Dynabeads® Mouse CD3/CD28 T Cell Expander From Invitrogen

For research studies involving T cells, it is essential to stimulate the cells in order to initiate activation, cytokine production, and proliferation. Without a stimulus, T cells in culture will remain inactive and eventually die. Common ways to stimulate T cells include treating with PMA, or exposing T cells to other cells or antigens that provide an actual or mimicked interaction between the T cell antigen receptor (TCR) and peptide-MHC complexes that reside on the surface of antigen presenting cells (APC).

A simple way to imitate this in vivo interaction between the T cell and APC is to use a product like the Dynabeads® Mouse CD3/CD28 T Cell Expander from Dynal Biotech (Prod. No. 114.11). These polystyrene beads are coated with a mixture of monoclonal antibodies against the CD3 and CD28 molecules that are expressed on the surface of T cells. During TCR-APC interaction, the T cell receives the transmitted signal through the CD3 complex within the TCR. For complete stimulation and proliferation however, the T cell requires additional signaling events such as cytokine binding and other growth and differentiation signals. An example of one of these necessary co-stimulatory signaling events is the interaction of CD28, found on T cells, with members of the B7 protein family expressed on the APC. Since the Dynabeads® contain antibodies against both CD3 and CD28, the beads, along with rIL-2 (not included with product), provide a complete stimulus for activation and proliferation.

I have used this product in my research to expand populations of T cells isolated from mouse spleen. Spleens are harvested, and T cells are isolated using the Pan T Cell Isolation Kit from Miltenyi Biotech (or a similar method). Cells are resuspended in media recommended in the Dynabeads® protocol: RPMI-1640 containing 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine. Cells are seeded at one million cells per well in a 24-well plate. Dynabeads® should be washed once prior to usage, and this is done by mixing an aliquot of beads and an equal volume (or at least 1 ml) of wash buffer (PBS with 0.1% BSA) in a tube. Because of the “superparamagnetic” properties of the beads, a magnetic stand (not provided) is used to remove beads from the wash buffer, and beads are resuspended in the original bead aliquot volume. After washing, beads are added to each well in a ratio of 10 ul of beads per 1 million cells, along with 10 U of rIL-2. Cells are allowed to culture for the desired time; cells should be counted twice per week.

In my experience, it has taken 2 days for each well to double (and sometimes triple) in cell number. When populations reach 2.5 million or when media gets yellow, cells should be split back to 0.5 to 1 million cells per ml of fresh media with 10 U/ml rIL-2. If cells appear small or just aren’t proliferating, cells can be re-stimulated using a magnetic stand to remove old beads, followed by centrifuging cells, resuspending in fresh media, and adding new beads and rIL-2. It is recommended in the protocol to first restimulate after 8 or 9 days of culturing, and then to add another day or two to this for subsequent stimulations.

For my experiments, I have followed this protocol very closely and I can’t seem to get my cells to survive past the first restimulation (10 days, which is much less than the 3-4 weeks described in the protocol). It even seems that when the old beads are removed during restimulation, cells go with these beads, leaving fewer cells to proliferate. I have noticed this by counting cells just before and after restimulation.

Overall, the product is very effective in expanding and maintaining a T cell population for just over a week, but I have yet to be successful culturing longer than this. Luckily, my experiments examine short-term adaptations to T cells and long-term culturing is not necessary. So, for longer culturing times, troubleshooting would be necessary.