A hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo expansion of human corneal epithelial stem cells

Abstract

PurposeTo develop a hyaluronan hydrogel scaffold-based xeno-free culture system for ex vivo cultivation of human corneal epithelial stem cells (CESCs).Patients and MethodsCESCs were cultivated from donor limbal explants on the HyStem-C Hydrogel bio-scaffold in 12-well plates for 3 weeks. Group A used the traditional supplemented hormonal epidermal medium (SHEM) and group B used the defined SHEM (without fetal bovine serum and toxin A, adding 20% serum replacement). The growth and morphology of the cultured cells were assessed by phase contrast microscope. The expressions of specific cell markers were assessed by immunofluorescence staining and quantitative real-time PCR (qRT-PCR).ResultsSuccessful cultures of CESCs were obtained in both groups, resulting in multilayered stratified epithelia. Comparing to group A, the cells in group B was grown slightly slower and formed less cellular layers at the end of culture. The corneal specific cytokeratin (K) 12 and differentiation markers, involucrin, and connexin 43, were mainly expressed in the superficial cellular layers in both groups. Interestingly, certain basal cells were immune-positive to proposed stem cell markers such as K19, ABCG2, and integrin β1 in both groups. There was no significant difference between the two groups with regard to the gene expression levels of all these selected corneal markers (all P>0.05).ConclusionsThe hyaluronan hydrogel scaffold-based xeno-free culture system may support the expansion of regenerative CESCs without the risk of xeno component contamination. The regenerated epithelium maintains similar characteristics of native corneal epithelium.

Figure 1

Ex vivo cultivation of human CESCs in a HA hydrogel scaffold-based culture system. Fresh limbal explant was trimmed and dissected into ~2 mm × 2 mm sized pieces and placed onto the HyStem-C Hydrogel-coated culture inserts in 12-well plates. Two culture media were used. Group A used traditional SHEM including toxin A and 5% FBS. Group B used defined SHEM without toxin A and 5% FBS, but included 20% serum replacement (Knockout SR, Thermo Fisher Scientific). The HyStem-C Hydrogel has a good stiffness and can be manipulated with forceps.

Figure 2

Demonstration of growth of limbal epithelial cells as observed under phase contrast microscope. The corneal epithelial cells were proliferating from the periphery of the explant onto the HyStem-C Hydrogel scaffolds within 7 days in both groups. The cells in group A appeared to grow faster than those in group B. The cultivated epithelial cells covered the whole hydrogel-coated insert at the end of culture at day 21. The regenerated cells in both media exhibited a cobblestone like morphology, whereas some cells showed fusiform in group B. Magnification: left, × 4; right, × 40.

Figure 3

The histology of the tissue generated on hydrogel scaffolds by cross-section with HE staining. At the early stage of cultivation around 1 week, a single cell layer cover the scaffold surface was observed in both media. At the end of cultivation, the cultured cells formed an artificial epithelium of 5–7 cellular layers in group A, whereas less cellular layers (3–5 layers) were observed in group B. The cellular layer(s) cultured in group B seemed less organized and striated than those cultured in group A.

Figure 4

Immunofluorescent staining of differentiation markers and stem cell-associated markers of corneal epithelial cells cultivated on hydrogel scaffolds in different media. The cytoplasmic expression of K12 was detected in the superficial cellular layers in both groups. Involucrin was positively expressed in the superficial and middle-upper layers in group A, and was mainly expressed in the superficial layer in group B. CX43 was positively expressed in the superficial and middle-upper layers in both groups. K19, Integrin β1 and ABCG2 were mainly expressed in the basal layer of the artificial corneal epithelium regenerated in both groups.

Figure 5

Gene expression of corneal epithelial cell differentiation markers and stem cell-associated markers in regenerated corneal epithelium on hydrogel scaffolds in different media. There was no significant difference in mRNA levels of all these markers between the two groups (all P>0.05).