter-

  Feron. In addition, in several interferon induction procedures, reagents are added to the culture to prevent protein synthesis. So,

  the growth stage of the culture process must be

  alized under conditions very different from the step of induction of interferon. If the substances used for the induction of interferon have a molecular weight

  greater than the upper limit of permeability of

  branes (as in the case of virus inductions), the induction process can not be

250 1715

  works in the encapsulated culture of cells. Thus, cells producing interferon, when they are

  formed in the capsule, must be released by fractional

* turation of the membrane so that they can undergo the process of induction.

  We now consider the fracture of the membranes.

  Cells confined within membranes of the type

  indicated above, may be released by a process

  utilizing commercially available reagents and having properties that do not significantly interfere with encapsulated cells. First, the capsules are separated from the suspending medium, washed thoroughly so that impurities that can be presented outside the microcapsules are

  hunted, then dispersed, with agitation, in a solution

  containing both monoatomic multivariate cations

  such as calcium ions, and a polymer with

  anionic units, for example a salt of a polysulphonic or polyphosphoric acid. Heparin, which is a natural sulfonated polysaccharide, is advantageous at this time. The density of the anionic charges of the polymer used should be equal to the charge density of the acid gum originally used for membrane formation or preferably higher. The molecular weight of the polymer must be at least comparable to that of the polymer having several cationic groups used for formation

  membrane, and preferably higher. In the

  board of capsules formed in the mixed solution, the

  Calcium ions compete with the poly-

  cationic mothers used for membrane formation.

  only for the occupation of the anionic sites formed on the water-soluble gum. Simultaneously, heparin or another polymer having several anionic units, dissolved in the solution, competes with the anionic gum

  present in the membrane for the occupation of

  present on the polymer chains. This results in

  the formation of a dispersible complex in water or preferably soluble in water, for example polylysine and heparin, with association of monoatomic cations

with gel molecules.

  This operation makes the membrane likely to dissolve when subsequently exposed

  to a sequestering agent that completes the process of breaking

  by taking monoatomic ions from the gel. The remains of the capsule membranes that remain in the middle of the

  where appropriate can be easily separated from the cells.

  The solution probably the most advantageous for

  the first step in the process of selective fracture

  contains 1.1% (weight / volume) of calcium chloride and 500 to 1500 units of heparin / cm3 of solution. A certain volume of microcapsules is added to this solution, in an amount sufficient to form about 20 to 30% of the total volume of the suspension. Calcium chloride and heparin are preferable because these two reagents are physiologically compatible with most cells and thus minimize the potential for cell damage. Mixtures of salts of strontium and other multivalent cations (other than Mg ions) can also be used with the polysulfonic acid or polyphosphoric acid salts of the type indicated above. The concentrations of monoatomic ions and anionic polymer used in this step can in

  general vary a lot. Optimal concentrations may

  can be easily determined empirically

  and they depend on the exposure time as well as the poly-

  particular mother used for membrane formation.

  The sequestering agent probably the most

  For the selective fracture, sodium citrate is preferred, although other salts in the form of alkaline citrate and salts of ethylenediaminetetraacetic acid and an alkali metal may also be used. When sodium citrate is used, the concentration is of the order of 50 to 60 mM. It is advantageous that the citrate or other sequestering agent is dissolved in isotonic saline so that the deterioration of the cells

be minimal.

  The examples which follow, given for purely illustrative and non-limiting purposes, illustrate the implementation of the invention. EXAMPLE: Human Fibroblasts Human fibroblasts obtained by treating a conventional monolayer culture with trypsin and ethylenediaminetetraacetic acid for 5 min at 370 ° C. in a known manner, are suspended in a complete growth medium ("CMLR 1969" Connaught Laboratories) supplemented with a 40% by volume solution of purified fetal calf serum, containing 0.8% sodium alginate ("Sigma") and 200 sig / cm3 purified calf skin collagen. The density of the cell suspension

7 3

  is of the order of 1.5 × 10 cells / cm

  Next, a 1.5% chlorine solution is used.

  Calcium chloride for the gelation of droplets formed using a suitable apparatus of the type described above. Droplets having a diameter of the order

  from 50 to 500 dam, leave the tip of the needle and freeze

  immediately, as soon as they enter the calcium solution. Afterwards, the supernatant solution is removed by suction. The gelled capsules are then transferred to a beaker containing 15 cm3 of a solution containing

  a buffer solution of 2- (cyclohexylamino) ethanesulphonic acid

  2% solution in a 0.6% solution of NaCl (isotonic solution, pH = 8.2) diluted with 20 parts of a 20% solution of CaCl 2. After 3 minutes of immersion, the capsules

  are washed twice in a 1% solution of CaCl 2.

  The capsules are then transferred into 32 cm3 of a

  solution containing 0.05% (by weight per volume) of poly-

  sine (average molecular weight of 43,000 daltons) in physiological saline. After 3 min, the polylysine solution is decanted. The resulting capsules, having "permanent" semi-permeable membranes, are then washed twice with a 1% solution of CaCl 2, twice with physiological saline and are mixed with 10 cm 3 of a solution.

at 0.03 - of alginic acid.

  The capsules are resistant to agglomeration and it may be noted that they all contain fibroblasts. The gel inside the capsules is delivered

  in the liquid state by immersing the capsules in a mixture of

  of saline and a citrate buffer solution (pH = 7.4) for 5 min. All operations

  above are implemented between 22 and 37WC.

  These capsules are then observed under the microscope and we note that they comprise a very thin membrane which

  surrounds cells. Molecules whose molecular weight

  can reach about 100,000 can cross

membranes.

  The resulting capsules are then suspended in the "CMLR-1969" culture medium supplemented with 10% fetal calf serum. After incubation at 37 ° C. for 4 to 5 days, it is found under the microscope that the capsules contain fibroblasts which have undergone

  karyokinesis and which have fibroblast morphology

  classic tick, inside the microcapsules.

  Capsule membranes can be broken

  without damaging the cells by sedimentation of a 10 cm3 fraction of the microcapsule suspension

  containing about 500 to 1000 capsules per cm. After

  medium, the capsules are washed twice with saline. The washed capsules are then mixed with a 3.0 cc fraction containing 1000 units / cm3 of heparin and CaCl at 1.1%. in weight by volume. The solution is stirred at 37WC for

  3 min, and the capsules can then separate by

  feed, the supernatant liquid is removed by suction

  tion, and the capsules are washed twice with 3.0 cm 3

  a 0.15 M solution of NaCl. After aspiration of the solution

  In the second wash, the capsules are mixed with 2.0 cc of a mixed solution containing equal volumes.

  of a solution of 110 mM sodium citrate and a solution of

  0.15 M NaCl (pH = 7.4). The mixture is then shaken by hand by swirling for 1 min to dissolve the membranes, and the cells are then washed twice in the medium. The fibroblasts are subjected to a "superinduction" technique of interferon B, according to the method of Vilcek. In an atmosphere containing 5% CO2 (95% air), the cell suspension is incubated at 37 ° C. for 1 hour in the presence of 100 μg / cm 3 of Poly I-Poly C, which is a two-ribonucleic acid. tablecloths (known inducer of interferon B) available from PL

  Biochemicals, Milwaukee, Wisconsin and 50 4g / cm3 of cyclo-

  heximide (Calbiochem Protein Synthesis Inhibitor,

  La Jolla, California). After 1 hour, the cells suspended

  are washed in a culture medium ("CMLR-1969")

  containing 50 g / cm3 of cycloheximide and then

  pension in the same solution for 3 hours at 37 C,

  mosphere containing 5% CO2. At the end of this incubation, the washing operation is repeated and the cells are resuspended in a liquid containing 50 g / cm 3

  of cycloheximide and 5.g / cm3 of actimomycin D (an inhibi-

  known producer of ribonucleic acid synthesis from Calbiochem),

  then incubated for 2 h at 37 ° C with

  mosphere containing 5% CO2. The cells are then washed twice in the medium and suspended in serum-free medium at 37 ° C. for 18 to 24 hours.

  and during this time the fibroblasts secrete from

  feron B which has a molecular weight of about 21,000 daltons and can be harvested in the external environment

to the capsules.

  In an experiment carried out with Poly I-Poly C (5S) (sedimentation value, substances Poly I and Poly C being treated so that they form

  ribonucleic acid with two layers), 2500

  Feron B are produced by 105 cells in culture. An identical yield is obtained in a second test with

2 501715

  Poly I-Poly C (two-ply, sold in this form).

  It is understood that the invention has been described and shown only as a preferred example and that

  may provide any technical equivalence in its

  constituent elements without departing from its

  which is defined in the appended claims.

Claims (10)

  1. A method of culturing cells requiring fixation, characterized in that it comprises A. suspending the cells in a culture medium containing all the ingredients (A)   necessary for the preservation of viability and the   the karyokinesis of cells and having a molecular weight   higher than a chosen value, B. the encapsulation of the cells with the medium in several semipermeable membranes having a limit   sufficient permeability for the ingredients to be   dients (A) can not cross the membrane and that molecules with a molecular weight lower than the chosen value can cross the membranes, C suspending the product of step B in a culture medium containing all the ingredients (B) necessary for maintaining the viability and maintenance of cell karyocytosis and having a molecular weight below a chosen value, and D. the natural karyokinesis of cells in   the volume delimited inside the membranes.   2. Method according to. claim 1, characterized   in that the fixing substrate having a molecular weight   greater than the chosen value, is incorporated in the suspension of step A. 3. Process according to claim 2, characterized   in that the binding substrate is a protein.   4. Method according to claim 2, characterized   in that the fixation substrate is collagen.   5. Method according to claim 2, characterized in that the fixing substrate is skin collagen.   of veal and is incorporated in the suspension at a concentration   between 10 g / cm3 and 1.0 mg / cm3 approximately.   6. Method according to claim 1, characterized in that it further comprises the selective fracture of the membranes after step B so that the cells are released. 7. Method according to claim 1, characterized   in that the cells are fibroblasts.   8. Process according to claim 1, characterized in that the ingredients (A) contain constituents of the serum. 9. The method according to claim 1, characterized   in that the cells are human fibroblasts capable of to secrete interferon.   10. The method of claim 1, wherein the cells are fibroblasts capable of secreting interferon, said method being characterized in that it comprises the following additional steps: E. the selective fracture of the membranes after step D so that the cells are released, F. the application to the cells of interferon induction treatment, G. the incubation of the cells obtained in step F, in a culture medium, and H. the harvesting interferon in the medium obtained in step G. il. Process according to Claim 1, characterized in that, during the encapsulation, spheroidal membranes with a mean diameter of 100 and 500 Name.   12. Process according to claim 2, characterized in that the fixing substrate is a protein having   several free cationic groups.   Method according to claim 1, characterized   in that the chosen value is less than 2.0.105 dal- about.   14. The method of claim 1, characterized in that the chosen value is less than approximately 1.105 daltons.