Oct 16, 2025
Long term cell culture of HeLa cells: Impact of Medium pH
- Ana Jared Tenorio-Salazar1,
- Miguel Ángel Vidal-Borbolla2,
- Luz Eugenia Alcántara-Quintana3
- 1Doctorado Institucional en Ciencia e Ingeniería de Materiales, Universidad Autónoma de San Luis Potosí, México;
- 2Coordinación para la Innovación y la Aplicación de la Ciencia y la Tecnología, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México;
- 3Unidad de Innovación en Diagnóstico Celular y Molecular, Coordinación para la Innovación y la Aplicación de la Ciencia y Tecnología, Universidad Autónoma de San Luis Potosí, México
Protocol Citation: Ana Jared Tenorio-Salazar, Miguel Ángel Vidal-Borbolla, Luz Eugenia Alcántara-Quintana 2025. Long term cell culture of HeLa cells: Impact of Medium pH. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21p4xg1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 16, 2025
Last Modified: October 16, 2025
Protocol Integer ID: 229919
Keywords: long term cell culture of hela cell, long term cell culture, hela cell, periods in hela cell, chronic acidosi, acidic ph, impact of medium ph, medium ph, cell, culture method
Disclaimer
The content of this article follows the recommendations of Official Mexican Standard NOM-052-SEMARNAT-2005, which establishes the characteristics, identification procedure, classification, and lists of hazardous waste. Mention of trade names or commercial products does not constitute endorsement or recommendations for use.
Abstract
This protocol describes long term cell culture of HeLa cells with impact of medium pH. We present a culture method capable of stably maintaining an acidic pH over extended periods in HeLa cells, providing a reproducible platform to study chronic acidosis and to evaluate pH-dependent therapies.
Guidelines
Researchers should have completed basic laboratory safety, chemical safety, bloodborne pathogens, and biosafety level 2 (BSL-2), training prior to conducting this protocol.
Materials
Reagents
- Dulbecco Modified Eagle Medium (DMEM, Thermo Fisher)
- Fetal Bovine Serum (FBS, Thermo Fisher)
- Penicillin-Streptomycin (Thermo Fisher)
- Trypsin (Gibco #25200-056)
Equipment/Supplies
- Biosafety cabinet
- Humidified tissue culture incubator with 5% CO₂
- Tissue culture dishes
- Vacuum-driven bottle filter with 0.2 µm pore
- Pipetaid
- 50mL conical tubes
- Tabletop centrifuge
- Pipettes
- Hemocytometer
Troubleshooting
Safety warnings
None.
Prerequisites
Reagents
- Dulbecco Modified Eagle Medium (DMEM, Thermo Fisher)
- Fetal Bovine Serum (FBS, Thermo Fisher)
- Penicillin-Streptomycin (Thermo Fisher)
- Trypsin (Gibco #25200-056)
Equipment/Supplies
- Biosafety cabinet
- Humidified tissue culture incubator with 5% CO₂
- Tissue culture dishes
- Vacuum-driven bottle filter with 0.2 µm pore
- Pipetaid
- 50mL conical tubes
- Tabletop centrifuge
- Pipettes
- Hemocytometer
Researchers should have completed basic laboratory safety, chemical safety, bloodborne pathogens, and biosafety level 2 (BSL-2), training prior to conducting this protocol.
Objective
Development of a culture method in the HeLa cell line for the evaluation of pH-dependent therapies.
Factors and levels
pH = [7.4, 7.0, 6.8]
Adaptation = [chronic ≥10 passages]
Seeding density 1x10^6^
Units of analysis
Independent culture
Controls
Neutral pH (7.4)
Buffer only (PBS 1X)
Inclusion/exclusion criteria
pH out of range
Contamination
Procedure
Dissolve 4.75g of DMEM powder without phenol red, without L-glutamine in 500mL of distilled water.
Prepare a sterile solution of 0.33M NaHCO3. Sterilize by filtration.
Sterilize the medium in an autoclave (15 min, 0.1-0.11 MPa, 212°C [liquid cycle]). Allow to cool to 25°C.
Add 0.5g glucose, 5mL penicillin-streptomycin, and 50mL fetal bovine serum to the bicarbonate-free DMEM.
For the control medium (pH 7.4), add 2.4mL of 0.33M NaHCO3 solution for every 100mL of bicarbonate-free DMEM.
Check the pH of the medium after 24 hours of incubation at 37°C with 5% CO₂.
Note: The amount of NaHCO3 solution must be adjusted according to the pH. Commercial products that can be used in this study are listed in the Materials table.
Add 150µL of 0.1M HCl to 100mL of control medium.
Check the pH of the medium after 24 hours of incubation at 37°C with 5% CO₂.
Note: The amount of HCl should also be adjusted according to the required pH.
Start culturing 1x10^6^ HeLa cells with control medium at 37°C with 5% CO₂ at least one week before starting treatment with acidic DMEM.
Change the control medium every 48 hours.
Pass the cells when they reach 70-80% confluence.
Wash the cells by adding 5mL of sterile 1X PBS.
Aspirate the PBS and add a detachable enzyme (TrypLE™ Express [1X]), 3mL.
Incubate at 37°C for 6 min. You should notice the cells rounding up and beginning to detach.
Add 3 mL of control DMEM culture medium to the detached cells to deactivate the enzyme.
Transfer the cell suspension to a 15 mL tube and centrifuge for 6 min at 1500 rpm.
Aspirate the supernatant and resuspend the cells with 5 mL of control DMEM culture medium.
Count viable cells using a Neubauer chamber and trypan blue.
Begin culturing HeLa cells from the standard culture.
Transfer (1x10^6^) HeLa cells to T25 flasks.
The final volume of the flask will be 5 mL of DMEM control (pH 7.4).
After 3 hours, verify that the cells begin to adhere to the substrate using a bright field microscope.
Aspirate the control medium and add 5 mL of medium at a lower pH.
Note: It is recommended to start the extracellular acidity treatment at pH 7.2 and renew the acidic medium every 48 hours.
Quality Control Rationale
Cells will be visually observed for culture quality prior to every passage.
Protocol references
Sutoo S, Maeda T, Suzuki A, Kato Y. Adaptation to chronic acidic extracellular pH elicits a sustained increase in lung cancer cell invasion and metastasis. Clinical 26 experimental metastasis. 2020;37(1):133-44.
Wu H, Estrella V, Beatty M, Abrahams D, El-Kenawi A, Russell S, et al. T-cells produce acidic niches in lymph nodes to suppress their own effector functions. Nature communications. 2020;11(1):4113.
Kondo A, Osawa T. Establishment of an extracellular acidic pH culture system. Journal of Visualized Experiments: JoVE. 2017(129):56660.
Pellegrini P, Serviss JT, Lundbäck T, Bancaro N, Mazurkiewicz M, Kolosenko I, et al. A drug screening assay on cancer cells chronically adapted to acidosis. Cancer Cell International. 2018;18(1):147.
Acknowledgements
N/A