Our resident dendritic cell expert, Benjamin Tjoa, was one of the first scientists in the world to develop dendritic cells for use in cell-based vaccines. Ben has traveled internationally to help labs prepare for developing dendritic cell-based therapies.
Monocyte-derived dendritic cells are a hot topic for many of our customers, and Ben has answered countless questions via our Ask A Scientist form. Here are some of the most popular and interesting questions Ben has answered about working with MDDCs, including recommendations for using them in a mixed lymphocyte reaction.
“What is the best way by flow to check that my MDDCs are truly MDDCs? Which is the best marker?”
Monocyte-derived dendritic cells are generally characterized by high expression of CD11c and low or no expression of CD14, as opposed to monocytes, which are CD11c+ CD14+/high. Low expression of CD14 can be affected by the culture medium (e.g., FBS versus human AB serum versus serum-free media). MDDC also typically contain a population of cells that express CD1a. In our experience, the proportion of CD1a+ population is dependent on the monocyte donor.
“You state that your human dendritic cells will survive for about 1 week. Do they start dying immediately, or will viability be >90% for the first few days, followed by a dropoff? Does activation change viability dynamics?”
Our cryopreserved dendritic cells have good viability upon thaw. The cell count, viability, and flow cytometry data in our Certificates of Analysis are all measured from a thawed vial.
We have thawed a vial of dendritic cells, cultured the cells both in the presence and absence of dendritic cell activation factors (e.g., poly I:C, LPS, CD40 ligand, etc.) for up to 48 hours and then completed flow cytometric analysis. Most cells maintained viability.
Adding GM-CSF (500 U/mL or about 10 ng/mL) to the culture seems to help with viability, although we have not done a quantitative comparison.
“Would culturing dendritic cells in plates that assist with adherence (like poly-lysine or gelatin) increase viability or length of culture?”
We have not used poly-lysine or gelatin-coated plates for dendritic cells. These cells adhered readily to regular tissue-culture treated plates.
“I want to use monocyte-derived dendritic cells for a mixed lymphocyte reaction. Are these the appropriate cells to use?”
Yes, you can use MDDCs for a mixed lymphocyte reaction. Follow our recommendations:
- Thaw dendritic cells 1–2 days prior to MLR and culture in the presence of maturation/activation factor (e.g., about 2 million cells in one well [24-well plate], in 2 mL X-VIVO 15Ⓡ media + 20 ug/mL poly I:C). Culture for 24–48 hours.
- On the day you plan to start the MLR, harvest the dendritic cells and thaw CD4+ effector T cells.
- For a 96-well round bottom plate, total culture volume is 200 uL/well. Media in the outer wells tend to evaporate excessively, so fill outer wells with 200 uL PBS.
- Plate about 100,000 effector cells/well.
- Titrate the number of dendritic cells/well to get the response you need (e.g., 1:4, 1:8, 1:16, 1:32, etc.), then pick one that suits your needs. If you want to test an agent that enhances MLR response, choose a condition that produces a sub-optimal response.
- Collect the supernatant for cytokine assays after 5–6 days.
- Alternatively, you can label the CD4+ T cells with CFSE and assess proliferation.
“Are your monocyte-derived dendritic cells mature DCs?”
Our monocyte-derived dendritic cells are immature DCs. CD14 expression varies from lot to lot, ranging from negative to a level much lower than monocytes.
On page 2 of the Certificate of Analysis for each dendritic cell product, you can find flow cytometric data, including CD14/CD11c, CD86/CD40, CD80/CD1a, and HLA-DR/CD83. We have seen that activation/maturation of these DCs prior to MLR increases the response considerably. HLA typing data is also presented in each Certificate of Analysis.