Efficient Isolation Of Bovine Keratocytes Utilizing Two Step Enzymatic Digestion

Background:Efficient and lowcost way to isolate keratocytes is helpful for research on cornea. Either relatively expensive or inefficient is the shortage of those means now applied, while raising the keratocytes through passage will change the phenotype of them quickly. Our aim is to approach the way getting keratocytes effectively utilizing modified two step enzymatic digestion by type I collagenase.Objective:To evaluate the effect of isolating the bovine keratocytes utilizing two step enzymatic digestion and observe the morphological changes of the keratocytes during cultivation in vitro.Methods:Keratocytes were isolated from bovine corneas using 0.5mg/mL and 1mg/mL type I collagenase digestion. The harvesting rate and viability rate of the primary keratocytes were evaluated. During the primary cultivation in vitro, the morphological changes of the keratocytes and their F-action distribution were observed.Results:(1) The extracellular matrix of the bovine corneas were almost dissolved by the two step enzymatic digestion, followed the keratocytes completely isolated from the solid matrix. The amount of the harvested keratocytes from each cornea was(2.109±0.142)×106 on average while the viability rate was (91.69±3.55)% and the inoculation rate was (81.20±1.21)%. (2) The primary keratocytes attached and spreaded out with dendritic and stellate morphology. After 3 days cultured, the branches of the keratocytes were contacting and formed networks. The F-actin detected by phalloidin binding showed a limited cortical localization.Conclusion:(1) The method of two step enzymatic digestion can make the extracellular matrix of bovine cornea stroma completely degraded with the advantages in high efficiency of harvesting keratocytes and high cell viability and relatively simple manipulation. (2) The primary bovine keratocytes have dendritic morphology and with limited F-action distribute in cellular cortex.