Cell invasion is a key process in tissue growth, wound healing, and tumor progression. Most invasion assays examine cells cultured in adherent monolayers, which fail to recapitulate the three-dimensional nuances of the tissue microenvironment. Multicellular cell spheroids have a three-dimensional (3D) morphology and mimic the intercellular interactions found in tissues in vivo, thus providing a more physiologically relevant model for studying the tissue microenvironment and processes such as cell invasion. Spheroid-based invasion assays often require tedious, manually intensive handling protocols or the use of robotic liquid handling systems, which can be expensive to acquire, operate, and maintain. Here we describe a digital microfluidic (DμF) platform that enables formation of spheroids by the hanging drop method, encapsulation of the spheroids in collagen, and the exposure of spheroids to migration-modulating agents. Collagen sol-gel solutions up to 4 mg mL(-1), which form gels with elastic moduli up to ∼50 kPa, can be manipulated on the device. In situ spheroid migration assays show that cells from human fibroblast spheroids exhibit invasion into collagen gels, which can be either enhanced or inhibited by the delivery of exogenous migration modulating agents. Exposing fibroblast spheroids to spheroid secretions from colon cancer spheroids resulted in a >100% increase in fibroblast invasion into the collagen gel, consistent with the cancer-associated fibroblast phenotype. These data show that DμF can be used to automate the liquid handling protocols for spheroid-based invasion assays and create a cell invasion model that mimics the tissue microenvironment more closely than two-dimensional culturing techniques do. A DμF platform that facilitates the creation and assaying of 3D in vitro tissue models has the potential to make automated 3D cell-based assays more accessible to researchers in the life sciences.
Digital (droplet) microfluidics (DµF) is a powerful platform for automated lab-on-a-chip procedures, ranging from quantitative bioassays such as RT-qPCR to complete mammalian cell culturing. The simple MEMS processing protocols typically employed to fabricate DµF devices limit their functionality to two dimensions, and hence constrain the applications for which these devices can be used. This paper describes the integration of vertical functionality into a DµF platform by stacking two planar digital microfluidic devices, altering the electrode fabrication process, and incorporating channels for reversibly translating droplets between layers. Vertical droplet movement was modeled to advance the device design, and three applications that were previously unachievable using a conventional format are demonstrated: (1) solutions of calcium dichloride and sodium alginate were vertically mixed to produce a hydrogel with a radially symmetric gradient in crosslink density; (2) a calcium alginate hydrogel was formed within the through-well to create a particle sieve for filtering suspensions passed from one layer to the next; and (3) a cell spheroid formed using an on-chip hanging-drop was retrieved for use in downstream processing. The general capability of vertically delivering droplets between multiple stacked levels represents a processing innovation that increases DµF functionality and has many potential applications.
Acute and chronic hydration status is important for athlete safety and performance and is frequently measured by sports scientists and performance staff in team environments via urinalysis. However, the time required for urine collection, staff testing, and reporting often delays immediate reporting and personalized nutrition insight in situations of acute hydration management before training or competition. Furthermore, the burdensome urine collection and testing process often renders chronic hydration monitoring sporadic or non-existent in real-world settings. An automated urinalysis device (InFlow) was developed to measure specific gravity, an index of hydration status, in real-time during urination. The device was strongly correlated to optical refractometry with a mean absolute error of 0.0029 (±0.0021). Our results show this device provides a novel and useful approach for real-time hydration status via urinalysis for male athletes in team environments with high testing frequency demands.
The Centers for Disease Control and Prevention (CDC) reports 47% of adults in the United States have hypertension [1]. Dietary risks constitute the largest disease risk factor in the US [2], whereby the largest single contributor to that risk is high dietary sodium intake [3].The World Health Organization (WHO) recommends that individuals reduce sodium (Na) and increase potassium (K) intake to lower blood pressure and improve cardiovascular health [4,5]. Emerging research has shown that urinary Na/K ratio is more strongly related to blood pressure, cardiovascular disease, and stroke than Na and K considered separately [6]. Urine Na guided dietary guidance enhances the ability to monitor sodium reduction and blood pressure [7]. Manual collection for chronic urine testing is too burdensome a process for practical everyday use. We have developed an accurate biosensor capable of being easily integrated into users’ daily routine for real-time and fully automated longitudinal Na/K analysis. Reference [1] Centers for Disease Control and Prevention, (2021),https://www.cdc.gov/bloodpressure/facts.htm#:~ :text=Nearly%20half%20of%20adults%20in,are%20taking%20medication%20for%20hypertension. [2] The US Burden of Disease Collaborators; A.H., Mokdad, JAMA, 319(14),1444-1472 (2018). [3] GBD 2017 Diet Collaborators, The Lancet, 393(10184), 1958-1972 (2019). [4] WHO Guideline Sodium intake for adults and children. Geneva: World Health Organization (WHO); 2012. [5] WHO Guideline Potassium intake for adults and children. Geneva: World Health Organization (WHO); 2012. [6] T. Iwahori; K. Miura; H. Ueshima; S. Tanaka-Mizuno; Q. Chan; H. Arima; A. R. Dyer; P. Elliott; J. Stamler, Hypertens Res, 42(10), 1590-1598 (2019). [7] T. Takada; M. Imamoto; S. Sasaki; T. Azuma; J. Miyashita; M. Hayashi; S. Fukuma; S. Fukuhara, Hypertens Res, 41(7), 524-530 (2018). Figure 1 (A) Urinalysis system installed in a urinal that can catch a stream of urine in real-time. (B) Biosensor cyclic voltammetry curve of human urine. Figure 1
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