Murine Model of CD40-activation of B cells

The protocol for the generation of murine CD40-activated B cells (mCD40B) from splenocytes is divided into two parts: Part A demonstrates the preparation of murine CD40 ligand (CD40L) expressing HeLa cells (tmuCD40L HeLa), which will be used as plate-bound feeder cells. Part B describes the actual murine CD40-B culture.

A. Preparation of feeder cells (tmuCD40L HeLa)

The tmuCD40L HeLa is an adherent human epithelial cell line, which should never become completely confluent. The cells are therefore splitted twice per week. Culturing over more than 6 weeks is not recommended.

1. Remove old medium from the primary culture with a sterile pipette and wash cells with 10 mL of 1x PBS. Aspirate the PBS after washing.
2. Add 4 mL of 1x Trypsin/EDTA in a 75 cm² flask for 10-15 minutes at 37°C. Use gentle tapping to detach the cells.
3. Add 10 mL of wild type medium and swivel gently to stop digestion with trypsin.
4. Transfer the cell suspension into a 50 mL tube with a sterile pipette and spin the cells down at 265 x g for 7 minutes.
5. Remove the supernatant and resuspend the cell pellet in 10 mL of wild type medium. Count the cell number of an aliquot of the cell suspension and prepare three 50 mL tubes with the appropriate number of cells:
   1. 2.5 x 10⁶ cells for subculture
   2. 0.4 x 10⁶ cells/well for irradiation used for the murine CD40-B cell culture
   3. remainder to freeze (if needed).
6. Spin the cells down at 265 x g for 7 minutes.
7. Remove the supernatant.
   1. For subculturing: resuspend 2.5 x 10⁶ cells in 10 mL selection medium in a 75 cm² cell culture flask (cell density 2.5 x 10⁵ cells/mL) and incubate the cells at 37°C with 5 % CO₂. Split the cells twice per week.
   2. For the murine CD40-B cell culture: You need 2.4 x 10⁶ cells for one 6-well plate. Resuspend the tmuCD40L HeLa cells in wild type medium at a density of 0.2 x 10⁶ cells/mL and irradiate them at 78 Gy. Plate 2 mL of the cell suspension into each well and incubate them at 37°C with 5 % CO₂. Use this prepared plates for B-cell stimulation when tmuCD40L HeLa cells are adherent (at least 4 hours: check adherence using the microscope; do not wait more than 24 h to start B-cell stimulation). (Continue with B.)

B. Murine CD40-B cell culture

I. Preparation of splenocytes for CD40-stimulation (day 0):

Please note: before you continue ascertain that feeder cells are adherent. Always add fresh solutions of murine interleukin-4, β-Mercaptoethanol and cyclosporin A to the growth medium immediately before use.

1. Dissect spleen of C57BL/6 mice (haplotype H-2b) and wash it twice in 10-20 mL DMEM supplemented with Gentamycin [15μg/mL] by transferring it with a 10mL pipette tip from one tube to another. Avoid transferring contaminants (e.g. hair) of the mouse. Mince spleen by passing it through a cell strainer (100μm) and rinse strainer with another 10 mL of medium. Spin down splenocytes at 265 x g for 7 min at room temperature, resuspend them in 7mL muCD40-B cell medium, and separate lymphocytes by Ficoll density centrifugation. To do so layer cells on 5 mL Mouse-Pancoll (preheated at room temperature) in a 15 mL tube and centrifuge at 450 x g for 30 min without brake at room temperature.
2. Draw off most of the upper layer, leaving the lymphocyte layer undisturbed at the interface. Transfer lymphocyte layer to a fresh 50 mL tube, wash once with 30mL DMEM with Gentamycin [15μg/mL] by spinning down at 265 x g for 7 min to remove other cells. Discard the supernatant and resuspend the cells in 10 mL of mCD40-B washing medium. Determine the cell number in an aliquot of the cell suspension. Determine cell number by counting an aliquot of the cell suspension.
3. Spin down the required amount of cells at 265 x g for 7 minutes. For a 6-well plate 5 x 10⁶ cells/well are needed, thus 30 x 10⁶ cells per plate.
4. Remove the supernatant and resuspend the lymphocytes at 1.25 x 10⁶ cells/mL in mCD40-B culture medium freshly supplemented with 1 U/mL of interleukin-4 as growth factor, 100 μM β-Mercaptoethanol and 0.63 μg/mL cyclosporin A to prevent outgrowth of T-cells (Given concentrations refer to one mL culture medium!).
5. Remove the supernatant from the 6-well plate pre-incubated with tmuCD40L HeLa cells.
6. Gently add 4 mL of the lymphocyte suspension (1.25 x 10⁶ cells/mL) to each well of the 6-well plate.
7. Incubate the plate at 37°C with 5 % CO₂.
8. On day 3 reculture the cells (Continue with II.1.).

II. Subculture of mCD40B cells (day 3 and then twice a week):

1. Harvest mCD40B cell cluster by vigorously pipetting up and down with a 10 mL pipette and pool them into a 50 mL tube.
2. Spin down at 265 x g for 7 minutes and completely replace the supernatant with mCD40-B washing medium. While counting the cell amount of an aliquot, spin the cells down at 265 x g for 7 minutes. Resuspend mCD40B cells in mCD40-B culture medium at a concentration of 0.75 x 10⁶ cells/mL.
3. Add fresh solutions of murine interleukin-4 in a concentration of 1 U/mL, 100 μM β-Mercaptoethanol and 0.63 μg/mL cyclosporin A to the medium.
4. Remove the supernatant from a 6-well plate pre-incubated with irradiated tmuCD40L HeLa cells.
5. Gently add 4 mL of the mCD40B cell suspension (0.75 x 10⁶cells/mL) to each well of the 6-well plate.
6. Incubate plates at 37°C with 5 % CO₂.
7. Again subculture the cells every 3-4 days to end up with highly pure murine CD40-activated B cells after a total of 14 days.

C. Trouble-shooting: What if CD40-B cells do not grow?

1. Have you checked for CD40 ligand expression of feeder cells?
2. Are the plate-bound feeder cells used for the stimulation older than 24h?
3. Is there a contamination with mycoplasma?
4. Was the interleukin-4 solution used for supplementation freshly thawed and did it have the appropriate biological activity?
5. Was β-Mercaptoethanol and cyclosporin A added in the correct concentration?

Figure 1a.

Figure 1b.

Figure 1d.

Figure 2.