生物工程

In recent years, the calcifying properties of some cyanobacteria have been used in the production of living building materials (LBMs), such as bio-concrete, as a CO₂-friendly alternative for cement. This microbially induced calcium carbonate precipitation (MICP) technique can act as a novel platform technology for carbon capture strategies. Consequently, various research articles have been conducted based on a diverse set of workflows, including several modifications, to manufacture LBMs. However, such articles contain only fragmentary descriptions of the materials and methods used. This protocol is meant to act as a detailed, step-by-step operational manual for the production of LBMs using the cyanobacterial model strain Picosynechococcus sp. PCC 7002. The process is divided into several steps, such as the activation of the cyanobacterial-gel solution with CaCl₂ × 2H₂O and NaHCO₃, casting standardized prisms (160 × 40 × 40 mm), and demolding LBMs. Subsequently, bending tensile and compressive strength tests are performed according to the procedures commonly used in concrete and material testing as proof of concept.

Intravenous hemostats have shown significant promise in prolonging survival for severe noncompressible and internal injuries in preclinical animal models. Existing approaches include the use of liposomes with or without procoagulant enzymes, as well as polymer nanoparticles or soluble biopolymers. While these methods predominantly target or mimic tissue components that are present during coagulation, such as activated platelets and collagen, they may not account for the loss of fibrinogen, which is not only key to clot formation but also the first protein to fall below critical levels in dilutional coagulopathy. This protocol describes the synthesis and in vitro or ex vivo characterization of a crosslinkable nanoparticle system that seeks to address dilutional coagulopathy by leveraging the critical gelation concentration and bioorthogonal click chemistry. The system was shown to only gel at high nanoparticle and crosslinker concentrations, increase the rate of platelet recruitment, and decrease the rate of clot degradation in a low-fibrinogen environment, providing a platform for treating severe hemorrhage in a coagulopathic environment. Ultimately, the contents of this protocol may assist researchers in the in vitro characterization and screening of other crosslinkable nanoparticle systems or hemostats, with potential expansions to other categories of coagulation dysfunction, such as embolism treatment.

Zebrafish offer numerous advantages as a vertebrate model because of their rapid development, high fecundity, transparent embryos, and ease of genetic manipulation. A wide variety of transgenic and mutant fish lines have been generated, and efficiently sharing these resources is crucial for advancing research. Zebrafish lines have typically been exchanged as early embryos, adult fish, or cryopreserved sperm, making transportation costly and logistically challenging. Here, we provide a protocol for preserving functional zebrafish sperm for more than 7 days at room temperature and subsequent in vitro fertilization using the preserved sperm. In this protocol, sperm collected either from the cloaca of an anesthetized male or from dissected testes is stored in L-15-based storage medium. Importantly, the storage medium, originally developed for zebrafish, is also applicable to medaka, another widely used vertebrate model. This sperm storage method allows researchers to ship sperm using low-cost methods and to investigate key factors for motility and fertilizing ability in those sperm.

Bottom-up tissue engineering using cell spheroids offers many advantages in recapitulating native cell–cell and cell–matrix interactions. Many tissues, such as cartilage, bone, cardiac muscle, intestine, and neural tissues, have been tissue-engineered using cell spheroids. However, previous methods for spheroid assembling, such as mold casting, hydrogel-based bioprinting, or needle array, either lack control over final tissue geometry or face challenges in scalability and throughput. In this protocol, we describe a robust and scalable tissue engineering method for assembling cell spheroids into a thin, planar spheroid sheet. The spheroids are sandwiched between two flexible meshes held by a frame, facilitating uniform spheroid fusion while ensuring nutrient exchange and ease of handling. We demonstrate this method by producing thin cartilage tissue from human mesenchymal stem cells undergoing chondrogenic differentiation. This approach offers a practical platform for producing thin membrane-like tissue constructs for many research and therapeutic applications.

Oxygen tension is a key regulator of early human neurogenesis; however, quantifying intra-tissue O₂ in 3D models for an extended period remains difficult. Existing approaches, such as needle-type fiber microsensors and intensity-based oxygen probes or time-domain lifetime imaging, either perturb the organoids or require high excitation doses that limit the measurement period. Here, we present a step-by-step protocol to measure intra-organoid oxygen in human cerebral organoids (hCOs) using embedded ruthenium-based CPOx microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM). The workflow covers dorsal/ventral cerebral organoid patterning, organoid fusion at day 12 with co-embedded CPOx beads, standardized FD-FLIM acquisition (470-nm external modulation, 16 phases at 50 kHz, dual-tap camera), automated bead detection and lifetime extraction in MATLAB, and session-matched Stern–Volmer calibration with Ru(dpp)₃(ClO₄)₂ to convert lifetimes to oxygen concentration. The protocol outputs per-bead oxygen maps and longitudinal patterns stratified by bead location (intra-organoid vs. gel) and sample state (healthy vs. abnormal), enabling direct linkage between developmental growth and oxygen dynamics.

Extracellular vesicles (EVs) have emerged as promising carriers for the targeted delivery of therapeutic proteins to specific cells. Previously, we demonstrated that genetically engineered EVs can be used for targeted protein delivery. This protocol details the generation of mannose receptor (CD206)-targeted EVs using a modular plasmid system optimized for production in HEK293T cells. Three plasmids enable customizable EV budding, cargo loading, and surface modification for targeting to antigen-presenting cells (APCs). EVs are isolated via differential centrifugation and chromatography, characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), and validated through functional uptake assays in primary human activated dendritic cells. Our approach combines flexibility in engineering required EVs with robust, reproducible isolation and characterization workflows. Its modularity allows easy adaptation to alternative targets or cargoes, which can be validated immediately through in vitro testing.

Micro milling is a subtractive manufacturing method for fabricating micro-scale three-dimensional features from hard substrates like acrylic, wood, or metal. It enables rapid prototyping of biomicrofluidic devices and master molds, offering advantages over traditional fabrication methods like photolithography. Micro milling is seldom applied in the fabrication of organs-on-a-chip, in part due to its requirement for knowledge of computer numerical machining techniques that are required to program and operate micro mills. This protocol provides practical guidelines for micro milling–based fabrication of organs-on-a-chip, including toolpath optimization, SolidWorks and Fusion workflows, and troubleshooting tips. A case study demonstrates the design and fabrication of master molds for a human airway-on-a-chip, validated in a recent publication. This resource supports the expansion of micro milling techniques into organs-on-a-chip, which will enhance capacity for rapid device prototyping and design of more complex 3D features that better recapitulate human physiology.

Lipid nanoparticles (LNPs) are powerful carriers for nucleic acid delivery, but plasmid DNA-loaded LNPs (pDNA-LNPs) have been limited by inflammation and toxicity. We showed that standard pDNA-LNPs activate the cGAS–STING pathway, leading to severe immune responses and mortality in mice. To overcome this, we co-loaded nitro-oleic acid (NOA), an endogenous STING inhibitor, into pDNA-LNPs. NOA-pDNA-LNPs mitigated inflammation, enabled safe in vivo delivery, and supported sustained gene expression for months. Here, we present a detailed protocol for producing and characterizing NOA-pDNA-LNPs to facilitate safer, long-term gene delivery applications.

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.