Cell Culture Techniques Flashcards
what is cell/tissue culture?
Lab method (in vitro). Cells are grown under controlled conditions outside their natural environment
HeLa cells were used in:
- To develop Polio Virus vaccine
- To discover 46 chrs in human somatic cells
- To develop IVF
Characteristics of Primary Cell Cultures
-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)
What are the methods of isolation?
1.Cells migrate out of an explant
Explant-
Transfer cells from animal to a nutrient medium
Cells isolate spontaneously - no disruption needed
- 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
Describe how we can isolate specific cell populations from the blood?
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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How does blood separate following centrifugation?
layers
(The least dense) Plasma PMBCs Density Gradient Medium Granulocytes , Erythrocytes (The most dense )
What is the buffy coat ?
Buffy coat:
- Fraction of an anticoagulated blood sample.
- Contains most WBCs + platelets following density gradient centrifugation.
Describe Immuno-purification
- Antibody-coated magnetic beads
- Mixed with heterogeneous cells
- Isolation of antigen-expressing cells
Describe Fluorescence Activated Cell Sorter
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.
Give examples of non-haematopoietic primary cells
Primary Cell = Cells taken directly from living tissue (e.g. biopsy material) + grown in vitro.
Liver Endothelial Muscle Skin Nerves Fibroblasts
Give examples of haematopoietic cells
Stem, progenitor cells T-lymphocytes, B-lymphocytes Monocyte, Macrophages Osteoblasts Dendritic cells Neutrophils Erythrocytes Megakaryocytes, Platelets
Outline the extraction of stem cells
Stem cells can be extracted from:
- Bone marrow aspirate
- Umbilical cord
- Fat
- Mobilised peripheral blood
- Embryonic tissue
- iPSCs
Disadvantages of Primary Cells
- Inter-patient variation
- Limited
- Finite lifespan
- Difficult molecular manipulation
- Phenotypic instability
- Aberrant expression of some genes (Abnormal expression)
- Variable contamination
What are cell line cultures?
- 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
Why would we need to use cell lines ?
- 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
What are the methods of cell line production?
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
Describe production of cell lines through genetic manipulation
To generate cell lines, we target processes that regulate cellular growth + ageing
How is a cell line produced?
- 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
Cells that propagate indefinitely =
tumour cells
Tumour suppressor genes encode …….
- 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
Telomerase =
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)
What do p53 and Rb do?
p53 and Retinoblastoma protein = tumour suppressor genes
-Highly regulate apoptosis
How are Rb and p53 manipulated in order to produce cell lines?
To produce cell lines, inhibit tumour suppressor proteins P53 and RB protein = inhibit apoptosis-activating mechanisms
Which 3 targets are manipulated to make immortal cell lines?
- p53
- Rb protein
- telomerase
inhibit p53 + Rb
OR
activate telomerase
A 3D cell culture is ………. created
3D cell culture = Artificially-created environment. Cells grow/interact with their surroundings in all three dimensions.
2D cell culture
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
3D cell culture
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
Growing cells in 2D means you lose …………
Growing cells in 3D retains ……….
the original phenotype + functional characteristics of cells
Some oncoviruses inhibit which proteins?
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
Which component of telomerase in inactive in somatic cells?
+ How is this introduced into the cell?
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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