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。孵育后，在显微镜下观察边缘收缩和发光的细胞，以确认消化终止。 注意：不建议使用基于胰蛋白酶的解离，因为它会导致细胞活力低下。</li>
	<li>用 1 mL 移液管轻轻上下移液 10 次以解离细胞，并用预热的培养基（不含 Y-27632）以 1：10 的比例稀释细胞悬浮液。然后，在室温下以 250 × g 离心细胞 3 分钟。</li>
	<li>离心后迅速倒出上清液，轻轻地将细胞沉淀重悬于 2 mL 培养基中，然后用移液管重悬细胞 10-15 次。</li>
	<li>用40μm细胞过滤器过滤细胞悬浮液，以获得单细胞悬浮液并计算细胞数。 注：单细胞悬浮液对于解冻后的准确细胞计数和均匀的细胞接种密度至关重要。</li>
</ol>2. 确定冷冻保存的最佳细胞阶段
注：由于分化方法和细胞系之间的细胞状态不同，因此应在冷冻前确定在不同实验室培养的RPE细胞的指数阶段。
<ol><li>在4°C的冰上解冻一小瓶基底膜基质溶液过夜。将基质稀释到 12 mL 冷 DMEM/F-12 中并充分混合。每孔添加一个盖玻片，并用 0.25 mL 稀释溶液包被 24 孔板的每个孔。使用前将板在室温下孵育1小时或在4°C下孵育过夜，并在接种细胞之前吸出包被溶液。 注：等分试样体积的说明基于蛋白质浓度的批次特异性说明，可在产品规格表中找到。</li>
	<li>将步骤 1.7 的 RPE 单细胞以 105/cm2 的密度接种在 1 mL 培养基中的基底膜基质包被盖玻片上。每 2-3 天更新一次培养基。</li>
	<li>在指定的时间点（传代后 1、3、5、7 和 11 天），将 RPE 细胞与 10 μM EdU 在培养基中孵育 24 小时。按照手册中所述，用反应混合物固定、透化和染色细胞，以检测掺入的 EdU。</li>
	<li>在荧光显微镜下从五个随机场捕获图像，并计算EdU阳性细胞的百分比。绘制相应的 EdU-正比例-时间曲线以生成生长曲线。然后，根据生长曲线确定每个细胞系的冷冻窗口或指数阶段。 注意：重建的六边形细胞形态表明细胞退出指数阶段。</li>
</ol>3. 冷冻保存
<ol><li>遵循步骤1.1至1.7，除了消化时间减少至5分钟外，将细胞悬浮液在室温下以250× g离心3分钟。</li>
	<li>离心后快速倒去上清液，将细胞沉淀轻轻重悬于冻存培养基中，密度为 2 × 106 个细胞/mL，并将 1 mL 细胞悬液转移到 1.2 mL 低温瓶中。</li>
	<li>立即将低温小瓶放入冷冻容器中，在-80°C下冷冻过夜以达到-1°C / min的冷却速率，然后将小瓶转移到液氮中长期储存。</li>
</ol>4. 解冻
<ol><li>在37°C水浴中加热培养基，并将10mL预热的培养基预填充到15mL管中。</li>
	<li>使用自动解冻系统快速解冻直接从液氮储存中取出的低温样品瓶。</li>
	<li>将0.5-1 mL预热的培养基滴入低温瓶中，以确保冷冻细胞逐渐适应新环境。然后，将 1.5-2 mL 细胞悬液转移到 15 mL 管中的 10 mL 培养基中，并在室温下以 250 × g 离心细胞 3 分钟。</li>
	<li>离心后快速倒去上清液，将沉淀重悬于 2 mL 预热的培养基中，并使用血细胞计数器计数细胞数，以使用标准台盼蓝排除物（0.4% 台盼蓝染色剂）确定回收率和存活率。</li>
	<li>在补充有Y-27632（终浓度：10μM）的培养基中，以105 活细胞/cm 2 的密度在基底膜基质包被表面上培养细胞。24小时后取出Y-27632。</li>
	<li>为了确定附着率，再次解离细胞并在解冻后24小时计数细胞数。</li>
</ol>5. 验证最佳冷冻阶段
<ol><li>为了验证步骤 2.4 中确定的最佳冷冻阶段，解冻在不同时间点冷冻的 RPE 细胞并培养 28 天。收获细胞进行 qPCR 和免疫染色分析，以评估 RPE 标志物的表达，如前所述17。</li>
</ol>

推进人干细胞来源的视网膜色素上皮细胞的低温储存

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作者没有要披露的利益冲突。

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

477 次观看.  Shanghai Jiao Tong University School of Medicine. 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?...

视频: 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

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.