Characterizing the Effects of VPA, VC and RCCS on Rabbit Keratocytes onto Decellularized Bovine Cornea

Abstract

To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH4OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.

Study on immortal conditions of chicken embryonic stem cells

“…Considerable studies have shown that appropriate concentrations of VC can promote cell proliferation in vitro. This method has been widely used in human bladder cancer T‐24 cells, fibroblasts, skeletal muscle myoblasts, and rabbit keratocytes . In this study, we observed that 10 μmol/L of VC could increase the proliferation of chicken ESCs and improve their survival rate.…”

Keeping an Eye on Decellularized Corneas: A Review of Methods, Characterization and Applications

“…Triton X-100 has frequently been used in corneal decellularization protocols [6,44,46,78,79,80]. However, its use has been criticized for the apparent failure to effectively reduce and remove cellular material from tissue [61].…”

Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor

“…After being pre-incubated with or without the test compounds for 1 h, the cells were exposed in the presence or absence of different concentrations of rhIL-6 (or IL-6). The number of viable cells was counted by an automated cell counter every 12 h over 3 d. After 72 h, the viabilities of the 7TD1 cells were measured by the CCK-8 system [26] . The U937 cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS in a 37 °C incubator with 5% CO 2 .…”