Two Clonal Cell Lines of Immortalized Human Corneal Endothelial Cells Show either Differentiated or Precursor Cell Characteristics

Abstract

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC.

Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea

“…The immortalized human corneal endothelial cell line (HCEC-B4G12, Ref-ACC 647; DCMZ, Germany) 17 was cultured in human endothelial serum-free medium (Gibco, Paisley, UK) and 10 ng/mL FGF-2 (Gibco) in tissue culture plates precoated with 10 lg/mL laminin (Sigma) and 10 mg/mL chondroitin sulphate (Sigma). At 70% confluence, the media were then changed to 10% CoM from LPS-or IFN-c-stimulated and 18 and nuclear counting within multiple high power fields was performed manually.…”

Temperature-Sensitive Transient Receptor Potential Channels in Corneal Tissue Layers and Cells

“…Modulation of their activity by temperature changes underlies essential homeostatic mechanisms, contributing to the support of corneal endothelial function under different ambient conditions. Interestingly, similar observations were made in the immortalized clonal cell populations HCEC-B4G12 and HCEC-H9C1 also under serum-free culture conditions [27]. The osmosensor TRPV4 was also identified in human corneal endothelial cells, because exposure to a hypotonic environment increased the intracellular Ca 2+ concentration and TRP-like whole-cell currents in these cells [28].…”

Electrospun nanofibrous SF/P(LLA-CL) membrane: a potential substratum for endothelial keratoplasty

“…Collagen type IV is expressed abundantly in the B4G12 cell line. 24 The mRNA levels for these two types of collagen decreased slightly on some of the blend ratio scaffolds when compared with the control group, but not in a consistent manner.…”

“…24 This clonal cell line was purchased from the Creative Bioarray Company (Shirley, NY, USA). HCEC-B4G12 cells were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and 100 U/mL penicillin-streptomycin (all from Gibco, Carlsbad, CA, USA).…”