Name: Autor em destaque: Desenvolvimento de um método eficiente e viável de congelamento de células epiteliais pigmentares da 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 trabalho foi financiado pela Fundação Nacional de Ciências Naturais da China (81970816) para Mei Jiang; a Fundação Nacional de Ciências Naturais da China (82201223) para Xinyue Zhu; e o Plano de Ação de Inovação em Ciência e Tecnologia da Comissão de Ciência e Tecnologia de Xangai (2014090067000) para Haiyun Liu.

1. Dissociação celular
<ol><li>Manter as células EPR conforme descrito anteriormente 17,22. NOTA: Todas as células são cultivadas a 37 ° C em uma atmosfera de 5% de CO2 durante toda a duração dos protocolos.</li>
	<li>Prepare a quantidade necessária de PBS e meio de cultura em banho-maria a 37 ° C e coloque o reagente de dissociação celular em temperatura ambiente.</li>
	<li>Descarte a solução de cultura e lave as pl...

Avanço do crioarmazenamento de células epiteliais pigmentares da retina derivadas de células-tronco 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>

No presente estudo, é descrito um protocolo de congelamento e descongelamento bem-sucedido para células RPE derivadas de hESC para pesquisa e necessidades clínicas. Ao contrário da linhagem celular RPE imortalizada, ARPE-19, as células RPE com fenótipo epitelial e função característicos adequados, como as células RPE derivadas de células-tronco, são mais sensíveis à criopreservação. Menos de 32% das células permaneceram 24 h após o descongelamento, se não forem adequadamente preservadas<sup class="xref...

O epitélio pigmentar da retina (EPR) é uma monocamada pigmentada de células necessárias para manter o funcionamento adequado da retina1. A disfunção e a morte do EPR estão intimamente associadas a muitas doenças degenerativas da retina, incluindo degeneração macular relacionada à idade, retinite pigmentosa e doença de Stargardt 2,3. A terapia de reposição do EPR é um dos esquemas de tratamento mais promissores para essas doenças 4,5,6,7. Um suprimento estável de células RPE do doador é vital para a terapia celular. As células RPE derivadas de células-tronco embrionárias humanas (hESC) são uma fonte celular ideal para terapia celular porque imitam a função das células primárias de RPE e podem produzir um suprimento teoricamente ilimitado8. No entanto, o processo de diferenciação é trabalhoso e a vida útil das células RPE obtidas é relativamente curta devido à subsequente transição epitelial-mesenquimal (EMT). Portanto, a criopreservação de células RPE derivadas de hESC é uma etapa indispensável necessária para armazenamento de longo prazo e distribuição sob demanda9.
O dano celular induzido pela criopreservação pode comprometer inadvertidamente a eficácia terapêutica10,11. Portanto, estudos recentes sobre criopreservação propuseram que as condições ideais de armazenamento criogênico devem ser determinadas ao projetar terapias celulares12. A criopreservação bem-sucedida garante recuperação celular eficiente, alta viabilidade e restauração da função celular após o ciclo de congelamento e descongelamento. No entanto, estudos anteriores sobre a criopreservação das monocamadas aderentes de células de mamíferos relataram taxas de sobrevivência altamente variáveis (35%-95%) após o descongelamento 13,14,15. Muitos fatores afetam consideravelmente os resultados da criopreservação, particularmente durante o estágio de congelamento16,17. Pesquisas recentes mostraram que as células RPE congeladas em diferentes momentos exibiram recuperação variada após o descongelamento17. Até onde sabemos, faltam estudos sobre a determinação da janela de tempo de congelamento ideal para células-tronco derivadas de células-tronco. Em diferentes estudos, as células foram congeladas em vários estágios: algumas células foram congeladas logo após a passagem ou antes da confluência ou pigmentação 8,15,18, enquanto outras foram congeladas em outros momentos 9,19,20,21. Além disso, não há evidências claras de se a fase ou estágio das células RPE usadas para criopreservação afeta a função do RPE após o descongelamento. Em nosso estudo anterior, demonstramos pela primeira vez que a fase exponencial do crescimento celular (P2D5) é o melhor estágio para a criopreservação de células RPE derivadas de hESC em termos de viabilidade celular e recuperação de propriedades e funções celulares17.
O método aqui estabelecido visa criopreservar o RPE derivado de hESC em um estágio ideal para obter a melhor preservação em termos de viabilidade e função celular após o descongelamento. Usando o ensaio de marcação de 5-etinil-2'-desoxiuridina (EdU) para detectar a fase exponencial da síntese de DNA antes da criopreservação, as células RPE descongeladas exibiram viabilidade e taxa de fixação >80%, expressão gênica específica de RPE, morfologia celular polarizada, secreção de fator derivado do epitélio pigmentar, resistência transepitelial apropriada e capacidade fagocítica 8,17,22. Embora este protocolo se concentre em células RPE derivadas de hESC e nem todas as células terapêuticas sejam igualmente criopreservadas, a estratégia de congelamento na fase exponencial pode ser aplicada a muitas outras células terapêuticas.

<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

As células epiteliais pigmentares da retina (EPR) derivadas de células-tronco embrionárias humanas (hESCs) são fontes celulares superiores para terapia de reposição celular em indivíduos com doenças degenerativas da retina; no entanto, estudos sobre o armazenamento estável e seguro dessas células terapêuticas são escassos. A viabilidade celular altamente variável e a recuperação funcional das células RPE após a criopreservação são os problemas mais comumente encontrados. No presente protocolo, nosso objetivo foi alcançar a melhor taxa de recuperação celular após o descongelamento, selecionando a fase celular ideal para congelamento com base nas condições experimentais originais. As células foram congeladas na fase exponencial determinada usando o ensaio de marcação com 5-etinil-2'-desoxiuridina, que melhorou a viabilidade celular e a taxa de recuperação após o descongelamento. Células estáveis e funcionais foram obtidas logo após o descongelamento, independentemente de um longo processo de diferenciação. Os métodos descritos aqui permitem a preservação e descongelamento simples, eficientes e baratos de células RPE derivadas de hESC. Embora este protocolo se concentre em células RPE, essa estratégia de congelamento pode ser aplicada a muitos outros 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 em destaque: Desenvolvimento de um método eficiente e viável de congelamento de células epiteliais pigmentares da retina

Criopreservação de células-tronco embrionárias humanas derivadas de células epiteliais pigmentares da retina no estágio ideal

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.

Aqui, células RPE derivadas de hESC em P1D35 foram passadas e semeadas a uma densidade de 105 / cm2. Dentro de uma semana após a semeadura, a morfologia hexagonal característica e a pigmentação foram perdidas durante a fase de latência (aproximadamente 2 dias). As células RPE gradualmente readotaram a morfologia hexagonal na fase exponencial (aproximadamente 5 dias, Figura 1A) e entraram na fase de desaceleração (aproximadamente 6 dias) com uma morfologia mais ...

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 estudo fornece um protocolo detalhado para a criopreservação eficiente de células epiteliais pigmentares da retina derivadas de células-tronco humanas.

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

Nosso laboratório visa entender melhor o mecanismo molecular das doenças da retina, com foco nas necessidades médicas não atendidas dos pacientes. Nosso objetivo é desenvolver tratamentos eficazes, como terapias baseadas em células-tronco, para eles. A criopreservação de células RPE derivadas de células-tronco é uma etapa importante para a terapia de reposição de RPE.Atualmente, as células são congeladas em vários estágios, resultando em taxas de sobrevivência altamente variáveis para descongelamento. Isso é necessário para determinar o estágio celular ideal para congelar essas células. Nosso protocolo fornece maneiras simples, eficientes e baratas de congelar células RPE, determinando a fase exponencial das células RPE para congelamento.É menos dependente de métodos diferenciados ou linhagens 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.

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.

Dissociação de células epiteliais pigmentares da retina derivadas de células-tronco embrionárias humanas e determinação ideal do estágio de criopreservação

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.