Abstract:

Objective To investigate the influence of serum in establishing a corneal stroma Pellet three-dimensional model by detecting cell growth and collagen fibers production, in order to offer a new culture pattern for extracellular matrix(ECM) fibrosis study in vitro. Design Experimental study. Participants Bovine keratocytes. Methods The primary bovine keratocytes were isolated and cultured, and the first generation  cells were used for building Pellet model. The constructions in Pellet systems were cultured in the DMEM/F12 without serum (control group) or with 10% fetal bovine serum (FBS) (group serum), respectively.  After 3 weeks cultured, the Pellets were observed. The state of cell growth and cell activity was assayed by HE and Calcein AM/PI staining. And then, MASSON staining and transmission electron microscopy(TEM) were used to detect the formation of collagen arrangement. Main Outcome Measures Cell growth, cell activity, collagen arrangement. Results Cultured for 3 weeks in Pellet model, cells in the control group didn’t assemble and could be broken up easily. It was difficult to do sequential experiment. Inversely, in group serum Pellet huddled growing and was in grain appearance, combining firmly. The majority of living cells in Group serum growed closely and remained structural integrity through the HE and Calcein AM/PI staining. MASSON staining showed many collagen fibers. TEM showed that extracellular collagen deposited and densely distributed. Conclusions Serum could assist in-vitro three-dimensional culture of keratocytes, and generate the ECM rich of collagen fibers. (Ophthalmol CHN, 2017, 26: 328-332)

Key words: pellet 3-D culture, keratocytes, serum, extracellular matrix, collagen fibers