BIO62004: Aseptic Techniques in Cell Culture

Instrumentation in Medical Diagnostic, Laboratory and Blood Banking

• Course: BIO62004
• Topic 2: Maintenance of Mammalian Cell Culture
• Section 2.1 (L4): Aseptic Techniques in maintenance of cell culture in vitro

Learning Objectives

• Discuss the principles of animal cell culture
• Demonstrate animal cell culture techniques
• Relate animal cell culture to specialized applications
• Understand the importance of aseptic technique (Sterile work area, Personal hygiene, Sterile reagents and media)
• Discuss the principles of cryopreservation and cell banking

Animal Cell Culture

• Cell culture: Process of growing cells in a controlled artificial environment
• In vitro maintenance: Cells can be maintained outside their original body
• Simpler than organ and tissue culture

Primary Cell Culture

• Outgrowth of cells: From a piece of tissue or disaggregated tissue (enzymatic/mechanical)
• Morphology: Similar to the parent tissue
• Limited cell divisions: Enter senescence and die
• Proliferative capacity: Extended by introducing viral transforming genes (e.g., SV40)
• Phenotype: Intermediate between finite and continuous cultures
• Finite cultures: Limited lifespan
• Continuous cultures: Proliferate indefinitely (similar to senescent primary cells)

Two Main Types of Cells

• Adherent cells: Anchorage dependent; grow as a monolayer attached to the culture vessel.
• Most tissue-derived cells are anchorage-dependent
• Suspension cells: Can survive and proliferate without attachment to a substrate.

Handling Cell Cultures

• Good laboratory practice (essential): Reduce exposure to infectious agents in the culture; prevent contamination by microbes
• Aseptic techniques and lab equipment: Vital for working with cell cultures
• Sterile equipment and reagents; wash hands, reagent bottles, and work surfaces with biocide/70% ethanol.

Cell Culture Conditions

• Culture media and serum: Different cell types require specific growth requirements. Media composition must be experimentally determined.
• Common basal media: Eagle minimal essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), RPMI 1640, and Ham F10. Contains amino acids, glucose, salts, vitamins, and other nutrients (powder or liquid form from suppliers).
• Media types: Balanced salt solutions (PBS, Hanks' BSS, Earle's salts, DPBS, HBSS, EBSS); Basal media (MEM, DMEM, GMEM); Complex media (RPMI 1640, Iscoves DMEM, Leibovitz L-15, TC 100, Graces, Schneider's, CHO, HEK293, Ham F10, DMEM/F12); Serum free media (Serum free insect Medium 1)

Basic Components of Media

• Bicarbonate buffering system (low cost, non-toxic): CO2 balances with HCO3- content; cultures need 5-10% CO2 atmosphere (CO2 incubator).
• HEPES buffering system: Superior buffering capacity in 7.2-7.6 pH range. Relatively expensive and toxic at higher concentrations for some cell types; increases media's sensitivity to phototoxic effects from fluorescent light.
• Phenol red: pH indicator; yellow at low pH, purple at higher; disadvantages of using phenol red?

Serum

• Growth factors and hormones: Promotes growth and specialized cell function
• Binding proteins: Albumin, transferrin that carry molecules into cells (lipids, vitamins, hormones)
• Attachment promotion: Proteins such as fibronectin; spreading factors allowing cells to spread out before division
• Protease inhibitors: Protect from proteolysis
• Minerals (Na+, K+, Zn2+, Fe2+, etc): Essential nutrients

Serum Inhibitors

• Heat inactivation: Removes complement proteins; reduces cytotoxic actions of immunoglobulins without damaging other growth factors; usually incubated for 30 minutes at 56°C.

Serum Advantages and Disadvantages

• Advantages: Growth factors, hormone attachment, spreading factor, buffering agent, binding protein, minimizes damage.
• Disadvantages: Batch variation, quality control needed, growth inhibiting properties, risk of contamination, interfere with purification and isolation.

Serum Free Media

• Serum-free media (SFM): Replaces serum, thus avoiding issues with animal-derived sera; Formulation exists for primary cultures and cell lines and includes those producing recombinant proteins (CHO, hybridoma cell lines, insect lines Sf9 and Sf21, and others for viral production.

Requirements of a Tissue Culture Lab

• Essential equipment: Cell culture hood, incubator (humid CO2 recommended), water bath, centrifuge, refrigerator & freezer (-20°C), cell counter, inverted microscope, liquid nitrogen freezer or cryostorage, sterilizer (autoclave)
• Beneficial equipment: Aspiration pump, pH meter, tube racks, pipettes, plate reader
• Additional equipment: Glassware washing machine, confocal microscope, flow cytometer, sterile containers, syringes, and needles

Cell Culture Hoods

• Air flow characteristics: Protects working environment from dust, airborne contaminants, by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area.
• Types: Horizontal (air flows towards the user) or vertical (air flows from top to bottom).

Cell Culture Conditions

• Incubation conditions: Incubator with controlled temperature and CO2 concentration is needed for optimal growth; includes dry and humid CO2 incubators.
• Cell culture vessels: Sterile, treated dishes and flasks for cell attachment; available commercially.
• Substrates and matrices: Cells attach to acid-washed glass, polystyrene, and plastics with a net positive charge. Cells secrete matrix products, such as collagen type IV, fibronectin, or laminin, to help adhere to the substrate and interacting with matrix receptors (e.g., integrins).