Pollok J M, Kluth D, Cusick R A, Lee H, Utsunomiya H, Ma P X, Langer R, Broelsch C E, Vacanti J P
Department of Surgery, Children's Hospital, Boston, USA.
Eur J Pediatr Surg. 1998 Aug;8(4):195-9. doi: 10.1055/s-2008-1071153.
Our laboratory has investigated heterotopic hepatocyte transplantation on biodegradable polymer matrices as an experimental treatment for end-stage liver disease. One of the limitations has been survival of sufficient cell mass after transplantation. We hypothesize that in vitro conditioning of cells within polymer matrices prior to implantation may increase hepatocyte survival and function. In this preliminary study we investigated the effect of continuous flow on hepatocytes and sinusoidal endothelial cells on poly-L-lactic acid (PLLA) discs in vitro. Highly porous PLLA discs were manufactured measuring 18 mm diameter by 1 mm thickness using previously described techniques. Hepatocytes were isolated from adult, male Lewis rats (200-300 g) using a two-step collagenase digestion. Sinusoidal endothelial cells were isolated using a two-step collagenase digestion, differential sedimentation, Percoll gradient centrifugation, and selective adherence. PLLA discs were seeded with hepatocytes alone or with co-cultures of hepatocytes and sinusoidal endothelial cells. Seeded discs were then secured within a flow bioreactor chamber and exposed to continuous flow of culture media at a rate of 20 ml/minute through the chamber. Seeded discs placed in static culture conditions served as controls. Specimens seeded with only hepatocytes were harvested at 24 hours, 48 hours, and 168 hours after seeding. Co-culture specimens were harvested after 168 hours. Specimens were viewed under phase-contrast microscopy and then formalin-fixed and prepared for histologic sectioning. Sections were stained with Hematoxylin and Eosin and then analyzed with light microscopy. Hepatocytes under flow conditions formed spheroidal aggregates of cells of 50 to 200 microns in diameter by 24 hours in culture. Hepatocytes in static conditions showed decreased aggregation of cells and spheroid formation was absent. Co-cultured specimens under flow also showed spheroid formation with endothelial cells lining the outside of hepatocyte spheroids. Co-cultured specimens in static culture showed no spheroid formation and no organization between sinusoidal endothelial cells and hepatocytes. These results suggest that continuous flow increases organization of hepatocytes cultured within biodegradable polymer matrices.
我们实验室研究了在可生物降解聚合物基质上进行异位肝细胞移植，作为终末期肝病的一种实验性治疗方法。其中一个限制因素是移植后要有足够数量的细胞存活。我们假设在植入前对聚合物基质内的细胞进行体外预处理可能会提高肝细胞的存活率和功能。在这项初步研究中，我们在体外研究了持续流动对聚-L-乳酸（PLLA）圆盘上的肝细胞和窦状内皮细胞的影响。使用先前描述的技术制造了高度多孔的PLLA圆盘，直径为18毫米，厚度为1毫米。使用两步胶原酶消化法从成年雄性Lewis大鼠（200-300克）中分离肝细胞。使用两步胶原酶消化、差速沉降、Percoll梯度离心和选择性贴壁法分离窦状内皮细胞。PLLA圆盘单独接种肝细胞或接种肝细胞与窦状内皮细胞的共培养物。然后将接种后的圆盘固定在流动生物反应器腔室内，并以20毫升/分钟的速度通过腔室使其暴露于连续流动的培养基中。置于静态培养条件下的接种圆盘用作对照。仅接种肝细胞的标本在接种后24小时、48小时和168小时收获。共培养标本在168小时后收获。在相差显微镜下观察标本，然后用福尔马林固定并准备进行组织切片。切片用苏木精和伊红染色，然后用光学显微镜分析。在流动条件下培养24小时后，肝细胞形成了直径为50至200微米的细胞球状聚集体。在静态条件下的肝细胞显示细胞聚集减少且没有形成球体。流动条件下的共培养标本也显示形成了球体，内皮细胞排列在肝细胞球体的外部。静态培养中的共培养标本未显示球体形成，且窦状内皮细胞与肝细胞之间没有组织化。这些结果表明，持续流动可增强在可生物降解聚合物基质内培养的肝细胞的组织化。
