HEAL: 3D Bioprinted Tissue Models

HEAL：3D 生物打印组织模型

• 批准号: 10259367
• 负责人: Marc Ferrer-Alegre
• 金额: $ 128.78万
• 依托单位: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
• 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/10259367
• 关键词: 3-Dimensional; 3D Print; Action Potentials; Address; Afferent Neurons; Animal Model; Architecture; Ascorbic Acid; Astrocytes; Biological Assay; Biosensor; Blood - brain barrier anatomy; Brain; Brain region; Calcium Binding; Calcium Oscillations; Cell Differentiation process; Cell Line; Cell model; Cell physiology; Cell secretion; Cells; Cellular Assay; Cellular biology; Chemical Agents; Chromosome Pairing; Clinic; Clinical Trials; Collagen Type IV; Complex; Crowding; Custom; Data; Deposition; Detection; Development; Disease; Disease model; Dopamine; Drug Evaluation; Drug Modelings; Drug Screening; Electrical Resistance; Endothelium; Engineering; Ensure; Evaluation; Extracellular Matrix; Failure; Fibronectins; Ficoll; Fluorescence; Genetic; Glutamates; Goals; Health; Human; Human Biology; Hydrogels; Immune; In Vitro; Infrastructure; Intervention; Laminin; Lead; Libraries; Light; Measurement; Measures; Mechanics; Medical; Medicine; Methods; Microfluidics; Modeling; Molecular Weight; Morphology; Neurons; Normal tissue morphology; Opiate Addiction; Opioid; Organoids; Overdose; Pain; Patients; Pattern; Penetrance; Penetration; Perfusion; Pericytes; Permeability; Pharmaceutical Preparations; Pharmacological Treatment; Pharmacology; Physiological; Physiological Effects of Drugs; Physiology; Play; Plug-in; Predictive Value; Process; Production; Property; Protocols documentation; Pruritus; Public Health; Publishing; Rewards; Risk; Role; Science; Signal Pathway; Signal Transduction; Skin; Skin Tissue; System; Techniques; Technology; Testing; Therapeutic; Tight Junctions; Time; Tissue Engineering; Tissue Microarray; Tissue Model; Tissues; Toxic effect; Translational Research; Tube; United States Food and Drug Administration; United States National Institutes of Health; Validation; Ventral Tegmental Area; Virus; Withdrawal; Work; addiction; angiogenesis; base; biocompatible polymer; biofabrication; bioprinting; brain endothelial cell; brain tissue; cell type; cerebral microvasculature; chemical addition; clinical development; design; disease phenotype; dopaminergic neuron; drug candidate; drug development; drug discovery; drug structure; efficacy study; experimental study; first-in-human; fluorescence imaging; gamma-Aminobutyric Acid; high throughput screening; human model; human tissue; improved; in vitro Model; in vivo; induced pluripotent stem cell; macromolecule; member; neuronal circuitry; neurovascular unit; novel; novel therapeutics; opioid epidemic; opioid misuse; opioid use; opioid use disorder; optogenetics; pain model; pre-clinical; preclinical development; preclinical efficacy; preservation; prevent; programs; relating to nervous system; release of sequestered calcium ion into cytoplasm; safety study; screening; sensor; small molecule libraries; stressor; therapeutic development; tool; vasculogenesis

Development of blood brain barrier models as a platform for compound testing A physiological relevant in vitro blood brain barrier (BBB) model is needed to establish the brain penetration potential of compounds being developed as drugs. Many of the compounds being developed for opioid use disorder or pain have targets in the brain and, in some cases, it would be beneficial to prevent brain penetration. Therefore, being able to establish brain penetrance during preclinical development is critical. Current in vitro models (e.g. PAMPA) are very simplistic and do not include the relevant endothelial, pericyte and astrocytes composing the BBB. The project team has established a 96-well transwell BBB model using human iPSC-derived human brain endothelial cells (ECs) and have used transepithelial electrical resistance (TEER) measurements to demonstrate tight junction and barrier formation. Using primary brain microvascular endothelial cells (BMEC), the team has also developed a BBB model that includes astrocytes and pericytes in a Mimetas microfluidic plate and have demonstrated low permeability to molecules of different molecular weights. This BBB model will allow evaluation of drug penetration in the brain, as well as to examine the effects of drugs on the physiology of the BBB in the context of pain and addiction. Biofabrication of a neurovascular unit (NVU) tissue model in multiwell format Brain tissue models that include brain microvasculature consisting of brain endothelial cells in a microenvironment of astrocytes, pericytes and neuronal cells is critically needed as an assay platform to assess the toxic effects of drugs in the brain, as well as to study efficacy of compounds on brain relevant pathophysiological disease phenotypes. Bioprinting techniques are being used to create a microvascularized brain tissue models made up of primary brain endothelial cells, pericytes and astrocytes in a 96-well plate format. Vasculogenesis and angiogenesis in these tissues has been shown by cell fluorescence imaging. The project team is also in the process of including neuronal cells and using a new multiwell plate platform that will enable perfusion though the vasculature. Neural spheroids models for opioid addiction screening 3D brain cellular models of relevant physiological complexity and amenable to HTS are needed as preclinical assay platforms to assess the toxicity and efficacy of compounds developed to treat OUD and pain. Spheroids are multi-cell type aggregates with physiologically functional activity and are being utilized as assay platforms for disease modeling and drug screening. Brain spheroids are produced using iPSC-derived neuronal cells and astrocytes, and cell composition is tailored to mimic that of different regions of the brain. Current work is focused on validating designer neural spheroids as a HTS platform for the discovery and validation of new treatments for OUD. The 3DTBL has neural 3D spheroid cultures in 384-well plates to use as a high throughput system to test compounds. The project team has developed a calcium flux (FLIPR) assay using these cortical spheroids and screened a library of 700 compounds targeting brain targets/signaling pathways involved in opioid use disorder. Team members are also further tailoring designer, self-organized brain organoids which are generated using controlled mixtures of iPSC-derived excitatory (glutamatergic), inhibitory (GABAergic) and dopamine neurons and astrocytes. These tailored neural spheroids assay platforms are being used to establish opioid-like activity signatures using detection methods commonly used in HTS, including fluorescence imaging calcium flux. The team has demonstrated that these neural spheroids have synchronized calcium waves depending on the neuronal composition. Developing optogenetic tools to create neuronal circuits in a multiwell plate format Opioids modulate reward circuits in the brain, including the ventral tegmental area (VTA), playing a central role in addiction and withdrawal, medically known as opioid use disorders (OUD). The ability to recreate neuronal circuits of addiction and reward in a test-tube would allow evaluation of the addictive potential of new pain medicines and facilitate the development of therapeutics to treat OUD. The project team is using bioprinting techniques to develope simplified ex vivo reward circuit. For example, the ventral tegmental area, is being constructed by bioprinting with the goal of testing the effects of compounds in reward and addiction neuronal circuits. Proof of principle experiments have shown that iPSC-derived dopamine and GABA neurons can be transduced with optogenetic sensors using AVV viruses. Further, cells can be bioprinted and action potential activities, following light activation, can be detected using microarrayelectrodes (MEA) and calcium binding fluorescence biosensors (GCamp) using confocal and epifluorescence, both in a 2D and 3D format. Innervated 3D skin models for pain sensing Pain drugs are being developed using engineered cell lines and animal models which are not very predictive of activity in humans, thus leading to a high number of failures in clinic in spite of good preliminary genetic evidence. There is a critical need to develop in vitro pain models that are more predictive of drug activity in the clinic. These in vitro cellular models should include human sensory neurons in the context on the tissue were pain is produced and in a format that is amenable to screening to ensure these models make an impact as preclinical assays for drug development. NCATS 3DTBL has established a robust protocol for the biofabrication of skin tissues in a HTS amenable multiwell platform. At the same time, NCATS SCTL has developed robust protocols for iPSC differentiation into sensory neurons. The two labs are working together towards the assembly of a functional innervated skin model that can use to quantitate pain or itch signals from the skin to the sensory neurons. The project team is working to evaluate formation of extensions from the sensory neurons into the skin tissue. Based on published data the team is also testing different approaches, including using DRG spheroids and designing custom wells that will allow DRG extension formation into the skin. Increasing endogenous matrix production in 3D Tissue models for improved physiological properties The extracellular matrix component (ECM) of tissues plays a critical role in tissue morphology and function by providing mechanical and physiological clues to the cells in the tissue. The native ECMs in each tissue are complex and generated by the cells in the tissue. Reproducing the complexity of the native ECMs in the biofabricated tissue is critical to ensure the correct physiological function of the tissue. Macromolecular crowding (MMC) is a technique that has been used to enhanced ECM secretion from cells. It is based on the addition of chemically inert macromolecules that increase ECM deposition by excluded volume effect. NCATS 3DTBL are applying MMC techniques to increase the native ECM in neuronal tissues and improve their neuronal network function. The project team has demonstrated that by treating astrocytes with Ficoll and ascorbic acid, ECM deposition increases, including collagen IV, fibronectin and laminin. Furthermore, when neurons are added to the astrocytes cultures treated with MMC, neuronal activities increase, as measured by calcium waves. We are currently investigating whether the MMC treated cultures induce a higher number of synapsis and more dense neuronal cultures to improve the quality of tissue models that will be used to more accurately model pain, addition, and overdose.

开发血脑屏障模型作为化合物测试平台 需要生理相关的体外血脑屏障（BBB）模型来确定正在开发的药物化合物的大脑渗透潜力。许多针对阿片类药物使用障碍或疼痛而开发的化合物都以大脑为目标，在某些情况下，这将有助于防止大脑渗透。因此，能够在临床前开发过程中建立脑渗透率至关重要。目前的体外模型（例如 PAMPA）非常简单，不包括组成 BBB 的相关内皮细胞、周细胞和星形胶质细胞。该项目团队使用人类 iPSC 衍生的人脑内皮细胞 (EC) 建立了 96 孔跨孔 BBB 模型，并使用跨上皮电阻 (TEER) 测量来证明紧密连接和屏障的形成。该团队还利用原代脑微血管内皮细胞 (BMEC) 开发了一种 BBB 模型，其中包括 Mimetas 微流体板中的星形胶质细胞和周细胞，并证明对不同分子量的分子具有低渗透性。该 BBB 模型将允许评估药物在大脑中的渗透，以及检查药物在疼痛和成瘾情况下对 BBB 生理学的影响。 多孔格式神经血管单元 (NVU) 组织模型的生物制造 脑组织模型包括由星形胶质细胞、周细胞和神经元细胞微环境中的脑内皮细胞组成的脑微脉管系统，作为评估药物在大脑中的毒性作用以及研究化合物对脑相关病理生理疾病表型的功效的分析平台，是至关重要的。生物打印技术被用来在 96 孔板中创建由原代脑内皮细胞、周细胞和星形胶质细胞组成的微血管化脑组织模型。 这些组织中的血管发生和血管生成已通过细胞荧光成像显示。该项目团队还正在纳入神经元细胞并使用新的多孔板平台，该平台将能够通过脉管系统进行灌注。 用于阿片类药物成瘾筛查的神经球体模型 需要具有相关生理复杂性且适合 HTS 的 3D 脑细胞模型作为临床前检测平台，以评估开发用于治疗 OUD 和疼痛的化合物的毒性和功效。 球体是具有生理功能活性的多细胞类型聚集体，被用作疾病建模和药物筛选的分析平台。大脑球体是使用 iPSC 衍生的神经元细胞和星形胶质细胞产生的，细胞组成经过定制以模仿大脑不同区域的细胞组成。目前的工作重点是验证设计的神经球作为 HTS 平台，用于发现和验证 OUD 新疗法。 3DTBL 在 384 孔板中具有神经 3D 球体培养物，可用作测试化合物的高通量系统。该项目团队使用这些皮质球体开发了一种钙流 (FLIPR) 测定法，并筛选了 700 种针对阿片类药物使用障碍所涉及的大脑靶标/信号通路的化合物库。团队成员还在进一步定制设计师自组织的大脑类器官，这些类器官是使用 iPSC 衍生的兴奋性（谷氨酸）、抑制性（GABA 能）和多巴胺神经元和星形胶质细胞的受控混合物生成的。这些定制的神经球体检测平台被用于使用 HTS 中常用的检测方法（包括荧光成像钙通量）建立类阿片活性特征。研究小组证明，这些神经球体具有同步的钙波，具体取决于神经元的组成。 开发光遗传学工具以多孔板形式创建神经元回路 阿片类药物调节大脑中的奖赏回路，包括腹侧被盖区 (VTA)，在成瘾和戒断中发挥核心作用，医学上称为阿片类药物使用障碍 (OUD)。在试管中重建成瘾和奖赏神经元回路的能力将有助于评估新止痛药的成瘾潜力，并促进 OUD 治疗方法的开发。该项目团队正在使用生物打印技术来开发简化的离体奖励电路。例如，腹侧被盖区正在通过生物打印构建，目的是测试化合物在奖赏和成瘾神经元回路中的作用。 原理实验证明表明，iPSC 衍生的多巴胺和 GABA 神经元可以通过使用 AVV 病毒的光遗传学传感器进行转导。此外，可以对细胞进行生物打印，并且可以使用微阵列电极 (MEA) 和钙结合荧光生物传感器 (GCamp) 使用共聚焦和落射荧光以 2D 和 3D 格式检测光激活后的动作电位活动。 Innervated 3D skin models for pain sensing 止痛药正在使用工程细胞系和动物模型进行开发，这些细胞系和动物模型不能很好地预测人类的活动，因此尽管有良好的初步遗传证据，但仍导致大量临床失败。 迫切需要开发能够更好地预测临床药物活性的体外疼痛模型。 这些体外细胞模型应包括产生疼痛的组织环境中的人类感觉神经元，并采用适合筛选的格式，以确保这些模型作为药物开发的临床前测定产生影响。 NCATS 3DTBL 已经建立了一个强大的协议，用于在 HTS 适合的多孔平台中进行皮肤组织的生物制造。与此同时，NCATS SCTL 开发了用于 iPSC 分化为感觉神经元的强大协议。这两个实验室正在共同努力组装功能性神经支配皮肤模型，该模型可用于量化从皮肤到感觉神经元的疼痛或瘙痒信号。该项目团队正在努力评估从感觉神经元到皮肤组织的延伸的形成。根据已发表的数据，该团队还在测试不同的方法，包括使用 DRG 球体和设计定制孔，以允许 DRG 延伸形成到皮肤中。 增加 3D 组织模型中的内源基质产量，以改善生理特性 组织的细胞外基质成分（ECM）通过为组织中的细胞提供机械和生理线索，在组织形态和功能中发挥着关键作用。每个组织中的天然 ECM 都很复杂，由组织中的细胞产生。在生物制造的组织中重现天然 ECM 的复杂性对于确保组织正确的生理功能至关重要。大分子拥挤 (MMC) 是一种用于增强细胞 ECM 分泌的技术。它基于添加化学惰性大分子，通过排除体积效应来增加 ECM 沉积。 NCATS 3DTBL 正在应用 MMC 技术来增加神经元组织中的天然 ECM 并改善其神经元网络功能。该项目团队已经证明，通过用聚蔗糖和抗坏血酸处理星形胶质细胞，ECM 沉积会增加，包括 IV 型胶原、纤连蛋白和层粘连蛋白。 此外，当将神经元添加到用MMC处理的星形胶质细胞培养物中时，通过钙波测量，神经元活动增加。 我们目前正在研究 MMC 处理的培养物是否诱导更多数量的突触和更密集的神经元培养物，以提高组织模型的质量，该组织模型将用于更准确地模拟疼痛、附加和过量。