BIOARTIFICIAL LIVERS FROM HEPATIC PROGENITOR CELLS

大鼠肝脏含有由每个门脉三联体定位的祖细胞 它产生的子细胞通过单向的， 分化过程在中央静脉结束。因此，这些车牌 每个腺泡内的实质细胞(在体内)的谱系是 成熟肝细胞的大小、倍性、生长和年龄相关 差异化潜力。我们建议使用这些祖细胞， 经多参数荧光激活细胞分选纯化后， 建立生物人工肝。这些细胞将被种植到一个凹陷的地方 纤维生物反应器，设计新颖，未定义体外培养 完全或大部分无血清的条件，包含明确的和 纯化的细胞外基质成分作为底物，并定义和 纯化的可溶性信号(激素、生长因子、营养素)。 已经开发出了协议，并通过这些协议定义了抗原谱 鉴定和分离肝祖细胞的三个亚群和 成熟实质细胞的两个亚群使用 平移和多参数荧光激活细胞分选(FACS)： 肝母细胞(多能肝祖细胞)；致力于胆汁和 肝细胞前体细胞；门脉周围实质细胞(假定年轻 实质)；以及5)中心周围的实质细胞(推测是陈旧的 薄壁组织)。还开发了用于命运研究的体内生物测定，例如 允许细胞扩张的活体条件和其他驱动 每个祖先亚群的分化。那只老鼠 生物人工肝将由5个人分别建立 接种成熟期实质细胞亚群的研究 将它们放置在涂有基质的多孔、可生物降解的微载体上 组件，制成一种新型的中空纤维生物反应器，并在 适宜的体外扩增条件。一个单独的生物反应器，用于 将建立饲养单元，并将包含两种饲养单元类型 发现产生旁分泌信号，这是生长的严格要求 祖细胞：1)FACS纯化的造血型OCAP细胞(髓系 携带卵圆细胞抗原3+，OC3+的细胞)和STO胚胎 新近发现的替代原代细胞的基质细胞系 年龄和肝脏特异的基质饲养层细胞(E14-E16)的培养 肝脏)。带有饲养单元的生物反应器将被耦合到 与含有肝祖细胞的细胞相结合；如果 喂食者产生的因子太不稳定，不能在困境中生存 连接生物反应器，饲养细胞将用丝裂霉素处理 C，并与肝祖细胞共同接种到相同的生物反应器中。 生物人工肝和对照单层培养将是 具有胎儿和成人肝脏特异性功能的特点，以及 强生干玻片中的造血标记物 免疫化学、生化分析、分子杂交分析 检测和流式细胞仪分析。此外，生物反应器 将使用核磁共振非侵入性地表征 和磁共振成像。细胞中细胞的命运 生物反应器将与体内鉴定的生物反应器进行比较 生物检测。

Rat liver contains progenitor cells located by each of the portal triads and which produce daughter cells that mature through a unidirectional, differentiation process ending at the central vein. Thus, the plates of parenchymal cells within each acinus (in vivo) are lineages of maturing liver cells with age-dependent size, ploidy, growth and differentiative potential. We propose to use these progenitor cells, purified by multiparametric fluorescence activated cell sorting, to establish a bioartificial liver. The cells will be seeded into a hollow fiber bioreactor, of novel design, and under defined ex vivo culture conditions that are entirely or mostly serum-free, contain defined and purified extracellular matrix components as substratum, and defined and purified soluble signals (hormones, growth factors, nutrients). Protocols have been developed, and antigenic profiles defined by which to identify and isolate three subpopulations of hepatic progenitors and two subpopulations of mature parenchymal cells using a combination of panning and multiparametric fluorescence activated cell sorting (FACS): hepatoblasts (pluripotent hepatic progenitors); committed biliary and hepatocytic progenitors; periportal parenchymal cells (presumptive young parenchyma); and 5) pericentral parenchymal cells (presumptive old parenchyma). Also developed are in vivo bioassays for fate studies, ex vivo conditions that permit cell expansion and others that drive differentiation of each of the progenitor subpopulations. The rat bioartificial liver will be established from each of the 5 subpopulations of maturationally staged parenchymal cells by seeding them onto porous, biodegradable mircocarriers coated with matrix components, into a novel form of hollow fiber bioreactor and under appropriate ex vivo expansion conditions. A separate bioreactor for feeder cells will be established and will contain two feeder cell types found to yield paracrine signals that are strict requirements for growth of the progenitors: 1) FACS-purified hemopoietic OCAP cells (myeloid cells that bear an oval cell antigen 3+, OC3+) and the STO embryonic stromal cell line, that has recently been found to replace primary cultures of age- and liver-specific stromal feeder cells (from E14-E16 livers). The bioreactors with the feeder cells will be coupled in tandem with the ones containing the hepatic progenitor cells; if the factors produce by the feeders are too labile to survive the tandemly connected bioreactors, the feeder cells will be treated with mitomycin C and co-seeded into the same bioreactors with the hepatic progenitors. The bioartifical livers and control monolayer cultures will be characterized for fetal and adult liver-specific functions and for hemopoietic markers by means of the Johnson and Johnson dry slide assays, by immunochemistry, biochemical assays, molecular hybridization assays and by flow cytometric analyses. In addition, the bioreactors will be characterized non-invasively using nuclear magnetic resonance and magnetic resonance imaging. The fates of the cells in the bioreactors will be compared with those identified from in vivo bioassays.