Biological Engineering

The skin microbiome, a diverse community of microorganisms, plays a crucial role in maintaining skin health and homeostasis. Traditional studies have relied on two-dimensional (2D) models, which fail to recreate the complex three-dimensional (3D) architecture and cellular interactions of in vivo human skin, and animal models, which have species-specific physiology and accompanying ethical concerns. Consequently, both types of models fall short in accurately replicating skin physiology and understanding its complex microbial interactions. Three-dimensional bioprinting, an advanced tissue engineering technology, addresses these limitations by creating custom-designed tissue scaffolds using biomaterial-based bioinks containing living cells. This approach provides a more physiologically relevant 3D structure and microenvironment, allowing the incorporation of microbial communities to better reflect in vivo conditions. Here, we present a protocol for 3D bioprinting an in vitro skin infection model by co-culturing human keratinocytes and dermal fibroblasts in a high-viscosity, fibrin-based bioink to mimic the dermis and epidermis. The bioprinted skin tissue was co-infected with Staphylococcus aureus and Staphylococcus epidermidis to mimic bacterial skin disease. Bacterial survival was assessed through colony-forming unit enumeration. By incorporating bacteria, this protocol offers the potential to serve as a more representative in vivo 3D bioprinted skin infection model, providing a platform to study host–microbe interactions, immune responses, and the development of antimicrobial therapeutics.

Every year, there is an increase in the number of cases of chronic kidney disease, and a delay in the initiation of adequate treatment can lead to kidney failure, which requires regular dialysis or transplantation. Intensive systemic therapy used to treat kidney diseases often has a negative impact on other weakened organs, making it crucial to ensure targeted delivery of medications directly to the kidneys and to minimize systemic side effects. In order to reduce the toxicity of medications and decrease dosages, innovative delivery methods are being developed, such as micro-sized targeted delivery systems, which ensure highly effective distribution of encapsulated drugs directly within the organs. In a recent article, we presented innovative emulsified microgels stabilized with whey protein isolate (WPI), specifically designed for targeted drug delivery to the kidneys. Our stability studies revealed that these microgels start to degrade after 72 h, with this degradation exhibiting a time-dependent profile. Furthermore, intravenous administration of the microgel suspension through the tail vein showed significant selective accumulation in both the liver and kidneys over a duration of 5 days. As part of our research, we present the protocol for synthesizing emulsion microgels derived from whey protein isolate. This article provides a comprehensive overview of the procedures for precursor preparation, along with an in-depth investigation of the emulsion system's stability over time. The protocol also includes the injection of an emulsion microgel suspension into the tail vein of mice, enabling the evaluation of their biocompatibility and potential therapeutic efficacy. This protocol outlines the precautions and important nuances that should be considered at each stage of the experiment.

Plastic pollution presents a looming danger to the environment and virtually all life on planet Earth. Especially pernicious are nanoplastics (NPs), which are plastic fragments with dimensions ≤1 μm. Conventional detection methods are ineffective for NPs, while their high specific surface area renders them efficient carriers of toxic substances; additionally, they may even be inherently toxic. Although NP waste chiefly arises from environmental weathering of larger plastic fragments, most published studies employed manufactured pristine NPs of uniform size and shape. Furthermore, almost all NP effects were studied using polystyrene (PS) as a convenient model material, despite PS accounting for <6% of all plastic pollution. There is thus an urgent need to expand investigations of environmental NP pollution and effects on biota. The present work provides a comprehensive roadmap for studying the effects of “real-world” NP pollution on living systems, using, for example, lung alveolar epithelial cells on which such NPs deposit by breathing ambient air. Herein, we describe detailed in-house methods to fabricate various NPs that are weathered with UV light and O₃ gas exposure to more closely mimic real environmental NPs. We also illustrate a simple and straightforward bioelectrical method for assessing passive and active ion transport properties of primary rat lung alveolar epithelial cell monolayers as a model for the distal mammalian lung exposed to one of the generated NPs. This protocol allows researchers to rapidly and more accurately assess the biological impact of various simulated environmental NPs on a vulnerable air–blood barrier in the lung.

Continuous and balanced bone remodeling is essential for maintaining mechanical integrity, mineral homeostasis, and hematopoiesis. Dysregulated bone metabolism develops pathological conditions, such as osteoporosis and bone metastasis. Functional and analytical recapitulation of bone remodeling in vitro is critical for advancing our understanding of bone mineral metabolism, disease mechanisms, and drug development. However, conventional models fail to replicate the essential complexity of the bone extracellular matrix (ECM) and the dynamic interplay between bone-forming osteoblasts and bone-resorbing osteoclasts. Recently, we developed an osteoid-mimicking demineralized bone paper (DBP) by thin-sectioning demineralized bovine compact bone matrix. DBP supports osteoblastic mineral deposition and the subsequent transition to bone-lining cells. When co-cultured with bone marrow mononuclear cells under biochemical stimulation, osteoblasts shift their regulatory secretion profiles and effectively induce osteoclastogenesis. The semi-transparent nature of DBP, combined with primary osteogenic cells retrieved from DsRed and eGFP reporter mice, enables longitudinal fluorescent monitoring of these multicellular processes and quantitative analysis. In this protocol, we describe the methods for DBP generation, reconstituting mineralized bone tissue complexity with osteoblasts, and recapitulating the bone remodeling cycle through bone marrow monocytes co-culture under biochemical stimulation, offering a useful platform for the related and broader research community.

Assessing thrombogenicity is crucial for evaluating biomaterials, especially those that interface with flowing blood, such as cardiovascular implants, including vascular stents, grafts, and stent-grafts. To standardize thrombogenicity assessments, we use human plasma and quantify the light absorbance associated with the biomaterial. For this evaluation, various tubular vascular implants from leading brands—such as bare-metal stents, drug-eluting stents, vascular grafts, and stent-grafts—are longitudinally sectioned into small pieces and placed in a low-adhesion 96-well plate. Using either platelet-rich plasma (PRP) or platelet-poor plasma (PPP), we measure the absorbance of light passing through the plate over an hour and plot the resulting curve. This method quantifies the thrombogenicity of a biomaterial under controlled conditions. Key factors examined include anticoagulants, platelet presence, and surface-coating molecules to assess their impact on thrombogenicity. Using this simple, reproducible protocol, we demonstrated (a) the relative efficacy of various anticoagulants in thrombogenicity assessments, and (b) the effectiveness of vascular coating molecules in reducing thrombogenicity on stents. This streamlined approach offers valuable insights for optimizing biomaterial performance in vascular implants. Unlike conventional clotting assays, which focus on standardized blood clotting mechanisms, this assay is tailored to evaluate biomaterials and external parameters influencing thrombogenicity.

A key goal in the bioengineering field is the development of surface patterning of proteins that guide and control cellular organization. To this end, we developed a method to create a microstructured hydrogel based on soft-lithography techniques using polydimethylsiloxane (PDMS) and polyethylene glycol diacrylate (PEGDA). This approach involves the design of microfluidic geometries using graphical software, employing PDMS as a mold and leaving PEGDA as a substrate for the fabrication of microstructures and, thus, patterning extracellular matrix (ECM) proteins to promote cell adhesion. The combination of these techniques allows the fabrication of hydrogel microstructures without following conventional photolithography methods, such as the use of a photomask, the alignment required to produce the patterns, and the associated expenses. This study highlights the versatility and potential of PEGDA-based hydrogels as platforms to advance tissue engineering strategies.

Pathological conditions of the cervix ranging from cervical cancer to structural dysfunction associated with preterm labor all have limited treatment options. Thus, there is a need for physiologically relevant preclinical models that recapitulate the structure and function of this human organ. Here, we describe a protocol for engineering and studying a highly functional in vitro model of the human cervix that is composed of a commercially available, dual-channel, microfluidic, organ-on-a-chip (Organ Chip) device lined by primary cervical epithelial (CE) cells interfaced across a porous membrane with cervical stromal cells. The provision of dynamic and customized media flow through both the epithelial and stromal compartments results in cell growth and differentiation, including the accumulation of a thick mucus layer overlying the epithelium. The resulting model closely mimics the structure, epithelial barrier, mucus composition and structure, and biochemical properties of the in vivo human cervix, as well as its responsiveness to female hormones, pH, and microbiome. This Cervix Chip protocol also includes noninvasive techniques for longitudinal monitoring of the live 3D tissue model. The Cervix Chip offers a powerful preclinical platform for replicating in vivo cervical physiology, studying disease mechanisms, and facilitating the development of new therapeutics and diagnostics.

Fluorescent protein biosensors (FPBs) that turn on—go from dark to bright upon binding their ligands—enable the detection of targets in living cells with high sensitivity and spatial localization. Several approaches exist for creating turn-on FPBs, most notably the method that gave rise to the GCaMP family of genetically encoded calcium indicators. However, it remains challenging to modify these sensors to recognize new ligands. We recently developed adaptable turn-on maturation (ATOM) biosensors, in which target recognition by a small binding domain triggers chromophore maturation in the fluorescent protein to which it is attached. ATOM sensors are advantageous because they are generalizable (by virtue of the monobody and nanobody binding domains) and modular (binding domains and fluorescent proteins of various colors can be mixed and matched for multiplexed imaging), capable of detecting endogenously expressed proteins, and able to function in subcellular compartments including the cytoplasm, nucleus, endoplasmic reticulum, and mitochondria. The protocols herein detail how to design, clone, and screen new ATOM sensors for detecting targets of choice. The starting materials are the genes encoding for a monobody or nanobody and for a cyan, yellow, or red fluorescent protein. We also present general guidelines for creating ATOM sensors using binding domains other than nanobodies and monobodies.

Dual-modal imaging, combining photoacoustic (PA) and ultrasound localization (UL) with microbubbles, holds substantial promise across biomedical fields such as oncology, neuroscience, nephrology, and immunology. The combination of PA and UL imaging faces challenges due to acquisition speed mismatches, limiting their combined efficacy. Here, we introduce a protocol that applies sparsity constraint optimization to accelerate dual-modal data acquisition, enabling in vivo super-resolution imaging of vascular and physiological structures at under two seconds per frame. The protocol provides detailed guidelines for constructing an interleaved PA/UL (PAUL) imaging system, covering material selection, system setup, and calibration, as well as methods for image acquisition, reconstruction, post-processing, and troubleshooting. This approach empowers the biomedical community to establish a rapid, dual-modal PAUL imaging platform, broadening biomedical applications and advancing imaging capabilities in clinical research.

Interstitial fluid (ISF) is a promising diagnostic sample due to its extensive biomolecular content while being safer and less invasive to collect than blood. However, existing ISF sampling methods are time-consuming, require specialized equipment, and yield small amounts of fluid (<5 μL). We have recently reported a simple and minimally invasive technique for rapidly sampling larger quantities of dermal ISF using a microneedle (MN) array to generate micropores in the skin from which ISF is extracted using a vacuum-assisted skin patch. Here, we present step-by-step protocols for fabricating the MN array and skin patch, as well as for using them to sample ISF from human skin. Using this technique, an average of 20.8 μL of dermal ISF can be collected within 25 min, which is a ∼6-fold improvement over existing ISF sampling methods. Furthermore, the technique is well-tolerated and does not require the use of expensive or specialized equipment. The ability to collect ample volumes of ISF in a quick and minimally invasive manner will facilitate the analysis of ISF for biomarker discovery and its use for diagnostic testing.