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.

项目概要用于癌症研究和药物发现的模型细胞目前使用低温方法储存，需要强大的冰箱和液氮。冷冻细胞需要定期维护，能源成本高，占用大量空间，并且如果冷却设备发生故障，性能会迅速下降。努力开发冻干方案模型细胞系已经存在了几十年，然而，将保护剂加载到有核细胞中已经事实证明具有挑战性。 DesiCorp 开发了一种新颖且创新的细胞装载装置，称为高流量声流体加载（HFAL），可以将保护性渗透剂、蛋白质和核苷酸传递到真核生物中细胞。与微流体加载技术不同，HFAL 使用大通道尺寸，可充分更大体积的细胞悬浮液立即流过它。将细胞与阳离子脂质包被混合与超声波共振的微泡，引起空化。这些空化引起的微射流将周围介质中的化合物物理注射到悬浮细胞中，然后修复任何膜损伤并将不渗透膜的化合物密封在细胞内。这项技术有以前被用来给人类红细胞装载保护性化合物，使它们能够冻干，室温保存，然后再水化，细胞回收率超过 90%。一项显着的创新 HFAL 平台的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用于冻干过程中真核细胞稳定的 HFAL 平台。该提案的具体目标是 1) 开发完整的无菌声流加载系统，加载 HepG2 和 HEK 293 细胞超过 60% 细胞回收率，将使用台盼蓝排除法进行测量； 2) 实现化合物加载到HepG2中和 HEK293 足以使其在干燥状态下稳定，这将使用酶法和比色分析； 3) 冻干 HepG2 和 HEK293 细胞，将其储存在环境温度下温度，并以足够高的回收率再水化它们以进行培养，并且它们的细胞器结构应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。这第一阶段提案成功的主要标准是制造完全集成的声流体负载成功地向真核细胞装载保护剂的平台，使它们能够被冻干、储存、再水化并培养。与低温保存的细胞相比，冻干细胞储液具有多种优势，例如缺乏持续的维护和能源成本。大型存储设备，例如冰箱或液体氮杜瓦瓶对于冻干产品来说是不必要的，因为它们具有热稳定性。如果这个提议工作成功，然后细胞库存可以存放在抽屉、活页夹中，甚至可以与详细说明的笔记本一起存放它们的创建、修改以及与资助或出版物相关的数据。