3D Bioprinting Solutions (Russia)

Preservatives, including antimicrobial compounds, are routinely added to home and personal care products such as dishwashing detergents to prevent microbial contamination during repeated consumer use. Traditional antimicrobial efficacy testing (T-AET) relies on colony counting methods, where microbial cocktails are inoculated into preservative-containing products and plated on agar to measure microbial survival. While considered a gold standard, T-AET is time-consuming, labor-intensive, costly, and prone to variability and error due to manual handling. To address these limitations, we developed a high-throughput, miniature, ATP-based, on-chip AET (O-AET) using a 384PillarPlate platform. Pseudomonas aeruginosa, Enterobacter cloacae, and Staphylococcus aureus were encapsulated in 3% agarose and exposed to test compounds and detergent formulations. ATP levels in viable microbes were quantified using the BacTiter-Glo™ luminescence assay to generate dose-response curves, IC50 values, and log reduction measurements. Two industrial antimicrobials, Acticide LG and Acticide MBR2, were evaluated, showing CV values below 8%. Nine detergent formulations from Colgate-Palmolive, including both fresh and aged samples (stored for 8 weeks at 40°C), were tested. Log reduction data from O-AET showed strong correlation with T-AET. Using a 3-log reduction cutoff, O-AET achieved 98% sensitivity, 44% specificity, and 71% overall predictivity. The lower specificity was partly attributed to limited true negative samples used and nutrient deficiencies. The O-AET enables significant reduction in reagent volume, rapid turnaround, and compatibility with standard plate readers, making it cost-effective and scalable. This ATP bioluminescence-based platform provides a robust, reproducible, and high-throughput alternative to T-AET, with high potential to streamline product development and preservative screening in consumer care applications.

Magnetic force and gravity are two fundamental forces affecting all living organisms, including bacteria. On Earth, experimentally created magnetic force can be used to counterbalance gravity and place living organisms in conditions of magnetic levitation. Under conditions of microgravity, magnetic force becomes the only force that moves bacteria, providing an acceleration towards areas of the lowest magnetic field and locking cells in this area. In this review, we consider basic principles and experimental systems used to create a magnetic force strong enough to balance gravity. Further, we describe how magnetic levitation is applied in on-Earth microbiological studies. Next, we consider bacterial behavior under combined conditions of microgravity and magnetic force onboard a spacecraft. At last, we discuss restrictions on applications of magnetic force in microbiological studies and the impact of these restrictions on biotechnological applications under space and on-Earth conditions.

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows.

Prenatal diagnostics holds great significance for pregnant women desiring healthy babies. Fetal nucleated red blood cells (fNRBCs), bearing the complete genome of the fetus, have been regarded as an important biomarker for noninvasive prenatal diagnostics (NIPD). The high-performance detection and enrichment of fNRBCs from maternal blood, especially during early pregnancy, is urgently needed for NIPD, which, unfortunately, remains a big challenge for early-pregnancy fNRBC isolation. In this study, we developed an innovative platform based on silica microbeads for fNRBC isolation and release in early pregnancy. Microbeads were coated with self-assembled MnO2 nanoparticles (SiO2@MnO2) and then modified with a specific antibody. Benefiting from the three-dimensional nanostructure of the MnO2 nanoparticles, the isolation efficiency of the fNRBCs was enhanced. Subsequently, fNRBCs were released via dissolving the MnO2-nanoparticle coating using oxalic acid. We successfully isolated fNRBCs from the maternal peripheral blood samples of 20 pregnant women in the early pregnancy period, ranging from 41 to 62 gestational days. More importantly, the fetal origin of isolated cells was confirmed via fluorescent in situ hybridization and short tandem repeat analysis. This platform based on SiO2@MnO2 microbeads has verified the existence of fNRBCs in early-pregnancy maternal blood and is a promising approach for NIPD in early pregnancy.

Chronic infections are associated with the formation of nonattached biofilm-like aggregates. In vitro models of surface-attached biofilms do not always accurately mimic these processes. Here, we tested a new approach to create in vitro nonattached bacterial aggregates using the principle of magnetic levitation of biological objects placed into a magnetic field gradient. Bacteria grown under magnetic levitation conditions formed nonattached aggregates that were studied with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) and characterized quantitatively. Nonattached aggregates consisted of bacteria submerged into an extracellular matrix and demonstrated features characteristic of biofilms, such as a polymeric matrix that binds Ruby Red and Congo red dyes, a prerequisite of bacterial growth, and increased resistance to gentamicin. Three quantitative parameters were explored to characterize strain-specific potential to form nonattached aggregates: geometric sizes, relative quantities of aggregated and free-swimming bacteria, and Congo red binding. Among three tested Escherichia coli strains, one strain formed nonattached aggregates poorly, and for this strain, all three of the considered parameters were different from those of the other two strains (P < 0.05). Further, we characterized biofilm formation on plastic and agar surfaces by these strains and found that good biofilm formation ability does not necessarily indicate good nonattached aggregate formation ability, and vice versa. The model and quantitative methods can be applied for in vitro studies of nonattached aggregates and modeling bacterial behavior in chronic infections, as it is important to increase our understanding of the role that nonattached bacterial aggregates play in the pathogenesis of chronic diseases.IMPORTANCE An increasing amount of evidence indicates that chronic infections are associated with nonattached biofilm-like aggregates formed by pathogenic bacteria. These aggregates differ from biofilms because they form under low-shear conditions within the volume of biological fluids and they do not attach to surfaces. Here, we describe an in vitro model that provides nonattached aggregate formation within the liquid volume due to magnetic levitation. Using this model, we demonstrated that despite morphological and functional similarities of nonattached aggregates and biofilms, strains that exhibit good biofilm formation might exhibit poor nonattached aggregate formation, suggesting that mechanisms underlying the formation of biofilms and nonattached aggregates are not identical. The magnetic levitation approach can be useful for in vitro studies of nonattached aggregate formation and simulation of bacterial behavior in chronic infections.

In traditional tissue engineering, synthetic or natural scaffolds are usually used as removable temporal support, which involves some biotechnology limitations. The concept of "scaffield" approach utilizing the physical fields instead of biomaterial scaffold has been proposed recently. In particular, a combination of intense magnetic and acoustic fields can enable rapid levitational bioassembly of complex-shaped 3D tissue constructs from tissue spheroids at low concentration of paramagnetic agent (gadolinium salt) in the medium. In the current study, the tissue spheroids from human bladder smooth muscle cells (myospheres) are used as building blocks for assembling the tubular 3D constructs. Levitational assembly is accomplished at low concentrations of gadolinium salts in the high magnetic field at 9.5T. The biofabricated smooth muscle constructs demonstrate contraction after the addition of vasoconstrictive agent endothelin-1. Thus, hybrid magnetoacoustic levitational bioassembly is considered as a new technology platform in the emerging field of formative biofabrication. This novel technology of scaffold-free, nozzle-free, and label-free bioassembly opens a unique opportunity for rapid biofabrication of 3D tissue and organ constructs with complex geometry.

Bioprinting is a rapidly emerging biomedical research field. Three-dimensional bioprinting is defined as a robotic additive, layer-by-layer biofabrication of functional tissues and organs from living cells, and biomaterials according to a digital model. Bioprinting can revolutionize medicine by automated robotic production of human tissues and organs suitable for transplantation. Bioprinting is based on sophisticated high technology, and it is obvious that only technologically advanced countries can make a real contribution to this rapidly evolving multidisciplinary field. In this paper, we present main Russia’s achievements in bioprinting. Here, we also discuss challenges and perspectives of bioprinting research and development in Russia. Russian researchers already made some impressive contributions with long-lasting impact and they have capacities, potential, and ambitions to continue contribute to the advancements of bioprinting.

The calcium phosphate particles can be used as building blocks for fabrication of 3D scaffolds intended for bone tissue engineering. This work presents for the first time a rapid creation of 3D scaffolds using magnetic levitation of calcium phosphate particles. Namely, tricalcium phosphate particles of equal size and certain porosity are used, which undergo the process of recrystallization after magnetic levitational assembly of the scaffold to ensure stitching of the scaffold. Label-free levitational assembly is achieved by using a custom-designed magnetic system in the presence of gadolinium salts, which allows the levitation of calcium phosphate particles. Chemical transformation of tricalcium- to octacalcium phosphate under the condition of magnetic levitation in non-homogeneous magnetic field is also demonstrated. This approach allows obtaining rapidly the octacalcium phosphate phase in the final 3D product, which is biocompatible.