组织工程皮肤构建中胎儿皮肤标本玻璃化冻存的实验

Experimental investigation on cryopreservation method of human fetal skin specimen by vitrification in construction of tissue-engineered skin

目的:建立一种用于细胞培养的皮肤标本新的保存方法。方法:实验于2004-05-01/2-31在第四军医大学西京医院烧伤外科实验室进行。①分别取流产胎儿头部及躯干部全层皮片(家属知情同意),按照标本保存方法不同分为常规组(4℃保存2h)、冻存1组(-196℃液氮保存1周组)、冻存2组(-196℃液氮保存1个月组)和冻存3组(-196℃液氮保存6个月组)。采用抗冻液4℃下预处理20min后直接快速置于-196℃液氮中的玻璃化冻存方法进行冻存。②分别进行胎儿角质形成细胞、成纤维细胞和毛乳头细胞的原代培养,进行细胞形态学观察,并采用锥虫蓝染色法、四唑盐比色法和克隆形成实验观察培养的第2代角质形成细胞、成纤维细胞和毛乳头细胞的活力。结果:①与常规组同种细胞相比,冻存1组、冻存2组和冻存3组的胎儿角质形成细胞、成纤维细胞和毛乳头细胞的形态学特点基本相同。②与常规组相比,冻存1组、冻存2组和冻存3组角质形成细胞、成纤维细胞和毛乳头细胞活细胞率、细胞存活率及克隆形成率差异均无显著性意义(P>0.05)。结论:利用液氮玻璃化保存用于细胞培养的皮肤标本不会影响细胞活力,是保持组织活性的一种有价值的组织标本保存方法,对组织工程皮肤的建立,乃至构建组织细胞库有着重要意义。

AIM：To establish a new conservancy method of skin specimen for cell culture. METHODS： The experiment was conducted at the laboratory for Department of Burn Surgery, Xijing Hospital, Forth Military Medical University of Chinese PLA from May 1^st, 2004 to Decefnber 31^th , 2004. Full-thickness fetal skin specimen of caput and trunk were obtained （Informed consent was obtained from the relatives） from aborted fetus and divided into routine group （stored at 4℃ for 2 hours）, frozen group 1 （stored in liquid nitrogen at -196℃ for 1 week）, frozen group 2（stored in liquid nitrogen at -196℃ for 1 month） and frozen group 3（stored in liquid nitrogen at -196℃ for 6 months）, and the vitrification cryopreservation （the skin specimen were put in liquid nitrogen at -196℃ directly and rapidly after pretreatment with cryopreservation agent DMSO for 4 minutes at 4℃） primary culture of fetal keratinocytes, fibroblasts and hair papilla cells were done with the above skin specimens. Cell morphologies were observed under an inverted phase contrast microscopy, and the viability of the cultured cells were determined by trypan blue staining, MTT chromatometry and clone formation experiment. RESULTS： ①Compared with the same kind of cells in routine group, the cultured fetal keratinocytes, fibroblasts and hair papilla cells from frozen group 1, frozen group 2 and frozen group 3 showed the same characteristics of cell morphologies; ② There was no significant difference in the rate of living cell, survival rates and cell cloning efficiency of fetal keratinocytes, fibroblasts and hair papilla cells in frozen group 1, frozen group 2 and frozen group 3 as comparedwith routine group （P 〉 0.05） CONCLUSION： The conservancy method of skin specimen for cell culture with liquid nitrogen would not affect the viabilities of cells. It is a valuable specimen conservancy method to keep the viabilities of tissue, and is of important significance to the construction of tissue-engineered skin and creation of human tissue and cell bank.