Many progenitor and stem cell-based therapies in pre-clinical development and in clinical trial have failed to achieve desired outcomes, in part, due to: low cell viability; dispersion of cells from the injection site; and/or failure of the cells to differentiate and engraft into host tissue. These problems may be exacerbated by the elevated shear stress applied to the cells as they pass through a small-bore needle. Our supposition is that these issues can be mitigated by replacing the phosphate buffered saline (PBS), which is likely employed as the cell carrier, with an injectable gelatin-based hydrogel incorporating an appropriate regulatory molecule (human epidermal growth factor). The first objectives of the study were to evaluate in vitro: the effects of injection through a 31-gauge needle on viability, proliferation, and phenotypic expression of human retinal progenitor cells (hRPCs), when employing PBS as the carrier and compare it to a gelatin-based gel as a carrier. The second objective of the work, conducted in vivo, in a rat model, was to: determine the degree of engraftment of injected cells; and evaluate markers of an immune response to the xenogeneic cells.Hydroxyphenyl propionic acid was conjugated to gelatin (Gtn-HPA) to enable in situ, enzyme-mediated covalent cross-linking, with independent control over the gelation time and degree of cross-linking. After 1 and 6 culture days, hRPC exposed to shear stress showed 50% or more reduction in viability of cells in PBS compared to cells in medium and in gel. Shear stress decreased proliferation and increased apoptosis in the PBS group by 50% or more, while cells in the gel group were protected from shear-induced apoptosis and their proliferation remained 2-fold higher than the PBS group. Also, of note was the preservation of or increase in stemness markers in the gel with hEGF group compared to cells in PBS and the gel alone, with or without shear stress. In vivo studies, performed 3 days after subretinal injection into non-immunosuppressed rats revealed: a) a greater number of cells at the injection site in the gel group, with signs of engraftment of cells to the retina; and b) a significantly lower immune response in the gel group, compared to cells injected in PBS.This work demonstrates that the shear stress experienced by hRPCs in PBS as a result of injection through a small-bore needle adversely affects cell viability, proliferation, apoptosis, phenotype, in vivo retention of cells at the injection site, migration, engraftment and immune reaction. These findings suggest the use of a biomaterial hydrogel as a replacement for PBS for retinal cell therapy.

在临床前开发和临床试验中，许多基于祖细胞和干细胞的疗法未能达到预期的结果，部分原因是：细胞生存力低；来自注射部位的细胞分散；和/或细胞不能分化并移植到宿主组织中。当细胞通过小孔针时，施加在细胞上的剪切应力升高可能会加剧这些问题。我们的假设是，可以通过使用掺入适当调节分子（人表皮生长因子）的可注射明胶基水凝胶代替磷酸盐缓冲盐水（PBS）（可能用作细胞载体）来缓解这些问题。该研究的首要目标是在体外进行评估：通过31号针头注射对生存能力，增殖，当使用PBS作为载体并将其与基于明胶的凝胶作为载体比较时，人视网膜祖细胞（hRPC）的表达和表型表达。在大鼠模型中进行的体内研究的第二个目的是：确定注射细胞的植入程度；并将羟苯基丙酸与明胶（Gtn-HPA）偶联以实现原位酶介导的共价交联，并独立控制胶凝时间和交联程度。在培养1天和6天后，与培养基和凝胶中的细胞相比，暴露于剪切应力下的hRPC在PBS中的细胞活力降低了50％或更多。剪切应力使PBS组的增殖减少，凋亡增加了50％或更多，而凝胶组的细胞受到保护，免受剪切诱导的细胞凋亡，其增殖仍比PBS组高2倍。同样，值得注意的是，与hEGF组的凝胶相比，在PBS或单独存在或不存在剪切应力的细胞中，干性标记物的保存或增加。向非免疫抑制的大鼠视网膜下注射后3天进行的体内研究显示：a）凝胶组注射部位的细胞数量更多，有向视网膜植入细胞的迹象；b）与注射PBS的细胞相比，凝胶组的免疫反应明显降低。这项研究表明，通过小口径针头注射的hRPCs在PBS中所经历的剪切应力会对细胞活力，增殖，细胞凋亡，表型，体内细胞在注射部位的滞留，迁移，植入和免疫反应。这些发现表明，将生物材料水凝胶替代PBS用于视网膜细胞治疗。