Morphological Characteristics and Proliferation of Keratocytes Cultured Under Simulated Microgravity

Abstract

This study probed the changes of keratocytes cultured under simulated microgravity. Keratocytes were isolated from rabbit corneas using collagenase digestion method. Cells were seeded in a 55-mL capacity high-aspect-ratio vessel (HARV) of rotary cell culture system (RCCS) at a density of 1 x 10(4) cells/mL. Dehydrated bovine acellular corneal stroma (5 x 5 x 1 mm, n = 30) was used as a carrier for keratocyte culture. Rotational speed was set at 15, 20, and 30 rpm in the first, second, and third week of culture, respectively. Histological evaluation showed that keratocytes in simulated microgravity culture grew into carriers, but those under conventional gravity grew on the surface of carriers. Scanning electron microscopic evaluation showed that after 19 days in culture, keratocytes on the carriers were spherical and spread in the spaces among the collagen fibers. Cells were dendritic or spindle shaped, and they developed many foot processes linked with surrounding cells. The absorbance values of the simulated microgravity group were significantly higher (P < 0.01) than that of the conventional group from 10 to 19 days of culture. The RCCS obviously enhanced the proliferation of rabbit keratocytes and facilitated the cells' growth into or on the dehydrated bovine acellular corneal stroma. Cells showed more natural morphology.

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“…This culture system seems to be ideal for overcoming some drawbacks of static culture, because the rotational motion can prevent sedimentation, and create a suspension culture environment and enhance cell-cell interactions. Several researches showed that RCCS contribute to cellular aggregation, intercellular adhesion and formation of 3D cell clumps [18].…”

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

“…Thus, comparison with the static culture, rotating simulated microgravity culture environment can show better cellular vitality and function for some cultivated cells. Our previous study found that SMG cultivation was propitious to proliferation of keratocytes for a rather long period of time [7]. Furthermore, SMG conditions are beneficial to culture stem cells.…”

“…Our previous study showed that bovine acellular stromal lamella appeared in favor of rabbit keratocyte growth in SMG condition. But the preparation process of acellular stromal lamella was rather long time, which underwent the dehydrated procedures of low-temperature preservation in glycerol for 6 months [7]. So, we here adopted short-term chemical-frozen to decellularizate bovine cornea and then prepared them as carriers to culture keratocytes.…”

“…The carriers of acellular corneal stroma could be obtained by dehydration of glycerol for 6 months, low-temperature preservation at −20°C or 4°C, and trypsinization for 10 min. After 19 days in SMG culture, rabbit keratocytes on the carriers were dendritic or spindle shaped and grew into the porous matrix of carriers [7]. In present study, we used a more rapid and convenient short-term chemical-frozen way to decellularize bovine cornea as carriers.…”

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“…Jiansu Chen et al. (99) of Jinan University (Guangzhou, China) studied the changes of keratocytes, isolated from rabbit corneas using a collagenase digestion method, cultured under simulated microgravity. Histological evaluations showed that the keratocytes grew into the carriers, but those under conventional gravity grew on the surface of carriers.…”