House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.
HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 °C to stimulate cell proliferation and incubation at 39 °C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.
House Ear Institute-Organ of Corti 1 (HEI-OC1) cells are derived from the auditory organ of a transgenic mouse 1,2. Incubation of any cell from this transgenic mouse at 33 °C/10% CO2 (permissive conditions) induces expression of an immortalizing gene that triggers de-differentiation and accelerated proliferation; moving the cells to 39 °C/5% CO2 (non-permissive conditions) lead to decreased proliferation, differentiation and, at least in the case of HEI-OC1, cell death 2,3.
HEI-OC1 cells were cloned and characterized in our laboratory over a decade ago, and initial studies indicated that they express specific markers of cochlear hair cells, such as prestin, myosin 7a, Atoh1, BDNF, calbindin and calmodulin, but also markers of supporting cells like connexin 26 and fibroblast growth factor receptor (FGF-R) 2. Therefore, it was suggested that HEI-OC1 could represent a common progenitor for sensory and supporting cells of the organ of Corti 2. Parallel studies provided strong evidence that archetypal ototoxic drugs like cisplatin, gentamicin and streptomycin induced caspase-3 activation in these cells, while drugs considered non-ototoxic, like penicillin, did not 2,3. Therefore, this cell line was proposed as an in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs. It is estimated that HEI-OC1 cells have been used in more than one hundred and fifty studies published in the last ten years.
Whereas looking at the potential pro-apoptotic effect of different drugs was the major goal of most of the studies involving this cell line, other important cell processes like autophagy and senescence have just started to be investigated in HEI-OC1 cells4-7. In a recent study from our laboratory 8, we used HEI-OC1 cells to collect a comprehensive set of data about cell death, survival, proliferation, senescence and autophagy induced by different pharmacological drugs frequently used in the clinic. We also compared some of the responses of HEI-OC1 cells with those from HEK-293 (human embryonic kidney cells) and HeLa (human epithelial cells) receiving identical treatment. Our results indicated that HEI-OC1 cells respond to the each drug in a characteristic way, with a distinctive dose- and time-dependent sensitivity to at least one of the mechanisms under study. We also emphasized in that study that a correct interpretation of the experimental results will require performing parallel studies with more than one technique 8.
In a different study we investigated the use of HEI-OC1 cells to evaluate the functional response of prestin, the motor protein of cochlear outer hair cells (OHCs) 9. We reported flow cytometry and confocal laser scanning microscopy studies on the pattern of prestin expression, as well as nonlinear capacitance (NLC) and whole cell-patch clamping studies in HEI-OC1 cells cultured at permissive (P-HEI-OC1) and non-permissive (NP-HEI-OC1) conditions. Our results indicated that both total prestin expression and plasma membrane localization increase in a time-dependent manner in NP-HEI-OC1 cells. Interestingly, we also found that the increase in prestin localization at the plasma membrane of NP-HEI-OC1 cells correlated with a decrease in Na+K+ATPase, which translocated from the plasma membrane to the cytoplasm without significant changes in total cell expression. In addition, we demonstrated that P-HEI-OC1 cells have a robust NLC associated to prestin motor function, which decreased when the density of prestin molecules present at the plasma membrane increased. Altogether, these results strongly support the usefulness of HEI-OC1 cells to investigate auditory proteins.
In this video article we describe how to culture HEI-OC1 cells, why it is convenient to use cells growing at permissive conditions (P-HEI-OC1) for cytotoxicity studies, how to evaluate the mechanism/s of drug-induced cytotoxicity and how to perform electrophysiological studies (e.g., patch-clamp, non-linear capacitance (NLC)) to investigate functional properties of prestin, the molecular motor of cochlear OHCs.
1. Cell Culture
Note: All cell culturing protocols must be performed using proper cell culture techniques (for reference see the first 3 Chapters of Cell Biology: A Laboratory Handbook, Volume I 10). HEI-OC1 cells do not require any additional coating or treatment of the cell culture dishes for proper adherence and growth. Very important: do not use glassware dishes for cell culture purposes; the phenotype and biological response of the cells to pharmacological drugs will change (G Kalinec & F Kalinec, unpublished); conventional plastic cell culture dishes are recommended (see Table of Materials/Equipment). Pay special attention to aseptic techniques to avoid contaminations, but never use antibiotics (e.g., ampicillin or streptomycin) with HEI-OC1 cells. If necessary, use amphotericin B. While HeLa and HEK-293 cells have been used as control in previous studies with HEI-OC1 8, any other cell line could be acceptable for this purpose.
2. Drug Cytotoxicity Studies
Note: HEI-OC1 cells grown at permissive conditions (P-HEI-OC1) are recommended for these studies (see Representative Results, Figure 1).
3. Electrophysiology Experiments with HEI-OC1 Cells
In a couple of recent publications we reported a comprehensive set of studies aimed at evaluating the response of HEI-OC1 cells to several commonly used pharmacological drugs as well as investigating prestin function 8,9. In these studies we made use of all the protocols described in the previous sections.
One of the results of these previous studies was that HEI-OC1 cells cultured at non-permissive conditions (39 °C/10% CO2 = NP-HEI-OC1) showed a highly significant decrease in cell viability, and an equally significant increase in cell death, respect to cells cultured at permissive conditions (33°C/10% CO2 = P-HEI-OC1) (Figure 1). Therefore, any cytotoxicity study performed on NP-HEI-OC1 cells should dedicate considerable efforts in discriminating drug effects from the effects of culture conditions. In addition, since drug effects on cells already dying or with a decreased viability could be different that the effects of the same drug on healthy cells, we recommend using P-HEI-OC1 cells for experiments that do not specifically require differentiated cells.
Our studies on the effect of different pharmacological drugs also produced interesting results. For example, cisplatin, a chemotherapeutic agent frequently used for the treatment of several human malignancies, and acetaminophen (APAP = N-acetyl-para-aminophenol), the most widely sold over-the-counter painkiller in the USA, were both toxic for HEI-OC1 cells (Figure 2). However, while APAP decreased cell viability and increased caspase 3/7 activation, it did not induce cell death. Cisplatin, in contrast, significantly increased the three responses (Figure 2). Interestingly, caspases 3/7 were mostly activated at lower cisplatin concentrations, suggesting that cell death induced by low doses of cisplatin was probably mediated by apoptosis, while high doses lethal effects could be associated with oncosis/necroptosis. While oncosis has been defined as a pre-lethal pathway leading to unregulated cell death accompanied by cellular and organelle swelling, blebbing, and increased membrane permeability 17,18, necroptosis is a regulated non-apoptotic cell death mechanism activated by the same stimuli that initiate apoptosis but with morphological features similar to those of oncosis 19-22.
Additional studies aimed at investigating the effects of APAP and cisplatin on P-HEI-OC1 cells showed a clear, dose-dependent effect of APAP on cell proliferation that could explain the intriguing decrease in cell viability without cell death described in the previous paragraph (Figure 3A). Cisplatin also decreased cell proliferation, and its effect augmented with time of exposure even for small doses (Figure 3A), but in addition decreased senescence at all the concentrations and time-points included in our study (Figure 3B). The significant decrease in cell senescence induced by cisplatin could be explained by the increase in cell death, assuming that senescent cells would be, probably, prone to die faster than healthy cells 23.
Importantly, these representative results suggest that HEI-OC1 cells respond to different pharmacological drugs with a distinctive dose- and time-dependent sensitivity to at least one of the mechanisms under study. Therefore, a correct interpretation of any experimental result will require a clear understanding of the particular cellular processes investigated and the information provided by each one of the techniques used to evaluate them.
Confocal and flow cytometry experiments, on the other hand, confirmed prestin expression in P-HEI-OC1 and NP-HEI-OC1 cells, with prestin immunolocalizing mostly at the cytoplasm of P-HEI-OC1 cells (Figure 4A, arrows), and more concentrated at the plasma membrane in NP-HEI-OC1 cells (Figure 4B, arrows). Flow cytometry studies showed a clear increase in prestin expression in NP-HEI-OC1 cells respect to P-HEI-OC1 cells (Figures 4C and 4D). The time-dependent increase in expression and plasma membrane localization of prestin in NP-HEI-OC1 cells suggests that translocation was accompanied by synthesis of more prestin molecules. However, these experiments did not provide clues to decide whether prestin molecules originally located in the cytoplasm move to the plasma membrane and new molecules replace them, if they remain at the cytoplasm and only the newly synthesized prestin moves to the plasma membrane, or a combination of both processes.
Electrophysiological studies showed a robust NLC in P-HEI-OC1 cells (Figure 5B), with a peak value that decreased and a voltage at peak capacitance that shifted to more depolarized values, when the cells were moved to non-permissive conditions for 1 and 2 weeks (Figures 5C and 5D). Because of the progressive translocation of prestin from the cytoplasm to the plasma membrane, we originally hypothesized that NLC would be higher in NP-HEI-OC1 than in P-HEI-OC1 cells, and that it would further increase with time of incubation at NP conditions. Surprisingly, the results of our studies showed exactly the opposite response. We speculated that the increase in the density of prestin molecules in the plasma membrane could be restricting their motor function.
Note in Figure 5 the depolarization of the voltage at peak capacitance in HEI-OC1 cells respect to the typical values reported in OHCs and in transfected HEK293 cells 24; moreover, observe that the depolarizing shift increases with time at NP conditions. The progressive depolarizing shift is similar to that described in OHCs during mouse and rat development 25,26, and it had been suggested that it might be related to the development of other cellular structures associated with the OHC's plasma membrane 25, or a maturation process intrinsic to prestin 24.
We hope this brief recount of a few studies with HEI-OC1 cells provide enough evidence of the potential usefulness of HEI-OC1 cells for investigating a wide spectrum of cell and molecular responses.
Figure 1: Cell Viability and Cell Death in P-HEI-OC1 and NP-HEI-OC1 Cells. (A) Cell viability evaluated with the MTT assay. Absorbance was measured with a Plate Reader, and average OD in control cells was taken as 100% of viability. (B) Cell death evaluated with a Cytotoxicity Assay. The number of positive (dead) cells was determined by flow cytometry with 488 nm excitation (blue laser) and FL1: 530 15 nm. Note the highly significant (P ≤0.001) decrease in cell viability (A) and increase in cell death (B) induced by non-permissive conditions. Bars represent standard error of the means (SEM). Modified from reference 8. Please click here to view a larger version of this figure.
Figure 2: Responses of HEI-OC1 Cells Exposed to APAP and Cisplatin when Analyzed by MTT, Caspases 3/7 and Cytotoxicity Assays. (A) Cell viability evaluated with the MTT assay, and absorbance measured with a Plate Reader. (B) Activation of executioner caspases 3/7. (C) Drug–induced cell death evaluated with a Cytotoxicity Assay; the number of positive (dead) cells was determined by flow cytometry. Data in every experiment was normalized to the respective control conditions (control = 1) to make possible simultaneous statistical analysis. * = P ≤0.05 respect to control. Bars represent standard error of the means (SEM). Modified fromreference 8. Please click here to view a larger version of this figure.
Figure 3: Effects of APAP and Cisplatin on HEI-OC1 Cells' Mitotic Rate and Senescence. (A) Drug-induced changes on the rate of mitotic division of HEI-OC1 cells, at 24 and 48 hr, was evaluated with a BrdU Assay. (B) Drug-induced senescence in HEI-OC1 cells was measured by flow cytometry and the senescence-associated β-galactosidase (SA-Bgal) technique. Data in every experiment was normalized to the respective control conditions (control = 1) to make possible simultaneous statistical analysis. * = P ≤0.05 respect to control. Bars represent standard error of the means (SEM). Modified fromreference 8. Please click here to view a larger version of this figure.
Figure 4: Prestin Expression in P-HEI-OC1 and NP-HEI-OC1 Cells – Cells were Labeled with Anti-Prestin Antibody and Observed by Confocal Microscopy and Flow Cytometry. (A) In P-HEI-OC1 cells prestin immunolocalized mostly at the cytoplasm (arrows). (B) In NP-HEI-OC1 cells, in contrast, prestin reactivity was concentrated at the plasma membrane (arrows). (C and D) Flow cytometry studies in HEI-OC1 cells cultured for 2 weeks at NP conditions (D) showed a clear increase in prestin expression respect to P-HEI-OC1 cells (C). (C) Inset is secondary ab only (negative control). Modified from reference 9. Please click here to view a larger version of this figure.
Figure 5: NLC studies in P-HEI-OC1 and NP-HEI-OC1 Cells. (A) Patch-clamped HEI-OC1 cell. (B–D) NLC in P-HEI-OC1 and NP-HEI-OC1 cells. Note the decrease in the peak of NLC and the progressive shift of the curves to more depolarized values in NP-HEI-OC1 cells. Modified from reference 9. Please click here to view a larger version of this figure.
In this report we describe how to culture HEI-OC1 cells and use them to evaluate mechanisms of drug-induced cytotoxicity and to investigate functional properties of prestin, the molecular motor of cochlear OHCs. The technical procedures, however, are general enough to be easily adapted to different studies.
All the protocols described here require the correct use of well-established cell culture techniques 10. Just like with any other cell line, working with HEI-OC1 cells requires a properly equipped cell culture facility, with a certified biological safety cabinet, a refrigerated centrifuge, a water bath, and one inverted microscope for examination of cell cultures and for counting cells if using hemocytometric techniques. A unique requirement, however, is the availability of two humidified incubators, one set at 33 °C/10% CO2 for P-HEI-OC1 cells and other at 39 °C/5% CO2 for NP-HEI-OC1 cells. If the planned studies involve only P-HEI-OC1 cells, the second incubator can be set at 37 °C/5% CO2 for different assays.
The use of plastic cell culture dishes is an important additional requisite for working with HEI-OC1 cells. In fact, HEI-OC1 cells are very robust and they will growth in different surfaces and under very different conditions. However, their phenotype and their response to drugs and other stimuli will strongly depend on the culture parameters (G Kalinec & F Kalinec, unpublished). As we argued in a previous paper 8, this plasticity of HEI-OC1 cells may be advantageously used to design novel experiments. For example, it is well known that the oxygen tension in the cochlear perilymph is much lower (~2 kPa) than in a typical incubator at sea level (~21.3 kPa) 27. Incubating HEI-OC1 cells at lower O2 concentrations will change their responses, perhaps making them a better model for auditory sensory cells.
It should be emphasized that the described protocols for culture and subculture of HEI-OC1 cells can also be used with other auditory cell lines developed in our laboratory from the same transgenic mouse, such as OC-k3 from organ of Corti 11,12 and SV-k1 from stria vascularis 13,14. Moreover, these cell lines could be useful as control for HEI-OC1 cells in selected experiments. For example, we have recently used SV-k1 cells to demonstrate that the inversely proportional immunoreactivity to prestin and Na+K+ATPase antibodies at the plasma membrane of HEI-OC1 cells was cell type-dependent response rather than associated with the transgenic mouse from which these cells were derived 9.
Other critical requirement for working with HEI-OC1 cells is the practice of appropriate aseptic techniques such as cleaning all work surfaces within the safety cabinet with 70% ethanol or isopropanol and the decontamination of any materials required for the procedures. However, while other mammalian cell lines are frequently protected against inadvertent bacterial contamination by using penicillin-streptomycin or other similar combinations, antibiotics must never be used with HEI-OC1 cells except if the purpose of the study is to investigate their effects. HEI-OC1 cells were generated in antibiotic-free conditions, and they must be avoided to reduce the emergence of antibiotic-resistant cells.
Finally, we want to re-emphasize a point already mentioned several times in this report: the phenotype and the response of HEI-OC1 cells to drugs and other stimuli will strongly depend on the culture parameters. Therefore, it is very important that the laboratories working with HEI-OC1 cells use similar protocols and cell culture conditions in order to make their results truly comparable with those provided by other groups. In addition, it is very important to remember that the HEI-OC1 cell line is only a model, with all the limitations that a model has. Expecting that in vitro experiments with HEI-OC1 cells will provide data accurately representing the in vivo responses of real auditory sensory cells is unrealistic. However, we strongly believe the HEI-OC1 cell line is a useful model for investigating functional responses of auditory sensory cells and the screening of the potential ototoxicity of pharmacological drugs.
The authors have nothing to disclose.
This work was supported by NIH Grants R01-DC010146 and R01-DC010397. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.
HEI-OC1 cells | ALL THE ASSAY KITS, EQUIPMENTS | ||
Class II Biological Safety cabinet | The Baker Company | Sterilgard III | AND COMPANIES INDICATED IN THE |
Refrigerated centrifuge | Eppendorf | 5810R | PREVIOUS 2 COLUMNS ARE ONLY |
Inverted microscope | Zeiss | Axiovert 25 | EXAMPLES, AND ANY OTHER SIMILAR |
Waterbath | Stovall | HWB115 | PRODUCT COULD BE USED. |
Cell counter | Nexcelom | Cellometer Auto T4 | |
Two (2) Cell incubators, one at 33°C/10% CO2 and other at 39°C/5% CO2 | Forma Scientific | 3110 | |
Cell culture dishes, PS, 100 x 20 mm with vents | Greinier Bio-One | 664-160 | |
Cell culture dishes , PS, 60 x 15 mm with vents | Greiner Bio-One | 628160 | |
Cellstar tissue culture flasks 250 mL | Greiner Bio-One | 658-175 | |
Cellstar tissue cultur flasks 550 mL | Greiner Bio-One | 660-175 | |
6 well cell culture plate, with lid-Cellstar | Greiner Bio-One | 657-160 | |
Microtest Tissue culture plate, 96 well,flat bottom with lid | Becton Dickinson | 353072 | |
Micro-Assay-Plate, Chimmey, 96-well white,clear botton | Greiner Bio-One | 655098 | |
50 ml Polypropylene conical tube with cap Cellstars | Becton Dickinson | 352070 | |
15 ml Polypropylene conical tubes with cap-Cellstars | Greiner Bio-One | 188-271 | |
PBS pH 7.4 (1X) | Life Technologies | 10010-023 | |
Dulbecco’s Modified Eagle’s Medium (DMEM) | Life Technologies | 11965-084 | |
Fetal bovine serum (FBS) | Hyclone | SH10073.1 | |
Leibovitz's L-15 Medium, no phenol red | Gibco/Invitrogen | 21083-027 | |
Trypsin, 0.25% | Life Technologies | 25200-056 | |
TACS MTT Cell Proliferation Assay Kit | Trevigen | 4890-25-K | |
Caspase-Glo 3/7 Assay kit | Promega | G8091 | |
BrdU Cell Proliferation Assay Kit | Cell Signaling | 6813 | |
Non-enzymatic cell dissociation solution | Sigma-Aldrich | C5789 | |
Cell-Tox Green Cytotoxicity Assay Kit | Promega | G8741 | |
FACSAriaIII instrument | BD Biosciences | FACSAriaIII | With 488 nm excitation (blue laser) |
Digital Blot Scanner | LI-COR | C-DiGit | |
Electrophoresis and Blotting Unit | Hoefer | SE300 miniVE | |
Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software | Molecular Devices | SpectraMax 5 | |
Patch-clamp amplifier | HEKA | EPC-10 | |
Puller for preparing patch electrodes | Sutter Instruments | P-97 |