Cell culture and maintenance protocol

This cell culture and maintenance protocol provides essential guidance for researchers working with cultured cells. Covering key stages such as cryopreservation, thawing, and ongoing maintenance, this protocol ensures optimal cell viability and reproducibility. It addresses the handling of both adherent and suspension cells, offering best practices for aseptic technique, storage, and monitoring. Designed for use in academic and industrial labs, the protocol helps minimize contamination risks and supports long-term cell line stability. Whether you’re working with finite or continuous cell lines, this resource is a reliable foundation for successful cell culture experiments.

Introduction

Cell culture is a cornerstone of biological research, enabling scientists to study cellular behavior, drug responses, and genetic modifications in a controlled environment. This protocol provides a simplified methodology for maintaining healthy cell lines, offering step-by-step instructions for cryopreservation, thawing, and routine care. The protocol covers various methods for culturing both adherent and suspension cells, addressing different approaches and considerations for each. It is tailored for both novice and experienced researchers, emphasizing aseptic techniques and proper equipment usage. By following this protocol, users can ensure consistent results and maintain the integrity of their cell lines across experiments. The guide is especially useful for labs handling a variety of cell types, including adherent and suspension cultures.

Background and principles

Cell culture involves growing cells under controlled conditions, typically outside their natural environment. This protocol is grounded in principles of sterility, viability, and reproducibility. It distinguishes between finite and continuous cell lines, each with unique growth characteristics. Cryopreservation is important for preventing genetic drift and contamination, using agents like DMSO and controlled freezing rates. Additionally, it is also important to monitor cell morphology and density to maintain healthy cultures. These foundational practices ensure that cells remain genetically stable and biologically relevant for downstream applications.

Cells require careful maintenance to prevent contamination, facilitate growth, and ensure long-term stability. The following pages provide general guidance for preserving, thawing, and maintaining cells in culture, including best practices for handling, storage, and monitoring to ensure optimal viability and reproducibility.

Cells in culture can broadly be classified into the following three types.

Adherent culture refers to the method where cells, known as adherent cultures, require attachment to a surface or extracellular matrix for growth and proliferation. These are typically anchorage-dependent cells, meaning they must attach to a substrate to survive and divide. Anchorage-dependent cell types are widely used in tissue engineering, research, and bioproduction, but their expansion is limited by the available surface area in culture vessels. Adherent platforms are commonly used in industry, especially for early-stage and certain commercial products, but their scalability is constrained by surface area and labor-intensive processes.

On the other hand, suspension cell culture involves growing cells that do not require attachment and can proliferate freely in the medium. Suspension cell cultures and suspension culture systems are highly scalable and are widely used in industrial applications such as vaccine, monoclonal antibody, and viral vector production.

Cells can also be classified according to their growth characteristics, and bring their own set of considerations for growth:

• Finite: Only proliferate and remain viable for a limited number of population doublings. Common examples are primary cells isolated directly from tissue, or cell lines that stop growing (senesce) after a certain number of passages.
• Continuous: Cells with the ability to divide indefinitely (immortalized). Often generated by undergoing transformation, to gain a cancer-like phenotype (e.g. Loss of contact inhibition, unlimited growth).

Selecting appropriate cell strains is crucial for optimizing growth, differentiation, and product yield in different culture systems.

Cell culture scalability and commercial viability are closely tied to the choice between adherent and suspension platforms, as these decisions impact cost, regulatory considerations, and product consistency.

Here, our focus will be on the most common cell types: adherent and suspension cells. Please note that all procedures involving the manipulation of cultured cells should be performed using an aseptic technique and the appropriate containment method(s).

Stage 1 - Cryopreservation

Genetic instability accumulates in cells that are continually cultured. Therefore, cell lines should be frozen and stored, or “banked down”, as soon as possible after receipt. This ensures that cell stocks are as genetically close as possible to the source material and reduces the risk of contamination. Cells should be frozen in a controlled manner (ideally at a rate of 1°C per minute) in the presence of cryoprotective agents (such as DMSO) to prevent the formation of ice crystals within the cells and a resulting loss in viability of the culture.

Note: our cell lines arrive frozen in cryoprotectant and should be immediately stored in liquid nitrogen upon receipt.

Materials required

• Cells in culture at an appropriate confluence
• Appropriate growth medium for the cells
• Enzymatic or chemical cell detachment agent (where required)
• Standard cell culture consumables (serological pipettes, pipette tips, centrifuge tubes, 70% ethanol, sterile PBS, etc.)
• Standard cell culture equipment (pipette boy, pipettes, containment facilities, centrifuge, light microscope, cell counter/haemocytometer, etc.)
• Freezing container to control the rate of freezing

Steps

Ensure your culture is healthy and in a logarithmic phase of growth

• Use a microscope to visualise the cells to check their general appearance and ensure there are no signs of microbial contamination.

Collect and count the cells as described for standard sub-culture

Wash and prepare cells

• Using the cell density information, transfer the required amount of cell culture to a suitable centrifuge tube.
• Centrifuge the cell suspension (200 x g, 5 mins), remove the supernatant, and resuspend the cell pellet in PBS.
• Centrifuge (200 x g, 5 mins) and discard the PBS.

Resuspend cells in cryoprotectant according to manufacturer’s instructions

• Gently resuspend cell pellet in chosen cryoprotectant.
• Transfer cell suspension into the appropriate number of cryovials, labeled with the date, name, cell number, passage number, and cell type

Pros and cons of different cryoprotectants:

Freeze cryovials

• Place cryovials in a freezing container and place at - 80°C overnight. This will bring the temperature down in a controlled manner.

Pros and cons of different freezing containers:

Transfer to liquid nitrogen (vapor phase) for long-term storage

Stage 2 - Cell line revival

When required for use, cells should be thawed as quickly as possible to minimize any adverse impact on cell viability. It is recommended that the cryopreservation agent is removed from the culture medium by centrifugation at time of revival.

Materials required

• Laminar flow hood
• Cryovial of frozen cells
• Water or bead bath
• Appropriate growth medium
• Falcon tubes, and culture flasks/vessels

Steps

Thaw cryovial in a water bath

• Place an appropriate volume of pre-warmed culture medium into a falcon tube
• Remove cryovial from liquid nitrogen storage and place in a 37°C water bath.
• Carefully monitor the vial and remove from the water bath when almost defrosted (a small amount of ice should remain).
• Transfer vial to the laminar flow hood and using a pipette, carefully transfer the contents of the vial into the falcon containing the pre-warmed culture medium

Remove cryopreservation agent and seed cells for culture

• Centrifuge samples at a speed suitable for your cells (200–250 x g, 5 min).
• Remove supernatant and resuspend in fresh pre-warmed growth medium.
• Transfer the cell suspension to an appropriate culture vessel and incubate as appropriate.

Stage 3 - Observation

Cells should be observed regularly using a microscope and with the unaided eye for signs of microbiological contamination. Microscopic examination should also be used to determine the general health of the cells and to establish whether subculture is required.

Materials required

• Cells
• Light microscope

Steps

Observe cell cultures with the naked eye for visible markers of growth or contamination

• Observe growth medium color (where media containing Phenol red is used). Production of metabolites from mammalian cells and/or contaminants and waste products from dying cells can decrease the pH of the media. The addition of pH indicators is a simple method for understanding whether the cell line requires a media change, subculture, or is contaminated

• Observe growth medium turbidity.

  • For adherent cells, the growth medium should be clear. If not, this can be a sign of contamination or that cells have surpassed confluence and are dying or detaching from the culture surface.
  • For suspension cells, the medium is more turbid due to the presence of cells suspended in the solution. A greater turbidity than expected, combined with an acidic medium, may indicate contamination or a high density of cells requiring subculture.

Observe cells under a light microscope for signs of growth or contamination

• Cell adherence. Ensure cells are adhered to the culture vessel or in suspension, as expected.
• Cell morphology. Check the culture to confirm the cellular morphology is as expected. Differing morphology can be a sign of contamination or differentiation.
• Confluency (for adherent cells) is the percentage of the cell culture surface covered by cells; 100% is complete cell coverage of the culture vessel surface. The percentage at which cells require sub-culturing is cell line-dependent; however, it is most commonly 70%. Refer to supplier protocols for your specific cell type.
• Cell Density. The health and growth characteristics of suspension cells are monitored by performing cell counts. Collect a sample of cells and perform a cell count using a hemocytometer or automated cell counter. The density at which cell lines require subculture is cell line dependent. Refer to supplier protocols for your specific cell type.

Stage 4 - Cell maintenance and subculture

Based on your observations, implement the appropriate course of action.

• If cells are growing healthily and are at the desired confluence, sub-culture and/or seed cells for your experiment.
• If cells are not yet at the desired confluence, and 2-3 days have passed since last subculture, exchange the media and continue until the desired confluence is reached.
• If the cells show signs of contamination, they should be discarded (see protocol on contamination).

Cells require regular media exchanges when in culture to prevent the build-up of toxic metabolites (eg, lactic acid) and ensure a continual supply of the growth medium components. The build-up of cell metabolites is usually monitored through pH indication (eg, phenol red), and this is used to determine a suitable time to complete a media change on your cells.

Materials required

• Cells
• Appropriate culture vessels (eg, plates or flasks)
• Appropriate growth medium (containing any serum, growth factors, or small molecules required for your specific cell type)
• Standard cell culture consumables (serological pipettes, pipette tips, centrifuge tubes, 70% ethanol, sterile PBS, etc.).
• Standard cell culture equipment (pipette boy, pipettes, containment facilities, centrifuge, light microscope, cell counter/haemocytometer, etc.)

Steps

Remove growth medium

• For suspension cells:

  • Transfer culture to centrifuge tube.
  • Centrifuge at 200–250 x g for 5 min.
  • Remove media

• For adherent cells:

  • Aspirate media and dispose

Add fresh growth medium

• For suspension cells:

  • Resuspend cell pellet in fresh growth medium.
  • Transfer to a new culture vessel.

• For adherent cells:

  • Add an appropriate volume of fresh growth medium

Incubate cells as required

Continue to monitor cells daily for growth and signs of contamination

If cells have grown to the desired confluence or density, they should be subcultured. Subculture, also known as passaging, is the transfer of cells from the previous culture to a new culture vessel with fresh media to allow the continuation of growth.

Materials required

• Cells
• Appropriate culture vessels (eg, plates or flasks)
• Appropriate growth medium (containing any serum, growth factors, or small molecules required for your specific cell type)
• Enzymatic or chemical cell detachment agent (for adherent or semi-adherent cells)
• Standard cell culture consumables (serological pipettes, pipette tips, centrifuge tubes, 70% ethanol, sterile PBS, etc.)
• Standard cell culture equipment (pipette boy, pipettes, containment facilities, centrifuge, light microscope, cell counter/haemocytometer, etc.)

Steps

Wash and collect cells.

• For suspension cells

  • Transfer culture to centrifuge tube.
  • Centrifuge at 200-250 x g for 5 min.
  • Remove media and resuspend cell pellet in an appropriate volume of PBS.
  • Centrifuge at 200-250 x g for 5 min.
  • Aspirate and discard PBS.
  • Resuspend cell pellet in fresh growth medium.

• For adherent cells:

  • Aspirate media and dispose.
  • Add an appropriate volume of PBS, ensuring the cell culture is covered.
  • Aspirate and discard PBS.
  • Add appropriate detachment reagent and incubate at 37 °C until cells are detached.
  • Add fresh culture media and transfer the cell suspension to a centrifuge tube.
  • Centrifuge at 200-250 x g for 5 min.
  • Remove media and resuspend the cell pellet in an appropriate volume of PBS.
  • Centrifuge at 200-250 x g for 5 min.
  • Aspirate and discard PBS.
  • Resuspend cell pellet in fresh growth medium.

Seed cells into new culture vessel(s) in fresh growth medium

• Count cells (if required), and record cell number and viability.
• Transfer the required volume of cells to the new culture vessel(s).
• Add growth medium to the required volume.

Label flasks with key information and incubate as required

• Include cell type, passage number, seeding density/split ratio, date, and initials.

Continue to monitor cells daily for growth and signs of contamination

The most common cause of contamination of cultures is with microorganisms such as bacteria, fungi, and yeasts, which can be observed under the microscope. If observed, you should discard the cell cultures and sterilize the work areas.

One particularly troublesome contaminant is mycoplasma, which is resistant to traditional cell culture sterilisation techniques, is not visible microscopically, and requires targeted testing for detection. It is good practice to routinely test cultures for mycoplasma contamination. All cell lines from Abcam are cultured in a mycoplasma-free environment.

Cross-contamination with other cell types can also be a serious issue. It is best practice to test cultures regularly for cross-contamination using STR (short tandem repeat) testing, which distinguishes the DNA profiles of cell lines. If you spot visual signs of cross-contamination, such as changes in cell morphology, you should discard the culture you’re working with.

Cell lines and cell morphology

Cell lines are the foundation of modern cell culture, providing researchers with consistent and reliable models for studying cellular processes, disease mechanisms, and therapeutic interventions. The morphology of a cell line, its shape, structure, and physical characteristics, plays a pivotal role in determining the most suitable culture technique and optimizing cell growth.

Adherent cell lines, such as many mammalian cell lines, require a surface to attach to to grow and divide. These cells, including epithelial cells with their classic polygonal shape and endothelial cells with their elongated appearance, rely on cell adhesion to the culture vessel for survival and proliferation. In contrast, suspension cell lines, like certain insect cells, are adapted to grow freely within the culture medium, forming a cell suspension that does not require attachment to a surface.

Understanding the unique cell morphology of each cell line is essential for successful cell culture. For example, neuronal cells display a distinctive morphology with long, projecting processes, reflecting their specialized function. Recognizing these morphological traits helps researchers select the appropriate culture medium, monitor cell health, and detect early signs of contamination or unwanted cell differentiation. By closely observing cell morphology, scientists can ensure optimal conditions for cell growth, maintain the integrity of their cultures, and achieve reproducible results in both research and industrial applications.

Adherent cell culture techniques

Adherent cell culture techniques are fundamental to the cultivation of most mammalian cell lines, including widely used models like CHO cells. These techniques involve growing cells on a suitable surface within a culture vessel, such as a culture flask, roller bottle, or microplate. The choice of vessel depends on the specific requirements of the cell line and the intended scale of production, ranging from small-scale research experiments to large-scale manufacturing processes using fixed-bed bioreactors.

To support the growth and differentiation of adherent cell cultures, the culture medium is carefully formulated with balanced salt solutions, essential nutrients, and growth factors tailored to the needs of the cell type. Regular monitoring of cell morphology and viability is crucial; healthy adherent cells should display consistent attachment and characteristic shapes, while changes in appearance may signal contamination or the need for passaging.

Passaging adherent cells at the right time prevents overgrowth, which can lead to cell death and reduced culture quality. The use of enzymatic dissociation reagents, such as trypsin, is common for detaching cells from the culture surface, but protocols must be optimized to minimize cell damage and maintain high viability. Selecting reputable suppliers for culture media and reagents further reduces contamination risk and supports reproducible results.

Adherent cell culture techniques are well established and widely used in research, gene therapy, and recombinant protein production. By understanding the specific growth requirements and morphology of each cell line, researchers can optimize cell growth, protect cells from contamination, and achieve high yields of healthy, functional cells for a variety of applications.

Applications

This protocol supports a wide range of applications in biomedical research, drug development, and molecular biology. It is suitable for maintaining cell lines used in cancer studies and immunology. Researchers can apply it to experiments involving gene expression, protein production, and cytotoxicity assays. The protocol’s emphasis on cryopreservation and aseptic handling ensures that cell lines remain viable for long-term studies. Whether you are preparing cells for transfection, imaging, or biochemical analysis, this guide provides the foundational steps needed for successful outcomes.

In addition, this protocol underpins the manufacturing process for gene therapies, particularly in the production of viral vectors. Early-stage or small-scale manufacturing often relies on adherent platforms, such as roller bottles, which are widely used for their simplicity and practicality in producing viral vectors for approved gene therapies. However, adherent platforms face scalability and labor-intensive limitations. During process development, manufacturers may transition to suspension-based systems, using shaker flasks, spinner flasks, and orbital shakers, to enable high yield production and meet commercial demands. Strategic considerations in choosing between adherent and suspension platforms depend on the specific gene therapy application, required scale, and regulatory requirements.

Limitations

While comprehensive, the protocol may require adaptation for specialized cell types or advanced experimental setups. It focuses primarily on adherent and suspension cells, with limited guidance for semi-adherent or organoid cultures. Additionally, the protocol assumes access to standard lab equipment, which may not be available in all settings. Users working with sensitive or rare cell lines might need to supplement the guide with cell-specific instructions. Finally, while troubleshooting tips are provided, complex contamination issues or genetic drift may require expert consultation beyond the scope of this protocol.

Troubleshooting

Common issues in cell culture include contamination, poor viability, and inconsistent growth. It is recommended to check the cell morphology and density using a microscope regularly. If contamination is suspected, discard affected cultures and sterilize equipment. For low viability, ensure cells are in the logarithmic growth phase before cryopreservation and use fresh medium during thawing. If cells fail to adhere, verify the use of appropriate detachment agents and surface coatings. Maintaining aseptic technique and monitoring environmental conditions like temperature and CO₂ levels are key to resolving most problems efficiently.