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Devices for Biological Systems: On-Chip Horizontal Gene Transfer and 3D-Printed Microfluidic Applications

机译:生物系统设备:片内水平基因转移和3D打印微流体应用

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摘要

This thesis covers the development of three devices for investigating biological systems: A 3D-printed oxygen control insert for a 24-well plate, an open source 3D-printable adjustable micropipette, and a droplet encapsulation device to isolate and investigate genetic transfer between pairs of S. pneumoniae..;Microfluidic platforms have been developed to provide more physiologically relevant oxygen environments for cell studies. Typically, building these platforms involves manual fabrication of microfluidic chips, a process that is time consuming, has a high failure rate and only produces limited geometries. Recently 3D printing has emerged as a method for directly printing complete microfluidic devices, although printing materials have been limited to oxygen-impermeable materials. We demonstrate the addition of gas permeable PDMS (Polydimethylsiloxane) membranes to a 3D-printed microfluidic devices as a means to enable oxygen control cell culture studies. The incorporation of a 3D-printed device and gas-permeable membranes was demonstrated on a 24-well oxygen control device for standard multiwell plates. The direct printing allows integrated distribution channels and device geometries not possible with traditional planar lithography. With this device, four different oxygen conditions were able to be controlled, and six wells were maintained under each oxygen condition. We demonstrate enhanced transcription of the gene VEGFA (vascular endothelial growth factor A) with decreasing oxygen levels in human lung adenocarcinoma cells. This is the first 3D-printed device that can be functionalized to control oxygen in cell culture.;Scientific communities are drawn to the open source model as an increasingly utilitarian method to produce and share work. Initially used as a means to develop freely available software, the open source model has been applied to hardware including scientific tools. Increasing convenience of 3D printing has fueled the proliferation of open labware projects aiming to develop and share designs for scientific tools that can be produced in-house as cheap alternatives to commercial products. We present our design of a micropipette that is assembled from 3D-printable parts and some hardware that works by actuating a disposable syringe to a user adjustable limit. Graduations on the syringe are used to accurately adjust the set point to the desired volume. Our open design printed micropipette is assessed in comparison to a commercial pipette and meets ISO 8655 standards for accuracy and precision.;In genetic transformation (GT), cells take up and incorporate DNA from the environment. GT allows rapid alterations of the genome resulting in persistent infections as polyclonal S. pneumoniae biofilms share genes to overcome immune response and transfer antibiotic resistance. In vitro, GT is studied in suspensions of billions of cells and can be used to assay transfer of traits between strains, such as antibiotic resistance and virulence, but does not reproduce the rapid and extensive of transfer observed in vivo. A flow focusing droplet generating device is demonstrated to encapsulate S. pneumoniae in droplets of M9 minimal medium. The addition of a coverslip layer to the microfluidic incubation chamber prevents evaporation of droplets which allows imaging of encapsulated cells on-chip that self organize into a monolayer. Imaging of cells in droplets confirms an occupancy distribution average of 2.5 cells per droplet and a mode of 2 cells per droplet.
机译:本论文涵盖了三种用于研究生物系统的设备的开发:用于24孔板的3D打印的氧气控制插件,开放源代码的3D打印的可调微量移液器以及用于隔离和研究成对的两对之间遗传转移的液滴封装设备。肺炎链球菌..微流体平台已经开发出来,可为细胞研究提供更多与生理相关的氧气环境。通常,构建这些平台涉及人工制造微流体芯片,该过程耗时,故障率高且仅产生有限的几何形状。尽管将打印材料限于不透氧的材料,但近来3D打印已经成为直接打印完整的微流体设备的一种方法。我们演示了向3D打印的微流控设备中添加透气的PDMS(聚二甲基硅氧烷)膜,作为使氧控制细胞培养研究成为一种手段的方法。在用于标准多孔板的24孔氧气控制设备上证明了3D打印设备和透气膜的结合。直接印刷允许使用传统的平面光刻无法实现的集成分配通道和设备几何形状。使用该设备,可以控制四种不同的氧气条件,并且在每种氧气条件下都可以维持六口井。我们证明了人类肺腺癌细胞中氧水平的降低,增强了基因VEGFA(血管内皮生长因子A)的转录。这是第一台可被功能化以控制细胞培养中氧气的3D打印设备。科学界被吸引到开源模型中,作为一种越来越实用的方法来生产和共享工作。开源模型最初被用作开发免费软件的一种手段,现已应用于包括科学工具在内的硬件。 3D打印便利性的提高推动了开放实验室软件项目的泛滥,这些项目旨在开发和共享科学工具的设计,这些工具可以在内部生产,作为商业产品的廉价替代品。我们介绍了微量移液器的设计,该移液器由3D可打印部件和一些硬件组成,这些硬件通过将一次性注射器驱动到用户可调节的极限来工作。注射器上的刻度用于将设定点准确地调整到所需的体积。我们的开放式设计印刷微量移液器经过了与商用移液器的比较评估,并符合ISO 8655标准的准确性和精密度。;在遗传转化(GT)中,细胞吸收并吸收了环境中的DNA。由于多克隆肺炎链球菌生物膜共享基因以克服免疫应答并转移抗生素抗性,因此GT可以快速改变基因组,从而导致持续感染。在体外,已在数十亿个细胞的悬浮液中研究了GT,可将其用于分析菌株之间的性状转移,例如抗生素抗性和毒力,但不能重现体内观察到的快速而广泛的转移。流动聚焦液滴产生装置被证明可将肺炎链球菌包裹在M9基本培养基的液滴中。在微流控培养箱中添加盖玻片层可防止液滴蒸发,从而使芯片上自组织成单层的封装细胞成像。液滴中细胞的成像确认平均每个液滴2.5个细胞的占用分布,每个液滴2个细胞的模式。

著录项

  • 作者

    Brennan, Martin D.;

  • 作者单位

    University of Illinois at Chicago.;

  • 授予单位 University of Illinois at Chicago.;
  • 学科 Biomedical engineering.;Cellular biology.;Microbiology.
  • 学位 Ph.D.
  • 年度 2017
  • 页码 171 p.
  • 总页数 171
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 遥感技术;
  • 关键词

  • 入库时间 2022-08-17 11:38:55

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