| 초록 |
Xyloglucan (XG) was used as a new synthetic extracellular matrix (ECM) for primary mouse hepatocyte attachment in Ca-alginate (AL) capsule. Hepatocyte adhesion onto collagen type I-, XG-coated and uncoated polystyrene (PS) surface was 89.1, 91.1 and 25.5 %, respectively, at 4 h after incubation at 37 ℃. From the inhibition study in a cell adhesion assay, the adhesion rate of freshly isolated hepatocytes and preincubated hepatocytes with 20 mM galactose onto the XG-coated surface were 55.7 and 17.3 %, respectively, after 30 min incubation at 37 ℃. Flow cytometric analysis (Fig. 1) shows that the internalization of XG by freshly isolated hepatocytes (C) is stronger than preincubated hepatocytes (B) with 20 mM galacotse. The concentration of XG in AL/XG capsules to perform the best liver-specific functions was 0.5 mg/ml, where the highest albumin secretion rates were obtained as shown in Fig. 2. The albumin secretion, ammonia elimination rate and cell viability of hepatocytes were slowly decreased with culture time in AL/XG capsules, whereas those were rapidly decreased in AL capsules, indication of the more rapid formation of hepatocyte spheroids in AL/XG capsules than in AL ones. More than 70 % of the seeded hepatocytes in AL/XG capsule participated in spheroid formation after 2 days, whereas most hepatocytes in AL ones remained as single cells and only a few cells began to form aggregates after 3 days. Intercellular molecule genes, such as connexin (Cx) 32 and E-cadherin, of hepatocyte spheroids in AL or AL/XG capsules were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) (Fig. 3). Cx32 and E-cadherin genes in AL/XG capsules were more rapidly reexpressed and expressed, respectively, than in AL ones. The results suggest that the multicellular spheroid formation of hepatocytes can enhance the liver-specific functions in the three dimensional space in the presence of XG as a new synthetic ECM owing to the specific interaction between the galactose moieties of XG and ASGPR of hepatocytes.
|
