Tools and Technologies I
Stem Cell Use:
Embryonic Stem Cell
We are proposing to optimize and scale up a highly advanced (microfluidic) cell culture system into manufacturable form. This system will allow researchers to: Identify stem cell culture and differentiation conditions Identify genes and small molecules effecting stem cell self-renewal and differentiation, and Identify genes and small molecules involving or effecting reprogramming of differentiated cells. ...much more rapidly and efficiently than they have been able to in the past. Reprogramming a patient's own differentiated cells (e.g. skin cells) into stem cells overcomes the ethical and immunological barriers to theraputic usage which are present with the use of embryonic stem cells. These stem cells can be used in cell based therapy, tissue or organ repair, and potentially even organ reconstruction. Understanding what controls stem cells to differentiate into a desired type of cell helps directly in the development of theraputic applications. Thus, this tool will help both to determine conditions to convert differentiated cells into stem cells, and to develop therapies using the resulting stem cells.
Statement of Benefit to California:
The system proposed here will allow us to Identify stem cell culture and differentiation conditions Identify genes and small molecules effecting stem cell self-renewal and differentiation, and Identify genes and small molecules involving or effecting reprogramming differentiated cells. These capabilities will accelerate stem cell research in California. Since the grant will support work done in South San Francisco and San Diego, and the end result may be the creation of a commercial product, there is a direct economic multiplier effect for the resources invested. More importantly, the identification of conditions which enable the reprogramming of differentiated cells will enable new therapies. Patient specific pluripotent stem cells can be used in cell based therapy, tissue or organ repair, and even organ reconstruction. The availability of powerful tools in California will help ensure that these new therapies are pioneered in California, leading both to job creation and the availability of the most advanced medical care in the world for California citizens.
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.