Name: 作者聚焦：开发一种高效可行的视网膜色素上皮细胞冷冻方法
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

这项工作由国家自然科学基金（81970816）资助，以梅江;国家自然科学基金面上基金面上项目（82201223年）朱新月;以及上海市科委科技创新行动计划（2014090067000）授予刘海云。

1. 细胞解离
<ol><li>如前所述维持RPE细胞17,22。 注意：在整个方案期间，所有细胞在37°C下在5%CO2 气氛中生长。</li>
	<li>在37°C水浴中制备所需量的PBS和培养基，并将细胞解离试剂置于室温下。</li>
	<li>丢弃培养溶液，每孔用 1 mL 预热的 PBS 洗涤板两次。</li>
	<li>将 1 mL 细胞解离试剂加入 6 孔板中，并在 37 °C 下消化细胞 15 分钟。孵?...

推进人干细胞来源的视网膜色素上皮细胞的低温储存

<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>

在本研究中，描述了用于研究和临床需求的 hESC 衍生的 RPE 细胞的成功冻融方案。与永生化的 RPE 细胞系 ARPE-19 不同，具有适当特征性上皮表型和功能的 RPE 细胞，如干细胞衍生的 RPE 细胞，对冻存更敏感。如果保存不当，不到 32% 的细胞在解冻后 24 小时内保留17。冷冻保存时间是一个关键参数。永生化细胞冻存的一个既定观点是在指数生长阶段冷冻细胞。分化的细胞离开细胞周期...

视网膜色素上皮 （RPE） 是维持视网膜正常功能所必需的色素沉着单层细胞1。RPE 功能障碍和死亡与许多视网膜退行性疾病密切相关，包括年龄相关性黄斑变性、视网膜色素变性和 Stargardt 病 2,3。RPE替代疗法是这些疾病最有前途的治疗方案之一4,5,6,7。供体RPE细胞的稳定供应对于细胞治疗至关重要。人胚胎干细胞 （hESC） 衍生的 RPE 细胞是细胞治疗的理想细胞来源，因为它们模仿原代 RPE 细胞的功能，并且可以产生理论上无限的供应8。然而，分化过程很费力，并且由于随后的上皮-间充质转化 （EMT），获得的 RPE 细胞的保质期相对较短。因此，hESC来源的RPE细胞的冻存是长期储存和按需分发所必需的必不可少的步骤9。
冷冻保存诱导的细胞损伤可能会无意中影响治疗效果10,11。因此，最近关于冷冻保存的研究提出，在设计细胞疗法时，应确定最佳的低温储存条件12。成功的冻存保证了高效的细胞回收、高活力和冻融循环后的细胞功能恢复。然而，先前关于哺乳动物细胞贴壁单层冻存的研究报告称，解冻后的存活率变化很大 （35%-95%）13,14,15。许多因素都会极大地影响冷冻保存的结果，尤其是在冷冻阶段16,17。最近的研究表明，在不同时间点冷冻的RPE细胞在解冻后表现出不同的恢复率17。据我们所知，目前缺乏关于确定干细胞来源的RPE细胞最佳冷冻时间窗口的研究。在不同的研究中，细胞在不同阶段被冷冻：一些细胞在传代后不久或汇合或色素沉着之前被冷冻 8,15,18，而其他细胞在其他时间点 9,19,20,21 被冷冻。此外，没有明确的证据表明用于冻存的RPE细胞的阶段或阶段是否会影响解冻后的RPE功能。在我们之前的研究中，我们首次证明了细胞生长的指数阶段（P2D5）是hESC衍生的RPE细胞冻存的最佳阶段，就细胞活力和细胞特性和功能的恢复而言17。
本文建立的方法旨在将hESC衍生的RPE冷冻保存在最佳阶段，以达到解冻后细胞活力和功能的最佳保存效果。使用5-乙炔基-2'-脱氧尿苷（EdU）标记试验检测冻存前DNA合成的指数阶段，解冻的RPE细胞表现出>80%的活力和附着率、RPE特异性基因表达、极化细胞形态、色素上皮衍生因子分泌、适当的跨上皮抵抗和吞噬能力8,17,22.尽管该方案侧重于hESC衍生的RPE细胞，并且并非所有治疗细胞都同样冷冻保存，但在指数期冷冻的策略可以应用于许多其他治疗细胞。

<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

源自人胚胎干细胞 （hESC） 的视网膜色素上皮 （RPE） 细胞是视网膜退行性疾病患者细胞替代疗法的优越细胞来源;然而，关于这些治疗性细胞的稳定和安全储存的研究很少。冻存后RPE细胞的细胞活力和功能恢复变化很大，这是最常遇到的问题。在本方案中，我们旨在通过根据原始实验条件选择最佳的细胞相进行冷冻，从而实现解冻后的最佳细胞回收率。使用5-乙炔基-2′-脱氧尿苷标记测定法测定的指数期冷冻细胞，提高了解冻后的细胞活力和恢复率。解冻后不久即可获得稳定且功能性的细胞，与漫长的分化过程无关。这里描述的方法允许简单、高效和廉价地保存和解冻 hESC 衍生的 RPE 细胞。尽管该协议侧重于RPE细胞，但这种冷冻策略可以应用于许多其他类型的分化细胞。

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.

作者聚焦：开发一种高效可行的视网膜色素上皮细胞冷冻方法

人胚胎干细胞来源的视网膜色素上皮细胞在最佳阶段的冷冻保存

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.

在这里，将 P1D35 的 hESC 衍生的 RPE 细胞传代并以 105/cm2 的密度接种。在播种后的一周内，特征性的六边形形态和色素沉着在滞后期（约 2 天）丢失。RPE细胞在指数阶段（约5天，图1A）逐渐重新采用六边形形态，并进入减速阶段（约6天），形态更多多边形。如果细胞培养再持续一周，细胞增殖会显着减少，细胞间连接起主要作用，边缘会亮起;此时，细胞不再?...

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

本研究为人类干细胞衍生的视网膜色素上皮细胞的有效冷冻保存提供了详细的方案。

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

我们的实验室旨在更好地了解视网膜疾病的分子机制，重点关注患者未得到满足的医疗需求。我们的目标是为他们开发有效的治疗方法，例如基于干细胞的疗法。干细胞来源的RPE细胞的冷冻保存是RPE替代疗法的重要步骤。目前，细胞在不同阶段被冷冻，导致解冻的存活率差异很大。这是确定冷冻这些细胞的最佳细胞阶段所必需的。我们的方案通过确定用于冷冻的RPE细胞的指数相位，提供了一种简单、高效和廉价的冷冻RPE细胞的方法。它对分化方法或细胞系的依赖性较小。

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

Research

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.

科研

医学

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.

人胚胎干细胞衍生的视网膜色素上皮细胞解离和最佳冷冻保存阶段的测定

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.