The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells

Abstract

Aim: To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE).

Methods: Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 microg/ml, 140 microg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically.

Results: The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm(2), 60 cells/cm(2), and 50 cells/cm(2), respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation.

Conclusions: The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.

Figure 1

Phase contrast appearance of conjunctival epithelial cell cultures. (A) Passage 2 conjunctival epithelial cells in 5% HS supplemented media, 6 days after plating. Colonies consisted of a collection of round or ovoid cells, surrounded by larger elongated cells. (B) 5% FBS supplemented culture, passage 2 conjunctival cells. (C) BPE supplemented (70 μg/ml) culture, and (D) basal medium, 6 days after plating (passage 2). Fibroblast proliferation was noted in (E) HS supplemented and (F) FBS supplemented cultures in passage 3. Ep: Epithelial cells; Fb: Fibroblasts. Bar = 125 μm.

Figure 2

Colony forming efficiency of conjunctival epithelial cells cultivated in HS, FBS, and BPE supplemented media (n = 12). HS: human serum; FBS: fetal bovine serum; BPE: bovine pituitary extract; BM: basal medium.

Figure 3

(A) BrdU ELISA cell proliferation assay of cells cultivated in HS, FBS, and BPE supplemented media (n = 16). The bars represent the mean values of BrdU absorbance in each culture condition. There was greater incorporation of BrdU in cells cultivated in FBS supplemented media, compared to HS or BPE supplemented media. (B) The number of cell generations achieved in the various culture conditions. Cells cultivated in FBS supplemented media demonstrated the greatest number of cell generations, followed by 5% HS supplemented media (n = 16). HS: human serum; FBS: fetal bovine serum; BPE: bovine pituitary extract; BM: basal medium.

Figure 4

Immunocytochemistry of cultivated conjunctival epithelial cells. Cells cultivated in HS supplemented media stained positively for (A) keratin 4 and (B) keratin 19. (C) HS supplemented culture, showing a pair of goblet cells stained positively with PAS. Immunofluorescence demonstrating a pair of MUC5AC positive cells (indicated by arrows) in (D) HS supplemented and (E) FBS supplemented cultures. (F) A cluster of MUC5AC stained goblet cells in HS supplemented media. (A–C) Bar = 125 μm. (D–F) Bar = 63 μm.

Figure 5

Identification of MUC5AC transcripts in normal conjunctiva and cultivated conjunctival epithelial cells by RT-PCR. MUC5AC transcripts were detected in cDNA from human forniceal conjunctiva (lane 1), conjunctival epithelial cells cultured in FBS supplemented media (lane 2), HS supplemented media (lane 3), and BPE supplemented media (lane 4). PCR was performed with water replacing cDNA as the negative control (lane 5). The similar signal for MUC5AC was present in the three culture conditions.

Figure 6

Human conjunctival epithelial equivalents 10 days following transplantation into SCID mice. (A) Basal medium. The conjunctival epithelial sheet consisted of 2–3 layers of cells. (B) HS supplemented culture, demonstrating a multilayered stratified epithelial sheet comprising 4–7 layers of cells. (C) FBS supplemented culture, demonstrating an epithelial sheet consisting of 5–8 layers of cells. (D) BPE supplemented culture, demonstrating 4–6 layers of flattened cells. The HAM is indicated by the area within the double headed arrow. Ep: epithelial sheet; S: stroma of SCID mouse. Bar = 100 μm.