Cell culture techniques Flashcards
Name some characteristics of Primary Cell Cultures
Characteristics of primary cell cultures include:
→ Cells derived directly from tissues (unmodified)
→ Finite lifespan (~6-7 divisions) - then they die
→ They can be grown in 2D or 3D
→ Cells divide and/or differentiate because there are some stem cells present
→ Cells carry out normal functions
→ Good for personalised medicine - e.g. biopsy –> grow patient cells in culture –> experiment on it to see reaction to treatments –> extrapolate findings to patient
What are examples of methods of isolation of cells? What is the exception?
- Cells allowed to migrate out of an explant - in vivo
→ e.g. chondrocytes naturally migrate away from cartilage explant whilst still maintaining morphological characteristics - natural way to separate cells - Mechanical - can’t happen naturally (mincing, sieving, pipetting) or/and enzymatic dissociation of cells in the tissue (trypsin, collagenase, hyaluronidase, protease, DNAase)
→ Different cell surface markers used to carry this out such as CD31
Exception: Haemopoietic cells
→ Do not need to be disaggregated because they already are, as individual cells circulating in blood
→ In which case, we can use density centrifugation, Immuno-purification (uses antibodies) or Fluorescence activated cell sorter (FACS) (uses fluorescent markers) to isolate specific cells
What are some examples of non-haematopoietic and haematopoietic primary cells?
Non-haematopoietic:
→ Liver
→ Endothelial cells
→ Muscle
→ Skin
→ Nerves
→ Fibroblasts
→ Prostate
Haematopoietic:
→ Stem, progenitor cells
→ T and B cells
→ Monocytes, macrophages
→ Osteoblasts
→ Dendritic cells
→ Neutrophils
→ Erythrocytes
→ Megakaryocytes, Platelets
What are some disadvantages of primary cells?
→ Inter-patient variation - difficult to reproduce results
→ Limited number (small amount at high cost)
→ Finite lifespan (ie don’t survive for longer than a week) - requires expensive medium e.g. hormones, cytokines etc
→ Difficult molecular manipulation - hard to CRISPR because division is limited
→ Phenotypic instability
→ Aberrant expression of some genes
→ Variable contamination - v. sensitive to contamination
What are some characteristics of Cell line cultures?
→ Immortalised cells of JUST ONE type of primary tissue cells
→ Less limited number of cell divisions (~30) or unlimited (whereas much more limited with primary cells)
→ They can be grown in 2D or 3D
→ Phenotypically stable, defined population Limitless availability
→ Easy to grow
→ Good reproducibility
→ Good model for basic science
What are some methods of production for cell line cultures?
- Isolated from cancerous tissues (e.g. HeLa cells)
- Immortalisation of healthy primary cultures usually through genetic manipulation:
a) Spontaneously from prolonged culture: multiple ill-defined mutations transformed phenotype
b) Through genetic manipulation:
→ Artificial transformation of healthy primary cells
→ Production through genetic manipulation
→ To generate cell lines we target processes that regulate cellular growth and ageing - As cells divide over time, telomeres shorten, and eventually cell division stops
→ Apoptosis (p53, pRb)
How can we inhibit the function of tumour suppressor proteins, or introduce telomerase in order to alter a cell’s capability for its finite number of divisions?
→ Taking advantage of viral ‘oncoproteins’
→ Oncoproteins are proteins encoded by oncogenes which are involved in the regulation or synthesis of proteins linked to cancer tumour cell growth
→ Current oncoprotein research focuses on antibodies that can directly target the oncoproteins inside cancer cells and suppress aggressive cancer growth
eg virus: Human Papilloma Virus (HPV) viral oncoprotein: → E6/E7 cellular targets: p53 pRb HPV’s E6 targets p53 for degradation, and E7 binds to pRb inactivating it Cell lines made using E6/ E7 oncoproteins are believed to maintain a differentiated phenotype
→ The telomerase gene can also be introduced into a target primary cell
→ Some cells need both introduction of the telomerase gene and inactivation of the pRb/p53 for “immortalisation”
basically nicola’s voicenote made sense but the above doesnt:
so looking at the second part of ‘finite’ so in a primary cell, there is a finite number that the cell can divide and if it divides more than that number, it will die-apoptosis and that number is 50(hayflicks constant!?) but that is what we want to prevent! we want to turn primary cells into cell lines so we can use as a culture add drugs etc becuase we can’t directly do that to primary cells as they will die (ie adding stuff to see how they respond etc),
we therefore need to do 2 things:
1. remove tumour supressors from primary cells
2.induce telomerase (you want to integrate telomerase into your primary cells) (which affects telomeres by adding a specific sequence to the end of the ) chromosome) but essentilaly telomerase is causing the cancer eg to be prominent- it allows the cancer to have that infinite number of divisions, and causes the tumour supressors to not supress the tumour.
the break and the accelerator analogy:
the break is the tumour suppressor- so you get rid of that (foot now on pedal) but you want the telomerase but essentially theres no stop to it- uncontrolled division
What conditions and requirements are required for growth in cultures?
→ Handled under aseptic conditions
→ Grown on tissue culture treated plastic flasks/dishes
→ Maintained in a warm (37°C) humidified atmosphere (5% CO2)
→ Ideal supplemented medium that needs replacing every 2/3 days! if medium is yellow=acidic, if medium is tomato red= neutral, if medium is red to purple in colour= basic
What are types of cell culture contamination that are a potential hazard?
→ 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)
What can cause cell line cross contamination?
→ Poor tissue culture technique
→ Culture of multiple cell lines at one time
→ Accidental mixing of cell lines
What are some differences between organoid and spheroid 3D cultures?
Cells are grown in suspension, which bunch together and form a bigger blob of cells (known as organoids or spheroids) organoids and spheroids are helpful for modelling specific organs in the body
Organoid: derived from stem cells multiple cell lineages long term culture eg so you can take a biopsy/sample of the tumour and grow organoid samples so you can test different drugs etc before giving it to the patient!
Spheroid: derived from cell line monoculture represent single/partial tissue components difficult to maintain long term
What are some advantages of organoids?
Advantages:
→ Gene expression as in vivo (87% phenotype and genotype similarity)
→ Cells-cell communication re-established
→ Cells are orientated in same ways as tissue
→ Ideal platform for individualised therapeutic screening
What are some limitations of organoids?
Limitations:
- Limited amount of tissue in some cases (e.g. prostate)
- Organoids in the same culture are heterogeneous
- Absence of immune cells in culture system
- Unable to mimic in vivo growth factor/signalling gradients
What is transfection?
Transfection is the process by which foreign DNA is deliberately introduced into a EUKARYOTIC CELL through NON-VIRAL methods including both CHEMICAL and PHYSICAL methods in the lab. e.g. a plasmid, a CRISPR/Cas9 complex
What does lipofection involve? (as a method of transfection)
→ Using cationic lipid transfection systems
→ Lipofection - introduces DNA into cells through liposomes (vesicles which can easily merge with the cell membrane of the eukaryotic cell because liposomes are made with phospholipid bilayers) liposomes have a net positive charge
- Interaction with the cell membrane
- Taken up by endocytosis
- Release from the endosome
- Transport to the nucleus
- Entry to the nucleus inefficient and may need mitosis so cell expresses gene/material we have introduced better used for when material is small so liposomes are used as potential drug delivery carriers
