Medicine

Obtaining articular cartilage-derived cells (chondroprogenitors) by explant methodology is a reliable approach for isolating migratory progenitor cells that retain strong chondrogenic potential. This method allows cells to emerge naturally from small cartilage fragments without enzymatic digestion. The procedure consists of plating cartilage explants on a plastic surface with culture medium, from which cells subsequently migrate and adhere to the substrate. Compared with enzymatic isolation, the explant approach minimizes cellular stress and better reproduces the physiological microenvironment of cartilage tissue. This protocol can be applied to both osteoarthritic and non-osteoarthritic samples, enabling comparative studies on disease-related phenotypic differences. Overall, this technique offers a reproducible, straightforward, and minimally invasive strategy for obtaining functional chondroprogenitor cells suitable for cartilage regeneration research.

Organoids are self-organizing 3D tissues representing an innovative technology with interesting implications and potential for the study of tumor biology. They can be developed from fine-needle biopsies or resection material from healthy or tumor tissues. Patient-derived organoids are able to retain most of the histological characteristics, the expression profile, and the genomic landscape of the corresponding primary tissues, making them suitable for translational studies and for the identification of molecular alterations in the field of personalized medicine. Here, we describe a detailed protocol for the preparation and in vitro expansion of tumor and non-tumor organoids from surgical resections or needle biopsies of patients with hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), enabling subsequent testing of small-molecule VDAC1 antagonists at different doses. In parallel, we developed a hepatic steatosis model by treating healthy liver organoids with oleic acid, recapitulating key features of lipid accumulation and metabolic dysfunction in vitro. This protocol enables the generation of patient-derived liver organoids that preserve the histological and molecular characteristics of their original tissue, providing a robust and versatile platform for translational studies, personalized drug testing, and the exploration of novel therapeutic strategies targeting tumor metabolism.

Extracellular vesicles (EVs) circulating in blood serve as non-invasive “liquid biopsies,” carrying molecular cargo that reflects the physiological and pathological state of distant cells. Their analysis is crucial for understanding disease mechanisms and discovering novel biomarkers. Clinically, blood EVs hold significant promise for early disease diagnosis, prognostic assessment, and monitoring treatment response in diverse areas such as organ transplantation, cancer, and neurological disorders. Current EV isolation techniques, beyond ultracentrifugation, include size exclusion chromatography (separation by size for high purity) and immunoaffinity capture (using antibodies for high specificity). Here, we present a simplified, rapid, and reproducible method for isolating EVs from small-volume blood samples. This protocol consistently yields a concentrated EV pellet covering 50–300 nm EVs, amenable to direct downstream analysis. Developed and validated in our laboratory using human, porcine, and murine blood samples, this method has proven instrumental in identifying EV-based biomarkers for predicting outcomes related to organ transplantation. The protocol’s adaptability and reliance on readily prepared, cost-effective reagents further enhance its utility. This scalable approach can be further integrated with subsequent purification or enrichment steps to optimize sample preparation for protein and nucleic acid assays.

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.

The pancreatic islet, the only type of tissue that secretes insulin in response to elevated blood glucose, plays a vital role in diabetes development and treatment. While various islet vascularization strategies have been developed, they have been hindered by major limitations such as relying on pre-patterning and the inability to span long distances. Furthermore, few strategies have demonstrated robust enough vascularization in vivo to support therapeutic subcutaneous islet transplantation. Using adaptive endothelial cells (ECs) reprogrammed by transient expression of the ETS Variant Transcription Factor 2 (ETV-2) gene, we have physiologically vascularized human islets within a generic microchamber and have achieved functional engraftment of human islets in the subcutaneous space of mice. Such adaptive ECs, which we term reprogrammed vascular ECs (R-VECs), have been proven to be a suitable tool for both in vitro disease modeling and in vivo transplantation of not only islets but also other organoids.

Following myocardial infarction (MI), myocardial cells undergo cell death, and the necrotic region is replaced by extracellular matrix (ECM) proteins such as collagens. Myofibroblasts are responsible for producing these ECM proteins. Cardiac myofibroblasts are differentiated from resident fibroblasts in response to inflammation. To date, genetically modified mice driven by the Periostin promoter and adeno-associated virus 9 (AAV9) carrying the Periostin promoter have been used for gene transfer into cardiac myofibroblasts. However, these methods require multiple steps and are time-consuming and expensive. Therefore, we developed a method for delivering genes into cardiac myofibroblasts using retroviruses. Specifically, the DNA of the target gene was transfected into Plat-E cells, which are packaging cells, to generate retroviruses. The virus-containing supernatant was then harvested, and the viruses were pelleted by centrifugation and suspended in PBS-containing polybrene. Subsequently, permanent occlusion of the left coronary artery was performed, and 20 μL of viral solution was immediately administered using a 29G needle at a position 1–2 mm below the ligation site in the heart of mice maintained in an open chest state. Using this method, we were able to introduce genes into the myofibroblasts of interest surrounding the MI site.

This protocol describes the isolation and flow cytometric analysis of extracellular vesicles (EVs) derived from red blood cells, endothelial cells, and platelets in human peripheral blood. The protocol includes steps for preparing platelet-free plasma, fluorescent antibody staining, gating strategies, and technical controls. This protocol was developed within a study on EV release in snakebite-associated thrombotic microangiopathy; the protocol addresses challenges such as variable autofluorescence and heterogeneity in EV origin. It is flexible and can be adapted for alternative antibody panels targeting different cell populations or EV subtypes, including leukocyte-derived EVs.

In the field of osteoarthritis (OA), the identification of reliable diagnostic and prognostic biomarkers in patients with hip lesions such as femoroacetabular impingement (FAI) could have an immeasurable value. Calcium crystal detection in synovial fluids (SFs) is one tool currently available to diagnose patients with rheumatologic disorders. Crystals, such as monosodium urate (MSU) and calcium pyrophosphate (CPP), are identified qualitatively by compensated polarized light, whereas basic calcium phosphate (BCP) crystals are visualized under conventional light microscopy by Alizarin red S (ARS) staining. Here, we present an efficient and straightforward protocol to quantify calcium crystals by spectrophotometric analysis in human osteoarthritic SFs after staining with ARS. The type and size of the different crystal species are confirmed by environmental scanning electron microscopy (ESEM).

Human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) provide a critical source for the study of motor neuron diseases (MNDs), which has been hindered by the lack of appropriate disease models for many years. Although many spinal MN differentiation protocols have been established by mimicking in vivo neurogenesis using extrinsic signaling molecules, substantial variations in the duration and efficiency persist due to inconsistencies in concentrations, timing, and delivery methods of these molecules. Here, we present an efficient monolayer culture differentiation strategy that enables the generation of enriched CHAT⁺ spinal MNs (sMNs) in 18 days and functional sMNs exhibiting extensive network activities, as confirmed by multielectrode array (MEA), within 28 days. Therefore, this optimized MN differentiation protocol facilitates the production of mature sMNs for MND research, high-throughput drug screening, and potential cell replacement therapies.

Cardiovascular disease, the current leading cause of death worldwide, is a multifactorial disorder that involves a strong contribution of both the innate and adaptive immune systems. Overactivation of the immune system and inappropriate secretion of pro-inflammatory cytokines lead to vascular impairments and the development of cardiovascular disorders, including hypertension, atherosclerosis, and peripheral artery disease. Lymphocytes, macrophages, and dendritic cells can all secrete pro-inflammatory cytokines. This makes it challenging to isolate a specific subset of immune cells, particularly cytokines, and their contribution to vascular dysfunction remains difficult to elucidate. To solve this problem, our laboratory has developed the novel “immune cell-aorta” co-culture system described herein. This experimental protocol enables investigators to isolate an immune cell of interest and identify the cytokine(s) at the origin of vascular alterations.