Animal Cell Culture Detailed Notes

Unit - III: Animal Cell Culture

Historical Background

• Ross Harrison: First attempt to culture animal cells (embryonic nerve cells of frogs using hanging drop method).
• Supplemented with chick embryo plasma, leading to better proliferation.
• 1940s: Rise of cell culture due to virus use in vaccine production, crucial for polio vaccine development.
• Cell lines available from various sources:
  • National Centre for Cell Science (NCCS), Pune, India
  • European Collection of Animal Cell Cultures (ECACC), UK
  • American Type Cell Culture Collection (ATCC), USA.

Principles of Sterile Techniques

Wet Heat Sterilization

• Sterilization method: Heat treatment under moist conditions.
  • Effective against various microorganisms at 60-80 °C (pasteurization).
• Resistance of spore-forming bacteria and prions.
• Pasteurization at 60 °C for 10 hours for viral contaminants in plasma products.
• Boiling/steam treatment: Effective for sterilizing equipment and fluids at 100 °C for 5-10 min.

Autoclaving

• Uses steam under pressure, effective at 121 °C.
• Prions are particularly resistant; require extreme conditions for inactivation.
• Air presence can reduce sterilization efficiency.
• Types of autoclaves vary from small units to large automated machines.

Dry Heat Sterilization

• Higher temperatures and longer times needed than wet methods.
• Incineration recommended for contaminated waste; efficient at 350 °C.
• Use of Bunsen burner for sterilizing openings of containers.

Hot Air Ovens

• Suitable for heat-resistant objects (glass, metal).
• Lower temperatures required for some plastics (e.g., 120 °C for 18 hours).
• Filtration systems may prevent recontamination during cooling.

Types of Filters

• Various materials (cellulose acetates, nylon, etc.) for removing microorganisms.
• 0.2 µm pore size standard for bacteria and fungi removal.
• 0.1 µm filters for mycoplasma removal.
• Membrane filters serve for gases (e.g., CO2) used in cultures.

Chemical Sterilization

• Formaldehyde/ethylene oxide: Effective fumigation for different microorganisms.
• Followed rigorous conditions during fumigation to ensure effectiveness.
• Alcohol (70-80% concentration) used for disinfecting surfaces but is flammable.
• Phenolic disinfectants: Not inactivated by organic matter but less effective against spores.

Cell Propagation

Primary Cultures

• Original cultures established from any tissue; choice of source is critical.
• Permits growth of specific cell types, but may have a finite life span.
• Must gain ethical permissions when working with human tissues.

Continuous Cell Cultures

• Dividing cell lines easily passaged and can be established indefinitely.
• Normal cells have a finite life span; transformed cells can grow indefinitely.
• Properties of continuous cells include aneuploidy and sometimes altered growth properties.

Establishment of Continuous Cell Lines

Methods

• Spontaneous transformations are rare but possible (e.g., fibroblast cell lines).
• Chemical transformation using carcinogens to induce immortal growth.
• Viral transformation (e.g., SV40) can lead to isolated transformed lines.

Cell Culture Techniques

Suspension Cultures

• Easier to propagate and harvest, but do not adhere well.
• Commonly utilized for cells from blood or bone marrow.

Cell Passage

• Maintain culture health, typically at 70-80% confluency.
• Use PBS washes, trypsin for cell release, resuspend in serum to deactivate trypsin.

Advantages and Disadvantages of Cell Cultures

Media for Cell Culture

Chemically Defined Media

• Essential for cell survival and proliferation.
• Growth Media: Provide necessary nutrients and supplements.

Types of Media

• Natural media: Biological fluids, tissue extracts.
• Artificial media: Balanced salt solutions, basal, and complex media.
• Common media examples: EMEM, DMEM, RPMI-1640.

Components of Media

• Basal salts: Maintain pH and osmotic pressure.
• Carbohydrates: Energy source; glucose is common.
• Amino Acids: Essential for cell growth; concentrations affect cell yields.
• Vitamins and proteins: Require for cell metabolic functions.
• Antibiotics: Cautiously used in cell culture; broad spectrum preferred.

Serum in Cell Culture

• Provides growth factors necessary for cell maintenance and proliferation.
• Variations in serum quality necessitate careful selection and testing before use.

Applications of Animal Cell Culture

• Cell Culture-Based Vaccines: Purified, safer than traditional vaccines.
• Drug Development: Essential for evaluating drug toxicity and efficacy.
• Cancer Research: Models for drug efficacy; tumor cell interactions studied.
• Genetic Engineering: Facilitate transfection and gene therapy applications.

Tissue Plasminogen Activator (t-PA)

• Enzyme for blood clot dissolution developed through CHO cell culture.
• Genentech cloned t-PA for therapeutic use in blood clots; branded as Activase.

3D Bioprinting

• Emerging technology utilizing living cells to create three-dimensional tissue constructs.
• Utilizes bioinks for building tissues and potentially addressing organ shortages.

Addressing Contamination in Cultures

• Chemical and biological contaminants are common issues in cell culture.
• Regular cleaning, strict aseptic practices help minimize contamination risk.

Characterization of Mammalian Cells

• Essential for verifying cell origins, species identification, chromosome content analysis (cytogenetics).
• Immunological tests confirm species-specific antigens.

Organ Culture

• Technique for culturing pieces of organs; often for embryonic organs which are easier to manipulate.

Artificial Tissues

• Artificial skin: Developed using fibroblasts and keratinocytes for burn patients.
• Regenerative Applications: Includes cartilage and complex organ development using engineered tissues.