公开(公告)号：US08609408B2

公开(公告)日：2013-12-17

申请号：US13258039

申请日：2010-02-08

Applicant: Tingjun Fan ,  Jun Zhao ,  Xiuxia Yang ,  Rishan Cong

Inventor： Tingjun Fan ,  Jun Zhao ,  Xiuxia Yang ,  Rishan Cong

IPC: C12N5/071 ,  C12N5/00

CPC classification number: C12N5/0621 ,  A61L27/3604 ,  A61L27/3808 ,  A61L27/3839 ,  A61L2430/16

Abstract: This invention relates to a method for the reconstruction of a tissue-engineered human corneal endothelium. Human corneal endothelial cells are cultured in vitro to logarithmic growth phase using 20% calf bovine serum-containing DMEM/F12 medium. Trypsin is used to digest epithelial layer of the freeze-dried human amniotic membrane in order to produce denuded human amniotic membrane as scaffold carriers. The scaffold carriers are tiled on the bottom of culture plate wells until they are dried and completely adhered to the bottom of wells. Human corneal endothelial cells at logarithmic growth phase are re-suspended in DMEM/F12 medium containing type-IV collagen and 20% calf bovine serum. Human corneal endothelial cell suspension is subsequently inoculated to amniotic membrane scaffold carriers that are tiled on the bottom of wells in culture plate to launch in vitro culture as well as in vitro reconstruction of tissue-engineered human corneal endothelium. This invention is scientific and rational. The reconstructed tissue-engineered human corneal endothelium can be produced on large scale to satisfy the great demand of tissue-engineered human corneal endothelium in clinical cornea transplantation for primary corneal endotheliopathy therapy. Meanwhile, costs for in vitro reconstruction of tissue-engineered human corneal endothelium and clinical therapy are low.

Abstract translation: 本发明涉及一种重组组织工程人角膜内皮的方法。 使用含有20％牛血清的DMEM / F12培养基将人角膜内皮细胞体外培养至对数生长期。 胰蛋白酶用于消化冷冻干燥人羊膜的上皮层，以产生裸露的人羊膜作为支架载体。 将支架载体平铺在培养板孔的底部，直到它们被干燥并完全粘附在孔的底部。 将人对角生长期角膜内皮细胞重新悬浮于含有IV型胶原和20％小牛血清的DMEM / F12培养基中。 随后将人角膜内皮细胞悬浮液接种到铺养在培养板的孔底部的羊膜支架载体上，以启动体外培养以及组织工程改造的人角膜内皮的体外重建。 本发明科学合理。 重建组织工程人角膜内皮可以大规模生产，以满足组织工程人角膜内皮在临床角膜移植中用于原发性角膜内皮细胞病变治疗的巨大需求。 同时，组织工程人角膜内皮和临床治疗的体外重建费用较低。

公开(公告)号：US20120015439A1

公开(公告)日：2012-01-19

申请号：US13258039

申请日：2010-02-08

Applicant: Tingjun Fan ,  Jun Zhao ,  Xiuxia Yang ,  Rishan Cong

Inventor： Tingjun Fan ,  Jun Zhao ,  Xiuxia Yang ,  Rishan Cong

IPC: C12N5/071

CPC classification number: C12N5/0621 ,  A61L27/3604 ,  A61L27/3808 ,  A61L27/3839 ,  A61L2430/16

Abstract: This invention relates to a method for the reconstruction of a tissue-engineered human corneal endothelium. Human corneal endothelial cells are cultured in vitro to logarithmic growth phase using 20% calf bovine serum-containing DMEM/F12 medium. Trypsin is used to digest epithelial layer of the freeze-dried human amniotic membrane in order to produce denuded human amniotic membrane as scaffold carriers. The scaffold carriers are tiled on the bottom of culture plate wells until they are dried and completely adhered to the bottom of wells. Human corneal endothelial cells at logarithmic growth phase are re-suspended in DMEM/F12 medium containing type-IV collagen and 20% calf bovine serum. Human corneal endothelial cell suspension is subsequently inoculated to amniotic membrane scaffold carriers that are tiled on the bottom of wells in culture plate to launch in vitro culture as well as in vitro reconstruction of tissue-engineered human corneal endothelium. This invention is scientific and rational. The reconstructed tissue-engineered human corneal endothelium can be produced on large scale to satisfy the great demand of tissue-engineered human corneal endothelium in clinical cornea transplantation for primary corneal endotheliopathy therapy. Meanwhile, costs for in vitro reconstruction of tissue-engineered human corneal endothelium and clinical therapy are low.

Abstract translation: 本发明涉及一种重组组织工程人角膜内皮的方法。 使用含有20％牛血清的DMEM / F12培养基将人角膜内皮细胞体外培养至对数生长期。 胰蛋白酶用于消化冷冻干燥人羊膜的上皮层，以产生裸露的人羊膜作为支架载体。 将支架载体平铺在培养板孔的底部，直到它们被干燥并完全粘附到孔的底部。 将人对角生长期角膜内皮细胞重新悬浮于含有IV型胶原和20％小牛血清的DMEM / F12培养基中。 随后将人角膜内皮细胞悬浮液接种到铺养在培养板的孔底部的羊膜支架载体上，以启动体外培养以及组织工程改造的人角膜内皮的体外重建。 本发明科学合理。 重建组织工程人角膜内皮可以大规模生产，以满足组织工程人角膜内皮在临床角膜移植中用于原发性角膜内皮细胞病变治疗的巨大需求。 同时，组织工程人角膜内皮和临床治疗的体外重建费用较低。