What happens in tissues and organs and how do they react to pharmaceutical substances? Three-dimensional cell cultures can reproduce reality far better than a single-cell layer can. With good reason. Reality is far from two-dimensional. A company called 300MICRONS GmbH develops films with tiny indentations that provide optimal conditions for cells to grow into 3D cell aggregates.
“For example, we observe that liver cells which grow in a monolayer on a plastic or glass substrate respond to a certain drug, whereas the human liver is insensitive to this particular compound,” says Prof. Dr. Eric Gottwald, one of the three founders of 300MICRONS. However, when grown in three-dimensional cell aggregates, the same cells are as sensitive to the drug as their natural counterparts.
“3D cell cultures have considerable advantages over 2D cell cultures as far as pre-selection of effective active ingredients in pharmaceutical research is concerned,” says Gottwald. The closer these organotypic culture conditions are to the in vivo situation, the more likely it is that pharmacologists will be able to use it to replace animal testing, which is still mandatory in the drug development value chain. However, 3D tests of this kind that could be alternatives to animal testing have only really been attracting broad interest for the past fifteen years or so.
Gottwald is sitting in a spacious office hidden away in the backyard of an industrial estate in Karlsruhe. The plate he is holding appears quite ordinary – a standard 96-well micotitre plate used by cell culture laboratories around the world for automated analyses. However, Gottwald’s plate is slightly different from traditional ones in that the bottom of the wells is made of a transparent film with microcavities in which up to 10,000 cells grow and form cell aggregates.
The company founders have chosen the diameter and depth of the miniature cavities wisely – 300 micrometers (around a third of a millimetre). “This is the natural maximal distance between two adjacent blood capillaries in a typical animal tissue. Oxygen supply can therefore be continuously maintained by simple diffusion without affecting the cells’ viability,” says Gottwald. The number 300 has proven to be key for cell culture, hence why it is part of the company name.
The conventional production method for spherical cell aggregates, so-called spheroids, involves attaching a drop of cells to the lid of a Petri dish. “At some stage, the spheroids exceed 300 micrometres in size and the cells inside die because they are no longer supplied with nutrient medium,” says Gottwald. In other cases, the cells are grown in a 3D scaffold, such as a gel, that contains extracellular matrix components of mice. These matrix components do not contain a defined mixture of growth factors and so the consistently high cell quality that the pharmaceutical industry requires is difficult to obtain with existing culturing systems.
Gottwald can grow more than 16,000 3D cell aggregates in a cell culture plate manufactured by his company. Liver cells, the main cell type he works with, have already been successfully grown in the plates into liver-like tissue together with other cell types that are also present in the liver. He has also been able to convert stem cells, which need to be grown, at least for a short period, in three-dimensional cultures in order to differentiate into tissue cells, into beating cardiomyocytes.
The miniature cavities have the potential to be used as artificial stem cell niches, which is an important prerequisite for producing viable stem cells for transplantation. Normally, cultured blood stem cells quickly lose their stem cell properties. “With our system, the stem cells maintain their capacity to differentiate into blood cells for about 21 days,” says Gottwald. This would enable doctors to transplant stem cells removed prior to chemotherapy back into the leukaemia patient.
The company’s 3D cell culture plates first originated in the Karlsruhe Institute of Technology (KIT) in the mid-1970s. When Gottwald joined the KIT’s Biological Institute in the 1990s, researchers there were already working on a chip-shaped predecessor of the current system, which cost an equivalent of 500 euros per unit. The relatively high price was due to the manufacturing process and in particular the postprocessing step involving micro-injection moulding. “Nobody would buy something like that at that price,” says Gottwald. All this changed when Stefan Giselbrecht joined the institute as a PhD student.
Together with Roman Truckenmüller, an engineer at the neighbouring Institute of Microsystems Engineering, Giselbrecht developed a cost-effective method to make thermoformed microcavities in the 300 micrometres size range in a polymer film. Up until then, microthermoforming had only been suitable for producing larger objects such as yoghurt pots or praline boxes. Gottwald, Giselbrecht and Truckenmüller established 300MICRONS in 2015.
Special machines were designed and are now used for producing microstructured polymer films with various geometries for use in standard microtitre plates and bioreactors that can house microcavity arrays in chip format. The number of wells can be changed, as can their diameter and shape. The film can also be made permeable for nutrient medium or certain substances.
In addition to running the company, Giselbrecht and Truckenmüller are carrying out research at the University of Maastricht in Holland. Prof. Gottwald is in charge of company operations in Karlsruhe, where he continues to teach and research at the KIT. In 2015, the company moved into a new office, and production, currently still at the KIT, will soon be moved to new premises as well. From his current office, Gottwald has a great view of a warehouse where the company’s production halls and laboratories are currently being built on an area of around 300 m².
In order to increase production and demand, the three company founders are currently looking for other suppliers and are also thinking about producing their film products on a reel. They have also plans to increase their service portfolio by offering precultured test plates. “3D cell culturing is booming. If we are not part of the game now, other systems will possibly be established,” says Gottwald who never had the slightest regret about establishing the company. “For a scientist, company foundation is extraordinarily exciting and always great fun.”