Effect of fetal bovine serum on the proliferation and differentiation of murine corneal epithelial cells in vitro
【摘要】 To investigate the effect of fetal bovine serum (FBS) on the proliferation and differentiation of murine corneal epithelial cells in vitro.METHODS: Mouse corneal epithelial cells(MCEs) were cultured in serumfree lowCa2+ medium(KSFM) and KSFM supplemented with 100mL/L FBS, respectively. Population doublings (PDs) were determined. The expressions of corneal epithelial cell markers p63, keratin 19 (K19) and involucrin were investigated by RTPCR and Western blotting analyses. RESULTS: Cells in KSFM were stably subcultured over 25 passages; however, none of the cell lines could pass P3 in KSFM with FBS. In KSFM, the cells showed typical cobblestone appearance and expressed p63, K19 and involucrin. After medium was supplemented with FBS, cells became homogeneous, large and squamous. Furthermore, both RTPCR and Western blotting analyses showed that the expression of involucrin was increased significantly.CONCLUSION: FBS has effects of inhibiting proliferation and triggering differentiation of MCEs.
【关键词】 serum cornea epithelium cell culture
INTRODUCTION
Cultivated limbal 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]. Maintaining progenitor cells with high proliferative potential is one of the keys of successful limbal epithelial cell culture. It is well known that serum triggers differentiation in several lines of epithelial cells[2,3]. We therefore hypothesized that serumfree medium could be used to enhance the expansion of epithelial progenitor cells from the murine limbus in vitro.
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. After sacrificing the mice, eye globes were enucleated with forceps and washed profusely in phosphate buffered saline (PBS). Eyes from each animal were kept separate throughout the culture procedure. Corneal buttons, including the limbus, were cut from the eye, and extraneous tissue was cleaned away (e.g., iris and ciliary body). Primary cell culture was performed using an explant culture method similar to Hazlett et al[4,5]. Briefly, the button was cut in half, and each explant with epithelium side up was plated flat on a 6well plate, one piece per well.After approximately 510 minutes to allow for attachment of the explant, 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 mouse corneal epithelial cells (MCEs) were subcultured by TrypLE Express (Invitrogen) at 1∶3 split ratio after small cells reached subconfluence until Passage 4 (P4) cultures. From P5, MCEs after subconfluence were subsequently serially passaged at a density of 5×104 per 75cm2 flask, 710 days per passage. The medium was changed every 3 to 4 days.
Medium In order to investigate the effect of serum on the differentiation of cells, we cultured the cells in serumfree lowCa2+ medium (designated "keratinocyte serumfree medium", KSFM; Invitrogen, Carlsbad, CA) consisting of 10ng/mL human recombinant epidermal growth factor (EGF)(Invitrogen), 100ng/mL cholera toxin (Calbiochem; Merck KGaA, Darmstadt, Germany), antibiotics, and growth supplement supplied by the manufacturer, and KSFM supplemented with 100mL/L fetal bovine serum (FBS)(KSFM+ FBS) in primary culture and subculture, respectively.
Cell Proliferation The population doublings (PDs) were calculated as log2 (D/D0), where D and D0 were defined as the density of cells at the time of harvesting and seeding,respectively.
Reverse TranscriptionPolymerase Chain Reaction Total RNA was extracted from MCEs cultured for 7 to 10 days using commercial RNA isolation kit (RNeasy, Qiagen, Valencia, CA), and cDNA was synthesized using the RevaTra Ace kit (Toyobo, Osaka, Japan). The same amount of cDNA was amplified by PCR (GeneAmp 9700; Applied Bioscience, Inc. (ABI), Foster City, CA) for each primer pair, as shown in Table 1. PCR products were analyzed by agarose gel electrophoresis.
Western Blotting Analysis MCEs were dissolved with lysis buffer (MPER, Pierce, Rockford, IL). The same amount of proteins were loaded on a 100mL/L BisTris gel (Ready Gel; BioRad Laboratories, Hercules, CA) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were immunostained with primary antibodies against p63 (1∶200), K19 (1∶50), involucrin (1∶3000) and actin (1∶500, mabcam 8226; Abcam Inc., Cambridge, MA), respectively. After the reaction with horseradishperoxidase conjugated secondary antibody, protein bands were visualized by ECL(GE Healthcare,Buckinghamshire, UK) and Xray film.
RESULTS
Figure 1 shows population doubling (PDs) of cells cultured in KSFM and KSFM+FBS. The cells were cultured from explant (P0), subcultured though 5 passages in KSFM for 11.53±1.74PDs (±s,n=6, Figure 1) and were stably subcultured though at least 25 passages without showing signs of replicative senescence (data not shown). However, none of the cell lines could pass P3 in KSFM+FBS(n=6, Figure 1).
With respect to morphology, the cells cultured in KSFM were homogeneous small cells(Figure 2A), while cells in KSFM+FBS were homogeneous, large and squamous (Figure 2B), indicating that FBS quickly increases the size of the cells.
In KSFM, both RTPCR and Western blotting analyses showed that cells expressed progenitor markers p63 and K19 and differentiation marker involucrin (IVL). After medium was supplemented with FBS, the expression of differentiation marker involucrin was increased significantly (Figure 3A,3B).
DISCUSSION
FBS is frequently added to the defined basal medium as a source of certain nutritional and macromolecular growth factors essential for cell growth. However, it is well known that FBS triggers differentiation in several lines of epithelial cells[2,3]. Therefore, in order to investigate the effect of FBS on proliferation and differentiation of corneal epithelial cells and optimize the culture method, we compared the culture of MCEs in serumfree medium (KSFM) and medium with FBS (KSFM+FBS).
The corneal epithelium is a rapidly regenerating stratified squamous epithelium. The epithelial progenitor cells, including limbal stem cells (LSCs) and transient amplifying cells (TACs)[6] with extensive proliferative potential, are crucial for maintaining the homeostasis of corneal epithelium,
which is an important prerequisite not only for the integrity of the ocular surface but also for visual function. Romano et al[7] have reported that the limbal basal epithelium has the smallest cell size in the corneal epithelial differentiation scheme. In our study, the cells cultured in KSFM appeared to have similar morphology, characterized by homogeneous small cell size with typical cobblestone appearance, which suggests that the cells maintain their progenitor cell state. However, after medium was supplemented with FBS, the size of the cells increased quickly and became homogeneous, large and squamous (Figure 2B), which suggested that FBS induces the differentiation of cells.Table 1Primers used for RTPCR（略）
Furthermore, we detected progenitor cell markers, including nuclear transcription factor p63 and K19, and differentiation marker involucrin[8]. The expression of p63[9] and K19[10] was localized to progenitor cells with high proliferative capacity, including the presence of both LSCs and TACs. The results of RTPCR and Western blotting showed that cells in KSFM expressed p63 and K19 strongly, which indicates that the phenotype of the cells might be equivalent to corneal epithelial progenitor cells. However, after medium was supplemented with FBS, the expression of differentiation marker involucrin was increased significantly, which confirmed that FBS triggers the differentiation of corneal epithelial cells.
In conclusion, we compared the proliferation and differentiation of mouse corneal epithelial cells in serumfree medium and medium supplemented with FBS. FBS has the effect of inhibiting proliferation and triggering differentiation of mouse corneal epithelial cells. Therefore, serumfree medium is a preferable option to culture mouse corneal epithelial cells.
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