Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 101
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To do so, tissuefragments are treated with a combination of a protease (e.g.,trypsin or the collagen-hydrolyzing enzyme collagenase orboth) and a divalent cation chelator (e.g., EDTA) that depletes the medium of usable Ca2 or Mg2. The releasedcells are then placed in dishes in a nutrient-rich, serumsupplemented medium, where they can adhere to the surfaceand one another.
The same protease/chelator solution is usedto remove adherent cells from a culture dish for biochemical studies or subculturing (transfer to another dish).Often connective tissue fibroblasts divide in culture morerapidly than other cells in a tissue, eventually becoming thepredominant type of cells in the primary culture, unless special precautions are taken to remove them when isolatingother types of cells. Certain cells from blood, spleen, or bonemarrow adhere poorly, if at all, to a culture dish but nonetheless grow well. In the body, such nonadherent cells are heldin suspension (in the blood) or they are loosely adherent (inthe bone marrow and spleen). Because these cells often comefrom immature stages in the development of differentiatedblood cells, they are very useful for studying normal bloodcell differentiation and the abnormal development ofleukemias.When cells removed from an embryo or an adult animalare cultured, most of the adherent ones will divide a finitenumber of times and then cease growing (cell senescence,Figure 6-38a).
For instance, human fetal fibroblasts divideabout 50 times before they cease growth. Starting with 106cells, 50 doublings can produce 106 250, or more than1020 cells, which is equivalent to the weight of about 105people. Normally, only a very small fraction of these cells areused in any one experiment. Thus, even though its lifetimeis limited, a single culture, if carefully maintained, can bestudied through many generations. Such a lineage of cellsoriginating from one initial primary culture is called a cellstrain.Cell strains can be frozen in a state of suspended animation and stored for extended periods at liquid nitrogen temperature, provided that a preservative that prevents theformation of damaging ice crystals is used.
Although somecells do not survive thawing, many do survive and resumegrowth. Research with cell strains is simplified by the ability to freeze and successfully thaw them at a later time for experimental analysis.Transformed Cells Can Grow Indefinitelyin CultureTo be able to clone individual cells, modify cell behavior, orselect mutants, biologists often want to maintain cell culturesfor many more than 100 doublings.
Such prolonged growthis exhibited by cells derived from some tumors. In addition,rare cells in a population of primary cells that undergo certain spontaneous genetic changes, called oncogenic transformation, are able to grow indefinitely. These cells are said tobe oncogenically transformed or simply transformed. A culture of cells with an indefinite life span is considered immortal and is called a cell line.The HeLa cell line, the first human cell line, was originally obtained in 1952 from a malignant tumor (carcinoma) of the uterine cervix.
Although primary cell culturesof normal human cells rarely undergo transformation intoa cell line, rodent cells commonly do. After rodent cellsare grown in culture for several generations, the culturegoes into senescence (Figure 6-37b). During this period,most of the cells stop growing, but often a rapidly dividing transformed cell arises spontaneously and takes over, orovergrows, the culture.
A cell line derived from such a6.7 • Growth and Use of Cultured Cellstransformed variant will grow indefinitely if provided withthe necessary nutrients.Regardless of the source, cells in immortalized lines oftenhave chromosomes with abnormal structures. In addition,the number of chromosomes in such cells is usually greaterthan that in the normal cell from which they arose, and thechromosome number expands and contracts as the cells continue to divide in culture.
A noteworthy exception is theChinese hamster ovary (CHO) line and its derivatives, whichhave fewer chromosomes than their hamster progenitors.Cells with an abnormal number of chromosomes are said tobe aneuploid.(a) Human cellsPhaseIPhaseIIIPhase IICell strainGrowthrate ofcultureCellsenescence125Cell generations50(b) Mouse cellsInitial lossof growthpotentialGrowthrate ofcultureEmergence ofimmortalvariant(cell line)Senescence3060Days after initiation of culture▲ FIGURE 6-37 Stages in the establishment of a cellculture. (a) When cells isolated from human tissue are initiallycultured, some cells die and others (mainly fibroblasts) start togrow; overall, the growth rate increases (phase I). If theremaining cells are harvested, diluted, and replated into dishesagain and again, the cell strain continues to divide at a constantrate for about 50 cell generations (phase II), after which thegrowth rate falls rapidly.
In the ensuing period (phase III), allthe cells in the culture stop growing (senescence). (b) In aculture prepared from mouse or other rodent cells, initial celldeath (not shown) is coupled with the emergence of healthygrowing cells. As these dividing cells are diluted and allowed tocontinue growth, they soon begin to lose growth potential, andmost stop growing (i.e., the culture goes into senescence). Veryrare cells survive and continue dividing until their progenyovergrow the culture. These cells constitute a cell line, which willgrow indefinitely if it is appropriately diluted and fed withnutrients: such cells are said to be immortal.237Most cell lines have lost some or many of the functionscharacteristic of the differentiated cells from which they werederived. Such relatively undifferentiated cells are poor models for investigating the normal functions of specific celltypes. Better in this regard are several more-differentiated celllines that exhibit many properties of normal nontransformedcells.
These lines include the liver tumor (hepatoma) HepG2line, which synthesizes most of the serum proteins made bynormal liver cells (hepatocytes). Another example consists ofcells from a certain cultured fibroblast line, which under certain experimental conditions behave as muscle precursorcells, or myoblasts. These cells can be induced to fuse to formmyotubes, which resemble differentiated multinucleatedmuscle cells and synthesize many of the specialized proteinsassociated with contraction. The results of studies with thiscell line have provided valuable information about the differentiation of muscle (Chapter 22).
Finally, as discussed previously, the MDCK cell line retains many properties of highlydifferentiated epithelial cells and forms well-defined epithelial sheets in culture (see Figure 6-6).Hybrid Cells Called Hybridomas ProduceAbundant Monoclonal AntibodiesIn addition to serving as research models for studies on cellfunction, cultured cells can be converted into “factories”for producing specific proteins.
In Chapter 9, we describehow it is done by introducing genes encoding insulin,growth factors, and other therapeutically useful proteinsinto bacterial or eukaryotic cells. Here we consider the useof special cultured cells to generate monoclonal antibodies,which are widely used experimental tools and increasinglyare being used for diagnostic and therapeutic purposes inmedicine.To understand the challenge of generating monoclonalantibodies, we need to briefly review how antibodies areproduced by mammals.
Each normal B lymphocyte in amammal is capable of producing a single type of antibodydirected against (can bind to) a specific chemical structure(called a determinant or epitope) on an antigen molecule. Ifan animal is injected with an antigen, B lymphocytes thatmake antibodies recognizing the antigen are stimulated togrow and secrete the antibodies. Each antigen-activatedB lymphocyte forms a clone of cells in the spleen or lymphnodes, with each cell of the clone producing the identicalantibody—that is, a monoclonal antibody. Because mostnatural antigens contain multiple epitopes, exposure of ananimal to an antigen usually stimulates the formation ofmultiple different B-lymphocyte clones, each producing adifferent antibody.
The resulting mixture of antibodies thatrecognize different epitopes on the same antigen is said tobe polyclonal. Such polyclonal antibodies circulate in theblood and can be isolated as a group and used for a variety of experiments. However, monoclonal antibodies arerequired for many types of experiments or medical applications. Unfortunately, the biochemical purification of any238CHAPTER 6 • Integrating Cells into TissuesInject mousewith antigen XMutant mousemyeloma cellsunable to growin HAT mediumMouse spleen cells;some cells (red) makeantibody to antigen X1Mix andfuse cells2Transfer toHAT mediumUnfused cells() dieFused cells() growTechnique Animation: Preparing Monoclonal AntibodiesMEDIA CONNECTIONS3Culture single cellsin separate wellsTest each well for antibody to antigen X▲ EXPERIMENTAL FIGURE 6-38 Use of cell fusionand selection to obtain hybridomas producingmonoclonal antibody to a specific protein.
Step 1 :Immortal myeloma cells that lack HGPRT, an enzymerequired for growth on HAT selection medium, are fusedwith normal antibody-producing spleen cells from an animalthat was immunized with antigen X. The spleen cells canmake HGPRT. Step 2 : When plated in HAT medium, theunfused cells do not grow; neither do the mutant myelomacells, because they cannot make purines through an HGPRTdependent metabolic “salvage” pathway (see Figure 6-41),and the spleen cells, because they have a limited life span inculture. Thus only fused cells formed from a myeloma celland a spleen cell survive on HAT medium, proliferating intoclones called hybridomas.
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