Cellular and molecular bioengineering最新文献

Purpose: Tissue dissociation is a critical but often overlooked step in single-cell analysis, impacting data quality, reproducibility, and biological insights. Conventional enzymatic and mechanical dissociation methods introduce variability, damage cells, and alter transcriptomic profiles, compromising downstream applications. While the initial innovation in electrical dissociation was published, this work introduces expanded characterization, including bulk RNA sequencing, diverse tissue types, and improved flow cytometry.

Methods: Here, we present a fully automated, enzyme-free method that integrates electric field-based dissociation with purification and centrifugation, providing a standardized, scalable alternative. A square wave oscillating electric field at 100 V/cm was used for dissociating tissue samples in 5 minutes or less.

Results: The system rapidly and gently dissociated glioblastoma spheroids and mouse spleen tissue, achieving a 10 × increase in live cell yield compared to automated enzymatic and mechanical dissociation (gentleMACS) and a 96 ± 2% single-cell recovery rate in glioblastoma spheroids. Transcriptomic analysis revealed minimal gene expression changes post-dissociation, with an R² value of 0.997 between conditions, indicating high consistency. Flow cytometry confirmed that key immune cell populations (B, T, NK cells) were preserved, with comparable distributions between manual and electrical dissociation.

Conclusions: By reducing operator variability, improving scalability, and maintaining cellular integrity, this technology offers a robust solution for high-throughput single-cell applications in diagnostics, drug discovery, and precision medicine.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00850-5.

Introduction: A significant obstacle to mesenchymal stem cell (MSC) potency and therapeutic utility is in vitro senescence, an irreversible cessation of replication associated with age-related complications. Senolytic drugs, such as quercetin, may be helpful in selectively culling senescent cells while leaving non-senescent cells unaffected, thereby increasing potency of high-passage MSCs.

Methods: The phenotypic, genotypic, and immunomodulatory effects of quercetin were assessed using in vitro models. Senescent cells, created through repeated subculturing of MSCs in vitro, and non-senescent cells were treated with 10 μM quercetin, differentiated into osteocytes, adipocytes, and chondrocytes, and analyzed to observe the effect of quercetin.

Results: Quercetin was not found to be beneficial to MSC function. It did not exhibit a consistent senolytic effect as evidenced by SAβ-gal and live dead staining, hindered proliferation in the short term in some donors, and lowered the expression of osteogenic markers COL1A1 and ALP. Quercetin treatment did not, however, negatively affect adipogenesis, chondrogenesis, or indoleamine 2,3 dioxygenase secretions.

Conclusion: This study contributes insight into the nature of quercetin and its effects on in vitro MSC culture and function.

Purpose: Kaposi's sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus that has a tropism for endothelial cells and leads to the development of Kaposi's sarcoma, especially in people living with HIV. The present study aimed to quantify morphological and mechanical changes in endothelial cells after infection with KSHV to assess their potential as diagnostic and therapeutic markers.

Methods: Vascular (HuARLT2) and lymphatic endothelial cells (LEC) were infected with recombinant KSHV (rKSHV) by spinoculation, establishing stable infections (HuARLT2-rKSHV and LEC-rKSHV). Cellular changes were assessed using mitochondria-tracking microrheology and morphometric analysis.

Results: rKSHV infection increased cellular deformability, indicated by higher mitochondrial mean squared displacement (MSD) for short lag times. Specifically, MSD at τ = 0.19 s was 49.4% and 42.2% higher in HuARLT2-rKSHV and LEC-rKSHV, respectively, compared to uninfected controls. There were 23.9% and 36.7% decreases in the MSD power law exponents for HuARLT2-rKSHV and LEC-rKSHV, respectively, indicating increased cytosolic viscosity associated with rKSHV infection. Infected cells displayed a marked spindloid phenotype with an increase in aspect ratio (29.7%) and decreases in roundness (26.1%) and circularity (25.7%) in HuARLT2-rKSHV, with similar changes observed in LEC-rKSHV.

Conclusions: The quantification of distinct KSHV-induced morpho-mechanical changes in endothelial cells demonstrates the potential of these changes as diagnostic markers and therapeutic targets.

Purpose: Extracorporeal membrane oxygenation (ECMO) is a life-saving critical care technology that presents significant risks of medical device-associated thrombosis. We developed a complete method for collecting membrane oxygenators (membrane lung) from patients receiving ECMO treatment and quantitatively analyzing the distribution of thrombus formation within the membrane.

Methods: We collected used membrane oxygenators from patients for processing and imaging with microcomputed tomography (microCT). We reconstructed the microCT data and performed image segmentation to identify regions of thrombus formation within these oxygenators. We performed density mapping to quantify thrombus volume across different regions of each oxygenator and within multiple oxygenator models.

Results: Our method yields two-dimensional and three-dimensional visualization and quantification of thrombus deposition in ECMO. Analysis of the spatial distribution of platelet deposition, red blood cell entrapment, and fibrin formation within the fouled device provides insights into the structural patterns of oxygenator thrombosis.

Conclusions: This method can enable quantification of oxygenator thrombosis which can be used for evaluating the effect of new biomaterial or pharmacological approaches for mitigating vascular device-associated thrombosis during ECMO.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00847-0.

Purpose: Rising rates of cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) make standard histopathology diagnostic methods a bottleneck. Using tissue molecular information for diagnostics offers a promising alternative. Faster specimen collection and high-throughput molecular identification can improve the processing of the increasing number of tumors. This study aims (i) to confirm the ability of e-biopsy technique to harvest metabolites, (ii) to obtain high-resolution metabolomic profiles of cSCC, BCC, and healthy skin tissues, and (iii) to perform a comparative analysis of the collected profiles.

Methods: Tumor specimens were collected with electroporation-based biopsy (e-biopsy), a minimally invasive sampling collection tool, from 13 tissue samples (cSCC, BCC, and healthy skin) from 12 patients. Ultra performance liquid chromatography and tandem mass spectrometry (UPLC-MS-MS) was used for molecular identification and quantification of resulting metabolomic profiles.

Results: Here we report measurements of 2325 small metabolites identified (301 with high confidence) in 13 tissue samples from 12 patients. Comparative analysis identified 34 significantly (p < 0.05) differentially expressed high-confidence metabolites. Generally, we observed a greater number of metabolites with higher expression, in cSCC and in BCC compared to healthy tissues, belonging to the subclass amino acids, peptides, and analogues.

Conclusions: These findings confirm the ability of e-biopsy technique to obtain high-resolution metabolomic profiles suitable to downstream bioinformatics analysis. This highlights the potential of e-biopsy coupled with UPLC-MS-MS for rapid, high-throughput metabolomic profiling in skin cancers and supports its utility as a promising diagnostic alternative to standard histopathology.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00846-1.

Background: Skin wound healing is a complex biological process involving cellular, molecular, and physiological events. Traditional treatments often fail to provide optimal outcomes, particularly for chronic wounds.

Objectives: This study aimed to develop a self-healing hydrogel loaded with Acteoside, a bioactive compound with antioxidant and anti-inflammatory properties, to enhance skin wound healing.

Methods: Using transcriptomic analysis, Rab31 was identified as a key target of Acteoside in regulating hair follicle stem cells (HFSCs). In vitro assays demonstrated that Acteoside promotes HFSC proliferation, migration, and differentiation by upregulating Rab31 expression. The self-healing hydrogel was prepared using quaternized chitosan derivatives, which exhibited excellent mechanical properties, antibacterial, and antioxidant activities.

Results: In vivo studies in a mouse model showed that Acteoside-loaded hydrogel significantly accelerated wound healing, promoting skin regeneration and improving wound closure.

Conclusions: This research highlights the potential of Acteoside-loaded self-healing hydrogels as an innovative therapeutic strategy for enhancing skin wound healing. By modulating HFSC activity, this hydrogel offers a promising solution for improving healing outcomes in challenging wound environments.

Graphical abstract: Schematic representation of an injectable self-healing hydrogel loaded with the phenylethanoid compound acteoside for regulating the proliferation and differentiation of HFSCs to mediate the healing of skin wounds.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00845-2.

Scope: The annulus fibrosus (AF), as an important component of the intervertebral disc (IVD), contributes to the structural integrity and functional normality of IVD. Degenerative disc diseases (DDD), due to AF impairment, are common problems that could lead to low back pain or neck pain, resulting in considerable disability and financial costs globally. The exact causes and underlying mechanisms of AF impairment, however, remain complex and unclear.

Methods: A literature search was conducted to identify relevant articles published between 1952 and 2024. We summarize the current literature on the potential etiologies of AF damage, while also providing a brief overview of the basic characteristics of the AF and current therapeutic strategies for AF impairment.

Results: The findings suggest that several factors could induce or exacerbate AF impairment. We categorize them into distinct groups as physical and chemical stimuli, nutritional or metabolic disorders, immune and inflammatory responses, and genetic abnormalities.

Conclusion: Various factors could lead to AF impairment, such as particular physical and chemical stimuli, nutritional or metabolic disorders, immune and inflammatory responses, and genetic abnormalities. Meanwhile, enhancing our understanding and management of AF impairment could help discover potential preventive or therapeutic interventions for DDD.

Background and objective: Hypertrophic cardiomyopathy (HCM) is associated with a significant risk of progression to heart failure (HF). Extensive experimental and clinical research has highlighted the therapeutic benefits of ranolazine in alleviating electrophysiological abnormalities and arrhythmias in the context of HCM and HF. Despite these findings, there is a shortage of studies examining the electromechanical responses of failing HCM cardiomyocytes to ranolazine and the impact of ranolazine dosage on outcomes across varying degrees of HF. This study aims to systematically address these issues.

Methods: A computational modeling approach was utilized to quantify alterations in electromechanical variables within failing HCM cardiomyocytes subsequent to ranolazine treatment. The model parameters were calibrated against extant literature data to delineate the spectrum of HF severities and the changes in ion channels following the administration of various doses of ranolazine.

Results: The inhibition of the augmented late Na⁺ current in failing HCM cardiomyocyte with an adequate amount of ranolazine was found to be effective in alleviating electrophysiological abnormalities (e.g., prolongation of action potential (AP), Ca²⁺ overload in diastole), which contributed to improving the diastolic function of the cardiomyocyte, albeit with a modest negative effect on the systolic function. A threshold drug dose was identified for achieving a significant normalization of the overall electromechanical profile. The threshold drug dose for effective therapy was observed to be contingent upon the severity of HF and the status of certain key ion channels. Furthermore, it was determined that an increase of the drug dose beyond the threshold did not yield substantial additional improvements in the principal electromechanical variables.

Conclusions: The study demonstrated the presence of a threshold dose of ranolazine for effective treatment of failing HCM cardiomyocyte, and further established that this threshold is influenced by the severity of HF and the functional status of key ion channels. These findings may serve as theoretical evidence for comprehending the mechanisms underlying ranolazine's therapeutic efficacy in treating failing HCM hearts. Moreover, the study underscores the potential clinical value of personalized dosing strategies.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00842-5.

Purpose: The purpose of this study was to boost the therapeutic effect of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) by overexpressing the gene TSG-6 through CRISPR activation, and assess the biological activity of EVs from these modified MSCs in vitro on human intervertebral disc (IVD) cells.

Methods: An immortalized human MSC line was transduced with a CRISPR activation lentivirus system targeting TSG-6. MSC-EVs were harvested by ultracentrifugation and particle number/size distribution was determined by nanoparticle tracking analysis. The efficiency of transduction activation was assessed by analyzing gene and protein expression. EV proteomic contents were analyzed by mass spectrometry. Human IVD cells from patients undergoing spinal surgery were isolated, expanded, exposed to IL-1β pre-stimulation and co-treated with MSC-EVs.

Results: MSC-EVs presented size distribution, morphology, and molecular markers consistent with common EV characteristics. The expression level of TSG-6 was significantly higher (> 800 fold) in transduced MSCs relative to controls. Protein analysis of MSCs and EVs showed higher protein expression of TSG-6 in CRISPR activated samples than controls. Proteomics of EVs identified 35 proteins (including TSG-6) that were differentially expressed in TSG-6 activated EVs vs control EVs. EV co-Treatment of IL-1β pre-Stimulated IVD cells resulted in a significant downregulation of IL-8 and COX-2.

Conclusions: We successfully generated an MSC line overexpressing TSG-6. Furthermore, we show that EVs isolated from these modified MSCs have the potential to attenuate the pro-inflammatory gene expression in IVD cells. This genomic engineering approach hence holds promise for boosting the therapeutic effects of EVs.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00843-4.