Lecture 3&4: 2D and 3D Tissues Flashcards
What could we use clinical samples to find out?
Disease onset or progression
Prognosis/diagnosis
How to make it better
Why do we need clinical samples?
To see:
Impact of a foreign substance on the body, causes destabilisation of cell homeostasis, toxic impact of a substance.
What is meant by ‘toxicology’?
The risk posed by a substance is a function of its potential to cause harm or its hazard, and the amount of substance a biological system is exposed.
Equation for risk
risk = hazard x exposure
what is meant by the term ‘hazard’?
something that is dangerous and likely to cause damage
What is meant by the term ‘risk’?
probability of an outcome having a negative effect
What are some types of toxicology?
chemical
forensic
regulatory
occupational
What is chemical toxicology?
effects of chemicals including drugs, pollutants, industrial chemicals, etc. on living organisms
What is forensic toxicology?
detection, identification, and interpretation of chemicals in biological specimens in the context of legal investigations – to determine cause of death etc. and identify substances contributing to the event
What is regulatory toxicology?
evaluating the safety and potential health risks of chemicals, products, etc. to develop regulations that protect public health and the environment
What is occupational toxicology?
harmful effects of chemicals, substances and env. hazards in the workplace
How can we assessing the toxicity of a substance?
in vitro
in vivo
ex vivo
Advantages of animal studies?
Improvements in human health
Genetically similar to humans (e.g. mice)
Disadvantages of animal studies
Expensive
Ethical issues
Humans not physiologically or genetically similar to animals (only a few similarities).
What is missed when we lose in vivo?
Alternative models (to animal models) require further improvement, relevance and validation in order to promote their development and use across disciplines.
Physiological and anatomical relevance (i.e. cell vs whole body interactions/effects)
Physiological relevance e.g. dynamic flow (air and liquid), breathing patterns, chemotaxis, mechanical stress
Heterogeneity of exposure to the whole biological system
advantages of in vitro methods
- Tight control of chemical and physical environment
- Reduced cost
- Higher throughput
- Reduced animal use
- Reduced need for specialist training
- No requirement to submit animal protocols to governing body
Disadvantages of in vitro methods
- Generally require the use of FBS (foetal bovine serum)
- Fail to replicate the conditions of cells in an organism
What is the 3Rs principle, and why are they essential?
- Framework for Humane Animal Research
- Integrated in national an international legislation regulating the use of animals in scientific procedures
- Funders look for evidence of 3Rs
- OECD guidelines on good in vitro methods
- Minimal reporting essentials support 3Rs
What are the 3Rs?
replacement
reduction
refinement
define replacement
Avoiding or replacing the use of animals in areas where they otherwise would have been used
define reduction
minding the number of animals used consistent with scientific aims
define refinement
minimising the pain, suffering, distress or lasting harm that research animals might experience
Why is refinement important?
- Example: certain methods of handling can cause mice stress or lasting harm which may influence their behaviour and physiology, and possibly lead to (unexplained) variability within/between studies.
- Beneficial for experimental results and animal welfare.
Explain monoculture systems (2D) as a reduction approach
2D cell culture = common approach
o Cells grown as a monolayer, attached to plastic (most cell types, e.g., epithelial cells), or grown in suspension (e.g. monocytes)
o OECD agreed and regulated for toxicology testing
o Cell lines or primary cells (immortalised, transformed)
advantages of monoculture systems (2D) as a reduction approach
o Well-established, extensive literature
o Inexpensive
o Easy to use and train researchers
disadvantages of monoculture systems (2D) as a reduction approach
o Simplistic system of human tissue
o No predictivity
Explain the general concept of multi-cellular systems (2D and 3D) as a reduction approach
- Multiple cell types in one culture system
- Enables cell-cell interactions but may lack a true 3D microarchitecture
Give examples of 3D systems
spheroids
3D reconstructed human skin micronucleus assay (RSMN)
organ on a chip
Explain spheroids as a 3D system
Small clusters of cells (e.g. 100-1000μm diameter)
Hanging drop technology (eliminates the need for cell contact with plastic/artificial matrix)
Explain 3D reconstructed human skin micronucleus assay (RSMN) as a 3D system
In vitro genotoxicity assay assessing potential of test items to cause genotoxicity in the form of chromosomal damage (clastogenecity and aneugenicity).
EpiDerm skin model (MatTek, Ashland, USA) (normal human epidermal keratinocytes derived from neonatal foreskin tissue on specifically prepared tissue culture inserts.
Predict potential of substance that are primarily associated with dermal exposure to cause DNA damage.
Explain organ-on-a-chip as a 3D system
Microfluidic cell culture device created with microchip manufacturing methods
Continuously perfused chambers inhabited by living cells arranged to stimulate tissue and organ-level physiology and organ-level function (e.g. circulatory system).
Still new (too many variables still unknown and inconsistent)
What is the aim of in silico toxicology?
to have predictive models that can define the risk of any substance
Explain how in silico toxicology can be used to move towards ‘replacement’
- Computer-based modelling of nanomaterial-cell interactions
- Designed to predict lab-based responses (a complement to in vitro and in vivo)
- May predict biological mode of action
- Fast, often free and 3Rs-compatible but requires large datasets – currently lacking in the nano field
- QSAR or QNAR (quantitative (nano)structure-activity relationships) – knowledge of current agents predict effects of new agents with similar chemical structure
