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人类细胞平台工程。DBM已经组建了一个专家联盟;我们共同寻求快速通道SBIR资金，为学术和行业市场建立和提供正常和ADPKD人类细胞平台的产品线菜单。现有的和感兴趣的DBM客户将根据产品线菜单中的哪些选项适合他们的研究需求进行介绍，正如我们的研究战略和商业化计划中所强调的那样。这一备受期待的产品线菜单将包括一系列：（a）特种细胞培养组分，（B）原代正常和ADPKD人原代培养物，（c）混合永生化培养物和（d）克隆永生化细胞系，这些细胞系经过充分表征、基因分型并定义为肾单位来源部分。将永生细胞系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、基因型和起源肾单位节段方面是否相似。DBM和合作者已开始在1期样试验研究中从2个人ADPKD（huADPKD）供体肾脏（从商业供应商处采购）中收集12个单囊组织来源的原代培养物和7个多囊组织来源的原代培养物并进行表征。将这些最初的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个不同供体的原代培养物组合，以便所有30个样本都可以由马约PKD中心的Peter Harris博士的核心进行基因分型。在组织培养塑料上的2D微量滴定板驱动的增殖生物测定以及3D Matrigel中对每个huADPKD原代培养物进行了分析。 基于膀胱生成生物测定。为支持上述试点工作，提供了初步数据。选择显示出令人信服的基因型并在2D和3D生物测定中表现出优异效用的原代培养物，将通过多种遗传方法永生化。DBM承诺在合作中也将在必要时使KUMC的初级文化不朽。在可能的情况下，将在huADPKD细胞中鉴定起源的肾单位节段。在我们的应用科学研究中将解决两个假设：（1）原代huADPKD成纤维细胞是否支持（共培养，条件培养基，两者）驱动人ADPKD囊性上皮细胞原代培养物更长寿命和/或在PKD研究的2D和3D生物测定中获得特定表型？和（2）给定huADPKD供体肾内的单个囊肿是否具有相同的基因型或它们的基因型在囊肿与囊肿之间是否不同（即，探索“一次击中”与“两次击中”假说或体细胞突变的出现和单倍不足的可能存在）？DBM等人为我们的工作设置了5个里程碑，以获得这个可能的快速通道SBIR奖的支持。里程碑1将是从每个huADPKD供体肾组织样本中建立单囊来源和多囊组织来源的原代培养物，最终目标是处理12-15个不同的供体。里程碑2将对每个单个囊肿来源和多囊性组织来源原代培养物进行基因分型，将基因型与ADPKD基因型数据库进行比较，并选择候选原代培养物进行永生化。里程碑3将是使用优化的关键路径使选择的原代培养物永生化。里程碑4将是表征永生克隆细胞系与原代培养物，其中细胞系起源于原代培养物以确定不同生物测定中的“可用性”。里程碑5将在该奖项的第2年和第3年以及本学期之后，从我们的产品线菜单中为PKD学术和行业研究社区提供人类正常和ADPKD肾细胞平台的定制包。

 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.