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 na ve T cells require the cytokine IL-7 and contact with self-MHC proteins for survival. | |
− | + | [[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.]] | |
− | Today you can stagger your arrivals to lab (see | + | 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. |
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+ | [[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 Hoechst is a popular dye for this purpose. Active proliferation can be monitored by addition of 5-bromo-2'-deoxyuridine (BrdU) to cell cultures. BrdU will be incorporated only in recently synthesized DNA (S-phase cells), and can be assessed by antibody-detection after a time lag. For tracking multiple cell divisions, long-lived fluorescent dyes such as the fluorescein derivative CFDA-SE are used: about 6-10 divisions can be seen by flow cytometry (see figure at right). | ||
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+ | Remember that cell death is just as important as cell life, and that the type of death also matters. Cells that die due to acute trauma or other tissue damage typically die by necrosis: they swell and finally burst, releasing their contents and often promoting inflammation. Under other circumstances, particularly in development and immunity, many cells undergo a programmed death called apoptosis. Unlike the more disruptive necrotic cells, apoptotic cells condense and then fragment, finally releasing membrane-contained cell bodies. Apoptosis gone awry is implicated in many diseases, and thus researchers are very interested in tracking apoptotic cells in various culture systems. Special dyes can be used to track nuclear fragmentation and other changes in early and late apoptotic cells. | ||
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+ | Your objective today is to determine the viabilities of your three different cell cultures, and to gain experience with fluorescence assays. You are likely to encounter fluorescence and other microscopy techniques in many fields of biological engineering research. | ||
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+ | ==Protocols== | ||
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+ | Today you can stagger your arrivals to lab (see today s talk page). Only one group at a time will be able to work on the microscope, and assuming that cell culture setup takes ~ 1.5 hours, you will each have ~25 minutes to spend on the microscope. '''Please be respectful of your labmates time.''' Reading the protocol in advance will help you work more quickly, and is strongly recommended. | ||
===Part 1: Cell preparation and counting by Trypan exclusion=== | ===Part 1: Cell preparation and counting by Trypan exclusion=== | ||
− | You will test one of your two replicates for each of your three cell samples. The cells in monolayer culture will be removed from the culture dish using trypsin (as we used for the MES cells in Module 1), while the cells in alginate must be isolated with an EDTA-citrate buffer. Otherwise, the cells can be treated the same way. Unless otherwise stated, all manipulations should be done with sterile technique. | + | You will test one of your two replicates for each of your three cell samples. The cells in monolayer culture will be removed from the culture dish using trypsin (as we used for the MES cells in Module 1), while the cells in alginate must be isolated with an EDTA-citrate buffer. Otherwise, the cells can basically be treated the same way. You and your partner may want to work in parallel, one on 2D prep and one on 3D prep. Unless otherwise stated, all manipulations should be done with sterile technique. |
− | #Aspirate the culture medium from each of your samples | + | ====2D culture prep==== |
− | #Rinse the cells with | + | #Aspirate the culture medium from each of your samples. |
− | #Add | + | #Rinse the cells with 6 mL of warm PBS, then aspirate the buffer. |
+ | #Add 1 mL of trypsin/EDTA, and incubate at 37 °C for 3 min. | ||
#Now recover your cells: | #Now recover your cells: | ||
− | #* | + | #*Add 4.5 mL of warm complete culture medium, pipet up and down, and transfer to a 15 mL conical tube. |
− | #* | + | #*Spin the cells down at 1900g for 6 min (using the centrifuge that is in the TC room). Time this spin with your partner. During the spin, you might observe what your remaining replicate of the 2D sample looks like - what is the cell morphology and density like? |
− | #Spin the cells down at | + | #Resuspend in ~5 mL of culture medium for the monolayer. You can adjust the volume a bit depending on the size of the pellet you see, but ''write down'' what you use. |
− | #Resuspend in | + | |
+ | ====3D culture prep==== | ||
+ | |||
+ | #Using a sterile spatula, transfer the beads from your bottom-well replicates into the next well over (to the right). This is to exclude any cells that are growing on the bottom of the plate (as opposed to actually in the beads) from analysis. | ||
+ | #Aspirate the culture medium from each of your samples. Be careful not to suck up the beads with the aspirating pipet, by using a serological pipet just as you did when washing your freshly synthesized beads. | ||
+ | #Rinse the cells with 4 mL of warm PBS, then aspirate the buffer. | ||
+ | #Add 3 mL of EDTA-citrate buffer, and incubate at 37 °C for 3 min. | ||
+ | #Now recover your cells: | ||
+ | #*Add 3 mL of warm complete culture medium, pipet up and down to break up the beads (you may find this easier with a 1 mL pipetman rather than a serological pipet), and transfer to a 15 mL conical tube. | ||
+ | #*Spin the cells down at 1900g for 6 min (using the centrifuge that is in the TC room). Time this spin with your partner. During the spin, you might observe what your remaining replicates of the 3D samples look like - what is the cell morphology and density like in each? | ||
+ | #Resuspend in ~5-10 mL of culture medium for the beads. (For high density beads, 10 mL tends to be good.) You can adjust the volume a bit depending on the size of the pellet you see, but ''write down'' what you use. | ||
+ | |||
+ | ====3D optional prep==== | ||
+ | |||
+ | #In a small Petri dish, set aside 2-3 beads for whole-construct staining if you wish. | ||
+ | |||
+ | ====Cell count for 2D and 3D samples==== | ||
#Take 90 μL of each cell sample you want to measure into an eppendorf tube. | #Take 90 μL of each cell sample you want to measure into an eppendorf tube. | ||
#Take the cell count samples out of the hood, and mix each one with 10 μL of Trypan blue solution. This is a toxic material, so please be careful not to spill it. | #Take the cell count samples out of the hood, and mix each one with 10 μL of Trypan blue solution. This is a toxic material, so please be careful not to spill it. | ||
− | #Count each sample on a hemacytometer | + | #Count each sample on a hemacytometer |