3 - Propagation of viruses II.

Question 1

Theory

Answer 1

In vitro maintenance and propagation of living cells

Question 2

1. Historically
2. More recently

Answer 2

1. Historically:
   
   • Organ pieces initially used, then surviving epithelial cells
2. More recently:
   
   • Monolayer cell cultures used from organs
     
     • Especially:
       
       • Kidneys
       • Testicles
       • Thymus
       • Embryo

Question 3

Processing

Answer 3

• In sterile conditions
• Steps:
  
  1. Removal of outer membranes; cut tissue to small pieces
  2. Separation of cells via digestion with Trypsin and EDTA
  3. Cell suspension removed regularly and replaced by renewed trypsin solution
  4. Trypsin blocked by an ice bed (0⁰C) and removed by centrifuge
  5. Cells resuspended in a culture medium – MEM – Minimal Essential Medium
     
     • Optimal environment needed – Iso-tonic/osmotic/ionic
     • Nutritive: amino acids, carbohydrates
     • Antibiotics, Indicator (Phenol red)
     • Foetal calf serum (FCS*) - *Protein source
  6. Cell counting in a burker chamber
  7. Cell culture transferred to culture flask – plates, roux flask, petri dish
  8. Incubation – 37⁰C, 5% CO2 –- within 3-5 days, cover bottom of flask
• Contact inhibition – don’t grow over each other
• ➔ Primary monolayer cell culture produced!

Question 4

Give the components of cell culture media

Answer 4

• Carbon source (​Source of energy)
• Amino acid​ (Building blocks of proteins)
• Vitamins (Promote cell survival and growth)
• Balanced salt solution
• Phenol red dye (pH inidcator)
  
  • The color of phenol red changes from orange/red at pH 7-7.4
  • Acidic: yellow
  • Basic: purple
• Bicarbonate (Maintains a balanced pH in the media)

Question 5

Give the typical growth conditions

Answer 5

• Temperature: 37ºC
• CO₂: 5%
• 3-5 days

Question 6

Use of monolayer

Answer 6

1. Propagation of viruses
2. Maintenance of cell cultures
3. Sub culturing ➔ Secondary culture – fresh, increased amount but can only be reused 2-3x (termed a “passage”)
4. Cellular cloning ➔ Cultivation of a single cell - 1cell/well

Question 7

Cloning

1. Cloning type
2. Cloning advatages
3. Cloning disadvantages

Answer 7

1. Cloning type: Diploidic or Aneuploidic
2. Cloning advatages:
   
   • Genetically homogenous, standardized, unlimited numbers of passages
   • Long-term storage -80ºC liquid nitrogen
3. Cloning disadvatages:
   
   • Sensitivity to infection varies
   • Contamination – virus, leptospira
   • Presence of active Oncogen, especially Tumour Cells

Question 8

Special virus propagation: Cell culturing methods

Answer 8

• Co-cultivation:
  
  • ​To isolate cell-associated and latent viruses and mix healthy and infected cells
  • White blood cell cultures - “Buffy coat culture”
    
    • WBC viruses, e.g. African swine fever
• Suspension cultures:
  
  • ​Continuous stirring stops settling → fewer more concentrated cells
  • This is preferable for vaccine production

(Most of the above is for diagnostics only!)