组织工程与重建外科杂志

组织工程与重建外科杂志 ›› 2019, Vol. 15 ›› Issue (3): 137-141.doi: 10.3969/j.issn.1673-0364.2019.03.003

• 论著 • 上一篇    下一篇

人耳软骨细胞冻存复苏后的体外成软骨能力及体内转归的研究

于瑶，殷宗琦，李丹，周广东，曹谊林，刘豫   

  1. 潍坊医学院；组织工程国家工程中心；上海市组织工程研究重点实验室
  • 收稿日期:2019-03-09 修回日期:2019-04-21 出版日期:2019-06-20 发布日期:2019-06-20
  • 作者简介:刘豫,E-mail:yuliu1211@163.com。

In Vitro Chondrogenesis and in Vivo Fate of Human Auricular Chondrocytes after Cryopreservation

YU Yao,YIN Zongqi,LI Dan,ZHOU Guangdong,CAO Yilin,LIU Yu   

  • Received:2019-03-09 Revised:2019-04-21 Online:2019-06-20 Published:2019-06-20
  • Contact: 上海市优秀技术带头人项目(19XD1431100)

摘要： 目的 以无支架软骨膜片为模型,研究冻存对人耳软骨细胞的细胞活力、增殖和体外/体内软骨再生能力的影响。方法 将小耳畸形患者术中废弃的残耳软骨,以胶原酶消化、分离,得到原代软骨细胞。将软骨细胞扩增至第1代(P1)后,分为实验组及对照组。实验组(Exp)以含有FBS和DMSO的冻存液,经程控降温仪和液氮冻存1个月后复苏,高密度接种,制备软骨膜片及可注射软骨;对照组(Ctrl)不经冻存继续扩增至第3代(P3),高密度接种,制备软骨膜片及可注射软骨。通过光学显微镜、CCK-8、活/死细胞染色、HE和阿利新蓝染色等,检测细胞形态、活力、增殖能力及软骨特异基质(ECM)的表达能力。通过每组体外软骨膜片的湿重、体积及生化成分定量检测,确定软骨细胞的体外成软骨能力。将每组软骨膜片制成新生软骨颗粒(可注射软骨),注射至裸鼠皮下(n=5),8周后取材,行大体观察、HE染色和生化成分定量检测,比较每组软骨细胞的体内成软骨能力。结果 两组细胞的形态、活力、增殖能力和软骨特异的糖胺聚糖(GAG)形成能力无明显差异。相比对照组,实验组体外再生软骨的湿重及总胶原表达升高;但体内软骨再生能力两组亦无明显差异。结论 细胞冻存复苏对人耳软骨细胞的细胞活力、增殖和体外/体内软骨再生能力无明显影响。

关键词: 冻存复苏, 人残耳软骨细胞, 无支架软骨膜片, 可注射软骨

Abstract: Objective To investigate the effect of cryopreservation on cell viability,proliferation,and in vitro/in vivo cartilage formation abilities of human auricular chondrocytes using a scaffold-free cartilage sheet model.Methods Human auricular cartilage was obtained from patients with microtia.Chondrocytes were isolated through collagenase digestion.The chondrocytes were cultured and expanded into passage 1(P1)before being divided into the experiment and the control groups.In the experiment group(Exp),the chondrocytes were collected,suspended with FBS and DMSO,then froze according to the cell cryopreservation steps and thawed after storage for 1 month in liquid nitrogen before being used to generate the cartilage sheet.In the control group(Ctrl),the cells were not cryopreserved and continued to be expanded until passage 3(P3)before being used for cartilage sheet generation.Cell morphology,viability,proliferation and cartilage specific extracellular matrix(ECM)production were examined by light microscopy,CCK-8 assay,live/dead,HE and Alcan blue staining.The in vitro scaffold-free cartilage sheets in each group were examined through wet weight,volume and biochemical contents to determine the in vitro cartilage formation abilities.Then the scaffold-free cartilage sheets were diced into neocartilage fragments(injectable cartilage)for nude mouse injection(n=5).After 8 weeks of injection,the explants were examined again by gross view,HE staining and biochemical contents to determine the in vivo cartilage formation abilities of the chondrocytes in each group.Results No significant differences were observed in morphology,viability,proliferation and cartilage specific GAG production abilities of the chondrocytes between the two groups.Compared with the Ctrl group,the wet weight and collagen content were slightly higher in the Exp group.However,there was no significant difference in the in vivo cartilage-forming ability between the two groups.Conclusion Cryopreserving and thawing process have no significant effect on the viabili

Key words: Cryopreservation, Human auricular chondrocytes, Sc

中图分类号: