3D Bioprinted skin models for drug screening

为了响应RFA-TR-17-007：NCATS使用生物打印皮肤组织进行药物合作发现研究的试点计划(U18)，启动了两个项目： 1)与哥伦比亚大学的安吉拉·克里斯蒂亚诺博士合作，利用3D生物打印技术将人类原始组织作为一种疾病模型进行药物发现。我们已经成功地开发了96孔跨孔板形式的全厚皮肤等价物(真皮和表皮)的生物制造协议，这将使化合物筛选产量显著增加。此外，我们一直在探索不同的细胞因子鸡尾酒，模拟牛皮癣的压力，并在皮肤组织上产生牛皮癣的表型。细胞因子分泌试验将被用作牛皮癣的表型分析，并用于目前计划的筛查。 2)利用洛克菲勒大学的Dan Gareau博士和纽约大学的John Carucci博士共同构建的新型3D打印皮肤癌的三模式共聚焦图像，非破坏性、高通量地用于药物发现。我们通过在全厚皮肤模型中引入荧光标记的皮肤鳞状细胞癌(SCC)肿瘤细胞并使用荧光监测肿瘤生长，开发了一种皮肤鳞状细胞癌(SCC)。组织学、显微镜和转录学分析表明，SCC皮肤组织模型模拟了许多天然肿瘤的特征。此外，皮肤鳞状细胞癌的标准护理药物5-氟尿嘧啶(5-FU)治疗鳞状细胞癌皮肤组织在减少组织中的肿瘤数量和大小方面显示出疗效。 我们还与NIAID的Ian Myles博士合作，展示了使用生物软化皮肤组织来证明Roseomonas粘膜治疗特应性皮炎的矫正效果。这项贡献是Myles博士实验室更大的工作的一部分，该工作证明特应性皮炎对Roseomonas粘膜的治疗反应涉及脂质介导的肿瘤坏死因子相关的上皮修复。 我们正在与NIAID的Yasmine Belkaid博士的实验室合作，探索微生物群对皮肤免疫反应的影响。 最后，我们与NCATS的Tox21小组建立了合作关系，由夏梦航博士领导，探索使用生物富含的皮肤组织和多路读出方案(细胞存活率、TEER和细胞因子分泌)来评估护肤品中使用的化合物的刺激性。从单层培养系统的初始筛选中鉴定出的46种有毒化合物在重建的人表皮(RHE)和全厚皮肤(FTS)三维(3D)组织模型上进一步测试皮肤刺激性潜力。通过测定组织活力、跨上皮电阻值(TEER)、细胞因子IL-1α(IL-1α)和IL-18(IL-18)的分泌来评估化合物的皮肤刺激性。在已知的刺激物中，高浓度的甲基紫和甲基罗桑胺降低了RHE和FTS模型的存活率，降低了TEER，并增加了IL-1的分泌，这与刺激性特性一致。然而，在低浓度下，在RHE或FTS模型中，这两种化合物增加了IL-18的分泌，而不影响IL-1的分泌水平，也不会降低组织存活率和TEER。这一结果表明，在低浓度下，甲基紫和甲基罗桑胺具有过敏潜力，不会引起刺激。使用HTS兼容的2D细胞和3D组织皮肤模型，以及刺激性相关活动终点，我们获得了有助于评估外用化合物的刺激性效果和识别潜在皮肤危害的数据。

In response to the RFA-TR-17-007: NCATS Pilot Program for Collaborative Drug Discovery Research using Bioprinted Skin Tissue (U18), two projects were on boarded: 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. In addition, we have been exploring different 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) Non-destructive, high throughput cytometry for drug discovery using trimodal confocal images of novel, 3D printed skin carcinoma construct with Drs.Dan Gareau at Rockefeller University and John Carucci at NYU. We have developed a skin squamous cell carcinoma (SCC) by introducing fluorescence labeled SCC tumor cells in the full thickness skin model and monitoring tumor growth using epifluorescence. Histological, microscopic and transcriptomics analysis showed that the SCC skin tissue model mimic many features of the native tumors. In addition, treatment of the SCC skin tissue with 5-fluorouracil (5-FU), standard of care for skin SCC, demonstrated efficacy in reducing tumor number and size in the tissue. We also collaborated with Dr. Ian Myles at NIAID to demonstrate the use of biofabricated skin tissues to demonstrate corrective effects Roseomonas mucosa treatments of atopic dermatatis phentypes. This contribution was part of a larger body of work by Dr. Myles lab that demonstarted that therapeutic responses to Roseomonas mucosa in atopic dermatitis involve lipid-mediated TNF-related epithelial repair. We have an on-going collaboration with the laboratory of Dr. Yasmine Belkaid at NIAID exploring the effects of microbiome on skin immune responses. Finally, we established a collaboration with the Tox21 group at NCATS led by Dr. Menghang Xia to explore the use of biofabricated skin tissues and a multiplex readout scheme (cell viability, TEER and cytokine secretion) to assess the irritation potential of compounds used in skin products. Forty-six toxic compounds identified from an initial screen with the monolayer culture systems were further tested for skin irritation potential on reconstructed human epidermis (RhE) and full thickness skin (FTS) three-dimensional (3D) tissue model constructs. Skin irritation potential of the compounds was assessed by measuring tissue viability, trans-epithelial electrical resistance (TEER), and secretion of cytokines interleukin 1 alpha (IL-1) and interleukin 18 (IL-18). Among known irritants, high concentrations of methyl violet and methylrosaniline decreased viability, lowered TEER, and increased IL-1 secretion in both RhE and FTS models, consistent with irritant properties. However, at low concentrations, these two compounds increased IL-18 secretion without affecting levels of secreted IL-1, and did not reduce tissue viability and TEER, in either RhE or FTS models. This result suggests that at low concentrations, methyl violet and methylrosaniline have an allergic potential without causing irritation. Using both HTS-compatible 2D cellular and 3D tissue skin models, together with irritation relevant activity endpoints, we obtained data to help assess the irritation effects of topical-use compounds and identify potential dermal hazards.