Chip-op basis van drie-dimensionale Cultuur van de Cel in doorbloed Micro-bioreactoren

Hi, I am Eric Gottwald and I'm research scientist at the Institute for Biological Interfaces of the Ware Institute of Technology. And today I would like to introduce you into our chip based perfused Micro bioactive system. Before we can use the chip In cell culture applications, we have to hydrolyze the polymer and have to de-rate the micro structured area.

This is achieved by just dipping the the chip into a different concentrations of isopropanol, beginning with 100%I'm dipping the chip just a few seconds into each concentration and the final step is just water. Afterwards, the chip is dried and finally we have to coat the chip with a collagen, one solution to facilitate the adhesion of the cells to the polymer. This is done by dripping just a few drops of a collagen, one solution on top of the micro structured area, and in this state, the chip is incubated at four degrees Celsius overnight.

After the overnight incubation with the collagen one solution, the solution is removed and the chip is washed with sterile PBS Afterwards, also, the PBS is removed and the chip is now ready for the inoculation of the cells. Now we'll inoculate The cell ship with a suspension of single cells, and this is done by just putting 150 microliters of cell suspension on top of the micro structured area. After the deposition of the cells, the chip is incubated for three hours at 37 degrees in an incubator.

After the three hour Incubation period, the chip is removed from the incubator and is now ready for the insertion into the ector. For this, the ector is taken out from its sterile packaging and the sterility is achieved by gamma irradiation of this transparency foil. So we just have to open the package and remove the ector.

What we will do next is we will insert the top of the bottle into this medium reservoir so that we can disassemble the bioreactor housing for the insertion of the of the chip. After having opened the bioreactor housing, we can insert the chip now, which is achieved by just placing the chip into the groove of the bioreactor housing and the bactor can now be assembled again. What we have to do finally is that we have to connect to sterile filters to the medium reservoir and one sterile sampling port.

For the measurement of the actual oxygen tension in the closed circulation loop, we will insert two oxygen sensors by just mounting them in between these two connectors. The bactor is now ready for filling with culture medium. The bactor system is now ready to be filled with cell culture medium, and this is achieved by simply connecting the closed circulation loop to the roller pump.

By this, we will simultaneously derate the whole bactor circulation. As you can see here in the Ector window, you can see the medium coming up from below rising and rising, and leaving the bactor at the top. Before we place the Bactor system into the incubator, we control for the bubble free filling of the circulation loop.

Now the system is completely filled with cell culture medium and we can transfer the bioreactor to the incubator. Now we will place the bi rector into the incubation chamber. And for this, at first, the bi rector is connected to the roller pump, as you have seen already shortly before, and now the ector is actively perfused by this medium from the bottle.

And to maintain the ambient oxygen concentration, we connect the medium reservoir to the gas supply. And finally, we'll connect two oxygen sensors to the circulation to online monitor the oxygen tension in the system. Now the setup is complete and can run as long as you have designed the experiment.

One of the most powerful tools during the analysis of the organotypic functions of the cells inside the MicroCon containers is the laser scanning microscope, which you can see here. The laser scanning microscope gives us the opportunity to look into each single MicroCon container without destruction. And because the MicroCon containers or the chip can be used for conventional immuno staining immunofluorescence methods, we are able to detect all kinds of, of markers and organotypic functions inside the micro containers.

As you can see here in the, in the animation of this, of this chip, the bottom is completely filled with cells as well as the MicroCon container walls. We've got plenty of cells that are quite alive, and we've got very, very few dead cells. The next picture shows you that we can detect all kind of organotypic functions inside the MicroCon container.

Like for example, for hepatocytes, the albumin production or the E could hear an expression in the cells, which you can see here nicely in the overlay. The nucle ir stained in blue. The eco herein is stained in green, and the albumin is expressed or stained in red.

And as you can see here nicely, all organotypic functions that you expect from hepatocytes are nicely expressed in this kind of 3D culture. Furthermore, we have looked at an another hepatocyte marker, and this was cytokeratin 18, and this picture shows you that Cytokeratin 18 is nicely expressed as well in those kind of cultures. And this hopefully convinces you that this kind of organotypic functions might be useful for other organotypic cultures as well.

After the end of the Biore experiment, the chip can be used for the isolation of all kinds of cellular components, like for example, total RNA. As you can see here, here we show you intake total RNA, that was isolated from the bioreactor system. This total RNA can then be used for downstream applications like for example, realtime PCR, or as you can see here, microarray analysis.

Furthermore, intact cells can be isolated from the system and those cells can then be used for conventional flow cytometry analysis. As you can see here, I hope that we could show you that the system might be useful for all kinds of applications where organotypic reactions are required and that the system might be useful for all those kind of applications that we have not considered so far, like for example, stem cell applications.