Seo Seog-Jin, Kim In-Yong, Choi Yun-Jaie, Akaike Toshihiro, Cho Chong-Su
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea.
Biomaterials. 2006 Mar;27(8):1487-95. doi: 10.1016/j.biomaterials.2005.09.018. Epub 2005 Sep 26.
Formation of primary hepatocyte spheroids in the hydrogel scaffold is a promising approach for enhancing liver-specific functions in liver tissue engineering as well as for developing bioartificial liver (BAL) devices. In the present study, a highly porous hydrogel scaffold composed of alginate (AL) and galactosylated chitosan (GC) as a synthetic extracellular matrix (ECM) for hepatocytes was fabricated with 150-200 microm pore size in diameter. Cell adhesion onto AL/GC and AL/chitosan film was 72.7 and 45% at 1 wt% of GC (or chitosan) to AL content whereas cell adhesion onto AL film was 28.5%. The optimal concentration of GC in AL/GC sponge was 1 wt% to AL content by the measurement of albumin secretion. Cell viabilities performed on AL and AL/GC sponges were 72.2+/-3.6 and 81.3+/-3.5% of control, respectively, after 10 days incubation. Hepatocytes were aggregated to form multicellular spheroids in AL/GC sponge with diameter enlarged up to about 100 microm, 36 h postseeding, whereas most of them in the AL sponge remained as single cells and only a few cells began to form aggregates. Intercellular molecules such as connexin32 and E-cadherin genes related with cell-cell contact were expressed in hepatocytes within AL/GC sponge at 36 h after incubation, but not in AL sponge. Treatment with a gap junctional intercellular communication (GJIC) inhibitor, 18beta-glycyrrhetinic acid, resulted in a 1.5-fold marked decrease in albumin secretion levels in AL/GC sponge. Specially, coculture of hepatocytes in AL and AL/GC sponges with NIH3T3 in a transwell insert resulted in enhanced increase of liver-specific functions, such as albumin secretion rates, ammonia elimination rates, and ethoxyresorufin-O-deethylase activity by cytochrome P4501A1, compared to those in hepatocyte monoculture. The results suggest that formation of hepatocyte spheroids in coculture system enhances liver-specific functions for the AL/GC sponge as a new synthetic ECM to design developed BAL devices.
在水凝胶支架中形成原代肝细胞球体是一种很有前景的方法，可增强肝组织工程中的肝脏特异性功能以及用于开发生物人工肝（BAL）装置。在本研究中，制备了一种由藻酸盐（AL）和半乳糖基化壳聚糖（GC）组成的高度多孔水凝胶支架，作为肝细胞的合成细胞外基质（ECM），其孔径直径为150 - 200微米。当GC（或壳聚糖）与AL的含量为1 wt%时，细胞在AL/GC和AL/壳聚糖膜上的粘附率分别为72.7%和45%，而细胞在AL膜上的粘附率为28.5%。通过测量白蛋白分泌，AL/GC海绵中GC的最佳浓度为AL含量的1 wt%。在培养10天后，在AL和AL/GC海绵上进行的细胞活力分别为对照的72.2±3.6%和81.3±3.5%。接种36小时后，肝细胞在AL/GC海绵中聚集形成直径增大至约100微米的多细胞球体，而它们在AL海绵中的大多数仍为单细胞，只有少数细胞开始形成聚集体。与细胞间接触相关的细胞间分子如连接蛋白32和E - 钙粘蛋白基因在培养36小时后在AL/GC海绵内的肝细胞中表达，但在AL海绵中不表达。用间隙连接细胞间通讯（GJIC）抑制剂18β - 甘草次酸处理导致AL/GC海绵中白蛋白分泌水平显著降低1.5倍。特别地，与肝细胞单培养相比，在transwell小室中肝细胞与NIH3T3在AL和AL/GC海绵中共培养导致肝脏特异性功能增强，如白蛋白分泌率、氨清除率以及细胞色素P4501A1的乙氧基异吩恶唑酮 - O - 脱乙基酶活性增强。结果表明，在共培养系统中形成肝细胞球体可增强AL/GC海绵作为一种新型合成ECM的肝脏特异性功能，以设计开发的BAL装置。
