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

基本信息

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
项目类别：
财政年份：
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

项目摘要

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测定平台项目：开发血脑屏障模型作为复合测试的平台：需要一个相关的体外血脑屏障（BBB）模型，以确定化合物作为神经疾病的药物开发的大脑渗透潜力。许多用于阿片类药物使用障碍或疼痛的化合物在大脑中具有靶标，但是在某些情况下，防止大脑渗透是有益的。因此，能够在临床前发育过程中建立大脑外观至关重要。当前的体外模型（例如PAMPA）非常简单，不包括组成BBB的相关内皮，周细胞和星形胶质细胞。该小组使用了脑微血管内皮细胞（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。在一个模型中，内皮细胞与含有星形胶质细胞和周细胞水凝胶接触的通道。我们已经证明对不同分子量分子的渗透性低。在另一个模型中，在两个内皮通道之间创建了微脉管系统，以评估微血管的灌注和渗漏。我们已经证明，我们可以创建一个至少两个星期的大型大分子的微脉管系统。目前，我们通过添加神经元，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模型将允许评估大脑中的药物渗透，并在疼痛和成瘾的背景下检查药物对BBB生理的影响以及其他神经炎性的神经退行性疾病。阿片类药物成瘾筛查的神经球体模型：相关生理复杂性和HTS适合的3D脑细胞模型是临床前测定平台，以评估为治疗OUD和疼痛而开发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，并被用作疾病建模和药物筛查的测定平台。使用IPSC衍生的神经元细胞和星形胶质细胞产生脑球体，并根据模仿大脑的不同区域量身定制细胞组成。这些量身定制的神经球体测定平台用于使用HTS中常用的检测方法（包括荧光成像钙通量）来建立类似阿片类药物的活性特征。该小组已经证明，这些神经球体根据神经元组成具有同步的钙波同步，并且能够证明模仿腹侧细饰区（VTA）的球体中的阿片类药物诱导的戒断样钙活性，并参与了大脑的面积。正在开发其他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到形成组合物，这应该使可能在体外产生成瘾和疼痛的神经回路。该团队还使用VTA神经球体开始在慢性MOR激动剂治疗模型中测试化合物，并测试MOR拮抗剂和开发其他化合物的作用以防止成瘾。多井板格式中成瘾功能性神经元回路的生物制作：阿片类药物调节大脑中的奖励电路，包括腹侧对接区域（VTA），在成瘾和戒断中起着核心作用，在医学上称为阿片类药物使用障碍（OUD）。在测试管中重现成瘾和奖励的神经元回路的能力将允许评估新的止痛药的成瘾潜力，并促进治疗OUD治疗的发展。该项目团队正在使用生物打印技术来开发体外奖励电路。我们使用IPSC衍生的多巴胺，谷氨酸能和GABA神经元，使用AVV病毒转导的遗传学或钙结合荧光生物传感器（GCAMP）生物传感器，并与水凝胶混合以形成空间排列，以模拟大脑不同区域的模拟连接性。我们正在使用光刺激电路的一端刺激神经活动，并且我们使用荧光共聚焦显微镜在另一端测量钙荧光。我们正在探索不同的电路形状如何影响mu阿片受体激动剂和拮抗剂的激活和药理调节。识别和筛查治疗剂的成瘾系统（辅助）：治疗阿片类药物使用障碍的3D生物工程方法：我们正在与Drs合作。塔夫茨大学的Nieland和Lovett使用其三维神经组织模型，该模型由神经祖细胞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，以生物效果3D的3D丝绸丝网组合在/近凝胶环境中，并占据了基于3D的丝绸coppocial，并占据了基于杂物的环境，并重复了整齐的环境。并实施毒品屏幕。 NCAT已在内部复制了辅助技术，并正在将其调整为高吞吐量筛查格式。神经感知的3D皮肤模型：使用工程的细胞系和动物模型开发疼痛药物，这些细胞系和动物模型对人类的活性不太预测，因此尽管有良好的初步遗传证据，但仍导致诊所的大量失败。迫切需要开发体外疼痛模型，这些模型更可预测诊所的药物活性。这些体外细胞模型应在产生疼痛的组织和可用于筛查的格式的组织上包括人类感觉神经元，以确保这些模型作为药物开发的临床前分析产生影响。 NCATS 3DTBL已建立了一个可靠的方案，用于在HTS Amenable Multiwell平台中对皮肤组织的生物制作。同时，NCATS SCTL已开发出鲁棒的IPSC分化为感觉神经元的方案。这两个实验室正在为组装功能神经支配的皮肤模型的组装而努力，该模型可用于定量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扩展形成。基于已发布的数据，团队还在测试不同的方法，包括使用DRG球体和设计可以允许DRG扩展形成的定制井。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

DOI：
10.1038/s42003-022-04177-z
发表时间：
2022-11-12
期刊：
Communications biology
影响因子：
5.9
作者：
通讯作者：

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

DOI：
10.1016/j.slasd.2022.01.003
发表时间：
2022-06
期刊：
SLAS DISCOVERY
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

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