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Cell cultivation: “You might describe it as gymnastics for cells“
Professor Martin Bastmeyer;
The cells of our body work within a three-dimensional environment. But under laboratory conditions, experiments are often still performed in Petri dishes, which provide a 2D environment.
Now a team surrounding Professor Martin Bastmeyer of the DFG Center for Functional Nanostructures in Karlsruhe, Germany was successful in cultivating cells specifically on three-dimensional structures. COMPAMED.de followed-up with the Professor on exactly how this was accomplished.
COMPAMED.de: Professor Bastmeyer, you and your team created a three-dimensional environment for cells. What advantage does this provide for research and why are the predominant two-dimensional environments in the Petri dish not sufficient? What kind of problems did this create in the past?
Martin Bastmeyer: It’s not that the past fifty years of cell culture research went wrong. During this time important, fundamental discoveries were made, which helped us learn a lot of new things about cells, cell differentiation and cell motion. That being said, a cell in an organism always exists in a three-dimensional environment. It also has been known for several years that this is an additional factor that can determine differentiation of cells. For this reason, currently several teams of scientists work on three-dimensional culture systems.
COMPAMED.de: So there are different procedures?
Bastmeyer: There are very many different procedures and by now there are also companies that offer prefabricated substrates for the three-dimensional cell culture. Our work differs from this in that our approach is aimed at specifically controlling the third dimension. We provide the possibility to change specific parameters as you wish to be able to read out on an individual cell level what impact this has on the cell. Many of the other methods don’t offer this option. There, only the pore size or the material composition for instance is variable or poorly defined. In the experiment you can tell that the cells react differently, but you don’t know why that is exactly.
COMPAMED.de: What kind of experiments with the new 3D cell cultures are meant to be conducted in the future?
Bastmeyer: For the moment, our work still depicts fundamental research. What we have accomplished is the fabrication of a cell scaffold, where we can specifically control factors such as three-dimensionality, flexibility and the three-dimensional distribution of cell adhesion points. In the near future we want to vary these parameters and for example see what impact this has on the cell behavior. What’s more, in fundamental research such results are being published, so others can build on this. It is the same with this experiment.
COMPAMED.de: Does this mean it is virtually always the same cell construct or that it keeps being adapted?
Bastmeyer: The method used to fabricate the cell scaffold, the so-called Direct Laser Writing, is a relatively complex method. It is intricate, so you can fabricate these three-dimensional substrates for laboratory use very well to be able to conduct biological experiments. However, I don’t think that you will be able to produce three-dimensional substrates for regenerative medicine with this method in the near future. The procedure is still too expensive for this. But our results can certainly be used to produce better materials for industrial applications in the future.
Laser-scanning microscopy (LSM)
of the cell in the two-component
polymer scaffold. The cytoskeleton
of the cell is colored green, parts
of the two-component polymer scaffold
are colored white, the “cell holds” are
colored red; © CFN
COMPAMED.de: What is Direct Laser Writing?
Bastmeyer: The Direct Laser Writing method originated in collaboration with the Institute of Applied Physics here in Karlsruhe. The team of Professor Martin Wegener further developed the Direct Laser Writing method technically, namely for their very specific problems. At first they fabricated so-called photonic crystals, which are three-dimensional structures that have periods in optical wavelength range with very interesting optical characteristics. I then approached Mr. Wegener because I thought it was very exciting that it is now possible to do something in 3D that’s more defined and I asked him if he was interested in collaborating. He was and we then modified Direct Laser Writing within the scope of joint dissertations for biological issues.
COMPAMED.de: What’s so special about your cell scaffold are the “adhesion points“, on which cells can adhere to. How did you succeed in constructing them?
Bastmeyer: The project has been going on for several years and we proceeded step-by-step. At first we developed three-dimensional structures for the cell culture. The next step was us changing the flexibility of the cell scaffold. To do this, we used materials that can be deformed by cells. This is important, because cells are found in different environments. For instance, a bone is firmer than cartilage and cartilage is firmer than muscle and a muscle in turn is firmer than nerve tissue. Cells can measure in what kind of environment they are in, meaning how firm it is for example. This in turn impacts how cells react. Stem cells are a good example for this. If you cultivate a stem cell and place specific growth factors in a specific ratio in the culture medium, the cell then differentiates in a specific direction.
Several years ago it turned out that mesenchymal stem cells that are cultivated under the same culture conditions on differing firm substrates, differentiate towards cartilage and bones on firm substrates, towards fibroblasts on medium hard substrates and towards nerve cells on soft substrates. Now this doesn’t mean that you can completely control cell behavior just by the flexibility of the environment – but it contributes to it. These experiments were conducted in 2D. The next step for us therefore was to design our three-dimensional structures in a way to where the cells can feel a natural flexibility. We also managed to accomplish this last year. The next step now was the development of the adhesion points, because there are specific proteins to which cells can adhere to. Among them for instance is the extracellular matrix which occurs in the connective tissue or collagen. Cells with very specific receptors hold on to the extracellular matrix – on other cells there are so-called adhesion molecules, which the cells can identify and interact with. For us it’s now important to understand, in what type of geometry these adhesion proteins affect the cells.
One example: Colon epithelial cells are cells which on one side, the apical side, border on the surroundings – meaning in the case of the colon on the lumen and for example on the border to the blood vessel. The job of the epithelial cells is to specifically absorb nutrients. They extract these from the digested food to release them on the other side, the basal side, into the blood vessel system. For these cells to properly function however, they need to know where the apical and the basal sides are. So they need a geometrical distribution of adhesion molecules. If you want to grow these cells in culture, you therefore need to offer them an asymmetrical distribution of adhesion proteins. We wanted to do this in 3D. That’s why we went to the next step and developed a method that works for this. At first we had to write a scaffold in 3D for this, which is protein-repellent. This means neither cells nor proteins can adhere to it. After we had developed the 3D structure, we coated the entire thing with a second photoresist which for instance is protein-binding. Now you polymerize it with a laser focus on a few holds on the 3D scaffold. This way we achieve a structure that consists of two different materials. If you then cover this with a solution that contains a protein, the protein then only adheres to those points that were written in the second photoresist. That is to say, we can control the cell in 3D by the adhesion points.
Cell in the two-component polymer scaffold. The photo composition is based on a scanning electron microscopy and laser scanning microscopy; © CFN
The next step will be for us to write a flexible protein-repellent scaffold and only provide defined adhesion points – you might describe it as ”gymnastics for cells“. The minute the cell “does gymnastics“, it starts to tug at its surroundings. We can measure this and study what difference it makes for example, if we change the geometry of the adhesion points. We then see how the different cell types react to this. Then the next step will be to offer two different proteins in two different 3D geometries.
COMPAMED.de: How important is the interdisciplinary collaboration for your research?
Bastmeyer: Generally, our research is only possible thanks to interdisciplinary collaboration. At the Center for Functional Nanostructures in Karlsruhe and generally at the KIT this works very well. Our project for instance could not have worked without the direct intensive collaboration with the physics department.
The interview was conducted by Simone Ernst and translated by Elena O’Meara