Supported in part by a previous CIRM Tools and Technologies Grant [REDACTED], we have optimized and scaled up highly advanced (microfluidic) cell culture chips into manufacturable form, produced prototype instruments to drive these chips, and demonstrated that we can culture cells, dose them with combinations of reagents, and export them back off the chip.
Since a cell’s state is controlled by multiple genes, experiments to control cell state (e.g. to turn skin cells into stem cells, or to turn stem cells into nerve cells) will almost always involve multiple factors as well. We believe the ability to do multi-factor experiments more quickly, easily, and reproducibly will be enabling for the stem cell field.
The research we propose here will push the capabilities of this system even further by producing a set of three complementary commercial instruments: a Controller (capable of full fluidic and environmental control on one chip), a Hotel (capable of limited fluidic and environmental control on multiple chips), and a Reader (capable of imaging the cells in the chip in phase contrast and fluorescence modes). The idea is to load cells and dose them with different drugs/chemicals on the Controller, transfer them to the Hotel for culture and maintenance, and transfer them to the Reader for periodic imaging, allowing therefore running multiple sets of experiments in parallel and increasing even more the throughput of the system.
We are also proposing two sets of experiments to demonstrate what the system can do: in the first one, we will develop new methods to turn IPS cells (stem cells obtained by reprogramming non-stem cells - skin cells for instance) into neural progenitor cells – cells which can become different types of neural cells. These cells could be used to study diseases such as Parkinson's or Alzheimer's. In the second set of experiments, we will develop methods to make these cells proliferate without turning into specific types of neural cells. Since these types of cells are potentially useful to treat neurodegenerative diseases (e.g. Parkinson's and Alzheimer's) and spinal cord injury, developing methods to make more of them could advance the field a step closer to clinical application. In both cases, we will avoid using serum and animal products, since methods which use these products cannot be used clinically.
The research proposed here will allow bringing to the broad stem cell community, in and out of California, a commercial system that will accelerate research aiming at
1.) Identifying genes and small molecules affecting stem cell self-renewal and differentiation
2.) Identifying stem cell differentiation and expansion conditions
Since the grant will support work done in [REDACTED] and [REDACTED], and the end result will be the creation of a set of commercial instruments, there is a direct economic multiplier effect for the resources invested. In particular, at least three positions will be created (2 engineer positions and one postdoctoral position) as soon as the project starts.
The availability of the system will accelerate discovery of cell differentiation and expansion conditions, multiplying the power of stem cell research, in which California is a leader. The more efficient identification of differentiation and expansion conditions should enable new therapies. More directly, the discovery of conditions for differentiation of IPS cells into neural progenitor cells should enable the use of those cells as disease models (e.g. for Alzheimer's or Parkinson's); the discovery of chemically-defined conditions for expansion of those neural progenitor cells could lead to cellular therapies for neurodegenerative diseases like Alzheimer's or Parkinson's or spinal cord injury.
The availability of powerful tools in California, such as those we will develop here, 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.
Determining the appropriate culture conditions for reprogramming, differentiation, and cell expansion is a challenge for the stem cell field, as these techniques rely on the targeting of multiple gene activities in order effectively regulate cell fate. With currently available tools, such multi-factor experiments are labor intensive and difficult to carry out reproducibly. To address these challenges, the applicant proposes to enhance an existing automated microfluidic cell culture platform to enable higher throughput and increased utility. First, a set of three complementary commercial instruments will be produced for: 1) setting up multi-factor culturing experiments on a microfluidic chip, 2) combining several chips and performing longer term incubation, and 3) performing on-chip cell imaging. The applicant will then demonstrate utility of the integrated system by developing xeno-free, chemically defined methods for differentiation of human induced pluripotent stem cells (iPSCs) into neural progenitors (Aim 2) and subsequent expansion of these derivatives (Aim 3).
Although reviewers were uncertain of the extent to which this project would improve throughput over existing platforms, they were confident that the resulting tools could offer advantages beyond those stated in the proposal. They praised the unique features and impressive flow rates of the applicant’s current instrumentation and agreed that the proposed improvements could reduce the time and technical effort that is necessary to conduct and analyze complex, multi-factorial experiments. Most importantly, compelling testimonials from leading stem cell experts convinced reviewers that the new technology could be enabling and therefore have major impact on the field of regenerative medicine.
Reviewers considered the instrumentation aspects of the proposal to be feasible based on demonstrated capabilities of the applicant team and their prior success in developing the prototype. They also considered the choice of neural progenitors as an initial model system to be appropriate. While the technological components were strong, reviewers were less certain about the proposed biological validation studies. Several noted that their confidence would have been boosted by inclusion of preliminary data demonstrating the capabilities of the current system for supporting growth of human iPSCs. In addition, they were not certain that conditions derived using microfluidic chambers would necessarily translate to the much larger scale that would be necessary to support therapeutic cell manufacturing. Finally, reviewers questioned whether the potential expense or complexity of operation might limit the availability of this tool to the scientific community. While these concerns were noteworthy, reviewers emphasized that the strength of this proposal lay in the instrumentation itself and they were confident that biological validation would ultimately be achieved if not by the applicant, then through collaborations with the stem cell community.
Reviewers acknowledged the strong engineering qualifications of the applicant team. The principal investigator (PI) was recognized as an expert in microfluidics with demonstrable productivity and appropriate experience in business and technology development. Although reservations were expressed about the team’s limited expertise in human iPSC/neural stem cell biology, reviewers acknowledged the enthusiastic support of three prominent leaders in the embryonic stem cell (ESC) field who elaborated both a need for the proposed technology as well as their desire to make use of it in their research programs.
In summary, reviewers believed that the proposed instrumentation could be highly useful for conducting complex, multi-factor experiments and could enable more efficient development of protocols for stem cell growth and manipulation. Despite some minor reservations about feasibility, reviewers appreciated the potential for broader impact and ultimately recommended this application for funding.
- A motion was made to move this application from Tier 2 to Tier 1, recommended for funding. Reviewers agreed that the proposed tool could be broadly useful. They recognized the applicant as a strong Californian for-profit entity with expertise in the project area. While they maintained reservations about scalability and impact with respect to achievable throughput, reviewers noted compelling broader advantages over existing 96- or 384-well plate platforms, such as higher resolution of temporal control and an ability to serve as a small-scale bioreactor. Additional concerns about a perceived lack of biological expertise were allayed by the strong endorsements of leading stem cell experts in their letters of support. The motion carried.