Biomimetic Hydrogels - Tools for Regenerative Medicine, Oncology, and Understanding Medical Gas Plasma Therapy.

Novel non-thermal food processing and preservation methods is in demand due to reduced nutritional modification after the processes. There are several non-thermal processing techniques are in practice, in that Cold Plasma (CP) technology is one of the low cost and energy efficient technique. Plasma is a form of ionized gas (containing balanced charges of ions and electrons) that is generated by a neutral gas. Plasma can interact and destruct microbial cells, spores and viruses by etching. Plasma can be generated in four different modes, using different techniques. Cold plasma technique is effective in pathogenic microbial decontamination which are present in food grains, meat products, milk, fruits and vegetables. It can also be used in effluent treatment in food industry because food industry sewage contains more reactive oxygen species. In addition to these cold plasma technique has some limitations while treating the sensitive food products due to degradation of oligosaccharides and lipid oxidation. These review article discuss the different cold plasma production techniques and its application in food processing industries.

In modern oncology, therapies are based on combining monotherapies to overcome treatment resistance and increase therapy precision. The application of microsecond-pulsed electric fields (PEF) is approved to enhance local chemotherapeutic drug uptake within combination electrochemotherapy regimens. Reactive oxygen species (ROS) have been implicated in anticancer effects, and cold physical plasma produces vast amounts of ROS, which have recently been shown to benefit head and neck cancer patients. PEF and cold plasma technology have been linked to immunogenic cell death (ICD) induction, a regulated cell death accompanied by sterile inflammation that promotes antitumor immunity. To this end, we investigated the combined effect of both treatments regarding their intracellular ROS accumulation, toxicity, ICD-related marker expression, and optimal exposure sequence in a leukemia model cell line. The combination treatment substantially increased ROS and intracellular glutathione levels, leading to additive cytotoxic effects accompanied by a significantly increased expression of ICD markers, such as the eat-me signal calreticulin (CRT). Preconditioned treatment with cold plasma followed by PEF exposure was the most potent treatment sequence. The results indicate additive effects of cold plasma and PEF, motivating further studies in skin and breast tumor models for the future improvement of ECT in such patients.

Event Abstract Back to Event Chondrogenic differentiation of human induced pluripotent stem cells in a photoclickable biomimetic PEG hydrogel Elizabeth Aisenbrey1*, Karin Payne2, Ganna Bilousova2 and Stephanie J. Bryant1* 1 University of Colorado, Chemical and Biological Engineering, United States 2 University of Colorado, Anschutz Medical Campus, United States Introduction: Cartilage lacks the ability to regenerate on its own, resulting in the necessity for alternative treatments such as tissue engineering[1]. The overall goal of this study was to develop a tissue engineering strategy for patient-specific repair of cartilage. Induced pluripotent stem cells (iPSCs) are an attractive cell source for cartilage tissue engineering because they can be obtained from a patient in a less invasive way than autologous chondrocytes or bone marrow derived MSCs, and unlike hMSCs, iPSCs have unlimited proliferation potential and the ability to differentiate into any cell in the body[2],[3]. In this study, human iPSCs were encapsulated into a photoclickable cartilage-like biomimetic poly(ethylene glycol) (PEG) hydrogel functionalized with chondroitin sulfate (ChS) and the cell adhesion peptide, RGD. Chondrogenesis was assessed in the cell-laden hydrogels and as a function of two growth factors TGFβ3 alone, BMP2 alone, or together. Materials and Methods: Human skin fibroblasts from a 50 year old female (ATCC) were reprogrammed under low oxygen (5%) conditions via mRNA transfection to generate iPSCs. These iPSCs were induced to become mesenchymal progenitors (iPSC-MPs) and were encapsulated in a hydrogel (9wt% 8-arm PEG(10kDa)norbornene, 1wt% thiolated ChS (16% conjugated), 1.4wt% PEG(1kDa)dithiol, and 0.1mM CYRGDS) via photopolymerization (7 minutes, 352 nm, 5 mW/cm2). The cell-laden hydrogels were cultured for 21 days in chondrogenic differentiation media without and with growth factors (+TGFB3 (2.5ng/ml), +BMP2 (25ng/ml), +TGFB3 (2.5 ng/ml) +BMP2 (25 ng/ml)). The iPSC-MPs were cultured as pellets in identical conditions or with media containing higher concentrations of growth factors ((+TGFB3 (10ng/ml), +BMP2 (100ng/ml), +TGFB3 (10ng/ml) +BMP2 (100ng/ml)). Differentiation was determined by qRT-PCR and immunohistochemistry for chondrogenic specific markers, sox-9, aggrecan, and collagen II and the hypertrophic marker collagen X. Results: After 21 days of culture, the expression of the chondrogenic markers sox9, aggrecan and collagen II were upregulated in the presence of TGFB3 and/or BMP2 compared to the constructs cultured without growth factors. Protein deposition of collagen II and aggrecan were found in all constructs cultured with TGFB3 and/or BMP2, although gene expression shows higher collagen II in constructs only cultured with BMP2. The relative gene expression of the hypertrophic marker collagen X was lower in the hydrogels cultured in BMP2 compared to those cultured in the presence of TGFB3, however, protein deposition was present. Similar results were obtained with the pellet culture when higher concentrations of growth factors were used. Chondrogenesis was not observed at the growth factor concentrations used with the hydrogel. Discussion and Conclusion: This study investigated if iPSCs, a novel cell source for articular cartilage tissue engineering, undergo chondrogenesis when cultured in a cartilage biomimetic hydrogel. The results from this study suggest that the presence of TGFB3 and/or BMP2 enhances chondrogenesis of iPSCs. Interestingly, lower concentrations of growth factors were needed for iPSCs to undergo chondrogenesis when in the hydrogel constructs compared to the pellet culture, suggesting that the biomimetic hydrogel is a promising scaffold for cartilage engineering. Studies are underway to investigate the effect of dynamic compression at physiological conditions on iPSCs. NSF Graduate Research Fellowship; NSF Career Award Grant # 0847390; Academic Enrichment Fund of the University of Colorado School of Medicine

Chronic wounds place a considerable burden on patients and healthcare systems. Cold plasma technology has become increasingly established for the treatment of people with chronic wounds in recent years. Currently, various cold plasma devices are available. These all generate body-temperature plasmas as well as reactive oxygen and nitrogen species (ROS/RNS). They have not only antimicrobial effects, but also increase dermal blood flow and enhance cell proliferation. Thus, cold plasma-mediated, accelerated wound healing is based on several biological effects that synergistically act within one treatment. Therefore, the AWMF S2k guideline on the rational therapeutic use of cold physical plasma, published in 2022, recommends wound care using cold plasma technology. This article provides an overview of the clinical evidence, potential mechanisms of action, and safety aspects of the therapeutic use of cold plasma technology in chronic wounds.

On behalf of the JALA scientific advisors and JALA editorial board, I am happy to present this year’s honorees of the prestigious JALA Ten. Each year, JALA seeks to highlight and honor the very best work of the year that will have a deep impact on how technology is used across a wide range of disciplines, including automation, life sciences and biomedical research, diagnostics, drug delivery, and regenerative medicine. Implementing the latest advances in microfluidics, nanotechnology, materials science, and other fields of research, this year’s JALA Ten honorees have and will continue to change the way research is performed and the way diseases are diagnosed and treated. As such, the work highlighted here should have far-reaching impact in our everyday lives. It demonstrates the promise that science brings toward a better future. While a number of areas of research will feel the impact of this year’s JALA Ten, one highlight of the collection is the diverse ways in which the honorees have advanced biological molecule detection and established foundations for tomorrow’s biosensors in life sciences research and medical diagnostics.1Lillehoj P.B. Kaplan C.W. He J. et al.Rapid, Electrical Impedance Detection of Bacterial Pathogens Using Immobilized Antimicrobial Peptides.J. Lab. Autom. 2014; 19: 42-49Google Scholar, 2Lee S.E. Chen Q. Bhat R. et al.Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules.Nano Lett. 2015; 15: 4564-4570Google Scholar, 3Yeo D. Wiraja C. Chuah Y.J. et al.A Nanoparticle-Based Sensor Platform for Cell Tracking and Status/Function Assessment.Sci. Rep. 2015; 5 (DOI: 10.1038/srep14768.): 14768Google Scholar For example, Peter Lillehoj at Michigan State University (USA) and his collaborators have developed a microfluidic biosensor using immobilized antimicrobial peptides for highly specific, multiplexed detection of bacterial pathogens.1Lillehoj P.B. Kaplan C.W. He J. et al.Rapid, Electrical Impedance Detection of Bacterial Pathogens Using Immobilized Antimicrobial Peptides.J. Lab. Autom. 2014; 19: 42-49Google Scholar Showing both high pathogen detection specificity and accurate pathogen quantification, this work is an example of how microfluidic technology is changing how medical professionals will track and diagnose infectious diseases. On the other end of the spectrum, Somin Eunice Lee at the University of Michigan (USA) has harnessed the power of nanotechnology to develop a gold nanoparticle–based plasmon ruler that is capable of single-molecule measurements.2Lee S.E. Chen Q. Bhat R. et al.Reversible Aptamer-Au Plasmon Rulers for Secreted Single Molecules.Nano Lett. 2015; 15: 4564-4570Google Scholar Even more impressive is the application of this plasmon ruler toward the accurate detection and measurement of secreted single molecules in the cellular microenvironment. Understanding how cells communicate with their cellular microenvironment remains a challenge to study. Tools such as Lee’s plasmon ruler vastly improve life sciences researchers’ abilities to learn more about the interplay between cells and their microenvironments. Another area of research highlighted this year is the advancement of technology toward customizable platforms for increased personalized clinical and research applications.4Griffin D.R. Weaver W.M. Scumpia P.O. et al.Accelerated Wound Healing by Injectable Microporous Gel Scaffolds Assembled from Annealed Building Blocks.Nat. Mater. 2015; 14 (DOI: 10.1038/nmat4294.): 737-744Google Scholar, 5Huang P.H. Nama N. Mao Z. et al.A Reliable, Programmable Acoustofluidic Pump Powered by Oscillating Sharp-Edge Structures.Lab Chip. 2014; 14: 4319-4323Google Scholar, 6Zhang Y.S. Ribas J. Nadhman A. et al.A Cost-Effective Fluorescence Mini-Microscope with Adjustable Magnifications for Biomedical Applications.Lab Chip. 2015; 15: 3661-3669Google Scholar, 7Bhargava K.C. Thompson B. Malmstadt N. et al.Discrete Elements for 3D Microfluidics.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 15013-15018Google Scholar The development of tunable injectable microporous gel scaffolds by researchers at the University of California, Los Angeles (USA) not only brings truly personalized medicine to regenerative medicine, but also allows researchers to quickly optimize culture conditions in research and drug development applications that require complex three-dimensional (3D) cell cultures.4Griffin D.R. Weaver W.M. Scumpia P.O. et al.Accelerated Wound Healing by Injectable Microporous Gel Scaffolds Assembled from Annealed Building Blocks.Nat. Mater. 2015; 14 (DOI: 10.1038/nmat4294.): 737-744Google Scholar The development of integrated platforms composed of modular technologies increases the diversity of clinical and research applications to which technology such as microfluidics can be applied and is highlighted here by work from Harvard University (USA)6Zhang Y.S. Ribas J. Nadhman A. et al.A Cost-Effective Fluorescence Mini-Microscope with Adjustable Magnifications for Biomedical Applications.Lab Chip. 2015; 15: 3661-3669Google Scholar and the University of Southern California (USA).7Bhargava K.C. Thompson B. Malmstadt N. et al.Discrete Elements for 3D Microfluidics.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 15013-15018Google Scholar Beyond building better biosensors and more effective and cost-efficient devices, work featured this year includes improvements in automation of large-cargo intracellular delivery, as well as innovating the use of inorganic and biomaterials in medical applications.8Wu Y.-C. Wu T.-H. Clemens D.L. et al.Massively Parallel Large Cargo Delivery into Mammalian Cells with Light Pulses.Nat. Methods. 2015; 12: 439-444Google Scholar, 9Dai X. Stogin B.B. Yang S. et al.Slippery Wenzel State.ACS Nano. 2015; 9: 9260-9267Google Scholar, 10Lu Y. Hu Q. Lin Y. et al.Transformable Liquid-Metal Nanomedicine.Nat. Commum. 2015; 6 (DOI: 10.1038/ncomms10066.): 10066Google Scholar This year’s honorees represent the best in research that advances translational science and technology, and we are proud to honor these researchers for their amazing work. JALA and SLAS would like to thank all the nominees as well as those who nominated them. We would also like to thank everyone who worked to discuss and select the 2016 JALA Ten. The last 6 years have allowed us to highlight innovative and exciting research breakthroughs that will greatly impact our lives. This year is no different and continues to raise the bar for innovation. We look forward to seeing what next year brings. Current sensing technologies for quantifying microbial pathogens rely on highly specific antibodies for molecular recognition that suffer from limited stability in high-temperature environments and can be difficult to obtain for similar pathogen species. Using synthetic antimicrobial peptides (AMPs) with species-specific targeting and binding capabilities, Peter Lillehoj of Michigan State University (USA) and his collaborators have developed a microfluidic biosensor for rapid, multiplexed detection of bacterial pathogens. AMP-coated sensors demonstrate strong preferential binding to their corresponding targeted cells with negligible cross-binding (Fig. 1) and impedance measurements correlated well to the cell concentration. With further development, this technology can provide a robust, portable platform for rapid pathogen detection. Tracking and studying single molecules in living cells is of great interest to life sciences researchers; however, the tools to do so are highly limited. Even more difficult is studying the function of single molecules following cell secretion in order to better understand how these molecules function in cellular microenvironments. Somin Eunice Lee at the University of Michigan (USA) and her colleagues have developed an aptamer-gold plasmon ruler that now achieves reliable detection and study of secreted single molecules (Fig. 2). Furthermore, this plasmon ruler is reversible, allowing for detection of multiple events. High specificity of this reversible plasmon ruler is demonstrated with the specific detection of the matrix metalloproteinase MMP3 over its family member MMP9. Implementation of this plasmon ruler into life sciences and biomedical research should allow for greater understanding of the functions of secreted molecules in the microenvironment. These plasmon rulers should also find use in phenotypic drug discovery and other translational applications. Even with the increasing popularity of phenotypic research methods such as high-content applications and the emergence of cell-based therapies, there is a dearth of cell labeling and tracking reagents that can reliably and quantifiably sense key cellular functions. Chenjie Xu and his colleagues at Nanyang Technological University (Singapore) have developed a nanoparticle-based platform for just this purpose. Encapsulating specific biosensor molecules in biodegradable polymeric nanoparticles, these nanosensors are able to serve as an intracellular source of sensor molecules for up to 30 days within cells (Fig. 3). This platform has a wide range of biomedical applications, as demonstrated by this team. Utilizing their nanosensors, they demonstrate real-time quantification of cellular processes such as nitric oxide production, as well as gene expression such as β-actin mRNA expression. The development of these nanosensors should serve as the foundation for a wide range of tools for molecular biology research, as well as phenotypic drug discovery. Scaffold materials for tissue engineering and 3D matrices for organ-on-a-chip technologies have been hampered by a fundamental limitation in the ability to control material porosity separately from mechanical properties. This limitation is especially exacerbated when considering flowable biomaterials, which are useful for in vivo delivery, filling of wounds, or encapsulation of cells. Dino Di Carlo and Tatiana Segura and their teams at the University of California, Los Angeles (USA) have overcome these limitations with the development of a tunable injectable microporous gel scaffold (Fig. 4). Microporosity is important in a biomaterial, as it improves transport within a scaffold and enables rapid cellular ingrowth. This work demonstrates the first approach to achieve an injectable microporous material for tissue engineering, and this material promoted cellular growth in vitro and accelerated healing in vivo. Because of the modularity and tenability of these microporous annealed particle (MAP) gels, they should impact a wide range of regenerative medical applications, as well as drug development applications that require 3D cell culture or organ-on-a-chip methods of study. Tony Jun Huang’s research group at the Pennsylvania State University (USA) demonstrate a microfluidic pump, so-called sharp-edge-based acoustofluidic pump, that utilizes the acoustic streaming effects induced by acoustically oscillating tilted sharp-edge structures. Their pump is simply composed of a quarter-sized piezoelectric transducer and a microfluidic channel (Fig. 5). Upon the oscillation, the tilted sharp-edge structures generate acoustic streaming effects, which in turn generate net forces in the direction that the sharp-edge structured oriented. Thus, fluid pumping takes place because the generated net forces push the bulk fluid to flow forward. By simply modulating the driving signals to the piezoelectric transducer, the pump can generate not only flow rates ranging from nanoliters to several microliters per minute, but also flow rates of various flow profiles. Thus, the acoustofluidic pump has the potential to be integrated into this kind of portable testing platform, because of its small size, easy fabrication, controllability, and tunability. Microscopes remain a key component of any life sciences research; however, they remain expensive and are often limited in specific functions when made more affordable. Y. S. Zhang from Harvard University (USA) has innovated a miniature microscope with built-in fluorescence capability for biomedical applications (Fig. 6). This mini-microscope has adjustable magnifications from 8× to 60× and achieves a high resolution of <2 μm. Using off-the-shelf components and a webcam, the mini-microscope system is inexpensive (<$10), and its modularity allows for convenient integration with a wide variety of preexisting platforms, such as cell culture plates and microfluidic devices. Therefore, this mini-microscope is likely to find widespread applications in cell biology, tissue engineering, biosensing, microfluidics, and organs-on-chips, which may potentially replace conventional benchtop microscopy where long-term in situ and high-throughput imaging and analysis are required. Traditionally, microfluidic systems have been designed as monolithic devices, each with a single dedicated application. Noah Malmstadt and his team at the University of Southern California (USA) have developed a modular microfluidic platform where each module can be designed with discrete fluid handling, routing, or analysis function (Fig. 7). These modules are assembled together to create an application-defined microfluidic circuit. A key benefit of this approach is design predictability. The behavior of the assembled system can be predicted by circuit design methodologies because each module has well-understood fluidic characteristics. This modular approach to microfluidic circuit design allows for a truly 3D microfluidic design rather than less efficient and more complex planar layer-by-layer arrangements of channels. Additionally, because individual models are manufactured by 3D printing, fabrication is simpler and cheaper. This work represents a shift in microfluidic design that should allow microfluidic devices to be incorporated in a wider range of commercial applications. While there are a number of well-established methods for delivery of small-scale cargo, such as kilobit-sized nucleic acids, reliably delivering large cargo into cells has remained elusive. Developing a method for large-cargo delivery in a high-throughput manner is an even more difficult hurdle to overcome. Eric Pei-Yu Chiou and his team at the University of California, Los Angeles (USA) have accomplished such a feat with their new intracellular delivery platform called biophotonic laser-assisted surgery tool (BLAST) (Fig. 8). BLAST enables the delivery of cargo up to several microns in size into 100,000 cells in 1 min, which is five orders of magnitude faster than prior methods. Cargo is delivered into the cytosol of cells directly without undesirable endosome trapping. High efficiency, high cell viability, and nearly simultaneous delivery of diverse types of cargo into numerous cells under constant physiological conditions allow reliable measurements in a variety of biological settings. Because of the reliability of the high-throughput platform and the increase in cargo size, BLAST can be applied to a broad range of applications, including delivery of large nanoparticle complexes, functional proteins, and large amounts of both DNA and RNA. As such, this platform should improve life sciences research, from basic molecular biological research to drug development and biomedical clinical research. The ability to repel liquids regardless of how they wet the surface has important technological implications for numerous industrial and biomedical processes, ranging from condensation heat transfer to water harvesting to antifouling of medical devices. However, maintaining liquid mobility on engineered surfaces under various conditions has been an engineering challenge for more than a decade. Now, researchers at the Pennsylvania State University (USA) led by Tak-Sing Wong have invented a new class of liquid-repellent surface, known as slippery rough surface, which can repel liquids in any state of wetness for the first time (Fig. 9). Their surfaces, modeled after lotus leaves and the pitcher plant, have been developed by engineering hierarchical nano- and microscale textures and infusing liquid lubricant into the nanotextures to create a highly slippery rough surface. The new surface may open up new opportunities for scientific studies and engineering applications related to wetting, adhesion, transport phenomena, and biofouling. While the use of inorganic nanomaterials in drug delivery remains an area of active research in nanomedicine, clinical translation of inorganic material-based drug delivery systems has been difficult due to their toxicity and clearance failures. A promising new approach that overcomes these hurdles was developed by Zhen Gu and his team at the University of North Carolina at Chapel Hill (USA) and North Carolina State University (USA). Utilizing a liquid-phase eutectic gallium-indium core and a thiolated polymeric shell, a transformable liquid-metal-based nanosphere drug delivery particle is formed (Fig. 10). When functionalized with hyaluronic acid, these drug delivery complexes are capable of efficiently delivering chemotherapeutics and more effectively inhibiting tumor growth than conventional chemotherapeutics. Degradable in mildly acidic environments, this new liquid-metal-based approach opens new avenues to exploring the use of inorganic materials in a variety of nanomedical applications.

In the rapidly evolving landscape of cell biology and biomedical research, three-dimensional (3D) cell culture has contributed not only to the diversification of experimental tools available but also to their improvement toward greater physiological relevance. 3D cell culture has emerged as a revolutionary technique that bridges the long-standing gap between traditional two-dimensional (2D) cell culture and the complex microenvironments found in living organisms. By providing conditions for establishing critical features of in vivo environment, such as cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper tissue-like architecture and differentiated function of cells. Since the early days of 3D cell culture in the 1970s, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. One particular 3D cell culture technique has caught the attention of many scientists and has experienced an unprecedented boom and enthusiastic application in both basic and translational research over the past decade - the organoid technology. This book chapter provides an introduction to the fundamental concepts of 3D cell culture including organoids, an overview of 3D cell culture techniques, and an overview of methodological- and protocol-oriented chapters in the book 3D Cell Culture.

The 2022 SLAS technology ten: Translating life sciences innovation.

The growing interest in biomimetic hydrogels is due to their successful applications in tissue engineering, 3D cell culturing and drug delivery. The major characteristics of hydrogels include swelling, porosity, degradation rate, biocompatibility, and mechanical properties. Poor mechanical properties can be regarded as the main limitation for the use of hydrogels in tissue engineering, and advanced techniques for its precise evaluation are of interest. The current research aims to demonstrate the suitability of scanning ion conductance microscopy (SICM) for assessing the stiffness of various hydrogels - Fmoc-FF peptide hydrogel, polyacrylamide and gelatin, - which differ by two orders of magnitude in Young's modulus (E). We provide a direct comparison between SICM measurements and atomic force microscopy (AFM) data, the latter being a widely used method for assessing the mechanical properties of scaffolds. The results of these methods showed good agreement, however, for materials with various stiffness two SICM-based approaches - application of hydrostatic pressure and application of intrinsic force - should be used. For hydrogels with Young's modulus of more than 2.5 kPa the application of SICM using hydrostatic pressure is recommended, whereas for soft materials with E ∼ 200-400 Pa the technique using intrinsic force can also be applied. We have shown that SICM and AFM methods can be used for the evaluation of the mechanical properties of soft hydrogels with nanometer resolution, while SICM is a completely non-invasive method, which requires a minimum influence on the sample structure.

Biomimetic hydrogels have garnered increased interest due to their considerable potential for use in various fields, such as tissue engineering, 3D cell cultivation, and drug delivery. The primary challenge for applying hydrogels in tissue engineering is accurately evaluating their mechanical characteristics. In this context, we propose a method using scanning ion conductance microscopy (SICM) to determine the rigidity of living human breast cancer cells MCF-7 cells grown on a soft, self-assembled Fmoc-FF peptide hydrogel. Moreover, it is demonstrated that the map of Young's modulus distribution obtained by the SICM method allows for determining the core location. The Young's modules for MCF-7 cells decrease with the substrate stiffening, with values of 1050 Pa, 835 Pa, and 600 Pa measured on a Petri dish, Fmoc-FF hydrogel, and Fmoc-FF/chitosan hydrogel, respectively. A comparative analysis of the SICM results and the data obtained by atomic force microscopy was in good agreement, allowing for the use of a composite cell-substrate model (CoCS) to evaluate the 'soft substrate effect'. Using the CoCS model allowed us to conclude that the MCF-7 softening was due to the cells' mechanical properties variations due to cytoskeletal changes. This research provides immediate insights into changes in cell mechanical properties resulting from different soft scaffold substrates.

Recent advancements in three-dimensional (3D) cell culture technologies, such as cell spheroids, organoids, and 3D bioprinted tissue constructs, have significantly improved the physiological relevance of in vitro models. These models better mimic tissue structure and function, closely emulating in vivo characteristics and enhancing phenotypic analysis, critical for basic research and drug screening in personalized cancer therapy. Despite their potential, current 3D cell culture platforms face technical challenges, which include user-unfriendliness in long-term dynamic cell culture, incompatibility with rapid cell encapsulation in biomimetic hydrogels, and low throughput for compound screening. To address these issues, we developed a 144-pillar plate with sidewalls and slits (144PillarPlate) and a complementary 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for dynamic 3D cell culture and predictive compound screening. To accelerate biomimetic tissue formation, small Hep3B liver tumor spheroids suspended in alginate were printed and encapsulated on the 144PillarPlate rapidly by using microsolenoid valve-driven 3D bioprinting technology. The microarray bioprinting technology enabled precise and rapid loading of small spheroids in alginate on the pillar plate, facilitating reproducible and scalable formation of large tumor spheroids with minimal manual intervention. The bioprinted Hep3B spheroids on the 144PillarPlate were dynamically cultured in the 144PerfusionPlate and tested with anticancer drugs to measure drug effectiveness and determine the concentration required to inhibit 50% of the cell viability (IC50 value). The perfusion plate enabled the convenient dynamic culture of tumor spheroids and facilitated the dynamic testing of anticancer drugs with increased sensitivity. It is envisioned that the integration of microarray bioprinting of tumor spheroids onto the pillar plate, along with dynamic 3D cell culture in the perfusion plate, could more accurately replicate tumor microenvironments. This advancement has the potential to enhance the predictive drug screening process in personalized cancer therapy significantly.

Objective: Pseudomonas aeruginosa infections pose a significant challenge due to their ability to resist various antibiotics and form biofilms. Excessive antibiotic use accelerates the development of multidrug-resistant P. aeruginosa, which is further enhanced by its ability to form biofilms. Therefore, alternative treatment options are needed to control antibiotic resistance and biofilm formation. Cold plasma contains a mixture of reactive oxygen and nitrogen species, which directly kill bacteria, modify virulence factors, and enhance innate immune responses. This study aimed to evaluate the effect of cold plasma on P. aeruginosa antibiotic resistance and ability to form biofilms. Methods: Clinical isolates of P. aeruginosa were exposed to cold plasma for different periods (3, 6, and 9 minutes). Changes in susceptibility to several antibiotics were assessed using the disk diffusion method, and biofilm formation was examined using crystal violet staining. Results: The results demonstrated the clear effectiveness of cold plasma against antibiotic resistance and the biofilm-forming ability of P. aeruginosa. The effectiveness of the tested antibiotics was increased, and the bacteria lost their ability to form biofilms by 100%. The results support the idea of using cold plasma technology as an alternative to antibiotics to eliminate pathogenic and antibiotic-resistant bacteria, thereby treating diseases associated with these bacteria. Conclusions: The results demonstrated the potential of using cold plasma technology as an alternative to antibiotics to treat diseases associated with this bacterium.

Huang-qin decoction increases the sensitivity of EGFR-TKIs to NSCLC cells by regulating stat3/GPX4 to induce redox ratio and ROS to inhibit CSCs

Owing to the high water content, porous structure, biocompatibility and tissue-like viscoelasticity, hydrogels have become attractive and promising biomaterials for use in drug delivery, 3D cell culture and tissue engineering applications. Various chemical approaches have been developed for hydrogel synthesis using monomers or polymers carrying reactive functional groups. For in vivo tissue repair and in vitro cell culture purposes, it is desirable that the crosslinking reactions occur under mild conditions, do not interfere with biological processes and proceed at high yield with exceptional selectivity. Additionally, the cross-linking reaction should allow straightforward incorporation of bioactive motifs or signaling molecules, at the same time, providing tunability of the hydrogel microstructure, mechanical properties, and degradation rates. In this review, we discuss various chemical approaches applied to the synthesis of complex hydrogel networks, highlighting recent developments from our group. The discovery of new chemistries and novel materials fabrication methods will lead to the development of the next generation biomimetic hydrogels with complex structures and diverse functionalities. These materials will likely facilitate the construction of engineered tissue models that may bridge the gap between 2D experiments and animal studies, providing preliminary insight prior to in vivo assessments.

Three-dimensional (3D) cell culture models are attracting significant attention by the researchers, especially in cancer therapy, as two-dimensional models have failed to replicate the 3D complexity of an in vivo tumor. Different 3D cell culture models available, including spheroids, polymer/ hydrogel scaffolds, hanging drop approach, bioreactors, organs-on-a-chip, and 3D bioprinting structures, are highlighted in this chapter. In drug discovery, new compounds are routinely screened for their anticancer efficacy using traditional 2D cell-based assays. A wide range of 3D cell culture models have been developed due to recent advances in cell biology, microfabrication methods, and tissue engineering. Scaffolds provide physical support for 3D cell culture growth. With recent advances in microfluidic fabrication technology, extensive and controlled 3D cell culture models have been designed to allow accurate microenvironmental parametric control by using biocompatible microfluidic chips fabricated by technologies, including photolithography and soft lithography.

It is of great value to develop reliable in vitro models for cell biology and toxicology. However, ethical issues and the decreasing number of donors restrict the further use of traditional animal models in various fields, including the emerging fields of tissue engineering and regenerative medicine. The huge gap created by the restrictions in animal models has pushed the development of the increasingly recognized three-dimensional (3D) cell culture, which enables cells to closely simulate authentic cellular behaviour such as close cell-to-cell interactions and can achieve higher functionality. Furthermore, 3D cell culturing is superior to the traditional 2D cell culture, which has obvious limitations and cannot closely mimic the structure and architecture of tissues. In this study, we review several methods used to form 3D multicellular spheroids. The extracellular microenvironment of 3D spheroids plays a role in many aspects of biological sciences, including cell signalling, cell growth, cancer cell generation, and anti-cancer drugs. More recently, they have been explored as basic construction units for tissue and organ engineering. We review this field with a focus on the previous research in different areas using spheroid models, emphasizing aqueous two-phase system (ATPS)-based techniques. Multi-cellular spheroids have great potential in the study of biological systems and can closely mimic the in vivo environment. New technologies to form and analyse spheroids such as the aqueous two-phase system and magnetic levitation are rapidly overcoming the technical limitations of spheroids and expanding their applications in tissue engineering and regenerative medicine.