In order to obtain enough number of lymphatic endothelial cells (LEC) in vitro study such as cell adhesion assay, gene expression analysis, FACS and S1P release assays, we need more than 1 x 10^6 cells. Thus we used embryo to purify LEC. We could not get enough number of cell from LN even if we used more than 10-20 mice.
Thank you Dr Mohr. The cell number we need is more than 1 x 10^6, FACS sorting and subsequent cell culture for expansion does not allow us to get what we need. Yes, immortalization is the practical approach, but it is the final option for us. Immortalized cell line is already available, however, the expression of signature marker is different from that from LN or fetus.
We use a lymph node stromal cell isolation procedure similar to the one outlined in Fletcher et al. Frontiers in Immunology 2011, 2:35. In that procedure, you prepare a a single cell suspension by enzymatically digesting the LN, then you plate 10^7 cells per well in a collagen and fibronectin-coated 6 well plate. If you change the media on days 1 and 3, you will remove most of the non-adherent CD45+ cells and by day 4-5, you have a mixture of mostly LECs and FRCs. You can split the stromal cells a couple more times for expansion, but ultimately you will have do FACS sorting to get a pure population of LECs.
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