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[MBD] NoNa > Cell Culture Techniques > Flashcards

Cell Culture Techniques Flashcards

1
Q

what is cell/tissue culture?

A

Lab method (in vitro). Cells are grown under controlled conditions outside their natural environment

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2
Q

HeLa cells were used in:

A
  • To develop Polio Virus vaccine
  • To discover 46 chrs in human somatic cells
  • To develop IVF
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3
Q

Characteristics of Primary Cell Cultures

A

-Cells are derived directly from patient tissues - unmodified
-They have a finite lifespan (not immortalised) - 6/7 divisions
-Cultured in vitro, Mimics in vivo conditions (body environment)
-They are initially heterogenous (tissue contains different cell types)
-Cells divide and/or differentiate – stem cells present
-Normal functions
-Good for personalised medicine -They come from the patient
(We can test drugs in vitro to see the reactions on the cells)

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4
Q

What are the methods of isolation?

A

1.Cells migrate out of an explant
Explant-
Transfer cells from animal to a nutrient medium
Cells isolate spontaneously - no disruption needed

  1. Mechanical/Enzymatic dissociation
    - Mechanical (Mincing, Sieving, Pipetting)
    - Enzymatic dissociation (Trypsin, Collagenase, Hyaluronidase, Protease, DNAase)

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

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5
Q

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

A

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

Depending on density medium used we can isolate populations.
E.g. ficcol

  • Centrifuge blood.
  • Higher density blood cells (granulocytes + erythrocytes) sediment through density gradient medium (ficcol) to bottom of tube.
  • Less dense mononuclear cells (PMBCs) + leukocytes remain at plasma-density gradient medium interface.
  • Alternative methods are more accurate - Immunopurification (antibodies) , FACS (antibodies, size/charge/polarity).

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6
Q

How does blood separate following centrifugation?

layers

A 
(The least dense)
Plasma
PMBCs
Density Gradient Medium
Granulocytes , Erythrocytes  (The most dense )
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7
Q

What is the buffy coat ?

A

Buffy coat:

  • Fraction of an anticoagulated blood sample.
  • Contains most WBCs + platelets following density gradient centrifugation.
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8
Q

Describe Immuno-purification

A
  1. Antibody-coated magnetic beads
  2. Mixed with heterogeneous cells
  3. Isolation of antigen-expressing cells
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9
Q

Describe Fluorescence Activated Cell Sorter

A

Specialized type of Flow Cytometry.

Method for sorting a heterogeneous mixture of biological cells into >=2 containers, 1 cell at a time, based on the specific light scattering and fluorescent characteristics of each cell.

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10
Q

Give examples of non-haematopoietic primary cells

A

Primary Cell = Cells taken directly from living tissue (e.g. biopsy material) + grown in vitro.

Liver 
Endothelial 
Muscle
Skin
Nerves
Fibroblasts
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11
Q

Give examples of haematopoietic cells

A 
Stem, progenitor cells 
T-lymphocytes, B-lymphocytes
Monocyte, Macrophages
Osteoblasts
Dendritic cells 
Neutrophils
Erythrocytes
Megakaryocytes, Platelets
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12
Q

Outline the extraction of stem cells

A

Stem cells can be extracted from:

  • Bone marrow aspirate
  • Umbilical cord
  • Fat
  • Mobilised peripheral blood
  • Embryonic tissue
  • iPSCs
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13
Q

Disadvantages of Primary Cells

A
  • Inter-patient variation
  • Limited
  • Finite lifespan
  • Difficult molecular manipulation
  • Phenotypic instability
  • Aberrant expression of some genes (Abnormal expression)
  • Variable contamination
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14
Q

What are cell line cultures?

A
  • Immortalised cells
  • Unlimited 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
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15
Q

Why would we need to use cell lines ?

A
  • Primary cells not ideal because they carry aberrant genes = dysfunctional protein expression
  • Can have variable contamination and short lifespan
  • Cannot carry out in vitro analysis using primary cells

So need to produce CELL LINES

  • From healthy/cancerous tissues
  • Spontaneous or genetic manipulation to make them immortal
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16
Q

What are the methods of cell line production?

A

1.Isolated from cancerous tissues(e.g. HeLa cells)
OR
2.Immortalisation of healthy primary cultures (e.g. by genetic manipulation of cellular growth/ageing - activate telomerase/inhibit p53+retinoblastoma)
-Spontaneously from prolonged culture
-Through genetic manipulation

17
Q

Describe production of cell lines through genetic manipulation

A

To generate cell lines, we target processes that regulate cellular growth + ageing

18
Q

How is a cell line produced?

A
  • Immortalise one type of primary tissue cell, then propagate in culture for limited divisions, but more than primary tissue cells
  • Maintain differentiation
  • Cell lines continue growing
19
Q

Cells that propagate indefinitely =

A

tumour cells

20
Q

Tumour suppressor genes encode …….

A
  • Proteins that slow/inhibit progression through a specific cell cycle stage
  • e.g. p53 and retinoblastoma protein are checkpoint control proteins that arrest cell cycle if DNA damage/abnormal chromosomes
21
Q

Telomerase =

A

Telomerase = elongates telomeres, increasing cell lifespan - no deterioration

-During DNA replication, DNA polymerase cannot duplicate the DNA at end of chromosomes = telomeres shorten with each cell division/DNA replication

  • Telomerase is active in some cells – germ cells, adult stem cells, cancers.
  • But not active in somatic cells = somatic cells die when telomeres reach critically short length (Hayflick Limit)

-As cells divide over time, telomeres shorten, and eventually cell division stops → Apoptosis (p53, pRb)

22
Q

What do p53 and Rb do?

A

p53 and Retinoblastoma protein = tumour suppressor genes

-Highly regulate apoptosis

23
Q

How are Rb and p53 manipulated in order to produce cell lines?

A

To produce cell lines, inhibit tumour suppressor proteins P53 and RB protein = inhibit apoptosis-activating mechanisms

24
Q

Which 3 targets are manipulated to make immortal cell lines?

A
  • p53
  • Rb protein
  • telomerase

inhibit p53 + Rb
OR
activate telomerase

25
Q

A 3D cell culture is ………. created

A

3D cell culture = Artificially-created environment. Cells grow/interact with their surroundings in all three dimensions.

26
Q

2D cell culture

A

Advantages:

  • Forced apical-basal polarity
  • High stiffness
  • Limited communication with other cells
  • No diffusion gradients
  • Results not relevant to human physiology

Disadvantages:

  • Simple, well established
  • Affordable
  • Reproducible results
27
Q

3D cell culture

A

Advantages:

  • Cells adhere to each other in all three dimensions
  • No forced polarity
  • Variable stiffness
  • Diffusion gradients of nutrients + waste products
  • More relevant to human physiology

Disadvantages:

  • More complex – require specific matrix, medium, nutrients, hormones
  • Expensive
28
Q

Growing cells in 2D means you lose …………

Growing cells in 3D retains ……….

A

the original phenotype + functional characteristics of cells

29
Q

Some oncoviruses inhibit which proteins?

A

p53 and Rb proteins

Viral oncoproteins target p53 and RB proteins

e. g:
- SV40’s T-antigen binds to p53 binding domain on DNA (not protein)
- SV40’s T-antigen also inactivates RB protein
- E6 targets p53 for degradation , E7 binds + inactivates pRb

30
Q

Which component of telomerase in inactive in somatic cells?

+ How is this introduced into the cell?

A

TERT - Transcriptase protein

-Activate/Introduce into cell via plasmid vector containing a selection marker.
-Neomycin = antibiotic = selection marker = antibiotic resistance marker.
+ sequence encoding telomerase.
-Gene for selection = neomycin + growth-promoting gene = telomerase, are both in a circular plasmid + transfected into primary cell pool isolated from patient tissue.
-Treat cells with antibiotic neomycin = only cells that have been transfected survive bc contain entire vector which contains gene selecting neomycin.
-Only cells that grow contain the vector. As they transcribe + translate their gene for selection (neomycin gene) , they use telomerase gene.
-Select only those that contain telomerase
-Introduce genes in cells
-This immortalises primary tissues, converting them into cell lines

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[MBD] NoNa (28 decks)

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