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.

An in situ hydrogel based on carboxymethyl chitosan and sodium alginate dialdehyde for corneal wound healing after alkali burn

“…27 LSC-based hydrogels which developed previously required additional surgical manipulation. 35 In the present study, hydrogel-encapsulated LSCs had a rapid effect on the restoration of corneal integrity compared to the untreated corneas after alkali burn. We believe that these effects are largely achieved by the combined use of LSC transplantation and the CMCTS/SAD hydrogel.…”

“…Besides that, transplanted LSCs can be used to complement the deficient epithelial cells in the wounded tissue and rapidly repair the cornea . LSC‐based hydrogels which developed previously required additional surgical manipulation . In the present study, hydrogel‐encapsulated LSCs had a rapid effect on the restoration of corneal integrity compared to the untreated corneas after alkali burn.…”

Epidermal Stem Cells in Wound Healing and Regeneration

“…EpiSCs express mainly keratin 15 and 19 (CK15 and CK19); TACs express keratin 5 and 14 (CK5 and CK14); and TDCs express keratin 1 and 10 (CK1 and CK10). Some scholars [32,33] conducted doublelabeling experiments, showing that cells with positive expression of CK19 had the following characteristics: (1) cells with positive expression of CK19 were (3H) label-retaining cells with slow-cycle characteristics of stem cells and (2) the expression of β1 integrin for cells with positive expression of CK19 was also positive, indicating that CK19 could be used as a specific marker for EpiSCs [34,35]. Lyle et al [36] found that EpiSCs in the hair follicle bulge showed high expression of CK15, and in the process of EpiSCs differentiation, the decrease in CK15 expression occurred earlier than the decrease in CK19 expression.…”

A Hyaluronan Hydrogel Scaffold for Culture of Human Oral Mucosal Epithelial Cells in Limbal Stem-Cell Therapy

“…The current study found that PEGDA crosslinker, one of the components in the HA hydrogel mixture supplied in the HyStem ® hydrogel kit, had a cytotoxic effect on cultured OMECs. No such effect was observed by Chen et al [18] when they used PEGDA during development of a HA hydrogel scaffold for ex vivo culture of limbal stem cells in a xenogeneic-free culture system. Crosslinkers are commonly used to overcome the poor solubility and physical properties of HA hydrogels, such as mechanical strength and gel formation time, but they may have a cytotoxic effect on some types of stem cells [27][28][29].…”

“…Additionally, a 3D HA hydrogel with and without collagen type I has been shown to enhance the growth and differentiation of human corneal epithelial cells (hCECs) when co-cultured with adipose stem cells (hASCs) [17]. Chen et al [18] have recently developed a HA hydrogel scaffold for cultivation of LSCs in a xenogeneic-free culture system. These authors used a commercially available HyStem ® -C hydrogel kit, which includes three main components: thiol-modified HA (Glycosil ® ), thiol-reactive polyethylene glycol diacrylate crosslinker (PEGDA; Extralink ® ) and thiol-modified collagen (Gelin-S ® ).…”