This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.
Retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2′-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.
The retinal pigment epithelium (RPE) is a pigmented monolayer of cells required for maintaining the proper function of the retina1. RPE dysfunction and death are closely associated with many retinal degenerative diseases, including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease2,3. RPE replacement therapy is one of the most promising treatment regimens for these diseases4,5,6,7. A stable supply of donor RPE cells is vital for cell therapy. Human embryonic stem cell (hESC)-derived RPE cells are an ideal cell source for cell therapy because they mimic the function of primary RPE cells and can produce a theoretically unlimited supply8. However, the differentiation process is laborious and the shelf-life of the obtained RPE cells is relatively short because of subsequent epithelial-mesenchymal transition (EMT). Therefore, the cryopreservation of hESC-derived RPE cells is an indispensable step required for long-term storage and on-demand distribution9.
Cryopreservation-induced cellular damage can inadvertently compromise therapeutic efficacy10,11. Therefore, recent studies on cryopreservation have proposed that optimal cryogenic storage conditions should be determined when designing cell therapies12. Successful cryopreservation guarantees efficient cell recovery, high viability, and cell function restoration after the freeze-thaw cycle. However, previous studies on the cryopreservation of the adherent monolayers of mammalian cells have reported highly variable (35%-95%) survival rates after thawing13,14,15. Many factors considerably affect the outcomes of cryopreservation, particularly during the freezing stage16,17. Recent research showed that RPE cells frozen at different time points exhibited varied recovery after thawing17. To the best of our knowledge, studies on the determination of the optimal freezing time window for stem cell-derived RPE cells are lacking. In different studies, the cells were frozen at various stages: some cells were frozen shortly after passaging or before confluency or pigmentation8,15,18, whereas others were frozen at other time points9,19,20,21. Furthermore, there is no clear evidence of whether the phase or stage of RPE cells used for cryopreservation affects RPE function after thawing. In our previous study, we demonstrated for the first time that the exponential phase of cell growth (P2D5) is the best stage for the cryopreservation of hESC-derived RPE cells in terms of cell viability and the recovery of cellular properties and functions17.
The method established here aims to cryopreserve hESC-derived RPE at an optimal stage to achieve the best preservation in terms of cell viability and function after thawing. Using the 5-ethynyl-2'-deoxyuridine (EdU) labeling assay to detect the exponential phase of DNA synthesis before cryopreservation, thawed RPE cells exhibited >80% viability and attachment rate, RPE-specific gene expression, polarized cell morphology, pigment epithelium-derived factor secretion, appropriate transepithelial resistance, and phagocytic ability8,17,22. Although this protocol focuses on hESC-derived RPE cells and not all therapeutic cells are equally cryopreserved, the strategy of freezing in the exponential phase may be applied to many other therapeutic cells.
1. Cell dissociation
2. Determination of the optimal cell stage for cryopreservation
NOTE: Because the cell state varies between differentiation methods and cell lines, the exponential phase of RPE cells cultured at different laboratories should be determined before freezing.
3. Cryopreservation
4. Thawing
5. Validation of the optimal freezing phase
Here, hESC-derived RPE cells at P1D35 were passaged and seeded at a density of 105/cm2. Within a week of seeding, the characteristic hexagonal morphology and pigmentation were lost during the lag phase (approximately 2 days). RPE cells gradually readopted the hexagonal morphology in the exponential phase (approximately 5 days, Figure 1A) and entered the deceleration phase (approximately 6 days) with a more polygonal morphology. If cell culturing was continued for another week, cell proliferation significantly decreased, and cell-cell junctions played a primary role, with the edges lit up; at this time, the cells were no longer in the exponential phase (Figure 1A, P2D11). In addition to cell morphology, the EdU cell proliferation assay was performed to help determine the phase. Figure 1B demonstrates that P2D5 cells exhibited a higher proliferation rate (exponential phase), whereas P2D11 cells had entered the deceleration phase.
After freezing in liquid nitrogen for 1-3 months, RPE cells were thawed. Upon thawing, some cells degraded into subcellular debris, some cells underwent apoptosis, and the remaining cells survived. After 24 h, RPE cells frozen at P2D5 exhibited a higher attachment rate than those frozen at other time points (Figure 2A). Furthermore, they exhibited the characteristic hexagonal morphology of mature RPE cells with distinct cell-cell adhesions (tight junctions) as early as 14 days after thawing. In contrast, cells frozen at other time points generally adopted a fibroblastic phenotype (Figure 2A), indicating that they were undergoing a relatively longer period of EMT transition. Twenty-eight days after thawing, RPE frozen at P2D5 showed higher expressed and more properly localized RPE cell markers, as shown by qPCR (Figure 2B) and immunofluorescence staining (Figure 2C,D).
To test the stability of this protocol in different cryopreservation media, the performance of two cryopreservation media applied in the protocol was compared. The result showed that the two cryopreservation media performed equally well in achieving high cell viability and attachment post thaw (Figure 3).
Figure 1: Representative images of ready-to-freeze RPE cells. (A) RPE cells at P2D5 exhibited less hexagonal morphology and pigmentation than those at P2D11. (B) RPE cells at P2D5 exhibited a higher proliferation rate than those at P2D11, as revealed by the EdU assay. Scale bars = 100 µm. Abbreviations: RPE = retinal pigment epithelium; BF = brightfield; EdU = 5-ethynyl-2'-deoxyuridine. Please click here to view a larger version of this figure.
Figure 2: Representative images of RPE cells after thawing. (A) RPE cells frozen at P2D5 exhibited better attachment 24 h after thawing (left column) and exhibited hexagonal morphology earlier-14 days after thawing (right column). Characteristic RPE cells with compacted morphology (arrowheads) in the P2D5 group, unsatisfactory fibroblast-like morphology (arrows) in the P2D11 group, and cell debris (yellow arrows) are highlighted. (B–D) These images are modified from Zhang et al17. (B,C) RPE cells frozen at P2D5 exhibited higher mRNA and protein expression levels of typical RPE marker genes 28 days after thawing. Statistical differences are evaluated with two-way ANOVA with Bonferroni's post hoc test. *P < 0.05. (D) Immunostaining analysis along the apical-to-basal axis 28 days after thawing showed better polarity of RPE cells frozen at P2D5. Nuclei were counterstained with DAPI. Scale bars = 50 µm (A), 25 µm (C,D). Abbreviations: RPE = retinal pigment epithelium; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 3: Performance of two cryopreservation media applied in the protocol. (A) The cell viability rate was measured upon thawing of hESC-derived RPE cells frozen at P2D5. (B) The brightfield images of RPE cells thawing from two different freezing media (#1 and #2) 24 h post thaw. Scale bar = 100 µm. Abbreviations: RPE = retinal pigment epithelium; hESC = human embryonic stem cell. Please click here to view a larger version of this figure.
In the present study, a successful freeze-thaw protocol for hESC-derived RPE cells for research and clinical needs is described. Unlike the immortalized RPE cell line, ARPE-19, RPE cells with proper characteristic epithelial phenotype and function, like stem cell derived-RPE cells, are more sensitive to cryopreservation. Less than 32% of the cells remained at 24 h post thaw if not properly preserved17. Cryopreservation timing is a critical parameter. An established view for immortalized cell cryopreservation is to freeze cells at the exponential growth phase. Differentiated cells exit the cell cycle. Therefore, the effect of the growth phase for cryopreservation has been seldom considered for differentiated cells. We demonstrated that the hESC-differentiated RPE cells regained the capacity to proliferate for a short time and entered an exponential stage after passaging. Improved outcomes can be achieved by cryopreserving RPE cells during the exponential phase17.
The limitation of this protocol is that only two types of cryopreservation media were tested. Different cryopreservation media may prevent cryoinjury in different ways23; therefore, cryopreservation media may affect the optimal stage of RPE cell cryopreservation. Whether cryopreservation media affect the optimal freezing time should be further evaluated.
The cryopreservation protocol presented in this study can easily be adapted for use at different laboratories by determining the exponential phase of RPE cells before freezing them, making it more independent of specific culturing periods, differentiation methods, or cell lines. In addition to assessing cell morphology, the EdU assay provides a straightforward assessment to ensure that the cells are preserved in the exponential phase.
The current protocol is a simple and efficient method for cryopreserving RPE cells. This strategy may be applied to cryopreserve other types of differentiated cells.
The authors have nothing to disclose.
This work was funded by the National Natural Science Foundation of China (81970816) to Mei Jiang; the National Natural Science Foundation of China (82201223) to Xinyue Zhu; and the Science and Technology Innovation Action Plan of the Shanghai Science and Technology Commission (2014090067000) to Haiyun Liu.
40 μm Cell strainer | Corning | 431750 | |
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 Dye | Thermo Fisher Scientific | C10337 | |
Cryo freezing container | Nalgene | 5100-0001 | |
CryoStor CS10 | Biolife Solutions | 07930 | cryopreservation medium #1 |
DPBS, no calcium, no magnesium | Thermo Fisher Scientific | 14190144 | |
Genxin | Selcell | YB050050 | cryopreservation medium #2 |
Human embryonic stem cells | provided by Wicell, USA | H9 cell line | |
Matrigel, hESC-Qualified Matrix | Corning | 354277 | basement membrane matrix |
ThawSTAR CFT2 Automated Cell Thawing System | BioLife Solutions | AST-601 | |
Trypan Blue solution 0.4% | Sigma | T8154 | |
TryPLE Select | Thermo Fisher Scientific | 12563029 | cell dissociation reagent |
XVIVO-10 medium | Lonza | BEBP04-743Q | RPE culture medium |
Y-27632 | Selleck | S1049 |