Cell Culture Techniques Flashcards
What is a cell/tissue culture?
Laboratory method (in vitro) by which cells are grown under controlled conditions outside their natural environment.
What are some advantages of cell cultures?
- Control physiochemical environment and physiological conditions
- Control micro-environment - Cells easily identified and visualised (staining and imaging techniques)
- Cells can be stored in liquid nitrogen for long periods
- Cells easily quantified
- Reduces use of animals in experiments
- Cheaper to maintain than growing mice
Give examples of physiochemical controls.
pH, temperature, osmolarity
Give examples of physiological factors
matrix, cell to cell interaction and cell substrate attachment
What is cryopreservation?
Storing cells in liquid nitrogen
What 2 types of cells can be cultured?
Primary tissue cells or immortalised cell lines
What are primary tissue cells?
Cells taken directly from patients/tissues and cultured in vitro to stimulate body environment
What are the characteristics of primary tissue cells?
- useful if directly from patient tissue - personalised medicine
- finite lifespan but retains cell identity
- cells carry out normal functions (as if still in tissue)
- varied donor (not just from one person)
What is immortalised cell lines?
Single donor cell line that has infinite lifespan
Advantages and disadvantages to immortalised cell lines
- infinite lifespan so can maintain for long but loses cell specificity
- from vial (not patient) with many mutations and clonal selections
- single donor studied repeatedly
What 2 ways can primary tissue cells be isolated by (how to extract cells)?
- Cells allowed to migrate out of explant naturally (only in some cells)
- Mechanical (biopsy) and/or enzymatic dissociation (purification via cell surface markers)
Give examples of mechanical dissociation
Mincing, sieving, pipetting
Give examples of enzymatic dissociation
Trypsin, collagenase, hyaluridanse, protease, DNAase
What is an explant
Transfer living cells/tissues to nutrient medium
Which primary tissue cell does not need to be disaggregated? and how is it isolated?
Haematopoietic cells - they are already individual cells circulating in blood.
Density centrifugation (specific cells separated into layers) followed by either immuno purification or FACS so cells of interest is separated
What are the disadvantages of primary tissue cells?
- Patient variation = difficult to reproduce results
- Limited number and high cost
- Finite lifespan and hard to maintain (due to specific medium)
- Difficult molecular manipulation (e.g. CRISPR to modify cells)
- Phenotypic instability (diff characteristics)
- Susceptible / variable contamination
Why might immortalised cell lines be better than primary tissue cells?
Good reproducibility Good model for basic science Phenotypically stable Easy to grow Limitless - available
Which 2 ways are “immortalised” cell lines are produced?
- Isolated from cancerous tissues (HeLa cells)
- Immortalisation of healthy primary cultures through genetic manipulation (transfection with either viral oncogenes that inhibit tumour suppressor genes or with telomerase gene introduction)
How does genetic manipulation produce cell lines?
Target processes that regulate cellular growth and ageing including inhibiting p53 and pRb and / or introducing telomerase
What does telomerase do?
Elongates telomere ends protecting ends from deterioration/fusion during DNA replication
What does p53 and pRb do?
Tumour suppressor proteins (from tumour suppressor genes)
p53 - control cell division and cell death
pRb - tumour suppressor protein that inhibits cell cycle progression until time to division
How do tumour suppressor genes help produce cell lines?
Inhibit function of tumour suppressor genes proteins such as p53 and pRb using viral oncoproteins e.g. SV40 and HPV
How does SV40 allow immortalised cell line productions?
SV40 is a virus with T antigen (the oncoprotein). T antigen interacts with p53 and pRb causing increased growth WITHOUT LOSS OF FUNCTIONS OF THOSE PROTEINS
How does HPV allow immortalised cell line production?
E6 oncoprotein in HPV targets p53 for degradation.
E7 oncoprotein in HPV binds to pRb inactivating it.
How is telomerase introduced to immortalise cell lines?
Telomerase gene introduced to target primary cell
Is telomerase enough to immortalise cell lines? explain
No, some cells may also need inactivation of pRb/p53 for immortalisation.
E6/E7 and telomerase transformation result in cell lines with different phenotypes.
What is the mechanism for introducing the telomerase gene into cell lines?
Plasmid containing gene for selection e.g. neomycin resistance is fused with (transfection) telomerase gene.
Culture and add selection pressure to primary cells (neomycin) so only cells with telomerase vector and selection gene survive and these colonies are selected.
What 3D cell cultures are there?
3D spheroid cultures from cell lines
3D organoid cultures from primary cultures
How are 3D cultures made?
Artificially created environment allows cells to grow and interact in all 3 dimensions
Explain advantages and disadvantages of 2D cultures
Explain advantages and disadvantages of 3D cultures
Describe spheroids
Describe organoids
Explain 1 use of organoids
What is transfection?
Process in which foreign DNA is introduced into EUKARYOTIC cell through NON VIRAL METHODS with both chemical and physical methods in labs. Viral methods also exist.
e.g. plasmid example where we introduce telomerase gene
Give chemical examples of cell transfection
Lipofection
Give physical examples of cell transfection
Electroporation
Nucleofaction
What is lipofection? - explain steps
Method of cell transfection by introducing DNA through liposomes.
Liposomes -ve charge and plasmids +ve charge so attraction.
Liposomes made of phospholipid bilayer so can interact with cell membrane. Liposomes taken up by endocytosis,
Plasmid release from endosome and transported to nucleus and expresses protein of interest
What is a medical use of lipofection?
Liposomes used as drug carriers drug delivery for both hydrophilic and hydrophobic drugs.
- delivered to cell of interest by attaching specific antigen to surface of liposome that will only recognise cell/organ of interest
What is electroporation?
Electric fields applied to cells of interest which increase permeability forming pores that DNA (plasmid or drugs) can enter cell through. Pores then reseal.
What affected pore sealing in electroporation?
Temperature effects rate of pore sealing.
What is nucelofaction? and why might it be used
Combination of electroporation and lipofection but is still a physical method.
- increased efficiency esp in non dividing cells
- if not known whether lipofection or electroporation will work.
What is viral transduction/infection?
Another method of cell transfection that isnt chemical or physical, its viral.
e.g. retrovirus, adenocirus and lentivirus
What are in the advantages and disadvantages of viral transduction cell transfection?
Exploits mechanism of viral infectio - highly efficient
Target cell need to express viral receptor to work
Biosafety
What is the use of 3D cultures?
Resemble in vivo cell, tissue and organ environments. Important in disease remodelling, drug screening and tissue regeneration.
