Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media

Arq Bras Oftalmol. 2016 Feb;79(1):37-41. doi: 10.5935/0004-2749.20160011.

Abstract

Purpose: To compare cryopreserved human corneal endothelial cells (HCECs) grown in human serum-supplemented media (HS-SM) with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM).

Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR) for endothelial cell markers, and detachment time.

Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages.

Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Cattle
  • Cell Count
  • Cell Culture Techniques / methods*
  • Cell Proliferation / drug effects
  • Cells, Cultured / drug effects
  • Child
  • Cryopreservation / methods*
  • Culture Media, Conditioned
  • Endothelial Cells / cytology*
  • Endothelium, Corneal / cytology*
  • Gene Expression
  • Humans
  • Immunohistochemistry
  • Reverse Transcriptase Polymerase Chain Reaction
  • Serum*
  • Statistics, Nonparametric
  • Time Factors

Substances

  • Culture Media, Conditioned