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Inserts or plates for your 3D cell culture?

By Frederique Tholozan, PhD
Cells under a fluorescent microscope

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 field1 or you hope it will make your drug discovery pipeline more effective2.

But how should you do it? If you are considering switching to a 3D culture system, we have described how best to achieve  this in the following article.

How do I 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 assay3, and can be teamed up with inserts for more complex models like epithelium-mucosa4 or air-liquid interface set-ups.

  • For genuine 3D cell invasion5 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. <7 days) culture or limited downwards invasion. Use suspended inserts for long-term (i.e. up to several weeks) culture or thorough substrate colonization.

  • Hanging drop and non-adherent micro-well plates are best for generating embryoid bodies6 or tumor spheroids7 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 cells8.

How to go 3D without losing all your 2D hard work

But what if you want to tag some 3D data at the end of an existing 2D study? When you need a 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?

  • Use your 2D set-up as a 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, e.g., 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.
  • Feel free 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. 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!

Download our Alvetex 3D Porous Subtrates Catalog (PDF)
(Includes detailed graphics of plates, inserts, and accessories)

Meet the inventor of Alvetex: Video Interview with Prof Stefan Przyborski

3D Cell Culture Systems available from REPROCELL

REPROCELL has a portfolio of tools for 3D cell culture that provide a physiologically relevant cellular environment for cell culture applications – all available worldwide:

  • Alvetex – our porous polystyrene scaffolds for 3D cell culture (REPROCELL)

  • EZSPHERE – cell culture plastic-ware plates for generating cellular clusters or spheroids that resemble embryonic bodies (Asahi Glass Co., Japan)

  • Koken Atelocollagen – bovine collagen-derived (natural) scaffolds in many configurations, offering in vitro growth capacity and biological compatibility to support animal implantation (Koken, Japan)

Further Reading

  1. Marina Simian, Mina J. Bissell; Organoids: A historical perspective of thinking in three dimensions. J Cell Biol 2 January 2017; 216 (1): 31–40
  2. Langhans SA (2018) Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front. Pharmacol. 9:6
  3. Swift, B., Pfeifer, N. D., & Brouwer, K. L. (2010). Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug metabolism reviews42(3), 446–471 
  4. Marrazzo, P., Maccari, S., Taddei, A., Bevan, L., Telford, J., Soriani, M., & Pezzicoli, A. (2016). 3D Reconstruction of the Human Airway Mucosa In Vitro as an Experimental Model to Study NTHi Infections. PloS one11(4), e0153985
  5. Koehler BC, Scherr AL, Lorenz S, Urbanik T, Kautz N, et al. (2013) Beyond Cell Death – Antiapoptotic Bcl-2 Proteins Regulate Migration and Invasion of Colorectal Cancer Cells In Vitro. PLOS ONE 8(10): e76446
  6. Sato, H., Idiris, A., Miwa, T. et al. Microfabric Vessels for Embryoid Body Formation and Rapid Differentiation of Pluripotent Stem Cells. Sci Rep 6, 31063 (2016)
  7. Zanoni, M., Piccinini, F., Arienti, C. et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep 6, 19103 (2016)
  8. Khan, O.F., Sefton, M.V. Endothelial cell behaviour within a microfluidic mimic of the flow channels of a modular tissue engineered construct. Biomed Microdevices 13, 69–87 (2011)

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