Hi, my name is Tanya Wig and I'm a PhD student and working in the laboratory for tumor immunology and transplantation immunology at the University Hospital of Cologne. My studies mainly focus on marine CD 40 activated B cells and their ability to induce immune responses in vivo. Originally, human CD 40 B cells were generated as alternative antigen presenting cells to dendritic cells.
Those cells are aimed to be used for immunotherapeutic purposes In clinical settings, CD 40 B cells are generated under CD 40 signaling and stimulation with IL four. According to this system, they can be expanded over several weeks. After optimizing the generation of human CD 40 B cells from PBMC in our lab, we transferred this principle successfully to mice and this is what I want to show you today.
Starting from spleen, we generate marine CD 40 activated B cells within 14 days of cell culture. The main steps are the preparation of feeder cells, therefore we use marine CD 40 ligand transfected heer cell line, which is adherent and becomes irradiated with 78 gray and plated onto six well plates. Second, we start marine CD 40 DB culture by using freshly purified SPOC cytes from black six mice, which become treated with marine interleukin four beta macab to ethanol and cyclosporine A.Those cells are then transferred on irradiated feta cells on six well plates.
This procedure is repeated every three to four days until day 14 of cell culture. To end up with highly pure marine CD 40 activated B cells, we begin the generation of marine CD 40 activated B cells using freshly purified SP cytes thromb LXi mice. First we wash the SP cytes by Resus suspending the cells in 50 milliliter of marine CD 40 B washing medium.
Then spin down the cells for seven minutes at 1300 RPM corresponding 260 5G for determination of the cell count. Thoroughly resuspend the cytes in 10 milliliter of marine CD 40 B washing medium, and finally mix the cells by vortexing. Then determine the cell count.
Spin down the required amount of cytes for seven minutes at 1300 RPM corresponding 260 5G using a six well plate. You need five times 10 to the six cells per well, thus 30 times 10 to the six cells per plate resource. Spend the required amount at one point 25 times 10 of the six cells per milliliter in marine CD 40 B culture media.
Then freshly supplement the cell suspension with marine IL four in a concentration of one unit per milliliter for stimulation cyclosporine A in a concentration of oh 0.63 microgram per milliliter to inhibit outgrowth of T cells and 100 micromolar Beamer CAPTA ethanol. Now the cell suspension is ready for stimulation with feer cells. We are using marine CD 40 ligand transfected healer cells to stimulate cell survival differentiation and proliferation as the heer cell line can survive in culture for several months.
Loss of marine CD 40 leg expression has to be excluded by fox regularly. CD 40 ligand transfected healer is an adherence cell line cultivated at 37 degrees with 5%CO2 in selection medium supplemented with icing B in a concentration of O 0.2 milligram per milliliter. For selection, this adherent epithelial cell line should never become completely confluent and should be split twice per week to split adherent cells.
Aspirate the medium wash once with 10 milliliter of PBS. Gently swivel and add four milliliter trypsin EDTA in a 75 square centimeter flak. Incubate the flask for five to 10 minutes at 37 degrees in the incubator.
Take the flask out again. Use gentle tapping to detach the cells from plastic. Then add 10 milliliter of biotype medium gently swivel and transfer the cells into a 50 milliliter tube.
Spin down the feeder cells for seven minutes at 1300 RPM corresponding 260 5G. Carefully resuspend pelleted cells in 10 milliliter of wild type medium to determine the cell count for he a subculture RESO spent 2.5 times 10 to the six cells in 10 milliliter selection medium and to transfer them into a 75 square centimeter flask and incubate the feeder cells at 37 degrees in 5%CO2 for CD 40 stimulation. Take the rest of the heer cells and lethally, irradiate them with 78 grade to stop them from proliferation.
Dilute the feeder cells in wild type medium with a cell density of O 0.2 times 10 to the six cells per liter and plate two milliliter per well on a six well plate. Let the cells become adherent again before using them as stimulator cells by incubating them for 24 hours in 37 degrees and 5%CO2 for stimulation of cytes with near CD 40 ligand. Check under the microscope if the feeder cells plated one day before are adherent.
If the feeder cells are adherent carefully remove the SUP natin from each well then gently transfer four milliliter of the SPOC side suspension, which had been adjusted to one point 25 times 10 to the six cells per milliliter shortly before to each well for stimulation and incubate the plate at 37 degrees and 5%CO2 on day four subculture marine CD 40 B cells again for the first subculture of marine CD 40, activated B cells on day four and following subcultures every three to four days. Gently harvest the clustered cells from the six well plate by reus spending with a 10 milliliter pipe. Pool the marine CD 40 B cells in a 50 milliliter tube and spin down the cells for seven minutes at 1300 RPM corresponding 260 5G completely exchanged the medium by marine CD 40 B washing medium.
And to determine the cell count of the marine CD 40 B cells, we suspend the marine CD 40 B cells at a cell density of oh point 75 times 10 to the six cells per milliliter in marine CD 40 B culture medium. Then again, supplement the cell suspension with fresh marine IL four in a concentration of one unit per milliliter, cyclosporine A in a concentration of oh point 63 microgram per milliliter and 100 micromolar beam up to ethanol. Of course, the irradiated feeder cells again have to be checked for the adherence under the microscope directly before use.
If the feeder cells are adherent, carefully remove the SUP natin from each. Well then transfer the marine CD 40 B suspension prepared shortly before to each well for stimulation. The subculture is repeated identically after three to four days twice a week to end up with highly pure marine CD 40 activated B cells on day 14 of culture.
At this point in time, Marine CD 40 B cells express co-stimulatory molecules such as CD 80, CD 86, and MHC molecules plus one and two, which can be checked by Fox during generation. The cells we generated right now can be used to do research on B-cell activation, differentiation and function, for example, to investigate their potential use for immunotherapeutic purposes.
