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Flashcards in Cell Culture Techniques Deck (42)
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1

What is a cell culture? 

Group of cells grown in a nutrient solution from a single original cell 

2

How can we isolate different cells? (summary) 

 

1) Blood 

  • Density centrifugation (used to isolate different blood cell populations) 
  • Immunopurification and FACS (used to isolare specific cells) 

2) From solid tissues 

  • Mechnical and enzymatic disruption to disrupt cells from solid tissue then magnetic immuno-purification technique used to extract cells of interest 
  • Mechanical and enzymatic disruption not required in explant culture where chondrocytes will migrate away from cartilage 

3

How do we isolate cell populations in blood? 

Density Centrifugation 

  • This takes advantage of the differential density of the cell population and the density gradient medium that is used.
  • The type of cell isolated will depend on the density gradient medium used

4

What do the layers look like following centrifugation? 

  • Granulocytes and erythrocytes are denser than mononuclear cells and will sedimeny through the density gradient medium 
  • Mononuclear cells will remain in the top layer 
  • Lymphocytes will remain in the buffy coat, these can be extracted through isolation of this specific coat 

5

Techniques used to isolate specific cells from blood 

  • Immuno-purification 
  • Fluorescence Activated Cell Sorter (FACS) 

6

How does immuno-purification work? 

  • Coat magnetic beads with an antibody which will bind to a specific cell surface receptor/ antigen present in the cell or interest 
  • Addition of magnetic field we can extract cells of interest 

7

Fluorescence-activated cell sorter (FACS) 

Uses antibodies to isolate cells of interest, but is also based on size 

8

How do we isolate cells from solid tissues

  • Mechanical and enzymatic disruption is carried out on solid tissues 
    • Mechanical (use a scalpel or passing tissue through needles) and enzymatic (through trypsin, collagenase) 
  • Once extraction has been carried out individual cells are extracted using magnetic immuno-purification 

9

What happens in an explant culture? 

  • In a cartilage explant culture, chondrocytes will migrate away from cartilage explant spontaneously 
  • The cartilage explant is set on a plate and chondrocytes will isolate by themselves 

10

What are cell lines and why are they used? 

Cell lines are immortalised cells that continue to grow and divide indefinitely in vitro for as long as the correct culture conditions are maintained 

Used as they pose fewer disadvantages compared to primary cells 

11

List some advantages and disadvantages of using primary cells? 

  • Advantages 
    • Unmodified 
    • Good for personalised medicine 
  • Disadvantages 
    • Aberrant expression of some genes 
    • Variable contamination 
    • Limited 
    • Short life-span 
    • Inter patient variation 
    • Difficult molecular manipulation 
    • Phenotypic instabillity 

12

Where are cell lines isolated from? 

  • Healthy or cancerous tissues 
    • (e.g HeLa cells) = HeLa cell line include cells derived from cell carcinoma 
  • Primary cultures 

13

Cell lines derived from primary cultures what can they either do? 

 

  • Survive spontaneously by themselves without manipulation 
  • Be genetically manipulated in order to transform them and make them immortal 

14

How do we make cell lines immortal? 

By genetic manipulation of three different proteins that are genetically manipulated in order to produce immortal cells 

  1. p53
  2. pRB 
  3. Telomerase enzyme 

15

What are p53 and pRB encoded by? 

What do they do? 

They are encoded by tumour supressor genes 

p53 and pRB both maintain genomic stabillity by mediating cell cycle checkpoints 

16

What is the role of telomerase? 

  • Reverse transcriptase enzyme carrying its own RNA molecule 3′-CCCAAUCCC-5′  which it uses as a template to elongate telomeres 
  • Prevents shortening of telomeres hence reducing point at reaching Hayflick limit where cell senescence is triggered 

 

 

17

What are the only cells with active telomerase? 

Stem cells, gametes and cancer cells 

18

How do we create an immortal cell line? 

  • Inhibit function of p53 and pRB 
  • Introduce/ over-express telomerase 

19

How do we inhibit the function of p53 and pRB? 

To inhibit tumour supressor activity (p53 and pRb) we use viral oncoproteins in SV40 (simian) and HPV which will target tumour supressor proteins. 

20

What is the mechanism of action of SV40 to inhibit tumour suppressor proteins? 

  • SV40 oncoviral large T antigen will interact with p53 and pRb protein indirectly 
  • It will interact with the cells protein DNA binding domain to which p53 and pRb bind to 
  • This prevents interaction of pRB and p53 with these domains 
    • However there are still levels of p53 and pRb in the cell due to indirect inactivation, they are still functional just unable to bind to their domains 

21

What is the mechanism of action of HPV to inhibit tumour supressor proteins? 

HPV = E6 will directly targets p53 for degradation, and E7 binds to pRb inactivating it

Cell lines made using E6/E7 are believed to maintain a differentiated phenotype

22

What is believed to be the most efficient way of producing cell lines? 

  • Immortalisation of cell lines has been proved most effective with inactivation of pRb /p53 through E6/E7 and telomerase transformation 
  • These are believed to result in cell lines with a differentiated phenotype 

23

How is telomerase transfected into primary cells to immortalise them? 

  1. Design a plasmid containing a gene for selection with selection marker e.g Antibiotic resistance marker
  2. Insert the telomerase sequence containing gene into the plasmid 
  3. Once plasmid construction is completed the primary cell will be transfected with those vectors 
  4. To check if the gene has been transfected a selection pressure is added (Antibiotic resistance to neomycin) 
  5. Only cells with AB resistance and telomerase gene will survive 

24

What are some advanatages of cell lines? 

  • Good growth characteristics 
  • Phenotypic stabillity 
  • Defined population 
  • Molecular manipulation readily achieved 
  • Good reproducibillity 
  • Good model for basic science 

25

What are some disadvantages of cell lines?

Often lose differentiated function 

Cell-substrate interactions dominate

Does not mimic real tumour conditions 

Lacks cells heterogeniety 

Phenotype needs to be validated 

26

What are conditions and requirements for cell growth in culture? 

  • Must be handled under aseptic conditions 
  • Grow on tissue culture treated plastic flasks/ dishes with enough space to add corresponding growth factors and supplements to medium 
  • Maintained in a warm incubator (37 degrees), humidified atmosphere (5% CO2) and a neutral pH (same cond. as human body) 

27

What is the significance of growth medium maintence in cell culture? 

  • Ideal supplemented medium needs to be replcaed by a fresh one every 2/3 days due to depletion of nutrients in the medium and due to release of waste products into medium after cell metabolism 
  • We will change/replace the growth medium when universal indicator will change colour 

28

Describe the difference between adherant and suspension cells? 

Adherant cells 

  • Cells which attacj to solid surface + proliferate 
  • Low yield 
  • Growth limited by surface area 
  • Most types of cell lines and primary cultures 

Suspension Cells 

  • Grow suspended (floating) in liquid medium 
  • High yield 
  • Some non-adhesive cell lines (e.g haematopoietic) 

29

What are the two ways in which cell cultures can become contaminated? 

  • Microbial Contamination
    • Bacteria (pH change, cloudiness/turbidity, precipitation, stink)
    • Yeast (cloudiness, pH change)
    • Fungus (spores furry growths, pH change)
    • Mycoplasma (often covert, poor cell adherent, reduced cell growth)
    • Virus (sometimes cytopathic)
  • Cell lines cross-contamination
    • Poor tissue culture techniques
    • Culture of multiple cell lines at one time
    • Accidental mixing of cell lines

30

What are new in-vitro cell line models and why are they used? 

3D cultures 

  • Spheroid cultures 
  • Organoid cultures 

These mimic cells growing in the body because they are 3D - unlike 2D cell line models 

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