关节软骨细胞库

Bank of articular cartilage chondrocytes

本文介绍了关节软骨细胞的分离、冷冻保存和复苏技术，并在阐明胶原酶的细胞毒性作用的基础上，提出了分阶段消化法，以减少分离细胞的损伤，由于低温保护剂ＤＭＳＯ也具有细胞毒性，因而应尽量缩短ＤＭＳＯ与软骨细胞在４℃以上的接触时间。降温和复温速度是影响细胞存活的关键因素。降温速度从４℃～－８０℃以Ｉ℃／ｍｉｎ为佳，之后迅速投入－１９６℃的液氮中长期保存，细胞复苏应采用３７℃水浴快速复温法，虽然检测冻存细胞的存活率可采用苔盼蓝拒染试验，但最可靠的方法是细胞培养，利用关节软骨细胞库进行细胞培养、电镜观察和激光流式细胞计量分析，结果证明冻存软骨细胞复苏后仍具有正常的结构和形态并保持新陈代谢和自我复制功能。

Cryopreservation of viable articular carti-lage chondrocytes has significance to bothclinic and basic scientific researches. In the present report,we introduced the techniquesof isolation,cryopreservation and reanimationof chondrocytes. To obviate the cell toxicity ofcollagenase,a new cell separation technique─the method of digestion by stages──had been proposed,which could reduce thedamage to the isolated chondrocytes.As thecryopreservative DMSO also has cell toxicity,it is justifiable to decrease the contact time be-tween DMSO and chondrocytes above 4 ℃.The key factor of cell survival is the rate offreezing and thawing. It is a better way tofreeze the cells at a rate of 1℃ /min from 4℃to─8℃,then rapidly put the cells into liquidnitrogen(─196℃)for long-term preservation.For cell reanimation we adopt the quick thaw-ing method in a constant-temperatute water-bath(37 ℃),Although the test of trypan-bluedye-exclusion can be used to determine the cellviability after thawing,the most reliablemethod is cell culture. Utilizing the bank ofarticular cartilage chondrocytes,we carried outthe cell culture,observation under transmis-sion and scanning electron microscope andanalysis with laser flow cytometery.The re-sults suggest that the chondrocytes,afterfreezing and reanimating,still show the nor-mal structure and morphology,and maintainthe function of metabolism and selfduplica-tion.