Organelle-Specific Antibodies Are Useful in Preparing Highly Purified Organelles Cell fractions remaining after differential and equilibrium density-gradient.

Organelle-Specific Antibodies Are Useful in Preparing Highly Purified Organelles

• Cell fractions remaining after differential and equilibrium density-gradient centrifugation usually contain more than one type of organelle.

• Monoclonal antibodies for various organelle-specific membrane proteins are a powerful tool for further purifying such fractions.

• One example is the purification of vesicles whose outer surface is covered with the protein clathrin; these coated vesicles, which participate in formation of endosomes from the plasma membrane.

• All cells contain a dozen or more different types of small membrane-limited vesicles of about the same size (50–100 nm in diameter) and density, which makes them difficult to separate from one another by centrifugation techniques.

• Immunological techniques are particularly useful for purifying specific classes of such vesicles.

• Fat and muscle cells, for instance, contain a particular glucose transporter (GLUT4) that is localized to the membrane of one of these types of vesicle.

• A gene encoding an organelle-specific membrane protein is modified by the addition of a segment encoding an epitope tag; the tag is placed on a segment of the protein that faces the cytosol.

Figure 9.27  Small coated vesicles can be purified by binding of antibody specific for a vesicle surface protein and linkage to bacterial cells.

Isolation, Culture, and Differentiation of Metazoan Cells

• fluorescence-activated cell sorter (FACS)• cells that express specific cell-surface proteins can be separated from a complex

mixture of cells.• an investigator can isolate homogenous populations of specific cell types for study. • culturing primary cells• Certain types of primary cells, especially those from embryos, can undergo

differentiation in culture.• muscle cell precursors grown in culture can differentiate and form apparently

normal muscle cells.• some accumulate cancer-causing (oncogenic) mutations can be cultured

indefinitely.• a single cell can be readily grown into a colony of identical cells, a process called

cell cloning.• Because these cells are genetically homogeneous, they are particularly suitable for

many types of biochemical and genetic studies. • Certain cloned cells can undergo differentiation into specific cells types such as

adipocytes (fat-storing cells), nerve, or muscle, allowing studies on the mechanism of cell differentiation to be conducted on homogenous cell populations.

Flow Cytometry Separates Different Cell Types

• Some cell types differ sufficiently in density that they can be separated on the basis of this physical property.

• White blood cells (leukocytes) and red blood cells (erythrocytes), for instance, have very different densities.

• Because most cell types cannot be differentiated so easily, other techniques such as flow cytometry must be used to separate them.

• A flow cytometer identifies different cells by measuring the light that they scatter and the fluorescence that they emit as they flow through a laser beam; thus it can quantify the numbers of cells of a particular type from a mixture.

• Indeed, a fluorescence-activated cell sorter (FACS), which is based on flow cytometry, can select one or a few cells from thousands of other cells and sort them into a separate culture dish.

• The FACS procedure is commonly used to purify the different types of white blood cells, each of which bears on its surface one or more distinctive proteins and will thus bind monoclonal antibodies specific for that protein.

• Only the T cells of the immune system, for instance, have both CD3 and Thy1.2 proteins on their surfaces.

• Other uses of flow cytometry include the measurement of a cell's DNA and RNA content and the determination of its general shape and size.

• The FACS can make simultaneous measurements of the size of a cell (from the amount of scattered light) and the amount of DNA that it contains (from the amount of fluorescence emitted from a DNA-binding dye).

Fluorescence-activated cell sorter (FACS) separates cells that are labeled differentially with a fluorescent reagent.

T cells bound to fluorescence-tagged antibodies to two cell-surface proteins are separated from other white blood cells by FACS

Culture of Animal Cells Requires Nutrient-Rich Media and Special Solid Surfaces

• animal cells require many specialized nutrients and often specially coated dishes for successful culturing.

• To permit the survival and normal function of cultured tissues or cells, the temperature, pH, ionic strength, and access to essential nutrients must simulate as closely as possible the conditions within an intact organism.

• Isolated animal cells are typically placed in a nutrient-rich liquid, called the culture medium, within specially treated plastic dishes or flasks.

• The cultures are kept in incubators in which the temperature, atmosphere, and humidity can be controlled.

• To reduce the chances of bacterial or fungal contamination, antibiotics are often added to the culture medium.

• Media for culturing animal cells must supply the nine amino acids that cannot be synthesized by adult vertebrate animal cells.

• most cultured cells require three other amino acids that are synthesized only by specialized cells in intact animals.

• vitamins, various salts, fatty acids, glucose, and serum• These factors include the polypeptide hormone insulin; transferrin, which supplies iron in a

bioaccessible form; and numerous growth factors. • certain cell types require specialized protein growth factors not present in serum. For instance,

progenitors of red blood cells require erythropoietin, and T lymphocytes require interleukin 2.• most animal cells will grow only on a solid surface.• cell-surface proteins, called cell-adhesion molecules (CAMs), that normally bind cells to other cells

or proteins such as collagen or fibronectin in the extracellular matrix surrounding many animal cells

• Some specialized blood cells and tumor cells can be maintained or grown in suspension as single cells.

Primary Cell Cultures Can Be Used to Study Cell Differentiation

• Normal animal tissues (e.g., skin, kidney, liver) or whole embryos are commonly used to establish primary cell cultures.

• To prepare tissue cells for a primary culture, the cell–cell and cell–matrix interactions must be broken.

• a combination of a protease (e.g., trypsin, the collagen-hydrolyzing enzyme collagenase, or both) and a divalent cation chelator (e.g., EDTA) that depletes the medium of usable Ca2+ and Mg2+.

• The same protease/chelator solution is used to remove adherent cells from a culture dish for biochemical studies or subculturing (transfer to another dish).

• Fibroblasts are the predominant cells in connective tissue and normally produce ECM components such as collagen that bind to cell-adhesion molecules, thereby anchoring cells to a surface.

• Several regions of vertebrate embryos contain myoblasts, precursors of muscle cells that both divide rapidly and migrate to other sites in the embryo.

• At some point myoblasts align with each other, cease division, and fuse to form a large multinucleate cell called a myotube.

• Similar mononucleated cells, termed satellite cells, are found in adult muscle and can fuse to form multinucleated myotubes and simultaneously induce synthesis of muscle-specific proteins such as myosin heavy chain.

• Certain cells from blood, spleen, or bone marrow adhere poorly, if at all, to a culture dish but nonetheless grow well in culture.

• In the body, such nonadherent cells are held in suspension (in the blood) or they are loosely adherent (in the bone marrow and spleen).

Figure 9.30  Differentiation in culture of primary mouse satellite (myoblast) cells into muscle cells.

Primary Cell Cultures and Cell Strains Have a Finite Life Span

• When cells removed from an embryo or an adult animal are cultured, most of the adherent ones will divide a finite number of times and then cease growing (cell senescence).

• For instance, human fetal fibroblasts divide about 50 times before they cease growth.

• Starting with 106 cells, 50 doublings can produce 106 × 250, or more than 1020 cells, which is equivalent to the weight of about 1000 people.

• a lineage of cells originating from one initial primary culture is called a cell strain.

• Research with cell strains is simplified by the ability to freeze and successfully thaw them at a later time for experimental analysis.

• at liquid nitrogen temperature

Figure 9.31  Stages in the establishment of a cell culture. 

Transformed Cells Can Grow Indefinitely in Culture

• rare cells in a population of primary cells may undergo spontaneous oncogenic mutations, leading to oncogenic transformation .

• Such cells, said to be oncogenically transformed or simply transformed, are able to grow indefinitely.

• 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 cultures of normal human cells rarely undergo transformation into a cell line, rodent cells commonly do.

• Regardless of the source, cells in immortalized lines often have chromosomes with abnormal DNA sequences.

• In addition, the number of chromosomes in such cells is usually greater than that in the normal cell from which they arose, and the chromosome number expands and contracts as the cells continue to divide in culture.

• A noteworthy exception is the Chinese hamster ovary (CHO) line and its derivatives, which have fewer chromosomes than their hamster progenitors.

• Cells with an abnormal number of chromosomes are said to be aneuploid.