Microfluidic platform with four orthogonal and overlapping gradients for soluble compound screening in regenerative medicine research

Abstract

We present here a screening method based on a microfluidic platform, which can generate four orthogonal and overlapping concentration gradients of soluble compounds over a monolayer of cells, in combination with automated and in situ image analysis, for use in regenerative medicine research. The device includes a square chamber in which cells are grown, and four independent supply channels along the sides of the chamber, which are connected through an array of small diffusion channels. Compounds flown through the supply channels diffuse through diffusion channels into the chamber to create a gradient over the cell culture area. Further, the chamber is connected to two channels intended for introduction of cells and in situ staining. In this study, the dimensions of the different channels were optimized through finite element modeling to yield stable gradients, and two designs were used with gradients spanning 2.9-2.4 μM and 3.4-2.0 μM. Next, overlapping gradients were generated using four rhodamine-derived fluorescent dyes, and imaged using confocal microscopy. Finally, the platform was applied to assess the concentration-dependent response of an osteoblastic cell line exposed to a hypoxia-mimicking molecule phenanthroline, using an in situ fluorescent staining assay in combination with image analysis, applicable to closed microfluidic devices. The on-chip assay yielded results comparable to those observed in conventional culture, where a range of concentrations was tested in independent microwells. In the future, we intend to use this method to complement or replace current research approaches in screening soluble compounds for regenerative medicine, which are often based on one-sample-for-one-experiment principle.

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics

“…Spatiotemporal information of high resolution is thereby an important advantage. Several devices have been proposed to study the effect of soluble species in cell culture media delivered gradient-wise including assays for bacterial or mammalian cell chemotaxis [56][57][58], cytotoxicity [59,60], transcription factor translocations [61] and differentiation [62]. This kind of platforms may enhance the power of assays as compared to conventional experimental set-ups by significantly decreasing the number of cells needed for robust and statistically relevant readout.…”

“…Image-based cytometry, which is frequently used for high-content assessment of cell responses in such devices, often employs fluorescent assays combined with automated microscopy image acquisition. For example, Harink et al [61] developed an end-point assay to quantify translocation of hypoxia-inducible factor 1 alpha in cells exposed to a diffusion-generated gradient in a closed, glass microfluidic system. A translocation assay was also used by Awwad et al [65] in a long perfused cell culture chamber where Chinese Hamster Ovary cells (CHO), hosting a reporter plasmid with a fluorescent fusion protein human NF-jBp65 and pHyg, were actuated by interleukin 1b gradients, demonstrating a linear response in the concentration range of 0.005-0.1 ng/ml (Fig.…”

“…A hydrodynamic junction can be used for diffusive mix, creating a progressive gradient over the length of the resulting channel [60,66]. Diffusion-based gradients are a result of a source-sink arrangement siding the chamber, whereby a directional movement by diffusion is observed from the compartment containing higher concentration to the one with lower concentration [61,67]. Image-based cytometry, which is frequently used for high-content assessment of cell responses in such devices, often employs fluorescent assays combined with automated microscopy image acquisition.…”

Simultaneous or Sequential Orthogonal Gradient Formation in a 3D Cell Culture Microfluidic Platform

“…Relying on pure chemokine diffusion, other designs, often consisting of rows of microgrooves that isolate the medium channels from the cell chambers, alleviate the issue of shear stress while conserving the presence of steep gradients . This approach was used to generate multidirectional gradients in solution for adherent, 2D cultures . A few groups proposed microfluidic devices capable of forming reversing, oscillatory or rotating gradients in solution, although they were exclusively used in the context of chemotaxis in suspended or on adherent cultures (reviewed in .…”

“…[13][14][15] This approach was used to generate multidirectional gradients in solution for adherent, 2D cultures. [ 16 ] A few groups proposed microfl uidic devices capable of forming reversing, [17][18][19] oscillatory [ 20 ] or rotating gradients [21][22][23] in solution, although they were exclusively used in the context of chemotaxis in suspended or on adherent cultures (reviewed in. [ 24,25 ] The recent development of microfl uidic devices capable of applying concentration gradients to 3D hydrogel-based cell culture systems not only increased the physiological relevance of such platforms, but it also made it possible to recapitulate tissue functions previously unattainable, such as the formation of a microvasculature.…”