Promoting appropriate cell life and death is a key part of tissue 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 naïve T cells require the cytokine IL-7 and contact with self-MHC proteins for survival.
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 modules 1 and 2; 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.
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).
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.
Your objective today is to determine the viabilities of your two 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.
Today you can stagger your arrivals to lab. Only one group at a time will be able to work on the microscope, and assuming that cell culture setup takes ~ 1 hour, 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.
Before or after performing the viability assay, and/or during incubation steps, you should work on Part 3 of today's protocol.
When observing your cells under fluorescence excitation, you should work with the room lights off for best results. You can turn on the working lamp at the microscope bench as you set up your samples, and otherwise when you need to see what you are doing.
Sample results from a student group, showing clustering of cells within the bead:
A related pair of images by Agi Stachowiak, showing cell suspensions isolated from the beads:
Before or after your fluorescence assay work, find a place (across the hall, in a coffeeshop, etc.) to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below.
Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you've proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you've gathered. Consider them based on:
It might be that not one of the topics you've identified is really suitable, in which case you should find some new ideas. It's also possible that through discussion with your lab partner, you've found something new to consider. Both of these outcomes are fine but by the end of today's lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic.
A few ground rules that are 20.109 specific:
Once you and your partner have decided on a suitable research problem, it's time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the "yeast rebuild" or the "T7.2" wiki pages on OpenWetWare for examples of research ideas in process. As part of your For Next Time assignment, you will have to print out your wiki page specifying your topic, your research goal and at least two helpful references that you've read and summarized.
You may find other helpful articles in this essay assignment from the Spring 2008 20.109 course.
You can organize your wiki page along these lines or however you feel is most helpful. For now, focus on coming up with a research problem and giving a little background about it. Print your user page(s) for next time, making sure it defines your topic, your idea and two or more references you've collected and summarized. Keep in mind that you're not committed to this idea - if you come up with something that you like better later on, that's fine.