WO1992009686A1

WO1992009686A1 - Supplement for hybridoma cell growth medium

Info

Publication number: WO1992009686A1
Application number: PCT/GB1990/001826
Authority: WO
Inventors: Lesley Torrance; Brian Reavy
Original assignee: The Scottish Crop Research Institute
Priority date: 1989-05-17
Filing date: 1990-11-26
Publication date: 1992-06-11
Also published as: GB9009950D0; GB2232998B; GB2232998A; GB8911356D0

Abstract

A supplement for use in the production and growth of hydridomas comprises supernatant fluid from a culture of a human cervical carcinoma cell line, HeLa, during the log growth period of the culture (usually at day 3, 4 or 5). The supplement avoids the need to use feeder cells during growth of fused cells and cloning; or serum during growth for antibody production.

Description

SUPPLEMENT FOR HYBRIDOMA CELL GROWTH MEDIUM

Field of the Invention

The invention relates to a supplement for culture medium for the production and growth of hybridoma cells, and which may be used as a replacement for feeder cells during cloning and/or growth at low cell density, and as a replacement for serum during growth at high cell density.

Prior Art

Hybrido as are obtained by fusion of a plasmacytoma (myeloma) and a lymphocyte, and are used for the production of monoclonal antibodies which find widespread uses in science and industry. The techniques for cell fusion and growth are well established (references 1 and 2) . At various stages in hybridoma production, hybrid cell lines must be grown at very low cell density. The usual practise when growing hybrid cell lines at low density, such as when cloning cells by limiting dilution or culturing cells after storage in liquid nitrogen, is to use a feeder layer of cells (for example mouse peritoneal macrophages, or other non-growing or slow growing cells) in order to stimulate growth of the hybrid cells. Such feeder cells supply growth factors which assist the growth of the hybridomas under such stressed conditions. Recently, conditioned medium supplements from mouse macrophage-like cell lines have been used as a substitute for feeder cells in such work. If feeder layers or other medium supplements are not used, such cell culture procedures tend to result in very little or no growth of cloned lines and probable loss of valuable cell lines on revival from storage. However, a major disadvantage of the use of feeder cells is the necessity to sacrifice animals every time cells are cloned (usually one mouse per cell line cloned) .

Moreover, hybridomas are generally cultured in the presence of serum, such as new born calf serum or foetal calf serum (FCS) which contains appropriate growth factors. However, the serum contains undefined species which can lead to non-reproducible results and variations in quality control.

A paper by A. Lazar, R. Wilson and R.E. Spier entitled "Growth Promoting Materials Derived from HeLa Cell Culture Supernatants" (Enzyme Microb. Technol. 1987 Vol. 9, May, 295 to 299) discloses the use of a serum-free concentrated conditioned medium derived from a human cervical carcinoma cell line, HeLa to reduce the serum requirement of cultures of baby hamster kidney and human fibroblast cells, or to replace serum completely. The medium is obtained by growing HeLa cells to confluency (i.e. when proliferation stops) , washing the cells and collecting the medium produced in the subsequent 48 hours. In other words, the medium used is that collected after confluency is established at the end of the cell growth stage.

It is an object of the present invention to provide a supplement for hybridoma growth media which removes the need to use feeder cells in fusion and cloning stages of hybridoma production, or the need to include serum in the growth medium of established hybridoma lines.

Suτππιarγ of the Invention

The present invention provides a supplement for use in the production and growth of hybridomas, which comprises the supernatant fluid withdrawn from a culture of a human cervical carcinoma cell line HeLa, during the log growth period of the culture.

Thus, in contrast to the Lazar et al reference, the present invention employs as a supplement the supernatant fluid withdrawn during the log growth period of the culture. It may be expected that different factors will be present in the supernatant fluid in the growth period and in the confluency period of the cell culture. Moreover, Lazar et al. is not concerned with the production or growth of hybridomas. According o the present invention, it is found that the supernatant withdrawn during the growth period is particularly valuable for the production and growth of hybridomas.

Preferably, the supernatant fluid is withdrawn at day 3, 4 or 5 of the culture period.

Whilst it is possible to withdraw the culture fluid continuously (adding fresh culture medium as desired) , better results are obtained if the cells are disrupted and sub-cultured after withdrawal of the supernatant fluid. Preferably, the cells are treated with enzyme (e.g. trypsin) to remove the cells from the walls of the culture vessel, diluted (e.g. to about 3 million cells per flask) , and reseeded in fresh growth medium in a fresh culture vessel. The supernatant may then be withdrawn once again at the optimum time (usually about day 4) during the log growth period of the reseeded culture. Reseeding may be repeated as desired.

Usually, the supplement is added to culture medium, such that the supernatant fluid constitutes 10 to 30% by volume of the culture medium.

It is found that the supplement according to the present invention may be used to wholly or partially replace feeder cells and serum, where these are used in the production and growth of hybridomas. The supplement may be used as a replacement for mouse macrophage feeder cells when cloning hybridoma cell lines, and to increase the survival of hybridomas when thawed after storage in liquid nitrogen. The supplement may also be used as a substitute for most of the serum requirement when growing hybridoma cultures for short periods of time before harvesting antibodies. The presence of very low amounts (e.g. up to 2%) of serum in the culture medium facilitates purification of the antibody preparations. Description of Preferred Embodiments

Embodiments of the present invention will now be described by way of example only in the following Examples.

Example 1 (Preparation of HeLa medium supplement (SCRICM) ) HeLa cells were grown in 260ml flasks as a monolayer in 25ml minimum essential medium (MEM) , (or Dulbecco's modified Eagle medium) containing 2mM extra glutamine, MEM non-essential amino acids (L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline and L-serine, 10% new born calf serum or foetal calf serum (FCS) , 100 units of penicillin and O.lmg streptomycin per ml. The supernatant fluid (medium supplement - SCRICM) was removed at day 4. The cells were treated with trypsin (to disrupt the cells and release them from the plastic flask surface), diluted to about 3,000,000 cells per flask, and reseeded in fresh medium in a fresh flask. Supernatant was collected at day 4 as before. The collected medium supplement was filtered through sterile 0.22 micron disposable filters (Millipore Millex-GS) and refrigerated (1-2 days) or stored frozen before use. The medium supplement was thawed and used at 10-30% (final concentration) in RPMI 1640 medium containing 15% FCS.

Growth of hybridomas in HeLa medium supplement. Example 2 (Cell cloning)

The mouse hybridoma cells used in the experiments were formed by fusing spleen cells from BALB/C or BALB/C x New Zealand black Fl hybrid mice with myeloma cell line X63/Ag8.653. The hybridoma cells were diluted to approximately 5 cells per ml in RPMI 1640 containing 15% FCS and distributed over the wells of microtitre plates (100 microlitres per well) , the wells of the microtitre plates contained either 1) a feeder layer of peritoneal macrophages, 2) 20-30 microlitres HeLa medium supplement (SCRICM), or 3) empty wells were used as a control i.e. no additions. After 10 days small colonies of cells were clearly seen in the plates containing the HeLa medium supplement. Because of increased numbers of cells and cell debris, it was more difficult to distinguish hybridoma colonies in the plates containing the feeder cells. The colonies were counted after 14-20 days and in experiment 1 where three hybridoma cell lines were cloned, the plates containing the HeLa medium supplement contained more growing colonies than the feeder cells or the controls (Table 1). In experiment 2, a further three hybridoma lines were cloned and the results obtained with HeLa medium supplement and with feeder cells were similar (Table 1) .

Table 1. Comparison of HeLa medium supplement and mouse peritoneal feeder cells to support the growth of cloned mouse hybridoma cells.

Treatment

Feeder cells Control 5 0 3 0 5 0

9 — II

39

7

' Numbers of wells containing growing colonies of hybridomas.

¹¹ Not done.

+ Wells seeded at approx 50 cells per ml.

Example 3 ( Survival after storage in liquid nitrogen) .

The HeLa medium supplement (SCRICM) has been successfully substituted for feeder cells when added at 20-30% to RPMI 1640 containing 15% FCS to aid survival of mouse hybridoma cells when thawed after storage in liquid nitrogen. Example 4 (Serum replacement and antibody production)

Two different mouse hybridoma cell lines which had been growing in culture for several generations and which were adapted to growth in relatively low levels of FCS (2-5%) were grown in RPMI containing 10% HeLa medium supplement (SCRICM) but without FCS. This composition supported cell growth and antibody production for 1-2 weeks. After three or four passages the cells began to die but the yield of specific antibodies as judged by their reactivity with homologous antigen in enzyme-linked immunosorbent assay (ELISA; Table 2) was as good as that from the medium containing FCS. The ability of the HeLa medium supplement to support growth in low serum conditions for several days is very useful when devising methods to purify monoclonal antibodies from hybridoma cell culture fluids, because the presence of very low levels of contaminating serum proteins simplifies purification.

Table 2. ELISA to check the specific activity of antibodies in cell culture fluids from hybridomas growing in two different media +

Hybridoma line 1 Hybridoma line 2 1 2 3 1 2 3 RPMI + 5%FCS 0.77" 0.45 0.78 0.74 0.89 0.99 RPMI + MS 0.86 0.60 0.68 0.81 0.82 0.86

+ Cell lines were growing in medium containing 5%FCS or 10% HeLa medium supplement (MS) . The cells were transferred to the respective media and culture supernatant fluid was harvested (1) at 4 days; (2) 8 (Line 1) or 9 (line 2) days; and (3) 14 (line 1) or 16 (line 2) days.

" Absorbance values A 405nm obtained in tests on specific antigen. The HeLa medium supplement may also be used to replace feeder cell layers in other cell culture procedures where cells are grown at very low density or when stressed such as during the fusion experiments when growing in HAZA, HAT or other form of selective medium.

Example 5 (Cell fusion experiment to produce hybridomas.) Brief experimental details

Materials: SCRICM lot 11.5.90 (prepared as generally indicated in Example 1) ; Igen lots 3 & 4 (a commercial supplement sold by Sigma Chemical Company Ltd. for comparison) ; spleen cells from immunised Balb/c mouse; and myeloma cell line X63/Ag8.653.

Method: The cell fusion was done by the usual methods using polyethylene glycol, fused cells were resuspended in 60 ml RPMI 1640 containing 20%FCS and HAZA (hypoxanthine-azaserine) . The mixture was distributed over six microtitre plates (100 microlitres per well) . Plates 1 & 2 contained 30 microlitres per well SCRICM; 3 & 4 contained 10 microlitres per well Igen material; and 5 & 6 contained no additives.

Results

The plates were checked for cell growth after 12 days (Table 3) . Many of the colonies observed were very small consisting of groups of only 10-12 cells, particularly those in plate 3 (Igen supplement) . The plates were screened again after 19 days (Table 3) and this result was more useful as some of the very small colonies had died. The results given in parenthesis in column three of Table 3 are for colonies secreting specific Ig.

The results in Table 3 show that use of either supplement gave a large increase in the number of hybridomas obtained after fusion. SCRICM gave the best results both in terms of number of hybridomas and the number which secreted specific Ig. Table 3. Comparison of supplements for use in cell fusion

Number of wells with hybridomas

Plate No. 12 Days 19 Days SCRICM 1 80 71 (33)* 2 66 58 f!8.

146 129 f51.

IGEN 3 60 42 (15)

4 78 69 .32.

(comparison. 138 Ill .47.

CONTROL 5 12 6 (2) 6 17 11 (5) 29 17 (7 )

* wells with cells secreting specific Ig.

Example 6. (Cloning experiment)

Brief experimental details

Materials: A mouse hybridoma cell line 2A1. SA5 diluted to 5 cells/ml with RPMI 1640 containing 15%FCS. Supplements used were (1) SCRICM lot 23.1.90 (2) SCRICM lot 26.3.90 (stored for 1 week at 4^,C) ; (3) Sigma hybridoma enhancing supplement lot no. 108F-0363 (expiry Nov 90) for comparison purposes; and (4) Medium from SCRI cell line grown in the normal medium but harvested continuously (cells not split - SCRI/CONT) . Supplement SCRICM as in Example 1.

For comparison, SCRI/CONT was prepared by removing the growth medium from the cells after 3-5 days as in Example 1 but (instead of trypsin treatment, dilution and reseeding) fresh medium was then added to the confluent cell layer and the culture was left for three days. The cells were therefore in a stationary phase of growth. The medium taken after the three days of stationary phase was SCRI/CONT.

Method: Each supplement was dispensed over three microtitre plates at 10 or 30 microlitres per well. Control plates received 30 microlitres RPMI 1640 containing 15%FCS. Hybridoma cell suspension added at 100 microlitres per well (0.5 cell/well). The number of wells containing hybridomas were counted after 12 and 18 days.

Results

Both lots of SCRICM whether used at 10 or 30 microlitres per well gave good results and were superior to the Sigma product (Table 4) . The results obtained with the medium supplement prepared in a different way (4) was poor, and the continuously harvested medium gave similar results to the Sigma product.

Table 4. Comparison of supplements for use in cell cloning. Plate Percent of wells with colonies

12 Days 18 Days

1. SCRICM 23.1 (30ul) 27 (78/288)* 32

2. SCRICM 26.3 (30ul) 28 (81/288) 33

(lOul) 26 (50/192) 34

3. Sigma (lOul) (comparison) 16 (45/288) 17

4. SCRI/CONT (30U1) ( " ) 11 (32/288) 17

5. Control 0.5 (1/192) 1 * Number of wells with colonies/number seeded.

(ul = microlitres) All of the proprietary media, amino acids, antibiotics and serum supplements were purchased from Gibco Ltd. Paisley PA3 4EF, Scotland and the cell lines (HeLa and myeloma) are readily available from cell culture collections or Flow Laboratories Ltd. Rickmansworth, Herts (ICN Biomedicals Ltd.). Although our tests were done with MEM, DMEM and RPMI 1640 culture media and mouse hybridomas it will be apparent to someone skilled in the art that the beneficial effects may also be found with other hybridoma cell types and/or synthetic culture media used for hybridoma culture, such as rat or human T- and B- hybridomas including heterohybrids (e.g. mouse x rat, mouse x human, rat x human) , myeloma and lymphoblastoid cells; or synthetic culture media types. These alternative media may or may not contain other additives such as insulin or various interleukins which are known to persons skilled in the art as beneficial to the growth of hybridomas.

REFERENCES

1. Goding, J.W. (1983) Monoclonal antibodies: principles and practice. Academic Press, London.

2. Campbell, A.M. (1987) Monoclonal antibody technology. Elsevier, the Netherlands.

Claims

1. A supplement for use in the production and growth of hybridomas, which comprises the supernatant fluid withdrawn from a culture of a human cervical carcinoma cell line, HeLa, produced during the log growth period of the culture.

2. A supplement according to claim 1 wherein the supernatant fluid has been withdrawn at day 3,4 or 5 of the culture period.

3. A supplement according to claim 1 diluted with culture medium, such that said supernatant fluid constitutes 10 to 30% by volume.

4. A supplement according to claim 1 having no added serum.

5. A method of producing a supplement for use in the production and growth of hybridomas, which comprises culturing a human cervical carcinoma cell line, HeLa; and withdrawing the supernatant fluid during the log growth period of the culture.

6. A method according to claim 5 wherein the supernatant fluid is withdrawn at day 3, 4 or 5 of the culture period.

7. A method according to claim 5 or 6, wherein after withdrawal of the supernatant fluid, the cells are treated with trypsin, diluted and reseeded in fresh culture medium; the reseeded cells being further cultured and further supernatant fluid being withdrawn during the log growth period.

8. A method of producing hybridomas which comprises growing fused cells in the supplement of claim 1.

9. A method of cloning hybridomas which comprises culturing the hybridomas in the presence of the supplement of claim 1.

10. A method of antibody production which comprises growing antibody-producing hybridomas in the presence of the supplement of claim 1.