A broad range of basic research applications, pharmaceutical industry and diagnostics applications rely on the selection of single cells from a large number of different cells. Tumours, for example, consist of different cell types that express different genes (tumour heterogeneity), which therefore gives them different characteristics. The individual cell subtypes can have a decisive influence on a patient’s prognosis and therapy selection. At present, cells are separated from each other manually under the microscope or using a so-called FACS (fluorescence-activated cell sorting) device, which is rather costly and only suitable for larger quantities of cell suspension. 

Microsystems engineering for application in cell biology

The Freiburg-based start-up company cytena GmbH has come up with a solution to this problem based on an idea developed by Dr. Peter Koltay and his team in the Department of Microsystems Engineering (IMTEK). Koltay’s team is specifically focused on research into the contact-free dispensing of fluids such as liquid metal and biological samples. 

“As our specific focus is the contact-free printing of fluids, we thought about whether our printing system would also be suitable for specifically isolating individual living cells from a sample,” says André Groß, CTO of the company. This question was answered as part of a doctoral thesis, and a European patent was filed for the resulting technology in 2010. 

But how does the single cell printer work? It consists of a basic unit and a print head into which a disposable cartridge containing a cell suspension is loaded. The basic unit controls the print head and guides the analysis substrate (e.g. microtitre plates) into the printer where individual cells from suspension samples will be deposited into the substrates. The cartridge contains a microchip that produces individual droplets from the cell suspension until a single cell is present in the nozzle.

Due to the special design of the microchip, it is possible to automatically monitor the printing process by way of a video microscope. A special computer algorithm detects and counts the cells in the chip, and also calculates when a cell – enclosed in a droplet – will leave the chip. In addition, the computer calculates the size and form of each cell. This enables the researchers to predict the number and type of cells contained in the individual droplets. The application of pneumatic suction only allows droplets that enclose a cell to actually reach the substrate.

“The individual cells are already recognisable within the chip, in fact we can see them before the droplet is actually generated. This enables us to predict precisely the droplets that contain cells, and we are also able to comment on the particular type of cell a specific droplet contains. So we can decide whether we want to print a particular droplet onto the substrate or not. This method gives us considerable flexibility in sample composition, cell isolation and substrate types,” says Groß explaining how the device works. 

The use of imaging technology enables the researchers to document the deposition of just one cell and makes the device suitable for creating clonal cell lines used for drug production. Another advantage is that only the cartridges come into contact with the cell suspension. They are disposable single-use cartridges that enable direct sample pipetting; cross-contamination is thus prevented and post-process cleaning is not needed. The device can handle quantities as small as 10 µl, making it suitable for basic research applications such as genetic single-cell analyses. 

“Our method is very gentle. More than 90% of the cells survive the procedure,” says Groß. “Compared to other laboratory equipment, the device is moderately priced. Operating costs are also quite low. It is also easy to operate and the workflow is quite simple. Users simply load the sample and the cartridge, and this starts a predefined programme. No extensive training is necessary. Everybody in the laboratory can quickly learn how the device works,” adds Benjamin Steimle, CFO of cytena.

Funding obtained under the EU’s FP7 enabled the researchers to develop their initial idea into a marketable device (name of the project: PASCA - Platform for advanced single-cell handling and analysis). When funding ended in 2013, the researchers had already produced seven prototypes that have since been tested in the laboratories of the researchers’ project partners. “The device worked extraordinarily well and our partners were very happy with the outcome. The idea of establishing a company was a logical consequence,” recalls Groß. The researchers received funds from the EXIST programme, which is co-financed by the BMWi (German Federal Ministry of Economics and Energy and the ESF (European Social Fund), to set up cytena GmbH in June 2014. 

“At present, our team consists mainly of individuals with excellent technological know-how. But from now on, we will also increasingly have to deal with administrative tasks such as sales, marketing and finances,” says Groß, talking about the typical challenges of technology-based start-ups. “It is extremely useful if you have someone who can pass on this special knowledge.” Here, cytena benefits from the experience of Dr. Koltay who previously successfully established a company called BioFluidix GmbH. Benjamin Steimle was taken on board as financing and marketing expert.

In addition, the cytena founders also rely on the advice of experienced external coaches. “We received EXI start-up vouchers – with the help of BIOPRO and bwcon – which put us in contact with an experienced coach who is now a member of cytena’s advisory board,” says Steimle. The company founders believe that they have also benefited considerably from their participation in the Science4Life Venture Cup 2014, in which they came 3rd.

The company now has four employees and manufactures devices and cartridges independently. Its next objectives are already clear: “We are currently working to obtain the required certifications to be able to offer the single-cell printer as a certified device rather than a prototype. In parallel, we are expanding production, marketing and sales,” said Groß, highlighting the next steps of the start-up. Another major focus is the acquisition of external funds: “We are open to cooperations and are actively looking for investors.”

Further information:
André Groß (CTO)
cytena GmbH
Georges-Köhler-Allee 103
D-79110 Freiburg
Tel.: +49 (0)761/20373257
E-mail: gross(at)cytena.com