专业英语细胞培养.pptx

Cell Culture Maolie liujing Outline nIntroductionnThe Physical Environment of Cell CulturenStandard Cell Culture TechniquesnContamination(污染污染)in Cell CultureintroductionnCell culture is the process by which prokaryotic,eukaryotic or plant cells are grown under controlled conditions.But in practice it refers to the culturing of cells derived from animal cellsnThe History of Cell Culture In 1912,Alexis Carrel,attempted to improve the state of the art of animal cell culture with experiments on the culture of chick embryo tissue.Carrel succeeded in expanding the possibilities of cell culture by keeping fragments of chick embryo heart alive and beating into the third month of culture and growing chick embryo connective tissue for over 3 months.This,along with Harry Eagles(1955)demonstration that the complex tissue extracts,clots,and so forth previously used to grow cells,opened up a new area of cell culture.The demonstration that each cell type has an optimal mix of nutrients that supports its function(Ham and McKeehan,1979;Waymouth,1981)has led to media derived to support specific cell types under specialized conditions.Finally,the advent of recombinant expression in mammalian cells and the creation of antibody-producing hybridoma(杂交瘤)cell lines,coupled with the use of large-scale culture techniques for culturing mammalian cells.A photograph of an industrial large-scale culture facility used to make recombinant proteins in mammalian cell culture.(A)Primary culture of isolated granulosa cells(粒层粒层细胞细胞);(B)a coculture of several cell types from neonatal(新生的新生的)rat lung;(C)a follicle(卵泡卵泡)derived from coculture of rat immature granulosa cells and oocytes(卵母细胞卵母细胞)that reassociate in vitro.The Physical Environment of Cell Culture LaboratorynThe essential idea of the entire panoply of the chemical solution and mechanical apparatus surrounding the cell in vitro culture is to recreate the physical,nutritional,and hormonal environment of the cell in vivo.nThis includes controlling the temperature,pH,osmolality,and gaseous environment providing a supporting surface,and protecting the cell from chemical,physical,and mechanical stress.nTemperature Most mammalian cell cultures are grown in incubators that are set at 37C.This was chosen because it is the core body temperature of Homo sapiens.One might therefore expect it to be the optimal temperature for growing many cell lines derived from human tissues.However,there is no reason to expect 37C to be optimal for all types of human cells,and certainly not for all cells from all mammals.Figure shows packed cell volume(a measure of total cell number)and protein secretion by CHO cells grown at various temperatures.Growing cells at too high a temperature is more detrimental(有害的有害的)than too low a temperature.npH Most media strive to achieve and maintain a pH between 7.0 and 7.4,with a median of 7.2.Different cell types may have an optimal pH slightly outside this range,and cells certainly differ widely in their ability to tolerate significant deviations from this level.As with temperature,slow changes in pH are tolerated better than rapid changes that cause apoptotic cell death.The actual pH in a culture at a given time is thus determined by the culture configuration,the medium the cells are grown in,the cell type,and the buffering capacity of the medium.nOsmolality（渗透压）（渗透压）The osmolality of the medium used is determined by the medium formulation.Salts and glucose are the major contributors to the osmolality of the medium,although amino acids may also contribute significantly.Altering the osmolality significantly(by more than 50 mOsm)will almost always affect cell growth and function in some manner.This should be taken into consideration when studying the effects of the addition of ions,energy sources,or large changes in amino acid levels on cell growth and function.The effect of increasing osmolality on cell growth.The optimal osmolality for the growth of these CHO cells is approximately 290 mOsm.nMedia in Cell Culture The medium provides essential nutrients that are incorporated into dividing cells,such as amino acids,fatty acids,sugars,ions,trace elements,vitamins,and cofactors,and ions and molecules necessary to maintain the proper chemical environment for the cell.nCO2,Oxygen,and other Gases The CO2 level is generally part of the buffering system of the medium and must be set accordingly.Oxygen levels in the incubator are provided by the 90-95%air in the incubator mix.nCell Culture Laboratory（incubator,culture flask,microscope）Standard Cell Culture TechniquesnCell subculturingnCell freezing nCell recovering nCell countingPROCESSRemove all medium Washing cells with Ca2+Mg2+-free PBSTrypsin/EDTA solution to dislodge cellsWash cells and count the cells numberComplete medium to inhibit the Trypsin activityAdd fresh medium to culture cells Thermostatwaterbath(恒温水浴恒温水浴锅锅)Freeze-stored cans(冻存罐冻存罐)Contamination in Cell CulturenHow to Avoid It,Recognize It,and Get Rid of It One occurrence that every person who tries to grow mammalian cells in vitro has to deal with sooner or later is contamination.As a problem,it can vary from irritating to catastrophic.The best solution?Avoid getting contamination in the first place.Failing this,the next best thing is to destroy all the contaminated cultures.One approach to contamination you cannot do is ignore it.Contamination in a cell culture will influence virtually any parameter you might wish to study,even if it does not immediately kill the cells.Do not ever use contaminated cultures to get numbers(we will not call it data)on the grounds that they are just a little contaminated or the cells are still alive.Any numbers you get will be misleading rather than helpful,and a waste of time.One approach to contamination you cannot do is ignore it.Contamination in a cell culture will influence virtually any parameter you might wish to study,even if it does not immediately kill the cells.Do not ever use contaminated cultures to get numbers(we will not call it data)on the grounds that they are just a little contaminated or the cells are still alive.Any numbers you get will be misleading rather than helpful,and a waste of time.