Preparing T Cell Growth Factor from Rat Splenocytes

Summary

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Abstract

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion ^1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization ³. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro ³. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2.

Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20°C.

Protocol

1. Take Lewis rat spleens (rats between 160-200g are best). Dilacerate on ice in a petri dish containing PBS + antibiotics (PBS-PS) using a cell strainer. Put in a 50 ml tube. Fill with PBS-PS.
2. Spin for 10 min at 4°C to pellet the cells.
3. Wash the cells twice.
4. Resuspend the pellet in NH₄Cl 0.15 M (5 ml per spleen). Mix gently and continuously with a pipet for 3 min on ice to lyse the erythrocytes. Fill the tube with medium.
5. Spin for 10 min at 4°C to pellet the cells.
6. Wash the cells twice.
7. Count the cells. A rat spleen gives 200-250 million cells.
8. Seed the cells at 2 million per ml in complete medium: 50 ml per 75 cm² flask.
9. Let grow for 48 hours in the incubator.
10. Spin 15 min at 4°C to pellet the cells.
11. Collect the supernatant; discard the cells.
12. Add 15 mg/ml a methyl mannoside to the supernatant. Mix thoroughly.
13. Filter (0.2 mm)
14. Aliquot and store at -20°C. Can be kept at 4°C for 10 days if necessary.

Discussion

We prepare TCGF from Lewis rat splenocytes since we regularly euthanize naive rats from this strain to harvest serum and thymi to stimulate Lewis rat T cell lines in vitro. This TCGF can be used to promote the growth and survival of T cell lines from other rat strains. TCGF can also be prepared from other strains of rats.

Materials

Material Name	Type	Company	Catalogue Number	Comment
PBS, 1x, sterile	Reagent	Gibco / Invitrogen	14040-182
Penicillin / Streptomycin 100x	Reagent	Sigma	P0781	Add 5 ml of 100x solution to 500 ml PBS to prepare PBS-PS
Cell strainer, 70 um diameter	Tool	Fisher	08-771-2
Ammonium Chloride (NH4Cl)	Reagent	Sigma	A0171	Prepare a 0.15 M solution in sterile distilled water, keep cold
RPMI 1640	Reagent	Gibco / Invitrogen	21870-092
Fetal Bovine Serum (FCS, FBS)	Reagent	Gibco / Invitrogen	16140-071	Heat-inactivated
Penicillin / Streptomycin / L-Glutamine	Reagent	Cambrex / Biowhittaker	17-718R	PSG
RPMI vitamins, 100x	Reagent	Sigma	R7256
Sodium pyruvate, 100x	Reagent	Sigma	S8636
Non-essential amino acids, 100x	Reagent	Sigma
Beta-mercaptoethanol	Reagent	Sigma	M7522
alpha methyl mannoside	Reagent	Sigma	M6882
Concanavalin A	Reagent	Sigma	M6882

To prepare complete culture medium add the following to a 500 ml bottle of RPMI 1640 and sterile-filter: 10% FCS; 1 bottle of PSG; 5 ml RPMI vitamins; 5 ml sodium pyruvate; 5 ml non-essential amino acids; 50 uM beta-mercaptoethanol; 2 ug/ml Concanavalin A.