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Frequently Asked Questions
1. Can T cells be passaged?
T cells cannot be passaged, but they can be expanded and activated upon addition of stimulants. Various methods can be used for activation and expansion, such as PHA, PMA, SEB, and CD3/CD28 antibodies. The standard method for activating and expanding T cells is using CD3/CD28-coated magnetic beads.
2. Why are all the T cells floating around—have they died?
T cells and all lymphocytes are suspension cells that do not adhere to surfaces; as long as the cell morphology appears uniform and translucent under the microscope, cell counting shows high viability, and the cells are in good condition, there is no issue.
3. What is the protocol for T-cell activation and expansion?
The medium consists of 1640 supplemented with 10% FBS, CD3/CD28 activation magnetic beads, and a specified concentration of IL-2; if feasible, serum-free medium is preferred.
4. Can PBMCs be passaged?
No, this is not possible. PBMCs are a heterogeneous mixture of multiple cell subtypes, including lymphocytes (T, B, and NK cells) and monocytes; they are not a single-cell population. None of the individual subtypes within PBMCs can be passaged or cultured indefinitely. However, certain cell types can be induced to differentiate, while others can be activated and expanded.
Inducible differentiating cells: monocytes. Monocytes in peripheral blood lack the capacity for proliferation, but can be induced by cytokines to differentiate into dendritic cells (DCs) and macrophages (including M0, M1, and M2 phenotypes).
Cells that can be activated and expanded include T cells and NK cells.
5. Can PBMCs be maintained in culture after receipt? How many days can they be cultured?
It is generally recommended to use PBMCs immediately after recovery; the longer PBMCs are cultured in vitro, the poorer their viability becomes, with many cells gradually undergoing apoptosis. Some customers choose to culture the cells overnight to restore their viability; a slight reduction in cell activity after an overnight incubation is typically considered normal. If there is a specific need to maintain the cultures, they can be continued in 1640 medium supplemented with 10% FBS; however, such maintenance should be completed within 2–3 days and not extended beyond one week. If a particular cell subtype is required, experiments can be conducted according to the corresponding experimental protocol.
6. T-cell activation fails
Batch-to-batch variability is expected; however, based on the experimental data accumulated to date by Heyousheng, most batches demonstrate robust expansion under CD3/CD28 magnetic-bead–mediated activation.
7. Monocytes do not adhere to the culture surface.
Cryopreservation can exert some effects on monocyte function, with substantial batch-to-batch variability. For applications involving the induction of DCs or macrophages, we recommend using fresh samples. HeyouSheng is continuously optimizing the monocyte cryopreservation protocol and will subsequently offer cryopreserved, induced-monocyte batches that have been rigorously screened to ensure receipt of samples with superior functional integrity.
8. Precautions and Yield in PBMC Isolation
The optimal temperature for Ficoll during density gradient centrifugation is 18–22°C; temperatures that are too high or too low can severely compromise the viability of the isolated cells, with typical recovery rates ranging from 60% to 80%.
9. Yield of Macrophages/DCs Induced by Monocytes
Around 50% to 60%.
10. Why are fresh cells yellow? What about cell debris and platelets in PBMCs?
A small amount of autologous plasma (at a concentration of approximately 15%, which imparts a yellowish hue) was added to the fresh PBMCs. The plasma contains abundant proteins that help maintain better cell viability; the plasma concentration will subsequently be reduced to about 10%. The presence of a small number of cellular debris in the cells is normal. As for platelets, variations in their yield among different apheresis devices are common and generally do not affect the experimental results.
11. Methods for Sterility and Mycoplasma Testing in the Laboratory
Sterility Testing: Plate Spread Method (Incubation for 14 Days)
Mycoplasma: Colorimetric Method
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