FR2572093A1 - NOVEL CELL CULTURE MEDIUM CONTAINING HUMAN RATE CELLS, CULTURE METHOD USING THE CULTURE MEDIUM, LYMPHOBLASTOID CELL LINES OBTAINED, INTERFERON PRODUCTION APPLICATION, INTERFERON PREPARATION METHOD, AND INTERFERON THUS PRODUCED - Google Patents

Abstract

THE INVENTION CONCERNS A NEW MEDIUM OF CELL CULTURE. THIS CELL CULTURE MEDIUM IS CHARACTERIZED IN THAT IT INCLUDES HUMAN RATE CELLS IN A CULTURALLY READY FORM, AND AN ADEQUATE CULTURE SUPPORT FOR THE CULTURE OF SAID HUMAN RATE CELLS, PREFERABLY CONTAINING SERIC PROTEINS PREFERENCE PREPARED AT LEAST IN PART FROM SERUM PURIFIED OR TREATED IN A MANNER KNOWN IN ITSELF TO MAKE THIS CULTURE SUPPORT APYROGENIC AND ATOXIC. THIS NEW CELL CULTURE MEDIUM CAN BE USED IN A CULTURE PROCESS TO PREPARE LYMPHOBLASTOID CELL LINES WHICH ARE THEN USED OR APPLIED TO THE PRODUCTION OF INTERFERON, SIMPLY AND AT THE INDUSTRIAL SCALE.

Description

The present invention relates to a new cell culture medium

  containing human spleen cells a culture method using this

  culture medium, lymphoblast cell lines

  toides, an application for the production of interferon, a process for the preparation of interferon and interferons

thus produced.

  Interferon is a protein molecule secreted by various cell types. It is supposed to block the multiplications of certain viruses and cancer cells. There are several types of interferons. Interferon alpha is secreted by lymphocytes and presumed beneficial in the treatment of common cold

  influenza and some cancers. Interferon is specific

  of the species, which means that only human interferon is effective for treating diseases in

the humankind.

  The production of interferon suitable for treatment

  Human disease is dependent on localized genes in cells of human origin. A line of research for the production of interferon lies in

  the excision of the discomfort and its reinsertion into a bacterium.

  The present inventor considers, however, that this attractive production technique per se is not

  not a panacea because the technique of excision and reinsertion

  This is difficult because the transformed bacteria do not necessarily produce an active interferon molecule, the production of interferon per bacterial unit and the passage of this production from the laboratory stage to the pilot and industrial stages is more than random. The present inventor has attached a mode of production of interferon alpha which is based on the

  culture of human cells. As a general rule,

  alpha feron is obtained by inoculation of human lymphocytes with Sendai virus, but this technique is laborious and provides a product that requires steps

  subsequent purification.

  The present inventor thus posed the new technical problem consisting of the development of a cell multiplication system amenable to industrial developments, and the development of a new medium. culture with minimal serum contamination so as to avoid

  laborious steps of purification of the product obtained.

  This new technical problem is solved for the

  first time by the present invention satisfactorily

  particularly simple and reproducible

  ductile, even at the industrial level.

  Thus, the present invention, according to a first of

  aspects, relates to a new cell culture medium.

  characterized in that it comprises human spleen cells in a form ready for culturing, and a

  culture support suitable for the cultivation of said cells

  human spleen, preferably containing serum proteins, further preferably prepared at least in part from serum purified or treated in a manner known per se to make this nonpyrogenic culture support

and nonpoisonous.

  According to a particular characteristic of this cell culture medium according to the invention, it is characterized in that the spleen cells

  are obtained from sterile minced human spleen

  without prior infusion, in a solution of

  Hank's iced ed passed on a sterile sieve.

  On the other hand, according to a particular advantageous embodiment, the culture medium is supplemented with a human serum fraction which is low in gamma globulin and in pre-albumin, and which is nontoxic to the mouse.

and pyrogen-free.

  According to a preferred embodiment, the human serum fraction is diluted to 5% protein, treated at -3 C and PH = 7.2 with ethanol in the amount necessary and sufficient to precipitate the fibrinogen, then brought to minus 6 C and pH 5.85 and an ethanol concentration of between 10% and 20%, the precipitate obtained being discarded and the supernatant being collected and used as

  protein supplement of said culture medium.

  According to another characteristic of the cellular environment

  according to the invention, the latter is brought to a concentration

  5.10 viable leukocytes per milliliter.

  According to another particular characteristic of the invention, this new cell culture medium is characterized in that it is in suspension in an RPMI medium, enriched with 2 to 10% of the following serum fraction: 80% by weight of albumin, 3% by weight alpha 1globulin; 6% by weight alpha 2-globulin; and 9% beta-globulin, not containing pre-albumin

nor detectable gamma globulin.

  The present invention also relates to a culture method characterized in that it comprises the

  culture of the aforementioned cell culture medium, preferably

  Preferably, the process comprises: incubating at 37 ° C. in a CO 2 enriched atmosphere;

  maintenance of the crop at this concentration

  cell treatment for 3 to 5 weeks, preferably with control of cell concentration; and

  - Isolation of lymphoblast cell lines

  suspended in the culture medium, preferably when the cell density is between 2.105 and

  5.105 cells per milliliter of culture medium.

  According to a preferred feature of this process, the isolated lymphoblastoid cell lines in suspension, substantially without feeder adherent cells,

  are multiplied in suspension in fermenters.

  The present invention also relates, according to a

  other aspects, lymphoblast cell lines

  toides obtained by this culture method. On the other hand, the present invention also relates to the application of the novel culture medium and cell lines

  lymphoblastoids to the production of interferon.

  The present invention also relates, according to yet another of its aspects, a method of preparation

  interferon, in particular alpha interferon,

  rised in that it includes the addition of an inductor of inter-

  feron to the aforementioned lymphoblastoid cell lines;

  and preferably the separation of interferon.

  According to a preferred characteristic of this interferon preparation process, the interferon inducer is chosen from (poly I: C) (polyosinic acid: cytidylic acid) added with protamine, in particular for the ST 1 and ST lines. 2; and the Sendai virus, in

  especially for ST 3, CA and CB lines. -

  The present invention finally relates to interferon, in particular alpha interferon produced by the method of

production mentioned above.

  Other objects, features and advantages of the invention will become apparent in light of the

  explanatory description of the invention which will follow, given

  simply as an illustration and therefore not

  in no way limit the scope of the invention. -

  I. CELL CULTURE MEDIUM ACCORDING TO THE INVENTION

  The cell culture medium according to the invention is characterized in that it comprises human spleen cells in a form ready for culture, and a culture support that is suitable for the culture of said spleen cells, preferably containing proteins. serum, more preferably prepared at least in part from serum purified or treated in a manner known per se

  to make this culture support pyrogen-free and non-toxic.

  For example, human spleen cells are obtained by splitting human spleen fragments obtained by autopsy of healthy donors. These pieces of human spleen are passed through a sieve (0.075 mm), the cells being

  collected in an ice-cold solution from Hank's.

  After washing, human spleen cells are

transferred to an RPMI medium.

  Preferably, as previously indicated, the

  culture port contains serum proteins or is supplemented with a serum fraction that is preferably human. Thus, preferably, the RPMI medium in which the human spleen cells are transferred, is supplemented respectively with 4% (weight / volume) of the two following preparations, in comparison with a medium supplemented with 10%

  of fetal calf serum as a control.

  1). A serum fraction depleted in gamma

  globulins (called AFF). The composition obtained after lyophilization is given in Table I:

TABLE I

(: :)

(: AFF: EPF

(: :)

(: :)

(Prealbumin: 1%: 0

(: :)

(Albumin: 74%: 82%

(: :)

(alpha 1-globulin: 9%: 3%

(: :)

(alpha 2-globulin: 2.5%: 6%

(: :)

(-globulin: 12.5%: 9%

(::

(gamma-globulin: 1%: 0

(: :)

()

(: 100%: 100%

____________________)

  This material was prepared after Goose et al. (Applied Microbiology: (1973), 25, 325-326), who had developed it to obtain interferon-induced cells

grown in vitro.

  21. A serum fraction called EPF, obtained as follows: - the plasma diluted in a phosphate isotonic solution at a protein concentration of 5%

  is treated at -30C, pH 7.2 with 1% ethanol.

  A precipitate of fibrinogen appears which is

  discarded by centrifugation or filtration.

  - The supernatant is supplemented with ethanol

  up to a concentration of 19%, the abat-

  at 5.85 per HCl 1N lowered temperature

at -6 C ..

  A big precipitate appears, which is spread by

centrifugation or filtration.

  The supernatant is collected, dialyzed at 4 ° C. against a 0.15 M NaCl solution to remove the ethanol and then lyophilized. In another mode of production, we immediately lyophilized the supernatant, without prior dialysis. In this case, the freeze-drying process is more delicate. This plasma purification process is known for a long time

  (Nischmann et al., Helv Chim Acta: (1954) 37, 866-873).

  The protein content of the EPF is given in Table I. A more detailed analysis of the respective components of these two preparations was carried out by gel electrophoresis

  polyacrylamide and revealed that the AFF contained

  still important IgG, IgA, C3, orosomucoid and

  traces of IgM and ceruloplasmin. In addition, the concentrations

  IgG, IgA and IgM analyzes of the EPF solution analyzed

  have been found to be less than 0.02%.

  The interest of this EPF preparation lies essentially

  in its apyrogenicity and nontoxicity in mice.

  3). Fetal calf serum: This is an ingredient

  commonly used cell culture media.

  These three preparations are then used to establish lymphoblastoid lines from the spleen

human.

  Since the EPF culture medium previously

  is most interesting, the establishment of cell lines from this nutritive agent will be mainly described, with the aim of obtaining cell strains capable of subsequently producing interferon after culture in a growth medium containing this product.

  I. CELL CULTURE OF LYMPHOBLASTE

  To do this, the previously mentioned culture method is used, which is characterized in that it comprises the culture of the cell culture media described above, advantageously comprising the steps

following: -

  incubation at 37 ° C. in an enriched atmosphere

in C02; -

  maintenance in culture for 3 to 5 weeks, preferably with control of the cell concentration; and - isolating the lymphoblastoid cell lines suspended in the culture medium, preferably when the cell density is between 2.105 and 5.

  viable per ml of culture medium.

  In this regard, the primary culture of human spleen cells requires supplies to an organ of origin

which must be avoided.

  The present inventor has succeeded in permanently cultivating "in vitro" spleen leukocytes from healthy donors in RPF 1640 medium supplemented with EPF, previously mentioned, which have been found to be excellent.

alpha interferon producers.

  As indicated previously, human spleens taken at autopsy are thinly sliced, without prior infusion, in ice-cold Hank's solution.

and passed on a sterile sieve.

  The cells washed in a Hank's solution are

  suspended in RPMI 1640 medium containing 2mK of L-glutamine and antibiotics, and enriched with 2-10%, according to

  the cultures, by weight, in EPF medium mentioned above.

2572O93

  The cell concentration is preferably brought

  at 5 × 10 5 viable leukocytes per ml of culture medium.

  Then 10 ml of this cell concentration

  are sown in 75 cm2 Falcon bottles.

  The cells are incubated at 37 ° C. in an atmosphere containing 5% CO 2, the remainder being air and maintained in culture with regular adjustment to a cell density of 5 × 10 5 viable cells per ml.

of culture medium.

  After one week of culture under these conditions, the cells adhering to the plastic actively multiply,

  to become confluent after three weeks of cultivation.

  At the same time, the number of small free-growing lymphocytes in suspension decreases and eventually disappears as lymphoblastoid cells grow on the surface of adherent cells that serve as

nanny, appear.

  Their number increases over time, for after about a month of cultivation under these conditions, pass

in suspension.

  The lymphoblastoid (LyCL) cell lines released from the dependence of the feeder feeder cells continue to multiply in suspension After obtaining a critical cell density, which has been found to be different for each cultured line but which is in general between 2.105 and 5.105 cells per ml of medium, the LyCL can be transferred and multiplied in another bottle or fermenter,

  the absence of nourishing adherent cells -

  They then multiply in a logarithmic mode and can, once this stage is reached, be transferred to

  fermenters and grown industrially.

  Lines which have been isolated by the present inventor according to this method are not all identical. The cells that compose them vary in appearance according to their apparent degree of maturity. The most mature cells are small lymphocytes. They are not present most frequently, the most common form being the

immature lymphoblast.

  However, an evaluation of average cell volume in the middle of the log phase of growth of each line showed that this volume could

  vary from 350, um3 to 950 pm3 depending on the line.

  The lymphocyte B character of these lines was analyzed for five of them, all five showing

  the presence of gamma globulin surface.

  The five lines analyzed also showed the presence of Epstein-Barr virus (EBV) antigen. This is not surprising since the majority of the population

  healthy adult is carrier of these antigens.

  Most lymphoblastoid lines analyzed

  have been shown to be diploid, with one line being hypotensive

  traploide. All had basophilic cytoplasm.

  Five Ly CL lines obtained by the technique described above, where EPF has been used in

  cultivation, were cultivated one last time in three

  different growing places by the parent compound

  serum used in the preparation.

  The five lineages are then tested for

production city of interferon.

  III. APPLICATION TO INTERFERON PRODUCTION

  Lymphoblastoid cell lines, according to a

  other aspects of the invention are used or applied.

  interferon production.

  The process for preparing interferon is, as previously mentioned, characterized in that it comprises adding

  from an interferon inducer to lymphocyte cell lines

  previous blastodes, and preferably the separation of interferon. The invention also relates to interferon produced

by this method.

  Preferably, the interferon inducer used is selected from the group consisting of poly (I: C) (poly (acetinosinic acid: cytidylic acid)) supplemented with protamine for the ST 1 and ST 2 lines; and Sendai virus for ST 3, CA and CB lines.

  The results that have been obtained are listed

Table II.

TABLE II

  (Lymphoblast lines: INTERFERON TITLE IN UNITS / nl) (human miceB (): calf serum) EPF: fetal AFF

(:: :)

(ST 1: 16000 8000: 2000)

(:: :)

(ST 2: 4,000,000,000,000)

(:: :)

(ST 3: 2,000: 4,000: 8,000)

(::: :)

(C A: 32,000: 8,000: 64,000)

(:: :)

(C B: 1: 2,0002,000: 2,000)

  (:: :) It can thus be seen from Table II that interferon is obtained, in variable quantities, from

  two induction techniques applied.

  Other inductors can of course be used.

  As can be seen in Table II, we also

  lymphoblastoid cells on the other two serum AFF adjuvants and fetal calf serum. It can be seen that the 3 adjuvants

  serum levels used for the growth of lymphocytes

  blastoids are adequate for this production, the one

  a minimum of contaminating material (i.e. EPF) being preferred for obtaining interferon for

for therapeutic use.

  Other formulations can of course be developed for culturing cells, such as human or bovine albumin or else a purified extract of the growth factors contained in the serum, without

  however, to depart from the scope or scope of the invention.

2572,093

R E V E N D ICAT I 0 N S

  1.- novel cell culture medium, characterized in that it comprises human spleen cells in a culture-ready form, and a culture support suitable for the culture of said human spleen cells, preferably containing serum proteins , still preferably prepared at least partly from serum purified or treated in a manner known per se to make this nonpyrogenic culture support

and nonpoisonous.

  2. The cell culture medium according to claim 1, characterized in that the human spleen cells are obtained from human spleen, sterilely sliced, without prior infusion, in a

  Hank's solution iced and passed on a sterile sieve.

  3. A cell culture medium according to claim 1 or 2, characterized in that the culture medium is supplemented with a human serum fraction low in gamma-globulin and pre-albulmin, non-toxic

for the mouse and pyrogen free.

  4. New cell culture medium according to claim 3, characterized in that the serum fraction comprises 82% by weight of albumin; 3%

  by weight of alpha 1-globulin; 6% by weight of alpha 2-

  globulin and 9% by weight beta-globulin, gamma-free

globulin and without pre-albumin.

  5. Cell culture medium according to one

  of the preceding claims, characterized in that

  is brought to a cell concentration of 5 × 10 5 leuco-

cytesviables per milliliter.

  6. Culture medium according to one of the claims

  1 to 3, 5, characterized in that the aforementioned serum fraction is a human serum fraction diluted to 5% of protein, treated at -3 C and pH 7.2 with ethanol in an amount necessary and sufficient to precipitate the

  fibrinogen, then brought to - 6 C and pH 5.85 and a

  concentration in ethanol of between 10% and 20%, the precipitate obtained being discarded and the supernatant being collected and used as protein supplement supra

in said culture medium.

  7.- Culture process, characterized in that it comprises the culture of the cell culture medium such as

  than previously defined according to any one of the

  1 to 6, advantageously comprising the following steps: incubation at 37 ° C. in a CO 2 -riched atmosphere; - maintenance in culture for 3 to 5 weeks,

  preferably with a control of the cell concentration

lar; and

  - isolation of lymphocyte cell lines

  bl-astoides in suspension in the culture medium, preferably

  when the cell density is between 2.105 and

  5.10 cells per milliliter of medium.

  8. A process according to claim 7, characterized

  in that the isolated lymphoblastoid cell lines are isolated, and substantially without adherent cells

* nourisher, are multiplied in suspension in fermenta-

  tors. 9. Lymphoblastoid cell lines obtained

  by the process according to claim 7 or 8.

  10. Application of the culture medium or lymphoblastoid cell lines to the production of interferon, as defined according to any one of the

  Claims 1 to 6 or 9, respectively.

25720 93

  11. A process for the preparation of interferon, in particular alpha interferon, characterized in that it comprises the addition of an interferon inducer to the lymphoblastoid cell lines according to claim 9 or obtained by the method according to the claim 7 or 8

  and advantageously the separation of interferon.

  12. The process according to claim 11, wherein

  characterized in that the interferon inducer is Sendai virus. 13.Interferon, in particular interferon alpha, characterized in that it is produced by the process according to

claim 11i or 12.