GB2166756A - Tissue culture using human spleen cells, especially for interferon production - Google Patents

Abstract

A tissue culture medium characterized in that it comprises the cells originating from human spleens under a form ready for cultivation and a support of culture adequate for the growth of these human spleen cells, preferentially containing seric proteins, still preferentially containing at least in part purified serum or serum treated in a known way to render this support of culture apyrogenic and atoxic. The medium may be used in a method of culture of lymphoblastoid cell lines which are used for the production of interferon. The cells are also used to produce gamma-globulins.

Description

SPECIFICATION Tissue culture using human spleen cells especially for interferon production This invention describes a new tissue culture medium containing human spleen cells, a process of culture using this medium, the lymphoblastoid cell lines obtained, the production of gamma-globulins, an application to the production of gamma-globulins or interferon, a process of preparation of interferon, the gamma-globulins and the interferon obtained by this method.

Interferon is a proteinic molecule secreted by various cellular types. This protein interferes with the multiplication of some viruses and also with the multiplication of cancerous cells. Several types of interferon exist. Interferon alpha is secreted by lymphocytes and is believed to be beneficial in the treatment of common colds, of influenza and of some cancers. Interferon is species specific, thereby restricting the use of human interferon to diseases occuring in the human species, and vice-versa.

One line of research aiming at the obtention of human interferon consists in the excision of the human gene specifying its synthesis and in the reinsertion of this gene into a bacterium through genetic engineering.

Such a process can be achieved but the so obtained synthesis of interferon by bacteria is not necessarily active, the yields are poor and the industrial scaie-up is aleatory.

We endeavoured to produce interferon alpha in human cells. In general, interferon alpha is obtained through the inoculation of human lymphocytes by a virus but this technique of production is extremely cumbersome because the obtention of human lymphocytes is arduous and yields a product that must undergo important purification steps. The aim of this invention is to provide an interferon secreting cell line that is amenable to a scale-up meeting industrial requirements and to choose a culture medium largely devoid of the common serum ingredients that are the reason for the laborious purification steps of the interferon obtained.

These technical problems have been, for the first time, resolved by the present invention. A new spleen cell line culture medium has been devised containing serum proteins, preferentially prepared from purified serum or from serum treated in a known way to render the culture medium apyrogenic and atoxic and of spleen cell lines fit for continuous cultivation in this medium.

A characteristic of the cellular culture medium resides in that the human spleen cells are obtained from human spleens minced under sterile conditions, in a sterile physiologic solution such as the Hank solution. Also, following a particularly advantageous mode of interferon production, the culture medium is supplemented by a human serum fraction depleted in prealbumin and in gamma-globulins, atoxic and apyrogenic.

A preferred mode of realisation resides in the dilution of human serum to 5% proteinic content in phosphate buffered saline (PBS), its subsequent treatment at -3 C and pH 7.2 by ethanol in quantities necessary and sufficient to precipitate fibrinogene, then brought to -6 C and pH 5.85 and a concentration of ethanol comprises between 10% and 20%.

The obtained precipitate is discarded and the supernatant collected and used as proteinic adjuvant to the culture medium.

According to another particular characteristic of this invention, this new tissue culture medium is characterized in that the cells are suspended in an RPMI tissue culture medium enriched to 2 to 10% by the following seric fraction: 80% in weight of albumin, 3% in weight by alpha 1-globulin; 6% in weight by alpha 2-globulin and 9% in weight by beta globulin, and containing no detectable prealbumin or gammaglobulin.

The present invention concerns also a process of culture characterized in that it comprises the previously mentioned cellular tissue culture medium comprising in an advantageous way the following steps: - an incubation at 37"C in a C02 enriched atmosphere, - the maintainance of the cellular culture during 3 to 5 weeks at a determined concentration, this concentration being regularly controlled and adjusted and - isolation of the lymphoblastoid cell lines growing in suspension in the tissue culture medium, preferentially when the cellular density is comprised between 2.105 and 5.105'cells/ml of culture medium.

The procedure allows preferentially the multiplication in suspension of the isolated lymphoblastoid cell lines substantially in the absence of feeding adherent cells, in suspension in fermentors.

The present invention concerns also, under another of its aspects, the lymphoblastoid cell lines obtained by this culture method.

The cell lines were found to spontaneously synthesize and produce gamma-globulins.

On the other side, the present invention concerns also the application of the new culture medium and the isolated lymphoblastoid cell lines to the production of gamma-globulins or of interferon.

The present invention concerns also, according to one of the other aspects of the procedure, the preparation of interferon, particularly interferon alpha, characterized in that it comprises the addition of an interferon inducer to the mentioned lymphoblastoid cell lines and preferentially the separation of the gained interferon.

According to a preferential characteristic of this process of preparation of interferon, the inducer of interferon is chosen among the poly]: C (polyinosinic acid : polycitidylic acid) additioned of protamin and interferon-inducing virus such as Sendai virus.

The present invention concerns finally the interferon, particularly interferon alpha, produced by the precited production technique. Other purposes, characteristics and advantages of the invention will more clearly appear at the lecture of the explicative description. This description is given simply as illustration and should not in any way limit the invention.

Principal embodiments I. Cellular tissue culture medium according to the invention The tissue culture medium is characterized in that it entails human spleen cells under a form ready for culture and an adequate support of culture for the culture of these human spleen cells, preferentially containing seric proteins, still preferentially prepared at least in part from purified serum or serum treated in a known way to render this support of culture apyrogenic and atoxic.

The human spleen cells are obtained by the slicing of pieces of a human spleen obtained at the autopsy of a healthy donor. These human spleen pieces are passed through a mesh (0.075 mm), the cells being collected in an ice cold solution of Hank. After washing, the human spleen cells are transferred in an RPMI medium.

Preferentially, as indicated hereabove, the support of culture contains culture seric proteins or is supplemented with a seric fraction of human origin. So, preferentially the RPMI medium in which the human spleen cells are transferred is supplemented respectively with 4% (weight"volume) of the 2 preparations described comparing them to a medium supplemented with 10% fetal calf serum serving as control.

1). A seric fraction poor in gamma globulins (denominated AFF). The obtained composition after lyophilisation 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 % ,8-GLOBULIN 12.5 % 9 % GAMMA-GLOBULIN 0 100% 100% This material was prepared after Goose et al. (Applied microbiology : (1973), 25, 325-326), who devised it for the obtention of interferon induced in in vitro cultivated cells.

2) A seric fraction named EPF obtained in the following way: Plasma is diluted in an isotonic phosphate buffered solution to a proteinic concentration of 5% and treated at -30C, pH 7.2 by 1% ethanol final concentration. A precipitate of fibrinogen occurs which is discarded by centrifugation or filtration.

The supernatant is supplemented by ethanol until a final concentration of 19% is reached, the pH is lowered to 5.85 by 1 N HC1 and the temperature is lowered to -60C.

A heavy precipitate appears which is discarded by centrifugation or filtration.

The supernatant is collected, dialysed at 4"C against an isotonic solution of NaC1 to discard the ethanol and is thereafter lyophilized. In another preparation way, we have immediately lyophilized the supernatant without prior dialysis. In this case, the process of lyophilization is more delicate to perform. This procedure to purify plasma is known since a long time (Nischmann et al.; Helv. Chim. Acta : (1954) 37, 866-873). The content in proteins of APF is given in table 1. A more detailed analysis of the respective components of the two cited preparations was performed by gel electrophoresis on polyacrylamide and revealed that AFF contained still important quantities of IgG, IgA, C3, orosomucoids and traces of IgM and ceruloplasmin.On the other hand, the concentrations in IgG, igA and IgM of the analysed solution of EPF proved to be inferior to 0.02%.

The interest of this EPF preparation resides essentially in its apyrogenicity and its non toxicity in the mouse.

3). Fetal calf serum. This is a usual ingredient included in tissue culture media.

These three preparations were used to establish lymphoblastoid lines starting from the human spleen.

Since the EPF medium is the most interesting, the establishment of cellular lines based on the use of this nutritive agent will more principally be described in the purpose to obtain cellular lines able to produce thereafter interferon after growth in this growth medium containing this EPF agent.

II. Cellular culture of the lymphoblast The culture of the lymphoblast entails the tissue culture medium described, advantageously compris ing the following steps: - an incubation at 37 C in an atmosphere enriched in C02; - a maintainance of the culture during 3 to 5 weeks, with preferentially a control of the cellular concentration; and - the isolation of lymphoblastoid cell lines suspended in the culture medium preferentially when the cellular density is comprised between 2.105 and 5.105 viable cells per ml of culture medium.

The primary culture of cells of human spleen origin demands materials and an organ of human origin which we intended to avoid. The present inventors have succeeded in the cultivation in vitro in a permanent way of spleen leucocytes originating from healthy donors in a medium RPMI 1640 supplemented by EPF previously mentioned and which, surprisingly, appeared to be excellent producers of interferon alpha.

As precedently indicated, the human spleens taken at autopsy are minced, under sterile conditions, without previous perfusion, in a solution of ice cold Hank solution and passed through a sterile mesh.

The cells are washed in a solution of Hank and resuspended in a RPMI 1640 medium containing 2mM of L-glutamine as well as antibiotics and enriched from 2 to 10%, depending on the cultures, in the EPF medium previously mentioned.

The cellular concentration is preferentially brought to 1.105 viable leucocytes per ml of culture medium.

Thereafter 10 ml of this cellular concentration are seeded in Falcon bottles of 75 cm2.

The cells are incubated at 37"C in an atmosphere enriched by 5% of C02, the remainder gas being constituted of air, and maintained in culture with regular adjustment to a cellular density of 5.105 viable cells per ml of culture medium.

After one week of culture under these conditions, the cells which adhere to the plastic bottom multiply actively to reach confluency after 3 weeks of culture.

Simultaneously the number of small lymphocytes growing freely in suspension diminish to finally disappear whereas the lymphoblastoid cells multiplying at the surface of adherent cells which serve them as feeding layer, appear.

Their number increases in the course of time to, after about one month of culture under these conditions, pass in suspension.

The lymphoblastoid cell lines (LyC1) freed from the dependence of feeding cells continue to multiply in suspension. After obtention of a critical cellular density which has been found to vary for each cultured line but which is in general comprised between 2.105 to 5.105 cells per ml of culture, the lymphoblastoid cells may be transferred and multiplied in another bottle or in a fermentor in the absence of feeding adherent cells.

They multiply then under a logarithmic mode and may, once this stage is attained, be transferred in fermentors and multiplied under industrial conditions.

The lines which have been isolated by the present inventor according to this method were not all identical. The cells which compose these lines vary in aspect according to their apparent degree of maturity.

The more mature cells are the small lymphocytes. They are not the most frequently present, the more common form being the immature lymphoblast.

However, an evaluation of the mean volume of the cells performed in the midst of the logarithmic phase of growth of each line has shown that this volume may vary from 350 Fm to 950 Am depending on the line.

The lymphocytic B character of these lines was analysed for 5 of them which are respectively named ST1; ST2; ST3; CA and CB. All five showed the presence of surface gamma globulins. Thus, all the lymphoblastoid cell lines are usable for producing gamma globulins. The five analysed lines showed also the presence of Epstein-Barr virus antigen (EBV). This is not astonishing since the majority of the healthy adult population bears these antigens. The majority of the lymphoblastoid lines analysed showed diploidy, one single line was hypotetraploid, all had a basophile cytoplasm.

The above five lymphoblastoid cell lines obtained by the described technique where EPF was used in the tissue culture media were cultured a last time in three different culture media, the difference bearing on the component of seric origin used in the preparation.

These 5 lines were thereafter tested for their capacity of production of interferon.

111- - Application to the production of interferon The lymphoblastoid cell lines according to another aspect of the invention are used or applied to the production of interferon.

The process of preparation of interferon is, as precedently mentioned, characterized in that it comprises the addition of an interferon inducer to the iymphoblastoid cell line and preferentially the separation of the secreted interferon.

The invention concerns also the interferon produced by this process.

Preferentially, the inducer of interferon used is chosen amidst the group consisting in poly (I : C) (poly (inosinic acid : cytidylic acid) additioned with protamine or Sendai virus.

The results which have been obtained are mentioned in table II.

TABLE II Lymphoblastoid Interferon titer in unitsiml human B cell lines EPF AFF Fetal calf serum ST 1 16000 8000 2000 ST 2 4000 4 000 4 000 ST 3 2 000 4 000 8 000 CA 32 000 8 000 64 000 C B 1 000 2 000 2 000 In this particular case poly l: C was applied to ST 1 and ST 2 and Sendai virus to ST 3, CA and CB. It is observed in table II that interferon is secreted in variable quantities by the two applied induction techniques.

Other inducers may of course also be used. As can be seen in table II, one has also grown the lymphoblastoid cells on the two other seric adjuvants mentioned, i.e. AFF and fetal calf serum. It is observed that the three seric adjuvants used for the growth of the lymphoblastoid cells are adequate for the production of interferon, the seric adjuvant containing the least contaminant material (i.e. EPF) being preferred for the obtention of interferon destinated to a therapeutic use. Other formulations may of course be developed for the culture of the cells such as human or bovine albumin or else a purified extract of the growth factors contained in the serum without therewith leave the frame and the value of this invention.

Claims (16)

1. New tissue culture medium characterized by human spleen cells under a form ready for continuous culture, by a support of culture adequate for the growth of these human spleen cells and preferentially containing seric proteins, preferentially still prepared at least in part from purified serum or serum treated in a way already known to make this support of growth apyrogenic and atoxic.

2. Tissue culture and medium according to Claim 1, characterized in that the human spleen cells are obtained from the human spleen sterilely minced without previous perfusion in an ice cold Hank solution and passed on a sterile mesh.

3. Tissue culture medium according to Claim 1 or 2, characterized in that the culture support is supplemented by a human seric fraction poor in gamma globulin and in prealbumin non toxic for the mouse and apyrogenic.

4. New tissue culture medium according to Claim 3, characterized in that the seric fraction comprises 82% in weight of albumin, 3% in weight of alpha 1-globulin; 6% in weight of alpha 2-globuiin and 9% in weight of beta-globulin, without gamma-globulin and devoid of prealbumin.

5. Cellular tissue culture medium according to one of the preceding Claims, characterized in that it is brought to a cellular concentration of 5.105 viable leucocytes per ml.

6. Growth medium according to Claims 1 to 3 and 5, characterized in that the seric fraction is a human seric fraction diluted to 5% weightlvolume of protein treated at -3"C and pH 7.2 by ethanol in a quantity necessary and sufficient to precipitate the fibrinogen then brought to -60C and to pH 5.85 and to a concentration in ethanol comprised betweeon 10% and 20%, the precipitate again being discarded and the supernatant being collected and used as above mentioned proteinic supplement in above mentioned tissue culture medium.

7. Process of cultivation characterized in that it entails the cellular tissue culture medium as precedently defined according to one of any Claims 1 to 6, comprising advantageously the following steps: - an incubation at 37"C in an atmosphere enriched in C02 - the maintenance of the culture during 3 to 5 weeks, with preferentially a regular control of the cellular concentration and an isolation of the lymphoblastoid cell lines growing in suspension in the medium preferentially when the cellular density is comprised between 2.105 and 5.105 cells per ml of medium.

8. Process according to Claim 7, characterized in that the-lymphoblastoid cell lines isolated in suspension and substantially without adherent feeding cells are multiplied in suspension in fermentors.

9. Lymphoblastoid cell lines obtained by the process described in Claim 7 or 8.

10. Application of the tissue culture medium or of the lymphoblastoid cell line as defined in any one of the Claims 1 to 6 or 9 respectively to the production of gamma-globulins.

11. Application of the tissue culture medium or of the lymphoblastoid cell lines as defined in any one of the Claims 1 to 6 or 9 respectively, to the production of interferon.

12. Process of preparation of interferon, particularly interferon alpha characterized in that it comprises the addition of an inducer of interferon to the lymphoblastoid cell lines according to Claim 9 or obtained by the process described in Claim 7 or 8 and advantageously the separation and the isolation of interferon.

13. Process according to Claim 12, characterized in that the inducer of interferon is Sendai virus.

14. Interferon, in particular interferon alpha characterized in that it is produced by the process accord ing to Claim 12 or 13.

15. Gamma globulins characterized in that they are produced from the lymphoblastoid cell lines as defined in Claim 9.

16. A tissue culture comprising human spleen cells in continuously culturable form, in a growth me dium thereof.