3D cell culture. You've heard about it, you've read about it. You're probably daunted by the plethora of techniques available out there, but you want to join the growing number of scientists publishing in the 3D field or you hope it will make your drug discovery pipeline more effective.
But how should you do it? What's the best for your cells?
How to choose the best 3D system? Get back to Biology.
We spend so much time looking at our 2D cells under a microscope, it's easy to forget what they look like in vivo. An epithelial sheet, a neuronal network, a vascular tube, a cancerous mass. Each a distinct form, with its particular function.
So, when planning a novel 3D project, put anatomy and physiology first. It will help you decide what 3D system is best.
- Extra-cellular matrix, from crude animal extracts to fully-defined synthetics, are popular as thinly-coated plates for simple epithelia, e.g. hepatocyte sandwich assay, and can be teamed up with inserts for more complex models like epithelium-mucosa or air-liquid interface set-ups.
- For genuine 3D cell invasion and network formation, thicker ECM gels and porous substrates offer greater Z-axis depth, but highly-porous synthetic substrates are best for endogenous ECM deposition. And don't forget the diffusional limits. Use plates for short-term (i.e. <7days) culture or limited downwards invasion. Use suspended inserts for long-term (i.e. up to several weeks) culture or thorough substrate colonisation.
- Hanging drop and non-adherent microwell plates are best for generating embryoid bodies or tumour spheroids and scale up well for high-throughput toxicity assays.
- Perfusion and microfluidic systems recapitulate not only the physical geometry, but also the mechanical forces important to the performance of some cell types, eg. vascular endothelial cells.
Here's a short video about various 3D cell culture techniques:
But what if you want to tag some 3D data at the end of an existing 2D study? When you need straightforward comparison with your 2D set-up? Or minimal disruption to your finely-tuned workflow? Or fast data to satisfy Reviewer #3 before the re-submission deadline?
How to go 3D without losing all your 2D hard work
You already have an assay. It works great. But it is in 2D and you feel the pressure to move it into 3D.
- Use your 2D set-up as template. What format do you use in 2D? If it isn't broken, don't fix it. And save yourself some scale-up/scale-down calculations.
- Some 3D systems are readily compatible with 2D formats. ECM coatings, after all, are simply poured onto a 2D surface. Porous substrates made as discs will fit standard plate sizes, or be sold as suspended inserts in sizes matching that of well-plate bottoms.
- Remember the effect of diffusional limits. Use plates for short-term (i.e. <7 days) and inserts for long-term (i.e. up to several weeks). For short-term assays, eg. a 24h drug toxicity test, consider growing your cells in 3D for a few passages before testing, so your cells will have had plenty of time to adapt to 3D but your seeding density and culture time during the actual testing can stay similar to your 2D set-up.
- Adjust your protocol where it matters. Good seeding technique can be crucial. Incomplete cell attachment in inserts can mean your cells could end up at the bottom of your well, not on top of your insert.
Here's a short video demonstrating cell seeding on Alvetex:
- Don't be afraid to contact Tech Support. They've spent years perfecting the best ways to handle their 3D brand product. A quick Tech call on study design might well save you a much longer call to troubleshoot your failed experiment. Here at REPROCELL, we have almost 60 protocols and more than 20 application notes and white papers on our website to consult and download.
It can be hard to strike the right balance between physiological relevance and experimental practicality. Hang on in there and Happy Experimenting!