Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Summary

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Abstract

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.

Introduction

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 disease^2,3. RPE replacement therapy is one of the most promising treatment regimens for these diseases^4,5,6,7. 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 efficacy^10,11. 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 thawing^13,14,15. Many factors considerably affect the outcomes of cryopreservation, particularly during the freezing stage^16,17. 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 pigmentation^8,15,18, whereas others were frozen at other time points^9,19,20,21. 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 ability^8,17,22. 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.

Protocol

1. Cell dissociation

1. Maintain RPE cells as previously described^17,22.
   NOTE: All cells are grown at 37 °C in a 5% CO₂ atmosphere throughout the duration of the protocols.
2. Prepare the required amount of PBS and culture medium in a 37 ° C water bath and place the cell dissociation reagent at room temperature.
3. Discard the culture solution and wash the plates twice with 1 mL of preheated PBS per well.
4. Add 1 mL of the cell dissociation reagent to the 6-well plates and digest the cells at 37 °C for 15 min. After incubation, observe the cells that shrink and shine at the edges under a microscope to confirm the termination of digestion.
   NOTE: Trypsin-based dissociation is not recommended as it gives low cell viability.
5. Pipette up and down gently 10x with a 1 mL pipette to dissociate the cells, and dilute the cell suspension with preheated culture medium (without Y-27632) at a ratio of 1:10. Then, centrifuge the cells at room temperature for 3 min at 250 × g.
6. Quickly pour out the supernatant after centrifugation, gently resuspend the cell pellet in 2 mL of the culture medium, and resuspend the cells 10-15x with a pipette.
7. Filter the cell suspension with a 40 µm cell strainer to obtain a single-cell suspension and calculate the number of cells.
   ​NOTE: A single-cell suspension is essential for accurate cell counting and uniform cell seeding density after thawing.

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.

1. Thaw one vial of basement membrane matrix solution on ice at 4 °C overnight. Dilute the matrix into 12 mL of cold DMEM/F-12 and mix well. Add one coverslip per well, and coat each well of a 24-well plate with 0.25 mL of the diluted solution. Incubate the plate for 1 h at room temperature or overnight at 4 °C before use and aspirate the coating solution just before plating the cells.
   NOTE: Instructions for aliquot volume are lot-specific based on the protein concentration and are found in the product specification sheet.
2. Seed the RPE single cells from step 1.7 on the basement membrane matrix-coated coverslips at a density of 10⁵/cm² in 1 mL of culture medium. Refresh the culture medium every 2-3 days.
3. At indicated time points (1, 3, 5, 7, and 11 days after passage), incubate RPE cells with 10 μM EdU in the medium for 24 h. Fix, permeabilize, and stain the cells with the reaction cocktail as described in the manual to detect incorporated EdU.
4. Capture images from five random fields under a fluorescence microscope and calculate the percentage of EdU-positive cells. Plot the corresponding EdU-positive proportion-time curves to generate a growth curve. Then, determine the freezing window or the exponential phase for each cell line according to the growth curve.
   ​NOTE: Reestablished hexagonal cell morphology indicates that cells exit the exponential phase.

3. Cryopreservation

1. Following steps 1.1 to 1.7, except that the digestion time decreases to 5 min, centrifuge the cell suspension at room temperature for 3 min at 250 × g.
2. Discard the supernatant by quickly pouring after centrifugation and gently resuspend the cell pellet in the cryopreservation medium to a density of 2 × 10⁶ cells/mL and transfer 1 mL of the cell suspension to 1.2 mL cryogenic vials.
3. Immediately place the cryogenic vials in a freezing container, freeze at −80 °C overnight to achieve a cooling rate of −1 °C/min, and then transfer the vials to liquid nitrogen for long-term storage.

4. Thawing

1. Warm the culture medium in a 37 °C water bath and prefill 10 mL of prewarmed culture medium into a 15 mL tube.
2. Rapidly thaw the cryogenic vials directly taken from the liquid nitrogen storage using an automated thawing system.
3. Drip 0.5-1 mL of preheated culture medium into the cryogenic vial to ensure that the frozen cells gradually adapt to the new environment. Then, transfer 1.5-2 mL of the cell suspension to 10 mL of the culture medium in a 15 mL tube and centrifuge the cells at 250 × g for 3 min at room temperature.
4. Discard the supernatant by quickly pouring after centrifugation, resuspend the pellet in 2 mL of preheated culture medium, and count the number of cells using a hemocytometer to determine recovery and survival rates using standard trypan blue exclusion (0.4% trypan blue stain).
5. Culture the cells on basement membrane matrix-coated surfaces at a density of 10⁵ viable cells/cm² in a culture medium supplemented with Y-27632 (final concentration: 10 µM). Remove Y-27632 after 24 h.
6. To determine the attachment rate, dissociate the cells again and count the number of cells 24 h after thawing.

5. Validation of the optimal freezing phase

1. To validate the optimal freezing phase determined in step 2.4, thaw RPE cells frozen at different time points and culture for 28 days. Harvest the cells for qPCR and immunostaining analysis to evaluate the expression of RPE markers as previously described¹⁷.

Representative Results

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.

Discussion

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.

Materials

Name	Company	Catalog Number	Comments
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