Characteristic Study Of Producing Human Dermal Fibroblasts And Human Epidermal Cells On A Large Scale By Rotary Cell Culture System

Owing to several recent developments, the culture methods of human skin cells, particularly epidermal keratinocytes of the human, has been greatly improved. Under the suitable culture, single cultured cells generate stratified colonies that ultimately fuse and form an epithelium that is a reasonable approximation of the epidermis. It will be shown a possibility that large amounts of cultured epithelium can be generated from a small piece of epidermis in a short time. In cilinic,surgeons and cell biologists use culture-gown epithelium derived from the same individual to restore defects in the epidermis. the cultural method of keratinocytes had made great progress , from primal serum and feeder layer culture to serum-free and feeder layer-free culture. This development had promoted clinical application of keratinocytes, from cell suspens ion transplantation to autografts transplantation, to cograft transplantation. These developments make people see the hope in the treatment of the large cutaneous deficiency.Because Cell culture techniques have become vital to the study of animal cell structure, function and differentiation, Microcarrier culture introduces new possibilities and, for the first time, makes possible the practical high-yield culture of anchorage-dependent cells for it. In microcarrier culture, cells grow as monolayers or multilayers on the surface of small spheres that are usually suspended in culture medium by gentle rotating or stirring. By using microcarriers in simple suspension culture, yields of up to 200 million cells per milliliter are possible.Cytodex microcarriers have been specifically developed by GE Healthcare for the high-yield culture of a wide range of animal cells in culture volumes ranging from a few milliliters to several thousand liters and are very suitable for large-scale expansion cells. Surface characteristics of the microcarriers have been optimized for efficient attachment and spreading of cells. Size and density are optimized to facilitate even suspension and give good growth and high yields for a wide variety of cells. Matrix is biologically inert and provides a strong but non-rigid substrate for sell cultures. Transparent and allows easy microscopic examination of the attached cells. Experience with Cytodex in a wide variety of applications has confirmed its importance and value in microcarrier techniques.The Rotary Cell Culture System bioreactor consists of a horizontally rotated culture vessel with a co-axial oxygenator in the center. When the vessel is filled with culture media and rotated, the fluid rotates as a solid body around a horizontal axis. These conditions produce minimal shear force inside the culture vessel. The cells are maintained in suspension by the resolution of the centrifugal, gravitational and Coriolis forces, so that cells placed in the RCCS bioreactor experience minimal mechanical stresses and high mass transport of nutrients, oxygen etc. Gas transfer is by means of diffusion through the silicone oxygenator, thereby avoiding bubble formation and turbulence.In clinic, rapid and efficient resurfacing of various skin defects by autologous dermal fibroblasts and epidermal cells transplantation is significant in skin wound healing. However, provide a sufficient number of seed cells to construct tissue engineering skin are very important. We attempted to produce human dermal fibroblasts (hDFBs) and human epidermal cells (hECs) on a large scale by Rotary Cell Culture System (RCCS). In this cultural system we used Cytodex-3 microbeads as microcarriers and high aspect ratio vessel RCCS as cultural system. Partâ… : The culture and identification of human derm fibroblasts and human epidermal cells in vitro1 Main methodsTake for the Foreskin tissue from newborn. The hECs were obtained from human skin by using the method of digesting human foreskin with Dispaseâ…¡and Trypsin-EDT and The hDFBs by using the method of tissue-adherence for primary culture and Subculture. Immunohistochemical method was used for vimentin staining and cytokeratin staining.2 Main resultsThe human derm fibroblasts were tested Vimentin positive and the human epidermal cells were tested cytokeratin positive.3 Main conclusionsThe cultured cells derived from foreskin tissue from newborn. They are the human derm fibroblasts and human epidermal cells. Partâ…¡: Using RCCS expansion and delivery of human dermal fibroblasts1 Main methodsThe hDFBs were obtained from human skin by using the method of tissue-adherence and cultured in vitro and labeled with DIO, then combined with Cytodex-3 in RCCS. The characteristics of fibroblasts combined with microcarriers were observed with fluorescent microscope, and scanning electronic microscope (SEM). The proliferative capacities of microgravity cultivation and routine cultivation were compared by cell cycle and group doubling time.2 Main resultsOur results showed that hDFBs rapidly attached to the microcarriers in the RCCS, achieving high cell densities. hDFBs cultured on the microbeads in the RCCS remained actively proliferating potentials.3 Main conclusionsUsing biological reactor and microcarriers culture human skin fibroblasts is a kind of effective method for preparation a large number of seed cells for tissue engineering. Partâ…¢: Using RCCS expansion and delivery of human epidermal cells1 Main methodsWe attempted to produce human epidermal cells (hECs) on a large scale by Rotary Cell Culture System (RCCS). In this cultural system we used Cytodex-3 microbeads as microcarriers and high aspect ratio vessel RCCS as cultural system. The hECs were obtained from human skin by using the method of digesting human foreskin with Dispase II and Trypsin-EDT and cultured in vitro and labeled with DIL, then combined with Cytodex-3 in RCCS. The characteristics of hECs combined with microcarriers were observed with Inverted microscope, and scanning electronic microscope ( SEM ) . The proliferative capacities of microgravity cultivation and routine cultivation were compared by group doubling time.2 Main resultsOur results showed that hECs rapidly attached to the microcarriers in the RCCS, achieving high cell densities. hECs cultured on the microbeads in the RCCS remained actively proliferating potentials.3 Main conclusionsUsing biological reactor and microcarriers culture human epidermal cells is a kind of effective method for preparation a large number of seed cells for tissue engineering. Partâ…£: Human Epidermal Cells in Coculture with Human Dermal Fibroblasts in a RCCS1 Main methodsThe experiments were trying to two different methods: Serum concentration gradient in reducing method and EpiLife culture method, use of Cytodex-3 microcarrier and High Aspect Ratio Vessel as a vessel bioreactor system try preparation granule skin. And using scanning electron microscopy (SEM) observe cell attached with a growth state.2 Main resultsIn EpiLife culture method, hECs attached on cytodex with polygons form, fibroblasts attached on cytodex with a poor state, partly sheded from microcarrier. Serum concentration gradient in reducing method have the polygons, polygons hECs and spindle hDFBs attached tightly, cells in a good condition, but with less cell density.3 Main conclusionsUsing RCCS and microcarrier suspension methods preparation granule skin, serum concentration gradient in reducing method is the effect good.