Technical Webinar

Advanced 3D Cell Culture Models: Your questions answered

Our webinar ‘Advanced 3D Cell Culture Models’ proved very popular and so we were unable to answer all questions in the time available. To further support you on this topic, our speakers have compiled answers to all the questions below.

Stefan Przyborski, PhD,
Professor of Cell Technology,
Durham University, UK.
Jim Cooper,
Cell Biology Applications
Scientist, Public Health England.

Questions & Answers

Could the speaker provide a link or citation to the 'dynamic diffusion' system mentioned?

I attach a slide (PDF 90 KB) with several examples of the dynamic systems that could be used. These range from microfluidics, chamber systems, to larger scale models.

Which cell types have been proven to not being suitable for 3D culture in scaffolds?
Some cell types naturally have orientation and structure such that they are not suitable to grow inside rigid scaffold materials. For example, myotube, tendons and cardiac muscle cells. Simple epithelia are also best grown on a flat substrate to form a monolayer rather then inside a scaffold.

What is a meaningful way to normalize cell density across 2D and 3D? In 2D the cells settle to the bottom in 3D culture they do not. So the cells per unit volume and therefore the density do not correspond.
It is possible to determine cell number indirectly using a variety of approaches. For example, determination of total protein is a useful general indication. A more accurate method is to determine the amount of double stranded DNA in both 2D and 3D cultures. This can be achieved using a PicoGreen® assay.

What is the best system to study the interactions between cell imunne system and endothelial cells?
The transwell model has been used previously for investigating interactions between immune cells and endothelia. It is possible that modern 3D cell cultures could also be adapted to improve this model.

For a 3D cell migration assay into scaffold, can you tell me how you would measure/image the migration?
Imaging in 3D culture is challenging but not impossible.  Confocal microscopy works successfully in 3D culture and provides unique information not achievable in 2D culture.  Cell migration can be determined by the movement of the cells through the 3D technology (scaffold or hyfrogels are possible).  Co-culture of additional cells in 3D can be used to setup cell invasion models - having the cells labelled to help distinguish them is essential for identification and tracking purposes.

Natural molecules for 3D cell culture, which already have growth factors and cytokines present, can lead to significant experiment to experiment variation due to batch variability. If I want do do reasearch and then procede to clincal applications, how can I ensure that I have 'real' results using products with uncontrollled additives?
There is a move towards Xeno-Free reagents - particularly with regard to regenerative medicine. I would suggest that good and accurate measures of cellular phenotype are the key to this. A lot of literature assumes that because the cells are in 3D they are physiologically relevant. You have to understand the in vivo situation and see how 3D culture systems compare. As Stefan said in his conclusion - 3D models are simply models - they don't as yet fully represent the in vivo state.

Can we consider matrigel cultures as genuine 3D culture? Or are cells only growing on matrigel surface? What can we consider be the mininum and maximun height of a 3D culture system?
Matrigel is effectively basal lamina and epithelial cells grown on its surface will be more epithelial like - even though effectively growing in two dimensions. Remember, in vivo, squamous, simple cuboidal, simple columnar and pseudo -stratified epithelia are in essence 'two dimensional' structures. Again, the important thing is to understand the in vivo situation and work with a culture system that best recapitulates it.  Matrigel is also used as a general substitute of 'extracellular matrix'.  In vivo the ECM varies significantly dependent on the cells in the tissue.  Matrigel is a good substitute in many ways since it is a natural product and contains many important factors and proteins.  It is however not perfect, it may not be suitable for the cells of interest and it does vary in its composition.  Matrigel can be used as a film, which is ideal for epithelial, or it can be 'poured' as a thicker layer in which cells can grow in 3D.  It is often used in this way for angiogenesis and ductal studies.

How well do nutrients and oxygen diffuse through hydrogels? For example, is there a maximum size of hydrogel beyond which diffusion will not be as efficient and cells will die? I sometimes notice that cells in the middle of the hydrogel are dead while the ones on the outside are fine.
O2 diffusion and concentration in tissues is not as high as we see in 2D cell culture. O2 gradients of this kind may be intrinsically more "in vivo-like". However, in vivo there is a vascular supply that provided oxygen and removes waste products. Diffusion distances and understirred layers therefore play an important role in 3D culture models. A setup is required where sufficient 3D culture can be achieved and also sufficient diffusion of oxygen etc. can occur. Optimisation of the model is therefore recommended in line with the needs of the experiment.

What would be the best synthetic hydrogel model to mimic brain ECM?
It is important to understand the structure of the brain and the ECM that the neural cells produce. This will various throughout the structure of the CNS. De-cellularised tissue maybe an option, there may also be some specialised hydrogel compositions/products that have been developed specifically to mimick CNS ECM. It is likely that more customised hydrogel products such as this will appear in the future.

Can cells eg neurons migrate within an Alvetex® scaffold?
Neurons can track through many different porous scaffold types including Alvetex.

How are cells seeded in the 3D scaffolds? Are hydrogels always used as a vehicle to seed cells in scaffolds? If yes, how would you obtain an homogeneous distribution of cells along all the scaffold volume, for example in a 1mm depth?
Cell seeding density depends on many factors including the size of the scaffold, proliferation rate of the cells, period of culture, cell motility, and cell type. Hydrogels and scaffolds can be used in combination. Cells can be seeded in suspension onto a hydrogel coated scaffold, the cells will settle onto the surface relatively homogeneously and depending on the cell-ECM interaction, may migrte into the 3D culture.

Has anyone tried the application of flow to any of these 3D systems, are they amenable to this?
Numerous dynamic perfusion systems are available, some of which also incorporate 3D culture models.

Since you mentioned that 3D grown cells can have different transcription and translation, do you think that growing the cells should also happen in 3D? (I'm not working with 3D cultures yet, I assume you grow them in 2D, then for your experiments you put them in your 3D model?) Is this feasible for cells that you can keep for lots of passages?
This is a very good question and Stefan and I have discussed this at some length. There is early work ongoing in attempting to passage cells in '3D' and continually maintain them in a 3D state. This work is currently ongoing at Durham University. It will be interesting to see in cells that have lost function and become de-differentated in continual 2D culture will regain function and phenotype through passaging in the 'correct' 3D system. As we've seen, just because a cell can be grown in 3D in one system does't mean that's the most relevant culture system to recapitulate the in vivo state.

Could scaffolds be used for nasal tissuel culture, including cells from the mucosa but also immune cells under it?

It is feasible to 'build' bespoke and customised models in 3D culture using different cells types and the creation of novel tissue-like constructs.

Would you say that the A549 cells in your research actually shows that it is a functional Type II cell?
No - more like a goblet cell - but still more pulmonary like than 2D monolayers

How was the CYP data in the liver model normalised to the cell number?
CYP data was normalised to total protein concentration. This can also be achieved using double stranded DNA (PicoGreen® assay).

Could you please comment on the use of ex vivo tissue as scaffold for cell culture?
Porous scaffolds have been used to maintain tissue fragments ex vivo. The tissue remains viable on the scaffold, it is extremely porous and enables cells to be fed from underneath. The surface of the scaffold is also 'rough' and this topography helps the tissue piece to adhere to the scaffold.

Is it possible to obtain some cell migration in the synthetic hydrogels?
Yes this is feasible if cells are encouraged to move into a thicker layer of the hydrogel.

I'm interested in creating a multilayer culture similar to the skin. (I need the squamous epithelial layer to differentiate.) What's the advantage of hydrogel versus scaffold?
A scaffold will provide a more robust 'backbone' upon which to build a skin construct. However, a skin model can be developed on a hydrogel-based system and the longer it matures the more ECM and internal natural scaffold is produced to create a more in vivo like system. A combination of scaffold and hydrogel models has advantages to enhance differentiation and increase the establishment of the model.

What do you think of PEG (polyethylene glycol) as hydrogel substrate?

Useful for transplantation studues, biodegradable. Has use in 3D culture, if biodegradation not an issue.

What media is used for culturing spheroids in Ultra-Low Attachment Surface Plates ? Is it the same media as 2D cultures or different?

The same media types can be used.

Would you recommend electrospun scaffolds for visualising cells? What about scaffolds made out of polymers?
It is possible to visualise cells on electrospun fibers, there is evidence for this using fluorescent microscopy. There are many scaffolds made from polymers that have successfully been used for 3D cell culture.

What is the significance of using 3D conditions when culturing suspension cells such as leukemic cells?
Suspension cells naturally reside in a 3D environment. Therefore growing them in a suspension model is most appropriate. Co-culture models with other cells, perhaps adherent cells, may be beneficial to enahnce the complexity of the in vitro micro-envionment.

Is it possible to use MaxGel™ or any other hydrogel matrix to study cutaneous melanoma in vitro and in turn possibly xenograft it onto mice?
The transplantation of in vitro 3D constructs into immunologically suppressed mice is feasible and has been reported.

Could MaxGel™ be used in a transwell set-up for the culturing of lung epithelial cells?

Several different types of hydrogel have been used in combination with transwell setup to create respiratory epithelial models.

Jim- with your A549 work in 2D vs 3D culture, did you see any difference in tight junction structure?
There are some differences between expression of tight junction specific mRNA between 2D and 3D - but the question is good. It is not just the level of expression of these genes but also the spatial organisation and localisation of the proteins in the cell membranes. This can only be seen in microscopic analysis / immunostaining - which is work yet to be done. However previous authors reported increased tight junction expression and localisation in A549 spheroids.

Alvetex is a registered trademark of and manufactured by Reinnervate.
PicoGreen is a registered trademark of Life Technologies
MaxGel is a trademark of Sigma-Aldrich Co. LLC
Matrigel is a TM product owned by BD Biosciences