Culture Of Epidermal Cells On Carboxymethyl Chitosan Membrane

Background and objectiveVitiligo is an acquired disorder of pigmentation characterized by areas of depigmented skin due to the loss of epidermal melanocytes. Surgical procedures are applicablefor stable vitiligo, such as punch minigrafting,suction blister epidermalgrafting,transplantation of autologous epidermal cell suspension (NCEAs) and cultured epithelialautografts (CEAs). However, curved areas often get inadequate pigment resulting from cells out flowed and piled. Therefore we have tried to find a chemically defined carrier to improve celladherence to the recipient site.Carboxymethyl chitosan (CM- chitosan),a dissolvable chitosan derivative,is carboxymethylated by chloroacetic acid. It possesses many desirable physiochemical and biological features,such as nontoxicity,instance gelating capability,good water solubilityand biocompatibility. CM-chitosan has been under investigation for various applications in the degradable biomedical and absorbable materials field,such as drug deliverycarriers and tissue engineering scaffolds.In our study, melanocytes and keratinocytes were cultured on the surface of carboxymethyl chitosan membrane which performed as the carrier, by observing and detecting the variation in morphology and function, we tried to explore the role of carboxymethyl chitosan membrane in melanocytes and keratinocytes. The object of this study was through testing whether carboxymethyl chitosan membrane was suitable for human epidermal cell growth to provide a safe and effective cell carrier for cell transplantation treatment of vitiligo.In order to investigate the feasibility of using carboxymethyl chitosan membrane to carry and transport epidermic cells, melanocytes and keratinocytes were cultured on the surface of the membrane which performed as a carrier, then the variation of morphology and function of these cells were studied.MethodsKeratinocytes and Melanocytes were cultured in vitro with KM and MCD254, and use the antibodies of HMB-45 to identify cultured melanocytes. Cells were cultured separately on normal plates and CM-chitosan membranecoated plates.Inverted microscopy was used to observe the cellular morphology; The MTT assay was used to determine the proliferation of epidermal cells; the sodium hydroxide-based lysis method was used to determine the content of melanin; the dopa oxidase assay was used to detect tyrosinase activity; the western blot was used to determine the protein expression of TRP-1 and TRP-2.Results1. After 6h of incubation, both keratinocytes and melanocytes attached to the CM-chitosan membrane were slightly less than those attached to the uncoated surface. After 3 days incubation, keratinocytes and melanocytes on the CM-chitosan membrane remained discrete andtheir morphology was similar to those on the uncoated surface.2. Keratinocytes and melanocytes were treated with CM-chitosan solution(0.5%,0.25%,0.125%,0.0625%) for 3 days,7 days and 10 days. The result showed that there no significantly difference between the different groups (P>0.05)3. Cells culture on CM-chitosan membrane for lOdays, it showed significantly lower cell proliferation(P<0.01). Bathed in cell medium for 8days, We notice that the CM-chitosan membrane began to desolve.4. The epidermal cells after cultured 3days and 7days resulted in no significant difference of melanin content and tyrosinase activity(P >0.05).5. TRP-1 and TRP-2 protein were strong expressed in the two groups with no significant difference.ConclusionsKeratinocytes and melanocytes can be cultured on CM-chitosan membrane while maintaining the normal biological activity.