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Updated: Dec 10, 2025
Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting
Published on: May 1, 2015
A novel method for isolating lymphatic endothelial cells from lymphatic malformations and detecting PIK3CA somatic
Hidehito Usui1, Yoshinori Tsurusaki2, Hiroko Shimbo2
1Department of Surgery, Kanagawa Children's Medical Center, 2-138-4, Mutsukawa, Minami-ku, Yokohama, Japan. usui9282@yahoo.co.jp.
Purpose:
Tissue disaggregation and the cell sorting technique by surface markers has played an important role in isolating lymphatic endothelial cells (LECs) from lymphatic malformation (LM). However, this technique may have the drawback of impurities or result in isolation failure because it is dependent on surface marker expressions, the heterogeneity of which has been found in the lymphatic system. We developed a novel method for isolating LM-LECs without using whole tissue disaggregation.
Methods:
Seven LM surgical specimens were collected from seven patients with LMs. LM-LECs were detached from the LM cyst wall by "lumen digestion" and irrigating the cystic cavity with trypsin, and maintained in culture.
Results:
The cells formed a monolayer with a cobblestone-like appearance. Immunohistochemistry and quantitative RT-PCR of these cells revealed high expression of lymphatic-specific genes, confirming their identity as LM-LECs. The whole-exome sequencing and PIK3CA sequencing of these cells revealed somatic mutations in PIK3CA in all cases.
Conclusions:
We established a novel technique for isolating LM-LECs from LM tissue by "lumen digestion" without whole-tissue disaggregation. The limited incorporation of non-LM LECs in the isolate in our method could make it an important tool for investigating the heterogeneity of gene expression as well as mutations in LM-LECs.
