Microfluidics in Stem Cell Research

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Author: Luke Harland Edited by: Sandra Ionescu

One of the most dynamic and important stages of human development occurs at its outset, long before a mother is even aware that she bears a child within her womb. This stage of development is called gastrulation. During gastrulation, an amorphous ball of cells, called a blastocyst, undergoes a series of dynamic morphological changes that ultimately results in the production of three distinct layers called the mesoderm, endoderm and ectoderm. These cellular layers undergo further division, growth, specification and remodelling, ultimately giving rise to all cell types of the human body. The cardiovascular and muscular systems are derived from the mesoderm, the gut tube and its associated organs come from the endoderm and the ectoderm produces the nervous system and the skin. In the 1980s, researchers demonstrated that under certain conditions mouse blastocyst cells can be cultured indefinitely in a petri dish whilst still retaining the capacity to give rise to all the future cell types of the mouse body. These cells were named embryonic stem cells (ESCs). In the decade that followed, we also learned how to grow and propagate ESCs from human blastocysts.  Since the initial derivation of ESCs, scientists around the world have tried to develop innovative strategies to coax ESCs into all human cell types for drug screening, regenerative medicine and to more deeply understand human development.

In order to convert an embryonic stem cell into a specific cell type, stem cell biologists must recapitulate the early stages of human development within a petri dish. During gastrulation and early human development, cells of the blastocyst grow, divide and migrate, encountering numerous microenvironments filled with different cell types, biomolecules, biophysical parameters and extracellular matrix proteins. Scientists try and mimic, in a stepwise fashion, these different environments to convert ESCs into desired cell types. If you wanted to produce a heart cell, for example, ESCs need to be converted first into mesodermal cells and then pushed towards a cardiac fate. Precisely manipulating the microenvironment of the ESC via the addition of exogenous chemicals at different times and in different concentrations is an essential requirement for directed differentiation of therapeutically relevant cell types.   

Initial differentiation strategies involved removal of the special factors that enabled the indefinite propagation of ESCs and the addition of serum. Different serum batches, however, produced specific cell types with varying efficiency. For example, one batch of serum might produce 20% cardiac muscle cells, whilst the next may only produce 2%. Serum-free methods using cytokines, recombinant proteins and small molecules were developed, enabling a more refined approach to determining the exogenous factors required for the production of certain cell types. These initial differentiation strategies, however, were performed at a large scale in flasks and petri dishes and did not enable precise control over the microenvironment. The use of microfluidic devices in stem cell research is changing that.

Microfluidic devices are miniature culture systems made up of numerous culture chambers that hold liquid volumes in the microlitre range. Microchannels are connected to the various chambers, producing inlets and outlets that allow tight spatiotemporal control of liquid and gas exchange. The chamber and microchannel networks are primarily produced from polydimethylsiloxane (PDMS), a biocompatible elastomer that is transparent and gas permeable. Microfluidics provides stem cell scientists multiple advantages over traditional petri dish/flask culture systems. Microfluidic cell culture devices enable more precise control of cellular microenvironments, require lesser amounts of expensive reagents, enable a highly efficient parallelisation (production of different cell types from ESCs in parallel) for high throughput screening, reduce technical variability and provide more precise control over paracrine and autocrine signaling effects. Due to the transparency of the chambers and microchannel networks, these devices can also be coupled to downstream analyses such as immunofluorescent imaging, RNA sequencing techniques and cell sorting via optical tweezers.

A microfluidic device for cell capture and pairing (a) Schematic of the device operation and structure. (b) Cell traps. (c) Magnified image of the device, showing the densely packed structures. (d) Detail of the trap structure (e) A 2 × 2 mm array containing ~750–1,200 traps; and an 8 × 4 mm array containing ~6,000 traps. Image courtesy of Nature Methods.

A microfluidic device for cell capture and pairing (a) Schematic of the device operation and structure. (b) Cell traps. (c) Magnified image of the device, showing the densely packed structures. (d) Detail of the trap structure (e) A 2 × 2 mm array containing ~750–1,200 traps; and an 8 × 4 mm array containing ~6,000 traps. Image courtesy of Nature Methods.

 

When microfluidic devices are utilised by stem cell scientists, each chamber that houses the differentiating ESCs is separated from its neighbour and is provided its own liquid media supply. The effects of spatiotemporal modulation of media ingredient concentrations, chamber slide extracellular matrix coating and cell density can all be tested within the same device. This allows for massive parallelisation in experimentation, expanding the number of parameters that can be monitored in any given experiment. For example, ESC lines that report the expression of a cardiac muscle cell-specific gene via expression of a fluorescent protein can be used to determine the upstream signals required to produce cardiac muscle cells. Stem cell scientists can begin to unravel, in a more versatile and technically refined manner, signals that drive the development of different cell types from ESCs.

In the future, microfluidic devices will likely become more automated and coupled to computational software that can control mixing devices that distribute liquid media into the cell-containing chambers. Machine learning algorithms may enable automated microfluidic systems, similar to the one described above, to learn how to make a cell type of interest. The use of microfluidic devices in stem cell research will allow scientists to more effectively determine ways of producing therapeutically relevant cell types from ESCs and, in the process, begin to more fully understand early stages of human development.