Hi, my name is Alexander Shiku Van Hagen. I'm a member of the Laboratory for two Immunology and Transportation Immunology at the University Hospital of Cologne. In this video, we are going to show you how we can generate human CD 40 activated B cells from peripheral blood mononuclear cells.
One of the main interests of our laboratory is the study of the antibody independent functions of BB cells. Beyond their role as producers of antibodies, B cells have an important role as antigen presenting cells both under physiologic conditions as well as in autoimmune disease. After activation, B cells can acquire potent antigen presenting and immunostimulatory capacities, especially when activated via CD 40.
CD 40. Activated B cells have several advantages when compared to dendritic cells. They can be easily expanded at relatively low cost from small amounts of peripheral blood, even from cancer patients.
Now our PhD student, Tanya Leic and our technician LER, will give you a step by step introduction on how to generate human CD 40 activated B cells from peripheral blood. Hello, my name is Tanya Livi. I'm a PhD student and I want to demonstrate to you how to generate human CD 40 activated B cells.
This procedure was optimized in our lab. The main steps are, first the preparation of feeder cells. Therefore, we use human CD 40 ligand transfected N IH 33 cells, which become irradiated with 78 gray and plated onto six well plates.
Second, we start the culture of human CD 40, activated B cells from purified PBMC, which become treated with human interleukin four and cyclosporine aim, those cells are then plated onto feta cells on six well plates. This procedure is repeated on day seven and then every three to four days until day 14 of cell culture. To end up with highly pure human CD 40 activated B cells, we start the generation of CD 40 activated B cells with freshly purified or appropriately thought human PBMC.
First, we wash the pbmc for two times, therefore re suspend the cells in 50 milliliter of PBS. Spin down the PBMC for seven minutes at 1300 RPM corresponding 260 5G. This car, the supinate and grease, suspend the cells again in 50 milliliter of PBS spinned down again for seven minutes at 1100 RPM corresponding 190 G.This card the supinate again for determination of the cell count reus.
Suspend the PBMC in 20 milliliter of PBS thoroughly mix the cells by vortexing and determine the cell count. Spin down the required amount of PBMC for CD 40 B culture for five minutes at 1200 RPM corresponding 220 5G. Using a six well plate, you need four times 10 to the six cells per well plus 24 times 10 to the six cells per plate.
Then re suspend the PBMC at one times 10 to the six cells per milliliter in CD 40 B culture medium. Supplement the cell suspension with fresh human IL four in a concentration of 50 units per milliliter for stimulation and cyclosporine A in a concentration of oh point 63 microgram per milliliter to inhibit outgrowth of T-cell. Now the PBMC suspension is ready for stimulation with CD 40 ligand.
To our knowledge, there is currently no functional GMP grade SOMAL CD 40 ligand available. Therefore, we use human CD 40 ligand transfected NIH three T three cells to stimulate cell survival differentiation and proliferation as the NIH three T three cell line can survive in culture for several months. Loss of CD 40 ligand expression has to be excluded by facts regularly.
CD 40 ligand transfected, NIH three three is an adherence cell line and cultivated at 37 degrees with 5%CO2 in selection medium, which is supplemented with G 4 1 8 in a concentration of oh 0.7 milligram per milliliter. This adherent fibroblast cell line should never become completely confluent and should be split twice per week To split the adherence cells aspirate the medium wash once with 10 milliliter PBS gently swivel. Aspirate the medium again and add four milliliter tripsin EDTA in a 75 square centimeter flask.
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 the wild type medium gently swivel and transfer the cells into a 50 milliliter tube. Spin down the NA eight three T three cells for five minutes at 1200 RPM corresponding to hundred and 20 5G. Carefully resuspend the paled cells in 10 millimeter wire type medium.
Determine the cell count for N IH three three subculture and resuspend 1.5 times 10 to the six feeder cells in 10 milliliter wildtype medium. Transfer them in a 75 square centimeter flask and add G 4 1 8 in a concentration of oh 0.7 milligram per milliliter to select for the transfected cells which carry neomycin resistance. Genes finally incubate the feeder cells at 37 degrees in 5%CO2 for CD 40 B stimulation.
Take the rest of the NI 8 3 3 cells and lethally, irradiate them with 78 grade to stop them from proliferation. Dilute the irradiated feeder cells in wire type medium with a cell density of oh 0.1 times 10 to six cells per milliliter plate two milliliter per well on a six well plate. Let the cells on six well plates become adherent again before using them as feeder cells by incubating them for 24 hours in 37 degrees and 5%CO2 for stimulation of bi lymphocytes with CD 40 ligand.
Check under the microscope if the feeder cells plated one day before are adherent. If the feeder cells are adherent, remove SUP natin from each well and quickly wash them two times. First, wash each well with two milliliter PBS and second with two milliliters.
CD 40 B washing Medium For stimulation at the PBMC suspension. Prepared shortly before with a cell density of one times 10 to the six cells per milliliter to the feeder cells. To do so, gently transfer four milliliter of the cell suspension to each.
Well incubate the plate at 37 degrees in 5%CO2 on day seven. CD 40 B cells can be sub cultivated for the first subculture of CD 40. Activated B cells on day seven and following subcultures every three to four days.
Gently harvest the clustered cells from the six 12 plate by Resus, suspending them with a 10 milliliter pipe. Pull the CD 40 B cells in a 50 milliliter tube. Spin down the CD 40 B cells for seven minutes at 1200 RPM corresponding 220 5G completely.
Exchange the medium by CD 40 B washing medium and determine the cell count of the CD 40. Activated B cells. Re suspend the CD 40 B cells at one times 10 to the six cells per milliliter in CD 40 B culture medium.
Then again, supplement the cell suspension with fresh human IL four in a concentration of 50 units per milliliter and cyclosporine A in a concentration of oh 0.63 microgram per milliliter for stimulation. Uc, irradiated feeder cells prepared one day earlier. Of course, the irradiated feeder cells again have to be checked for adherence under the microscope directly before use.
If adherent, wash the feeder cells quickly for two times like before one time with PBS. The other time with CD 40 B washing media then carefully played four milliliter of the CD 40 B suspension adjusted to one times 10 to the six cells per milliliter on each well of the six well plate. The subculture is repeated identically after three to four days twice a week to end up with cell clusters of highly pure human CD 40 activated B cells on day 14 of culture.
At this point in time, CD 40 B cells express co-stimulatory molecules such as CD 80, CD 86, CD 83 MHC molecules, and the so-called lymph node homing triad of CD 62 ligand CCR seven and LFA one, which can be checked by Fox during generation For troubleshooting. Please refer to to text version of this protocol. We hope that in the future CD 40 activated B cells will serve as an important tool for cancer immunotherapy.
Good luck with your experiments.
