Cell culture

Question 1

What are the 5 universal considerations of cell culture?

Answer 1

1. Aseptic technique - only culture what you choose to culture
2. Temperature - 37 degrees for mammals, lower for insects, can be very high for archea
3. Atmosphere - Co2 or O2
4. Nutrients - solid or liquid medium
5. Safety

Question 2

What conditions must Schizosaccharomyces pombe be gown under?

Answer 2

• Agar, however different nutrients than bacteria
• Atmospheric gases
• Different temperature

Question 3

What can yeast be used for?

Answer 3

S. pombe - Model for the cell cycle, as have simple eukaryotic genome and similar components and cycle
S. cervisiae - beer and studies of cell trafficking

Question 4

What conditions must Haloferax be grown in?

Answer 4

45 degrees with high salt concentrations, has red colour due to lycopene

Question 5

How are cell cultures of multicellular organisms usually conducted?

Answer 5

• Liquid culture
• Usually red, indicating pH of 7.4. Bi-products such as lactic acid and pyloric acid can lower pH and turn indicator yellow

Question 6

What was Alexis Carrel the first to produce?

Answer 6

A cell line of chick embreyonic fibroblasts described as “immortal”, living for a long time under aseptic conditions when fed with nutrients.
However, doubt over whether a malignant transformation was present or if accidental contamination with fresh cells had occurred

Question 7

How can one produce a primary cell line?

Answer 7

• Isolate cells from tissue by dissection, mincing or enzymatic dissociation
• Dealing with cells directly from the organisms, although these are frequently short lived and it’s hard to maintain consistency

Question 8

How can one produce a transformed (immortal) cell line?

Answer 8

• Take cells from a tumor
• Expression of viral proteins such as Large T-antigen (SV40)
• Express genes that confer immortality such as telomerase
• However, not as representative of cells in the body

Question 9

What is 2D culture?

Answer 9

Use of glass/plastic flask where cells adhere to the base and culture on the medium

Question 10

How is 2(1/2)D culture different from 2D culture?

Answer 10

Extracellular matrix at the base of the flask which cells can modify to create “organs”. Also contains different components.

Question 11

What is 3D culture?

Answer 11

Where a 3D agar gel is used to culture stem cells/mammalian cells in order to produce precise patterning

Question 12

How does mechanically supported culture exploit the air/liquid barrier?

Answer 12

Grows organ explant at air/liquid interface to exploit O2 perfusion

Question 13

Name 3 reasons why cell culture is important

Answer 13

1. In order to reduce, refine and replace animal studies
2. Scarce availability of human tissues and ethics problems in obtaining them
3. Usage for regenerative medicine e.g stem cells