Cell culture is a fundamental technique in biological research, particularly in virology, immunology, molecular biology, and pharmacology. It involves growing and maintaining cells in a controlled artificial environment.
1. Types of Cell Culture
- Primary Culture
- Obtained directly from animal or plant tissues.
- Cells have a limited lifespan and maintain many characteristics of the original tissue.
- Secondary (Subcultured) Cells
- Derived from primary cultures after the cells are transferred to a new vessel.
- Longer lifespan compared to primary cells but can undergo senescence.
- Continuous Cell Lines
- Derived from a single cell and can grow indefinitely (e.g., HeLa, Vero, and MDCK).
- Often modified or transformed to achieve immortality.
- 3D Cultures
- Simulate the in vivo environment better than 2D monolayer cultures.
- Include spheroids and organoids.
2. Equipment and Materials
- Biosafety Cabinet: To ensure a sterile working environment.
- Incubator: Maintains temperature (typically 37°C), humidity, and CO₂ levels (5% for most mammalian cells).
- Sterile Consumables: Flasks, petri dishes, multi-well plates, pipettes.
- Culture Media: Nutrient-rich solutions like DMEM, RPMI, or MEM supplemented with serum (e.g., fetal bovine serum, FBS).
- Trypsin-EDTA: For detaching adherent cells during subculturing.
- Cryoprotective Agents: Like DMSO, for freezing cells for long-term storage.
3. Steps in Cell Culture
a. Initiation
- Isolation: Tissue or organ is enzymatically or mechanically dissociated into single cells.
- Seeding: Cells are seeded into culture vessels with the appropriate medium.
b. Maintenance
- Feeding: Replace spent medium regularly to replenish nutrients and remove waste.
- Monitoring: Regularly check cells under an inverted microscope for growth, contamination, or morphology changes.
c. Subculturing
- Wash cells with PBS (phosphate-buffered saline).
- Detach adherent cells using trypsin-EDTA or other enzymatic solutions.
- Resuspend cells in fresh medium and transfer to a new vessel.
d. Cryopreservation
- Suspend cells in a cryoprotective medium.
- Freeze gradually (e.g., in -80°C freezer) before transferring to liquid nitrogen (-196°C).
Step-by-Step Procedure for Culturing Adherent Cells
Materials and Equipment Needed
- Cell Culture Equipment
- Biosafety cabinet (sterile workspace)
- CO₂ incubator
- Inverted microscope
- Centrifuge
- Reagents and Supplies
- Cell culture medium (e.g., DMEM, RPMI-1640) supplemented with fetal bovine serum (FBS) and antibiotics (if needed)
- Trypsin-EDTA solution (for detachment)
- Phosphate-buffered saline (PBS)
- Sterile cell culture flasks or plates
- Sterile pipettes, tubes, and pipette tips
- Personal protective equipment (PPE)
Step-by-Step Procedure
1. Preparation
1.1. Sterilize all tools and reagents under a biosafety cabinet.
1.2. Warm culture media and trypsin-EDTA solution to 37°C.
2. Thawing Frozen Cells (if starting from cryopreserved stock)
2.1. Quickly thaw the cryovial in a 37°C water bath (2–3 minutes).
2.2. Transfer the cells to a centrifuge tube with 10 mL pre-warmed medium.
2.3. Centrifuge at 200–300 × g for 5 minutes.
2.4. Discard the supernatant and resuspend the cell pellet in fresh culture medium.
2.5. Transfer to a flask and incubate at 37°C, 5% CO₂.
3. Subculturing (Passaging) Adherent Cells
3.1. Observation
- Examine cells under an inverted microscope to check confluency (~70–80%).
3.2. Remove Old Media
- Aspirate spent culture medium using a sterile pipette.
3.3. Wash the Cells
- Add 5–10 mL PBS to the flask to wash away residual media.
- Aspirate the PBS.
3.4. Detachment
- Add 1–2 mL of trypsin-EDTA solution to cover the cells.
- Incubate at 37°C for 2–5 minutes or until cells start detaching.
3.5. Neutralize Trypsin
- Add 8–10 mL culture medium containing FBS to neutralize the trypsin.
3.6. Collect Cells
- Pipette the cell suspension to ensure detachment and transfer it to a centrifuge tube.
3.7. Centrifuge
- Spin at 200–300 × g for 5 minutes.
3.8. Resuspend Cells
- Discard the supernatant and resuspend the pellet in fresh medium.
3.9. Replate
- Seed cells into new flasks or plates with appropriate volumes of medium.
4. Maintenance and Observation
4.1. Place the culture flask in the incubator (37°C, 5% CO₂).
4.2. Monitor daily for confluency, contamination, and morphology.
4.3. Change the medium every 2–3 days.
5. Cryopreservation (Optional)
5.1. Harvest cells at 70–80% confluency.
5.2. Resuspend cells in freezing medium (e.g., 10% DMSO in FBS).
5.3. Transfer to cryovials and freeze gradually at -80°C (use a cryo-freezing container).
5.4. Transfer to liquid nitrogen storage for long-term preservation.
Step-by-Step Procedure for Culturing Suspension Cells
Materials and Equipment
- Cell Line: The suspension cells to be cultured.
- Culture Medium: Suitable for the specific cell line (e.g., RPMI-1640, DMEM, etc.), supplemented with fetal bovine serum (FBS), antibiotics, and other supplements as needed.
- Sterile Flasks: Vent-cap culture flasks or spinner flasks.
- Pipettes: Sterile serological and micropipettes.
- Laminar Flow Hood: To maintain sterility.
- Centrifuge and Tubes: For cell harvesting, if needed.
- Hemocytometer or Cell Counter: To assess cell density and viability.
- CO2 Incubator: Set at optimal conditions for the cell line (typically 37°C with 5% CO2).
- Reagents: Trypan blue or another viability dye.
- Sterile PBS: For washing cells if necessary.
Procedure
1. Prepare the Workspace
- Work in a biosafety cabinet (BSC) to maintain sterility.
- Disinfect surfaces with 70% ethanol.
- Gather all materials and reagents before starting.
2. Thawing Frozen Suspension Cells (if applicable)
- Quickly thaw frozen cells in a 37°C water bath.
- Immediately transfer the thawed cells to a centrifuge tube containing pre-warmed culture medium to dilute the cryoprotectant (e.g., DMSO).
- Centrifuge at 200–300 × g for 5 minutes.
- Discard the supernatant and resuspend the cell pellet in fresh culture medium.
3. Seed the Cells
- Transfer cells into a sterile culture flask containing the appropriate volume of pre-warmed culture medium. Use the recommended cell density for the specific cell line (e.g., 2–5 × 10⁵ cells/mL).
- Use a vented-cap flask to allow gas exchange or tightly seal it if the flask supports non-vented use.
4. Incubate the Cells
- Place the flask in a CO2 incubator at 37°C (or the temperature optimal for the cell line) with 5% CO2.
- Avoid disturbing the flask unnecessarily.
5. Monitor the Cells
- Observe the cells daily under an inverted microscope to check cell density, morphology, and absence of contamination.
- Ensure the medium is not overused (change color due to pH shifts) and cells remain in suspension.
6. Subculturing Suspension Cells
- Suspension cells are typically subcultured every 2–4 days or when the density reaches the upper limit (e.g., 1–2 × 10⁶ cells/mL).
- Determine the cell density using a hemocytometer or an automated cell counter.
- Dilute the cells into fresh, pre-warmed medium to the recommended starting density (e.g., 2–5 × 10⁵ cells/mL).
- For spinner flasks, maintain stirring at appropriate speeds to prevent cell clumping or damage.
7. Cryopreserving Cells (Optional)
- To preserve cells for long-term storage:
- Harvest cells by centrifugation.
- Resuspend in freezing medium (typically 10% DMSO in FBS).
- Aliquot into cryovials and freeze gradually at -80°C using a controlled-rate freezing method (e.g., freezing container) before transferring to liquid nitrogen.
Maintenance of Adherent and Suspension Cells
Maintenance Procedure
- Daily monitoring -Observe cells daily under a microscope to assess morphology, confluency/growth, and contamination
- Medium Changes or feeding the cells -Replace the culture medium every 2-3 days to provide fresh nutrients and remove waste
- Monitoring Confluency -Subculture (passage) cells when they reach 70-90% confluency to prevent overcrowding and nutrient depletion
- Passaging (Subculturing) -adherent cells must be passaged when they reach confluency (often at 70-90%) to avoid overcrowding and senescence
- -Transfer a portion of the culture to a new flask with fresh medium when cell density becomes high
Harvesting of Adherent Cells
- Prepare Equipment and Reagents: Pre-warm trypsin-EDTA, PBS, and medium. Use sterile pipettes, centrifuge tubes, and culture vessels
- Remove Medium and Wash: Aspirate the medium from the flask and wash cells with PBS to remove serum (which inhibits trypsin)
- Detaching Cells with Trypsin: Add enough trypsin-EDTA solution to cover the cell layer
- Incubate at 37°C for 2-5 minutes, observing under a microscope until cells begin to round up and detach
- Neutralizing Trypsin: Once cells detach, add fresh medium to neutralize the trypsin. Pipette gently to further detach and collect cells into a sterile tube
- Centrifugation and Resuspension: Centrifuge at 300-500g for 5 minutes to pellet the cells.
- Aspirate the supernatant, resuspend the cell pellet in fresh medium or buffer, and proceed with further steps
Harvesting of Suspension Cells
1.Preparing Reagents and Equipment
- Pre-warm the medium and prepare sterile centrifuge tubes.
2.Centrifugation
- Transfer the culture to centrifuge tubes and centrifuge at 300-500g for 5 minutes.
- Aspirate the supernatant carefully without disturbing the cell pellet.
3.Resuspension
- Resuspend the cell pellet in fresh medium or a suitable buffer for downstream applications.
- If needed, count cells using a hemocytometer or automated cell counter before further use.
4.Harvesting for High-Density Cultures
- If cell density is very high, dilute with fresh medium before centrifugation to reduce clumping and improve yield