细胞保存技术手册

CryopreservationManualNalge NuncInternational9Written by Frank P.Simione,M.S.of the American Type Culture Collection(ATCC)in cooperation with Nalge Nunc International Corp.Nalge Nunc International Corp.1998ContentsIntroduction.1Seed Lot System.1Cryoprotective Agents.2Preparation of Cells.2Equilibration.4Ampoules and Vials.4Rate of Cooling.4Storage.5Reconstitution (Thawing).6Determination of Recovered Cells.6Inventory Control.6Culture Collection Practices.7Safety Considerations.7Step-by-Step.8Selected References.81More intracellular iceLess osmotic imbalanceLess intracellular iceMore osmotic imbalanceFig.1Use a cooling rate of 1C/minute and acryoprotective agent to minimize damage due toosmotic imbalance and ice crystal formation.IntroductionTo ensure reproducible results and continuity in research andbiomedical processes,todays scientists are faced with the task ofgenetically stabilizing living cells.Serial subculturing is timeconsuming and can lead to contamination or genetic drift assmaller and smaller portions of a population are selected.However,a population of cells can be stabilized by subjecting them tocryogenic temperatures which,for all practical purposes,stopstime.Stabilizing cells at cryogenic temperatures is calledcryopreservation,an applied aspect of cryobiology,or the studyof life at low temperatures.Advances in cryopreservationtechnology have led to methods that allow low-temperaturemaintenance of a variety of cell types.Techniques are available forthe preservation of microorganisms,isolated tissue cells,smallmulticellular organisms,and even more complex organisms such asembryos.The freezing process involves complex phenomena that,evenafter decades of research,are not fully understood.Cryobiologicalstudies have led to speculation on what occurs during the freezingof living cells and how adverse phenomena can be overcome.Since water is the major component of all living cells and must beavailable for the chemical processes of life to occur,cellularmetabolism stops when all water in the system is converted to ice.Ice forms at different rates during the cooling process.Duringslow cooling,freezing occurs external to the cell before intracellularice begins to form(1).As ice forms,water is removed from theextracellular environment and an osmotic imbalance occurs acrossthe cell membrane leading to water migration out of the cell.Theincrease in solute concentration outside the cell,as well asintracellularly,can be detrimental to cell survival(1).If too muchwater remains inside the cell,damage due to ice crystal formationand recrystallization during warming can occur.The rate of cooling has a dramatic effect on these phenomena.Rapid cooling minimizes the solute concentration effects as iceforms uniformly,but leads to more intracellular ice.Slow cooling,on the other hand,results in a greater loss of water from the celland less internal ice,but increases the solution effects.Cellpermeability affects the rate of water loss;more permeable cells areable to tolerate rapid cooling better than less permeable cells(2).Mazur et.al.(3)have postulated that ice crystal formation andsolution effects both play a role in cell inactivation,and that anoptimum cooling rate minimizes the effect of each.With fewexceptions,a cooling rate of 1C per minute is preferred.Usingcryoprotective additives or chemicals that protect the cells duringfreezing can also minimize the detrimental effects of increasedsolute concentration and ice crystal formation.The most com-monly used cryoprotective agents are dimethylsulfoxide(DMSO)and glycerol.Additionally,maintaining frozen cells at the properstorage temperature and using an appropriate warming rate willminimize damage to frozen cells.Seed Lot SystemTo ensure the genetic stability of a culture,the number of pas-sages from the original must be minimized.When freezing cells,use a system that ensures that early passage material is alwaysavailable for producing new working stock.One method ofpreserving early passage material is to use a seed lot system(4).When preparing the first frozen lot of a culture,a portion ofthe lot is set aside as seed material.The vials designated asseed material are maintained separately from the working stocksto ensure that they remain unused and are not handled duringretrieval operations.When the first working stock lot is de-pleted,a vial is retrieved from the seed lot and used to prepare asecond working stock.This continues until all seed vials,exceptone have been depleted.The last seed vial is then used toprepare a second seed lot.The second seed lot remains onlyone or two passages from the original material,but may beseparated by many years if the lots are adequately sized.In addition to seed material,a small portion of the original lotshould be segregated and maintained in a location remote fromall other material.Preferably,this reserve material should bestored in an off-site location,similar to the practice of theAmerican Type Culture Collection(ATCC).Rese