Silk Fibers-Assisted 3D System for Large-Scale Culture of Human Urine-Derived Stem Cells Suitable for Late Mitotoxicity Testing

用于大规模培养人尿液干细胞的丝纤维辅助 3D 系统，适用于晚期有丝分裂毒性测试

• 批准号: 10417268
• 负责人: YUANYUAN no ZHANG
• 金额: $ 7.75万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-03 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10417268
• 关键词: 3-Dimensional; Address; Alginates; Anti-HIV Agents; Antiviral Agents; Apoptosis; Biocompatible Materials; Biological; Biological Assay; Biopolymers; CASP3 gene; Caliber; Cell Adhesion; Cell Density; Cell Survival; Cells; Cellular Assay; Characteristics; Chitosan; Collagen; Complex; Consumption; Diffusion; Dose; Evaluation; Excision; Fiber; Fibrinogen; Fibroins; Gelatin; Geometry; Glutathione; Goals; Growth; HIV; Human; Hyaluronic Acid; In Vitro; Infiltration; Longevity; Metabolism; Microspheres; Mitochondria; Mitochondrial DNA; Modeling; Nucleosides; Nutrient; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen; Pharmaceutical Preparations; Ploidies; Porosity; Production; Reactive Nitrogen Species; Regenerative Medicine; Reverse Transcriptase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Scanning Electron Microscopy; Seeds; Silk; Site; Spiders; Structure; System; Testing; Therapeutic; Thick; Time; Tissue Engineering; Toxic effect; Toxicity Tests; Toxicology; Urine; Zalcitabine; antiretroviral therapy; biomaterial compatibility; cell growth; cost; cost effective; cost effectiveness; drug testing; flexibility; histological stains; human model; human stem cells; large scale production; nanosized; nephrotoxicity; new technology; novel; novel strategies; process optimization; scale up; stem cells; three dimensional cell culture; three dimensional structure; tissue repair; wasting

PROJECT SUMMARY In vitro 3-D cultures are a promising way to assess delayed mitochondrial toxicity (MtT) caused by antiretroviral therapy. Standard MtT testing requires large numbers of cells for serial assessments. However, there are no 3D platforms available for mass production of primary human cells for long-term culture. In currently accessible 3D spheroid models, ability to control the geometry of the structures is limited, which does not support reliable and scaled-up cell production. Furthermore, creating large numbers of primary human cells in 3D spheroid systems is time-consuming, labor-intensive, and expensive for MtT assessment. Thus, a novel approach to address each of these issues is urgently demanded. Cells seeding in porous biomaterials used for tissue repair might provide a solution to these current barriers to progress. Various biomaterials with porous microstructure have been used in 3D culture for tissue engineering, such as natural materials (spider silk, chitosan, microspheres made from collagen, gelatin, fibrinogen, hyaluronic acid, alginate) and synthetic materials (PGA, PLGA, PLLA). However, silk fibroin as a natural biopolymer possesses outstanding characteristics with biocompatibility, biodegradability, durability, and flexibility for regenerative medicine. Fiber biomatrices with high porosity have interconnected pore networks, which provide anchoring sites to hold the cells together and facilitate nutrient and oxygen diffusion and waste removal for efficient cell growth in long-term 3D culture. Our recent study demonstrated that an in vitro 3D spheroid model of human urine-derived stem cells (USCs) can be used for nephrotoxicity assays. To extend our ongoing study and bridge the gap between current 3D spheroid cultures and MtT testing, we will explore a novel technology to provide large numbers of primary human cells. Thus, the overall goal of this R03 study is to develop a strategy for large-scale production of human USC in 3D culture suitable for late MtT testing. We hypothesize that an in vitro 3D culture system with porous silk fiber matrix (SFM) will support adequate USCs (≥ 3 x 106/sample) for long-term growth (≥ 8 weeks), with stable mitochondrial copy number and function, to eventually be used in late MtT testing. To test this hypothesis, we propose these aims: Aim 1. Optimize experimental strategies for 3D culture system of silk fiber network for large-scale production of human USCs; Aim 2. Validate a 3D culture system of USC-SFM and compare it to 3D spheroid culture. This will be the first study to test a silk fiber network with human USCs for MtT testing. We will use this newly developed 3D system in our existing study (3D Culture Systems of USCs for NTRI-Induced Mitotoxicity; R21 AI152832). We expect that 3D culture of USC-SFM as a less labor- intensive, more efficient, and cost-effective approach will be able to maintain large amounts of human stem cells within silk fibers with stable mitochondrial quantities and function over time. This proposed 3D cultures of USC-SFM, in contrast to 3D spheroids, may yield a reliable platform suitable for late MtT assays in antiviral drug testing.

项目摘要 体外3-D培养物是评估抗逆转录病毒引起的延迟线粒体毒性（MTT）的有前途的方法 治疗。标准MTT测试需要大量的细胞进行连续评估。但是，没有 3D平台可用于大规模生产原代人类细胞，用于长期培养。在当前可访问中 3D球体模型，控制结构几何形状的能力有限，这不支持可靠 并扩大细胞的生产。此外，在3D球体中创建大量原代人细胞 系统耗时，劳动密集型，用于MTT评估昂贵。那是一种新颖的方法 迫切需要解决这些问题。细胞在用于组织修复的多孔生物材料中播种 可能可以解决这些当前进步障碍的方法。多孔微观结构的各种生物材料 已在3D培养物中用于组织工程，例如天然材料（蜘蛛丝，壳聚糖， 微球由胶原蛋白，明胶，纤维蛋白原，透明质酸，藻酸盐）和合成材料（PGA，PGA， PLGA，PLLA）。然而，丝绸纤维作为天然生物聚合物具有出色的特征 再生医学的生物相容性，生物降解性，耐用性和灵活性。带有纤维生物组合 高孔隙率具有相互关联的孔网络，该网络提供锚定位点以将细胞固定在一起，并且 在长期3D培养物中，促进营养和氧扩散和废物清除，以有效地细胞生长。我们的 最近的研究表明，人尿液衍生的干细胞（USC）的体外3D球体模型可以 用于肾毒性评估。扩展我们正在进行的研究并弥合当前3D之间的差距 球体培养物和MTT测试，我们将探索一种新型技术，以提供大量主要的主要技术 人类细胞。这是这项R03研究的总体目标是制定一种大规模生产的策略 3D文化中的人类USC适用于晚期MTT测试。我们假设一个体外3D培养系统具有 多孔丝纤维基质（SFM）将支持足够的USC（≥3x 106/样品）长期生长（≥8周）， 使用稳定的线粒体拷贝数和功能，最终将用于MTT晚期测试。测试这个 假设，我们提出了以下目的：目标1。优化丝绸纤维3D培养系统的实验策略 大规模生产人类USC的网络；目标2。验证USC-SFM的3D培养系统和 将其与3D球体培养物进行比较。这将是第一项与人类USC一起测试丝绸纤维网络的研究 MTT测试。我们将在现有研究中使用这个新开发的3D系统（USC的3D培养系统 用于NTRI诱导的有丝毒性； R21 AI152832）。我们期望USC-SFM的3D文化是一种较少的劳动力 - 密集，更高效，更具成本效益的方法将能够维持大量的人类茎 随着时间的推移，线粒体量和功能稳定的丝纤维中的细胞。这个拟议的3D文化 与3D球体相比，USC-SFM可能会产生一个可靠的平台 药物测试。