介绍 Introduction

Cell lines in continuous culture are prone to genetic drift, finite cell lines are fated for senescence, all cell cultures are susceptible to microbial contamination, and even the best-run laboratories can experience equipment failure. Because an established cell line is a valuable resource and its replacement is expensive and time consuming, it is vitally important that they are frozen down and preserved for long-term storage. 连续培养的细胞系容易发生遗传漂变，有限的细胞系注定会衰老，所有的细胞培养物都容易受到微生物污染，即使是运行最好的实验室也会遇到设备故障。因为一个已经建立的细胞系是一个宝贵的资源，它的替换是昂贵的和费时的，它们是至关重要的冷冻和保存为长期储存

As soon as a small surplus of cells becomes available from subculturing, they should be frozen as a 一旦有少量剩余的细胞可以通过继代培养获得，它们就应该被冷冻成为seed stock 种子库, protected, and not be made available for general laboratory use. ，受保护，不得提供给一般实验室使用Working stocks 工作股 can be prepared and replenished from frozen seed stocks. If the seed stocks become depleted, cryopreserved working stocks can then serve as a source for preparing a fresh seed stock with a minimum increase in generation number from the initial freezing. 可以从冷冻的种子储备中提取和补充。如果种子库变得枯竭，低温保存的工作库就可以作为准备新鲜种子库的来源，从最初的冷冻开始以最小的代数增加

The best method for cryopreserving cultured cells is storing them in liquid nitrogen in complete medium in the presence of a cryoprotective agent such as dimethylsulfoxide (DMSO). Cryoprotective agents reduce the freezing point of the medium and also allow a slower cooling rate, greatly reducing the risk of ice crystal formation, which can damage cells and cause cell death. 培养细胞冷冻保存的最佳方法是在液氮中加入二甲基亚砜(DMSO)等低温保护剂进行冷冻保存。低温保护剂可以降低介质的冰点，同时降低冷却速度，大大降低冰晶形成的风险，而冰晶形成会损伤细胞并导致细胞死亡

注： Note:DMSO is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with local regulations. 二甲基亚砜是众所周知的促进有机分子进入组织。处理含有二甲基亚砜的试剂时，使用适合于此类材料所造成危险的设备和做法。按照当地规定处理试剂

Freezing Medium 冷冻介质

Always use the recommended freezing medium for cryopreserving your cells. The freezing medium should contain a cryoprotective agent such as DMSO or glycerol. You may also use a specially formulated complete cryopreservation medium such as 永远使用推荐的冷冻介质来冷冻保存你的细胞。冷冻介质应该包含一个低温保护剂，如 DMSO 或甘油。你也可以使用一种特殊配方的完整的深低温保存培养基，如Recovery™ Cell Culture Freezing Medium 恢复细胞培养液 or 或Synth-a-Freeze Cryopreservation Medium 合成器-a-freeze 深低温保存介质.

• Recovery™ Cell Culture Freezing Medium 恢复细胞培养液 is a ready-to-use complete cryopreservation medium for mammalian cell cultures, containing an optimized ratio of fetal bovine serum to bovine serum for improved cell viability and cell recovery after thawing. 是哺乳动物细胞培养的即用完全深低温保存培养基，含有优化比例的胎牛血清和牛血清，以提高细胞活力和解冻后的细胞恢复

• Synth-a-Freeze Cryopreservation Medium 合成器-a-freeze 深低温保存介质 is a chemically defined, protein-free, sterile cryopreservation medium containing 10% DMSO that is suitable for the cryopreservation of many stem and primary cell types with the exception of melanocytes. 是一种化学定义的、无蛋白质、无菌的深低温保存培养基，含有10% DMSO，除黑素细胞外，适用于多种干细胞和原代细胞的深低温保存

实验材料 Experimental material

• Culture vessels containing cultured cells in log-phase of growth 含有培养细胞的培养血管处于对数生长期

• Complete growth medium 完全生长培养基

• Cryoprotective agent such as DMSO (use a bottle set aside for cell culture; open only in a laminar flow hood) or a freezing medium such as Synth-a-Freeze Cryopreservation Medium or Recovery™ Cell Culture Freezing Medium 低温保护剂，如 DMSO (使用细胞培养的瓶子，只能在层流过滤器中打开)或冻结介质，如合成 a-freeze 深低温保存或 RecoveryTM 细胞培养冻结介质

• Disposable, sterile 15-mL or 50-mL conical tubes 一次性无菌15毫升或50毫升锥形管

• Reagents and equipment to determine viable and total cell counts (e.g., 测定活细胞和总细胞计数的试剂和设备(例如:Countess Automated Cell Counter 自动细胞计数器伯爵夫人, or hemacytometer, cell counter and Trypan Blue) 或血球计数器，细胞计数器和台盼蓝)

• Sterile cryogenic storage vials (i.e., cryovials) 无菌低温贮存瓶(即地窖贮存瓶)

• Controlled rate freezing apparatus or isopropanol chamber 控速冷冻装置或异丙醇室

• Liquid nitrogen storage container 液氮贮存容器

For freezing adherent cells, in addition to the above materials, you need: 为了冷冻附着细胞，除了上述材料，你还需要:

• Balanced salt solution such as Dulbecco’s Phosphate Buffered Saline (D-PBS), containing no calcium, magnesium, or phenol red 平衡盐溶液，如 Dulbecco’s 磷酸盐缓冲生理盐水(D-PBS) ，不含钙、镁或酚红

• Dissociation reagent such as trypsin or TrypLE™ Express, without phenol red 离解试剂，如胰蛋白酶或 TrypLETM Express，不含苯酚红

Protocol for Cryopreserving Cultured Cells 细胞冻存方案

细胞的冷冻 Cell freezing

该视频展示了细胞冻存的同时保持细胞最佳健康状态所需的关键步骤。 We review the equipment required, how to prepare for freezing and each step performed in a careful way at the right pace to prevent damage to your cells. The video shows the key steps needed to keep cells in optimal health while they are frozen. We review the equipment required, how to prepare for freezing and each step performed in a careful way at the right pace to prevent damage to your cells.

The following protocol describes a 下面的协议描述了一个general procedure 一般程序 for cryopreserving cultured cells. 用于冷冻保存培养细胞For detailed protocols, always refer to the cell-specific product insert. 对于详细的协议，始终参考特定于单元格的产品插入

1. Prepare freezing medium and store at 2° to 8°C until use. Note that the appropriate freezing medium depends on the cell line. 配制冷冻培养基，存放在摄氏2至8度的环境中直至使用。请注意，适当的冷冻培养基取决于细胞系

2. For adherent cells, gently detach cells from the tissue culture vessel following the procedure used during the subculture. Resuspend the cells in complete medium required for that cell type. 对于粘连细胞，按照继代培养过程中使用的程序，轻轻地将细胞从组织培养血管中分离出来。将细胞悬浮在该细胞类型所需的完全培养基中

3. Determine the total number of cells and percent viability using a hemacytometer, cell counter and Trypan Blue exclusion, or the Countess Automated Cell Counter. According to the desired viable cell density, calculate the required volume of freezing medium. 使用血球计数器、细胞计数器和台盼蓝排除法或伯爵夫人自动细胞计数器测定细胞总数和百分比活力。根据所需的活细胞密度，计算所需的冷冻培养基体积

4. Centrifuge the cell suspension at approximately 100–200 × g for 5 to 10 minutes Aseptically decant supernatant without disturbing the cell pellet. 离心细胞悬浮液大约100-200 × g 5-10分钟无菌倾倒上清液不干扰细胞颗粒
   
   注意： Note: Centrifugation speed and duration varies depending on the cell type. 离心速度和持续时间取决于细胞类型
   
   

5. Resuspend the cell pellet in cold freezing medium at the recommended viable cell density for the specific cell type. 按照特定细胞类型的建议活细胞密度，将细胞颗粒悬浮在冷冻培养基中

6. Dispense aliquots of the cell suspension into cryogenic storage vials. As you aliquot them, frequently and gently mix the cells to maintain a homogeneous cell suspension. 将电池悬浮液分配到低温贮存小瓶中。当你分离它们时，要经常轻轻地混合细胞，以保持细胞悬浮的均匀性

7. Freeze the cells in a controlled rate freezing apparatus, decreasing the temperature approximately 1°C per minute. Alternatively, place the cryovials containing the cells in an isopropanol chamber and store them at –80°C overnight. 将细胞冷冻在速率控制的冷冻设备中，使温度降低大约每分钟1摄氏度。或者，将含有这些细胞的隐窝细胞放在异丙醇室中，在零下80摄氏度的环境中过夜

8. Transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid nitrogen. 将冷冻的细胞转移到液氮中，并将它们储存在液氮上方的气相中

Guidelines for Cryopreservation 深低温保存指南

Following the guidelines below is essential for cryopreserving your cell lines for future use. 遵循下面的指导方针是必不可少的冷冻保存您的细胞系，以供将来使用As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cell line for best results. 与其他细胞培养程序一样，我们建议您严格按照细胞系提供的说明进行操作，以获得最佳效果

• Freeze your cultured cells at a high concentration and at as low a passage number as possible. Make sure that the cells are at least 90% viable before freezing. Note that the optimal freezing conditions depend on the cell line in use. 将培养的细胞冷冻在高浓度和尽可能低的通道数上。在冷冻之前，确保细胞至少有90% 的存活率。注意，最佳的冷冻条件取决于使用的细胞系

• Freeze the cells slowly by reducing the temperature at approximately 1°C per minute using a controlled rate cryo-freezer or a cryo-freezing container such as “Mr. Frosty,” available from NALGENE labware (Nalge Nunc) 使用低温冷冻机或低温冷冻容器，如 NALGENE 实验室(Nalge Nunc)提供的“ Frosty 先生”，将温度降低到大约每分钟1摄氏度，从而缓慢地冷冻细胞

• Always use the recommended freezing medium. The freezing medium should contain a cryoprotective agent such as DMSO or glycerol (see 始终使用推荐的冷冻介质。冷冻介质应包含低温保护剂，如 DMSO 或甘油(见What is Subculture? 什么是亚文化？).

• Store the frozen cells below –70°C; frozen cells begin to deteriorate above –50°C. 冷冻细胞存放在摄氏零下70度以下; 冷冻细胞在摄氏零下50度以上开始变坏

• Always use sterile cryovials for storing frozen cells. Cryovials containing the frozen cells may be stored immersed in liquid nitrogen or in the gas phase above the liquid nitrogen (see 总是使用无菌的冰冻细胞来储存冷冻的细胞。含有冷冻细胞的 Cryovials 可以浸泡在液氮中或液氮上方的气相中(见Safety Note 安全须知 below). 以下)

• Always wear personal protective equipment. 始终佩戴个人防护装备

• All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood. 所有与细胞接触的溶液和设备必须是无菌的。始终使用适当的无菌技术和工作在层流罩