Name: Autor destacado: Desarrollo de un método eficiente y factible de congelación de células epiteliales pigmentarias de la retina
Uploaded: 2023-11-03T13:20:00
Description: Watch this Scientific Journal Video about Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage at JoVE.com

Este trabajo fue financiado por la Fundación Nacional de Ciencias Naturales de China (81970816) a Mei Jiang; la Fundación Nacional de Ciencias Naturales de China (82201223) a Xinyue Zhu; y el Plan de Acción de Innovación Científica y Tecnológica de la Comisión de Ciencia y Tecnología de Shanghái (2014090067000) a Haiyun Liu.

1. Disociación celular
<ol><li>Mantener las células RPE como se ha descrito anteriormente17,22. NOTA: Todas las células se cultivan a 37 °C en una atmósfera con un 5% deCO2 durante toda la duración de los protocolos.</li>
	<li>Prepare la cantidad requerida de PBS y medio de cultivo en un baño de agua a 37 ° C y coloque el reactivo de disociación celular a temperatura ambiente.</li>
	<li>Deseche la solución de cultivo y l...

Avance del crioalmacenamiento de células epiteliales pigmentarias de la retina derivadas de células madre humanas

<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>

En el presente estudio, se describe un protocolo exitoso de congelación-descongelación para células RPE derivadas de hESC para necesidades clínicas y de investigación. A diferencia de la línea celular de EPR inmortalizada, ARPE-19, las células EPR con el fenotipo epitelial y la función característicos adecuados, como las células EPR derivadas de células madre, son más sensibles a la criopreservación. Menos del 32% de las células permanecieron a las 24 h después de la descongelación si no se conservaban ad...

El epitelio pigmentario de la retina (EPR) es una monocapa pigmentada de células necesarias para mantener el correcto funcionamiento de la retina. La disfunción y la muerte del EPR están estrechamente asociadas con muchas enfermedades degenerativas de la retina, como la degeneración macular relacionada con la edad, la retinosis pigmentaria y la enfermedad de Stargardt 2,3. La terapia de reemplazo del EPR es uno de los esquemas de tratamiento más prometedores para estas enfermedades 4,5,6,7. Un suministro estable de células RPE del donante es vital para la terapia celular. Las células EPR derivadas de células madre embrionarias humanas (hESC) son una fuente celular ideal para la terapia celular porque imitan la función de las células primarias EPR y pueden producir un suministro teóricamente ilimitado8. Sin embargo, el proceso de diferenciación es laborioso y la vida útil de las células EPR obtenidas es relativamente corta debido a la posterior transición epitelio-mesenquimal (EMT). Por lo tanto, la criopreservación de células EPR derivadas de hESC es un paso indispensable necesario para el almacenamiento a largo plazo y la distribución bajo demanda9.
El daño celular inducido por la criopreservación puede comprometer inadvertidamente la eficacia terapéutica 10,11. Por lo tanto, estudios recientes sobre criopreservación han propuesto que las condiciones óptimas de almacenamiento criogénico deben determinarse al diseñar terapias celulares12. La criopreservación exitosa garantiza una recuperación celular eficiente, alta viabilidad y restauración de la función celular después del ciclo de congelación-descongelación. Sin embargo, estudios previos sobre la criopreservación de las monocapas adherentes de células de mamíferos han reportado tasas de supervivencia altamente variables (35%-95%) después de la descongelación 13,14,15. Muchos factores afectan considerablemente los resultados de la criopreservación, particularmente durante la etapa de congelación16,17. Investigaciones recientes mostraron que las células EPR congeladas en diferentes puntos de tiempo exhibieron una recuperación variada después de la descongelación17. Hasta donde sabemos, faltan estudios sobre la determinación de la ventana de tiempo de congelación óptima para las células RPE derivadas de células madre. En diferentes estudios, las células se congelaron en varias etapas: algunas células se congelaron poco después del paso o antes de la confluencia o pigmentación 8,15,18, mientras que otras se congelaron en otros puntosde tiempo 9,19,20,21. Además, no hay pruebas claras de si la fase o el estadio de las células EPR utilizadas para la criopreservación afectan a la función del EPR después de la descongelación. En nuestro estudio anterior, demostramos por primera vez que la fase exponencial de crecimiento celular (P2D5) es la mejor etapa para la criopreservación de células EPR derivadas de hESC en términos de viabilidad celular y recuperación de propiedades y funciones celulares17.
El método establecido aquí tiene como objetivo criopreservar el EPR derivado de hESC en una etapa óptima para lograr la mejor preservación en términos de viabilidad y función celular después de la descongelación. Utilizando el ensayo de marcaje de 5-etinil-2'-desoxiuridina (EdU) para detectar la fase exponencial de la síntesis de ADN antes de la criopreservación, las células de EPR descongeladas mostraron una tasa de viabilidad y adhesión del >80%, expresión génica específica de EPR, morfología celular polarizada, secreción de factor derivada del epitelio pigmentario, resistencia transepitelial adecuada y capacidad fagocítica 8,17,22. Aunque este protocolo se centra en las células EPR derivadas de hESC y no todas las células terapéuticas son igualmente criopreservadas, la estrategia de congelación en la fase exponencial puede aplicarse a muchas otras células terapéuticas.

<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

Las células epiteliales pigmentarias de la retina (EPR) derivadas de células madre embrionarias humanas (hESCs) son fuentes celulares superiores para la terapia de reemplazo celular en individuos con enfermedades degenerativas de la retina; Sin embargo, los estudios sobre el almacenamiento estable y seguro de estas células terapéuticas son escasos. La viabilidad celular altamente variable y la recuperación funcional de las células EPR después de la criopreservación son los problemas más comunes. En el protocolo actual, nuestro objetivo fue lograr la mejor tasa de recuperación celular después de la descongelación seleccionando la fase celular óptima para la congelación en función de las condiciones experimentales originales. Las células se congelaron en la fase exponencial determinada mediante el ensayo de marcaje con 5-etinil-2′-desoxiuridina, que mejoró la viabilidad celular y la tasa de recuperación después de la descongelación. Las células estables y funcionales se obtuvieron poco después de la descongelación, independientemente de un largo proceso de diferenciación. Los métodos descritos aquí permiten la conservación y descongelación simples, eficientes y económicas de las células RPE derivadas de hESC. Aunque este protocolo se centra en las células EPR, esta estrategia de congelación puede aplicarse a muchos otros tipos de células diferenciadas.

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.

Autor destacado: Desarrollo de un método eficiente y factible de congelación de células epiteliales pigmentarias de la retina

Criopreservación de células epiteliales pigmentarias de la retina derivadas de células madre embrionarias humanas en la etapa óptima

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.

Aquí, las células RPE derivadas de hESC en P1D35 fueron pasadas y sembradas a una densidad de 105/cm2. A la semana de la siembra, la morfología hexagonal característica y la pigmentación se perdieron durante la fase de retraso (aproximadamente 2 días). Las células EPR readoptaron gradualmente la morfología hexagonal en la fase exponencial (aproximadamente 5 días, Figura 1A) y entraron en la fase de desaceleración (aproximadamente 6 días) con una morfología m...

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

Este estudio proporciona un protocolo detallado para la criopreservación eficiente de células epiteliales pigmentarias de la retina derivadas de células madre humanas.

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

Nuestro laboratorio tiene como objetivo comprender mejor el mecanismo molecular de las enfermedades de la retina, centrándose en las necesidades médicas insatisfechas de los pacientes. Nuestro objetivo es desarrollar tratamientos eficaces, como las terapias basadas en células madre, para ellos. La criopreservación de células RPE derivadas de células madre es un paso importante para la terapia de reemplazo de RPE.Actualmente, las células se congelan en varias etapas, lo que resulta en tasas de supervivencia muy variables para la descongelación. Esto es necesario para determinar la etapa celular óptima para congelar estas células. Nuestro protocolo proporciona una forma simple, eficiente y económica de congelar células RPE mediante la determinación de la fase exponencial de las células RPE para la congelación.Es menos dependiente de métodos diferenciados o líneas celulares.

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JoVE Journal

Medicine

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.

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

Investigación

Medicina

Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Dissociation and Optimal Cryopreservation Stage Determination

To begin, dilute a vial of thawed cold basement membrane matrix with 12 milliliters of DM EM F12. Mix the suspension well. Add one cover slip into each well of a 24-well plate.Then, pipette 250 microliters of the dilute matrix solution into each well. Discard the culture medium of the cultured hESC derived retinal pigment epithelial cell plates. Wash the plates two times with one milliliter of preheated PBS per well.Next, add 0.5 milliliters of the cell dissociation reagent to the wells of the culture plates, and incubate them at 37 degrees Celsius for 15 minutes to initiate digestion. After incubation, observe the cells under the microscope to confirm the end of digestion. Now with a one milliliter pipette, gently pipette the cell suspension up and down 10 times.Add preheated culture medium to dilute the suspension. Then, centrifuge it at 250 G for three minutes at room temperature. Pour out the supernatant from the centrifuge tube.Then, re-suspend the cell pellet in two milliliters of the culture medium. Use a pipette to mix the cells 10 to 15 times. Filter the cell suspension through a 40 micrometer cell strainer.Next, count the retinal pigment epithelial cells. Aspirate the coating solution before plating. Then, add one milliliter of the culture medium into each well and seed the cells on the cover slips.After EDU incorporation and staining, capture the images of five random fields with a fluorescence microscope. Calculate the percentage of EDU positive cells. Then, plot the corresponding proportion time curves to generate a growth curve.Lastly, determine the freezing window from the exponential phase for each cell line. The retinal pigment epithelial cells initially lost their characteristic hexagonal morphology, but later regained it in the exponential phase of growth. When the cells were cultured for an additional week, cell proliferation decreased.EDU proliferation assay confirmed that P2 day five cells exhibited a higher proliferation rate, whereas P2 day 11 cells had entered the deceleration phase.

Disociación de células epiteliales pigmentarias de la retina derivadas de células madre embrionarias humanas y determinación óptima de la etapa de criopreservación

Preservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells with Optimized Cryopreservation

To begin, dissociate the hESC-derived retinal pigment epithelial cells, prepare the cell suspension, and then count the cells. Now, centrifuge the cells at 250g for three minutes at room temperature. Pour out the supernatant, then, resuspend the cell pellet in cryopreservation medium.Next, transfer one milliliter of the cell suspension to a 1.2-milliliter cryogenic vial. Place the cryogenic vials in a freezing container and freeze overnight at minus 80 degrees Celsius. Transfer the vials to liquid nitrogen for long-term storage.To thaw the frozen vials, first heat the culture medium in metallic beads heated to 37 degrees Celsius, pre-fill 10 milliliters of the warmed culture medium into a 15-milliliter tube. Now, remove the cryogenic vials from the liquid nitrogen and place them in an automated thawing system for rapid thawing. Drop 0.5 to one milliliter of the preheated culture medium into the cryogenic vial to ensure gradual cell adaptation.Then, pipet 1.5 to two milliliters of the cell suspension to the 15-milliliter tube with medium. Centrifuge the cells at 250g for three minutes at room temperature. Then, discard the supernatant.Resuspend the pellet in two milliliters of warm medium. Load the cells in a hemocytometer and count them to determine the recovery and survival rates. Culture the thawed cells on basement membrane coated plates in a culture medium with Y27632.The retinal pigment epithelial cells that were frozen on day five in their exponential phase displayed a higher attachment rate after thawing. Relative to their characteristic hexagonal morphology, the cells frozen at other time points had a fibroblastic phenotype. P2 day-five cells displayed higher expression and more properly localized cell markers 28 days after thawing.It was noted that different cryopreservation media performed equally well in achieving high cell viability and attachment post-thawing.