Fundamentals of tissue culture Flashcards
What is a tissue culture?
Cultivation of eukaryotic tissues outside of the organism
- in a growth media with the necessary nutrients
Which nutrients are required in a tissue culture to function in a physiologically normal manner?
- Inorganic salts
- pH (potential of hydrogen, acid/basic)
What is a ‘cell culture’?
Culturing of dissociated cells rather than pieces of tissue (tissue culture)
Why is tissue culture useful?
> Model system for studying basic processes of cell biology
> Clinical applications
How did Kohler and Milstein (1975) clinically apply tissue culture?
Generation of monoclonal antibodies
- production of vaccines as a result of the development of hybridoma cells
How did Steptoe and Edwards (1977) clinically apply tissue culture?
In vitro fertilisation
- through techniques developed for culture of early embryo
Who achieved short term maintenance of tissue outside an organism in 1885?
Wilhelm Roux:
- short term maintenance of neural folds from early chick embryos in a saline solution
- BUT media did not support long term growth
Who achieved the first maintenance of animal cells in a substrate supporting growth AND long-term survival in 1907?
How?
Ross Granville Harrison:
- removed small section of frog embryos and embedded them in blood clots on underside of coverslips to allow microscopic evaluation
- good aseptic technique
-> he observed the outgrowth of nerve cells over several weeks
Who achieved the first long-term cell cultures in 1911?
Carrel and Burrows:
- generated the first ‘cell line’ from embryonic chicken heart
How can cell lines grow indefinitely in culture?
Through genetic mutations and chromosomal abnormalities
Which problems rose from media based upon blood products?
Why?
Problems with reproducibility of results
- due to its poorly defined nature
Who made the first defined liquid media to overcome the problems of blood-based media in 1911?
How?
Margaret Reed Lewis and Warren H. Lewis:
- cultivation of tissues from chick embryos in simple defined liquid media
- solutions of NaCI, CaCI2, KC1 and NaHCO3
In what kind of serum are cell types typically grown with nowadays?
Media containing serum
- e.g. human fibroblasts
Who first dissociated tissues into individual cells for culture in 1916?
How?
Francis Peyton Rous and F.S. Jones:
- enzymatic dissociation
- use of proteolytic enzyme trypsin
How do most cell types - with the exception of blood cells - grow?
Attached to an extracellular matrix (ECM)
What is an extracellular matrix (ECM) composed of?
A complex mixture of polysaccharides and proteins
- e.g. collagens and laminin
How do tissue cultures support attachment and normal functioning of many types of adherent cells?
> Tissue culture vessels are coated with purified or unpurified components of the ECM
> Binding of cell adhesion molecules to components of the ECM needs to be disrupted to detach cells without causing cell death
> Use of trypsin allows the passaging (re-plating) of cells grown attached to a substrate
- essential for dividing cells to grow and occupy all space in a cell culture vessel
What happens to dividing cells when they no longer have room to grow in a cell culture vessel?
They undergo contact inhibition:
- stops the cells from dividing further
- can alter characteristics of the examined cells
What is the current use of trypsin?
Still used to enzymatically dissociate tissues into single cells
What is the limitation of trypsin?
For the passaging of cells, trypsin can cause a degree of cell death
What are the alternative methods to trypsin?
What do they tend to result in?
> Gentler enzymes such as Accutase
Non-enzymatic methods such as EDTA solutions (ethylenediamine tetraacetic acid)
=> reduced cell death
How do non-enzymatic methods work for the passaging of cells?
They chelate ions (e.g. Ca2+) that are essential for the function of cell adhesion molecules
What are chelating agents?
Organic compounds capable of linking together metal ions to form complex ring-like structures called chelates
What is the passaging of cells?
Passaging, or subculturing of cells:
- common procedure wherein cells from a given culture are divided into new cultures
- and fed with fresh media to facilitate further expansion
What are the 4 steps to freeze and thaw cells?
- Cells are detached using same methods used for passaging cells; and re-suspended in a solution with cyroprotectant (e.g. DMSO - dimethyl sulfoxide)
- Cells are frozen down in -80°C freezer
- at rate of 1°C/minute using a vessel filled with isopropanol - Cells are transferred to a liquid nitrogen vessel to be stored indefinitely
- Cells are revived by rapidly thawing in a 37°C water-bath
- minimises ice crystal formation before replacing in growth media
What is the point of using a cryoprotectant in the solution where detached cells are re-suspended in?
Reduces formation of ice crystals, which would cause cell death
What are the problems created from Ross Granville Harrison’s tissue culture method - ‘hanging drop technique’?
> Cells in the blood clots were difficult to view under the microscope
Are unable to grow to a larger size
How did Alexis Carrel and Lillian E. Baker address in 1923 the problems of Harrison’s ‘hanging drop technique’?
Developed new vessel for tissue culture: the Carrel Flask
- angled neck to prevent airborne particles from settling into the flask when open
- allows for sterilisation with a flame, further reducing the risk of airborne contaminants infecting the culture
How are most modern tissue culture vessels made?
- Made of plastic
- sterile
- intended for single use
-> reduces the risk of microbial contamination AND cross-contamination
In what is most tissue culture performed nowadays?
In plates or flasks, which come in varying sizes
- plates range from single dishes up to 15cm diameter, to plates with 384 wells
- flasks typically range in size from 25 to 175cmsq, often with vented lids
What is the function of vented lids in flasks?
To prevent airborne particles entering, whilst allowing the free exchange of gasses
What was the effect of the widespread use of the antibiotics, penicillin and streptomycin in the 1940s onwards?
Reduced the problem of microbial contamination of cultures
What are antibiotics, penicillin and streptomycin ineffective to?
Against a certain common strains of bacteria
- e.g. mycoplasma
What are mycoplasma?
> Very small (<1 micron in length)
> Can be a significant problem in long-term culture
How to fight against mycoplasma?
> Some antibiotics are effective against mycoplasmas
- e.g. Citoprofloxacin
> Best practice is to prevent contamination by employing an aseptic technique
What do biological safety cabinets represent historically?
One of the most important developments in improving aseptic techniques
For what purpose were class I biological cabinets developed in 1909?
Preparation of tuberculin for mycobacterium tuberculosis
What are the characteristics of class I biological cabinets?
> Protect both the user and the environment from the sample, from hazardous microbes
> Does not protect the sample from airborne particles
For what purpose were class II biological cabinets developed in the 1960s?
Manipulation of biological materials (e.g. eukaryotic cells)
- to subsequently be grown in culture for extended periods of time without microbial contamination
What are the characteristics of class II biological cabinets?
> Protect sample from outside contamination
> Relies on continuous uniform flow of clean filtered air travelling down over sample
What are class II biological cabinets used for? Why?
Most routine tissue culture
- they’re very effective at reducing microbial contamination from airborne particles when manipulating the sample
For what purpose were class III biological cabinets developed?
For samples requiring greater level of confinement than class I or II cabinets
What are the characteristics of class III biological cabinets?
Completely encloses the sample, which can only be accessed through the gloves integrated into the cabinet
What are the characteristics of tissue culture incubators?
Maintain critical parameters to allow optimal growth and survival:
- constant levels of temperature, humidity, CO2 and O2
- specific incubators can reduce oxygen levels with displacement by nitrogen, for cell types that grow better under low oxygen conditions
What is the atmosphere required for most media buffers in use for tissue culture?
Atmosphere of 5% CO2 to maintain a physiological pH
Who derived the first strains of human fibroblasts (WI-38) in 1961?
Leonard Hayflick and Paul Moorhead
What is the distinction between primary cells, cell lines and cell strains made by Leonard Hayflick and Paul Moorhead in 1961?
> Primary cells are derived from normal tissue and grown without passaging
> Cell strains are derived from primary cells which have limited capacity for growth and division, but retain a normal karyotype
> Cell lines have the capacity to grow indefinitely, AND have abnormal karyotypes invariably
What is a karyotype?
The size, shape, and number of chromosomes in a cell
Who developed immortal human cell lines in 1951?
How?
George Otto Gey
- cultured cells from Henrietta Lacks, who had cervical cancer
- cells derived from the cervical tumour could grow and divide indefinitely
=> HeLa cells
What was, and is the use of HeLa cells?
> Aided the development of the first Polio vaccine
> Still being used for research today
What did Martin Evans achieve regarding embryonic stem cells in 1982?
Established cultures of cells derived from mouse blastocysts
- can in principle generate any cell type of the body in a cell culture dish
Who first generated human embryonic stem cells from human blastocysts in 1998?
How?
Jamie Thomson:
- allowed for the generation of inaccessible cell types (e.g. neurons) in large numbers for the first time
- ethical issues: requires destruction of human embryos
Who developed induced pluripotent stem cells (iPSCs) in 2006-2007?
Sinya Yamanaka:
- directly generated ES cells by directly manipulating fibroblasts in a ‘reprogramming’ process
- produced iPSCs
- used to study genetic diseases and inaccessible cell types
- no ethical issues as those surrounding embryonic stem cells
What are the 4 Yamanaka factors of pluripotency, highly expressed in embryonic stem cells?
- Oct3/4
- Sox2
- Klf4
- c-Myc
What are neural ‘rosettes’?
What are they composed of?
> Considered as 2D-cell culture model of cortical neural tube development
> Composed of radial glial cells, which in turn generate intermediate progenitors, which in turn generate neurons
What is the prospect of using iPSCs?
Personalised regenerative medicine
What is the process of regenerative medicine using iPSCs?
Human biopsy -> somatic cell -> reprogramming into iPSCs
- differentiation: neurons, thymus epithelial cells, hematopoietic cells, B-cells
- transplantation
What is the major advantage of regenerative medicine through iPSCs?
Transplanted cells will be genetically identical to the biopsied individual
- eliminating the risk of rejection or the use of immunosuppressive drugs
