Björn Harink

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

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.

1.19 Calcium Phosphate Ceramics With Inorganic Additives

The use of inorganic compounds as synthetic growth factors is a promising approach for improving the biological properties of existing synthetic bone graft substitutes such as calcium phosphates. In this article we have described some of the inorganic additives that may improve the capabilities of calcium phosphates, and help bridge the gap toward autograft׳s performance known as gold standard for bone regeneration. This article focuses on the specific roles of bioinorganics in processes related to bone formation and resorption and how these modify the biological properties of calcium phosphates, and finally provides insight into the future of this field.

Development of a shear stress-free microfluidic gradient generator capable of quantitatively analyzing single-cell morphology

Microfluidics, the science of engineering fluid streams at the micrometer scale, offers unique tools for creating and controlling gradients of soluble compounds. Gradient generation can be used to recreate complex physiological microenvironments, but is also useful for screening purposes. For example, in a single experiment, adherent cells can be exposed to a range of concentrations of the compound of interest, enabling high-content analysis of cell behaviour and enhancing throughput. In this study, we present the development of a microfluidic screening platform where, by means of diffusion, gradients of soluble compounds can be generated and sustained. This platform enables the culture of adherent cells under shear stress-free conditions, and their exposure to a soluble compound in a concentration gradient-wise manner. The platform consists of five serial cell culture chambers, all coupled to two lateral fluid supply channels that are used for gradient generation through a source-sink mechanism. Furthermore, an additional inlet and outlet are used for cell seeding inside the chambers. Finite element modeling was used for the optimization of the design of the platform and for validation of the dynamics of gradient generation. Then, as a proof-of-concept, human osteosarcoma MG-63 cells were cultured inside the platform and exposed to a gradient of Cytochalasin D, an actin polymerization inhibitor. This set-up allowed us to analyze cell morphological changes over time, including cell area and eccentricity measurements, as a function of Cytochalasin D concentration by using fluorescence image-based cytometry.