Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable

Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo

Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN2) storage has been confirmed for one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.

Age-related macular degeneration (AMD) affects over 190 million people worldwide1 and is the leading cause of blindness in the elderly population of European descent. Vision loss due to non-neovascular AMD is associated with the degeneration of the retinal pigmented epithelial (RPE) cells and the consequent dysfunction of the overlying photoreceptors in the macula, the region of the retina responsible for high acuity vision. In an effort to ameliorate the effects of non-neovascular AMD and to delay the progression of this disease, for which no approved treatment currently exists, one therapeutic strategy involves the delivery of exogenous RPE cells into the subretinal space to replace the degenerated native epithelium4. Recent advancements in regenerative medicine have identified human pluripotent stem cells (PSC) as a potentially unlimited source of therapeutic RPE cells. Pre-clinical animal studies have demonstrated that PSC-derived RPE cells may survive and integrate with the host RPE when injected as a cellular suspension and that survival and retinal interaction is improved when RPE cells are implanted as an epithelial monolayer supported by a substrate.

Therapeutic implants comprised of PSC-derived RPE cells supported by a proteinaceous sheet or an implantable scaffold also offer the advantage of delivering a monolayer of mature RPE cells specifically to a diseased region in the eye. Results from multiple Phase I clinical trials suggest that such implants represent a promising approach for the treatment of degenerative eye diseases. In 2018, Kashani et al. reported interim results from a Phase I/IIa clinical trial, which found that vison loss did not progress in AMD patients who received human embryonic stem cell (hESC)-derived RPE cells seeded on a parylene-C scaffold. Parylene-C is an inert, biostable polymer commonly used in biomedical applications such as coating stents, pacemakers, and retinal prostheses and has been engineered to support RPE cells. Furthermore, post-operative data from the Kashani et al. study suggest that implanted RPE cells can support the host neural retina and improve the fixation capabilities of the implanted eye compared to the untreated, contralateral eye.

RPE implants currently employed in clinical trials are maintained in culture until the time of administration, which severely limits shelf life, restricts transport to treatment sites, and requires the coordination of surgical procedures with product manufacture. Cryopreservation of RPE-seeded implants would significantly increase product availability by extending shelf-life, increasing the flexibility of manufacturing and clinical schedules, and enabling wide-spread on-demand distribution. The capacity to cryopreserve these implants and administer an adherent cellular therapy immediately post-thaw has been identified as a crucial milestone for the commercialization of such therapies4. However, previous attempts to cryopreserve or vitrify adherent monolayers of mammalian cells have resulted in highly variable (35–89%) post-thaw survival rates and no clinical application of a cryopreserved, adherent RPE cell-based therapy has yet been reported.

Here they describe a novel, xeno-free method for the successful cryopreservation and thaw of an adherent monolayer of hESC-derived Retinal pigmented Epithelial cells on a Parylene Scaffold (REPS) and perform in vitro and in vivo characterization of REPS produced using this protocol. Several cryopreservation parameters were investigated including freeze rate, the concentration of the cryoprotective agent, and post-thaw rinse solution. Surprisingly, the most important factor for the successful cryopreservation of REPS was the duration of adherent cell culture prior to the time of the freeze. Clinical-grade REPS cryopreserved using the optimized parameters reproducibly exhibit ≥ 95% post-thaw survival, robust expression of RPE-specific genes, neurotrophic factor secretion, as well as demonstrating stability through one year of storage in liquid nitrogen (LN2). Furthermore, indications of RPE cell survival, maturation, and phagocytic function in vivo were observed for REPS that were immediately implanted into the subretinal space post-thaw. Together, these findings provide a path toward the clinical manufacture and implementation of cryopreserved adherent RPE-based therapeutics.

Pigmented REPS exhibit poor cell survival in response to cryopreservation.

(a) Schematic of overall process to seed, cryopreserve, and thaw a mature monolayer of Retinal pigmented Epithelial cells adhered to a Parylene Scaffold (REPS). (b) Representative image of REPS exhibiting pigmentation at 28 days post-seeding (DPS) (Bright field; scale bar, 1 mm). (c–e) REPS were cryopreserved at 30 DPS. (c) Cryopreservation of pigmented REPS results in significantly reduced viability one day post-thaw (DPT) compared to the non-cryopreserved control. (d) Pigmented REPS exhibit significantly reduced metabolic activity 1 DPT compared to levels measured prior to cryopreservation. Relative Fluorescence Units (RFU). (e) Cryopreservation of pigmented REPS results in severe hyperpigmentation within the first 24 hours post-thaw (Bright field; scale bar, 100 µm). (f) Optimized parameters to cryopreserve REPS were identified by analysis of (i) post-seeding culture period, (ii) cryopreservation medium and freeze rate, and (iii) post-thaw rinse solution. Optimal parameters are identified in bold (see also Supplementary Figs. S1, S2). Error bars indicate standard deviation. ***P < 0.001, unpaired two-tailed t-test.

Cryopreserved REPS retain high viability, cellular identity, and secretory function post-thaw. (a) Schematic of overall research design indicates the characterization assays conducted on the designated day post-seeding (DPS) or day post-thaw (DPT). Non-cryopreserved REPS were maintained in culture to provide an age-matched control for each day of sample collection. (b) Cryopreserved REPS exhibit 95 ± 2.3% viability as measured by propidium iodide exclusion at 1 DPT (n = 26); viability of non-cryopreserved control REPS (n = 7) averaged to 97 ± 0.89% (mean ± SD., *P < 0.05, unpaired two-tailed t-test). (c) REPS exhibit epithelial, cuboidal morphology prior to cryopreservation (Pre-Cryo), immediately post-thaw (PT), and 1 DPT. (Phase contrast, scale bar, 100 µm.) (d) Amount of secreted PEDF by non-cryopreserved control REPS (black triangles) and cryopreserved/thawed REPS (gray circles) as measured by ELISA. Thawed REPS exhibited a significant increase in secreted PEDF at 7 DPT compared to Pre-Cryopreservation, and also at 21 DPT compared to 7 DPT (***P < 0.001). A significant difference was detected between thawed REPS and age-matched non-cryopreserved controls at 7 DPT (**P < 0.01) and at 21 DPT (*P < 0.05). (Pre-Cryo, n = 93; Non-Cryo, n = 10; Cryo, n = 33. Each data point represents the mean of two technical replicates. Horizontal red bar indicates mean of the biological replicates. Independent samples Kruskal–Wallis test with pairwise comparisons.) (e) Non-cryopreserved control REPS (Non-Cryo) and cryopreserved/thawed REPS (Cryo) display uniform pigmentation and similar coverage of the parylene scaffold over the course of the analysis (Bright field; scale bar, 1 mm). (f) RT-qPCR analysis of RPE marker genes (TYRP1, RPE65) and an EMT marker (S100A4) for non-cryopreserved control REPS (black triangles) and cryopreserved/thawed REPS (gray circles). No significant differences were observed between cryopreserved REPS and age-matched non-cryopreserved controls for each target gene (P > 0.05). A significant increase in RPE65 was detected for both cryopreserved/thawed REPS and non-cryopreserved control REPS by 21 DPT or 28 DPS, respectively, compared to prior to cryopreservation and immediately post-thaw (**P < 0.01, independent samples Kruskal–Wallis test with pairwise comparisons). (g) En face (left, middle) and orthogonal (right) renderings of immunostained apical (ZO-1) and basolateral (BEST1) markers in cryopreserved REPS after 7–10 DPT. (Confocal Z-stacks, scale bar, 50 µm.) Error bars indicate standard deviation1.

  1. Pennington, B.O., Bailey, J.K., Faynus, M.A. et al. Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo. Sci Rep11, 6286 (2021).