In the past century, we have learned a tremendous amount by studying the behavior of mammalian cells maintained in the laboratory. Tissue culture was originally developed about 100 years ago as a method for learning about mammalian biology. The term tissue culture was coined because people were doing exactly that, extracting tissue and letting it live in a dish for a short time. Today, most tissue culture experiments are done using isolated cells rather than whole tissues. Much of what we know about cancer, heritable diseases, and the effects of the environment on human health has been derived from studies of cultured cells.
Cells that are isolated from tissue are called primary cells, because they come directly from an animal. It is very difficult to culture primary cells, largely because primary cells that are grown in culture divide only a limited number of times. This limitation on the lifespan of cultured primary cells, called the Hayflick limit, is a problem because it requires a researcher to constantly remove tissues from animals in order to complete a study. Cell isolation processes can be quite labor-intensive, and also can complicate data analysis due to inherent animal-to-animal variation. To get around the first of these problems, researchers use cells that are immortal, which means they can divide indefinitely, though some inherent cell-to-cell variation still exists in such cells.
One familiar type of immortalized cell is the cancer cell. Tumor cells continuously divide, allowing cancer to invade tissues and proliferate. Cancer cells behave the same way in culture, and under the right conditions, cells can be taken from a tumor and divide indefinitely in culture. Another type of immortalized cell is the embryonic stem cell. Embryonic stem cells are derived from an early stage embryo, and these cells are completely undifferentiated and pluripotent, which means that under the right conditions, they can become any mammalian cell type.
To test the effect of the putative small molecule binders on TDP43-RRM12 stability, you will Cath.-a-differentiated (CAD) cells. This cell line originated from a mouse (strain B6/D2 F1 hybrid) catecholaminergic neuronal tumor. In the exercise that you complete today, you will consider how to ensure that this particular cell line is appropriate for your experiment.
Part 1: Learn cell culture best practices
One major objective for this experimental module is for you to learn best practices for cell culture using correct sterile techniques. Pay close attention to the demonstration provided by the Instructor!
Review the following resource before you complete the tasks detailed in this exercise: Guidelines for working in the tissue culture room
To ensure the steps included below are clear, please watch the video tutorial linked here: [Cell Culture]. The steps are detailed below so you can follow along!
Preparing tissue culture hood
- The tissue culture hood is partly set up for you. Finish preparing your hood according to the demonstration, first bringing in any remaining supplies you will need, then obtaining the pre-warmed reagents from the water bath, and finally retrieving your cells from the 37 °C incubator.
- Don't forget to spray everything (except cells) with 70% ethanol!
- One of the greatest sources for tissue culture contamination is moving materials in and out of the hood because this disturbs the air flow that maintains a sterile environment inside the hood. Think about what you will need during your experiment to avoid moving your arms in and out of the hood while you are handling your cells.
- Obtain one ~48 h cultures of CAD cells in T75 flask from the 37 °C incubator.
- Examine your cell cultures after you remove the flask from the incubator.
- Look first at the color and clarity of the media. Fresh media is reddish-orange in color and if the media in your flask is yellow or cloudy, it could mean that the cells are overgrown, contaminated, or starved for CO2.
- Next, look at the cells using the inverted microscope. Note their shape, arrangement, and how densely the cells cover the surface of the flask.
- After you look at your cells, take the flask to your tissue culture hood to begin the seeding procedure.
- Aspirate the media from the cells using a sterile Pasteur pipet.
- Wash the cells by adding 5 mL PBS using a 5 mL pipet. Slightly tip the flask back and forth to rinse the cells then aspirate the PBS with a fresh Pasteur pipet.
- To dislodge the cells from the flask, you will add trypsin, a proteolytic enzyme.
- With a 2 mL pipet, add 1 mL of trypsin to the flask.
- 2 mL pipets are tricky! They fill up quickly. Be careful not to pull up the liquid too quickly or it will go all the way up your pipet into the pipet-aid! If this happens, please alert the teaching faculty rather than returning the pipet-aid to the rack.
- Tip the flask in each direction to distribute the trypsin evenly then incubate the cells at 37°C for 2 minutes using a timer.
- This is a great time to clear out your trash and read ahead!
- Retrieve your flask from the incubator and firmly tap the bottom to dislodge the cells.
- Check your cells using the microscope to ensure they are dislodged. They should appear round and move freely.
- If your cells are not detached from the flask, incubate at 37 °C for an additional minute.
- When your cells are dislodged, move your flask back into the tissue culture hood and add 5 mL of media to the cells then pipet the liquid up and down (“triturate”) to break up cells that are clumped together and suspend them in the liquid.
- Note: do not take up or release all the liquid, in order to avoid bubbles.
- Transfer the suspended cells into a labeled 15 mL conical tube.
- Transfer 90 μL of your cell suspension from the 15 mL conical tube into a labeled eppendorf tube.
During your work in tissue culture, you will use a hemocytometer to count mammalian cells. More importantly, you will use the cell count information to determine the density of your cultures. A hemocytometer is a modified glass microscope slide that has a chamber engraved with a grid. Stained mammalian cells are loaded into the chamber, which is manufactured such that the area within the gridlines is known and the volume of the chamber is known. These features enable researchers to count the number of cells within a specific volume of liquid.
Counting cells using a hemocytometer.
Using a hemocytometer, you can determine the density (cells per mL) of cell culture.
- Carry the tube with your 90 μL cell suspension aliquot to the center microscope bench and add 10 μL of trypan blue cell stain. Mix by pipetting up and down.
- Carefully pipet 10 μL of the stained cells between the hemocytometer and (weighted) glass cover slip.
- Count the cells that fall within the four corner squares (with a 4x4 etched grid pattern), average (i.e. divide by 4), and then multiply by 10,000 to determine the number of cells/mL.
In your laboratory notebook, complete the following:
- Use your cell counts to calculate the density of the cell culture.
- Calculate the total number of cells present in the cell suspension.
- Hint: use the density calculated and the total volume for this calculation.
- Calculate the volume of cell suspension that contains 1 M cells.
Part 2: Seed cells for localization experiment
- Obtain one T75 flasks from the center laboratory bench.
- Clearly label the flasks with the date and your team name. Include the name of the cell line seeded!
- Mix the cell suspension from Part 2.
- Cells settle quickly in conical tubes. It's important you mix before adding cells to the flasks.
- Add 1 M cells into the flask.
- If the cell suspension does not contain 1 M cells, just add the entire the volume of the cell suspension to the flask.
- Label the flask such that the number of cells that were added is easily visible.
- Give any extra cell suspension to the Instructor.
- Add fresh media to the flask such that the final volume equals 12 mL.
- Hint: subtract the volume of cell suspension added from 12 to calculate the volume of fresh media needed.
- Carefully move your flask to the 37°C incubator
- Clean out the tissue culture hood:
- Aspirate any remaining aliquots.
- Dispose of all vessels that held cells in the biohazard waste box and be sure that all sharps are in the sharps jar.
- Remove any equipment or supplies that you transferred into the hood and return to the appropriate location.
- Please leave the equipment that was already there.
- Spray the TC hood surface with 70% ethanol and wipe with paper towels.
- Be sure the paper towels are disposed of in the biohazard waste box!
- Empty the benchtop biohazard bucket into the biohazard waste box.
Part 3: Research CAD cell line
Though 109ers in a previous semester purified TDP43-RRM12 and conducted the SMM screen that you studied in the first days of this module, you are the first class to test the identified 'hits' in this cell-based assay. Because this is a new experiment, we needed to acquire new tools! Specifically, we needed to find an appropriate cell line for the experiment. It was important to address two specifications when considering which cell line to use:
- Is the cell line appropriate for the experiment?
- Do the cells generate the protein of interest?
To address these questions, you will work with your laboratory partner. The following resources may be useful to you as you address the above questions:
- The publication in which the cell line was first described is linked here.
- The American Type Tissue Collection (ATCC) product information is linked here.
- Pubmed and Google (or your favorite search engine) are also great resources!
In your laboratory notebook, complete the following:
- Is the CAD cell line appropriate to use in experiments associated with this module? Why or why not?
- Design an experiment that will allow you to confirm that the CAD cells generate the TDP43 protein.
- Mouse Cath.-a-differentiated (CAD) cell line (a gift from the Gertler Laboratory, MIT)
- DMEM / F12 (1:1) (from Gibco), supplemented with:
- 10% fetal bovine serum (FBS) (from Atlanta Biologicals)
- 100 U/mL of antibiotic solution, containing penicillin and streptomycin (from Gibco)
- trypan blue (from VWR)
- trypsin-EDTA (0.25%) (from Gibco)
- incubator maintains 37°C, 5% CO2 and 95% relative humidity
Next day: Complete staining for TDP43-localization assay
Previous day: Perform aggregation assay using TDP43 protein and draft data slide for Research summary