Microfluidic scaffolds for tissue engineering

Nak Won Choi, Mario Cabodi, Brittany Held, Jason P. Gleghorn, Lawrence J. Bonassar & Abraham D. Stroock

Nature Materials 6, 908 - 915 (2007)

Summary:
This paper proposes a method to control the microenvironment of cultured cells in 3-dimensions. Choi et al achieve this by creating microfluidic networks inside of a cell-seeded hydrogel to help distribute desired chemicals throughout the scaffold. This system allows for temporal and micrometer-scale spatial control of the biochemical environment. In simple terms, this tissue scaffold is a cell-seeded biomaterial made to have a network of small channels running through it, allowing for solutions to flow through by some external pressure force. Solute exchange may then occur by convective mass transfer between the flowing solution and the microchannel wall, and by diffusion from the channel walls through the rest of the gel.

To ensure efficient transport of solute through the channels and hydrogel, the scaffold must be suitable for different types of solutes. Here, they are categorized as: reactive (e.g. metabolites and waste) and non-reactive (e.g. signaling molecules and buffers). For reactive solutes, variations in solute concentration should be minimized; this is accomplished by setting a maximum spacing between channels (Figure 1a). This distance is determined using a characteristic diffusion-reaction variable (Krogh length). For non-reactive solutes, channels in the forms of sources and sinks are necessary for controlled gradients at steady state (Figure 1b).

A 3D scaffold made of calcium alginate containing microfluidic structures is populated with different cell types. The fabrication of the microfluidic scaffold is shown in Fig 2 and detailed in the Methods and Supplementary sections; in short, soft lithography of silicone creates a mould to create microchannels in the hydrogel, which is then cross-linked to aluminum jigs.

[proof of concept]

In 3D cell cultures, diffusion of both small and large molecules must be unobstructed. Here, diffusivity is measured by injecting a fluorescent dye comprised of both small and large fluorescent-labeled molecules into the microfluidic scaffold. The solute distributes throughout the gel by pure diffusion; the amount of fluorescence is quantified along with time and distance diffused. Diffusivity of both large and small solutes is said to be constant since fickian diffusion and exponential decay is observed. This result leads the group to conclude that diffusion of different sized molecules is unhindered within the pores of the gel.

Besides transport within the gel (diffusion), there is also a flow of solutions through the scaffold (convective mass transfer). To optimize the system, flow speed needs to be high enough so that the concentration of solute remains constant (i.e. equal to the inlet concentration) through the length of the channel; this ensures that the overall mass transfer process is diffusion-limited, and dependent on a controllable variable—the inlet concentration.

Choi et al. also demonstrate the ability to change out the solutes in the scaffold such that solute concentration is uniform across the microfluidic network. Figure 3f shows fluorescent micrographs of the scaffold at different time points after sequential delivery of green (fluorescein) and red (rhodamine) solutes to the scaffold. The dyes are observed to fill the microchannels and then spread out to cover the scaffold uniformly.

[other operational considerations]

A microfluidic scaffold also allows control of the metabolic environment of the cell culture. Choi et al. opt to obtain a uniform distribution of reactive solutes (e.g. metabolites and toxic waste products) across the length of the scaffold. To this end, they try to find the maximum separation between microchannels that satisfies the above criteria. Their experiment involves the metabolite calcein-AM, which forms a fluorescent product when taken up by the cell. The metabolites are delivered via a microfluidic network with variable spacing between channels (Fig 4b). The result proved the possibility of a uniform metabolic environment, and that scaffold geometry is an important factor to consider.

More than one independent microfluidic network may be used to allow steady-state gradients in the solute concentrations (Fig 5). This would prove useful and convenient in studying cellular response to different concentrations of solute.

Comments:

Overall, the paper does a good job of proving the viability of the microfluidic scaffold in providing a means of controlling the biochemical environment. A wide range of solute sizes is proven to be effectively exchanged with the scaffold. Also this concept has useful applications including: growing thick tissue; providing a means of inducing certain cell development; and providing a basis for bioreactors.

It seems that this approach could be time-consuming and difficult to create for different combinations cell types and biomaterials. It would be interesting to see how many of these combinations are useable with the microfluidic scaffold approach, and if this is a limiting factor in experimentation. Also, since cell cultures tend to be affected by fluid shear forces, the microfluidic scaffold approach may induce some unwanted differentiation in the cells close to the microchannels.

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