Section 1: Cell culture

Question 1

What is the purpose of a cell culture?

Answer 1

• To grow cells or tissue outside of an organism under strictly controlled conditions
  
  • We know exactly what is in the medium
  • We know exactly what the cells are

Question 2

How are cells obtained from tissue?

Answer 2

• By breaking down the cell-cell and cell-matrix (protein) interactions
• 2 methods:
  
  1. Mechanical fragmentation
     
     • Add protease (ex., trypsin) to cleave cell-cell and cell-matrix bonds
     • Protease must be well regulated; must be able to turn it off
  2. Add EDTA
     
     • EDTA binds cations, metals, etc.
     • Used to absorb metals (ex., calcium) to break down the bonds

Question 3

What sort of medium are cells grown in?

Answer 3

• A mixture of proper nutrients (amino acids, minerals, vitamins, salts, glucose, etc.)
• Serum (insulin, growth factors) is added
  
  • Serum is obtained from centrifuging (usually bovine) blood

Question 4

What environment are cells grown in and why?

Answer 4

• Cells are grown at 37^oC (for mammal cells) in a CO₂ incubator
  
  • CO₂ addition mimics the normal tissue environment

Question 5

Describe the two ways cells can be grown (adherent vs. suspension)

Answer 5

• Cells can grow as adherent cell cultures or suspension cell cultures
  
  • Adherent = on a flat surface
    
    • Most common
  • Suspension = in constantly moving media

Question 6

What is a ‘primary cell culture’?

Answer 6

• Primary cells are cells taken directly from an organism
  
  • They are mortal: they divide a limited number of times
    
    • This is due to contact inhibition: primary cells cannot grow on top of each other
      
      • Without replating, they will run out of space and no longer divide
      • This is the Hayflick limit (they will divide until ~50 generations)

Question 7

What is the solution to the problem of primary cell cultures?

Answer 7

• Cell lines! These are transformed cells that grow indefinitely
  
  • They are immortal
  • They are less likely to exhibit contact inhibition
  • Transformation can include making the cells cancerous so they divide forever

Question 8

What is the first human cell line?

Answer 8

HeLa = first human cell line from a cervical carcinoma biopsy

Question 9

Compare the morphology of a normal vs. transformed cell

Answer 9

The example are normal fibroblasts (connective tissue cell) vs. transformed fibroblasts (with Rous sarcoma virus that makes it cancerous)

Question 10

Compare symmetric vs. asymmetric cell division

Answer 10

• Symmetric = progeny are identical to each other
• Asymmetric = progeny are not identical to each other

Question 11

How do stem cells divide?

Answer 11

• Through symmetric and asymmetric cell division

Question 12

Describe embryonic stem cells

Answer 12

• A.k.a. inner cell mass; makes all types of cells
• Require a niche to grow
  
  • Niche: adjacent cells and extracellular proteins which provide signals to either self-renew or differentiate, and keep the stem cells immortal
• In cell culture, we can grow the three primary cell layers/germ layers (endoderm, ectoderm, mesoderm), but we cannot get defined tissues

Question 13

Describe adult stem cells

Answer 13

• Most tissues contain adult stem cells
• Required to maintain and repair tissue
  
  • Capable of generating a limited number of different cell types
  • Ex., adult stem cells replace the intestinal epithelium every 5 days
• Located in a stem-cell niche as well