The overall goal of this procedure is to rapidly expand large numbers of human NK cells. Ex vivo begin by isolating p BMCs or NK cells from buffy coat or whole blood sample. Then co-culture pbmc with artificial antigen presenting cells in NK cell expansion.
Media supplemented with IL two maintain the culture of NK cells for three weeks with weekly stimulations with a a PCs taking care to keep NK cell numbers under one times 10 to the sixth per milliliter of culture. Media finally validate the functional efficacy of the expanded NK cells by using a non-radioactive cytotoxicity assay. A typical NK cell yield by this expansion scheme results in a median 21, 000 fold expansion of NK cells at the end of 21 days that have a high cytotoxicity against various tumors as determined through a calcium release assay.
This medical can help answer a key question in the NK cell biology field because large numbers of NK cells have historically been very difficult to obtain. This technique has implications towards the immunotherapy of human cancer because generating enough NK cells for such therapy has historically been very difficult. Demonstrating this technique will be Cecily, the senior research assistant for my laboratory To obtain P BMCs blood from healthy donors.
Typically as blood bank Buffy coat is used as a source. First, add PBS to adjust the volume of the Buffy coat to 140 milliliters. Then layer 35 milliliters of sample onto 15 milliliters of fial PA for the gradient separation centrifuge at 400 G for 20 minutes.
Without the break. Carefully recover the P BMCs from the Fial PA plasma interface and set aside the tube of RBCs to use Later. Wash the P BMCs three times with PBS centrifuging each time at 400 G for 10 minutes.
Use this fraction of P BMCs for NK cell expansion. Now return to the gradient tube. Aspirate off the fi.
Call and transfer the RBCs into two 50 milliliter centrifuge tubes wash with PBS added to the 50 milliliter mark. After centrifugation, aspirate the supernatant by skimming the top of the RBC layer. To remove granulocytes.
Perform three washes, then resuspend the RBCs in an equal volume of else's solution and store at four degrees Celsius for up to one month. This RBC fraction may be used for roset SEP purification of NK cells at the end of two weeks of expansion. To efficiently expand functional NK cells ex vivo K 5 62 clone nine cells with membrane-bound IL 21 referred to hereafter as K 5 62.
Clone nine IL 21 are used as an artificial antigen presenting cell first count pbmc and calculate volume for five times 10 to the six cells to be seated for NK cell expansion for each five times 10 to the sixth pbmc count and irradiate 10 times 10 to the sixth K 5 62 clone nine IL 21 cells. Using a gamma irradiator at 100 gras irradiated cells can also be frozen for future use. Now wash the pre irradiated cells with PBS and RESUSPEND in NK cell expansion media.
Now see a two to one ratio of irradiated cells with pbmc in 40 milliliters of NK cell expansion media and place the T 75 flask upright in an incubator at 37 degrees Celsius and 5%carbon dioxide on day three. Recover the stimulated cells by centrifugation at 400 G for five minutes and replace half of the media with fresh NKEM and continue to culture. Repeat a change of medium on day five At the end of one week, count the number of cells set aside five times 10 to the fifth cells for phenotyping by flow cytometry for the second stimulation count and irradiate a one to one ratio of K 5 62.
Clone nine IL 21 cells to expand its cells and then combine the cells in NK cell expansion media at 2.5 times 10 to the fifth total cells per milliliter. Now see the cells in T 75 flasks and continue to culture on days 10 and 12 passage cells in NK cell expansion media at 2.5 times 10 to the fifth cells per milliliter at day 14 of expansion. Again, enumerate the cells and phenotype a sample by flow cytometry.
Purify this PBMC derived cell expansion using the rosette SEP purification protocol and verify purity of NK cells by flow cytometry phenotyping. Perform a third stimulation on the purified NK cells using a one-to-one ratio of irradiated K 5 62 clone nine IL 21 cells. Finally at day 21, analyze the amplified NK cells by flow cytometry using a full phenotyping antibody panel.
Freeze cell stocks in FBS containing 10%DMSO at a maximum density of five times 10 to the seventh cells per vial. Use either fresh or frozen NK cells for the cytotoxicity assay. If using frozen NK cells first thaw vial of NK cells and seed in NKEM for 24 hours to allow cell recovery on the day of the assay, resus suspend 10 to the six target cells in one milliliter of NKEM containing calcium am and incubate for one hour at 37 degrees Celsius with occasional shaking in a UBO 96 well plate add in triplicate 200 microliters of NK cells suspended at one times 10 to the six cells per milliliter and perform serial dilution of the cells as indicated.
After one hour of calcium loading. Wash the target cells in NK cell expansion media twice centrifusion for five minutes at 400 G.Now recount the target cells and resuspend at one times 10 to the fifth cells per milliliter. Add 100 microliters of target cells to each well of the 96 well plate and centrifuge for one minute at 100 G to initiate cell contact, incubate at 37 degrees Celsius and 5%carbon dioxide for four hours.
In order to uniformly suspend the released calcium, mix the culture gently by pipetting pellet cells at 100 G for five minutes and transfer 100 microliters of the supernatant to a new flat. Bottom 96 well plate taking care to avoid bubbles if necessary, pop any bubbles that may form using a fine needle. Read the plate using a fluorescent plate reader set at excitation filter 485 nanometers and emission filter at 530 nanometers.
Calculate percent specific lysis. Typically the expansion of five times 10 to the sixth pbmc isolated from buffy coat yields between one times 10 to the ninth and 10 to the 10th. NK cells, the expanded NK cells express various NK cell receptors that are comparable to the unexpanded primary NK cells in the buffy coat.
NK cells may comprise two to 18%of the pbmc at day 14. Rosette SEP purification of expanded cells can recover 40 to 70%of NK cells present in the culture at an NK cell purity of 99%indicated by CD three. Negative CD 56 positive cells expanded NK cells have demonstrated cytotoxicity against a range of tumor cell lines, including neuroblastoma, A ML osteosarcoma and melanoma.
Here the representative A ML killing is shown as percent specific lysis. The live BIOT video shows NK cell killing of neuroblastoma cell line CHP 1 34 loaded with calcium am After this procedure. Other methods like analysis for cytokine production by NK cells and adaptive transfer in animal tumor models can be performed, you know, to answer additional questions regarding the functions of the NK cells.
After watching this video, you should have a good understanding of how to expand and purify large numbers of NK cells. This can be accomplished directly from peripheral blood mononuclear cells and then the NK cells purified or the expansion can be initiated using NK cells purified by the rosette method. At the very beginning, a typical buffy coat will yield from 15 to 40 million NK cells, all of which can be used to expand very large numbers of NK cells using the scheme described in this method.
