Biological Engineering

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.

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.

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.

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.

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.