Simultaneous generation of chemical concentration and mechanical shear stress gradients using microfluidic osmotic flow comparable to interstitial flow

Abstract

Cells are very sensitive to various microenvironmental cues, including mechanical stress and chemical gradients. Therefore, physiologically relevant models of cells should consider how cells sense and respond to microenvironmental cues. This can be accomplished by using microfluidic systems, in which fluid physics can be realized at a nanoliter scale. Here we describe a simple and versatile method to study the generation of chemical concentration and mechanical shear stress gradients in a single microfluidic chip. Our system uses an osmotic pump that produces very slow (<a few microm/s) and controlled flow, allowing a wide and stable diffusion of specific chemical concentration. We also established a shear stress gradient passively via a circular channel in the interstitial level. For evaluation of the system, we used L929 mouse fibroblast cells and simultaneously exposed them to a mechanical stress gradient and a chemical nutrient gradient. The interstitial shear stress level clearly affected cell alignment, mobility velocity, and attachment. At the same time, cell proliferation reflected nutrient concentration level. Our system, which enables continuous and long-term culture of cells in a combined chemical and mechanical gradient, provides physiologically realistic conditions and will be applicable to studies of cancer metastasis and stem cell differentiation.

Stem cells in microfluidics

“…It was reported that the cell density in the higher FBS concentration area was twice as high as that in the pure culture medium. Similarly, a microfluidic chip which can provide physiologically 145 This work realized a culture platform capable of producing chemical and mechanical stimuli similar to realistic physiological conditions. The performance of the system was evaluated by monitoring L929 mouse fibroblast cells for a continuous and long-term culture, which can be applied to the differentiation of stem cells.…”

“…Many processing tools for HSCs are introduced and reviewed in this section including purification and separation, 145,[154][155][156][157][158] signaling analysis, [159][160][161][162][163][164] microchip electrophoresis assays, and microfluidicbased, digital, reverse transcription-polymerase chain reaction ͑RT-PCR͒ assays. [165][166][167] …”

Responses of endothelial cells to extremely slow flows

“…1͒. Flow was generated by osmotic pumps and the flow rate was controlled by altering concentrations of polyethylene glycol ͑PEG͒ ͑Sigma-Aldrich, St. Louis, MO͒ in aqueous solution: [14][15][16] briefly, PDMS cubic chambers ͑inner space of 5 ϫ 5 ϫ 5 mm͒ with a window of cellulose membrane ͑5 ϫ 5 mm, used as the osmotic membrane͒ were connected to tubing and filled with de-ionized ͑DI͒ water. An osmotic pressure was generated as a result of the solute differential between the inner chamber filled with water and the outer chamber filled with PEG solution, resulting in flow.…”

“…Here, we utilize a previously reported osmotic pump [14][15][16] to generate the required extremely slow flows over ECs attached to the surface of a microfluidic channel rather than embedded in hydrogels and analyze their responses; modeling the internal lumen of developing blood vessel. Slow flows with extremely low shear stresses ͑10 −2 −10 −4 dyn/ cm 2 ͒, which cover a diffusiondominant flow regime which is useful for evaluating the role of the indirect mechanism, were achieved and applied to the single layered endothelial cells.…”

An integrated microfluidic device for characterizing chondrocyte metabolism in response to distinct levels of fluid flow stimulus

“…According to the results, the described device generated shear stresses spanning over at least four orders of magnitude (0.007-15.4 dyne/cm 2 ). This range of shear stresses started from an extremely low level comparable to interstitial shear stress level (Park et al 2009;Chen et al 2012;Rutkowski and Swartz 2007) to a high level which was deemed to be injurious for chondrocytes (Lee et al 2003; Healy et al 2005;Zhu et al 2010b). It also had a broader scope than previous methods and had the advantage of controlling stimulus intensity in a particular range.…”