In vitro culture of human corneal epithelial stem cells using modified explant culture

doi:10.3969/j.issn.2095-4344.2097

Abstract

Abstract:

BACKGROUND: Corneal epithelial stem cells, also known as limbal stem cells, are distributed in the basal layer of limbal epithelium. It is extremely difficult to deal with limbal stem cell deficiency or dysfunction that is caused by severe thermal burn, chemical burn, and chronic inflammation of ocular surface. At present, in vitro culture of corneal epithelial stem cells using tissue engineering technology followed by clinical transplantation is a new and effective therapeutic direction.

OBJECTIVE: To explore the feasibility of serum-free culture of human corneal epithelial stem cells in vitro using modified explant culture method.

METHODS: The remaining donor corneal tissues after keratoplasty (less than 8 mm in diameter) were obtained from Henan Eye Bank, and the outer and middle limbus were dissected under surgical microscope. Two culture methods were used to culture human corneal epithelial stem cells. In the conventional explant culture group, the limbal tissues were adhered to the dish with the epithelium being upward, then Keratinocyte-serum free medium (K-SFM) was added into dishes, followed by incubation at 37 °C in a 5% CO₂ incubator. In the modified explant culture group, limbal tissues were dissected to immerse in the K-SFM culture medium and incubated at 37 °C in the 5% CO₂ incubator for 12 hours. The limbal tissues were then adhered to the dish with the epithelium being downward. The day whenever the cells from the limbal tissues adhered to the dish was marked as the 1^st day of culture, and changes in cell morphology and growth were recorded by phase contrast microscopy every day. Immunofluorescent staining was used to detect the expression of K3 and p63 in primary cells on the 5^th, 10^th and 14^th day of the modified explant culture.

RESULTS AND CONCLUSION: The mean early stage of growth in the modified explant culture group was shorter than that of the conventional explant culture group (P < 0.05), and the mean growth rate of the modified explant culture group was higher than that of the conventional explant culture group (P < 0.05). In the modified explant culture group, cells had a good growth state, and many cells with small size gathered together on the 10th day of culture. On the 14^th day, cell clones were formed, and the cells in the clone showed uniform morphology. On the 5^th day, K3 highly expressed, while p63 lowly expressed in primary cells. On the 10^th day, both of K3 and p63 had an increased expression. On the 14^th day, there was no significant increase in the K3 expression, but the expression of p63 increased significantly. In the in vitro serum-free culture condition, the modified explant culture could significantly promote the growth of corneal epithelial stem cells, and expand corneal epithelial stem cells in vitro, which could provide sufficient seed cells for enriching corneal epithelial stem cells and constructing human limbal multilayered epithelial sheets.

Key words:

corneal epithelial stem cells, serum-free culture, explant culture, modified, modification, K3, p6, marker

CLC Number:

Xu Zhongzhong, Yu Xiaofei, Wang Liya. In vitro culture of human corneal epithelial stem cells using modified explant culture[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(25): 4012-4017.

Figures/Tables 3

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