Droplets for Gene Editing Using CRISPR-Cas9 and Clonal Selection Improvement Using Hydrogels.

Gene editing tools have triggered a revolutionary transformation in the realms of cellular and molecular physiology, serving as a fundamental cornerstone for the evolution of disease models and assays in cell culture reactions, marked by various enhancements. Concurrently, microfluidics has emerged over recent decades as a versatile technology capable of elevating performance and reducing costs in daily experiments across diverse scientific disciplines, with a pronounced impact on cell biology. The amalgamation of these groundbreaking techniques holds the potential to amplify the generation of stable cell lines and the production of extracellular matrix hydrogels. These hydrogels, assuming a pivotal role in isolating cells at the single-cell level, facilitate a myriad of analyses. This study presents a novel method that seamlessly integrates CRISPR-Cas9 gene editing techniques with single-cell isolation methods in induced pluripotent stem cell (hiPSC) lines, utilizing the combined power of droplets and hydrogels. This innovative approach is designed to optimize clonal selection, thereby concurrently reducing costs and the time required for generating a stable genetically modified cell line. By bridging the advancements in gene editing and microfluidic technologies, our approach not only holds significant promise for the development of disease models and assays but also addresses the crucial need for efficient single-cell isolation. This integration contributes to streamlining processes, making it a transformative method with implications for enhancing the efficiency and cost-effectiveness of stable cell line generation. As we navigate the intersection of gene editing and microfluidics, our study marks a significant stride toward innovative methodologies in the dynamic landscape of cellular and molecular physiology research.

Simple Microcontact Printing Technique to Obtain Cell Patterns by Lithography Using Grayscale, Photopolymer Flexographic Mold, and PDMS.

Microcontact printing using PDMS embossing tools and its variations have aroused the interest of a wide spectrum of research fields, hence the feasibility of defining micro and nanoscale patterns. In this work, we have proposed and demonstrated a novel lithography method based on grayscale patterns printed in a flexographic photopolymer mold and transferred to epoxy resin and a single PDMS stamp to obtain different microprint pattern structures. The geometry of the patterns can be modified by adjusting the layout and grayscale of the stamp patterns. The functionality of this contact printing methodology was validated by generating human induced pluripotent stem cells (hiPSC) patterns. These specific micropatterns can be very useful for achieving complex differentiation in cell lines such as hiPSC. Microfabrication through the new technique provides a promising alternative to conventional lithography for constructing complex aligned surfaces; these structures could be used as components of biological patterns or microfluidic devices.

GEMA-An Automatic Segmentation Method for Real-Time Analysis of Mammalian Cell Growth in Microfluidic Devices.

Nowadays, image analysis has a relevant role in most scientific and research areas. This process is used to extract and understand information from images to obtain a model, knowledge, and rules in the decision process. In the case of biological areas, images are acquired to describe the behavior of a biological agent in time such as cells using a mathematical and computational approach to generate a system with automatic control. In this paper, MCF7 cells are used to model their growth and death when they have been injected with a drug. These mammalian cells allow understanding of behavior, gene expression, and drug resistance to breast cancer. For this, an automatic segmentation method called GEMA is presented to analyze the apoptosis and confluence stages of culture by measuring the increase or decrease of the image area occupied by cells in microfluidic devices. In vitro, the biological experiments can be analyzed through a sequence of images taken at specific intervals of time. To automate the image segmentation, the proposed algorithm is based on a Gabor filter, a coefficient of variation (CV), and linear regression. This allows the processing of images in real time during the evolution of biological experiments. Moreover, GEMA has been compared with another three representative methods such as gold standard (manual segmentation), morphological gradient, and a semi-automatic algorithm using FIJI. The experiments show promising results, due to the proposed algorithm achieving an accuracy above 90% and a lower computation time because it requires on average 1 s to process each image. This makes it suitable for image-based real-time automatization of biological lab-on-a-chip experiments.

Novel Reproducible Manufacturing and Reversible Sealing Method for Microfluidic Devices.

Conventional manufacturing methods for polydimethylsiloxane (PDMS)-based microdevices require multiple steps and elements that increase cost and production time. Also, these PDMS microdevices are mostly limited to single use, and it is difficult to recover the contents inside the microchannels or perform advanced microscopy visualization due to their irreversible sealing method. Herein, we developed a novel manufacturing method based on polymethylmethacrylate (PMMA) plates adjusted using a mechanical pressure-based system. One conformation of the PMMA plate assembly system allows the reproducible manufacture of PDMS replicas, reducing the cost since a precise amount of PDMS is used, and the PDMS replicas show uniform dimensions. A second form of assembling the PMMA plates permits pressure-based sealing of the PDMS layer with a glass base. By reversibly sealing the microdevice without using plasma for bonding, we achieve chip on/off configurations, which allow the user to open and close the device and reuse it in an easy-to-use way. No deformation was observed on the structures of the PDMS microchannels when a range of 10 to 18 kPa pressure was applied using the technique. Furthermore, the functionality of the proposed system was successfully validated by the generation of microdroplets with reused microdevices via three repetitions.

Single cell transfection of human-induced pluripotent stem cells using a droplet-based microfluidic system.

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research in fields such as physics, engineering, chemistry and biology have combined to produce innovation in microfluidics which has positively impacted diverse areas such as nucleotide sequencing, functional genomics, single-cell studies, single molecules assays and biomedical diagnostics. Among these areas, regenerative medicine and stem cells have benefited from microfluidics since these tools have had a profound impact on their applications. In this study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We will demonstrate that this system has great efficiency in single cells and captured droplets, like other microfluidic methods but with lower cost. Moreover, this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations and cell therapy, among other potential applications.