Year 3

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.