荷斯坦奶牛乳腺组织冻存及乳腺上皮细胞原代培养技术改进

Cryopreservation of the Mammary Gland and Improvement on the Culture of the Primary Epithelial Cells in Holstein Dairy Cows

为了改进奶牛乳腺上皮细胞原代培养技术和探究乳腺组织冻存方法,选用24月龄无泌乳史荷斯坦奶牛,采用组织块种植法,通过侧置和倒置两种方式,分别从新鲜和冻存（8个月）的乳腺组织中分离培养奶牛乳腺上皮细胞。结果表明,侧置处理能使乳腺细胞爬出时间缩短1-2d,并改善组织块贴壁速度和效果;回收于前次培养后的乳腺组织块再分离培养,贴壁第2天即有乳腺上皮细胞爬出;一步冷冻（-80℃）液氮保存较大奶牛乳腺组织块,冻存液A（FBS∶DMSO∶DMEM/F12=7∶2∶1）冻存的组织块存活率为50%,冻存液B（FBS∶DMEM/F12∶DMSO∶HEPES/丙酮酸钠贮存液=10∶7∶2∶1）冻存的组织块存活率为56%。可见,作者建立的侧置处理法和组织块回收法可以用于奶牛乳腺上皮细胞的分离培养,可以缩短组织块种植法实验周期,冻存液中加入缓冲液HEPES/丙酮酸钠后有利于组织块生物活性保持。

The present study was performed to improve the process of tissue explant method and explore the cryopreservation method of bovine mammary gland. The mammary epithelial cells were isolated and cultured from the fresh and eryopreserved （8 mouths） mammary gland that col- lected from a 24 months＇ Holstein dairy cow without history of lactation by tissue explant method including the side-place and inversion pattern respectively. Side-place method could shorten 1-2 days for appearance of cell, and improve the speed and adherence effect of tissue pieces, and the cells could climb out on the second day from the tissue pieces that were cultured and moved an- other flask. The survival rate was 50% for the tissue mass by liquid nitrogen storage followed by one-step cryopreservation （-80 ℃） with freeze-stored solution FBS＂ DMSO＂ DMEM/F12=7 ： 2 ： 1 while the survival rate was 56% with freeze-stored solution FBS ： DMEM/F12 ： DMSO ： HEPES/sodium pyruvate：10 ： 7 ： 2： 1. The od were established for mammary epithelial side-place method and tissue pieces recycling meth- cells culture in thepresented study ,which could Ashorten the cycle of tissue explant method. And the buffer HEPES/sodium pyruvate was benefit for maintaining the tissue bioactivity.