Many immune cells are versatile and can be easily manipulated in the lab to proliferate or perform the way you want. Culturing immune cells is relatively simple, but it’s easy to go wrong without the proper procedures, materials, and equipment.
When it comes to securing immune cells, some scientists think fresh cells are best. But freshness is not necessarily the best measure of quality. In fact, samples that are frozen within hours of collection have been shown to be more viable and functional than those delivered “fresh” up to 24 hours after collection.
If using frozen cells, the first step in a successful culture is proper thawing. For simple cell thawing, follow these instructions:
Once your cells are sufficiently thawed, you can begin your cultures. Below are tips for culturing some of the most commonly used immune cells.
Depending on the culture medium used (RPMI 1640, DMEM, X-VIVO 15™), the proportion of adherent and non-adherent monocytes grown in your culture will vary. If your mixture has a high percentage of adherent cells, you may have difficulty changing culture vessels or collecting cells intact. To prevent these issues, setup your culture in the same configuration you want to use in your experiment. If that is really not feasible, try culturing in Teflon (PTFE) or Ultra-Low Attachment culture ware from Corning®.
Note that after 5 days of culture in serum or M-CSF, your monocytes will be considered macrophages.
Just like monocytes, dendritic cells can be difficult to collect following culture depending on the mixture of adherent and non-adherent cells. Dendritic cells can survive in culture for approximately one week. To maintain the dendritic cell phenotype during culture, be sure to add GM-CSF at 500 U/mL and IL-4 at 500 U/mL when you start the culture after thaw. If you plan to use the dendritic cells after overnight culture, you don’t need to add any GM-CSF or IL-4.
Note that dendritic cells will not divide but can be stimulated to mature and secrete cytokines such as IL-12.
The procedures for maintaining a BC3 cell line are quite tedious and should be followed carefully, but the payout is worth it. This cell line can proliferate vigorously when properly stimulated with APC and antigen, resulting in all the cells you could ever use.
When measuring cytokine production from your culture medium, the optimum culture time will vary depending on the cytokines you want to measure. Typical culture time for cytokine production will be 24–48 hours.
You can repeat the culture process every 10 days to generate more cells. Cells can be frozen for later use.
B-LCL are relatively easy to grow, but the instructions should be followed carefully to ensure success. Make sure you freeze additional vials of cells after you start your cultures. If you notice the growth rate begins to slow after 6–8 weeks of culture, you should start up a new culture from your backup vials. We have noticed that when this slow down occurs, the cells usually are not as good at presenting antigen.
If you notice any accumulation of cell debris in your culture, don’t be alarmed. This is quite normal for B-LCL cultures. The debris may look like a bacterial contamination, but it’s actually a harmless material that seems to be shed by the cells.
Note that B-LCL express CD19, HLA-DR, and CD86.
Mouse bone marrow cells are some of the most versatile, multipurpose cells you can use. You can culture mouse bone marrow cells to create macrophages, dendritic cells, or mast cells.
To create macrophages, incubate your bone marrow cells for 3–4 days with M-CSF added until you begin to see colonies of macrophages. From this point, change the medium every 3–4 days until the macrophages fill the available surface.
When following the procedure to create dendritic cells, you will see a mix of adherent and non-adherent cells by day 9. The non-adherent cells should all be dendritic cells, while the adherent cells will be macrophages.
The mast cell procedure is simple, but requires repetitive centrifuging and fresh medium. Be sure to follow the protocol and centrifuge the non-adherent cells every 3–4 days three times.
If you’re using chondrocytes for joint repair or other procedures, you will need to accurately measure chondrocyte proliferation. When culturing chondrocytes, be sure to passage the cells with 0.25% tryspin-EDTA just before they reach confluence. Then you can transfer cells to a new vessel, incubate for 1–3 days, and continue with your culture.
Note that certain growth factors, like FGF-18, can increase chondrocyte proliferation. If you’re measuring type II collagen production or proteoglycan synthesis, you can use culture supernatant.
Culturing immune cells in your lab is a great option to ensure your cells are grown to meet your exact specifications. Just be sure to follow all protocols closely and use the recommended materials. For complete culture procedures, materials needed, and additional tips, see our full list of immune cell protocols.