Culturing Purifies Murine Lymph Node Lymphatic Endothelium

Abstract

Background: Lymph node (LN) lymphatic sinuses transport lymph, cells, and antigens from the periphery through the LN. The lymphatic endothelium lining these sinuses appears to be an important contributor to the lymph node immune response. It has been challenging to obtain sufficient LN lymphatic endothelial cells for investigation of their functions, as they are minor constituents of LNs. Methods and Results: A procedure was developed to purify lymphatic endothelial cells (LEC) from murine LNs, which yields large numbers of primary LN LEC. Two-dimensional in vitro cultures of dissociated LN stromal cells initially consist of multiple cell types, and then rapidly evolve to produce pure cultures of lymphatic endothelium within a few passages. One million LEC can be harvested after 4 weeks of culture, and much larger cell numbers can be obtained by continued culturing over long periods. The LEC cultures maintain endothelial morphology and expression of LEC markers, and preserve the same slow growth characteristics over at least 20 passages. The LEC cultures readily form tubes in Matrigel at early and at late passages, resembling those formed by LEC lines. Conclusions: A simple and economical approach to obtain purified primary murine LN LEC was developed for in vitro studies of their function. The morphology, growth characteristics, and functional behavior of these cells in tube formation assays did not change between initial and long-term passages. Large numbers of these cells can be harvested after long-term passage, so that they can be studied in biochemical and biological assays.

The Lymphatic Endothelial mCLCA1 Antibody Induces Proliferation and Growth of Lymph Node Lymphatic Sinuses

“…LN stromal cells were isolated from pooled LNs by collagenase digestion, and adherent cells were cultured on chamber slides either in the presence of 10.1.1 Ab or control Hamster IgG for 5 days. This isolation and culture method results in a mixed culture consisting of LECs, BECs, FRCs, and DN stromal cells [ 39 , 40 ]. While the morphology of cells in these cultures was similar, mitotic figures were common in the 10.1.1 Ab-treated cultures, prompting development of an assay to measure proliferation.…”

“…LN stroma was isolated by digesting LNs with Collagenase P (Roche) and Dispase I (Worthington Biochemical) as previously described [ 39 , 40 ]. Single cell suspensions were stained with anti-CD45, anti-CD31, and anti-Podoplanin and data collected on a BD CantoII cytometer.…”

“…LNs were digested as described above [ 39 , 40 ] or with Collagenase D (Worthington) and DNAse I (BD Biosciences) as described [ 2 , 41 ]. The cells from each mouse were plated into a 4-well chamber slide (Lab-tek, Nunc) in DME (Invitrogen) plus 10% fetal calf serum (Hyclone).…”

Simultaneous isolation of endothelial and alveolar epithelial type I and type II cells during mouse lung development in the absence of a transgenic reporter

“…There were 2 populations of CD31 pos (endothelial) cells: podoplanin (PDPN) neg literature and robust markers for these cell types (30). In the CD326 neg gated cells, CD31 pos cells were split in two groups based on PDPN expression, CD31 pos PDPN pos being the putative lymphatic endothelial cell population and CD31 pos PDPN neg being the vascular endothelial cells (17). The CD31 neg cells were analyzed for the expression of CD326 and markers of AEC type I (PDPN) and II (MHCII and LT) (Fig.…”

Regulation of T-cell Tolerance by Lymphatic Endothelial Cells

“…The small numbers of LECs makes many biochemical techniques impractical on ex vivo samples. As a result, several groups have optimized protocols to expand LNSC and LEC in culture [ 99 , 100 ]. However, the phenotype of LEC changes rapidly in culture, as the surrounding microenvironment contributes to the maintenance of LEC differentiation in vivo [ 59 , 101 – 103 ].…”