Ask-the-Expert: 3D Cell Culture Questions | Mimetas

Ask-the-Expert: 3D Cell Culture Questions

Ask-the-Expert: 3D Cell Culture Questions

In this post, the answers to your most common questions about 3D cell culture, presented by Gwenaëlle Rabussier, Early Stage Researcher at MIMETAS.

What is the benefit of 3D cell culture compared to 2D cell culture? Is it easy to use?

As you know, most cell cultures are is done in 2D. You grow cells as a monolayer in a culture flask or dish, typically made of plastic. These systems are easy and low cost but have a lot of limitations. The problem is that 2D cultured cells don’t mimic the natural structures of tissues or tumors because the interactions between cells and with the ECM are not the same as they would be in vivo. So you can have cells with abnormal expression patterns resulting in cells behaving differently from how they would in vivo.

On the other hand, in 3D cell culture systems, the cells attach to one another and to a 3D ECM in a way that is more similar to the in vivo situation. So, you can imagine that cells grown in 3D represent physiological conditions more accurately. They have the same morphology as in vivo. They also behave more similar to in vivo in terms of proliferation, migration, differentiation, and gene expression.  These are all features lacking 2D cell culture.

The biggest advantage of 3D cell culture over 2D cell culture is its ability to better represent in-vivo processes in term of gene expression and response to toxic compounds, allowing for better translation of in vitro-obtained data to in vivo.

Now, are 3D cell cultures easy to use?

Well, that depends. Most systems are complicated. Some systems need tubes and pumps. Some don’t permit to separate the access to apical and basal compartments. In some it’s difficult to collect cells or secreted factors for biochemical assays. Analyzing with microscopy can also be challenging with certain ECM gels.

At MIMETAS, we have developed the OrganoPlate®.

This platform looks like a standard 384-well plate but it integrates 40 to 96 microfluidic chips. Here you can grow your cells in 3D but do much more than you would normally do in a 2D culture.

For instance, you can have intricate co-cultures or create perfused tubules.

In this system, you can easily access cell-conditioned medium on both apical and basal sides of the tissue, perfuse nutrients and oxygen, give mechanical stimuli to the cells, and define concentration gradients of soluble factors. The limit here is only your imagination. With all these features, you can see how it’s much easier to create more physiologically relevant tissue models.

How can I do transport studies in your system?

Once you have made tubules in the OrganoPlate, it’s pretty straightforward to do transport studies.
Let’s see what kind of assays you can do:

  • If you want to study Paracellular flux and transcellular transport, you can assess it indirectly by performing a Barrier Integrity assay in which the lumen of the tubule is perfused with a fluorescent dye and then monitor the movement of this dye through the barrier tissue into the adjacent gel channel  with a standard fluorescent microscope. Another alternative is the TEER measurement which use transepithelial/endothelial electrical resistance to measure the integrity of the tight junction dynamics.  By the way, we have a new product called the OrganoTEER that allows to quickly assess TEER on the OrganoPlate. You can check it in our product page.
  • The OrganoPlate 3-lane enables you to study transport functionality You can add a substrate for a transporter to a tissue, for example in the basal compartment, and then the samples can be taken from the apical compartment by simply pipetting the medium out of the wells of the channel. The content can be assessed using for example mass spectrometry, ELISA, metabolomics, … Besides that, we have set-up several imaging-based transport assays that look at the transport of fluorescent molecules, such as the SGLT-2 transporter, the P-gp transporter, the GLUT-1 transporter and MRP transporter.
  • If you want to check the presence and localization of your transporters, you can also perform immunohistochemistry. The OrganoPlate allows immunofluorescent detection of biomolecules inside the plate. You can measure the light intensity of the tagged antibodies by confocal fluorescence microscopy and quantify it by using image analysis software. 
  • And besides looking at your transporter protein expression, you can also go further and detect and quantify RNA expression in your sample by performing quantitative RT-PCR. The process is really easy, you lyse the cells directly in the OrganoPlate, you take the medium out, pulled together and samples are performed following the standard process.
     

I want to study angiogenesis. Are there benefits in using the OrganoPlate instead of a 2D Assay?

You know that studying angiogenesis in vitro can be pretty tricky. With 2D in vitro models you can study fundamental EC biology such as migration and proliferation. However, since these models lack a 3D environment, the endothelial cells fail to show many of their typical hallmarks. Also, if lumina are formed in these types of models, its often very difficult to perfuse them. Also, using the standard angiogenesis-on-Matrigel assay, the vessels are stable for a short period of time, like 1 or 2 days, giving a short window for experiments and assays.

In the OrganoPlate you can easily study gradient-driven angiogenesis and you can do that in high-throughput. In fact, you can grow 40 identical lumenized and perfusable microvessels. Our protocol has been optimized to trigger angiogenic sprouting that reproduces all the angiogenic events that occur in vivo, such as the differentiation of the endothelial cells into tip, stalk, and phalanx cells and the formation of perfusable lumen. You can create reproducible gradients that you can maintain for up to 7 days, allowing for a much longer window to run assays.

Since gradients and perfusion are two important thing during the initial sprouting and the stabilization phase in angiogenesis, their integration in our platform makes our model uniquely suited to perform physiologically relevant studies on the formation and regression of the microvasculature in vitro.

You can find the protocol for angiogenesis in OrganoPlate here.

How many cells do I need to culture in the OrganoPlate?

This depends on the cell type and whether you culture your cells inside an ECM gel, such as neurons or liver cells, or against an ECM gel to create tubular structures such as endothelial and epithelial tubules.

  • For in-gel seeding, you can usually go from a range of 10.000 cells to 80.000 cells per chip which means that you would need approximately 500.000 cells to 3.200.000 cells to seed a 3-lane OrganoPlate, which doesn’t constitute a lot of biological materials for the modeling of 40 tissues.
  • For against-gel seeding, we usually go for a range between 10.000 to 40.000 cells per chips, which constitute a total of around 500.000 to 1.6 million cells per 3-lane OrganoPlate.

What type of gel do you usually use?

In the OrganoPlate you can use any type of ECM such as natural and synthetical extracellular matrices. Which one to use, depends on the cell type you want to grow and the assays you want to perform. Among these different types of ECM, Type I collagen matrix is the one we commonly use within our lab, as well as Matrigel, Bovine Collagen and Fibronectin. You can find the details on how to prepare collagen-I and Matrigel-base ECMs here.

Conclusions

This was the last question for today. Thank you for your questions. It was good to see that many people are interested in 3D cell culture and in the OrganoPlate. I hope I could inform you enough but should you have any questions, don’t hesitate to send an e-mail to the e-mail address you see in the slide.

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