Cell culture and maintenance protocol

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

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).

Here our focus will be on the most common cell types; adherent and suspension cells.  Please note: all procedures involving the manipulation of cultured cells should take place using 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 as close as possible to the source material and reduces 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 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 rate of freezing

Steps

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

• Use a microscope 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 culture by naked eye for visible markers of growth or contamination

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

• Observe growth medium turbidity.

  • For adherent cells, 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 media 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 that they are in suspension, as expected.
• Cell morphology. Check culture to confirm the cellular morphology is as expected. Differing morphology can be a sign of contamination or differentiation.
• Confluency (for adherent cells). This 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 sub-culture 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 exchange when in culture, to prevent the build-up of toxic metabolites (e.g. lactic acid) and to ensure a continual supply of the growth medium components. The build-up of cell metabolites is usually monitored through pH indication (e.g. phenol red), and this is used to determine a suitable time at which 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, the cells should be sub-cultured. Sub-culture, also known as passaging, is the transfer of cells from the previous culture to a new culture vessel with fresh media to allow 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 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 cell suspension to a 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.

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

• Count cells (if required), and record cell number and viability.
• Transfer 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 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, isn’t 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 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.