Biophysics

Device-induced thrombosis remains a major complication of extracorporeal life support (ECLS). To more thoroughly understand how blood components interact with the artificial surfaces of ECLS circuit components, assessment of clot deposition on these surfaces following clinical use is urgently needed. Scanning electron microscopy (SEM), which produces high-resolution images at nanoscale level, allows visualization and characterization of thrombotic deposits on ECLS circuitry. However, methodologies to increase the quantifiability of SEM analysis of ECLS circuit components have yet to be applied clinically. To address these issues, we developed a protocol to quantify clot deposition on ECLS membrane oxygenator gas transfer fiber sheets through digital and SEM imaging techniques. In this study, ECLS membrane oxygenator fiber sheets were obtained, fixed, and imaged after use. Following a standardized process, the percentage of clot deposition on both digital images and SEM images was quantified using ImageJ through blind reviews. The interrater reliability of quantitative analysis among reviewers was evaluated. Although this protocol focused on the analysis of ECLS membrane oxygenators, it is also adaptable to other components of the ECLS circuits such as catheters and tubing.

Key features

• Quantitative analysis of clot deposition using digital and scanning electron microscopy (SEM) techniques

• High-resolution images at nanoscale level

• Extracorporeal life support (ECLS) devices

• Membrane oxygenators

• Blood-contacting surfaces

Graphical overview

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue’s vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 µm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2–3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies.

When performing renal biopsy, it is necessary to identify the cortex, where glomeruli are exclusively distributed, to ensure the quality of the specimen for histological diagnosis. However, conventional methods using a stereomicroscope or magnifying lens often fail to clarify the quality of the specimen. We have established a fluorescent-based imaging technique for the on-site assessment of renal biopsy specimens. The fluorescent images can be easily obtained by adding an optical filter to the microscope and with a short incubation of an activatable fluorescent probe. This novel imaging technique can be applied to renal biopsy specimens for distinguishing the renal cortex.

The design of effective nanoformulations that target metastatic breast cancers is challenging due to a lack of competent imaging and image analysis protocols that can capture the interactions between the injected nanoparticles and metastatic lesions. Here, we describe the integration of in vivo whole-body PET-CT with high temporal resolution, ex vivo whole-organ optical imaging and high spatial resolution confocal microscopy to deconstruct the trafficking of injectable nanoparticle generators encapsulated with polymeric doxorubicin (iNPG-pDox) in pulmonary metastases of triple-negative breast cancer. We describe the details of image acquisition and analysis in a step-wise manner along with the development of a mouse model for metastatic breast cancer. The methods described herein can be easily adapted to any nanoparticle or disease model, allowing a standardized pipeline for in vivo preclinical studies that focus on delineating nanoparticle kinetics and interactions within metastases.

Synthetic nanoparticle-based drug delivery system is widely known for its ability to increase the efficacy and specificity of loaded drugs, but it often suffers from relatively higher immunotoxicity and higher costs as compared to traditional drug formulations. Contrarily, plant-derived nanoparticles appear to be free from these limitations of synthetic nanoparticles; they are naturally occurring biocompatible vesicles that do not generate immunotoxicity and are easy to obtain. Additionally, lipids isolated from plant-derived nanoparticles have shown the capability of assembling themselves to spherical nano-sized liposomal particles. Herein, we employ lipids extracted from ginger-derived nanoparticles and load them with therapeutic siRNA (CD98-siRNA) to create CD98-siRNA/ginger-lipid nanoparticles. Characterization of the CD98-siRNA/ginger-lipid nanoparticles showed that they present a spherical shape, with a diameter of around 189.5 nm. The surface zeta potential of the nanoparticles varies from -18.1 to -18.4 mV. Furthermore, in recent research, the CD98-siRNA/ginger-lipid nanoparticles have shown specific colon targeting capability and excellent anti-inflammatory efficacy in a Dextran Sodium Sulfate (DSS) induced mouse model of colitis.

Biomaterial-associated thrombosis is still a major concern for blood-contacting implants. After the medical device is implanted and comes in contact with blood, several complex reactions occur, which may lead to thrombus formation and failure of the device. Therefore, it is essential to evaluate the biomaterial interaction with the whole blood. Several studies have been reported in the literature that evaluate different steps in the coagulation cascade, such as protein adsorption, plasma activation, and platelet adhesion in vitro, however, evaluation of whole blood clotting on biomaterial surfaces is not widely reported. Here, a protocol to evaluate whole blood clotting in vitro on 2D biomaterials surfaces via a simple and fast hemolysis assay is presented. Whole human blood is placed onto the biomaterial surfaces and is allowed to clot for different time periods. After the specific time intervals, the surfaces are transferred into deionized (DI) water to release the free hemoglobin and the absorbance of this solution is measured. The absorbance value is proportional to the free hemoglobin concentration in the DI water due to lysis of red blood cells and gives an indirect correlation to the extent of blood clotting on the biomaterial surfaces. This protocol provides a fast, facile and effective method to measure the anti-thrombogenic properties of biomaterials.

After silk fiber is degummed in boiling 0.2% Na₂CO₃ solution, the degummed fibroin fiber could be dissolved in highly concentrated neutral salts such as CaCl₂. The partially degraded polypeptides of silk fibroin, commonly called as regenerated liquid silk, could be obtained via water dialysis. The silk fibroin nanoparticles (SFNs) or enzyme-entrapped SFNs are prepared rapidly from the liquid silk by using acetone. The globular particles with a range of 35-125 nm in diameter, are water-insoluble but well dispersed and stable in aqueous solution. The nanoparticles are potentially useful in biomaterials such as cosmetics, anti-UV skincare products, and surface improving materials, especially in enzyme/drug delivery system as vehicle. Here, a detailed protocol for SFNs and enzyme-entrapped SFNs preparation is described.