Cell Culture Techniques

Question 1

What are primary cell cultures ?

Answer 1

-They are derived directly from tissues
-They have a finite lifespan
-They are initially heterogenous (tissue contains different cell types)
-Normal functions
-Good for personalised medicine -They come from the patient
(We can test drugs in vivo to see the reactions on the cells )

Question 2

What are the methods of isolation?

Answer 2

1.The cells are allowed to migrate out of an explant

Explant-
Cell which has been transferred from animals to nutrient medium
Allow to isolate spontaneously- no disruption needed

2. Mechanical (Mincing ,sieving, pipetting)
   - Enzymatic dissociation (Trypsin,collagenase,hyaluronidase,protease, DNAase)

Some cells do not need to be disaggregated because they already are e.g. hemopoietic

Question 3

Describe how we can isolate specific cell populations from the blood?

Answer 3

Density medium centrifugation
-Blood - Plasma,PMBCs,Density gradient medium,Granulocytes,Erythrocyte

Depending on density medium used we can isolate populations.
An example is secol

Question 4

How does blood separate following centrifugation?

layers

Answer 4

(The least dense)
Plasma
PMBCS
Density Gradient Medium
Granulocytes ,Ertyhrocytes  ( The most dense )

Question 5

What is the buffy coat ?

Answer 5

fraction of an anticoagulated blood sample that contains most of the white blood cells and platelets following density gradient centrifugation.

Question 6

Describe Immuno-purification

Answer 6

1. Antibody-coated magnetic beads
2. Mixed with heterogeneous cells
3. Isolation of antigen expressing cells

Question 7

Describe Fluorescence activated cell sorter

Answer 7

Specialized type of flow cytometry.
Method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.

Question 8

Give examples of non-haematopoietic primary cells

Answer 8

Liver 
Endothelial 
Muscle
Skin
Nerves
Fibroblasts

Question 9

Give examples of haematopoietic cells

Answer 9

Stem, progenitor cells 
T/B cells
Monocyte, Macrophages
Osteoblasts
Dendritic cells 
Neutrophils
Erythrocytes
Megakaryocytes, Platelets

Question 10

Outline the extraction of stem cells

Answer 10

They can be extracted from bone marrow aspirate,fat,mobilised peripheral blood ,umbilical cord,embryonal tissue or iPSCS

Question 11

What are the disadvantages of primary cells

Answer 11

• Inter-patient variation
• Limited
• Finite lifespan
• Difficult molecular manipulation
• Phenotypic instability
• Aberrant expression of some genes (Abnormal expression)
• Variable contamination

Question 12

What are cell line cultures?

Answer 12

• Immortalised cells
• Less limited number of cell divisions
• They can be grown 2D or 3D
• Phenotypically stable, defined population
• Limitless availability
• Easy to grow
• Good reproducibility
• Good model for basic science

Question 13

What are the methods of cell line production?

Answer 13

1. Isolated from cancerous tissues
2. Immortalisation of primary cultures
   - Spontaneously from prolonged culture
   - Through genetic manipulation

Question 14

Why would we need to use cell lines ?

Answer 14

• Primary cells not ideal because they carry aberrant genes
• So unfunctional protein expression sometimes
• Can have variable contamination and short lifespan
• Cannot carry out in vitro analysis using primary cells
• So need to produce CELL LINES
• From healthy or cancerous tissues
• Spontaneous or genetic manipulation to make them immortal

Question 15

Describe production of cell lines through genetic manipulation

Answer 15

To generate cell lines we target processes that regulate