EX VIVO EXPANSION OF RPE CELLS FOR TRANSPLANTATION

视网膜色素上皮细胞（Retinal pigment epithelial cells，RPE）是维持视网膜功能的重要细胞。视网膜色素上皮的功能障碍导致年龄相关性黄斑变性（ARMD）的发病机制，在西方国家，年龄相关性黄斑变性是55岁以后永久性视力丧失的主要原因。传统的治疗，如激光光凝有一个有限的成功和光动力治疗仍然姑息。一个令人兴奋的和潜在的治愈性治疗是健康RPE的视网膜下移植。我们的初步研究表明，冻存的人羊膜（AM）在培养中可促进兔RPE的生长和适当分化。因此，我们设想通过包括以下步骤来建立新的RPE移植：步骤1：经玻璃体活检以收获自体RPE;步骤2：体外扩增收获的RPE， 步骤3：用AM进行扩张的RPE的视网膜下移植。在第一阶段SBIR中，我们建议解决这三个步骤的关键要素。目标1：在步骤2中，我们希望通过确定在离体扩增期间维持正常形态和表型所需的最小接种密度，建立在人AM上培养兔RPE的可重现方案。目标二：在步骤1中，我们希望建立一个可重复的方案，用于进行经玻璃体活检，以便可以获得足够数量的RPE，以匹配目标1中建立的接种密度。目的3：在第3步中，我们希望建立一个可重复的程序，将人AM与RPE植入兔视网膜下腔。完成 这三个目的将使我们能够在兔子中进行临床前研究，以证明将这种离体扩增的自体RPE与AM一起移植到兔子的视网膜下空间并在II期SBIR期间移植后保持正常的RPE形态和功能的安全性和有效性。成功实现这些目标将使我们能够设计这种新的基于组织的“生物制剂”，以恢复患有ARMD或其他视网膜疾病的患者的视力，其中RPE功能失调。

Retinal pigment epithelial cells (RPE) play a pivotal role in maintaining retinal function. Dysfunction of RPE contributes to the pathogenesis of age-related macular degeneration (ARMD), which accounts for the major cause of permanent visual loss after age 55 years in Western countries. Conventional treatment such as laser photocoagulation has a limited success and photodynamic treatment remains palliative. One exciting and potentially curative treatment is subretinal transplantation of healthy RPE. Our preliminary studies showed that growth and proper differentiation of rabbit RPE can be promoted by cryopreserved human amniotic membrane (AM) in culture. Thus we envision to establish new RPE transplantation by including Step 1: Transvitreal Biopsy to Harvest Autologous RPE; Step 2: Ex Vivo Expansion of Harvested RPE on AM; and Step 3: Subretinal Transplantation of Expanded RPE with AM. In the Phase I SBIR, we propose to resolve key elements of these three steps. Aim 1: In Step 2, we would like to establish a reproducible protocol for culturing rabbit RPE on human AM by determining the minimal seeding density needed to maintain normal morphology and phenotype during ex vivo expansion. Aim 2: In Step 1, we would like to establish a reproducible protocol for performing transvitreal biopsy so that sufficient numbers of RPE can be obtained to match the seeding density established in Aim 1. Aim 3: In Step 3, we would like to establish a reproducible procedure of implanting human AM with RPE into the subretinal space in rabbits. Completion of these three Aims will allow us to launch a pre-clinical study in rabbits to demonstrate the safety and eficacy of transplanting such ex vivo expanded autologous RPE together with AM to the rabbit's subretinal space and maintaining normal RPE morphology and functions after transplantation during Phase II SBIR. Success in accomplishing these goals will allow us to engineer this new tissue-based "Biologies" to restore sights in patients suffering ARMD or other retinal diseases in which RPE is dysfunctional.