University of Ferrara, Ferrara, Italy.
Veneto Eye Bank Foundation, Venice, Italy.
BMJ Open Ophthalmol. 2022 Nov;7(Suppl 2):A9. doi: 10.1136/bmjophth-2022-EEBA.21.
Recent clinical studies suggest that RPE-cell replacement therapy may preserve vision and restore retinal structure in retinal degenerative diseases. New developments enabled the differentiation of RPE cells from pluripotent stem cells. Scaffold-based methods are being tested in ongoing clinical trials for delivering these cells to the back of the eye. Borrowed materials from donor tissues can be used as cell supports in subretinal transplantation. These biological matrices resemble the extracellular matrix microenvironment of the native tissue. The Descemet's membrane (DM) is an example of high collagen-rich basement membrane (BM). The potential of this tissue in retinal repair remains to be uncovered.
To investigate human embryonic stem cell-retinal pigment epithelium (hESC-RPE) cells survival and behaviour on a decellularized DM, which may be of clinical relevance in retinal transplantation.
DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope and histology. hESC-RPE cells were seeded onto the endothelial-side surface of acellular DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene, protein expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate.
Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. hESC-RPE cell attachment 6 days post-seeding and proliferation rates over the acellular DM were similar to hESC-RPE cells cultured on tissue culture inserts.On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell graft showed the characteristic RPE morphology. The expression of typical RPE genes, proper protein localization and key growth factor secretion further confirmed the correct RPE phenotype. The viability of the cells was maintained for up to 4 weeks in culture.
Acellular DM was shown to be capable of sustaining hESC-RPE cells growth, thus confirming to be potentially a valid alternative to the Bruch's membrane.Further in vivo studies will need to verify if this product can represent a feasible tool to deliver RPE cells in the back of the eye.Our study highlights the possibility of recycling unsuitable corneal tissues, which would otherwise be discarded by the eye banks for clinical application.
最近的临床研究表明，RPE 细胞替代疗法可能在视网膜退行性疾病中保持视力并恢复视网膜结构。新的发展使多能干细胞分化为 RPE 细胞成为可能。基于支架的方法正在进行中的临床试验中进行测试，以将这些细胞递送到眼睛的后部。从供体组织中借用的材料可作为视网膜下移植的细胞支持物。这些生物基质类似于天然组织的细胞外基质微环境。Descemet 膜 (DM) 就是富含高胶原蛋白的基底膜 (BM) 的一个例子。该组织在视网膜修复中的潜力尚待发掘。
研究人胚胎干细胞-视网膜色素上皮 (hESC-RPE) 细胞在去细胞化 DM 上的存活和行为，这可能与视网膜移植有关。
从人供体角膜中分离出 DM，并使用胰蛋白酶处理。通过原子力显微镜和组织学评估 DM 表面拓扑结构和去角质方法的效率。将 hESC-RPE 细胞接种到无细胞 DM 的内皮表面上，以确定该膜支持 hESC-RPE 细胞培养的潜力，同时保持其活力。通过测量跨上皮电阻来评估 hESC-RPE 单层的完整性。评估 RPE 特异性基因、蛋白质表达和生长因子分泌，以确认细胞在新基质上的成熟和功能。
胰蛋白酶处理不影响组织的完整性，从而确保了一种可靠的方法来标准化去细胞化 DM 的制备。hESC-RPE 细胞在接种后 6 天的附着和在无细胞 DM 上的增殖率与在组织培养插入物上培养的 hESC-RPE 细胞相似。在新基质上，hESC-RPE 细胞成功形成了具有成熟紧密连接的完整单层。所得细胞移植物表现出典型的 RPE 形态。典型 RPE 基因的表达、适当的蛋白质定位和关键生长因子的分泌进一步证实了正确的 RPE 表型。细胞在培养中可保持活力长达 4 周。
无细胞 DM 能够维持 hESC-RPE 细胞的生长，因此证实它可能是 Bruch 膜的有效替代品。需要进一步的体内研究来验证该产品是否可以作为在眼睛后部递送 RPE 细胞的可行工具。我们的研究强调了回收不合适的角膜组织的可能性，否则这些组织会被眼库丢弃用于临床应用。
