As nonbiodegradable plastics continue to pollute our land and oceans, countries are starting to ban the use of single-use plastics. In this paper, we demonstrated the fabrication of wood-based microfluidic devices and their adaptability for single-use, point-of-care (POC) applications. These devices are made from easily sourced renewable materials for fabrication while exhibiting all the advantages of plastic devices without the problem of nonbiodegradable waste and cost. To build these wood devices, we utilized laser engraving and traditional mechanical methods and have adapted specific surface coatings to counter the wicking effect of wood. To demonstrate their versatility, wood microfluidic devices were adapted for (i) surface plasmon coupled enhancement (SPCE) of fluorescence for detection of proteins, (ii) T-/Y-geometry microfluidic channel mixers, and (iii) devices for rapid detection of microbial contamination. These provide proof of concept for the use of wooden platforms for POC applications. In this study, we measured the fluorescence intensities of recombinant green fluorescent protein (GFP) standards (ranging from 1.5−25 ng/μL) and 6XHis-G-CSF (ranging from 0.1−100 ng/μL) expressed in cell-free translation systems. All tested devices perform as well as or better than their plastic counterparts.
Several groups have recently reported on the utility of cell-free expression systems to make therapeutic proteins, most of them employing CHO or E. coli cell-free extracts. Here, we propose an alternative that uses human blood derived leukocyte cell extracts for the expression of recombinant proteins. We demonstrate expression of nano luciferase (Nluc), Granulocyte-colony stimulating factor (G-CSF) and Erythropoietin (EPO) in cell-free leukocyte extracts within two hours. Human blood is readily available from donors and blood banks and leukocyte rich fractions are easy to obtain. The method described here demonstrates the ability to rapidly express recombinant proteins from human cell extracts that could provide the research community with a facile technology to make their target protein. Eventually, we envision that any recombinant protein can be produced from patient-supplied leukocytes, which can then be injected back into the patient. This approach could lead to an alternative model for personalized medicines and vaccines.
Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any high-performance liquid chromatography (HPLC) system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared with current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was used to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte colony stimulating factor (G-CSF) expressed using a cell-free CHO in vitro translation (IVT) system and were compared to a conventional 1 ml IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin, and purification time for the μCols compared with conventional columns for similar protein yields.
K E Y W O R D Schromatography, granulocyte colony stimulating factor, immobilized metal affinity chromatography, microfluidics, protein purification
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Historically, some of the first cell-free protein expression systems studied in vitro translation in various human blood cells. However, because of limited knowledge of eukaryotic translation and the advancement of cell line development, interest in these systems decreased. Eukaryotic translation is a complex system of factors that contribute to the overall translation of mRNA to produce proteins. The intracellular translateome of a cell can be modified by various factors and disease states, but it is impossible to individually measure all factors involved when there is no comprehensive understanding of eukaryotic translation. The present work outlines the use of a coupled transcription and translation cell-free protein expression system to produce recombinant proteins derived from human donor peripheral blood mononuclear cells (PBMCs) activated with phytohemagglutinin-M (PHA-M). The methods outlined here could result in tools to aid immunology, gene therapy, cell therapy, and synthetic biology research and provide a convenient and holistic method to study and assess the intracellular translation environment of primary immune cells.
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