Synthetic hydrogels for biomanufacturing of iPSC-derived neural cells for precision medicine

使用患者来源的诱导多能干细胞(IPSCs)制造的人类神经细胞具有很好的 有望为神经发育障碍建模，发现新的精确疗法，并筛查 环境毒素的潜在风险1-4。在过去的十年中，在协议方面取得了重大进展 以及为分化成特定神经细胞类型而开发的商业媒体系统5-8。然而，在那里 在生产、制造和检测工作流程中仍然是需要克服的重大技术挑战。 IPSCs通常是在动物来源的底物上分化出来的，这种底物引入了内在的可变性和缺乏控制 超过机械硬度和生化成分。这通常导致低产量和高变异性，这 在建立疾病的细胞模型时可能更加明显。迫切需要发展 促进IPSCs分化为成熟神经细胞的商业工具在受控、高效和 可复制的时尚，并消除动物衍生产品。生成的单元格、基于关联单元格的 化验和细胞疗法将对神经疾病建模、药物和 治疗发现和毒素筛选。 我们的第一阶段研究确定了有效分化的化学定义和坚固的合成水凝胶 IPSC来源的神经前体细胞(NPC)分化为皮质神经元，随后成熟到有丝分裂后， 功能成熟的神经元。这项工作的极具创新性的方面是底物被用作 使用我们专有的表面局部化聚合方法的薄型水凝胶涂层提供了几种 技术和商业化优势。为了将这些新型底物推向市场，我们建议 以下是我们第二阶段提案的具体目标：具体目标1将进一步验证所展示的工作 我们优化的合成薄型水凝胶涂层支持神经分化和成熟。包括进一步 微电极阵列分析及细胞功能表征 差异转录分析比较培养在底物上的细胞。我们将描述身体的特征 和优化的薄水凝胶的机械性能，并开发用于涂布平板的方法 自动化系统。特定目标2将在概念验证演示中应用底物，使用 从重度抑郁症患者样本中评估皮质神经元的底物与 控制。具体目标3将通过优化微载体上的涂层技术来扩展技术平台 适用于生物反应器的规模，这是证明这些底物适用于 生物制造应用。这项工作意义重大，因为迫切需要更好的工具来优化产量 并减少IPSCs分化为特定神经亚型的可变性，支持其长期培养， 减少达到功能成熟所需的时间，并在工作流程中消除动物源性产品。

Human neural cells manufactured using patient-derived induced pluripotent stem cells (iPSCs) hold great promise for modeling neurodevelopmental disorders, discovering new precision therapies, and screening for potential risks from environmental toxins1-4. There have been significant advances in the last decade in protocols and commercial media systems developed for differentiation into specific neural cell types5-8. However, there remain significant technical challenges to overcome in their generation, manufacturing and assay workflows. iPSCs are typically differentiated on animal-derived substrates that introduce intrinsic variability and lack control over mechanical stiffness and biochemical composition. This often results in low yields and high variability, which may be more pronounced when generating cellular models of diseases. There is a critical need to develop commercial tools that promote differentiation of iPSCs into mature neural cells in a controlled, efficient, and reproducible fashion and that eliminate animal derived products. The resulting cells, associated cell-based assays and cellular therapeutics will have a transformative impact on neural disease modeling, drug and therapeutic discovery and toxin screening. Our Phase I study identified chemically defined and robust synthetic hydrogels for efficient differentiation of iPSC-derived neural progenitor cells (NPCs) into cortical neurons and subsequent maturation to post-mitotic, functionally mature neurons. The highly innovative aspects of this work are that the substrates are employed as thin hydrogel coatings using our proprietary surface-localized polymerization methods which provides several technical and commercialization advantages. In order to bring these novel substrates to market we propose the following specific aims for our Phase II proposal: Specific Aim 1 will further validate the work that demonstrated our optimized synthetic thin hydrogel coatings support neural differentiation and maturation. Including further functional characterization of cells cultured on the substrates by employing microelectrode array analysis and differential transcriptional analysis to compare cells cultured on the substrate. We will characterize of the physical and mechanical properties of the optimized thin hydrogels and develop methods for coating plates using automated systems. Specific Aim 2 will apply the substrates in a Proof-of-Concept demonstration utilizing the substrates to assess cortical neurons from Major Depressive Disorder patient-derived samples compared with controls. Specific Aim 3 will expand the technology platform by optimizing coating techniques on microcarriers suitable for bioreactor scaling, which is a critical step to demonstrate these substrates are applicable to biomanufacturing applications. This work is significant, as there is a critical need for better tools to optimize yields and reduce variability in the differentiation of iPSCs to defined neural subtypes, support their long-term culture, reduce the time needed to reach functional maturity and eliminate animal-derived products in the workflow.