Phase I: High throughput fabrication of fractal cell culture substrates

目前，还没有能够高保真培养关键肾细胞：足细胞的细胞培养技术。单层培养不能概括它们的分枝表型。我们已经开发了一种全新的方法：通过忠实地再现细胞培养基质上的原生肾小球的分形地形线索，我们微工程化了一个环境，足细胞表现出与体内足细胞相似的分支形态和基因表达。与常规单层相比，这种改进的细胞培养在新化合物测试和冠状病毒感染研究中具有上级优势。本提案的主要目标是开发一种用于生产具有分形形貌线索的商业孔板的高通量制造工艺，从目前需要> 20小时生产一个2x2 cm 2基底的手动工艺到每个板需要15分钟的工艺。在目标1中，我们将通过热压印和注塑成型扩大生产规模，以生产商业化的24孔和96孔板。在目标2中，我们将为这些接骨板制定灭菌方案，在目标3中，我们将确认其生物相容性。为了达到所需的生产量并制造非吸收性设备，我们需要克服从聚二甲基硅氧烷实验室原型插件扩展到玻璃层压非吸收性热塑性弹性体的主要工程挑战。 我们的主要客户将是制药公司，例如填写订单的Regeneron，以及需要高保真足细胞培养的学术实验室。我们的合作者Quaggin、Barua和Rogers博士是领先的肾脏研究人员，他们渴望在实验室中测试用于足细胞培养的新平板。新的制造工艺也可用于制造其他细胞的分形板，例如脂肪细胞和肺细胞。我们的首选模式是将平板授权给现有的成熟公司，该公司制造和分销用于生命科学研究的创新工具，例如提供支持信的公司-干细胞技术公司和uFluidix。通过我们团队成员将致力于的新初创公司，这些平板还将有助于发现治疗肾脏疾病的新化合物。

Currently, there are no cell culture techniques that enable high fidelity cultivation of the key kidney cells: podocytes. Monolayer culture fails to recapitulate their branching phenotype. We have developed a completely new approach: by faithfully recreating fractal topographical cues of the native kidney glomerulus on cell culture substrates, we microengineered an environment where podocytes exhibit their branching morphology and gene expression similar to the podocytes in vivo. Such improved cell culture was superior in new compound testing and coronavirus infection studies compared to the conventional monolayers. The main goal of this proposal is to develop a high throughput manufacturing process for production of commercial well plates with fractal topographical cues, from the current manual process that takes >20hr to produce one 2x2cm2 substrate, to a process that takes 15min per plate. In Aim 1, we will scale-up production by hot embossing and injection molding to create commercial 24- and 96-well plates. In Aim 2, we will develop sterilization protocol for these plates and in Aim 3, we will validate their biocompatibility. To reach the desired production throughput and create non-absorbent devices, we need to overcome the main engineering challenge of scaling from polydimethylsiloxane lab prototype inserts to non-absorbent thermoplastic elastomer laminated on glass. Our main customers will be pharmaceutical companies, e.g. Regeneron that filled an order form, and academic laboratories who have a need for high fidelity culture of podocytes. Our collaborators, Drs. Quaggin, Barua and Rogers, are leading kidney researchers, eager to test the new plates for podocyte cultivation in their laboratories. The new manufacturing process can also be used to fabricate fractal plates for other cells e.g. adipocytes and lung cells. Our preferred model is licensing the plates to an existing established company that manufactures and distributes innovative tools for life science research, such as companies that provided support letters-Stem Cell Technologies and uFluidix. The plates will also enable discovery of new compounds for kidney disease, through a new start-up that our team members will work on.