介紹?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.?連續(xù)培養(yǎng)的細胞系容易發(fā)生遺傳漂變，有限的細胞系注定會衰老，所有的細胞培養(yǎng)物都容易受到微生物污染，即使是運行最好的實驗室也會遇到設(shè)備故障。因為一個已經(jīng)建立的細胞系是一個寶貴的資源，它的替換是昂貴的和費時的，它們是至關(guān)重要的冷凍和保存為長期儲存

As soon as a small surplus of cells becomes available from subculturing, they should be frozen as a?一旦有少量剩余的細胞可以通過繼代培養(yǎng)獲得，它們就應(yīng)該被冷凍成為seed stock?種子庫, protected, and not be made available for general laboratory use.?，受保護，不得提供給一般實驗室使用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.?可以從冷凍的種子儲備中提取和補充。如果種子庫變得枯竭，低溫保存的工作庫就可以作為準備新鮮種子庫的來源，從最初的冷凍開始以最小的代數(shù)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.?培養(yǎng)細胞冷凍保存的最佳方法是在液氮中加入二甲基亞砜(DMSO)等低溫保護劑進行冷凍保存。低溫保護劑可以降低介質(zhì)的冰點，同時降低冷卻速度，大大降低冰晶形成的風險，而冰晶形成會損傷細胞并導致細胞死亡

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.?二甲基亞砜是眾所周知的促進有機分子進入組織。處理含有二甲基亞砜的試劑時，使用適合于此類材料所造成危險的設(shè)備和做法。按照當?shù)匾?guī)定處理試劑

Freezing Medium?冷凍介質(zhì)

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?永遠使用推薦的冷凍介質(zhì)來冷凍保存你的細胞。冷凍介質(zhì)應(yīng)該包含一個低溫保護劑，如 DMSO 或甘油。你也可以使用一種特殊配方的完整的深低溫保存培養(yǎng)基，如Recovery? Cell Culture Freezing Medium?恢復細胞培養(yǎng)液?or?或Synth-a-Freeze Cryopreservation Medium?合成器-a-freeze 深低溫保存介質(zhì).

• Recovery? Cell Culture Freezing Medium?恢復細胞培養(yǎng)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.?是哺乳動物細胞培養(yǎng)的即用完全深低溫保存培養(yǎng)基，含有優(yōu)化比例的胎牛血清和牛血清，以提高細胞活力和解凍后的細胞恢復

• Synth-a-Freeze Cryopreservation Medium?合成器-a-freeze 深低溫保存介質(zhì)?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.?是一種化學定義的、無蛋白質(zhì)、無菌的深低溫保存培養(yǎng)基，含有10% DMSO，除黑素細胞外，適用于多種干細胞和原代細胞的深低溫保存

實驗材料?Experimental material

• Culture vessels containing cultured cells in log-phase of growth?含有培養(yǎng)細胞的培養(yǎng)血管處于對數(shù)生長期

• Complete growth medium?完全生長培養(yǎng)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?低溫保護劑，如 DMSO (使用細胞培養(yǎng)的瓶子，只能在層流過濾器中打開)或凍結(jié)介質(zhì)，如合成 a-freeze 深低溫保存或 RecoveryTM 細胞培養(yǎng)凍結(jié)介質(zhì)

• Disposable, sterile 15-mL or 50-mL conical tubes?一次性無菌15毫升或50毫升錐形管

• Reagents and equipment to determine viable and total cell counts (e.g.,?測定活細胞和總細胞計數(shù)的試劑和設(shè)備(例如:Countess Automated Cell Counter?自動細胞計數(shù)器伯爵夫人, or hemacytometer, cell counter and Trypan Blue)?或血球計數(shù)器，細胞計數(shù)器和臺盼藍)

• Sterile cryogenic storage vials (i.e., cryovials)?無菌低溫貯存瓶(即地窖貯存瓶)

• Controlled rate freezing apparatus or isopropanol chamber?控速冷凍裝置或異丙醇室

• Liquid nitrogen storage container?液氮貯存容器

For freezing adherent cells, in addition to the above materials, you need:?為了冷凍附著細胞，除了上述材料，你還需要:

• Balanced salt solution such as Dulbecco’s Phosphate Buffered Saline (D-PBS), containing no calcium, magnesium, or phenol red?平衡鹽溶液，如 Dulbecco’s 磷酸鹽緩沖生理鹽水(D-PBS) ，不含鈣、鎂或酚紅

• Dissociation reagent such as trypsin or TrypLE? Express, without phenol red?離解試劑，如胰蛋白酶或 TrypLETM Express，不含苯酚紅

Protocol for Cryopreserving Cultured Cells?細胞凍存方案

細胞的冷凍?Cell freezing

該視頻展示了細胞凍存的同時保持細胞最佳健康狀態(tài)所需的關(guān)鍵步驟。 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?下面的協(xié)議描述了一個general procedure?一般程序?for cryopreserving cultured cells.?用于冷凍保存培養(yǎng)細胞For detailed protocols, always refer to the cell-specific product insert.?對于詳細的協(xié)議，始終參考特定于單元格的產(chǎn)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.?配制冷凍培養(yǎng)基，存放在攝氏2至8度的環(huán)境中直至使用。請注意，適當?shù)睦鋬雠囵B(yǎng)基取決于細胞系

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.?對于粘連細胞，按照繼代培養(yǎng)過程中使用的程序，輕輕地將細胞從組織培養(yǎng)血管中分離出來。將細胞懸浮在該細胞類型所需的完全培養(yǎng)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.?使用血球計數(shù)器、細胞計數(shù)器和臺盼藍排除法或伯爵夫人自動細胞計數(shù)器測定細胞總數(shù)和百分比活力。根據(jù)所需的活細胞密度，計算所需的冷凍培養(yǎng)基體積

4. Centrifuge the cell suspension at approximately 100–200 × g for 5 to 10 minutes Aseptically decant supernatant without disturbing the cell pellet.?離心細胞懸浮液大約100-200 × g 5-10分鐘無菌傾倒上清液不干擾細胞顆粒
   
   注意：?Note:?Centrifugation speed and duration varies depending on the cell type.?離心速度和持續(xù)時間取決于細胞類型
   
   

5. Resuspend the cell pellet in cold freezing medium at the recommended viable cell density for the specific cell type.?按照特定細胞類型的建議活細胞密度，將細胞顆粒懸浮在冷凍培養(yǎng)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.?將電池懸浮液分配到低溫貯存小瓶中。當你分離它們時，要經(jīng)常輕輕地混合細胞，以保持細胞懸浮的均勻性

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.?將細胞冷凍在速率控制的冷凍設(shè)備中，使溫度降低大約每分鐘1攝氏度?；蛘?，將含有這些細胞的隱窩細胞放在異丙醇室中，在零下80攝氏度的環(huán)境中過夜

8. Transfer frozen cells to liquid nitrogen, and store them in the gas phase above the liquid nitrogen.?將冷凍的細胞轉(zhuǎn)移到液氮中，并將它們儲存在液氮上方的氣相中

Guidelines for Cryopreservation?深低溫保存指南

Following the guidelines below is essential for cryopreserving your cell lines for future use.?遵循下面的指導方針是必不可少的冷凍保存您的細胞系，以供將來使用As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cell line for best results.?與其他細胞培養(yǎng)程序一樣，我們建議您嚴格按照細胞系提供的說明進行操作，以獲得最佳效果

• 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.?將培養(yǎng)的細胞冷凍在高濃度和盡可能低的通道數(shù)上。在冷凍之前，確保細胞至少有90% 的存活率。注意，最佳的冷凍條件取決于使用的細胞系

• 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)?使用低溫冷凍機或低溫冷凍容器，如 NALGENE 實驗室(Nalge Nunc)提供的“ Frosty 先生”，將溫度降低到大約每分鐘1攝氏度，從而緩慢地冷凍細胞

• Always use the recommended freezing medium. The freezing medium should contain a cryoprotective agent such as DMSO or glycerol (see?始終使用推薦的冷凍介質(zhì)。冷凍介質(zhì)應(yīng)包含低溫保護劑，如 DMSO 或甘油(見What is Subculture??什么是亞文化？).

• Store the frozen cells below –70°C; frozen cells begin to deteriorate above –50°C.?冷凍細胞存放在攝氏零下70度以下; 冷凍細胞在攝氏零下50度以上開始變壞

• 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?總是使用無菌的冰凍細胞來儲存冷凍的細胞。含有冷凍細胞的 Cryovials 可以浸泡在液氮中或液氮上方的氣相中(見Safety Note?安全須知?below).?以下)

• Always wear personal protective equipment.?始終佩戴個人防護裝備

• 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.?所有與細胞接觸的溶液和設(shè)備必須是無菌的。始終使用適當?shù)臒o菌技術(shù)和工作在層流罩