Difference between revisions of "20.109(S08):Testing cell viability (Day3)"
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==Introduction== | ==Introduction== | ||
− | Promoting appropriate cell life and death is a key part of tisssue engineering. When cells are put into contact with a biomaterial (or into any novel culture condition), their viability may be affected. Some materials are cytotoxic, i.e., deadly to cells. Often, cytotoxicity varies with the concentration of one or more of the chemical components (such as a cross-linker) comprising the biomaterial, and is more or less severe for different cell types. Cell density within a culture is another factor affecting cell livelihood, notably when the number of cells exceeds the nutrient concentrations available in the culture medium. In a 3D culture such as an alginate bead, sufficient nutrients and even oxygen may not be able to diffuse to the center of the bead prior to depletion by cells on the outer rim, even when at a high concentration in the bulk fluid. Finally, note that most cells require certain soluble and/or contact-dependent signals to remain viable. For example, immune cells called | + | Promoting appropriate cell life and death is a key part of tisssue engineering. When cells are put into contact with a biomaterial (or into any novel culture condition), their viability may be affected. Some materials are cytotoxic, i.e., deadly to cells. Often, cytotoxicity varies with the concentration of one or more of the chemical components (such as a cross-linker) comprising the biomaterial, and is more or less severe for different cell types. Cell density within a culture is another factor affecting cell livelihood, notably when the number of cells exceeds the nutrient concentrations available in the culture medium. In a 3D culture such as an alginate bead, sufficient nutrients and even oxygen may not be able to diffuse to the center of the bead prior to depletion by cells on the outer rim, even when at a high concentration in the bulk fluid. Finally, note that most cells require certain soluble and/or contact-dependent signals to remain viable. For example, immune cells called na ve T cells require the cytokine IL-7 and contact with self-MHC proteins for survival. |
− | [[Image: 20.109_live-dead-example. | + | [[Image: 20.109_live-dead-example.png |thumb|right|275px| '''LIVE/DEAD® assay example.''' Cell viability was monitored using fluorescent dyes that differ in their cell permeance and nucleic acid affinity. Fluorescence emission in the green (left) and red (right) channels is shown for the same field of cells.]] |
− | Many assays are available to monitor the numbers of live and dead cells in a culture. The kit you will use today is made by Molecular Probes, a company (now partnered with Invitrogen) that makes a plethora of fluorescent cell stains for various purposes. The principle exploited by the LIVE/DEAD® kit is the relative permeability of cell membranes when the cell is live (intact membrane) or dead (damaged membrane). Ethidium is a nucleic acid stain that you are familiar with from running agarose gels in module 1; the ethidium homodimer-2 variant emits red fluorescence, and cannot diffuse past intact cell membranes. The dye SYTO 10, on the other hand, is membrane-permeant, and thus enters both live and dead cells; it emits fluorescence in the green channel. SYTO 10 has lower affinity for nucleic acids than does ethidium, and thus is excluded from dead cells over time, enabling one to distinguish between live (green) and dead (red) cells. | + | Many assays are available to monitor the numbers of live and dead cells in a culture. The kit you will use today is made by Molecular Probes, a company (now partnered with Invitrogen) that makes a plethora of fluorescent cell stains for various purposes. The principle exploited by the LIVE/DEAD® kit is the relative permeability of cell membranes when the cell is live (intact membrane) or dead (damaged membrane). Ethidium is a nucleic acid stain that you are familiar with from running agarose gels in module 1; the ethidium homodimer-2 variant emits red fluorescence, and cannot diffuse past intact cell membranes. The dye SYTO 10, on the other hand, is membrane-permeant, and thus enters both live and dead cells; it emits fluorescence in the green channel. SYTO 10 has lower affinity for nucleic acids than does ethidium, and thus is excluded from dead cells over time, enabling one to distinguish between live (green) and dead (red) cells. Viability can be inferred by monitoring parameters other than cell permeability. For example, some membrane-permeable dyes are only activated to a fluorescent form inside cells that have active esterase enzymes, thus indicating their metabolic activity. Assays that measure cell potentials or redox activity are also available. In general, fluorescence assays are more sensitive than colorimetric assays. Along with sensitivity, stability, toxicity, and ease of scale-up are important factors to consider when choosing an assay. |
− | + | [[Image: 20.109_CFSE-example.tiff|right|thumb|225px| '''Cell proliferation assay example.''' Cells were stained with CFDA-SE and monitored by flow cytometry after several days.]] | |
− | + | Cell vitality (or lack thereof) tells only one part of a cell culture s story. For example, kits like the one we are using today cannot determine whether the cells assayed have divided or not. However, other dyes are available that specifically test for cell proliferation, or even distinguish cells based on what part of the cell cycle they are presently in. Proliferation assays are important for drug development, cancer research, and in tissue engineering. Total nucleic acid content is sometimes used as a measure of proliferation |