Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system
Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system
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DOI：
10.1167/iovs.03-1361
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发表时间：
2004-06-01
影响因子：
4.4
通讯作者：
Lavker, RM
中科院分区：
文献类型：
--
作者：
Ang, LPK;Tan, DTH;Lavker, RM
目的。研究使用多步无血清培养系统开发具有改善的体外和体内增殖特性的结膜上皮等同物，并评估血清补充和培养条件对这些细胞增殖能力的影响。方法。在多步无血清培养系统中，在浸没和气升条件下，在人羊膜（HAM）上培养结膜上皮细胞。将溴脱氧尿苷 (BrdU) ELISA 增殖测定、集落形成效率 (CFE) 和细胞代数与含血清培养基中的进行比较。通过异种移植至 SCID 小鼠来评估组织构建体的体内增殖能力。评估培养细胞的角蛋白-4、-19和-3以及MUC5AC杯状细胞粘蛋白的表达。结果。在无血清培养基中培养的上皮细胞（BrdU吸光度，1.91 +/- 0.08；细胞代数，25.6 +/- 4.5）比在含血清培养基中培养的上皮细胞（BrdU吸光度，1.06 +/- 0.08；细胞代数，12.1 +/- 3.0）更具增殖性。无血清来源的上皮等同物显示移植后增殖和分层显着增加。与深层培养相比，空气提升 6 天和 12 天的细胞在体外和体内的增殖能力均降低。培养细胞表达角蛋白4和-19，RTPCR检测MUC5AC mRNA。电子显微镜显示具有大量半桥粒的基底层。结论。这是一种多步无血清培养系统，用于开发具有改善的增殖和结构特性的结膜上皮等同物，这对于临床移植后提高移植物存活和结膜表面再生至关重要。
PURPOSE. To investigate the use of a multistep serum-free culture system in developing a conjunctival epithelial equivalent with improved in vitro and in vivo proliferative properties and to evaluate the effect of serum supplementation and culture conditions on the proliferative capacity of these cells.METHODS. Conjunctival epithelial cells were cultivated on human amniotic membrane (HAM) in a multistep serum-free culture system, under submerged and air-lifted conditions. The bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and number of cell generations were compared with those in serum-containing medium. The in vivo proliferative capability of the tissue-constructs were evaluated by xenotransplantation to SCID mice. Cultured cells were evaluated for the expression of keratin-4, -19, and -3, as well as MUC5AC goblet cell mucin.RESULTS. The epithelial cells cultivated in serum-free medium (BrdU absorbance, 1.91 +/- 0.08; cell generations, 25.6 +/- 4.5) were more proliferative than those cultivated in serum-containing medium (BrdU absorbance, 1.06 +/- 0.08; cell generations, 12.1 +/- 3.0). The serum-free-derived epithelial equivalents demonstrated a significant increase in proliferation and stratification after transplantation. Cells that were air lifted for 6 and 12 days had a reduced proliferative capacity in vitro and in vivo compared with submerged cultures. Cultured cells expressed keratin-4 and -19, and MUC5AC mRNA was detected by RTPCR. Electron microscopy demonstrated a basal lamina with numerous hemidesmosomes.CONCLUSIONS. This is a multistep serum-free culture system for developing a conjunctival epithelial equivalent with improved proliferative and structural properties, which are crucial for enhancing graft survival and regeneration of the conjunctival surface after clinical transplantation.