Search Results (446)

Lentiviral vector (LV) production for CAR-T therapy remains challenging due to the limited scalability of adherent HEK293 cell cultures. To address this, we adapted HEK293FT cells directly to serum-free FreeStyle 293 Expression Medium, generating a novel suspension cell line, HEK293FT-DS. We characterized growth kinetics, stability over 35 passages, and transient transfection parameters (PEI:DNA ratio, cell density). The suspension-adapted cells grew with >93% viability and a specific growth rate of 0.54 day⁻¹, and maintained stable viable cell density (1.51 ± 0.03 × 10⁶ cells/mL) over 35 passages. The optimal PEI:DNA ratio was 2.5:1, and increasing the transfection cell density to 16 × 10⁶ cells/mL boosted functional titers to approximately 9 × 10⁶ TU/mL in clarified supernatant. Scaled production (1 L culture supernatant per batch) yielded up to 5 × 10⁹ TU. The resulting vectors efficiently transduced primary human T cells (57% CAR-positive cells) in a CliniMACS Prodigy-based process. The platform based on suspension-adapted HEK293FT-DS cells enables high-density transfection and provides a cost-effective, scalable alternative to commercial LV production systems, particularly suited for academic CAR-T cell manufacturing. Full article

Live-attenuated rotavirus (RV) vaccines are the most effective interventions for preventing RV gastroenteritis (RVGE) in young children. However, the molecular basis of attenuation remains not well understood. Here, we describe a compact but comprehensive strategy to identify RV virulence determinants by combining low-passage bidirectional selection, sequence analysis, and segment-level phenotype testing via a reverse genetics infectious system. Using the virulent murine RV strain EW, virulence was quantified by diarrhea severity/duration and body-weight gain. Serial passaging in cell culture selected an attenuated population, which regained virulence after passaging in suckling mice. Sequence comparison of the virulent and attenuated EW populations revealed only seven amino acid differences. We summarized literature describing attenuation/virulence-associated mutations in various RV group A (RVA) strains and found previous findings identical or similar to four of the seven mutations: NSP4-T45M, VP4-S470L, VP4-T612A, and VP7-T75P. Virulent- and attenuated-type EW variants of VP2, VP4, VP7, and NSP4 were introduced individually, or as NSP4/VP7 or VP4/VP7 pairs, into a simian SA11-L2 backbone using an 11-plasmid reverse genetics system. Phenotyping of rescued viruses consistently linked cell-culture–adapted VP4 to enhanced replication in vitro and reduced virulence in suckling mice. In vivo passaging strongly favored VP4 residue S470 over cell-culture-selected L470. More generally, our findings (i) underscore VP4 and VP7 as key determinants of EW virulence, (ii) provide a practical framework for identifying driver mutations underlying RVA attenuation, and (iii) highlight attenuation-associated substitutions shared across diverse RVAs. Full article

Porcine group A rotavirus (PoRVA) is a significant cause of viral diarrhea in piglets, necessitating urgent global implementation of effective control strategies. This review assesses advancements in PoRVA in vitro cultivation and amplification, crucial for PoRVA vaccine development. Traditional PoRVA cultivation commonly employs primary porcine kidney cells or finite cell lines like MA-104, posing well-documented challenges in scalability, production cost, and their ability to recapitulate the natural intestinal microenvironment. Consequently, research has increasingly focused on adapting PoRVA to alternative systems, particularly immortalized porcine cell lines or physiologically relevant porcine intestinal organoids. This adaptation process, involving serial passaging, can induce genomic alterations and virulence attenuation in piglets, essential for generating live attenuated vaccine (LAV) candidates. Modern biotechnological tools, such as reverse genetics and synthetic genomics, have expedited the creation of recombinant PoRVA strains with defined antigenic profiles and enhanced in vitro growth characteristics. However, a significant concern regarding LAV candidates derived from cell culture adaptation is the risk of virulence reversion upon pig back-passage, necessitating thorough safety and genetic stability evaluations. Nevertheless, utilizing stable cell lines or organoid platforms presents a feasible and cost-effective approach for large-scale PoRVA vaccine production. Future research should focus on identifying vaccine candidates that provide broad protection and exceptional safety, with an emphasis on cross-protection against divergent epidemic genotypes, while ensuring the economic feasibility of innovative manufacturing approaches. Full article

Background/Objectives: The corpus luteum (CL) in the dog is the sole source of progesterone (P4) during diestrus and pregnancy, making it a key regulator of reproductive function. However, robust and functionally validated in vitro models of canine luteal cells remain limited. This study aimed to establish and characterize a reproducible primary culture system of canine luteal cells with preserved steroidogenic activity and regulatory responsiveness. Methods: Ovaries containing CLs were collected from five clinically healthy bitches undergoing routine ovariohysterectomy (OHE). Luteal tissue was mechanically fragmented and enzymatically digested using collagenase type II. Primary cultures were established using an explant-based approach and maintained in Dulbecco’s Modified Eagle Medium/Ham’s F-12 nutrient mixture (DMEM/F12) or Roswell Park Memorial Institute medium 1640 (RPMI 1640) supplemented with 20% fetal bovine serum (FBS). Cellular morphology, proliferation, expression of steroidogenic markers—steroidogenic acute regulatory protein (STAR) and 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1), P4 secretion, and responsiveness to forskolin stimulation were evaluated. Results: Cultured luteal cells exhibited stable attachment, proliferation, and a predominantly spindle-shaped morphology. Both media supported maintenance of a steroidogenic phenotype, while RPMI 1640 enabled enhanced proliferation, allowing expansion up to passage three and efficient cryobanking. Cells remained functionally active, secreting progesterone for up to 28 days in vitro. Forskolin stimulation increased progesterone secretion up to 2.7-fold, confirming preserved cyclic AMP-dependent steroidogenic responsiveness. Conclusions: The canine CL is a reliable source of functionally competent luteal cells, and the established culture system represents a physiologically relevant in vitro model. To our knowledge, this is the first functionally validated in vitro model of the canine CL. This platform enables controlled investigations of luteal function, endocrine regulation, and mechanisms of P4 synthesis, supporting its application in mechanistic and translational reproductive research. Full article

iPSC technology is well established in mammals but remains underdeveloped in non-mammalian species. A major barrier to generating avian iPSCs has been the lack of species-specific reprogramming factors and culture conditions capable of supporting self-renewal in avian pluripotent stem cells. Here, we report the generation of chicken iPSCs (ciPSCs) using a cocktail of seven chicken transcription factors (T7: Oct4, Sox2, Sox3, Klf4, c-Myc, Nanog, and Lin28B) combined with an optimized avian culture system. Transcriptomic and functional analyses identified Sox3, rather than Sox2, as the predominant SoxB1 factor in avian reprogramming. The resulting ciPSCs exhibited stable self-renewal for over 40 passages, expressed core pluripotency markers, differentiated into all three germ layers, and were transcriptionally similar to chicken ESCs. In chimera assays, ciPSCs contributed to somatic, extra-embryonic, and germline lineages, giving rise to gonadal PGC-like cells that did not acquire full germline competence. We further demonstrate that the T7 system generates iPSCs from quail, duck, peacock, zebra finch, and pigeon, and that duck iPSCs can form interspecies chimeras with donor cells detected in the host gonads. These findings establish a generalizable platform for avian iPSC generation with applications in developmental biology and germline preservation of endangered species. Full article

Mesenchymal stromal cells (MSCs) are central to regenerative medicine and advanced therapies. However, the absence of consensus on reporting kinetic parameters, such as population doubling level (PDL), population doubling time (PDT), and the reliance on passage number alone obscures biological age and manufacturing history, and limits correlation of potency with expansion dynamics. Here, we clarify the distinctions among passages, PDL, PDT, and replication rate; we synthesize evidence that identical passage numbers can conceal multifold differences in cumulative doublings, with downstream effects on transcriptomic stability, and immunomodulatory performance. We further highlight culture determinants, oxygen tension, seeding density, media formulation, surface/bioreactor systems, and early niche mimetic stimuli, that shape proliferative kinetics and cellular aging trajectories in WJ-MSCs. Critically, we propose extracellular vesicles (EVs) as sensitive functional readouts of bioprocess stress and expansion history: EV quantity can increase while functional bioactivity declines, and EV miRNA cargo captures cell state programs not evident from minimal identity markers. To address these gaps, we recommend a reporting framework that incorporates: (1) culture conditions, (2) passage number and PDL at harvest, and (3) functional consequences of expansion. Adopting kinetic metrics beyond passage number will harmonize data capture and enable pooled analyses, accelerating clinical translation while safeguarding patient outcomes. Full article

Lentiviral vectors (LVs) are indispensable tools in cell and gene therapy. Rising demand has created a global shortage of LVs, driving the development of novel packaging approaches. We report a novel vector packaging approach using autonomously replicating cytoplasmic RNAs (replicons) to express packaging proteins. Yellow fever virus (YFV) was used as a source of replicons encoding the HIV-1 Gag–Pol polyprotein together with reporter or selectable markers. YFV replicons were able to establish chronic infection in HEK293FT cells. Replicons expressing HIV-1 Gag–Pol containing the wild-type HIV-1 protease caused strong cytotoxicity, which prevented the selection of polyclonal cell pools harboring the replicon. In contrast, a replicon carrying the T26S mutation in the HIV-1 protease gene showed no measurable cytotoxic effects, enabling the generation of stable replicon-containing cell pools. The replicon cell pools were established using antibiotic selection and maintained Gag-Pol expression for at least ten passages under selection pressure. Using these first-generation replicon cell pools as packaging cells, LV production required only transient transfection of a transfer vector, a Tat/Rev plasmid, and an envelope plasmid. Yields reached ~10⁶ TU/mL prior to concentration and ~10⁹ TU from multilayer cell stacks, which fall within the range typically reported for conventional transient transfection systems under similar culture conditions. The resulting vectors efficiently transduced target cells, and no replication-competent lentivirus (RCL) was detected using a two-phase RCL assay with p24 ELISA detection. This demonstrator platform utilizing replicon cell pools represents a novel approach for LV packaging. Full article

The isolation of fibroblast-like cells from crocodile skeletal muscle provides a valuable platform for studies in comparative physiology, reptilian biology, regenerative medicine, and tissue engineering. In this article, we present an optimized protocol for isolating and characterizing fibroblast-like cells derived from the embryonic skeletal muscle of the Siamese crocodile (Crocodylus siamensis). The procedure improved cell yields and viability while maintaining phenotypic and genetic stability. Dorsal and tail skeletal muscle tissue was cultured in flasks pre-coated with collagen. The cells attached and began migrating from the explants within one day. Optimal culture conditions were achieved using Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 or Minimum Essential Medium Alpha supplemented with 10% fetal bovine serum, 2% crocodile serum, and growth-promoting factors, incubated at 28 °C without CO₂. These conditions supported a shorter population doubling time and enhanced cell proliferation. The established cells displayed a spindle-shaped fibroblastic morphology, expressed the fibroblast-specific marker fibronectin, and maintained a stable karyotype through passage 20. The cell line remained stable and proliferative for at least 30 passages under standard culture conditions. Our study established the first standardized fibroblast-like cell line derived from C. siamensis, thus providing a foundation for future studies in reptilian physiology, cytogenetics, and regenerative biotechnology. Full article

A549 cells are widely used as an in vitro model of alveolar type II (ATII) epithelial cells; however, their phenotype and metabolic state are highly sensitive to culture conditions, cell density, and the duration of static, non-passaged cultivation. Here, we examined how prolonged static culture affects lipid metabolism, mitochondrial bioenergetics, and viability in A549 cells. A549 cultures were maintained without passaging for up to 25 days in DMEM or Ham’s F-12 and analyzed using lipid secretion assays, targeted lipidomics, [¹⁴C]-acetate incorporation, Seahorse bioenergetic profiling, and transcriptional analysis of stress-associated markers. Several surfactant-associated readouts were highest during early culture, peaking on day 7, as evidenced by elevated expression of ABCA3 and SP-A and maximal secretion of surfactant-associated phospholipids. With prolonged cultivation and increasing culture density, cellular phosphatidylglycerol levels declined progressively and became nearly undetectable by day 25, accompanied by reduced anabolic lipid metabolism, lower oxygen consumption, and impaired glycolytic activity. These changes coincided with increased reactive oxygen species, elevated intracellular Ca²⁺ levels, and increased expression of stress-associated transcripts, including CASP1, IL1B, and C3. Later stages were also associated with reduced mitochondrial respiration and decreased viability. Collectively, our findings show that prolonged static culture is associated with metabolic remodeling and reduced bioenergetic capacity in A549 cells. Full article

Cultured urine-derived cells (UDCs) have been proposed as a source of material for the RNA-based molecular diagnosis of genetic disorders. Previous studies have shown that UDCs can be clonally expanded, passaged, frozen, regrown and have some stem cell characteristics, but their anatomic origin and diagnostic utility remain insufficiently explored. In this study, we cultured UDCs from 40 individuals (aged 4 to 20 years; 21 females) and extracted RNA for sequencing. We compared UDC gene expression to that of marker genes of the kidney and urinary tract segments. UDC gene expression most closely matched marker genes of parietal epithelial cells that line the inner surface of Bowman’s capsule in the kidney glomerulus. UDCs expressed VCAM1 (CD106) and POUF51 (OCT4), consistent with a progenitor cell type. UDCs also expressed 54.4% of 3125 OMIM-listed disease-causing genes. This indicated that UDCs can be used to diagnose a similar number of genetic disorders as skin fibroblasts and a wider range of genetic disorders than can be analysed by RNA extracted from whole blood. In conclusion, UDCs are a non-invasive cell source for RNA sequencing that is suitable for investigating a broad range of conditions. Full article

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease with high case–fatality rates in East Asia, yet no licensed vaccines are currently available. Here, we generated attenuated severe fever with thrombocytopenia syndrome virus (SFTSV) strains through serial passage in Huh-7 cells and evaluated their pathogenicity, immunogenicity, and protective efficacy. Attenuation candidates were selected based on reduced pathogenicity, estimated based on the median lethal dose (LD₅₀), and genetic sequencing was performed to identify mutations associated with attenuation. In C57BL/6 IFNAR⁻/⁻ mice, the attenuated strain exhibited markedly reduced virulence and viral loads while inducing robust virus-specific IgG, neutralizing antibody, and cellular immune responses. Notably, immunization with the attenuated strain conferred complete protection against lethal challenge with heterologous SFTSV genotypes. Genomic analysis revealed nonsynonymous mutations in the RNA-dependent RNA polymerase (RdRp), glycoprotein, and NSs genes, implicating alterations in viral replication, entry, and immune evasion. Collectively, these findings demonstrate that serial cell-culture passage can generate attenuated SFTSV strains that retain strong immunogenicity and cross-protective efficacy, supporting their potential as live-attenuated vaccine candidates for SFTS. Full article

Food allergies are increasing worldwide, yet the early epithelial mechanisms that initiate allergic sensitization remain incompletely defined. As the intestinal epithelium governs both allergen translocation and epithelial–immune crosstalk, it constitutes a critical but underutilized model for predicting allergenicity. In this study, we used Caco-2 and T84 intestinal epithelial monolayers cultured on Transwell^® inserts to compare barrier properties and responses to peanut protein extract. Phenotypic characterization included biomarker profiling, transepithelial electrical resistance (TEER) measurements, tight junction integrity assessment, and analysis of cytokine levels as well as oxidative and nitrosative stress. Peanut exposure caused moderate TEER reductions without overt tight junction disruption while allowing translocation of the major allergen, Arachis hypogaea allergen 1 (Ara h 1), likely via transcellular pathways. Peanut protein extracts also induced epithelial stress responses, characterized by increased reactive oxygen species and nitric oxide production, alongside time-dependent secretion of innate and type 2-associated mediators, including IL-1β, TSLP, IL-25, and IL-33, indicating epithelial activation in the absence of complete barrier breakdown. Notably, basolateral supernatants from peanut-exposed epithelial monolayers activated THP-1-derived macrophages and enhanced IL-6 secretion, demonstrating that limited allergen passage across an otherwise intact epithelial barrier is sufficient to elicit early innate immune responses. Collectively, these findings indicate that peanut extract induce subtle functional perturbations in the intestinal epithelium while promoting downstream immune activation, highlighting Caco-2 and T84 cells as complementary in vitro platforms for studying barrier-dependent mechanisms of allergic sensitization. Full article

Metabolic dysfunction-associated steatotic liver disease (MASLD) and its consequences represent a growing global health burden that urgently requires physiologically relevant in vitro models beyond conventional 2D culture systems. In this study, we report the successful establishment of 45 patient-derived liver organoid lines. These organoids were generated from healthy, steatotic and cirrhotic tissues collected from 207 liver surgeries at RWTH University Hospital Aachen, with an initiation success rate of 82%. The organoids were propagated for at least six passages using an optimized protocol. Multiplex immunofluorescence analysis revealed highly proliferative structures with approximately 40% Ki-67-positive cells expressing hepatocyte (Albumin and HNF4α) and cholangiocyte (CK19) markers. Intermittent LGR5 staining suggested the presence of liver progenitor cell features. Quantitative PCR results confirmed variable HNF4α expression, indicating inter-patient heterogeneity in differentiation status. Time-lapse imaging combined with mathematical modeling uncovered a biphasic growth dynamic with an initial linear expansion in the first 15 h, followed by exponential growth (doubling time ≈ 20.6 h) between 30 and 72 h. Overall, our workflow produced genetically and phenotypically stable liver organoids that recapitulate essential features of various hepatic conditions. This provides a solid foundation for disease modeling, potential drug testing, and quantitative systems biology. Full article

Glioblastoma (GBM) is an extremely aggressive and incurable primary tumor of the brain. GBM is characterized by interpatient and intratumoral heterogeneity, making this cancer particularly resistant to therapy and likely to recur. Mapping the complex dynamics that underpin the development and evolution of gliomas with human-based in vitro models is difficult. This study aimed to generate 3D glioma patient-derived tumor constructs (PTCs) using a clinically relevant, Matrigel-free, hyaluronic acid system, evaluate their suitability in drug screening assays, and determine the stability of their genetic profiles compared to originating tumors. In this study, we utilized a synthetically modified hyaluronic acid and gelatin hydrogel system to generate tumor constructs containing cells from clinical glioma biospecimens. PTCs were characterized phenotypically, after which they were deployed in chemotherapy drug screens using temozolomide (TMZ) and a P53 activator compound. Drug responses of these 3D cultures were compared with 2D cultures, as well as PTCs that were generated after passaging in 2D. RNA sequencing was used to evaluate genetic parity between PTCs or 2D cultures with originating tumor tissues, using The Cancer Genome Atlas (TCGA) GBM subpopulations for subcategorizing. PTCs were created successfully from five World Health Organization (WHO) grade 4, two grade 3, and two grade 2 gliomas. PTCs were maintained with high viability. Chemotherapy drug screens demonstrated that expected TMZ responses were observed for Isocitrate dehydrogenase (IDH) mutant diffuse gliomas while drug response was variable for IDH wildtype GBM PTCs. PTCs demonstrated stable drug response over time, while 2D passaging resulted in significant shifts in drug sensitivity. RNA sequencing revealed maintenance of subpopulation signatures for PTCs which clustered with their originating patient tumor tissue. In contrast, 2D cultures largely clustered together regardless of the patient. Our PTC approach utilizes a defined hydrogel biomaterial system that maintains the genotypic and drug response characteristics of patient tumors making this an ideal ex vivo model for translational applications. Full article

Cancer-associated fibroblasts (CAFs), the major constituent of the tumor microenvironment, are considered the most active cells and key contributors to tumor resistance, recurrence, and metastasis. Therefore, we have investigated here the potential normalization of the active features of breast CAFs with decorin (DCN), a small leucine-rich proteoglycan that acts as an oncogene suppressor. We have first shown that rhDCN modulates the expression of a plethora of proteins involved in different signaling pathways, including STAT3/NF-κB and ERK. Consequently, rhDCN repressed the important active CAF biomarkers α-SMA, IL-6, and SDF-1 through inhibition of the STAT3/AUF-1 pathway, in cells grown as 2D and 3D cultures. Furthermore, rhDCN had a strong downregulation effect on FAP-α, a key biomarker of active CAFs, and suppressed their proliferative and invasive capacities through upregulation of p16 and p21, and downregulation of MMP-2 and MMP-9. Furthermore, rhDCN suppressed the paracrine effects of active CAFs in promoting epithelial-to-mesenchymal transition (EMT) and cancer stem cells in breast cancer cells, both in vitro and in orthotopic tumor xenografts. Importantly, rhDCN-related normalization of active CAFs features was persistent through cellular passaging, and was not accompanied by cytotoxicity. Together, these findings have revealed rhDCN as a promising anti-breast cancer therapeutic cytokine through suppression of the non-cell-autonomous cancer-promoting effects of active CAFs. Full article