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

眼睛前面的角膜是我们观察世界的窗口。如果透明度受损，可能会出现视力障碍甚至失明。全世界有1000万人因角膜瘢痕而失明，并且由于供体组织可用性不足而被拒绝进行挽救视力的角膜移植。在实验室中“生长”角膜成分的能力将代表一个非常重要的科学和医学进步，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称为角膜缘上皮干细胞（LESC）缺乏的情况，这种情况会导致疼痛，失明的角膜表面衰竭。据估计，英国每年发生240例LESC缺乏症。我们培养并移植LESC片，以改善化学烧伤患者的视力。然而，许多患者很难控制需要用基质治疗的疾病，以帮助细胞存活，在我们的情况下是人类羊膜。这种方法可以显着提高生活质量和工作场所的生产力（患者个人通信），但是，我们发现长期治疗益处是可变的。我们怀疑，部分原因是培养的LESC片不能恢复被疾病破坏的正常LESC微环境或“生态位”。使用一种新的组织工程方法，我们最近使用蛋白质，上皮细胞（包括LESC）和支持它们的成纤维细胞制作了人类角膜表面的简单结构。使用新技术（3D组织的真实的架构- RAFT）制备具有模拟体内干细胞龛的表面拓扑学的成纤维细胞接种的胶原构建体，然后将角膜上皮细胞接种在表面上。我们的数据非常有希望，上皮细胞成功生长并产生上皮。RAFT构建体是可重复的，并且对于LESC培养而言比人羊膜具有显著优势。羊膜在生物学上是可变的，通常（40%的病例）不支持健康LESC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后在Moorfields眼科医院（MEH）进行首次人体研究。我们已经知道RAFT的强度足以被处理，并且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。还将测量透明度，因为RAFT理想情况下应至少与我们旨在替换的羊膜一样透明，如果不是更好的话。该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缺陷的角膜表面。没有这些信息，我们就无法在人类身上测试RAFT。在这里，我们将与同事合作，评估RAFT在LESC缺陷的既定模型中的安全性和有效性。我们生产RAFT结构的技术和方法，将它们以可访问的设备呈现给外科医生，并在我们的模型中测量临床结果，这些技术和方法将得到严格的验证。如果在临床前测试期间，RAFT结构可以恢复角膜透明度并保持健康的眼表，我们的数据将用于制定符合法规的标准操作程序，用于生产RAFT，用于未来的人体测试。因此，该项目对于将我们的研究成果应用于临床实践至关重要。如果该项目按计划进行，IoO有经验和能力在“视觉细胞治疗研究单元”中制造RAFT构建体。这种由申请人领导的最先进的洁净室设施用于为患者生产细胞疗法。如果该项目成功，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与在羊膜上培养的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.