細(xì)胞培養(yǎng)(英文版)

1、Paras Yadav1, Annu Yadav1, P. Kumar1, J.S. Arora11, S. De1, S.L. Goswami1, Mukesh Yadav2, Shalini Jain3, Ravinder Nagpal4 and Hariom Yadav31Department of Animal Biotechnology, 3Animal Biochemistry Division and 4Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana),

2、India; 2SOS in Chemistry, Jiwaji University, Gwalior-474011, M.P., IndiaBasics of Cell CultureIntroductionCell 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 cells.C

3、ell culture was first successfully undertaken by Ross Harrison in 1907Roux in 1885 for the first time maintained embryonic chick cells in a cell cultureHistorical events in the development of cell culture1878: Claude Bernard proposed that physiological systems of an organism can be maintained in ali

4、ving system after the death of an organism.1885: Roux maintained embryonic chick cells in a saline culture.1897: Loeb demonstrated the survival of cells isolated from blood and connective tissue in serumand plasma.1903: Jolly observed cell division of salamander leucocytes in vitro.1907: Harrison cu

5、ltivated frog nerve cells in a lymph clot held by the hanging drop method andobserved the growth of nerve fibers in vitro for several weeks. He was considered by some asthe father of cell culture.1910: Burrows succeeded in long term cultivation of chicken embryo cell in plasma clots. He made detaile

6、d observation of mitosis.Contd.1911: Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract, salts and peptones. They observed limited monolayer growth.1913: Carrel introduced strict aseptic techniques so that cells could be cultured for long periods.1916: Rous and

7、 Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells.1923: Carrel and Baker developed Carrel or T-flask as the first specifically designed cell culture vessel. They employed microscopic evaluation of cells in culture.1927: Carrel and Rivera produced the first viral vacci

8、ne - Vaccinia.1933: Gey developed the roller tube techniqueContd.1940s: The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem of contamination in cell culture.1948: Earle isolated mouse L fibroblasts which formed clones from single cells. Fischer developed a

9、chemically defined medium, CMRL 1066.1952: Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells. Dulbecco developed plaque assay for animal viruses using confluent monolayers of cultured cells.1954: Abercrombie observed contact inhibition: motility

10、of diploid cells in monolayer culture ceases when contact is made with adjacent cells.1955: Eagle studied the nutrient requirements of selected cells in culture and established the first widely used chemically defined medium.1961: Hayflick and Moorhead isolated human fibroblasts (WI-38) and showed t

11、hat they have a finite lifespan in culture.1964: Littlefield introduced the HAT medium for cell selection.1965: Ham introduced the first serum-free medium which was able to support the growth of some cells.Contd.1965: Harris and Watkins were able to fuse human and mouse cells by the use of a virus.1

12、975: Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal antibody.1978: Sato established the basis for the development of serum-free media from cocktails of hormones and growth factors.1982: Human insulin became the first recombinant protein to be licensed as a therape

13、utic agent.1985: Human growth hormone produced from recombinant bacteria was accepted for therapeutic use.1986: Lymphoblastoid IFN licensed.1987: Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially available.1989: Recombinant erythropoietin in trial.1990: Recomb

14、inant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF, EGF, mAbs, IL-2).Major developments in cell culture technologyFirst development was the use of antibiotics which inhibits the growth of contaminants.Second was the use of trypsin to remove adherent cells to subculture furth

15、er from the culture vesselThird was the use of chemically defined culture medium.Why is cell culture used for? Areas where cell culture technology is currently playing a major role.Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs

16、on cells, process and triggering of aging & nutritional studiesToxicity testing Study the effects of new drugsCancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells Contd.Virology Cultivation of virus for vaccine production, als

17、o used to study there infectious cycle. Genetic Engineering Production of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measles Gene therapy Cells having a functional gene can be replaced to cells which are having

18、non-functional geneTissue cultureIn vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue cultureDifferent types of cell grown in culture includes connective tissu

19、e elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells. Primary cultureCells when surgically or enzymatically removed from an organism and placed in suitable culture environment will attach and grow are called

20、 as primary culturePrimary cells have a finite life spanPrimary culture contains a very heterogeneous population of cellsSub culturing of primary cells leads to the generation of cell linesCell lines have limited life span, they passage several times before they become senescentCells such as macroph

21、ages and neurons do not divide in vitro so can be used as primary culturesLineage of cells originating from the primary culture is called a cell strainContinous cell linesMost cell lines grow for a limited number of generations after which they ceasesCell lines which either occur spontaneously or in

22、duced virally or chemically transformed into Continous cell linesCharacteristics of continous cell lines -smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio -Fast growth and have aneuploid chromosome number -reduced serum and anchorage dependence and grow more in suspension

23、conditions -ability to grow upto higher cell density -different in phenotypes from donar tissue -stop expressing tissue specific genesTypes of cells On the basis of morphology (shape & appearance) or on their functional characteristics. They are divided into three.Epithelial like-attached to a subst

24、rate and appears flattened and polygonal in shapeLymphoblast like- cells do not attach remain in suspension with a spherical shapeFibroblast like- cells attached to an substrate appears elongated and bipolarCulture mediaChoice of media depends on the type of cell being culturedCommonly used Medium a

25、re GMEM, EMEM,DMEM etc.Media is supplemented with antibiotics viz. penicillin, streptomycin etc. Prepared media is filtered and incubated at 4 CWhy sub culturing.?Once the available substrate surface is covered by cells (a confluent culture) growth slows & ceases.Cells to be kept in healthy & in gro

26、wing state have to be sub-cultured or passagedIts the passage of cells when they reach to 80-90% confluency in flask/dishes/platesEnzyme such as trypsin, dipase, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surfaceCulturing of cellsCells are cultured a

27、s anchorage dependent or independentCell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate e.g. tissue cellsSuspension cells are anchorage-independent e.g. blood cellsTransformed cell lines either grows as monolayer or as suspensionAdherent ce

28、llsCells which are anchorage dependent Cells are washed with PBS (free of ca & mg ) solution. Add enough trypsin/EDTA to cover the monolayer Incubate the plate at 37 C for 1-2 mts Tap the vessel from the sides to dislodge the cells Add complete medium to dissociate and dislodge the cells with the he

29、lp of pipette which are remained to be adherent Add complete medium depends on the subculture requirement either to 75 cm or 175 cm flaskSuspension cellsEasier to passage as no need to detach themAs the suspension cells reach to confluencyAsceptically remove 1/3rd of mediumReplaced with the same amo

30、unt of pre-warmed mediumTransfection methodsCalcium phosphate precipitationDEAE-dextran (dimethylaminoethyl-dextran)Lipid mediated lipofectionElectroporationRetroviral InfectionMicroinjectionCell toxicityCytotoxicity causes inhibition of cell growthObserved effect on the morphological alteration in

31、the cell layer or cell shape Characteristics of abnormal morphology is the giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleusCytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc. at atimeWorking with cryo

32、preserved cellsVial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously until medium is thawedCentrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial with 70% ethanol and discard the supernatantResuspend the cell pellet in 1 ml of complete medium with 20% FBS an

33、d transfer to properly labeled culture plate containing the appropriate amount of mediumCheck the cultures after 24 hrs to ensure that they are attached to the plateChange medium as the colour changes, use 20% FBS until the cells are establishedFreezing cells for storageRemove the growth medium, was

34、h the cells by PBS and remove the PBS by aspirationDislodge the cells by trypsin-verseneDilute the cells with growth mediumTransfer the cell suspension to a 15 ml conical tube, centrifuge at 200g for 5 mts at RT and remove the growth medium by aspirationResuspend the cells in 1-2ml of freezing mediu

35、mTransfer the cells to cryovials, incubate the cryovials at -80 C overnightNext day transfer the cryovials to Liquid nitrogenCell viabilityCell viability is determined by staining the cells with trypan blueAs trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable t

36、o this dyeStain the cells with trypan dye and load to haemocytometer and calculate % of viable cells - % of viable cells= Nu. of unstained cells x 100 total nu. of cellsCommon cell linesHuman cell lines-MCF-7 breast cancerHL 60 LeukemiaHEK-293 Human embryonic kidneyHeLa Henrietta lacksPrimate cell l

37、inesVero African green monkey kidney epithelial cellsCos-7 African green monkey kidney cellsAnd others such as CHO from hamster, sf9 & sf21 from insect cells Contaminants of cell culture Cell culture contaminants of two typesChemical-difficult to detect caused by endotoxins, plasticizers, metal ions

38、 or traces of disinfectants that are invisibleBiological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell linesEffects of Biological ContaminationsThey competes for nutrients with host cellsSecreted acidic o

39、r alkaline by-products ceses the growth of the host cellsDegraded arginine & purine inhibits the synthesis of histone and nucleic acidThey also produces H2O2 which is directly toxic to cellsDetection of contaminantsIn general indicators of contamination are turbid culture media, change in growth rat

40、es, abnormally high pH, poor attachment, multi-nucleated cells, graining cellular appearance, vacuolization, inclusion bodies and cell lysisYeast, bacteria & fungi usually shows visible effect on the culture (changes in medium turbidity or pH) Mycoplasma detected by direct DNA staining with intercal

41、ating fluorescent substances e.g. Hoechst 33258Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycoplasmal RNAThe best and the oldest way to eliminate contamination is to discard the infected cell lines directlyBasic equipments used in

42、 cell cultureLaminar cabinet-Vertical are preferableIncubation facilities- Temperature of 25-30 C for insect & 37 C for mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx. 38.5 CRefrigerators- Liquid media kept at 4 C, enzymes (e.g

43、. trypsin) & media components (e.g. glutamine & serum) at -20 CMicroscope- An inverted microscope with 10 x to 100 x magnificationTissue culture ware- Culture plastic ware treated by polystyreneRules for working with cell culture Never use contaminated material within a sterile area Use the correct

44、sequence when working with more than one cell lines.Diploid cells (Primary cultures, lines for the production of vaccines etc.)Diploid cells (Laboratory lines)Continous, slow growing lineContinous, rapidly growing linesLines which may be contaminatedVirus producing linesBasic aseptic conditionsIf working on the bench use a Bunse