Helping to End Addiction Long-term (HEAL): 3D Bioprinted Tissue Models

帮助戒除长期成瘾 (HEAL)：3D 生物打印组织模型

• 批准号: 10907365
• 负责人: Marc Ferrer
• 金额: $ 57.73万
• 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/10907365
• 关键词: 3-Dimensional; 3D Print; Acceleration; Action Potentials; Acute; Address; Affect; Afferent Neurons; Agonist; Animal Model; Architecture; Area; Astrocytes; Behavior; Biological Assay; Biomedical Engineering; Biosensor; Blood - brain barrier anatomy; Brain; Brain region; Calcium; Calcium Binding; Calcium Oscillations; Cell Differentiation process; Cell Line; Cell model; Cell physiology; Cells; Cellular Assay; Cellular biology; Chemical Agents; Chronic; Clinic; Clinical Trials; Collaborations; Confocal Microscopy; Custom; Data; Development; Disease; Disease model; Dopamine; Drug Evaluation; Drug Modelings; Drug Screening; Endothelial Cells; Endothelium; Engineering; Ensure; Environment; Evaluation; Failure; Fluorescence; Genetic; Glutamates; Goals; Health; Helping to End Addiction Long-term; Human; Human Biology; Hydrogels; Immune; In Vitro; Induced pluripotent stem cell derived neurons; Infrastructure; Intervention; Lead; Libraries; Light; Measures; Medical; Medicine; Methods; Microfluidics; Microglia; Modeling; Molecular Weight; Morphology; National Center for Advancing Translational Sciences; Neurodegenerative Disorders; Neurons; Normal tissue morphology; Oligodendroglia; Opiate Addiction; Opioid; Opioid agonist; Pain; Patients; Pattern; Penetrance; Penetration; Perfusion; Pericytes; Permeability; Pharmaceutical Preparations; Pharmacological Treatment; Pharmacology; Physiological; Physiological Effects of Drugs; Physiology; Play; Predictive Value; Printing; Process; Protocols documentation; Pruritus; Public Health; Publishing; Rewards; Risk; Role; Science; Shapes; Signal Transduction; Silk; Skin; Skin Tissue; Supporting Cell; System; Techniques; Technology; Testing; Therapeutic; 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; Universities; Ventral Tegmental Area; Virus; Withdrawal; Work; addiction; antagonist; biocompatible polymer; biofabrication; biomaterial compatibility; bioprinting; brain endothelial cell; cell type; clinical development; design; detection method; drug candidate; drug development; drug discovery; drug structure; efficacy 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; manufacture; mu opioid receptors; nerve stem cell; nervous system disorder; neural; neural circuit; neural stimulation; neuroinflammation; neuronal circuitry; neurotransmitter release; novel; novel therapeutics; opioid epidemic; opioid exposure; opioid misuse; opioid use; opioid use disorder; optogenetics; pain model; pharmacologic; pre-clinical; preclinical development; preclinical efficacy; preservation; prevent; programs; release of sequestered calcium ion into cytoplasm; safety study; scaffold; screening; small molecule libraries; stem cells; stressor; therapeutic development

The work on this program is focusing on the development of six assay platform projects: Development of blood brain barrier models as platforms 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 for neurological diseases. Many of the compounds being developed for opioid use disorder or pain have targets in the brain, but 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 Team has used brain microvascular endothelial cells (BMEC), human astrocytes and pericytes to develop two models of the BBB on microfluidic tissue plates. In one model endothelial cells create a channel in contact with a hydrogel containing astrocytes and pericytes. We have demonstrated low permeability to molecules of different molecular weights. In another model, a microvasculature is created between two channels of endothelial to assess perfusion and leakiness of microvessels. We have demonstrated that we can create a microvasculature that is perfusable with large macromolecules for at least two weeks. We are currently increasing the physiological relevance of this model by adding neurons, oligodendrocytes, and microglia. These two BBB models 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, and other neuroinflammatory, neurodegenerative diseases. 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. 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 and have been able to demonstrate an opioid-induced, withdrawal-like calcium activity in the spheroids mimicking the ventral tegmental area (VTA), and area of the brain involved in addiction behavior. Additional biosensors to detect action potential and neurotransmitter release are being developed to further explore the physiology of these neural spheroids. The Team has also showed that neural spheroids mimicking different brain regions can be functionally connected to form assembloids, which should enable the possibility of creating neural circuits of addiction and pain in vitro. The Team has also started testing compounds in a chronic mOR agonists treatment model using VTA neural spheroids and testing the effects of mOR antagonists and other compounds being developed to prevent addiction. Biofabrication of functional neuronal circuits of addiction 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 will 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 develop in vitro reward circuits. We are using iPSC-derived dopamine, glutamatergic and GABA neurons transduced with optogenetic or calcium binding fluorescence biosensors (GCamp) biosensors using AVV viruses and mixed with hydrogels to create spatial arrangements to mimic connectivity of different regions of the brain. We are using light to stimulate neural activity at one end of the circuit, and we are measuring calcium fluorescence at the other end using fluorescence confocal microscopy. We are exploring how different circuit shapes affect activation and pharmacological modulation by mu-opioid receptor agonists and antagonists. Addiction-in-a-Scaffold System to Identify and Screen Therapeutics (ASSIST): a 3D bioengineering approach to treating opioid use disorder: We are collaborating with Drs. Nieland and Lovett at Tufts University to use their 3-dimensional neural tissue model composed of neuronal progenitor stem cells-derived neurons and support cells (glutamatergic and GABAergic neurons and astrocytes) in a biocompatible 3D silk-based scaffold matrix enveloped in a hydrogel environment, to recapitulates the acute, chronic and/or repeated opioid exposure, and implement drug screens. NCATS has reproduced the ASSIST technology in-house and is working on adapting it to high throughput screening 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 despite 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 where 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 be used to quantitate pain or itch signals from the skin to the sensory neurons. The project team is working to evaluate the 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.

该计划的工作重点是开发六个检测平台项目： 开发血脑屏障模型作为化合物测试平台： 需要一个生理相关的体外血脑屏障（BBB）模型来确定正在开发的神经疾病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，但在某些情况下，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。该团队使用脑微血管内皮细胞 (BMEC)、人星形胶质细胞和周细胞在微流体组织板上开发了两种 BBB 模型。 在一种模型中，内皮细胞创建了一个与含有星形胶质细胞和周细胞的水凝胶接触的通道。 我们已经证明对不同分子量的分子具有低渗透性。在另一个模型中，在内皮的两个通道之间创建微脉管系统，以评估微血管的灌注和渗漏。 我们已经证明，我们可以创建一种可以用大分子灌注至少两周的微脉管系统。 我们目前正在通过添加神经元、少突胶质细胞和小胶质细胞来提高该模型的生理相关性。 这两个 BBB 模型将允许评估药物在大脑中的渗透，并检查药物在疼痛、成瘾以及其他神经炎症、神经退行性疾病的背景下对 BBB 生理学的影响。 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。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。研究小组已经证明，这些神经球体具有取决于神经元组成的同步钙波，并且能够在模仿腹侧被盖区（VTA）和大脑区域的球体中展示阿片类药物诱导的、类似戒断的钙活动参与成瘾行为。正在开发额外的生物传感器来检测动作电位和神经递质释放，以进一步探索这些神经球体的生理学。该团队还表明，模仿不同大脑区域的神经球体可以在功能上连接形成组合体，这应该能够在体外创建成瘾和疼痛的神经回路。该团队还开始使用 VTA 神经球体在慢性 mOR 激动剂治疗模型中测试化合物，并测试 mOR 拮抗剂和其他正在开发的预防成瘾化合物的效果。 多孔板形式的成瘾功能神经元回路的生物制造： 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体外奖励回路。我们使用 iPSC 衍生的多巴胺、谷氨酸和 GABA 神经元，通过使用 AVV 病毒的光遗传学或钙结合荧光生物传感器 (GCamp) 生物传感器进行转导，并与水凝胶混合以创建空间排列，以模拟大脑不同区域的连接。 我们使用光刺激电路一端的神经活动，并使用荧光共聚焦显微镜测量另一端的钙荧光。 我们正在探索不同的电路形状如何影响μ阿片受体激动剂和拮抗剂的激活和药理调节。 用于识别和筛选治疗方法的支架成瘾系统 (ASSIST)：一种治疗阿片类药物使用障碍的 3D 生物工程方法： 我们正在与博士合作。塔夫茨大学的 Nieland 和 Lovett 使用他们的 3 维神经组织模型，该模型由神经祖干细胞衍生的神经元和支持细胞（谷氨酸能和 GABA 能神经元和星形胶质细胞）组成，位于水凝胶环境中的生物相容性 3D 丝基支架基质中，概括急性、慢性和/或反复阿片类药物暴露，并实施药物筛查。 NCATS 已在内部复制了 ASSIST 技术，并正在努力使其适应高通量筛选形式。 用于疼痛感知的神经支配 3D 皮肤模型： 止痛药正在使用工程细胞系和动物模型开发，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，并采用适合筛选的形式，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。

项目成果

High throughput 3D gel-based neural organotypic model for cellular assays using fluorescence biosensors.

基于高通量 3D 凝胶的神经器官模型，用于使用荧光生物传感器进行细胞测定。

• 发表时间: 2022-11-12
• 影响因子: 5.9
• 作者: Kundu, Srikanya;Boutin, Molly E;Strong, Caroline E;Voss, Ty;Ferrer, Marc
• 通讯作者: Ferrer, Marc

Enhancement of Neuroglial Extracellular Matrix Formation and Physiological Activity of Dopaminergic Neural Cocultures by Macromolecular Crowding.

大分子拥挤增强神经胶质细胞外基质的形成和多巴胺能神经共培养物的生理活性。

• 发表时间: 2022-07-06
• 影响因子: 6
• 作者: Vo, Andy N;Kundu, Srikanya;Strong, Caroline;Jung, Olive;Lee, Emily;Song, Min Jae;Boutin, Molly E;Raghunath, Michael;Ferrer, Marc
• 通讯作者: Ferrer, Marc

A multiparametric calcium signal screening platform using iPSC-derived cortical neural spheroids.

使用 iPSC 衍生的皮质神经球体的多参数钙信号筛选平台。

• DOI: 10.1016/j.slasd.2022.01.003
• 发表时间: 2022-06
• 期刊: SLAS discovery : advancing life sciences R & D
• 影响因子: 3.1
• 作者: Boutin, Molly E.;Strong, Caroline E.;Van Hese, Brittney;Hu, Xin;Itkin, Zina;Chen, Yu-Chi;LaCroix, Andrew;Gordon, Ryan;Guicherit, Oivin;Carromeu, Cassiano;Kundu, Srikanya;Lee, Emily;Ferrer, Marc
• 通讯作者: Ferrer, Marc