Topic 7: Cell Culture Techniques

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

1. Primary Culture
   • Obtained directly from animal or plant tissues.
   • Cells have a limited lifespan and maintain many characteristics of the original tissue.
2. 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.
3. 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.
4. 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

1. Isolation: Tissue or organ is enzymatically or mechanically dissociated into single cells.
2. Seeding: Cells are seeded into culture vessels with the appropriate medium.

b. Maintenance

1. Feeding: Replace spent medium regularly to replenish nutrients and remove waste.
2. Monitoring: Regularly check cells under an inverted microscope for growth, contamination, or morphology changes.

c. Subculturing

1. Wash cells with PBS (phosphate-buffered saline).
2. Detach adherent cells using trypsin-EDTA or other enzymatic solutions.
3. Resuspend cells in fresh medium and transfer to a new vessel.

d. Cryopreservation

1. Suspend cells in a cryoprotective medium.
2. 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

1. Cell Culture Equipment
   • Biosafety cabinet (sterile workspace)
   • CO₂ incubator
   • Inverted microscope
   • Centrifuge
2. 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

1. Cell Line: The suspension cells to be cultured.
2. 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.
3. Sterile Flasks: Vent-cap culture flasks or spinner flasks.
4. Pipettes: Sterile serological and micropipettes.
5. Laminar Flow Hood: To maintain sterility.
6. Centrifuge and Tubes: For cell harvesting, if needed.
7. Hemocytometer or Cell Counter: To assess cell density and viability.
8. CO2 Incubator: Set at optimal conditions for the cell line (typically 37°C with 5% CO2).
9. Reagents: Trypan blue or another viability dye.
10. 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

1. Prepare Equipment and Reagents: Pre-warm trypsin-EDTA, PBS, and medium. Use sterile pipettes, centrifuge tubes, and culture vessels
2. Remove Medium and Wash: Aspirate the medium from the flask and wash cells with PBS to remove serum (which inhibits trypsin)
3. 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

3. Neutralizing Trypsin: Once cells detach, add fresh medium to neutralize the trypsin. Pipette gently to further detach and collect cells into a sterile tube
4. 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