Developing a High-Flow Acoustofluidic Loading Platform for Research Cell Stabilization in the Anhydrous State

开发用于研究细胞在无水状态下稳定的高流量声流体加载平台

• 批准号: 10603701
• 负责人: Brett R Janis
• 金额: $ 28.19万
• 依托单位: DESICORP, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10603701
• 关键词: 3D Print; Address; Animals; Antineoplastic Agents; Area; Asphyxia; Biological Assay; Cancer Model; Cell Count; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell Survival; Cell Volumes; Cell model; Cells; Cellular Structures; Complex; Cryoprotective Agents; Culture Media; Dangerousness; Data; Desiccation; Devices; Dimensions; Dryness; Effectiveness; Equipment; Erythrocytes; Eukaryotic Cell; Exclusion; Exposure to; Failure; Fluorescence Microscopy; Freeze Drying; Freezing; Future; Gases; Goals; Grant; Hazardous Substances; HepG2; Hour; Human; Hydration status; Induction of Apoptosis; Lipids; Liquid substance; Longevity; Maintenance; Measures; Medical; Medical Research; Membrane; Methods; Microbubbles; Microfluidics; Microscopic; Mitochondria; Modification; Mothers; Nitrogen; Nucleotides; Organelles; Phase; Plants; Process; Protective Agents; Proteins; Protocols documentation; Publications; Qualifying; Records; Recovery; Reproducibility; Research; Research Personnel; Risk; Sampling; Sterility; Structure; Suspensions; System; Techniques; Technology; Temperature; Toxic effect; Treatment Efficacy; Trehalose; Trypan Blue; Ultrasonic wave; Vendor; Water; Work; anticancer research; cancer cell; cancer therapy; cold temperature; cost; cryogenics; daughter cell; design; drug discovery; drug efficacy; efficacy study; evaporation; experience; experimental study; fabrication; innovation; novel; novel therapeutics; preservation; pressure; prototype; repaired; safety study; seal; sonoporation; success; tool; ultrasound

Project Summary Model cells for cancer research and drug discovery are currently stored using cryogenic methods that necessitate powerful freezers and liquid nitrogen. Frozen cells require regular upkeep, have high energy costs, take up a lot of space, and rapidly degrade if cooling devices fail. Efforts to develop a lyophilization protocol for model cell lines have been ongoing for decades, however, loading protective agents into nucleated cells has proven challenging. DesiCorp has developed a novel and innovative cell loading device, termed the high flow acoustofluidic loading (HFAL) that can deliver protective osmolytes, proteins, and nucleotides into eukaryotic cells. Unlike microfluidic loading techniques, HFAL uses large channel dimensions that allow substantially larger volumes of cell suspension to flow through it at once. The cells are mixed with cationic lipid-coated microbubbles that resonate with ultrasound waves, inducing cavitation. These cavitation-induced microjets physically inject compounds from the surrounding media into the cells in suspension, which then repair any membrane damage and seal membrane-impermeant compounds inside of the cell. This technology has previously been utilized to load human erythrocytes with protective compounds, allowing them to be lyophilized, stored at ambient temperatures, then rehydrated with over 90% cell recovery. A notable innovation of the HFAL platform is the ability to load cells at high rates, exceeding 500 billion erythrocytes per minute. However, the unique challenges of protecting organelles requires additional work to design and validate the HFAL platform for eukaryotic cell stabilization during lyophilization. The specific aims for this proposal are 1) to develop a complete, sterile acoustofluidic loading system that loads HepG2 and HEK 293 cells with over 60% cell recovery, which will be measured using trypan-blue exclusion; 2) to achieve compound loading into HepG2 and HEK293 that is sufficient for their stabilization in the desiccated state, which will be measured using enzymatic and colorimetric assays; and 3) to lyophilize HepG2 and HEK293 cells, store them at ambient temperature, and rehydrate them with high enough recovery for culturing and their organelle structure should resemble that of frozen and thawed model cells, which will be measured using fluorescence microscopy. The main criterion for success of this phase I proposal is the fabrication of a fully integrated acoustofluidic loading platform that successfully loads eukaryotic cells with protective agents that allow them to be lyophilized, stored, rehydrated, and cultured. Lyophilized cell stocks have several advantages over cryogenically preserved cells, such as the lack of ongoing maintenance and energy costs. Large storage devices, such as freezers or liquid nitrogen dewars, are unnecessary for lyophilized products because they are thermally stable. If this proposed work is successful, then cell stocks could be stored in drawers, binders, or even with the notebooks detailing their creation, modification, and data associated with grants or publications.

项目摘要 用于癌症研究和药物发现的模型细胞目前是使用低温方法储存的，这种方法 需要强大的冰柜和液氮。冷冻细胞需要定期维护，能源成本很高， 会占用大量空间，如果冷却设备出现故障，会迅速降级。努力开发一种用于冷冻干燥的方案 模型细胞系已经持续了几十年，然而，将保护剂加载到有核细胞中已经 事实证明是具有挑战性的。DesiCorp开发了一种新颖而创新的细胞加载设备，称为高流量 声流控负载(HFAL)可将保护性渗透压、蛋白质和核苷酸输送到真核生物中 细胞。与微流控加载技术不同，HFAL使用较大的通道尺寸，基本上允许 更大体积的细胞悬浮液一次通过它。细胞与阳离子脂膜混合。 与超声波共振的微气泡，导致空化。这些由空化引起的微喷流 将周围介质中的化合物以悬浮液的形式注入细胞，然后细胞修复任何 膜损伤和密封膜--细胞内不必要的化合物。这项技术已经 以前被用来向人类红细胞装载保护性化合物，使它们能够 冷冻干燥，常温储存，然后复水，细胞回收率超过90%。一项值得注意的创新 HFAL平台的最大特点是能够高速加载细胞，每分钟超过5000亿个红细胞。 然而，保护细胞器的独特挑战需要额外的工作来设计和验证 冷冻干燥过程中真核细胞稳定的HFAL平台。这项建议的具体目标是1) 开发一套完整的无菌声流加载系统，使HepG2和HEK 293细胞的载药量超过60% 细胞回收率，将用台盼蓝排斥法测量；2)实现复合负载到HepG2 和HEK293，这足以使它们在干燥状态下稳定，这将使用以下方法测量 酶法和比色法；3)冻干HepG2和HEK293细胞，在常温下保存 温度，并以足够高的回收率进行复水培养，它们的细胞器结构应该 类似于冷冻和解冻的模型细胞，将使用荧光显微镜进行测量。这个 第一阶段方案成功的主要标准是制造完全集成的声流加载 一个平台，成功地将真核细胞装载到保护剂中，使它们能够被冷冻干燥，储存， 补充水分，培养。与冷冻保存的细胞相比，冷冻干燥的细胞库存有几个优势， 比如缺乏持续的维护和能源成本。大型存储设备，如冰柜或液体 对于冻干产品来说，氮气杜瓦是不必要的，因为它们是热稳定的。如果这项提议 如果工作成功，那么电池库存就可以储存在抽屉、活页夹，甚至可以与笔记本一起详细说明 它们的创建、修改以及与赠款或出版物相关的数据。