发布日期：2020-08-03 16:10:00浏览次数：5452
〈1044〉CRYOPRESERVATION OF CELLS
细胞的冷冻保存
INTRODUCTION
引言
Cryopreservationis the process of cooling and storing cells, tissues, or organs at very lowtemperatures to maintain their viability. The purpose of cryopreservation is tobank the cells and allow their future use in in vitro or in vivo applicationsfor which post-thaw function is sufficiently representative of the cells'prefreeze function. Cryopreservation also minimizes the risk of geneticmutation or development of subpopulations due to cell replication. Depending onthe application, sufficient postcryopreservation function may beassessed by the ability to divide, proliferate, differentiate, express genes,or to produce proteins, or by another specific functional property.
冷冻保存是将细胞、组织或器官在超低温环境下存储来维持其活力的过程。目的是将细胞储存起来并应用到后续的体内外实验中，因为复苏后的细胞仍可以保持冻存前的功能。冷冻保存还可以最大限度地降低由于细胞复制而导致的基因突变或亚群发育的风险。根据不同的应用，充分的冷冻保存后的功能可通过细胞分裂、增殖、分化、表达基因或产生蛋白质的能力或其他特定的功能特性来评估。
PRINCIPLESOF CRYOPRESERVATION
Overview
冷冻保存的原理概述
Understandingthe role of water and the need to adequately remove it from cells or abrogateits ability to form ice crystals, which damage the cell membrane, is criticalto successful cryopreservation. When cells are frozen in aqueous suspension,often they are destroyed. However, in the 1940s Polge and others discovered thecryoprotective properties of glycerol. Since then several chemicals,generically called cryoprotectant agents (CPAs), have been identified. Themechanism of action of CPAs is complex and is not fully understood. However,according to the commonly accepted theory of colligative action, CPAs increasesolute concentration both within the cell and extracellularly, therebysuppressing ice formation. For this purpose, the so-called penetrating (orintracellular) CPAs [e.g., dimethylsulfoxide (DMSO), glycerol, propanediol, andmethanol] must be able to cross the cell membrane readily and penetrate thecell without significant toxicity.There also is a group of nonpenetrating (orextracellular) CPAs (e.g., sucrose and trehalose) whose mechanism of action isthought to be related at least in part to their stabilizing interaction withcell membranes. This property also may explain the cryoprotective activities ofcertain large molecular weight compounds such as hydroxyethyl starch andpolyvinylpropylene. Theoretical models of cryoprotection typically evoke thecolligative theory, but full explanation of CPA action is yet to beestablished.
了解水的作用以及将水从细胞中充分去除或消除其形成冰晶的能力以减少损坏细胞膜损伤，这对于成功进行冷冻保存至关重要。当细胞在水性悬浮液中冷冻时，通常会被破坏。在1940年代，Polge等人发现了甘油的防冻特性。从那时起，陆续鉴定出了几种通常称为冷冻保护剂（CPA）的化学物质。 CPA的作用机制很复杂，尚未完全了解。但是，根据公认的依数作用理论，CPA会增加细胞内和细胞外的溶质浓度，从而抑制冰晶的形成。因此，所谓的穿透性（或细胞内）CPAs [例如二甲亚砜（DMSO）、甘油、丙二醇和甲醇]必须能够轻松穿过细胞膜而没有明显的毒性。还有一组非渗透性（或细胞外）CPAs（例如蔗糖和海藻糖），其作用机制被认为至少部分与细胞膜的稳定相互作用有关。这种性质也可以解释某些大分子化合物例如羟乙基淀粉和聚乙烯丙烯的防冻活性。冷冻保护的理论模型通常唤起依数理论，但尚未建立对CPA作用的完整解释。
Analternative form of cell preservation, commonly called vitrification,wherebythe cell suspension is loaded with high levels of penetrating CPAs (oftenseveral in combination), induces a glass-like state in which cellular andextracellular water cannot readily form ice crystals. When cell suspensionsprepared in this way then are cooled very rapidly (cooling rates of100°–1000°/min or more) the extreme viscosity prevents osmosis, and the watermolecules are unable to form ice. This procedure has been widely used forcomplex structures including a variety of human, plant, and animal tissues andmay help preserve those cell preparations that have variable degrees ofcellular permeability or when standard cryoprotection cannot deliver the rangeof conditions required to optimally preserve viability in all the tissues'component cell types.
另一种细胞保存方式，通常称为玻璃化，即在细胞悬浮液中加入高浓度的穿透性CPA（通常是几种），诱导形成玻璃化状态，诱导细胞和细胞外水不易形成冰晶。当以这种方式制备的细胞悬液快速冷却（冷却速率为100°–1000°/ min或更高）时，极高的粘度可防止渗透，并且水分子无法形成冰晶。这个过程已广泛用于各种复杂结构的保存过程，包括各种人类、植物和动物组织在内，并且可以帮助保存具有不同程度的细胞渗透性的细胞制剂，或者也用于在所有组织的组成细胞类型标准冷冻保护程序无法提供其生存能力的条件范围内的最佳保存方案。
CPAs havebiological activities beyond their cryoprotective properties. Some, like DMSO,can affect the cell membrane, cytoskeleton, and gene expression and may betoxic to cells following prolonged exposure. Therefore, during development ofnew cryopreservation protocols analysts should perform a toxicity assay inwhich the cells are exposed to the CPA over a range of time intervals toevaluate loss of viability or alteration of functionality.
CPA具有超出其防冻特性的生物学活性。诸如DMSO等，可能会影响细胞膜、细胞骨架和基因表达，并且在细胞长时间暴露后可能对细胞有毒。因此，在开发新的低温保存方案的过程中，分析人员应进行毒性试验，将细胞在一定时间间隔内暴露于CPA，以评估活力丧失或功能改变。
Key Elements of Cryopreservation Practice
低温保存的关键因素
For anycellular sample or therapeutic product being cryopreserved, method developmentshould address the following elements:
对于任何冷冻保存的细胞样品或治疗产品，方法开发应解决以下要素:
PREFREEZE PROCESSING AND CHARACTERIZATION
预冷冻处理和鉴定
Optimizingthe condition of the cells immediately before cryopreservation is critical to asuccessful outcome. The nature and extent of prefreeze processing depends onthe state of the original cells harvested for preservation, the composition ofthe cell suspension, and the specific processing steps leading into cryopreservation.Prefreeze processing may include selection of subpopulations, ex vivoexpansion, or incubation with activating or priming factors.
冷冻保存之前对细胞条件进行优化是获得成功的关键。预冻处理的性质和程度取决于为保存而收获的原始细胞的状态、细胞悬液的组成及冷冻保存的特定处理步骤。预冷冻过程可能包括亚种群的选择、体外扩增或激活或启动因子孵育。
Precryopreservationcharacteristics and identity should be established during early processdevelopment. For cell banks in particular, the cell status and optimal growthconditions, as well as documented history (with traceability to a qualifiedcell bank or acceptable source), characteristics, and authenticity should be documented.Cell status and history typically are described in terms of the nature andnumber of manipulations and culture passages from the primary cells or originalisolate. Finite or primary cells usually are cryopreserved at an early passageto maintain integrity of the original tissue, but continuous cell lines may be clonedand expanded, ensuring a homogeneous cell population. It is recommended toprepare cell banks from a single preparation or expanded population of cells sinceit is often necessary to pool cells for freezing from multiple culture vessels.Cells from cultures with different passage histories and certainly from differentdonors should not be pooled. In both cases, analysts should maintain detailedrecords of the procedures.
在早期工艺开发过程中应确定好低温保存的特性。尤其是细胞库，应记录细胞状态和最佳生长条件，以及历史记录(可追溯到合格的细胞库或可接受的来源)、特征和真实性。通常根据来自原代细胞或原始分离株的操作或培养传代的性质和数量来描述细胞的状态和历史。有限细胞系或原代细胞通常会在早期传代时进行冷冻保存来保持原始组织的完整性，而连续细胞系可以通过克隆和扩增保持细胞群体同质性。建议从单一制备物或扩大的细胞群中制备细胞库，因为通常需要将细胞从多个培养容器中汇集起来进行冷冻。来自不同传代历史的培养细胞和来自不同捐赠者的细胞当然不应该混合在一起。在这两种情况下，分析人员均应保留操作过程的详细记录。
Toprepare for cryopreservation of cultured cells, cells should be harvestedduring exponential or the most rapid phase of growth and before the cultureenters stationary phase. Harvesting cells during this phase ensures that thecells are most viable and uniform. The optimal concentration of cells willdepend on the cell type, purpose, and best recovery. Typically this liesbetween 10 and 10 /mL for manufacturing cell banks but may be different forother purposes. Complete growth medium renewal a day before cell harvest alsocan be beneficial. Additionally, most cell suspensions benefit from washing bycentrifugation and resuspension in an isotonic medium to a specific cellconcentration. Prefreeze processing should not result in cells that arestressed before the start of the freezing process, or cell losses duringfreezing or after thaw will be higher than expected.
准备低温保存的细胞应该在指数生长或最快速生长阶段和培养进入平台期之前进行收集。在此阶段收集细胞可确保细胞最具活力和均一性。冻存细胞的最佳浓度取决于细胞类型、用途和最佳回收率。对于制造细胞库来说，这个值通常在106到107 /mL之间，但是出于其他目的可能会有所不同。在细胞收获的前一天完全更新生长培养基也会有益处。此外，大多数细胞悬浮液通过离心洗涤和在等渗介质中重悬来达到特定的细胞浓度。在冷冻过程开始前，预冷冻处理不应使细胞受到压力，否则在冷冻过程中或解冻后细胞损失将会高于预期。
Optimizingthe growth conditions of a cell line or primary cells is important to maintainhigh viability of the cells in culture. Typically, cells growing actively andin exponential phase have a low cytoplasm to nuclear volume ratio, which isconducive to successful cryopreservation with penetrating cryoprotectants.Suboptimal or improper culture conditions may result in lower viability andcell states that will be less robust for preservation and recovery. The culturemedium should be optimized and the same medium should be used throughout allexperiments, and each batch of animal-derived materials (e.g., serum) and otherculture reagents should be qualitied (e.g., see the 2010 WHO guidance and theFDA 2010 guidance referenced in the Appendix). If possible, it is recommendedto not use animalderived components in the culture medium particularly forcells used for therapy or as manufacturing substrates.
优化细胞系或原代细胞的生长条件对保持细胞在培养中的高活力很重要。通常情况下，活跃且处于指数生长期的细胞具有较低的细胞质与细胞核的体积比，这有利于穿透性冷冻保护剂成功进行冷冻保存。欠佳或不合适的培养条件可能会导致存活率降低和细胞状态变差，不利于冻存复苏。需要优化培养基，并且在所有实验中都应使用相同的培养基，对每批动物来源的材料（例如血清）和其他培养试剂进行鉴定（如见在附录中引用的2010年 WHO指南和2010年FDA指南）。如果可能的话，建议不要在培养基中使用动物来源的成分，尤其是用于治疗或作为生成底物的细胞。
Per the WHO 2010 guidance and based on a risk assessment, either theMaster Cell Bank (MCB) or the Working Cell Bank (WCB) must be tested foradventitious
agents. Ideally, samples of cells should be tested for adventitious agentsbefore freezing. The specific testing regimen for potential microbial or viralcontamination of cells depends on the donor source, the culture history, andthe intended use. Detailed records of the cell history should be maintained asa basis for appropriate risk assessment to direct supplementary testing thatmay be required (e.g., exposure to bovine viruses in bovine serum albumin).Speci