Correction: Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing.

Correction for 'Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing' by Ketaki Bachal et al., Lab Chip, 2023, 23, 261-271, https://doi.org/10.1039/D2LC00879C.

Design and validation of a flowless gradient generating microfluidic device for high-throughput drug testing.

Drug testing is a vital step in the identification of the potential efficacy of any new/existing drug and/or combinations of drugs. The conventional methods of testing the efficacy of new drugs using multiwell plates are time consuming and prone to evaporation loss and manual error. Microfluidic devices with automated generation of concentration gradients provide a promising alternative. The implementation of such microfluidic devices is still limited owing to the additional expertise and facilities required to fabricate and run these devices. Conventional microfluidic devices also need pumps, tubing, valves, and other accessories, making them bulky and non-portable. To address these problems, we have developed a method for fabricating microfluidic structures using a nonconventional technique by exploiting the Saffman-Taylor instability in lifted Hele-Shaw cells. Multi-channel structure molds with varying dimensions were fabricated by shaping ceramic polymer slurry and retaining the shape. Further using the mold thus made, polydimethyl siloxane (PDMS) devices offering static, stable, diffusion-based gradients were casted using soft lithography. We have demonstrated with COMSOL simulation, as well as using fluorescein isothiocyanate (FITC), a fluorescent dye, that the concentration gradient can be generated in this device, which remains stable for at least 5 days. Using this multichannel device, in vitro drug efficacy was validated with two drugs namely, temozolomide (TMZ) and curcumin, one FDA approved and one under research, on glioblastoma cells (U87MG). The resulting IC50 values were consistent with those reported in the literature. We have also demonstrated the possibility of conducting molecular assays post-drug testing in the device by microtubule staining after curcumin treatment on cervical cancer cells (HeLa). In summary, we have demonstrated a i) user-friendly, ii) portable, static drug testing platform that iii) does not require further accessories and can create iv) a stable gradient for a long duration. Such a device can reduce the time, manual errors, fabrication and running expenditure, and resources needed to a great extent in drug testing.

Scalable large-area mesh-structured microfluidic gradient generator for drug testing applications.

Microfluidic concentration gradient generators are useful in drug testing, drug screening, and other cellular applications to avoid manual errors, save time, and labor. However, expensive fabrication techniques make such devices prohibitively costly. Here, in the present work, we developed a microfluidic concentration gradient generator (µCGG) using a recently proposed non-conventional photolithography-less method. In this method, ceramic suspension fluid was shaped into a square mesh by controlling Saffman Taylor instability in a multiport lifted Hele-Shaw cell (MLHSC). Using the shaped ceramic structure as the template, µCGG was prepared by soft lithography. The concentration gradient was characterized and effect of the flow rates was studied using COMSOL simulations. The simulation result was further validated by creating a fluorescein dye (fluorescein isothiocanate) gradient in the fabricated µCGG. To demonstrate the use of this device for drug testing, we created various concentrations of an anticancer drug-curcumin-using the device and determined its inhibitory concentration on cervical cancer cell-line HeLa. We found that the IC50 of curcumin for HeLa matched well with the conventional multi-well drug testing method. This method of µCGG fabrication has multiple advantages over conventional photolithography such as: (i) the channel layout and inlet-outlet arrangements can be changed by simply wiping the ceramic fluid before it solidifies, (ii) it is cost effective, (iii) large area patterning is easily achievable, and (iv) the method is scalable. This technique can be utilized to achieve a broad range of concentration gradient to be used for various biological and non-biological applications.

Compartmentalized microfluidic device for in vitro co-culture of retinal cells.

The investigation is focused on the development of a compartmentalized microfluidic device for coculturing the cells of crucial retinal cellular layers and assessing cell-to-cell interactions. A perfusion-based microfluidic co-culture device was employed and computationally validated for determining the pressure drop and fluid flow rate within the device microchannels. Fabrication was performed using PDMS polymer and coating of fibronectin and collagen facilitated adherence of the cells over the glass surface. Microfluidic device successfully supported cell proliferation, under continuous perfusion of 1 µl min-1 flow rate. The barrier integrity of this coculture was confirmed by evaluating the permeability of fluorescently labeled molecules. The coculture expressed characteristic phenotypic protein markers like recoverin, PAX6, for retinal precursor cells, and RPE65 for retinal epithelial cells. The coculture also exhibited basal expression of TNF-α under normal conditions. Differentiated photoreceptor cells positively expressed rhod inherently possess sensitivity toward violet/blue light, which was validated in R28 cells by exposure to light having a wavelength of 405 nm, which significantly decreased cell viability via increased TNF-α production and reduced rhodopsin expression. This proof-of-concept investigation proved the functionality of the retinal coculture, which may be used as an appropriate perfusion-based, preclinical tool for the evaluation of novel retinal drugs and delivery systems.

Biomimicked large-area anisotropic grooves fromDracaena sanderianaleaf enhances cellular alignment and subsequent differentiation.

Cellular alignment is important for the proper functioning of different tissues such as muscles or blood vessel walls. Hence, in tissue engineering, sufficient effort has been made to control cellular orientation and alignment. It has been shown that micro-and nanoscale anisotropic topological features on cell culture substrates can control cellular orientation. Such substrates are fabricated using various lithography techniques such as photolithography and soft lithography. Although such techniques are suitable for creating patterns in small areas to establish a proof-of-concept, patterning large areas with intricate features is an unsolved problem. In this work, we report that a replica of the groove-like anisotropic patterns of the abaxial side of aDracaena sanderiana(bamboo) leaf can be used for large-area patterning of cells. We imprinted the leaf on polydimethylsiloxane (PDMS) and characterised its surface topography using scanning electron microscopy. We further cultured bone marrow human mesenchymal cells (BM-hMSCs), skeletal muscle cells (C2C12), and neuroblastoma cells (SHSY5Y) on the patterned PDMS on which the cells orient along the direction of the grooved pattern. Further, we observed enhanced neuronal differentiation of SHSY5Y cells on biomimicked pattern compared to flat PDMS as measured by percentage of cells with neurites, neurite length and the expression of neuronal differentiation marker beta-III tubulin (TUJ1). This process is simple, frugal, and can be adopted by laboratories with resource constraints. This one-step technique to fabricate large-area anisotropic surface patterns from bamboo leaves can be used as a platform to study cellular alignment and its effect on various cellular functions, including differentiation.

Biomimicked hierarchical 2D and 3D structures from natural templates: applications in cell biology.

Intricate structures of natural surfaces and materials have amazed people over the ages. The unique properties of various surfaces also created interest and curiosity in researchers. In the recent past, with the advent of superior microscopy techniques, we have started to understand how these complex structures provide superior properties. With that knowledge, scientists have developed various biomimicked and bio-inspired surfaces for different non-biological applications. In the last two decades, we have also started to learn how structures of the tissue microenvironment influence cell function and behaviour, both in physiological and pathological conditions. Hence, it became essential to decipher the role and importance of structural hierarchy in the cellular context. With advances in microfabricated techniques, such complex structures were made by superimposing features of different dimensions. However, the fabricated topographies are far from matching the complexities presentin vivo. Hence, the need of biomimicking the natural surfaces for cellular applications was felt. In this review, we discuss a few examples of hierarchical surfaces found in plants, insects, and vertebrates. Such structures have been widely biomimicked for various applications but rarely studied for cell-substrate interaction and cellular response. Here, we discuss the research work wherein 2D hierarchical substrates were prepared using biomimicking to understand cellular functions such as adhesion, orientation, differentiation, and formation of spheroids. Further, we also present the status of ongoing research in mimicking 3D tissue architecture using de-cellularized plant-based and tissue/organ-based scaffolds. We will also discuss 3D printing for fabricating 2D and 3D hierarchical structures. The review will end by highlighting the various advantages and research challenges in this approach. The biomimickedin-vivolike substrate can be used to better understand cellular physiology, and for tissue engineering.