BACKGROUND: Under the cellular physiological condition, 15.3% N-acetylated chitosan has poor solubility which limits cellular microencapsulation process.OBJECTIVE: This study was designed to prepare 50% N-acetylated chitosan with 15.3% N-acetylated chitosan and investigate its feasibility for preparing chitosan microcapsule used for hepatocyte embedding after in conjunction with methacrylic acid-hydroxyethyl methacrylate-methyl methacrylate (MAA-HEMA-MMA) copolymer under the cellular physiological condition.DESIGN, TIME AND SETTING: This study, a control observation experiment, was performed at the Central Laboratory, First Hospital Affiliated to Jinan University, Guagnzhou, Guangdong Province, China between January and October 2006.MATERIALS: 15.3% N-acetylated chitosan was provided by Sigma-Aldrich Company, Singapore. Hepatocytes were acquired from male Wistar rats, weighing 250-300g, by two-step collagenase digestion method.METHODS: Hepatocytes were microencapsulated using 50% N-acetylated chitosan and MAA-HEMA-MMA copolymer at 25℃ under aseptic condition.MAIN OUTCOME MEASURES: Microcapsule permeability was expressed with the diffusion degree of fluorescein isothiocyanate (FITC)-dextran in the empty microcapsule. The homeostasis of microencapsulated hepatocytes was measured by mechanical shearing-crush tests. The albumin-synthesizing capability of microencapsulated hepatocytes was determined by enzyme-labeled immunosorbent assay (ELISA). Urea-synthesizing capability was tested using kits (Sigma Diagnostics, USA) at the wavelength of 540 nm by colorimetric assay. Cytochrome P450 activity was determined using confocal laser microscopes and quantitated by the fluorescence intensity of products of 0-dealkylated 7-ethoxy resorufin, a substrate of cytochrome P450IA1. The plate-cultured hepatocytes were taken as controls.RESULTS: With increasing mass concentration of 50% N-acetylated chitosan, the diffusion degree of Mr 20000 and Mr 40000 FITC-dextran presented with a decreasing tendency, while the diffusion degree of Mr 70000 FITC-dextran was low and did not alter markedly. When the microencapsulated hepatocyte density was 2×109 L-1, the broken rate of microcapsule was only about 6% after 24 hours of shaking. Under the same condition of shaking, empty microcapsules were all broken after 4 hours of shaking. After 1 day of culture, approximately 30μmol/d urea could be synthesized among 1×106 bepatocytes that was markedly higher than the plate culture experimental result, 15μmol/d. After 7 days of culture, urea-synthesizing capability was close between in the microencapsulated hepatocytes and in the plate cultured hepatocytes. In the first 3 days of culture, 10μg/d albumin was acquired from 1×106 hepatocytes, and after 7 days of culture, only about 5μg/d albumin was obtained. The albumin level in the whole culture process was higher than plate culture results. After 1 day of culture, cytochrome P450 activity in the microencapsulated hepatocytes was higher approximately 8 times compared to plate culture results. After 1 week of culture, cytochrome P450 activity still retained at 50% of 1-day culture level.CONCLUSION: Microcapsule prepared by 50% N-acetylated chitosan under the physiological condition has good permeability and structural stability during the process of culture. Compared with in vivo surface culture test, degenerated chitosan microcapsule is better favorable to in vitro function of hepatocytes.

Prepared from 15.3% N-acetylated chitosan (FNC), half N-acetylated chitosan (HNC) possesses a good solubility in a weak basic solution, guaranteeing the formation of microcapsules by the coacervating reaction between HNC and methacrylic acid (MAA)-hydroxyethyl methacrylate (HEMA)-methyl methacrylate (MMA) (MAA-HEMA-MMA) terpolymer under physiological conditions. When hepatocytes were encapsulated in such 3-dimensional microenvironment, as compared to monolayer culture, cell functions, including P450 activity, urea production and albumin release, were well supported. The prepared microcapsules have good mechanical stability and permeability.
Animals ; Capsules ; Cell Culture Techniques ; methods ; Cells, Cultured ; Chitosan ; chemistry ; pharmacology ; Hepatocytes ; cytology ; drug effects ; Male ; Methacrylates ; Methylmethacrylate ; Polymers ; Rats ; Rats, Wistar ; Tissue Engineering ; methods