Culture Guidelines of Adherent Cells using Trypsin

Purpose
The following protocol is suggested for the basic instruction of culturing adherent cell lines.

Responsibility
It is the responsibility of all trained laboratory personnel to perform and adhere to all aspects of the procedure. It is management’s responsibility to verify qualifications of personnel to perform the technical aspect of the stated procedure.

Definitions

• Refrigerator capable of maintaining 2 to 8°C, optional
• Confluence – the percentage of the surface area covered by the cells. A confluent culture is when all the cells are in contact and the entire surface of the culture vessel is covered.
• Epithelial-like – Cells that are polygonal in shape with more regular dimension, and grow attached to a substrate in discrete patches.
• Fibroblastic (or fibroblast-like) – Cells grown on a substrate that appear elongated and bipolar, frequently forming swirls in heavy cultures
• Hybridomas – Cell line formed by fusing two different but related cells.
• Immortal or Continuous cell line – Cells that have been adapted to grow indefinite in culture.
• Lymphoblast-like – Cells that are spherical in shape and are usually grown in suspension without attaching to a surface.
• Monolayer culture system – Cells that primarily grow attached to glass or treated plastic in a uniform layer.
• Passage number – The number of sub-cultures the cells have undergone. Passage number should be recorded with each sub-culture.
• Primary culture – Cells that are derived from and organism and placed into a suitable culture environment.
• Senescence – cumulative changes to molecular and cellular structure that disrupts metabolism with the passage of time, resulting in deterioration and eventually leading to cell death.
• Subculture or passaging – removal of medium and transfer of cells from a previous culture into fresh growth medium that enables continued propagation of the cell line.
• Suspension culture system – Free floating cells in a culture system.
• Tissue culture – The general term for removal of cells, tissues, or organs from an animal or plant and subsequent placement into an artificial environment conducive to growth.
• Quiescence – A state of quietness or inactivity, a state of a cell when it is not dividing.

Equipment
Equipment will be of appropriate design for designated use and will be suitably located and installed to facilitate operations, including cleaning and maintenance. Equipment will be cleaned, sanitized, maintained and calibrated according to established procedures and schedules.

• Auto-Pipettor filler/dispenser
• Micropipettes
• Thermometers
• Inverted Light Microscope
• Centrifuge
• CO2 water jacketed incubator capable of maintaining 37°C with humidified atmosphere of 5% CO2

Materials
Culture vessels containing cells in healthy phase of growth or cryopreserved cells that are at least 90% viable prior to cryopreservation. Consumables and disposables used in cell culture operations including but not limited to; liquid media and additives, serum, sterile disposable culture vessels and liquid handling utensils, gloves, etc.

• Lab task wipes
• Disinfectant
• Lens tissue paper
• Cell culture media
• Dissociation media such as Trypsin
• 0.4% Trypan blue
• Antibiotic and appropriate cell culture additives
• Balanced salt solution without calcium and magnesium
• Sterile 15 and 50mL conical tubes
• Sterile serological pipets
• Sterile culture vessels appropriate for cell type
• Bovine serum appropriate for cell type

Specifications
Cultures should be maintained in the log phase of growth (70-80% confluent). The optimal culture condition is depended on the cell line. All solutions, equipment and consumables that come in contact with cell cultures must be sterile. Different cell lines should never be processed at the same time. All working areas should be thoroughly cleaned between the preparations of different cell types.

Precautions
Wear personal protective equipment. Use proper aseptic technique.

Procedure

1. Preparation of cell growth medium.
   1. Check the information pertaining to the cell line to identify what media type, additives and recommendations should be used. Follow manufacturer recommendation for working volume of culture vessels.
   2. Basal media is supplemented with 5-20% bovine serum, and often antibiotic, growth factors, amino acids, or sugars are added. Once basal media is supplemented to create complete media, it is generally stable for 6 weeks at 4°C.
2. Examination of cells
   1. Cells should be checked microscopically often to ensure they are healthy and growing as expected. Attached cells should be mainly attached to the bottom of the flask, round and plump or elongated in shape and refracting light around their membrane. Check the information pertaining to the cell line to identify what media type, additives and recommendations should be used. Follow manufacturer recommendation for working volume of culture vessels.
   2. Observe culture for health of cells, morphology, % confluence, cell debris, contamination, etc.
   3. Discard cells if they are detaching in large number and look shriveled and grainy or dark in color, if they do not appear to be growing at all, or appear to have been contaminated.
3. Split-Ratios
   1. Split ratios can be used to ensure cells should be ready for an experiment. Check the guidelines for the cell lines in use. Some slow growing cells may not grow if a high split ratio is used. Some fast growing cells may require a high split ratio to make sure they do not overgrow. As a general guide:
      1. 1:2 split should be 70-80% confluent and ready for an experiment in 1-2 days
      2. 1:5 split should be 70-80% confluent and ready for an experiment in 2-4 days.
   2. If cells are less than 70-80% confluent but the need to be subcultured, then they should be split at a lower ratio in order to seed the cells at a high enough density to survive.
4. Sub-culturing or passaging
   1. Remove and discard the spent cell culture media from the culture vessel.
   2. Wash cells using a balanced salt solution without calcium and magnesium.
      1. Approximately 2mL per 10cm2 cultured surface area. Gently add wash solution to the side of the vessel opposite the attached cell layer to avoid disturbing the cell layer. Gently rock the vessel back and forth several times to wash the cell layer.
      2. Remove and discard the wash solution from the culture vessel.
      3. Repeat the wash step for a total of two washes.
   3. Add pre-warmed dissociation reagent to the side of the flask. Use enough reagent to cover the cell layer, approximately 0.5ml per 10cm2. Gently rock the culture vessel to get complete coverage of the dissociation reagent to cover the cell layer.
      1. Incubate the culture vessel at room temperature for approximately 2-3 minutes. The actual incubation time varies with the cell line used. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
      2. Observe the cells under the microscope for detachment. If the cells are less than 90% detached, increase the incubation time a few more minutes, checking for dissociation every 30 seconds.
         1. Detached cells will be round and in suspension. Depending on the cell line culture vessel may be gently tapped on the side of the flask. Note: to avoid clumping do not agitate the cells by tapping while in trypsin.
         2. Do not allow cells to sit in dissociation media for more than 10 minutes.
      3. Aspirate cell suspension and transfer to a conical tube.
      4. Add the equivalent of 2 volumes of pre-warmed complete growth medium. Disperse the medium by pipetting over the cell layer surface several times. Transfer cell suspension to the tube containing cells in the dissociation media. Cell suspension may be counted for cryopreservation or cells may be centrifuged for sub-culturing.
      5. Centrifuge cells at 200-1000xg for 5 to 10 minutes. Centrifuge speed and time will vary based on the cell type.
      6. Resuspend the cell pellet in a minimal volume of pre-warmed complete growth medium and remove a sample for counting.
      7. Dilute cell suspension to the seeding density or split ratio recommended for the cell line. Pipet the appropriate volume into the new culture vessel and return the cells to the incubator.
5. Changing media
   1. If cells have been growing well for a few days but are not yet confluent then a media change to replenish nutrient may be required. If there are a lot of cells in suspension or the media pH is starting to become acidic change the cell culture media.
   2. Warm complete media to 37°C. Carefully aspirate media from culture vessel and discard. Replace with fresh pre-warmed complete media and return to incubator.