Topic 2: Laboratory Setup and Equipment for Cell and Tissue Culture

A well-equipped and carefully organized laboratory is crucial for successful cell and tissue culture work. Proper planning ensures sterility, minimizes contamination, and facilitates efficient workflow.

1. Laboratory Setup

Key Considerations

1. Space Design
   • Separate areas for cell culture, reagent preparation, and waste disposal.
   • Clear demarcation of sterile (aseptic) and non-sterile zones.
2. Environmental Controls
   • Temperature: Maintain a stable temperature for equipment and culture.
   • Humidity: Control moisture to prevent drying of culture media.
   • Air Quality: Use High-Efficiency Particulate Air (HEPA)-filtered ventilation to minimize airborne contamination.
3. Workflow Layout
   • Unidirectional workflow to reduce cross-contamination.
   • Logical arrangement: reagent storage → preparation → culture work → disposal.
4. Lighting
   • Ensure proper illumination for microscopic observations.
   • Avoid direct exposure of light-sensitive reagents to intense light.

2. Essential Equipment

Primary Equipment

1. Biosafety Cabinet
   • Provides a sterile environment to handle cultures
   • Types: 
     • Laminar Flow Cabinets: These provide the most sterile environment for the cells, but offer no protection to the operator. Filtered air enters at the back of the cabinet and is directed to the front, directly at the operator. The most sterile part of the cabinet is at the back.
       
     • Class II: Most common for cell culture, protects both samples and user. The air is directed downwards from the top of the cabinet to the base. When working in these cabinets it is important not to pass non-sterile objects over sterile ones
       because air is also drawn in from the front of the cabinet, this area is not sterile. Maintain all the cell lines in class II containment. Most work with Human or Primate cells must be done in Class II containment
       
     • Class III: For hazardous materials or higher safety requirements.
2. Incubator:
   • Maintains optimal temperature (usually 37°C) and humidity.
   • CO₂ incubators regulate the CO₂ level (typically 5%) for pH control.
   • Carbon dioxide incubators: The incubators run at 37℃ or with a range of temperatures and 5% CO2 to keep the medium at the correct pH.
   • Incubators without CO2: The incubators run at 37℃ or with a range of temperatures and without CO2, the medium has been conditioned to keep the correct pH.
   • They all have meters on them to register temperature and gas level for CO2 incubators
   • There are alarms to indicate when these deviate from set parameters
   • Keep the door open for as short a time as possible

1. Microscope
   • For monitoring cell morphology and confluency.
   • Types:
     • Inverted Microscope: Common for observing adherent cultures.

1. • • Phase Contrast Microscope: Enhances contrast without staining.
2. Centrifuge:
   • Bench centrifuges-For separating cells, removing debris, and washing cell pellets.
   • Tabletop models are typically sufficient for most culture applications.
   • There are centrifuges in each cell culture area which are refrigerated
   • Human derived cells must be centrifuges in sealed rotors
   • 100 x g is hard enough to sediment cells, higher g forces may damage cells
   • If a tube breaks in the centrifuge, take the whole bucket into a cabinet and clean it there

1. Water Bath:
   • Used to warm media and reagents to 37°C before use.

1. Refrigerator and Freezer:
   • 4°C Refrigerator: For short-term storage of reagents and media.
   • -20°C or -80°C Freezer: For long-term storage of enzymes, sera, and cell stocks.

1. Autoclave:
   • Sterilizes equipment and media to ensure aseptic conditions.

Support Equipment

1. Pipetting Systems:
   • Manual or electronic pipettes with sterile disposable tips.
2. Vacuum Pump:
   • For aspirating media or washing cultures.

1. pH Meter:
   • To ensure culture media has the correct pH.
2. Sterile Waste Disposal System:
   • Dedicated containers for biohazardous waste.
3. Timer and Thermometer:
   • For precise incubation and monitoring conditions.

3. Consumables

• Culture Vessels: Flasks, Petri dishes, multi-well plates, and tubes.
• Pipettes and Pipette Tips: Sterile, disposable options to avoid contamination.
• Filters: For sterilizing media and reagents.
• Sterile Gloves: Prevent contamination during handling.
• Parafilm and Aluminum Foil: For sealing and protecting cultures.

4. Sterility and Contamination Control

1. Surface Disinfection:
   • Use 70% ethanol or other disinfectants to wipe surfaces.
2. UV Sterilization:
   • Some laminar hoods are equipped with UV light for pre-use sterilization.
3. Aseptic Techniques:
   • Minimize open handling of cultures.
   • Work near the flame or within the sterile airflow.

5. Additional Equipment for Advanced Techniques

1. Cryopreservation System:
   • Liquid nitrogen storage tanks for long-term cell storage.
   • Cryovials and freezing equipment.
2. Flow Cytometer:
   • For cell sorting and analysis.
3. Microplate Reader:
   • For assays such as cell viability, proliferation, or fluorescence.

Maintenance of Equipment

• Regular calibration of pipettes, microscopes, and incubators.
• Routine cleaning and decontamination.
• Periodic servicing by certified technicians.

A properly equipped lab not only enhances productivity but also ensures reproducibility and reliability in cell and tissue culture experiments.