The overall goal of this protocol is to describe the basic procedures to work with HEI-OC1 cells, which can be used to study the effects of pharmacological drugs on auditory cells, and assist in the functional characterization of the motor protein prestin. HEI-OC1 cells are used by many researchers around the world in different experiments with different goals and using different techniques. Hopefully, the procedures described here will provide a common base to make these studies comparable.
Of course, these experimental procedures may be further improved. But the differences with those described here could be key to understand and explain possibly contradictory results. In this demonstration we will describe how to work with HEI-OC1 cells starting from a frozen aliquot to having samples ready to perform different studies with widely available techniques.
To begin this procedure remove a vial of cryopreserved HEI-OC1 cells from liquid nitrogen and place it in a water bath at 37 degrees Celsius. Submerge only the lower half of the vial and allow it to thaw until only a small amount of ice remains in the vial. In a biological safety cabinet, wipe the outside of the vial with 70%alcohol.
Then, pipette the cells from the vial and deliver the entire volume drop by drop, slowly, into a 15 milliliter conical tube containing 10 milliliters of prewarmed growth medium such as DMEM supplemented with 10%FBS. Next, remove the DMSO by centrifuging the tube for five minutes. After five minutes, discard the supernatant and resuspend the cells in nine milliliters of fresh growth medium.
Disaggregate the clumps or sheets of cells by fluxing and refluxing the suspended cells between the pipette and the medium three to four times with the tip of the pipette touching the bottom of the tube. Then, place the total volume of cells suspended in growth medium in a 100 millimeter diameter untreated plastic cell culture dish. Incubate the cells at 33 degrees Celsius with 10%CO2.
In this step, remove the old medium and wash the cells with two milliliters of PBS. Then, cover the cell monolayer with 0.25%trypsin. Examine the cells using an inverted microscope when 40%of them have been detached.
If necessary, gently rock the culture dish to release any remaining attached cells Resuspend the cells and remove the trypsin by centrifuging the tube for five minutes. Afterward, discard the supernatant and resuspend the cells in nine milliliters of fresh growth medium. Next, disaggregate the clumps or sheets of cells by fluxing and refluxing the suspended cells between the pipette and the medium, three to four times with the tip of the pipette touching the bottom of the tube.
Proceed to seed the cells in 100 millimeter diameter cell culture dishes. Incubate them at 33 degrees Celsius with 10%CO2 and divide them as many times as necessary for the planned experiments, or for generating a new stock. For differentiation, incubate HEI-OC1 cells at the permissive conditions until they reach about 80%confluence.
Then, incubate the cells at 39 degrees Celsius with 5%CO2 for two to three weeks and change the medium every other day to remove dead cells. Begin this procedure by counting the cells using either an automatic cell counter or a hemocytometer and adjust the cell concentration to the required value. Next, seed the cells in a 96 well clear flat bottom plate, a white walled 96 well plate, or a six well plate.
If required by the particular assay, incubate the cells overnight for substrate attachment before treating them with the drugs of interest. Then, measure the outcome of the treatment by flow cytometry or another adequate technique. To harvest the cells, wash them twice with 10 milliliters of calcium-and magnesium-free PBS.
Add two milliliters of the enzyme-free detacher solution and incubate the cells for three minutes at 37 degrees Celsius with 5%CO2. Use an inverted microscope to check the detachment of the cells. If necessary, move the cell culture dish gently to detach more cells, and be careful not to shake it.
Next, add 10 milliliters of DMEM plus 10%FBS and pipette the cells up and down, gently, five times. After that, look at the cells under a microscope. If more than 80%of the cells are already separated, no further pipetting is needed.
If the cells are still in clusters, continue gently pipetting until more than 80%of the cells are no longer in clusters. An optical control of the cells should show many single round cells with smooth membrane edges. Afterwards, proceed with patch-clamp and NLC measurements, as described in the text protocol.
HEI-OC1 cells cultured at nonpermissive conditions showed a highly significant decrease in cell viability, and an equally significant increase in cell death in respect to cells cultures at permissive conditions. Since drug effects on cells already dying or with a decreased viability could be different than the effects of the same drug on healthy cells, considerable efforts should be dedicated to discriminate drug effects from the effects of culture conditions in any cytotoxicity study performed on NP-HEI-OC1 cells. Therefore, we recommend using HEI-OC1 cells cultured at permissive conditions for experiments that do not specifically require differentiated cells.
The confocal images shown here confirm the expression of the auditory motor protein prestin in HEI-OC1 cells cultured at 33 degrees Celsius and 39 degrees Celsius. Note that prestin immunolocalizes mostly at the cytoplasm of HEI-OC1 cells growing in the permissive conditions, and it is more concentrated at the plasma membrane in the more differentiated cells. Flow cytometry studies showed a clear increase in prestin expression in both conditions, suggesting that translocation was accompanied by synthesis of more prestin molecules.
On the other hand, the electrophysiological studies showed a robust NLC in HEI-OC1 cells growing at 33 degrees Celsius, with a peak value that decreased and a voltage at peak capacitance that shifted to the more depolarized values as the cells were moved to the nonpermissive conditions for one and two weeks. Intriguingly, these results suggest that motor function could be compromised if the density of prestin molecules in the plasma membrane is too high. It is important to remember that the protocols described here require the correct use of well-established cell culture techniques.
Also, remember to avoid the use of antibiotics. In addition, remember that the phenotype and response of HEI-OC1 cells will strongly depend on the culture parameters.
