人血管内皮細胞に関する研究 : 1.初代細胞培養法の基礎的検討

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人血管内皮細胞のin vitro cultureの方法に関する培養条件を検討した。内皮細胞層を3種類の蚕白分解酵素処理-trypsin, collagenase, trypsin+collagenase, bacterial neutralprotease (Dispase)-によって分散し,preclostrum new born calf serumを最終濃度20%に添加した合成培地199中で開放系静置培養をした。35 mm plastic culture dishに初代培養され,24時間後にcell sheetをtrypsin処理し細胞数を算定した。臍帯静脈からの内皮細胞剥離をしらべるために酵素処理前後の切片を作製し組織学的に検討した。分娩から臍帯処理までの時間,及び胎児のApgar Scoreと得られた細胞数との関連について検討した結果,分娩後1.5時間以内にcollagenase処理を行った実験群が,細胞の収量及び培養の成績において最も効果的であった。collagenaseは内皮細胞層下の基底膜に働き内皮細胞層を効率よく剥離し,そのうえtrypsinなどとちがって細胞膜への障害も比較的少ないためと推察された。

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Various conditions of establishing the human endothelial cells in vitro culture were investigated. The endothelial cells derived from umbilical cord veins were collected by enzymatic digestions -with trypsin, collagenase, a mixture of trypsin and collagenase, bacterial neutral protease -and cultured in the synthetic Medium, 199 with 20% preclostrum new born calf serum. In primary culture the endothelial cells were seeded in 35 mm culture dishes for 24 hours and viable cells were counted after dispersing with trypsin. Section specimens of umbilical cords were histologically examined in order to confirm the isolation of endothelial cells from the umbilical cord veins before and after enzyme treatment. The relationship among the elapsed time after delivery till enzyme treatment, fetal Apgar Score and viable cell counts for primary culture were investigated. The treatment with collagenase within 1.5 hours after the delivery was most effective for collecting the endothelial cells. It is supposed that collagenase may act mainly on the subendothelial basement membrane, and damage the membrane of endothelial cells less than trypsin and other enzyme used in the experiment.