Microfabrication for Biomedical Research

生物医学研究的微加工

• 批准号: 10008866
• 负责人: Nicole Y Morgan
• 金额: $ 47.04万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10008866
• 关键词: 3-Dimensional; Adhesives; Aluminum; Axon; Biochemical; Biomedical Research; Bioreactors; CCR; Cells; Chemotaxis; Collaborations; Collagen; Complement; Complex; Consult; Custom; Decision Making; Deposition; Development; Device Designs; Device or Instrument Development; Devices; Dimensions; Discipline; Elements; Equipment; Exposure to; Film; Fluorescence; Fluorescence Resonance Energy Transfer; Glass; Harvest; Human Resources; Hybrids; Hydrogels; Hypoxia; Image; Immune; Institutes; Intramural Research Program; Knowledge; Laboratories; Lateral; Light; Liquid substance; Measurement; Measures; Membrane; Metals; Methods; Microdissection; Microfabrication; Microfluidic Microchips; Microfluidics; Modeling; Modification; National Heart, Lung, and Blood Institute; National Institute of Allergy and Infectious Disease; National Institute of Biomedical Imaging and Bioengineering; National Institute of Child Health and Human Development; National Institute of Diabetes and Digestive and Kidney Diseases; National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; National Institute on Deafness and Other Communication Disorders; Neoplasm Metastasis; Neurons; Operating System; Oxygen; Pattern; Polymers; Polymethyl Methacrylate; Printing; Process; Protocols documentation; Pump; Reproducibility; Research; Research Personnel; Resolution; Resource Sharing; Sepharose; Silicon; Spatial Distribution; Stains; Standardization; Structure; Surface; Syringes; Techniques; Thinness; Time; Tracer; Training; Traumatic Brain Injury; UV Radiation Exposure; United States National Institutes of Health; Validation; Variant; Work; Zebrafish; base; biological systems; cell motility; chemokine; cost; design; experience; experimental study; high resolution imaging; in vitro Model; in vivo; instrument; instrumentation; interest; matrigel; millisecond; operation; polycarbonate; pressure; programs; prototype; simulation; three dimensional cell culture; transmission process; two-photon

The use of microfabricated and microfluidic structures in biomedical research has been rapidly expanding in recent years, but most research applications of these devices require customization, and new applications typically require several design iterations for troubleshooting. In an effort to bring the necessary technical capabilities and knowledge to do so into biomedical laboratories, we have developed a basic in-house microfabrication facility accessible to researchers across the intramural program. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. We are able to reliably pattern single- and multi-layer template features with lateral dimensions of less than 2 microns, and with heights ranging from a few microns to a few hundred microns. We have developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels, including techniques for making and manipulating thin (<200 micrometer) PDMS layers for use in multilayer devices and bottomless structures. We can also perform surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass, and have developed techniques for connecting devices to flow-control instruments such as syringe pumps and pressure controllers. We also have protocols for generating micropatterns of biomolecules on surfaces using microcontact printing or selective UV exposure, as well as protocols for generating monodisperse droplets and hydrogel beads. In addition, we have the ability to deposit and pattern metal layers, as well as a heated hydraulic press for hot-embossing thermoplastics, including PMMA, polycarbonate, and COC. We have also used a programmable razor cutter and pressure-sensitive adhesive to directly make thin film structures with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This is a low-cost and convenient method for several applications, including the ready fabrication of flow cells with two glass walls, or for fluidic confinement over already-functionalized surfaces. Finally, we continue to work on finite element modeling of transport in microfabricated structures, developing models for oxygen delivery in a bioreactor with micropillars and for chemokine concentration in a microfluidic hydrogel device. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NEI, NIAID, NHLBI, NCI, NICHD, NINDS, NIDCD, NIDDK, and NIBIB. 1) A collaborative effort with LSB, NIAID, to study chemotaxis, currently focusing on the decision-making of single cells exposed to competing solution gradients. Earlier efforts with this group looked at chemotaxis of primary immune cells in 3-D collagen matrices, using a microfluidic agarose device to generate reproducible time-varying spatial gradients on a platform compatible with high-resolution fluorescence and two-photon imaging. 2) The design, fabrication, modeling, and use of an oxygen-transmissive membrane, patterned with a micropillar array to deliver oxygen to three-dimensional cell culture volumes with in vivo-like spatial distribution, in collaboration with LCB, CCR, NCI and SPIS, CIT. Because the pillar spacing is approximately equal to typical intercapillary distances (200 microns), cells in a Matrigel layer surrounding the pillars can be maintained under hypoxic conditions in an extended 3D volume. We have recently started working with researchers in LAMB, NHLBI to employ their recently developed FLIM-FRET probe for intracellular oxygen to directly image spatial and cellular variations in oxygenation in these structures. 3) The fabrication and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, light-activated microdissection. This work was begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD; more recent efforts with NCI and CIT have focused on refining the instrumentation and developing standardized protocols for a variety of targets and stain localizations. 4) The use of a microfluidic channel together with custom instrumentation developed in LCMB, NICHD in order to separate the effects of pressure and rapid (msec) shear transients on cultured neural cells, in order to gain a better understanding of the cellular mechanisms of traumatic brain injury. Our group has fabricated the microchannels, performed modelling aimed at understanding the shear transients experienced by the cells as a function of the bulk flow, and consulted extensively on how best to interface the microchannel with the existing instrumentation. 5) At the instigation of LCIMB, NIBIB, the collaborative design and fabrication of an aluminum-patterned window for use in calibrating the measured settling distances in analytical ultracentrifuges. NIST researchers are currently working on the development and validation of a low-volume, commercially available, certified standard, based on the prototype windows made here. 6) In collaboration with LCB, NCI, development of an in vitro model to complement measurements on cell migration in zebrafish to advance understanding of the mechanisms for cell migration and metastasis. 7) In collaboration with researchers in NINDS and NICHD, the development of devices to enable selective harvesting of axons from cultured neurons for biochemical analysis, in order to readily be able to generate enough material for downstream measurements. 8) In collaboration with researchers from the LIG, NIAID, the development of PDMS/glass microfluidic devices for studying chemotaxis. The first device design enables measurements of chemotaxis in 2D chambers on a platform compatible with high-resolution imaging; the generated gradients can be directly measured with a fluorescent tracer, and the system operates without pumps.

近年来，微加工和微流体结构在生物医学研究中的使用迅速扩大，但这些设备的大多数研究应用都需要定制，并且新应用通常需要多次设计迭代来进行故障排除。为了将必要的技术能力和知识带入生物医学实验室，我们开发了一个基本的内部微加工设施，供整个校内项目的研究人员使用。尽管分辨率、器件良率和复杂性略低于专用洁净室所能达到的水平，但它们仍然足以进行许多细胞实验。此外，仪器复杂性、制造成本和周转时间都大大降低，从而能够根据需要快速循环设计参数。 我们能够可靠地图案化单层和多层模板特征，横向尺寸小于 2 微米，高度范围从几微米到几百微米。我们开发了使用这些模板生成微结构 PDMS、琼脂糖和 PEGDA 水凝胶的协议，包括制作和操作薄（<200 微米）PDMS 层以用于多层器件和无底结构的技术。我们还可以对 PDMS 和其他聚合物进行表面改性，包括将 PDMS 与玻璃不可逆粘合，并开发了将设备连接到注射泵和压力控制器等流量控制仪器的技术。我们还有使用微接触印刷或选择性紫外线曝光在表面上生成生物分子微图案的方案，以及生成单分散液滴和水凝胶珠的方案。 此外，我们还能够沉积和图案化金属层，以及用于热压印热塑性塑料（包括 PMMA、聚碳酸酯和 COC）的加热液压机。我们还使用可编程剃须刀和压敏粘合剂直接制造高度范围从25微米到几百微米、横向尺寸为亚毫米的薄膜结构。这是一种低成本且方便的方法，适用于多种应用，包括准备制造具有两个玻璃壁的流通池，或用于在已功能化的表面上进行流体限制。 最后，我们继续致力于微制造结构中运输的有限元建模，开发带有微柱的生物反应器中的氧气输送和微流体水凝胶装置中的趋化因子浓度模型。 这些功能已在许多项目中得到应用，代表了广泛的利益和机构。除了下面讨论的代表性项目和其他仍处于早期阶段的项目外，我们还对基础微加工技术的研究人员进行了培训，包括来自 NEI、NIAID、NHLBI、NCI、NICHD、NINDS、NIDCD、NIDDK 和 NIBIB 其他实验室的人员。 1) 与 LSB、NIAID 合作研究趋化性，目前重点关注暴露于竞争溶液梯度的单细胞的决策。 该小组的早期工作着眼于 3-D 胶原基质中初级免疫细胞的趋化性，使用微流体琼脂糖装置在与高分辨率荧光和双光子成像兼容的平台上生成可重复的时变空间梯度。 2) 与 LCB、CCR、NCI 和 SPIS、CIT 合作，设计、制造、建模和使用透氧膜，采用微柱阵列图案，将氧气输送到具有体内空间分布的三维细胞培养体积。由于柱间距约等于典型毛细管间距离（200 微米），因此柱周围基质胶层中的细胞可以在缺氧条件下维持在扩展的 3D 体积中。我们最近开始与 LAMB、NHLBI 的研究人员合作，采用他们最近开发的细胞内氧 FLIM-FRET 探针，直接对这些结构中氧合的空间和细胞变化进行成像。 3）通过旋涂制成的混合聚合物薄膜的制造和表征，用于独立于操作员的高分辨率光激活显微切割。这项工作是由 NIMH、NCI 和 NICHD 的研究人员参与的研究所间主任挑战项目的一部分；最近与 NCI 和 CIT 的合作重点是改进仪器并为各种目标和染色定位开发标准化协议。 4) 使用微流体通道以及 LCMB、NICHD 开发的定制仪器来分离压力和快速（毫秒）剪切瞬变对培养神经细胞的影响，以便更好地了解创伤性脑损伤的细胞机制。我们的团队制作了微通道，进行了建模，旨在了解细胞所经历的剪切瞬变作为总体流量的函数，并就如何最好地将微通道与现有仪器连接进行了广泛咨询。 5) 在 LCIMB、NIBIB 的推动下，合作设计和制造了铝制图案窗口，用于校准分析超速离心机中测量的沉降距离。 NIST 研究人员目前正在基于此处制造的原型窗户开发和验证小批量、商用的认证标准。 6) 与 LCB、NCI 合作，开发体外模型来补充斑马鱼细胞迁移的测量，以促进对细胞迁移和转移机制的理解。 7) 与 NINDS 和 NICHD 的研究人员合作，开发了能够从培养的神经元中选择性收获轴突进行生化分析的设备，以便能够轻松生成足够的材料用于下游测量。 8) 与 LIG、NIAID 的研究人员合作，开发用于研究趋化性的 PDMS/玻璃微流体装置。 第一个设备设计能够在与高分辨率成像兼容的平台上测量二维​​室中的趋化性；产生的梯度可以用荧光示踪剂直接测量，并且系统无需泵即可运行。