Journal of bioscience and bioengineering最新文献

Monoclonal antibodies (mAbs) are key therapeutics for diseases like cancer and autoimmunity. The production of mAbs relies on cell culture, in which the culture medium for high productivity and activity is essential. Despite the traditional manual and advanced computational methodologies for medium optimization, it remains challenging to incorporate biological insights gained during cell culture experimentation into the optimization process. To address this issue, an active learning strategy that sequentially integrates machine learning predictions with experimental observations of biological meaningfulness was developed in the present study. Medium design and prediction were conducted with the combination of the design of experiment and two different machine learning models, to optimize the culture medium for Chinese hamster ovary (CHO) cells producing increased immunoglobulin G (IgG) titer. Using this approach, we iteratively adjusted the concentrations of 44 components in a serum-free medium and achieved a significant improvement in IgG monoclonal antibody production. Biological insights such as osmolality control and amino acid composition, which were not initially considered, were progressively incorporated into the data-driven optimization process. The proposed strategy is practical and effective, even under limited experimental resources, and offers a new direction for rational medium design in biopharmaceutical manufacturing.

Type I diabetes is a chronic disease that affects people worldwide. When insulin administration is no longer effective, transplantation of pancreatic islets represents an alternative for diabetics. However, islet grafting carries limitations, which include poor availability of donors, surgery risks, and lifelong immunosuppressive therapy. To address this, novel approaches, such as the use of soft hydrogels as vehicles of cells are being developed for tissue grafting applications. Self-assembling peptide hydrogels (SAPHs) are biocompatible and versatile materials widely used for both, three-dimensional (3D) cell culture and regenerative medicine applications. Therefore, in this study, we explored the effect of the functionalization of the SAPH FEFEFKFKK (FEK9) with extracellular matrix (ECM) motifs - RGD, GFOGER and IKVAV - to support the directed differentiation of human dental pulp stem cells (hDPSCs) into insulin-producing cells (IPCs). The resulting ECM-functionalized FEK9 hydrogel was formed under mildly acidic conditions (pH 5-6). Infrared spectroscopy confirmed that ECM-FEK9 adopts a β-sheet secondary structure and forms a dense nanofibrillar network, while rheological measurements demonstrated the formation of a soft hydrogel. hDPSC cultured in hydrogel displayed steady viability and metabolism. Moreover, under directed induction, cells in ECM-FEK9 expressed β-cell markers, such as PDX-1 and Glut-2, as well as synthetized insulin within 10 days of 3D culture in vitro, as evidenced through fluorescence confocal microscopy and spectrophotometry evaluations, respectively. Therefore, ECM-FEK9 could be a promising candidate to support the culture of hDPSCs and the generation of IPCs after refinement of directed induction under 3D cell culture conditions.

The development of an effective vaccine against noroviruses remains a major public health priority. Norovirus GII.4 capsid protein VP1 as a promising vaccine candidate was produced by the methylotrophic yeast Ogataea minuta production system. It was intracellularly expressed and subsequently purified by immobilized metal affinity chromatography and anion exchange chromatography, yielding 13.4 mg of highly purified VP1 protein from 50 mL of culture supernatant. The formation of VP1-based virus-like particles (VLPs) that retained their structure even after freeze-thawing was confirmed by transmission electron microscopy. The VP1 protein purified in the form of VLPs displayed strong antigenicity and specific, dose-dependent binding to histo-blood group antigens, as determined by the enzyme-linked immunosorbent assay (ELISA). Immunogenicity studies in BALB/c mice demonstrated that intramuscular administration induced robust serum IgG responses across all tested doses, with no significant dose-dependent differences. Furthermore, mucosal administration intranasally or sublingually induced systemic IgG, systemic IgA, and mucosal IgA responses. These responses were significantly enhanced by the lipid A adjuvant. These findings showed that the O. minuta production system is capable of producing immunogenic Norovirus VLPs as a vaccine candidate.

Induced pluripotent stem cells (iPSCs) have significant potential for regenerative medicine, particularly for bone tissue engineering. While three-dimensional spheroid cultures enhance iPSC differentiation by better mimicking physiological conditions, spheroid size critically affects cell viability and differentiation ability. Microwell plates enable large-scale production of uniform spheroids and would be especially useful for regenerative medicine and tissue engineering. Here, we investigated the effect of spheroid size on the osteogenic differentiation of iPSCs using microwell plates to generate spheroids under the following conditions: Elp200 (microwell plate with 200/100 μm diameter/depth) and Elp900 (microwell plate with 900/700 μm). We observed that larger Elp900 spheroids promoted mesodermal differentiation more effectively, likely due to enhanced cell-cell interactions and altered internal microenvironments. However, Elp900 spheroids exhibited increased apoptosis in their core regions, evidenced by viability staining, transmission electron microscopy, and TUNEL staining. Upon dissociation and adherent culture, Elp900-derived cells demonstrated significantly higher expression of osteogenic markers (Runx2, Ibsp) and mineralization compared to Elp200-derived cells. Proteomic analysis revealed that apoptosis- and extracellular matrix (ECM)-related proteins, such as SERPINH1 and COL4A1, were upregulated in Elp900 cultures. These findings suggest that controlled apoptosis within larger spheroids may activate stress-related pathways, promote ECM formation, and enhance osteogenic differentiation by activating the TGF-β signaling pathway. Our findings highlight optimal spheroid sizing as a key factor for maximizing the efficiency and reproducibility of osteogenic differentiation of iPSCs, providing a foundation for improved strategies in iPSC-based bone tissue regeneration.

Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are membrane-bound vesicles secreted by cells. They play essential roles in intercellular communications and are involved in numerous physiological processes. Given their functional importance, EVs have emerged as promising tools for diagnosing and treating various diseases. In this study, we focused on the utility of EVs and explored their application in the analysis of contact-dependent cell-cell interactions, which are essential for the control of cell differentiation and induction of immune responses. Although several methods have been developed to evaluate these interactions, they often require complex procedures and advanced optimization, limiting their broad applicability. To overcome these limitations, we developed a novel method utilizing EVs to present membrane proteins in their native conformations. Our strategy involved producing fluorescently labeled EVs with target antigens and quantitatively assessing their binding to target cells via flow cytometry. Using fluorescently labeled EVs presenting with either an N-terminal pro-brain natriuretic peptide or interleukin-2 receptor, we successfully detected specific interactions with corresponding hybridoma B cell receptors. This simpler method requires no advanced optimization and effectively analyzes cell-cell interactions under physiological conditions in a high-throughput and quantitative manner. Our findings highlight the potential of this EV-based system as a valuable tool for studying membrane protein-mediated cell-cell interactions in bioscience research.

Synthetic biology approaches enable the creation of promising chassis for practical application in various fields, though engineering of microbial metabolism often imposes a metabolic burden, potentially driving adaptive evolution during long-term cultivation. A previously established phosphite (Pt)-dependent metabolic system has proven to be an effective strategy for the containment of genetically engineered microorganisms, although its implementation accompanied a slight growth retardation. Here, we investigated the effect of long-term serial passaging cultivation on the Pt-dependent strain of Synechococcus elongatus PCC 7942, RH714. Compared with the originally constructed RH714, the passaged population of RH714 exhibited improved growth and a higher rate of Pt consumption in culture medium. Sequence analysis revealed point mutations within the introduced htxBCDE transporter genes, which are required for selective incorporation of Pt as a phosphorus nutrition. Introduction of the mutated gene cluster into S. elongatus PCC 7942 reproduced the traits of the passaged RH714 population, suggesting that these genetic changes enhance Pt transport activity and account for the observed phenotypes. Disruption of endogenous phosphate (Pi) transporter genes in the strains expressing the mutated htxBCDE-ptxD cluster abolished growth in Pi-containing medium, suggesting that the mutations in the transporter genes did not alter substrate specificity toward Pi. These results indicated that long-term passage cultivation developed an optimized mutant capable of efficient proliferation under the Pt metabolizing conditions without compromising its biocontainment capability.

Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in initiating and shaping adaptive immune responses. Targeting antigens to DCs has emerged as a promising strategy to enhance vaccine efficacy and tailor desired immune responses. While antibodies are well established as targeting molecules for DCs, the use of peptides remains underexplored despite their favorable tissue penetration and their ability to offer design flexibility. Here, we report the identification of dectin-1-binding peptides using a ribosome display-based in vitro directed evolution system. Dectin-1 is a C-type lectin receptor expressed on murine CD11b⁺ and human CD1c⁺ DCs, which plays a key role in antigen uptake and in directing immune responses toward specific pathways, particularly the Th17 pathway. Using recombinant murine dectin-1 as bait, we performed four rounds of ribosome display selection and obtained peptides with high affinity. Selected peptides fused to enhanced green fluorescent protein showed binding to recombinant dectin-1 and native dectin-1-expressing cells, including bone marrow-derived DCs. These results demonstrated the feasibility of peptide-based molecular targeting toward dectin-1⁺ DCs, which would not only provide a versatile platform for the development of next-generation vaccines and immunotherapies, but also a valuable tool for dissecting the functional roles of dectin-1⁺ DCs in immune regulation.

Single-cell genomics (SCG) complements culture-independent metagenomics for accessing fungal genomes, particularly from lineages that remain uncultured. We contrast metagenomics, which excels when profiling community composition and metabolic potential but often underrepresents low-abundance fungi, with SCG, which first isolates individual cells or nuclei to generate single-amplified genomes (SAGs) and can recover rare or microdiverse taxa. We then organize existing fungal SCG applications into three subgroups: spore-level sequencing from host-enriched or environmental material; single-nucleus genomics for multinucleate fungi; and single-spore sequencing of haploid progeny for diploid linkage and chromosome phasing. Across studies, pooling and co-assembly of cognate cells improves completeness; key hurdles persist in wall lysis, whole-genome amplification bias, and contamination control. Practical advances include shallow sequencing for QC triage, nuclei pooling with normalized co-assembly, and hybrid long- and short-read assembly. SCG adds unique value where strain resolution and genotypic context matter, including host-to-mobile-element linkage, recovery of large biosynthetic gene clusters, and karyotype validation against telomere-to-telomere references. Used alongside metagenomics, SCG enables a strain-resolved view of fungal biodiversity and function, with incremental improvements across the SCG pipeline promising routine access to genomes from early-diverging and other environmentally embedded fungi.

Microcarrier surface topography and fluid shear stress (FSS) critically regulate cellular behavior. An edible zein/fucoidan microcarrier with 200 μm grooves was designed for this study. Computational fluid dynamics (CFD) simulations and experiments were combined to analyze surface microfluidic characteristics during dynamic culture and their cellular effects. The research demonstrates controlled myogenic differentiation through groove topography and FSS modulation for scalable cultured meat production. The results demonstrated that the grooved microcarriers exhibited excellent cell attachment and proliferation capacity in spinner flask dynamic culture, achieving a maximum cell density of 1.16 × 10⁶ cells/mL, comparable to commercial Cultispher-S microcarriers. The groove structure promoted cell alignment through contact guidance, significantly enhancing the gene expression of myogenic differentiation markers (myogenic differentiation 1, MyoD1; α-actinin; myosin heavy chain, MHC) and cell fusion (myomaker, MYMK). CFD simulations revealed that the grooves created a low-shear microenvironment (minimum average FSS: 3.93 × 10^-2 Pa, maximum: 1.18 × 10^-1 Pa), which effectively avoided high FSS-induced damage while maintaining mechanical stimulation. This optimal mechanical microenvironment further activated the expression of key genes involved in early-stage (MyoD1; myogenin, MyoG; myocyte enhancer factor 2C, MEF2C) and late-stage (α-actinin; myosin heavy chain 2, Myh2) myogenic differentiation. Flat and spherical microcarriers showed lower myogenic differentiation efficiency. This study elucidates the synergistic mechanism between groove structures and the low FSS microenvironment within grooves, providing a novel scaffold design rationale that combines biomimetic topology with fluid dynamics compatibility for large-scale cultured meat production.