A novel method of isolation, preservation, and expansion of human corneal endothelial cells

Abstract

Purpose: To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs).

Methods: Human corneal Descemet's membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to 16 hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve growth factor (NGF), or basic fibroblast growth factor (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67.

Results: Digestion with collagenase A, but not Dispase, of the stripped Descemet's membrane generated HCEC aggregates, which preserved cell-cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell-cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell-cell junctions.

Conclusions: Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.

Figures

Figure 1

Isolation of HCECs as cell aggregates by collagenase A in a serum-containing medium. Descemet’s membrane was stripped from the peripheral donor cornea and most of the HCECs were adherent to the Descemet’s membrane, whereas some cells were detached in some areas (dotted lines and asterisks) (A). Dispase II digestion at 37°C for 1.5 hours, HCECs started to aggregate but still did not detach from the Descemet’s membrane (B). In contrast, collagenase A digestion for 1.5 hours resulted in frank aggregation of HCECs into clusters and complete detachment from Descemet’s membrane (C). After 16 hours’ digestion in collagenase A, most of the HCEC aggregates became compact and varied in size and shape (D) with some not forming tight aggregates (D, E, arrows). The cell-viability assay showed that the compact aggregates were composed of viable cells; however, those loosely held and disintegrated aggregates contained some dead cells (F, arrows; E is a phase-contrast micrograph of F). Bars, 100 μm.

Figure 2

Maintenance of cell–cell junctions and basement membrane components in HCEC aggregates. Immunofluorescence staining showed ZO-1 (A), connexin-43 (B), type IV collagen α1 (C) and α2 (D) chains, laminin 5 (E), and perlecan (F) were present in HCEC aggregates. The TUNEL assay revealed only a few apoptotic cells present in the center of the aggregate (G, arrowheads; H shows the nuclear counterstaining of G). Bar, 100 μm.

Figure 3

Incubation of HCEC aggregates in a high- or low-calcium, serum-free medium. HCEC aggregates harvested from collagenase A digestion (A, E) were incubated in a high-calcium, serum-free medium (A–C) or a low-calcium, serum-free medium (E–G) continuously for 1 week (B, F) and 3 weeks (C, G). At the end of the third week, aggregates were transferred to the SHEM and further cultured for 4 days. Aggregates incubated in a high-calcium, serum-free medium generated an intact HCEC monolayer (D). However, aggregates incubated in a low calcium, serum-free medium generated few scattered single cells (H). Bar, 100 μm.

Figure 4

Expansion of HCECs from aggregates in SHEM. HCEC aggregates from a 24-year-old donor were subsequently treated with trypsin/EDTA for 10 minutes, and seeded in the SHEM. This treatment dissociated HCEC aggregates into small clusters and single cells (A). Most cells attached and spread out within 24 hours (B), and grew into patches and sheets 4 days later (C). After 1 week, the cells reached confluence and maintained a typical hexagonal shape (D). Immunostaining showed that confluent cells expressed tight junction ZO-1 (E) and gap junction connexin-43 (F). Bars, 100 μm.

Figure 5

Ki67 expression of HCECs cultured under different conditions. HCEC aggregates harvested from two donors, 48 and 53 years old, without (A, C, E) or with (B, D, F) a brief trypsin/EDTA treatment were seeded on plastic in SHEM (A, B), SHEM+BPE (C, D), or SHEM+NGF (E, F) for 1 week. Immunostaining showed sporadic Ki67-positive nuclei in the periphery of HCEC sheets when aggregates were cultured in SHEM (G). In contrast, very few Ki67-positive nuclei were found in SHEM+BPE (I) and SHEM+NGF (K). Ki67-positive nuclei increased dramatically when aggregates were pretreated with trypsin/EDTA (H, J, L). However, there were more Ki67-positive nuclei in SHEM (H) than those in SHEM+BPE (J) and SHEM+NGF (L). Bar, 100 μm.