3D Bioprinting of human native-like tissues as disease-in-a-dish models for drug discovery

组织生物打印计划的活动包括：i）开发生物打印方案以制造类似天然的功能性人体组织。 Ii）打印组织的标记：通过使用显微镜、组织学、基因测序、电生理学和其他方法来开发组织结构和功能的形态学和生理学生物标志物。 iii）使用打印的组织来筛选用于药物发现的化合物的聚焦文库。 iv）开发一个共享验证协议的框架，作为研究人员交换生物墨水优化条件、培养技术、细胞类型和软件工具以及量化和验证打印组织技术的数据的现成资源。 皮肤是一种复杂的，分层的和分层的组织，它通过充当进入体内的主动物理屏障并调节水和其他代谢物的运输出体外来提供保护。 生物打印的人类天然皮肤模型可以为皮肤疾病的病因学提供基本见解，并阐明皮肤疾病进展和发现治疗方法的病理生理机制。 目前的努力涉及使用正常人成纤维细胞和角质形成细胞生物打印皮肤的一致且可再现的人天然样真皮和表皮层。 一旦实现这一目标，我们将引入可用的疾病患者细胞来复制疾病皮肤组织，这些组织将用作筛选的疾病培养皿模型。 用于湿性年龄相关性黄斑变性（湿性AMD）的疾病模型的血液视网膜屏障的生物打印：视网膜变性疾病是发达国家中不可逆视力丧失的主要原因。在许多情况下，这些疾病起源于眼睛后部的稳态单元，该稳态单元包含视网膜、视网膜色素上皮（RPE）和脉络膜毛细血管。 在像年龄相关性黄斑变性（AMD）这样的疾病中，认为RPE功能障碍引起疾病起始事件，并且随着RPE退化，光感受器开始死亡，并且患者开始丧失视力。患者特异性诱导多能干（iPS）细胞衍生的RPE提供了直接访问患者遗传学，并允许识别RPE相关退行性疾病的起始事件的可能性。 RPE，神经视网膜和脉络膜毛细血管的三维模型正在使用组织生物打印结合iPS细胞技术和基础发育生物学开发。在整个稳态单位水平上分析疾病过程可能会对视网膜退行性疾病的分子机制提供更多的见解，并为发现AMD的新治疗方法提供天然疾病模型。 这是与NEI的Kapil Bharti博士团队的合作。 生物打印血管壁模型用于模拟早衰症： Hutchinson-Gilford早衰症（HGPS）是一种遗传性疾病，虽然罕见，但对受影响的儿童具有破坏性后果。那些患有HGPS的人会加速衰老，平均预期寿命只有13.4岁。患有HGPS的患者患有加速的血管疾病，并且死亡几乎总是由冠状动脉疾病或中风引起。先前的研究表明，在HGPS患者和动物模型中，大血管中层平滑肌细胞（SMC）大量丢失，这表明这种SMC丢失表型与HGPS相关的致命心血管功能障碍之间可能存在联系。 3D生物打印技术正在被用于构建和表征组织工程血管壁系统，该系统使用HGPS和正常对照iPSC衍生的SMC作为用于发现HGPS治疗方法的disease-in-a-dish模型。 多孔板中血管壁的3D生物打印组织模型将使用先前描述的治疗选项进行生物学验证和生物学验证。 我们期望HGPS的这种3D血管系统对于HGPS和年龄相关心血管疾病的未来药物筛选和治疗开发具有重要意义，特别是因为导致HGPS的毒性蛋白质也在正常个体中少量产生，并且随着细胞接近衰老而增加。 这是与马里兰州大学帕克分校的Can Kao博士和NIH/NHGRI的弗朗西斯柯林斯博士的合作。 用于建模卵巢癌转移的网膜模型的生物打印：转移是肿瘤细胞扩散到身体不同部位的过程，在大多数情况下，它是导致癌症最终死亡的病理学。 一项使用来自接受手术的卵巢癌患者的细胞重建人类网膜的3D测定成功地用于发现可以防止肿瘤细胞附着在网膜上的化合物，网膜是卵巢癌转移的早期部位。 我们目前正在使用组织生物打印技术，通过引入对网膜转移部位的肿瘤-微环境相互作用重要的其他细胞类型，包括血管系统，脂肪细胞和白细胞，来增加卵巢癌转移网膜模型的相关性。 一旦重建了天然网膜模型，我们将研究癌细胞的生长并筛选预防肿瘤转移的药物。 这是与芝加哥大学Ernst Lengyels博士团队的合作。

The activities of the Tissue Bioprinting program include: i) Develop bioprinting protocols to fabricate native-like, functional human tissues. Ii) Validate of printed tissues: develop morphological and physiological biomarkers of tissue architecture and function by using microscopy, histology, gene sequencing, electrophysiological and other methodologies. iii) Use printed tissues for the screening of focused libraries of compounds for drug discovery. iv) Develop a framework for the sharing of validated protocols as a readily available resource for researchers to exchange data on optimized conditions for bioinks, culture techniques, cell types, and software tools as well as techniques to quantify and validate printed tissues. Bioprinting of dermal tissue for modeling skin diseases: Skin is a complex, hierarchical and stratified tissue that provides protection from the external environment by acting as an active physical barrier into the body and regulating transport of water and other metabolites out of the body. Bioprinted human native skin models can provide fundamental insights into the etiology of skin diseases as well as elucidate the pathophysiological mechanisms in skin disease progression and discovery of treatments. Current efforts are directed to the bioprinting of consistent and reproducible human native-like dermis and epidermis layers of the skin using normal human fibroblasts and keratinocytes. Once this is achieved, we will introduce available disease patient cells to reproduce disease skin tissues that will be used as disease-in-dish models for screening. Bioprinting of blood retinal barrier for a disease model of wet age related macular degeneration (wet AMD): Retinal degenerative diseases are the leading cause of irreversible vision loss in developed countries. In many cases the diseases originate in the homeostatic unit in the back of the eye that contains the retina, retinal pigment epithelium (RPE) and the choriocapillaris. In diseases like age-related macular degeneration (AMD), it is thought that RPE dysfunctions cause disease-initiating events and as the RPE degenerates photoreceptors begin to die and patients start losing vision. Patient-specific induced pluripotent stem (iPS) cell-derived RPE provides direct access to a patients genetics and allow the possibility of identifying the initiating events of RPE-associated degenerative diseases. Three-dimensional models of the RPE, neuroretina, and the choriocapillaris are being developed using tissue bioprinting combined with iPS cell technology and fundamental developmental biology. Analysis of disease processes at the level of this entire homeostatic unit will likely provide more insight into molecular mechanisms of retinal degenerative diseases, as well as providing a native disease model for the discovery of new treatments for AMD. This is a collaboration with the group of Dr. Kapil Bharti at NEI. Bioprinting of a blood vessel wall model for modeling Progeria: Hutchinson-Gilford Progeria syndrome (HGPS) is a genetic disorder that, although rare, has devastating consequences to the affected children. Those with HGPS undergo accelerated aging and have an average life expectancy of just 13.4 years. Patients with HGPS suffer from accelerated vascular disease, and death almost always results from coronary artery disease or stroke. Previous studies have shown a massive loss of smooth muscle cells (SMCs) in the medial layer of large vessels in HGPS patients and animal models, suggesting a possible link between this SMC loss phenotype and the deadly cardiovascular malfunction associated with HGPS. 3D bioprinting techniques are being used to build and characterize a tissue engineered blood vessel wall system using HGPS and normal control iPSC-derived SMCs as disease-in-a-dish models for the discovery of treatments for HGPS. The 3D bioprinted tissue models of blood vessel walls in multi-well plates will be validated biologically and pharmacologically using treatment options previously described. We expect that this 3D vessel system of HGPS will be of great importance for future drug screening and therapeutic development for HGPS and age-related cardiovascular diseases, especially since the toxic protein that causes HGPS is also made in small quantities in normal individuals, and increases as cells approach senescence. This is a collaboration with Dr. Can Kao at University of Maryland, College Park, and Dr. Francis Collins, at NIH/NHGRI. Bioprinting of an omentum model for modeling ovarian cancer metastasis: Metastasis is the process of spreading of tumor cells to different parts of the body and in most cases it is the pathology that leads to ultimate death in cancer. A 3D assay that recreated the human omentum using cells from ovarian cancer patients undergoing surgery was successfully used to discover compounds that would prevent attachment of tumor cells to the omentum, an early site of metastasis in ovarian cancer. We are currently using tissue bioprinting techniques to increase the relevance on the ovarian cancer metastasis omentum model by introducing additional cell types that are important for the tumor-microenvironment interaction in the omentum metastatic site, including vasculature, adipose cells and leukocytes. Once a native omentum model is recreated, we will study the growth of cancer cells and screen for pharmacological agents that prevent tumor metastasis. This is a collaboration with Dr. Ernst Lengyels group at the Universtiy of Chicago.