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.

1. Cell dissociation
<ol>
	<li>Maintain RPE cells as previously described17,22. 
	NOTE: All cells are grown at 37 &#176;C in a 5% CO2 atmosphere throughout the duration of the protocols.</li>
	<li>Prepare the required amount of PBS and culture medium in a 37 &#176; C water bath and place the cell dissociation reagent at room temperature.</li>
	<li>Discard the culture solution and wash the plates twice with 1 mL of preheated PBS ...

Advancing Cryostorage of Human Stem Cell-Derived Retinal Pigment Epithelial Cells

<ol>
	<li>Lakkaraju, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+the+retinal+pigment+epithelium.">The cell biology of the retinal pigment epithelium.</a> Progress in Retinal and Eye Research. 78, 100846 (2020).</li><li>Mcbain, V. A., Townend, J., Lois, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+retinal+pigment+epithelial+atrophy+in+stargardt+disease.">Progression of retinal pigment epithelial atrophy in stargardt disease.</a> American Journal of Ophthamology. 154 (1), 146-154 (2012).</li><li>George, S. M., Lu, F., Rao, M., Leach, L. L., Gross, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+pigment+epithelium:+Development,+injury+responses,+and+regenerative+potential+in+mammalian+and+non-mammalian+systems.">The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems.</a> Progress in Retinal and Eye Research. 85, 100969 (2021).</li><li>Rizzolo, L. J., Nasonkin, I. O., Adelman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+cell+transplantation,+biomaterials,+and+in+vitro+models+for+developing+next-generation+therapies+of+age-related+macular+degeneration.">Retinal cell transplantation, biomaterials, and in vitro models for developing next-generation therapies of age-related macular degeneration.</a> Stem Cells Translational Medicine. 11 (3), 269-281 (2022).</li><li>Bertolotti, E., Neri, A., Camparini, M., Macaluso, C., Marigo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+as+source+for+retinal+pigment+epithelium+transplantation.">Stem cells as source for retinal pigment epithelium transplantation.</a> Progress in Retinal and Eye Research. 42, 130-144 (2014).</li><li>Da Cruz, ., L, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+1+clinical+study+of+an+embryonic+stem+cell-derived+retinal+pigment+epithelium+patch+in+age-related+macular+degeneration.">Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.</a> Nature Biotechnology. 36 (4), 328-337 (2018).</li><li>Mehat, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+embryonic+stem+cell-derived+retinal+pigment+epithelial+cells+in+macular+degeneration.">Transplantation of human embryonic stem cell-derived retinal pigment epithelial cells in macular degeneration.</a> Ophthalmology. 125 (11), 1765-1775 (2018).</li><li>Buchholz, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+directed+differentiation+of+human+pluripotent+stem+cells+into+retinal+pigmented+epithelium.">Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium.</a> Stem Cells Translational Medicine. 2 (5), 384-393 (2013).</li><li>Li, Q. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+cryopreserved+clinical+grade+hESC-RPE+cells+as+a+qualified+cell+source+for+stem+cell+therapy+of+retinal+degenerative+diseases.">Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.</a> Experimental Eye Research. 202, 108305 (2021).</li><li>Francois, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+mesenchymal+stromal+cells+display+impaired+immunosuppressive+properties+as+a+result+of+heat-shock+response+and+impaired+interferon-gamma+licensing.">Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-gamma licensing.</a> Cytotherapy. 14 (2), 147-152 (2012).</li><li>Moll, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+cryopreserved+mesenchymal+stromal+cells+display+impaired+immunomodulatory+and+therapeutic+properties.">Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties.</a> Stem Cells. 32 (9), 2430-2442 (2014).</li><li>Ekpo, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporating+cryopreservation+evaluations+into+the+design+of+cell-based+drug+delivery+systems:+an+opinion+paper.">Incorporating cryopreservation evaluations into the design of cell-based drug delivery systems: an opinion paper.</a> Frontiers in Immunology. 13, 967731 (2022).</li><li>Bailey, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+osmolytes+in+cryopreserving+adherent+neuroblastoma+(Neuro-2a)+cells.">Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells.</a> Cryobiology. 71 (3), 472-480 (2015).</li><li>Okumura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+of+a+cryopreservation+of+cultured+human+corneal+endothelial+cells.">Feasibility of a cryopreservation of cultured human corneal endothelial cells.</a> PLoS One. 14 (6), e0218431 (2019).</li><li>Pennington, B. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xeno-free+cryopreservation+of+adherent+retinal+pigmented+epithelium+yields+viable+and+functional+cells+in+vitro+and+in+vivo.">Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo.</a> Scientific Reports. 11 (1), 6286 (2021).</li><li>Woods, E. J., Thirumala, S., Badhe-Buchanan, S. S., Clarke, D., Mathew, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off+the+shelf+cellular+therapeutics:+Factors+to+consider+during+cryopreservation+and+storage+of+human+cells+for+clinical+use.">Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use.</a> Cytotherapy. 18 (6), 697-711 (2016).</li><li>Zhang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+optimal+stage+for+cryopreservation+of+human+embryonic+stem+cell-derived+retinal+pigment+epithelial+cells.">Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells.</a> Stem Cell Research &amp; Therapy. 13 (1), 454 (2022).</li><li>Leach, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell-derived+retinal+pigmented+epithelium:+a+comparative+study+between+cell+lines+and+differentiation+Methods.">Induced pluripotent stem cell-derived retinal pigmented epithelium: a comparative study between cell lines and differentiation Methods.</a> Journal of Ocular Pharmacology and Therapeutics. 32 (5), 317-330 (2016).</li><li>Reichman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+storable+retinal+organoids+and+retinal+pigmented+epithelium+from+adherent+human+iPS+cells+in+Xeno-free+and+feeder-free+conditions.">Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in Xeno-free and feeder-free conditions.</a> Stem Cells. 35 (5), 1176-1188 (2017).</li><li>Brandl, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+characterisation+of+retinal+pigment+epithelium+(RPE)+cells+derived+from+human+induced+pluripotent+stem+cells+(hiPSC).">In-depth characterisation of retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC).</a> Neuromolecular Medicine. 16 (3), 551-564 (2014).</li><li>Hongisto, H., Ilmarinen, T., Vattulainen, M., Mikhailova, A., Skottman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xeno-+and+feeder-free+differentiation+of+human+pluripotent+stem+cells+to+two+distinct+ocular+epithelial+cell+types+using+simple+modifications+of+one+method.">Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method.</a> Stem Cell Research &amp; Therapy. 8 (1), 291 (2017).</li><li>Foltz, L. P., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+directed+differentiation+of+retinal+pigment+epithelial+cells+from+human+embryonic+or+induced+pluripotent+stem+cells.">Rapid, directed differentiation of retinal pigment epithelial cells from human embryonic or induced pluripotent stem cells.</a> Journal of Visualized Experiments: JoVE. (128), 10.3791/56274 (2017).</li><li>Marcantonini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+cryoprotective+and+cytoprotective+agents+in+cryopreservation:+a+focus+on+melatonin.">Natural cryoprotective and cytoprotective agents in cryopreservation: a focus on melatonin.</a> Molecules. 27 (10), 3254 (2022).</li></ol>

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 cryop...

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&#39;-deoxyuridine (EdU) labeling assay to detect the exponential phase of DNA synthesis before cryopreservation, thawed RPE cells exhibited &#62;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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40 &#956;m Cell strainer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10337</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo freezing container</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>Biolife Solutions</td>
			<td>07930</td>
			<td>cryopreservation medium #1</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Genxin</td>
			<td>Selcell</td>
			<td>YB050050</td>
			<td>cryopreservation medium #2</td>
		</tr>
		<tr>
			<td>Human embryonic stem cells</td>
			<td>provided by Wicell, USA</td>
			<td>H9 cell line</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, hESC-Qualified Matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td>basement membrane matrix</td>
		</tr>
		<tr>
			<td>ThawSTAR CFT2 Automated Cell Thawing System</td>
			<td>BioLife Solutions</td>
			<td>AST-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 0.4%</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>TryPLE Select</td>
			<td>Thermo Fisher Scientific</td>
			<td>12563029</td>
			<td>cell dissociation reagent</td>
		</tr>
		<tr>
			<td>XVIVO-10 medium</td>
			<td>Lonza</td>
			<td>BEBP04-743Q</td>
			<td>RPE culture medium</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells at the optimal stage

Retinal pigment epithelial&#160;(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&#160;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&#8242;-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.

Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing

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

Our lab aims to better understand the molecular mechanism of retinal diseases, with a focus on unmet medical needs of patients. We aim to develop effective treatments, such as stem-cell-based therapies, for them. Cryopreservation of stem-cell-derived RPE cells is an important step for RPE replacement therapy.Currently, the cells are frozen at various stages, resulting in highly variable survival rates for thawing. This is needed to determine the optimal cell stage for freezing these cells. Our protocol provides a simple, efficient, and inexpensive ways of freezing RPE cells by determining the exponential phase of RPE cells for freezing.It's less dependent of differentiated methods or cell lines.

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 anothe...

Watch this Scientific Journal Video about Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage at JoVE.com

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

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...

cryopreservation-human-embryonic-stem-cell-derived-retinal-pigment