Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

Abstract

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs.

Keywords: cell therapy; corneal stroma; fibroblasts; keratocytes; morphology; serum.

Figure 2

Still photographs of the human corneal tissue dissection. (A–D) The first dissection steps involve the removal of the corneal epithelial and endothelial cells, and trabecular meshwork from the corneas, by scraping with a surgical blade no. 10. (E,F) The corneal stroma is then separated from the scleral tissue by cutting ~2 mm from the sclera. (G–I) Finally, the corneal stroma is cut into smaller pieces by leaving the edges of each piece still attached to the adjacent pieces.

Figure 1

Overview of human corneal stromal keratocyte (CSK) cell culture procedure. In the propagation phase, the culture medium is supplemented with 0.5% fetal bovine serum (FBS) to support the proliferation of CSKs, which are otherwise quiescent. In the stabilization phase, the FBS is removed from the culture medium to allow the cells to regain bona fide CSK phenotypes.

Figure 3

Proliferative capacity of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and stromal fibroblasts (SFs). The representative images of CSKs (A), A-CSKs (B), and SFs (C) were captured from cells at P5, which were expanded from the same donor. (D) The proliferative capacity (indicated by Ki-67-positive cells/total number of cells × 100%) of the A-CSKs was 5.6 ± 6.1%. On day 14, following medium switching to serum-free conditions, the proliferative capacity of CSKs was 0%. The SFs had a significantly higher proliferation rate of 20.1 ± 7.2% compared to both the CSKs (p = 3.55 × 10⁻⁸) and the activated CSKs (p = 3.78 × 10⁻⁵). Group comparisons were statistically determined using one-way ANOVA and Tukey comparison tests. Scale bars = 100 μm.

Figure 4

Morphology of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and stromal fibroblasts (SFs). The representative images were captured from cells at P5, which were expanded from the same donor. (A–F) Brightfield images at low and high magnification revealed the loss of thin, dendritic morphology and long cellular processes, typically seen in the CSKs, in the SFs. The SFs also featured larger cell bodies compared to the CSKs and A-CSKs. (G–I) Phalloidin staining showed the stellate morphology of the CSKs, which was progressively lost in the A-CSK and SF cell culture. Scale bars = 100 μm.

Figure 5

Protein expression of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and corneal fibroblasts (SFs). (A,D,G,J) Typical CSK markers, such as ALDH1A1, ALDH3A1, keratocan, and lumican were strongly expressed in the CSKs following 14 days of culture media switching to serum-free conditions. (B,E,H,K) In the propagation medium, the A-CSKs exhibited an attenuated expression of the CSK markers. (C,F,I,L) In contrast, in medium supplemented with 5% FBS, the SFs did not express or express only a little of the CSK markers. (M,N,O) All three cell types were not immunoreactive with α-smooth muscle actin (α-SMA), the cell marker of corneal stromal myofibroblasts (see inset in pane O). Scale bars = 50 μm.

Figure 6

Gene expression of corneal stromal keratocytes (CSKs), activated CSKs (A-CSKs), and corneal fibroblasts (SFs). The gene expression was detected using real time-polymerase chain reaction. Similar to the protein expression, CSK-associated genes, such as ALDH1A1 (A), ALDH3A1 (B), KERA (C), and LUM (D) were strongly expressed in the CSKs following 14 days of culture media switching to serum-free conditions and were significantly upregulated when compared to the A-CSKs and SFs. (E) The corneal stromal myofibroblast (MyoFB)-associated gene, ACTA2, was significantly downregulated in the CSKs, A-CSKs, and SFs compared to the MyoFBs. For the analysis of differentially expressed genes, CSKs was used as the reference group for comparison, whereas GAPDH was used as the housekeeping gene. Group comparisons were statistically determined using one-way ANOVA and Tukey comparison tests.