3D Bioprinted skin models for drug screening

为了响应RFA-TR-19-020：使用生物制造的3-D皮肤病组织模型（U18）进行药物筛选，正在实施两个项目： 1)人类天然组织的3D生物打印作为药物发现的培养皿中疾病模型与哥伦比亚大学的Angela Christiano博士合作。 我们已经成功地开发了用于96孔transwell板形式的全厚度皮肤等同物（具有真皮和表皮）的生物制剂方案，这将使得化合物筛选通量显著增加。 我们已经确定了一个鸡尾酒的细胞因子，模拟银屑病的压力，并产生银屑病表型的皮肤组织。 细胞因子分泌试验将用作银屑病的表型试验，并用于目前计划的筛查。 2)华盛顿大学朱佳博士的生物制造3-D皮肤模型用于抗人类HSV感染的抗病毒药物发现。高通量筛选，以确定在生物制造的3-D全厚度皮肤模型中阻断HSV感染的潜在抗病毒化合物。 我们已经使用HSV-GFP报告病毒感染96孔板形式的生物打印的全厚度皮肤等同物，并使用荧光显微镜测量病毒感染和复制。我们已经实现了700种化合物的库的HTS。 来自HTS的选定数量的活性化合物现在正在血管化3-D皮肤模型中进行验证，该模型由来自不同HSV结果队列的患者特异性原代角质形成细胞、成纤维细胞和内皮细胞制成。 我们继续使用生物打印的全层皮肤等同物和荧光标记的癌细胞，以96孔板形式开发皮肤癌模型（皮肤鳞状细胞癌和黑色素瘤）。 这些检测方法对HTS具有鲁棒性，我们现在已经完成了对900种化合物的肿瘤学收集的筛选，以在3D组织模型中识别这些癌症的新化疗药物。 我们已经启动了一个项目，将人类巨噬细胞纳入生物制造的皮肤组织模型中，以研究免疫细胞在皮肤疾病中的作用，包括纤维化，伤口愈合等。 我们与NIAID的Yasmine Belkaid博士实验室正在进行合作，通过测量炎症细胞因子的分泌来探索微生物组对血管化皮肤等效物的皮肤免疫反应的影响。 我们与FDA/NCTR的夏梦航博士和路易莎·卡马乔博士领导的NCATS的Tox 21小组建立了合作关系，探索使用生物制造皮肤组织研究药物渗透性。 我们与FDA/CBER的Kyung Sung博士合作，评估间充质干细胞在特应性皮炎生物制造皮肤模型中的治疗效果。

In response to the RFA-TR-19-020: Drug Screening with Biofabricated 3-D Skin Disease Tissue Models (U18), two projects are being implemented: 1) 3D Bioprinting of human native-like tissues as disease-in-a-dish models for drug discovery with Dr. Angela Christiano at Columbia University. We have been successfully developed biofabrication protocols for full thickness skin equivalents (with dermis and epidermis) in a 96-well transwell plate format which will enable a significance increase in compound screening throughput. We have identified a cocktails of cytokines that mimic a psoriatic stress and produces a psoriatic phenotype on the skin tissue. A cytokine secretion assay will be used as a phenotypic assay for psoriasis and used for the screen which is now planned. 2) Biofabricated 3-D skin model for antiviral drug discovery against human HSV infection with Dr. Jia Zhu at University of Washington. A High-throughput screen to identify potential antiviral compounds that block HSV infection in biofabricated 3-D full thickness skin models. We have used a HSV-GFP reporter virus to infect a bioprinted full thickness skin equivalents in a 96-well plate format and measured viral infection and replication using fluorescence microscopy. We have implemented an HTS of a library of 700 compounds. A selected number of active compounds from the HTS are now being validated in a vascularized 3-D skin models fabricated with patient-specific primary keratinocyte, fibroblast and endothelium cells from a cohort of diverse HSV outcomes. We have continued to develop skin cancer models (cutaneous squamous cell carcinoma and melanoma) using bioprinted full thickness skin equivalents and fluorescently labeled cancer cells, in a 96-well plate format. The assays have shown to be robust for HTS and we have now completed screens of an oncology collection of 900 compounds to identify new chemotherapeutic agents for these cancers in a 3D tissue model. We have initiated a project to incorporate human macrophages into biofabricated skin tissue models to investigate the effect of immune cells in skin diseases, including fibrosis, wound healing and others. We have an on-going collaboration with the laboratory of Dr. Yasmine Belkaid at NIAID exploring the effects of microbiome on skin immune responses on vascularized skin equivalents by measuring secretion of inflammatory cytokines. We have established a collaboration with the Tox21 group at NCATS led by Dr. Menghang Xia and Dr. Luisa Camacho at the FDA/NCTR, to explore the use of biofabricated skin tissues to study drug permeability. We have initiated a collaboration with Dr. Kyung Sung at the FDA/CBER to assess the therapeutic effects of mesenchymal stem cells in a biofabricated skin model of atopic dermatitis.