作者：马小力,孔珺,刘汉强，赵江月，张劲松 作者单位：1110001 中国辽宁省沈阳市，中国医科大学第一附属临床医院眼科；2110001 中国辽宁省沈阳市，中国医科大学第四附属临床医院眼科
【摘要】目的：探讨滋养细胞在小鼠角膜上皮细胞复层化中的作用，并研究构建组织工程化角膜上皮的理想方法。方法：在Transwell气液界面培养系统中，分别采用接触滋养层培养法、分离滋养层培养法、复式滋养层培养法以及无滋养层培养法等4种方法进行组织工程化小鼠角膜上皮的重建。HE染色进行组织学观察，免疫荧光法检测p63、角蛋白19以及involucrin的表达。结果：接触滋养层培养法、分离滋养层培养法中，角膜上皮复层化为3 4层；复式滋养层培养法中，角膜上皮复层化达57层；而无滋养层培养法中，复层化仅为23层。复式滋养层培养法构建的小鼠角膜上皮，基底细胞层和基底细胞上层表达祖细胞标记p63和角蛋白19，角膜上皮全层均表达分化标记involucrin。结论：复式滋养层培养法为构建组织工程化小鼠角膜上皮的理想方法。
【关键词】小鼠；角膜；上皮；细胞培养；组织工程
Comparative study of four culture methods to engineer murine corneal epithelial sheet
XiaoLi Ma, Jun Kong , HanQiang Liu, JiangYue Zhao, JinSong Zhang
Foundation item:Supported by Liaoning Provincal Department of Education (No. 05L564)
1Department of Ophthalmology, the First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
2Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China
Correspondence to:JinSong Zhang.
Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China. [email protected]
AbstractAIM: To investigate the roles of feeder cells in stratification of murine corneal epithelial cells and build an ideal method to engineer stratified epithelial sheet.METHODS: Using contact feeder culture, separated feeder culture, compound feeder culture and culture without feeder cells by Airlifting method in Transwell chamber culture system, tissue engineered corneal epithelium was reconstructed. Corneal sheets were stained with hematoxylin and eosin (HE) for histological observation. The expression of p63 and keratin 19 (K19) and involucrin (IVL) was investigated by immunocytochemistry analysis.RESULTS: Stratification was limited to three to four layers in the contact feeder group, whereas separate feeder sheets were slightly more stratified. The compound feeder group produced a stratified epithelium with five to seven layers of cells. The group without 3T3 feeder cells formed only two to three layers of cells. Immunostaining images in the compound feeder group showed expression of progenitor markers p63 and K19 in the basal and suprabasal layer, as well as differentiation marker involucrin in all layers. CONCLUSION: The remarkable stratification as well as the limbal phenotype makes the compound feeder system a candidate tool for cultivating transplantable epithelial sheets.
KEYWORDS: mouse; cornea; epithelium; cell culture; tissue engineering
INTRODUCTION
C ultivated epithelial cell sheets are used clinically for reconstructing the ocular surface in blinding diseases that destroy the corneal epithelial stem cell niche located in the limbus[1,2]. The preparation of epithelial sheets requires feeder cells to expand progenitor cells and to produce stratified sheets. Using feeder cells is believed to reproduce several aspects of the stem cell niche, although molecular mechanisms involved in interactions between epithelial and feeder cells are unclear. Direct celltocell contact and soluble factors secreted by viable feeder cells both seem to be involved in promoting the proliferation and differentiation of epithelial cells and the formation of stratified epithelial sheets.
In the current study, we sought to find an ideal method to use feeder cells to engineer a fully stratified epithelial sheet with a limbal phenotype. Therefore, we compared several culture conditions with or without 3T3 feeder cells and devised a novel “compound feeder system” to achieve the optimal cultivated sheets.
MATERIALS AND METHODS
Tissue Preparation and Cell Culture C57BL/6 mice (CLER, Tokyo, Japan)，aged 810 weeks, were handled according to the guidelines in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Eye globes were enucleated from the mice with forceps after death, washed profusely in phosphate buffered saline (PBS). Eyes from each animal were kept to separate throughout the culture procedure. Corneal buttons including the limbus were cut from the eye and cleaned of extraneous tissue (e.g. iris, ciliary body, etc.). Primary cell culture was performed using explants culture method similar to Hazlett et al[3]. Briefly, the button was cut in half and each explant with epithelium side up was plated flat on 6well plate, one piece per well. After approximately 510 minutes to allow for attachment of the explant, serumfree lowCa2+ medium (defined keratinocyte serumfree medium, KSFM; Invitrogen, Carlsbad, CA) consisting of 10ng/mL human recombinant EGF (Invitrogen), 100ng/mL cholera toxin (Calbiochem; Merck KGaA, Darmstadt, Germany), antibiotics, and growth supplement supplied by the manufacturer was supplemented. The cultures were incubated at 37℃, under 95% humidity and 50mL/L CO2 with the medium changed every 3 to 4 days. Within 10 days, the explant was carefully transferred to a new dish and cultured as described above.
Subculture The epithelial sheets were subcultured by TrypLE Express (Invitrogen) at 1∶3 split after small cells reached subconfluence until Passage 4 (P4) cultures. From P5, cells after subconfluence were subsequently serially passaged at a density of 5x104 per 75cm2 flask, 710 days per passage. The cultures were incubated at 37℃, under 95%
Preparation of Feeder Cells NIH/3T3 cells were purchased from American Type Culture Collection (Manassas, VA) and cultured with Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) containing 100mL/L fetal bovine serum (FBS). Confluent cells were treated with mitomycin C (4mg/mL, SigmaAldrich) at 37℃ for 2 hours. Dissociated cells were cryopreserved until use.
Stratification Ability Analysis To evaluate the effect of direct celltocell contact with feeder cells, 3T3 fibroblasts feeder cells were seeded on a cell culture inserts (Transwell, cat no 3450, Corning, Corning, NY) at a density of 2.5×104/cm2. On the following day, 3×105 epithelial cells were seeded on the insert. After epithelial cells reached confluence, cells were airlift cultured for an additional week to allow stratification (Contact feeder culture, Figure 1A).
To evaluate the effect of soluble factors secreted by feeder cells, 3×105 epithelial cells were seeded on the insert. After epithelial cells reached confluence, 2.5×104/cm2 3T3 feeder cells were seeded in the bottom of the well. On the following day, epithelial cells were airlift cultured for an additional week to allow stratification (Separate feeder culture, Figure 1B).
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