Q2. What have the authors stated for future works in "Three-dimensional cell culture: the missing link in drug discovery" ?
3D cell culture is an evolving field and requires further research for its optimisation. Ideally, such 3D cultures would include co-culturing with other relevant cells that are present in human tumours and would be further developed to represent in vivo situations in as best a manner as is possible. While the Please cite this article in press as: S. Breslin,, Three-dimensional cell culture: the missing link in generation of 3D culture can be more labour-intensive that 2D culture, the routine incorporation of these multi-cellular spheroids into in vitro drug efficacy and toxicity testing during development will very probably generate more accurate results than the use of monolayer cultures alone, and should better indicate in a timely manner which candidate compounds will/will not have the desired effects on target cells. It is feasible to propose that the routine inclusion of 3D cultures will effectively bridge the gap between in vitro 2D assessment and animal models of disease, fasttracking drug screening and, hopefully, yielding more effective and less toxic drugs as future therapies.
Q3. Why is it important to dismiss compounds that are potentially ineffective or have an unacceptable toxicity profile?
Because of the high costs (typically 1 billion US dollars) in getting new drugs approved and the fact that many oncology drugs fail during clinical testing, especially during phase III – the most expensive phase of clinical development [5,6], it is imperative that compounds that are potentially ineffective or have an unacceptable toxicity profile are dismissed as early in the evaluation process as possible.
Q4. What are the common substrates for cell culture?
Microcarriers can be coated with cell adhesion promoting substrates (e.g. gelatin, collagen) and are generally compatible for use in typical bioreactors for cell culture.
Q5. What is the hypothesis that differences in culture environment can affect how cells will respond to drug treatment?
The hypothesis that differences in culture environment can affect how cells will respond to drug treatment is supported by findings that cells grown as spheroids exhibiting polarised 3D architecture and show difference in resistance to apoptosis and chemotherapeutic drugs (in addition to those mentioned above) when compared to 2D controls [54].
Q6. What is the method for generating 3D spheroids?
A relatively simple method for generating 3D spheroids is to prevent their attachment to the vessel surface by modifying the surface, resulting in forced-floating of cells (Fig. 1a).
Q7. What are the advantages and limitations of the NASA bioreactor?
Other advantages and limitations of this bioreactor are similar to those of the spinner flask; it follows a relatively simple method, enables large-scale production of 3D cultures, and allows for long-term culture of 3D spheroids.
Q8. What is the reason why the 3D spheroid tumour model is relevant for investigating?
This further supports the 3D spheroid tumour models as relevant for investigating effects of drugs intended for use on solid tumours, as hypoxia has many effects in tumour biology, some of which can affect drug responses.
Q9. How many steps would be required to produce 3D spheroids?
For drug screening purposes 3D spheroids would, of course, have to be re-plated into suitable dishes (e.g. 96- or 384-well plates), overall resulting in a number of necessary consecutive steps before drug screening (i.e. 96-well plates for forced-floating to form uniform spheroids; then spinner flasks; then 96- or 384-well plates for drug screening), which adds to the labour involved.
Q10. Why is it necessary to improve in vitro cell-based testing methods?
It is, therefore, necessary to improve in vitro cell-based testing methods for a more informed prediction of drug candidate efficacy and safety, and thereby sieve out poorly functioning compounds while prioritising promising candidates [6].
Q11. What are the advantages and disadvantages of different methods for 3D cell culture?
The authors propose the necessity to incorporate 3D cell methods into drug development for human therapy, based on their ability to mimic tissue-like structures more effectively than 2D cell culture.
Q12. How many spheroids are produced in a 96-well plate?
As these 3D spheroids are typically generated in a 96-well plate, large numbers of morphologically homogenous spheroids are easily produced.
Q13. What is the way to represent in vivo situations?
such 3D cultures would include co-culturing with other relevant cells that are present in human tumours and would be further developed to represent in vivo situations in as best a manner as is possible.
Q14. What is the way to test a drug candidate using cell-based assays?
when drug candidates are being tested using cell-based assays, the culture methods used should mimic the most natural in vivo representative form possible.