Microfabrication for Biomedical Research

生物医学研究的微加工

• 批准号: 8933892
• 负责人: Nicole Y Morgan
• 金额: $ 32.78万
• 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/8933892
• 关键词: 3-Dimensional; Adhesives; Aluminum; Area; Biomedical Research; Biomedical Technology; Bioreactors; Brain; CCR; Cell Culture Techniques; Cells; Chemotaxis; Chondroitin Sulfate Proteoglycan; Coculture Techniques; Collaborations; Collagen; Complex; Computer software; Consult; Cultured Cells; Custom; Cytoplasmic Granules; Deposition; Development; Device Designs; Devices; Dimensions; Discipline; Elements; Equipment; Familiarity; Fertilization; Film; Fluorescence; Generations; Glass; Heating; Height; Housing; Human Resources; Hybrids; Hydrogels; Hypoxia; Image; Image Analysis; Immune; Institutes; Institution; Laboratories; Lateral; Light; Measures; Membrane; Metals; Methods; Microdissection; Microfabrication; Microfluidics; Microscopy; Modeling; Modification; Molecular Classification of Tumors; Mus; 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 Drug Abuse; National Institute of Mental Health; Neurons; Oxygen; Pattern; Perfusion; Phase; Play; Polymers; Polymethyl Methacrylate; Presynaptic Terminals; Process; Protocols documentation; Publications; Pump; Relative (related person); Research; Research Personnel; Research Project Grants; Resolution; Resource Sharing; Role; Sepharose; Silicon; Slice; Spatial Distribution; Staging; Staining method; Stains; Structure; Surface; Syringes; System; Techniques; Technology; Testing; Time; Tissue Model; Training; Traumatic Brain Injury; United States National Institutes of Health; Validation; Work; axon growth; axon guidance; base; biological systems; cell type; chemokine; cost; design; egg; experience; fluorescence imaging; in vivo; instrument; instrumentation; interest; matrigel; millimeter; millisecond; nanofabrication; neurite growth; next generation; operation; polycarbonate; pressure; prototype; research study; simulation; submicron; two-photon

Although there has been extensive work at many institutions to develop microfabrication and microfluidic technology for biomedical applications, the technology has yet to move broadly into biomedical research laboratories. Part of the issue is a lack of familiarity on the part of many biomedical researchers, but in addition many potential research projects require capabilities or customization absent from the limited number of commercially available products. In an effort to lower these barriers, we have developed a basic in-house microfabrication facility. 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. This year, we have continued to refine techniques for incorporating track-etched polymer membranes into multilayer structures for co-culture of different cell types, and worked with SPIS, DCB, CIT to incorporate them into perfusion bioreactors for 3D tissue models. 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. 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, and NIBIB. 1) A collaborative effort with LSB, NIAID, to study chemotaxis of primary immune cells in 3-D collagen matrices, for which we have been developing and refining a microfluidic agarose device compatible with high-resolution fluorescence and two-photon imaging. A mixing tee on a separate platform, together with programmable syringe pump flow control, enables the formation of reproducible time-varying spatial gradients. We have also developed finite element models of the original device and used these in combination with quantitative image analysis to assist in characterization and modeling of the 3-D device for different temporal inputs. 2) 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, and more recent efforts with NICHD and CIT have focused on refining the instrumentation and microdissection protocols for a variety of targets and stain localizations, from whole-cell and region-of-interest based captures, to subcellular targets, such as axon terminals in brain slices (in collaboration with NIDA). 3) The continuing development, in collaboration with LCE, NHLBI, of gratings for phase-contrast x-ray imaging, which involves the use of the NIST nanofabrication facilities as well as our own equipment. This year our efforts have focused on publication of imaging results from the current generation of gratings. Preliminary work on the next generation of gratings is underway. 4) In collaboration with MDP, NIDDK, the use of coverslip-mounted, bottomless PDMS microwells to confine eggs in order to study fertilization with fast, high-resolution microscopy. For this application, the wells need to be compatible with existing fluorescence imaging and culture systems, and also to enable capture of the eggs with minimal handling. The addition of microchannels connecting the wells has provided space for the cumulus cells as they separate, enabling the eggs to be gently captured and imaged at the coverslip surface. 5) The development and implementation of a PDMS microfluidic gradient generator for the deposition of chondroitin sulfate proteoglycans on substrates for neural cell culture that we are using, in collaboration with DN, CBPC, NHLBI, to gain better understanding of the role these molecules play in axon growth and guidance. We have successfully used these removable devices for deposition of CSPG gradients over an extended area (1.3 mm x 20 mm) of PLL-coated glass, and have used these patterned substrates in the culture of mouse cerebellar granule neurons. Recent efforts have focused on the development of customized image analysis software to extract relevant neurite growth parameters from statistically meaningful numbers of cultured cells (a few thousand cells per substrate). 6) 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. Current efforts are focused on the design, fabrication, validation and implementation of a higher throughput system that incorporates the membranes into a multiwell plate format. 7) 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. 8) 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. A first generation prototype has been made and tested, and we have begun discussions with researchers at NIST about developing a low-volume, commercially available, certified standard.

尽管许多机构已经开展了大量工作来开发生物医学应用的微加工和微流体技术，但该技术尚未广泛进入生物医学研究实验室。部分问题是许多生物医学研究人员缺乏熟悉度，但此外，许多潜在的研究项目需要有限数量的商用产品所缺乏的功能或定制功能。 为了降低这些障碍，我们开发了基本的内部微加工设施。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。今年，我们继续完善将径迹蚀刻聚合物膜纳入多层结构以共培养不同细胞类型的技术，并与 SPIS、DCB、CIT 合作，将其纳入 3D 组织模型的灌注生物反应器中。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。 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，我们还培训了基础微加工技术的研究人员，包括来自 NEI、NIAID、NHLBI、NCI、NICHD 和 NIBIB 其他实验室的人员。 1) 与 LSB、NIAID 合作，研究 3-D 胶原基质中原代免疫细胞的趋化性，为此我们一直在开发和完善与高分辨率荧光和双光子成像兼容的微流控琼脂糖装置。独立平台上的混合三通与可编程注射泵流量控制一起，能够形成可重复的时变空间梯度。我们还开发了原始设备的有限元模型，并将其与定量图像分析结合使用，以帮助针对不同时间输入对 3D 设备进行表征和建模。 2）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。这项工作是作为 NIMH、NCI 和 NICHD 研究人员的研究所间主任挑战项目的一部分开始的，最近与 NICHD 和 CIT 的合作重点是完善各种目标和染色定位的仪器和显微解剖协议，从基于全细胞和感兴趣区域的捕获，到亚细胞目标，例如脑切片中的轴突末端（与 NIDA 合作）。 3) 与 LCE、NHLBI 合作，持续开发用于相衬 X 射线成像的光栅，其中涉及使用 NIST 纳米加工设施以及我们自己的设备。今年我们的工作重点是发布当前一代光栅的成像结果。 下一代光栅的初步工作正在进行中。 4) 与 MDP、NIDDK 合作，使用盖玻片安装的无底 PDMS 微孔限制卵子，以便通过快速、高分辨率显微镜研究受精。 对于此应用，孔需要与现有的荧光成像和培养系统兼容，并且能够以最少的处理捕获卵。 添加连接孔的微通道为卵丘细胞分离时提供了空间，使卵能够在盖玻片表面被轻轻捕获和成像。 5) 与 DN、CBPC、NHLBI 合作开发和实施 PDMS 微流体梯度发生器，用于将硫酸软骨素蛋白多糖沉积在我们正在使用的神经细胞培养基质上，以更好地了解这些分子在轴突生长和引导中发挥的作用。 我们已成功使用这些可移动装置在 PLL 涂层玻璃的扩展区域 (1.3 mm x 20 mm) 上沉积 CSPG 梯度，并在小鼠小脑颗粒神经元的培养中使用这些图案化基质。 最近的工作重点是开发定制的图像分析软件，以从具有统计意义的培养细胞数量（每个基质数千个细胞）中提取相关的神经突生长参数。 6) 与 LCB、CCR、NCI 和 SPIS、CIT 合作，设计、制造、建模和使用透氧膜，该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。 目前的工作重点是设计、制造、验证和实施更高通量的系统，该系统将膜整合到多孔板中。 7) 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。 8) 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。 第一代原型机已经制造出来并进行了测试，我们已经开始与 NIST 的研究人员讨论开发小批量、商用的认证标准。