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: 10326588
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10326588
• 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.

项目总结