Genotyped and Single Cyst-derived Human ADPKD Cell Platforms for Industry and Academia

 描述(由申请人提供)：DiscoveryBioMed，Inc.(DBM，Inc.或DBM)专门从事正常和患病的人类细胞平台工程。DBM已经组建了一个专家联盟；据此，我们共同寻求Fast Track SBIR资金，以建立和提供面向学术和行业市场的正常和ADPKD人体细胞平台的产品线菜单。现有的和感兴趣的DBM客户将按照我们的研究战略和商业化计划中强调的那样，按照产品线菜单中的哪些选项适合他们的研究需求进行展示。这一备受期待的产品线菜单将包括一系列：(A)特殊细胞培养成分，(B)正常原代和ADPKD人类原代培养，(C)混合永生培养和(D)克隆永生细胞系，这些细胞系具有良好的特性、基因分型，并被定义为起源于肾单位的片段。永生细胞系将与其来源的原代培养物进行比较，以确定它们在生物测定性能、基因型和起源的肾单位节段是否相似。DBM及其合作者已经开始收集和鉴定来自2个人类ADPKD(HuADPKD)捐赠者肾脏(从商业供应商获得)的12个单囊来源的原代培养物和7个多囊组织来源的原代培养物，进行类似第一阶段的试点研究。这些最初的19个huADPKD原代培养物与来自堪萨斯大学医学中心(KUMC)Darren Wallace博士的PKD研究生物材料和细胞模型核心的11个不同捐赠者的原代培养物相结合，以便所有30个样本都可以由梅奥PKD中心的Peter Harris博士的Core进行基因分型。每个huADPKD原代培养物都已在组织培养塑料上的2D微量平板驱动的增殖生物测定中以及在3D Matrigel中进行了描述 以成囊生物测定为基础。提供了支持上述试点工作的初步数据。选择表现出引人注目的基因和极好的实用性的原代培养物，2D和3D生物分析将通过多种遗传方法永垂不朽。DBM还承诺在合作中必要时将KUMC的主要文化永垂不朽。在可能的情况下，将在huADPKD细胞中确定起源于肾单位的片段。在我们的应用科学研究中将提出两个假设：(1)原代HuADPKD成纤维细胞支持(共培养，条件培养液，两者)是否能使原代培养的人ADPKD囊性上皮细胞活得更长和/或在用于PKD研究的2D和3D生物检测中获得特定的表型？以及(2)在一个特定的huADPKD供体肾内的单个包囊是否具有相同的基因，或者它们是否在不同的包囊中具有不同的基因(例如，探索‘一击’与‘两个Ht’的假说或出现体细胞突变和可能存在的单倍体功能不全)？DBM等人。为我们的工作设定5个里程碑，以支持这个可能的快速跟踪SBIR奖。里程碑1将是建立从每个HuADPKD捐赠者肾脏组织样本中获得的单囊和多囊组织来源的原代培养物，最终目标是处理12-15个不同的捐赠者。里程碑2将是对每个单独的囊源性和多囊组织来源的原代培养物进行分型，与ADPKD基因数据库进行比较，并选择候选原代培养物进行永生化。里程碑3将是使用一条优化得很好的关键路径使选定的初级文化永垂不朽。里程碑4将是对永生克隆细胞系与细胞系起源的原代培养物进行比较，以确定其在不同生物检测中的可用性。里程碑5将是在获奖的第二年和第三年以及之后作为一项自给自足的业务，从我们的产品线菜单向PKD学术和行业研究社区提供人类正常和ADPKD肾脏细胞平台的定制包。

 DESCRIPTION (provided by applicant): DiscoveryBioMed, Inc. (DBM, Inc. or DBM) specializes in normal and diseased human cell platform engineering. DBM has assembled a consortium of experts; whereby, collectively, we seek Fast Track SBIR funding to establish and offer a Product Line Menu of normal and ADPKD human cell platforms for the academic and industry marketplaces. Existing and interested DBM clients will be presented in terms of which options in the Product Line Menu fit their research needs, as highlighted in our Research Strategy and Commercialization Plan. This highly anticipated Product Line Menu will include an array of: (a) specialty cell culture components, (b) primary normal and ADPKD human primary cultures, (c) mixed immortal cultures and (d) clonal immortal cell lines that are well-characterized, genotyped, and defined as to nephron segment of origin. Immortal cell lines will be compared to the primary cultures from which they were derived to determine whether they are similar in bioassay performance, in genotype, and in nephron segment of origin. DBM and collaborators have begun the collection and characterization of 12 single cyst-derived primary cultures and 7 multicystic tissue-derived primary cultures from 2 human ADPKD (huADPKD) donor kidneys (procured from commercial vendors) in pilot Phase 1-like studies. These initial 19 huADPKD primary cultures were combined with primary cultures from 11 different donors from Dr. Darren Wallace's PKD Research Biomaterials and Cellular Models Core at Kansas University Medical Center (KUMC) so that all 30 samples could be genotyped by Dr. Peter Harris' Core at the Mayo PKD Center. Each huADPKD primary culture has been profiled in 2D microtiter plate-driven proliferation bioassays on tissue culture plastic as well as in 3D Matrigel based cystogenesis bioassays. Preliminary data are presented in support of the above pilot efforts. Select primary cultures that display a compelling genotype and exhibit excellent utility i 2D and 3D bioassays will be immortalized by multiple genetic methods. DBM has pledged to also immortalize KUMC primary cultures as necessary in collaboration. Where possible, nephron segment of origin will be identified in huADPKD cells. Two hypotheses will be addressed within our applied science research: (1) Does primary huADPKD fibroblast support (co-culture, conditioned medium, both) drive human ADPKD cystic epithelial cell primary cultures to be longer-lived and/or to attain specific phenotypes in 2D and 3D bioassays for PKD research? and (2) Do individual cysts within a given huADPKD donor kidney possess the same genotype or can they differ in genotype from cyst to cyst (i.e., exploring the 'one hit' vs. 'two ht' hypothesis or the emergence of somatic mutations and the possible presence of haploinsufficiency)? DBM et al. set 5 milestones for our work to be supported by this possible Fast Track SBIR award. Milestone 1 will be the establishment of single cyst-derived and multicystic tissue-derived primary cultures from each huADPKD donor kidney tissue sample and with a final goal of 12-15 different donors processed. Milestone 2 will be to genotype each individual cyst-derived and multicystic tissue-derived primary culture, compare genotypes against the ADPKD genotype database, and select candidate primary cultures for immortalization. Milestone 3 will be to immortalize select primary cultures using a well-optimized Critical Path. Milestone 4 will be to characterize immortal clonal cell lines versus primary cultures wherefrom the cell lines originated to determine 'usability' in different bioassays. Milestone 5 will be to offer custom packages of human normal and ADPKD kidney cell platforms from our Product Line Menu to the PKD academic and industry research communities in Years 2 and 3 of the award and beyond this term as a self-sustaining business.