Scaleup of Versatile, Fully Automated, Microfluidic Cell Culture System

Researchers in the laboratory of Professor Stephen Quake at Stanford University applied microfluidics to stem cell biology, creating a system that enables very precise control of cell culture conditions. Adult cells can be reprogrammed into induced pluripotent stem (IPS) cells by treating them with the right combination of factors; stem cells can be induced to differentiate into desired cell types by treating them with a different combination of factors. Both reprogramming and differentiation require searching for the right combination of factors, so a system which can culture cells with different combinations of factors should be very useful to stem cell scientists. Fluidigm and Stemgent jointly applied for and received a CIRM Tools and Technologies grant to scale up and commercialize this technology, in order to make it more broadly available to the stem cell community. In the first year of this grant, we have: • Built a manufacturable version of the microfluidic cell culture chips used in the Quake lab (including several improvements) • Built a breadboard instrument system capable of loading, culturing, and dosing cells in the chip, as well as automatically imaging the cells at pre-programmed timepoints • Built a prototype of a commercial chip controller instrument • Demonstrated the ability to culture multiple cell types on chip (including both cell lines and stem cells) • Demonstrated the ability to transfect cultured cells (insert genes) using viruses • Exported live cells out of the chip Having done these things, we are well positioned to carry out the rest of the work called for in the grant: replicating literature experiments in cell reprogramming, and screening combinations of small molecules, proteins, and nucleic acids for differentiation or reprogramming.

We have optimized and scaled up an advanced (microfluidic) cell culture system into manufacturable form. The types of experiments required to convert normal cells into cells which can function as stem cells (induced pluripotent stem cells), or convert stem cells into cells of a desired type (e.g. neural cells) require multiple factors (i.e. chemicals). These types of multifactor experiments are necessary for applications of stem cells to medical research, but they are difficult and laborious. Following and expanding on previous work, we have created a cell culture system which allows multifactor experiments to be carried out under computer control, with unprecedented levels of control over the timing and amount of dosing of the cells with different factors. We utilized advanced microfluidics technology (Multilayer Soft Lithography) to create cell culture chips which could load cells, culture them, and treat them with any combination and permutation of 8 factors. These chips are built in our commercial microfluidics fab, and mounted on plastic carriers which serve as an i/o interface. In the course of the grant, we also built several pieces of instrumentation to control the chips. These instruments control the microfluidics, maintain the correct environment for cell culture, and allow automated imaging of the cells. The controller instrument has been developed to the level of a commercial prototype. We demonstrated the ability to culture multiple types of cells in these microfluidic chips. We also demonstrated the ability to dose the cells with factors (converting them into stem cells), as well as to dose different chambers of cells with different combinations and permutations of factors. We believe this system will be a useful tool for the stem cell research community in their search for methods for producing cells useful for medical applications.