Corneal Regeneration Lab, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Immunologic Monitoring and Cellular Products Laboratory, Hillman Cancer Centre, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Stem Cell Res Ther. 2024 Jan 8;15(1):11. doi: 10.1186/s13287-023-03626-8.
Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities.
A total of 24 donor corneal rims with informed consent were used-18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC's conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring.
By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters-stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)-were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring.
We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC-in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.
成人角膜基质中的间充质干细胞（称为角膜基质干细胞，CSSCs）可抑制角膜炎症和瘢痕形成，并在临床前角膜损伤模型中恢复角膜透明度。这种细胞疗法可以减轻对角膜移植治疗角膜混浊的供体材料的严重依赖。在此，我们建立了 CSSC 分离、扩增和低温保存的良好生产规范（GMP）方案，并开发了体外质量控制（QC）指标，用于评估 CSSC 在治疗角膜混浊中的抗瘢痕形成能力。
共使用 24 个有知情同意的供体角膜边缘-18 个用于 CSSC 培养的 GMP 优化和 QC 测定开发，而其余 6 个供体角膜边缘的 CSSC 在 GMP 优化条件下培养，并用于 QC 验证。通过使用 xCELLigence 实时细胞分析仪监测电阻抗变化来检测细胞活力、生长、基质附着、干细胞表型和向基质角膜细胞的分化。通过使用鼠巨噬细胞 RAW264.7 细胞的破骨细胞生成测定法测试 CSSC 条件培养基的抗炎活性。使用 Algerbrush 打磨器机械消融引起的前基质损伤的小鼠模型验证体内抑制瘢痕形成的结果。
通过比较评估各种符合 GMP 的试剂与实验室基础方案中使用的相应非 GMP 研究级化学品，我们最终确定了涵盖供体角膜缘基质组织处理、酶消化、原代 CSSC 培养和冷冻保存的 GMP 方案。在建立体外 QC 指标时，将 ABCG2 和巢蛋白的稳定性和抗炎能力（炎症率）这两个参数纳入到一个新的公式中，为每个 CSSC 批次计算一个瘢痕指数（SI）。与体内抑制瘢痕形成的结果相关，SI<10 的 CSSC 批次具有 50%的瘢痕减少潜力，而 SI>10 的细胞无效抑制瘢痕形成。
我们建立了一个完整的符合 GMP 的供体 CSSC 培养方案，这对于临床级细胞制造至关重要。开发了一种新的体外 QC-体内效力相关性，以预测供体 CSSC 在治疗角膜混浊中的抗瘢痕形成功效。该方法适用于其他基于细胞的疗法和药物治疗。
