Streamlined Specimen Purification for Rapid COVID-19 Diagnosis Using Positively Charged Polymer Thin Film-Coated Surfaces and Chamber Digital PCR.

The ongoing coronavirus disease 2019 (COVID-19) pandemic demands rapid and straightforward diagnostic tools to prevent early-stage viral transmission. Although nasopharyngeal swabs are a widely used patient sample collection method for diagnosing COVID-19, using these samples for diagnosis without RNA extraction increases the risk of obtaining false-positive and -negative results. Thus, multiple purification steps are necessary, which are time-consuming, generate significant waste, and result in substantial sample loss. To address these issues, we developed surface-modified polymerase chain reaction (PCR) tubes using the tertiary aminated polymer poly(2-dimethylaminomethylstyrene) (pDMAMS) via initiated chemical vapor deposition. Introducing the clinical samples into the pDMAMS-coated tubes resulted in approximately 100% RNA capture efficiency within 25 min, which occurred through electrostatic interactions between the positively charged pDMAMS surface and the negatively charged RNA. The captured RNA is then detected via chamber digital PCR, enabling a sensitive, accurate, and rapid diagnosis. Our platform provides a simple and efficient RNA extraction and detection strategy that allows detection from 22 nasopharyngeal swabs and 21 saliva specimens with 0% false negatives. The proposed method can facilitate the diagnosis of COVID-19 and contribute to the prevention of early-stage transmission.

Long-Term Culture of Human Pluripotent Stem Cells in Xeno-Free Condition Using Functional Polymer Films.

Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies.

Heparin-mediated electrostatic immobilization of bFGF via functional polymer films for enhanced self-renewal of human neural stem cells.

Preserving the self-renewal capability of undifferentiated human neural stem cells (hNSCs) is one of the crucial prerequisites for efficient hNSC-based regenerative medicine. Considering that basic fibroblast growth factor (bFGF) is one of the key contributing factors in maintaining the self-renewal property of hNSCs, the bioactivity and stability of bFGF in the hNSC culture should be regulated carefully. In this study, we developed a functional polymer film of poly(glycidyl methacrylate (GMA)-co-N,N-dimethylaminoethyl methacrylate (DMAEMA)) (coGD, or p(GMA-co-DMAEMA)) via initiated chemical vapor deposition (iCVD), which facilitated a stable, electrostatic adsorption of heparin and subsequent immobilization of bFGF. The bFGF-immobilized coGD surface substantially enhanced the proliferation rate and neurosphere forming ability of hNSCs compared to tissue culture plate (TCP). The expression of the stemness markers of hNSCs such as NESTIN and SOX-2 was also upregulated prominently on the coGD surface. Also, the hNSCs cultured on the coGD surface showed enhanced neurogenesis upon spontaneous differentiation. The immobilized bFGF on the coGD surface stimulated the expression of bFGF receptors and subsequently activated the mitogen-activated protein kinase (MAPK) pathway, attributed to the increase in self-renewal property of hNSCs. Our results indicate that the coGD surface allowed in situ heparin-mediated bFGF immobilization, which served as a robust platform to generate hNSC neurospheres with enhanced self-renewal and differentiation capabilities and thereby will prompt an advance in the field of therapeutics of neurodegenerative diseases.

Three-Dimensional Spheroid Culture on Polymer-Coated Surface Potentiate Stem Cell Functions via Enhanced Cell-Extracellular Matrix Interactions.

The aggregation of mesenchymal stem cells (MSCs) into three-dimensional (3D) spheroids has emerged as a promising therapeutic candidate for the treatment of a variety of diseases. In spite of the numerous 3D culture methods suggested recently for MSC spheroid generation, it is still elusive to fully reflect real stem cell niches; this effort majorly suffers from a lack of cell-extracellular matrix (ECM) interactions within the 3D spheroids. In this study, we develop a simple but versatile method for generating human MSC (hMSC) spheroids by culturing the cells on a functional polymer film surface, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4). Interestingly, the pV4D4-coated surface allows a dynamic cell adhesion to the polymer surface while developing the formation of 3D spheroids. The corresponding mechanotransduction promotes the expression of the endogenous ECM and, in turn, results in a remarkable improvement in self-renewal abilities, pro-angiogenic potency, and multilineage differentiation capabilities. This observation highlights the significance of our method compared to the conventional spheroid-generating methods in terms of recreating the ECM-rich microenvironment. We believe the developed surface can serve as a versatile but reliable method for stem cell-based tissue engineering and regenerative medicine.

A Surface-Tailoring Method for Rapid Non-Thermosensitive Cell-Sheet Engineering via Functional Polymer Coatings.

Cell sheet engineering, a technique utilizing a monolayer cell sheet, has recently emerged as a promising technology for scaffold-free tissue engineering. In contrast to conventional tissue-engineering approaches, the cell sheet technology allows cell harvest as a continuous cell sheet with intact extracellular matrix proteins and cell-cell junction, which facilitates cell transplantation without any other artificial biomaterials. A facile, non-thermoresponsive method is demonstrated for a rapid but highly reliable platform for cell-sheet engineering. The developed method exploits the precise modulation of cell-substrate interactions by controlling the surface energy of the substrate via a series of functional polymer coatings to enable prompt cell sheet harvesting within 100 s. The engineered surface can trigger an intrinsic cellular response upon the depletion of divalent cations, leading to spontaneous cell sheet detachment under physiological conditions (pH 7.4 and 37 °C) in a non-thermoresponsive manner. Additionally, the therapeutic potential of the cell sheet is successfully demonstrated by the transplantation of multilayered cell sheets into mouse models of diabetic wounds and ischemia. These findings highlight the ability of the developed surface for non-thermoresponsive cell sheet engineering to serve as a robust platform for regenerative medicine and provide significant breakthroughs in cell sheet technology.

Microfluidic acoustophoretic force based low-concentration oil separation and detection from the environment.

Detecting and quantifying extremely low concentrations of oil from the environment have broad applications in oil spill monitoring in ocean and coastal areas as well as in oil leakage monitoring on land. Currently available methods for low-concentration oil detection are bulky or costly with limited sensitivities. Thus they are difficult to be used as portable and field-deployable detectors in the case of oil spills or for monitoring the long-term effects of dispersed oil on marine and coastal ecosystems. Here, we present a low-concentration oil droplet trapping and detection microfluidic system based on the acoustophoresis phenomenon where oil droplets in water having a negative acoustic contrast factor move towards acoustic pressure anti-nodes. By trapping oil droplets from water samples flowing through a microfluidic channel, even very low concentrations of oil droplets can be concentrated to a detectable level for further analyses, which is a significant improvement over currently available oil detection systems. Oil droplets in water were successfully trapped and accumulated in a circular acoustophoretic trapping chamber of the microfluidic device and detected using a custom-built compact fluorescent detector based on the natural fluorescence of the trapped crude oil droplets. After the on-line detection, crude oil droplets released from the trapping chamber were successfully separated into a collection outlet by acoustophoretic force for further off-chip analyses. The developed microfluidic system provides a new way of trapping, detecting, and separating low-concentration crude oil from environmental water samples and holds promise as a low-cost field-deployable oil detector with extremely high sensitivity. The microfluidic system and operation principle are expected to be utilized in a wide range of applications where separating, concentrating, and detecting small particles having a negative acoustic contrast factor are required.