BIOARTIFICIAL LIVERS FROM HEPATIC PROGENITOR CELLS

来自肝祖细胞的生物人工肝

• 批准号: 6177720
• 负责人: LOLA M REID
• 金额: $ 30.55万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-24 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177720
• 关键词: acinar cell; bioassay; bioengineering /biomedical engineering; biomarker; biomaterial development /preparation; bioreactors; biotechnology; cell differentiation; cell growth regulation; cell proliferation; deficient growth media; embryonic stem cell; flow cytometry; hematopoietic stem cells; hepatocyte growth factor; laboratory rat; liver; liver cells; magnetic resonance imaging; nuclear magnetic resonance spectroscopy; organ culture; stem cells; tissue engineering; tissue support frame

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.

大鼠肝脏包含每个门户三合会位置的祖细胞 并产生通过单向成熟的子细胞， 区分过程以中央静脉结束。 因此，板 每个兴奋剂（体内）内的实质细胞的谱系是 将肝细胞与年龄相关的大小，倍状，生长和 区分潜力。 我们建议使用这些祖细胞， 通过多参数荧光激活的细胞排序纯化 建立生物人工肝。 细胞将被播种成一个空心 纤维生物反应器，新颖的设计和定义的离体培养物 完全或大部分无血清的条件，包含定义和 纯化的细胞外基质成分作为基质，并定义和 纯化的可溶性信号（激素，生长因子，养分）。 已经开发了协议，并定义了抗原剖面 识别和隔离肝祖细胞的三个亚群 两种成熟实质细胞的亚群，结合 平移和多参数荧光激活的细胞分选（FACS）： 肝细胞（多能肝祖细胞）；承诺的胆道和 肝细胞祖细胞；外周实质细胞（推定年轻人 实质）; 5）腹膜实质细胞（推定旧 实质）。 还开发了用于命运研究的体内生物测定。 可以允许细胞扩展的体内条件和其他驱动 每个祖细胞亚群的分化。 大鼠 生物人工肝将从5个 通过播种 它们在涂有基质的多孔，可生物降解的MiRDRADARRIS 成分，成为一种新型的空心纤维生物反应器和下方的形式 适当的离体扩展条件。 一个单独的生物反应器 将建立馈线电池，并包含两种馈线电池类型 发现会产生严格生长要求的旁分泌信号 祖细胞：1）FACS纯化的血有性OCAP细胞（髓样 带有椭圆形细胞抗原3+，OC3+）的细胞和sto Embryonic 基质细胞系，最近发现它取代了主要 年龄和肝脏特异性基质馈物细胞的培养物（来自E14-E16 肝）。 带有馈线电池的生物反应器将耦合 与含有肝祖细胞的其中串联；如果是 馈线产生的因素过于不稳定，无法生存 连接的生物反应器，馈送细胞将用丝裂霉素处理 C并与肝祖细胞共同种子。 生物体会肝和控制单层培养物将是 胎儿和成人肝特异性功能的特征以及 通过约翰逊和约翰逊干幻灯片的血压标记 测定，通过免疫化学，生化测定，分子杂交 测定和通过流式细胞仪分析。此外，生物反应器 将使用核磁共振表征非侵入性的表征 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 生物测定。