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Cell Isolation and Cell Culture
Update time:2019-05-15 00:55:33   【 Font: Large  Medium Small

Regardless of the species to be studied, growing large populations of isolated cells for biochemical analysis and microscopic observation is helpful.This is straightforward for the unicellular organisms such as fungi or bacteria, which can be grown suspended in a nutrient medium. These organisms can also be grown on the surface of gelled agar in a petri dish.When single cells are dispersed widely on an agar surface,each multiplies to form a macroscopic colony, all descendents of a single cell. This family of cells is called a clone.
For multicellular organisms, it is often possible to isolate single live cells by dissociating a tissue with proteolytic enzymes and media that weaken adhesions between the cells.Tissues provide the most realistic source of material. Several approaches are used to separate the different cell types from solid tissue or liquid tissue. For solid tissue, the first step in isolating individual cells is to get mixed cell suspension through proteolytic enzymes digestion to disrupt the extracellular matrix and cell-cell junctions. After that, a flow cytometer, also known as fluorescence activated cell sorter (FACs), can be used to isolate target cells from the mixed cell suspension. For isolating cells, an antibody coupled to a fluorescent dye is used to specifically bind to the surface of specific cells, then the labeled cells are chosen from the unlabeled ones using the flow cytometer. 'The flow cytometer can sort 20 thousand cells each second, the purity of selected cell can reach to 95%. Immunomagnetic separation(lMS) is another useful tool that can efficiently isolate viable and functional cells, ln one approach, cells are incubated with immunomagnetic beads precoating antibodies which will bind to antigens present on the surface of cells, thus these bead-attached cells will be captured by a magnet placed on the side of the test tube. Compared with FACs, this technique is easy to work and its procedure is very rapid. In addition, a technique, lase capture microdissection (LCM), can be used to separate cells even a single cell accurately from the tissue.'Tissue sections (frozen section is generally used) are prepared for lase capture microdissection. LCM is a method to obtain the cells of interest in tissue under direct microscopic visualization and allow the properties or molecular composition of selected cells to be examined. It is especially useful to isolate cells dispersive in tissue.
Many but not all isolated cells can be grown in sterile media, a method called tissue culture or cell culture. Terminally differentiated cells such as muscle or nerve cells do not reenter the cell cycle and grow. Cells that are predisposed to grow in the body including fibroblasts and endothelial cells from blood vessels will grow if the nutrient medium is supplemented with growth factors to drive the cell cycle. This is accomplished by adding fetal calf serum, which contains a particularly rich mixture of in suspension, but most prefer to grow on a surface of plastic or glass, often coated with extracellular matrix molecules for adhesion. This is the origin of the term in vitro, meaning in glass, used to describe cell culture. Normal cells grow until they cover the artificial surface, when contacts with other cells arrest further growth. Dissociation and dilution of the cells onto a fresh surface allow growth to resume.Cultured prepared directly from the tissues of an organism are called primary cultures. Most primary cells isolated directly from tissues divide a limited number of times. After primary culture, cells can be removed from the culture dish and recultured repeatedly, this process is called secondary culture. Primary cells can become immortal, either through mutations or transformation by a tumor virus that over comes cell cycle controls. Such immortal cells are called cell lines.Similar changes allow cancer cells to grow indefinitely. HeLa cells are a famous cell line derived from Henrietta Lax, an African-American patient with cervical cancer. HeLa cells have been growing in laboratories for more than half a century.

A variation on cell culture is to grow a whole organ or part of an organ in vitro. The requirements for organ culture are often more stringent than those for growing individual cells,but the method is used routinely for experiments on slices of brain tissue and for studying the development of embryonic organs.

From liu jia adapted from medical cell biology

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