MICA: Proof of Concept for Real Architecture for 3D Tissues in the Cornea

眼睛前表面的角膜是我们通向世界的窗口。如果透明度受损，可能会导致视力受损，甚至失明。全世界有1000万人因角膜疤痕致盲，由于供体组织供应不足，无法获得挽救视力的角膜移植。在实验室中“培养”角膜成分的能力将代表着一项非常重大的科学和医学进步，可以提高生活质量。作为实现这一目标的第一步，我们正在努力解决一种名为角膜缘上皮干细胞(LESC)缺陷的疾病，这种疾病会导致痛苦、失明的角膜表面衰竭。据估计，英国每年新增240例LESC缺乏症。我们已经培养和移植了LESCs薄片，以改善化学烧伤患者的视力。然而，许多患者很难处理需要用底物来帮助细胞存活的疾病，在我们的病例中是人羊膜。这种方法可以显著提高工作场所(患者个人交流)的生活质量和生产力，然而，我们发现长期的治疗益处是可变的。我们怀疑，部分原因是因为培养的LESC薄片不能恢复正常的LESC微环境或被疾病破坏的‘生态位’。使用一种新的组织工程学方法，我们最近利用蛋白质、上皮细胞(包括LESCs)和支持它们的成纤维细胞构建了简单的人类角膜表面。利用新技术(Real Architecture for 3D Organities-RAFT)制作成纤维细胞种植的胶原结构，其表面拓扑学模拟体内干细胞壁龛，然后在表面种植角膜上皮细胞。我们的数据非常有希望，上皮细胞成功生长并产生上皮细胞。在LESC培养中，筏子结构是可重复性的，并且比人羊膜有显著的优势。羊膜在生物学上是可变的，通常(40%的病例)不支持健康的LESCs的生长。我们现在希望a)评估RAFT的物理和功能特性，b)进行安全性和有效性研究，以便我们可以在该项目之后在Moorfield眼科医院(MEH)进行首例人体研究。我们已经知道，木筏足够结实，可以通过它阅读文本。在这里，我们将测试实际的机械强度，因为这必须足以承受手术。透明度也将进行衡量，理想情况下，RAFT应该至少与我们计划取代的羊膜一样透明(如果不是更好的话)。这项建议背后的关键驱动因素是需要确定RAFT是否可以安全使用，以及它是否可以改善LESC缺乏症患者的角膜表面。如果没有这些信息，我们就不能在人体上测试木筏。在这里，我们将与同事们合作，在已建立的LESC缺陷模型中评估RAFT的安全性和有效性。我们的技术和方法将在我们的模型中得到严格的验证，这些技术和方法用于生产筏子结构，在可访问的设备中将它们呈现给外科医生，并在我们的模型中测量临床结果。如果在这项临床前测试中，RAFT构造可以恢复角膜透明度并维持健康的眼表，我们的数据将被用于开发符合法规的RAFT生产标准操作程序，用于未来的人类测试。因此，这个项目对于将我们的研究成果转化为临床实践至关重要。如果该项目按计划进行，IOO将有经验和能力在“视力细胞治疗研究单位”中制造木筏结构。这个新的最先进的洁净室设施，由申请者领导，用于为患者生产细胞疗法。如果项目成功，IOO和MEH将设计一项I/II期安全性和有效性临床试验，以比较RAFT和羊膜上培养的LESC。

The cornea on the front surface of the eye is our window to the world. If transparency is compromised, visual impairment and even blindness can occur. There are 10 million people worldwide who are blinded by scarring of the cornea and are denied a sight saving corneal graft due to insufficient donor tissue availability. The ability to 'grow' components of a cornea in the laboratory would represent a very significant scientific and medical advance that could improve quality of life. As a first step towards this goal, we are working to address a condition called limbal epithelial stem cell (LESC) deficiency of the cornea, which causes painful, blinding corneal surface failure. An estimated 240 new cases of LESC deficiency occur annually in the UK. We have cultured and transplanted sheets of LESCs to improve vision in patients with chemical burns. However, many patients have very difficult to manage diseases requiring treatment with a substrate to aid cell survival, in our case human amnion. This approach can significantly improve quality of life and productivity in the workplace (patient personal communications) however, we have found the long-term therapeutic benefit to be variable. In part, we suspect, because cultured LESC sheets do not restore the normal LESC microenvironment or 'niche' destroyed by disease. Using a novel tissue engineering approach we have recently made simple constructs of the human corneal surface using protein, epithelial cells (including LESCs) and the fibroblasts that support them. Novel technology (Real Architecture for 3D Tissues - RAFT) is used to make a fibroblast-seeded collagen construct with surface toplology that mimics the in vivo stem cell niche, then corneal epithelial cells are seeded on the surface. Our data are very promising, the epithelial cells grow successfully and produce an epithelium. RAFT constructs are reproducible and have significant advantages over human amnion for LESC culture. Amnion is biologially variable and often (40% of cases) does not support the growth of healthy LESCs. We now wish to a) asses the physical and functional properties of RAFT and b) perform safety and efficacy studies so that we may proceed to first in man studies at Moorfields Eye Hospital (MEH) following this project. We already know that RAFT is strong enough to be handled and that text can be read through it. Here we will test actual mechanical strength as this must be sufficient to withstand surgery. Transparency will also be measured as RAFT should ideally be at least as transparent as the amnion we aim to replace if not better. The key driver behind this proposal is the need to establish if RAFT is safe to use and if it can improve the surface of a cornea with LESC deficiency. Without this information we cannot test RAFT in humans. Here we will work with colleagues to assess RAFT safety and efficacy in an established model of LESC deficiency. Our techniques and methods for producing RAFT constructs, presenting them to the surgeon in an accessible device and measuring clinical outcome in our model will be rigorously validated. If during this pre-clinical testing RAFT constructs can restore corneal transparency and maintain a healthy ocular surface, our data will be used to develop regulatory compliant standard operating procedures for the production of RAFT for future testing in man. This project is therefore essential to progress our research findings into clinical practice. If the project goes as planned, IoO have the experience and capacity to manufacture RAFT constructs in the 'Cells for Sight Cell Therapy Research Unit'. This new state of the art cleanroom facility, led by the applicant, is used to produce cell therapies for patients. If the project is successful, IoO and MEH will design a phase I/II safety and efficacy clinical trial to compare RAFT with LESC cultured on amnion.