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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要目前，使用低温方法来存储癌症研究和药物发现的模型细胞需要强大的冰柜和液氮。冷冻细胞需要定期保养，具有高能源成本，占用很多空间，如果冷却设备失败，则会迅速降级。为制定冻干协议的努力但是，模型细胞系一直在进行数十年，但是，将保护剂加载到成核细胞中已有被证明具有挑战性。 Desicorp开发了一种新颖而创新的电池加载装置，称为高流量可将保护性渗透剂，蛋白质和核苷酸递送到真核生物中细胞。与微流体负载技术不同，HFAL使用大通道尺寸，可以实质上大量的细胞悬浮液立即流过它。将细胞与阳离子脂质涂层混合与超声波共鸣的微泡，引起空化。这些空化引起的微射流从周围培养基物理注入化合物中的悬浮液中的细胞，然后修复任何膜损伤和密封膜覆盖化合物在细胞内部。这项技术有以前被用来用保护性化合物加载人类红细胞，使其成为冻干，储存在环境温度下，然后以超过90％的细胞回收率再水化。一项著名的创新 HFAL平台的能力是以高速率加载电池，每分钟超过5000亿个红细胞。但是，保护细胞器的独特挑战需要额外的工作来设计和验证冻干期间真核细胞稳定的HFAL平台。该建议的具体目的是1）开发一个完整的无菌浮力载荷系统，该系统加载HEPG2和HEK 293个细胞，超过60％细胞恢复，将使用锥虫蓝色排除进行测量； 2）将复合加载到HEPG2中和HEK293足以在干燥状态下稳定，这将使用酶和比色测定； 3）要冻干hepg2和hek293细胞，将它们存储在环境中温度，并通过足够高的恢复以培养和细胞器结构来补充它们类似于冷冻和融化的模型细胞，该模型细胞将使用荧光显微镜进行测量。这该阶段I提案成功的主要标准是制造完全集成的Acoustofluidic负载平台成功地加载了具有保护剂的真核细胞，使它们能够被冻干，存储，补充并培养。冻干的细胞库存比紧急保存的细胞具有多个优点，例如缺乏持续的维护和能源成本。大型存储设备，例如冰柜或液体氮露水是冻干产品的不必要的，因为它们是热稳定的。如果提出了工作成功，然后可以将细胞库存存储在抽屉，粘合剂中，甚至用笔记本上的细节存储它们的创建，修改和与赠款或出版物相关的数据。