Development and Application of Versatile, Automated, Microfluidic Cell Culture System
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.
We have made significant progresses in designing, developing, and optimizing the commercial level instrumentation of an automated microfluidic cell culture system, and have demonstrated an application of the system in direct converting human skin cells (fibroblasts) into neurons on the microfluidic chip.
A fundamental challenge for stem cell technology is finding the right cell culture conditions for growing cells and steering cells into the desired lineage. Cell fate is determined by timely coordination of many genes and environmental factors. With currently available tools, however, multifactor experiments are often labor intensive and difficult to carry out reproducibly. Using the core technology of Multilayer Soft Lithography at Fluidigm, we have developed a microfluidic chip and an automated instrument that can culture cells on the chip for extended period of time and automatically deliver multiple combinations of different factors to cells controlled by arrays of on-chip polymer microvalves. Cells can also be harvested from the chip for continued off-chip culturing, single-cell genomic analysis, and/or functional assays. We have updated several key components of the system (including hardware, firmware and software), and significantly improved the system performance and reliability. We have validated the thermal, pressure and fluidic performance of the system controller, and are planning to develop and optimize the other parts of the commercial instrumentation in the next period.
To demonstrate the biology application of the system, we have developed a novel method for long-term dosing of different micro RNAs and their permutations/combinations to human skin fibroblasts on chip and succeeded in direct conversion of the fibroblasts into neurons. Recently several laboratories have reported direct conversion of other type of cells into functional mouse and human neurons and specific neuronal subtypes without cells being reprogrammed to a stem cell stage first. Compared to differentiation from stem cells, direct conversion is much faster and efficient, and may reduce genetic/epigenetic aberrations associated with stem cell induction. Our new non-viral safe dosing approach generated neurons with high efficiency and cell viability. The results were in agreement with published reports, and were confirmed in large well-dish culture format.
The microfluidic cell culture system allows for precise control of microenvironment of cells, and is advantageous in screening multi-factorial conditions for cell maintenance, differentiation and reprogramming with minimal reagent consumption. We believe this system will be a valuable tool for the stem cell research community in both understanding of basic biological signaling mechanisms and developing of cell-based therapies.
We have further developed and optimized the commercial level instrumentation of an automated microfluidic cell culture system, and have deployed a prototype system to a collaborator site for testing. We have demonstrated that we can change cells into different types of neurons using only chemically-defined factors – i.e. only reagents that could be used in a clinical protocol. We have shown that we can do this in several different ways (either with synthetic nucleic acids (RNA) or small molecules like drugs). Furthermore, we have shown that we can get cells out of the system and measure the gene expression of single cells individually.
Finding the right culture conditions to grow cells and transform them into desired types of cells is central to stem cell research and cell-based therapies. It is also very challenging: experiments in traditional culture systems are often labor intensive and difficult to reproduce due to the need to screen many factors involved in the cellular processes. Using advanced microfluidic technologies at Fluidigm, we have developed a prototype automated instrument that can culture cells on microfluidic chips for more than 2 weeks and automatically dose different cells with different permutations and combinations of multiple factors. In the last year, we have further optimized key components of the system to improve the thermal, pressure and fluidic delivery performance. We have also redesigned the environmental control system so that it can be an add-on module to a commercial instrument (the C1 Single Cell Auto Prep System), which will make it easier for researchers to do these types of cell culture condition screening experiments. Two new versions of the microfluidic chips have been designed and tested to offer more flexibility of cell dosing scheme and longer duration of unattended experiments. Cells can be stained with fluorescently-labeled cell-type specific markers on chip and images scanned with an automated microscope. We have also optimized a workflow to export live cells from the culture chip and analyze the gene expression profiles of single cells which may shed lights on cell-cell variability within a cell population.
A desired target cell type may be derived in vitro by either direct conversion from another committed cell type or differentiation from stem cells or progenitor cells. Direct conversion is usually much faster and more efficient, but stem cells, especially induced pluripotent stem cells, are more versatile and proliferative and easier to generate millions of cells. We have demonstrated both methods on chip by converting human fibroblasts to neurons with combinations of micro RNAs and messenger RNAs, and by differentiating human induced pluripotent stem cells into pain receptor neurons with small molecules. The results were in agreement with published reports, and were confirmed in large well-dish culture format. We have established chemically-defined conditions for both protocols and are currently optimizing the protocols for higher induction efficiency and better cell health.
Due to its precise control and easy set-up of multifactorial screening experiments, we believe this automated system will be a valuable tool for the stem cell and the general cell biology research community, and we plan to continue our efforts in optimizing the instrumentation and exploring the biological applications.
Using advanced microfluidic technologies at Fluidigm, we have developed a commercial-level automated cell culture system. We have made significant progress in system development and biology applications over the course of the CIRM grant. We have been working towards making the technology available to the broader stem cell community, and optimizing instrumentation, chips, and biology protocols.
Cell cultures are invaluable platforms for understanding basic biology mechanisms and for biomedical applications such as diagnostics, drug screening, biologics- and cell- based therapies. However finding the right culture conditions to grow cells and direct them into desired types is often very challenging: because every cellular process requires multiple factors, experiments in traditional culture systems are labor intensive and often difficult to reproduce. To tackle this challenge we have developed a prototype automated system that enables culturing cells on microfluidic chips for more than 3 weeks and automatically dosing cells with multiple combinatorial conditions. 32 different conditions using different permutation, combinations, and ratios of 16 different reagents can be carried out in parallel; cell treatment conditions can also be programmed to change at different times. We have demonstrated dosing cells with miRNAs, mRNAs, DNAs, proteins, viruses, small molecules, etc, and have developed streamlined protocols to stain and image cells on chip or breaking the cells apart on chip and export genetic content out for downstream analysis. We also demonstrated harvesting live cells out of each microchamber to analyze the gene expression of single cells individually.
Over last year, we have redesigned and manufactured a new version of environmental control (EC) module that has much better performance and is very close to commercial production. The new EC works together with a modified Fluidigm commercial C1 Single Cell Auto Prep system to provide stable and precise temperature, humidity and pressure control for long-term cell culture and combinational dosing. We also developed and tested several new versions of the cell culture chips with incremental improvements; and started developing an intuitive graphic user interface based software for users to plan experiments, control run-time protocols, and analyze data.
For biology applications, previously we demonstrated two ways to change cells into different types of neurons using only chemically-defined factors –by converting human skin cells to neurons using combinations of micro RNAs and messenger RNAs, and by differentiating human induced pluripotent stem cells (hiPSCs) into pain receptor neurons with small molecules. Over last year we developed a chemically-defined method to induce hiPSCs to neural progenitor cells (NPCs) –cells that give rise to neurons and other neural cells types.
We have also spent significant efforts on developing a chemically-defined method for testing pluripotency of human iPSCs on chip. hiPSCs are generated directly from adult cells; they can propagate indefinitely and give rise to every cell type in the body. Due to the tremendous therapeutic potentials of hiPSCs, thousands of new hiPSC lines are generated each year; however hiPSCs are not all created equal: due to variations in the original cells and the reprogramming methods used, there is enormous variability in cell lines in terms of how well they can be differentiated into different cell types. The standard test for differentiation potential is time consuming, expensive, and requires animal testing. We believe a standardized in-vitro functional pluripotency test (testing the direct differentiation potentials of human iPSCs/ESCs to all three germ layers on one chip) will be more useful for the stem cell community. We have made significant progress in method development and generated proof-of-concept results showing our customized conditions using chemically-defined media and signaling factors can direct hiPSCs to all three germ layers. We are currently optimizing the protocols and developing automated data analysis tools and plan to continue this work beyond the CIRM grant period.
Based on these works, Fluidigm has decided to commercialize the automated microfluidic cell culture system. Because its precise control and automation of multi-factorial screening experiments, we believe the system will be a valuable tool for the stem cell and the general cell biology community.