Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine

Abstract

Keratinocyte culture medium (KCM) has been used for the in vitro culture of keratinocytes and other types of epithelial cells, and the medium includes various ingredients. In this study, two modified KCMs were prepared. In the first, insulin, hydrocortisone and antibiotics that are normally included in KCM were replaced with clinically approved pharmaceutical agents, except transferrin and selenium; in the second, cholera toxin (CT) was replaced by L-isoproterenol (ISO). The modified KCMs were then compared to conventional KCM containing laboratory-grade reagents. Induced cell colony formations of canine oral mucosal epithelial cells cultured in both modified KCMs were found to be nearly equivalent to that in the control KCM, and there was no significant difference between the effect of CT and ISO. Canine oral mucosal cells proliferated to confluence in all three KCM formulations, with or without the use of 3T3 feeder layers. Cultured epithelial cells were harvested from temperature-responsive culture surfaces as an intact cell sheet, and the immunohistochemical analysis of the sheets showed that p63 and cytokeratin were expressed in the epithelial cell sheets cultured in all KCMs. Eventually, in the modified KCM formula, fetal bovine serum was replaced by autologous human serum, and the formula was found to be able to fabricate human oral mucosal epithelial cell sheets. These results indicated that the modified KCM was equally efficient as conventional KCM in the fabrication of transplantable stratified epithelial cell sheets.

Superficial Dermal Fibroblasts Enhance Basement Membrane and Epidermal Barrier Formation in Tissue-Engineered Skin: Implications for Treatment of Skin Basement Membrane Disorders

“…Passage 4 SF or DF were seeded onto crosslinked C-GAG discs at a density of 0.5 · 10 6 cells/cm 2 and cultured (at 37°C, 5% CO 2 ). Two days later, K were seeded on top of the fibroblast-populated matrices at a density of 1.0 · 10 6 cells/ cm 2 and the medium was replaced with a coculture medium containing serum (DMEM-HG and nutrient mixture F-12 Ham [3:1], 2 nM triiodothyronine, 5% FBS, 0.5% insulintransferrin-selenium-G supplement, 1 nM cholera toxin, 10 ng/mL EGF, 0.4 mg/mL hydrocortisone, 5 mg/mL transferrin, 1% antibiotic-antimycotic 26 ). Matrices with fibroblasts alone were used as controls, wherein the number of cells was adjusted so as to make the number of cells on the matrices comparable to those with K. The submerged culture was continued for five additional days and then lifted to the airliquid interface on a steel platform to enable epidermal stratification.…”

Regenerative medicine of cornea by cell sheet engineering using temperature-responsive culture surfaces

“…In fact, our clinical trials using autologous oral mucosal cell sheets prepared using temperature-responsive culture surfaces have demonstrated significant improvement in visual acuity and successful long-term maintenance of healthy conditions at the ocular surface in all patients after transplantation ( Figure 5) [36]. In addition, we recently established more optimized culture conditions in the preparation of cell sheets [57][58][59] and the validation system for tissue-engineered cell sheets [60,61]. Moreover, we also developed an autologous transplantation technique for patients with inherited corneal disorders by genetic modification of corneal epithelial stem cell sheets using lentiviral vectors [62].…”

Preparation of hyperbranched polystyrene-g-poly(N-isopropylacrylamide) copolymers and its application to novel thermo-responsive cell culture dishes

“…The cell sheets with a large surface area can be easily recovered from such a smart bio-interface. This class of cell culture dishes has been commercialized as UpCell ® and used for tissue engineering [11,12]. In the manufacturing of these dishes, PNIPAM is polymerized on the surface of the polystyrene dish by electron beam (E-beam) polymerization of N-isopropylacrylamide (NIPAM), which requires complicated equipment and tends to be expensive.…”