freezingcell20.pptVIP

Freezing and thawing cells Rational for freezing Cell lines in continuous culture are prone to variation Even the best-run laboratory is prone to equipment failure and contamination and cross-contamination There are many reasons, therefore, for freezing down a validated stock of cells; these reasons can be summarized as follows: (1) Genotypic drift基因型漂移 due to genetic instability (2) Senescence衰老 and the resultant extinction of the cell line (3) Transformation of growth characteristics and acquisition of malignancy-associated properties肿瘤相关的属性 (4) Phenotypic instability due to selection and dedifferentiation去分化 (5) Contamination by microorganisms (6)Cross-contamination by other cell lines (7)Misidenti?cation due to careless handling (8)Incubator failure (9)Saving time and materials maintaining lines not in immediate use 10)Need for distribution to other users Acquisition of cell lines for cryopreservation Validation验证 Cell lines should be shown to be free of contamination and authentic可靠的 before cryopreservation. When to freeze. If it is a ?nite cell line, it is grown to around the ?fth population doubling in order to create a suf?cient number of cells for freezing. Continuous cell lines should be cloned ，a characterized clone selected, and suf?cient stocks grown for freezing. Benefits of Freezing Cells Benefits of Freezing a Validated Stock of Cells Having a frozen bank of cells provide a backup in case cells are lost due to: Contamination Equipment failure such as incubator Cross-contamination by other cell lines Carelessness Senescence(衰老，normal human cells) leading to extinction of cell line Distribution to others Saving reagents, time Theoretical Background of Cell Freezing Optimal Cell Freezing Is Characterized By Maximum number of viable cells upon thawing Minimum intracellular crystal细胞内结晶 formation Minimum formation of foci集中 of high solute concentration Optimal Freezing Is Accomplished By Cooling slowly so water escapes Cooling fast enough to avoid