Filamentous microorganisms play a central role in industrial biotechnology, where pellet morphology affects mass transfer, rheology, and process performance. However, time-resolved monitoring of filamentous biomass and pellet size dynamics in stirred-tank reactors remains challenging due to offline sampling limitations. We present a noninvasive in situ imaging approach to observe biomass-related light scattering and projected pellet size. A custom-built camera and ring-light setup with MATLAB-based image acquisition and processing was used to quantify image luminosity as a proxy for biomass content and to determine projected object area (POA) and the number of detected objects per image above a POA threshold of 0.5 mm[2]. To characterize systematic biases, polyoxymethylene spheres of 1-4 mm diameter served as a model system. Luminosity scaled with object concentration, whereas object detection and apparent size estimation were increasingly affected by object size and concentration. In a bench-scale cultivation of malic acid-producing Aspergillus oryzae, image luminosity followed biomass content, while in situ POA analysis revealed increasing projected pellet size coinciding with Zn[2+] limitation. These observations differed from ex situ sampling, indicating that image-based in situ monitoring provides time-resolved information on biomass formation and pellet size dynamics that may not be captured by offline sampling alone.

Recombinant adeno-associated virus (rAAV) vectors have emerged as leading platforms for gene therapy owing to their safety, long-term transgene expression, and broad tissue tropism. However, rAAV production in HEK293 cells via triple-plasmid transfection remains limited by low yield, partly due to inefficient nuclear import of plasmids. In this study, we enhanced rAAV production by promoting plasmid nuclear delivery. DNA-targeting sequence (DTS) insertion alone did not improve plasmid nuclear import or rAAV yield, whereas combining DTS insertion with tubacin, an HDAC6 inhibitor, significantly increased nuclear plasmid accumulation and rAAV productivity. Among the three plasmids, inserting the 5NF sequence into pHelper produced the greatest effect. Under tubacin treatment, pHelper-5NF increased rAAV5 yield from 1.08 × 10[11] to 2.44 × 10[11] vg/mL (2.26-fold), without altering the full capsid percentage. This improvement was associated with increased Rep/Cap expression and viral DNA replication. Moreover, the pHelper-5NF enhanced rAAV production across multiple capsid serotypes, including rAAV1, rAAV2, rAAV6, rAAV8, rAAV9, and rAAVrh10. Unexpectedly, insertion of 5NF into two or three plasmids did not further increase rAAV yield via triple-plasmid transfection. These results identify pHelper-5NF combined with HDAC6 inhibition as an effective strategy to enhance triple-plasmid transfection-based rAAV manufacturing.

Retinal degenerative diseases, including age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and inherited retinal dystrophies, are among the leading causes of irreversible vision loss and blindness worldwide. Current treatments primarily slow disease progression and are unable to restore lost retinal cells or visual function, highlighting the need for regenerative therapeutic strategies. Stem cell-based therapies have emerged as a promising approach for retinal repair through multiple mechanisms, including retinal cell replacement, stimulation of endogenous regeneration, cellular reprogramming, immunomodulation, and paracrine signaling. This narrative review provides a comprehensive overview of the major stem cell sources investigated for retinal regeneration. In addition, the roles of extracellular vesicles and retinal organoids as emerging therapeutic platforms are discussed. Particular emphasis is placed on comparing the biological rationale, therapeutic potential, current preclinical and clinical evidence, and translational readiness of each approach. Despite encouraging progress, several challenges continue to limit routine clinical application, including poor survival and integration of transplanted cells, immune rejection, tumorigenicity, genomic instability, variability in differentiation efficiency, manufacturing standardization, and long-term safety. Continued advances in stem cell biology, tissue engineering, gene editing, and clinical trial design are expected to accelerate the development of safe and effective regenerative therapies for retinal diseases.

The lysosomal enzyme Arylsulfatase A (ARSA) plays a critical role in hydrolyzing sulfatides from within cells. While gene therapies for ARSA replacement have recently been approved, challenges remain in recombinantly producing large amounts of active ARSA, especially for research purposes where generating stable cell lines is cumbersome. A major bottleneck is the need to convert the active-site residue Cysteine at position 69 to formylglycine by a formylglycine-generating enzyme (FGE). Further, transporter mannose-6-phosphate receptors (M6PR) are required to mediate active transport of ARSA out of the cell. To address these issues, we have optimized a three-plasmid transient transfection approach that produces ARSA with active site conversion of over 70% as measured by mass spectrometry and is highly active in assays with a model substrate. We find the optimal ARSA:M6PR:FGE plasmid ratio to be 8:1:1, which balances active site conversion with total protein yield. This optimized protocol will assist in producing highly active ARSA for biochemical studies in research labs with access to standard cell culture facilities.

Continuous monitoring of therapeutic cell cultures via Raman spectroscopy traditionally relies on product-specific calibration models, which often fail to generalize across diverse commercial processes when process parameter or cell lines change. To overcome the need for extensive recalibration, an integrated generic Raman modeling framework was developed by aggregating in-line spectra from four distinct 1500-L commercial Chinese hamster ovary fed-batch processes. Fifteen combinations of regression algorithms and feature selection strategies were first evaluated under sample-level splitting, and representative pipelines were subsequently assessed through repeated batch-level validation splits. A Support Vector Machine combined with the interval whale optimization algorithm demonstrated competitive performance on validation and test sets. The model achieved high prediction accuracy for glucose, lactate, and viable cell density, with R2 values exceeding 0.94 and low prediction errors observed on the independent test set. Shapley Additive Explanations and permutation importance analyses indicated that the retained intervals were consistent with chemically plausible vibrational regions. Transfer to a fifth unseen commercial process further demonstrated the applicability limits of the global model and the potential of local updating with limited labeled spectra. The integration of machine learning algorithms and feature selection provides a practical strategy for multiproduct Raman PAT development in commercial biopharmaceutical manufacturing.

Head and neck squamous cell carcinoma (HNSCC) progression is profoundly influenced by dynamic cellular crosstalk within the tumor microenvironment (TME), where interactions between cancer cells, stromal components, and immune populations orchestrate therapeutic resistance, metastasis, and immune evasion. This review examines the utility of three-dimensional (3D) spheroid models as physiologically relevant platforms for investigating these cellular interactions compared with conventional two-dimensional (2D) cultures. By incorporating cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), endothelial cells, and cancer stem cells (CSCs) into multicellular spheroid systems, these models recapitulate several key features of the TME and provide insights into how signaling mediated by cytokines, exosomes, and direct cell-cell interactions may influence tumor progression. Studies utilizing spheroid models have highlighted processes associated with CAF-mediated CSC enrichment, TAM-driven immune modulation, and endothelial cell-supported niche formation, which have been linked to epithelial-mesenchymal transition (EMT), hypoxic adaptation, and metabolic reprogramming. Furthermore, 3D spheroid systems can reproduce aspects of spatial organization, extracellular matrix (ECM) remodeling, and microenvironmental gradients that are difficult to achieve in traditional 2D cultures and are relevant to the study of therapeutic resistance. The review also discusses recent advances, including microfluidic technologies and co-culture systems, which enhance the investigation of stromal-immune-tumor interactions and may facilitate the identification of potential therapeutic targets. Overall, this review highlights the value of 3D spheroid models as versatile preclinical tools for advancing our understanding of HNSCC biology and supporting the development and evaluation of personalized therapeutic strategies targeting pro-tumorigenic communication networks.

Expansion of human mesenchymal stromal cells (hMSCs) remains a bottleneck during manufacturing, where high cell yields must be achieved without compromising quality. Microcarriers provide elevated surface areas but often rely on synthetic materials. Here we use heparin/collagen Layer-by-Layer (LbL) coatings on microcarriers to enhance dynamic hMSC culture. HMSCs were cultured on LbL-coated and untreated microcarriers in up to 500 mL flasks, and were assessed over time, up to 18 days across three seeding densities (2600-20,000 cells/cm[2]), while yield was assessed at the end of culture. Metabolic analysis suggests no induction of a generalized stress response, increased glycolytic activity, and a shift toward a more active secretory state for cells grown on coated surfaces, as measured by noninvasive assays (Luminex multiplex and Raman spectroscopy). At medium and high densities, the cost (18.6%) and yield (60% and 30%, respectively) increases (coated: untreated) were moderately favorable. However, at the lower density, the cost increase remained similar, despite a yield increase of 110%. Together, the findings of this study demonstrate that our LbL surface coatings enhance hMSC functional performance and scalability of microcarrier-based culture, especially at lower initial cell densities, representing a promising approach for scalable cell manufacturing with the potential for GMP adaptation.

Over the past 50 years, the Drosophila melanogaster S2 cells have been valued for their ability to synthesize therapeutic molecules at high yield. To further increase protein expression, it is imperative to improve cellular performance, which is intrinsically linked to cell metabolism. Nevertheless, information on S2 metabolism, including pathways, components, and cellular compartments, remains limited, hindering advances in S2 cellular performance. Herein, using a genetically modified S2 cell line expressing the recombinant rabies virus glycoprotein (RVGP), we investigated the stress caused by RVGP production on S2 cells. Batch cultures using wild- and rec-types were performed, and 27 compounds were quantified over 192 h. The extracellular metabolome affected the rec-S2 growth kinetics after RVGP expression was activated. Although RVGP was produced in high amounts, we identified a substrate limitation for rec-S2 cell growth (glutamine), changes in amino acid routes due to RVGP biosynthesis (leucine, serine, glycine, and valine), and metabolites that might be affecting rec-S2 cell growth (acetate, pyruvate, citrate, and malate). Organic acid analysis indicated that malate and acetate production are correlated with RVGP production. This work revealed metabolic correlations in S2 cells that may have direct implications for media optimization and yield maximization, thereby improving S2 performance for scale-up.

The biocatalytic synthesis of UDP-glucuronic acid (UDPGA), a key precursor for glucuronides and high-value carbohydrates, is limited by an unstable UDP-glucose (UDPG) supply in coupled systems. In this study, through an extensive screening of 10 sucrose synthases derived from both eukaryotic and prokaryotic microorganisms, we identified a broad-spectrum, highly efficient enzymatic scaffold suitable for the synthesis of UDP-sugars. A rational design integrating PROSS and ThermoMPNN algorithms with molecular dynamics (MD) analysis yielded the V353L mutant, which retained 97% of wild-type activity and extended the half-life at 55°C from 0.4 to 5.5 h. Tetrameric MD simulations indicated that V353L strengthens hydrophobic stacking in the GT-B linker region, reducing conformational fluctuations and driving the enzyme toward a stable low-energy state. In a dual-enzyme cascade synthesizing UDPG and UDPGA, the mutant sustained UDP consumption and increased the accumulated UDPGA concentration from 0.60 to 2.09 mM after 8 h. Engineering sucrose synthase thermostability thus improves UDPG supply in cascade biocatalysis and offers a viable enzymatic strategy for UDPGA-centered synthesis of high-value carbohydrate derivatives.

S-Adenosylmethionine (SAM) is a high-value biomolecule with critical applications in nutraceuticals, pharmaceuticals, and health supplements. However, the detailed metabolic mechanism by which sodium citrate promotes SAM production in Pichia pastoris has not yet been elucidated. Here, a comprehensive time-series transcriptomic analysis revealed that sodium citrate profoundly influences gene expression across multiple pathways. Sodium citrate supplementation redirects carbon flux toward oxidative energy metabolism by upregulating amino acid biosynthesis, translation, and glycolysis. Crucially, the transcriptional upregulation of argininosuccinate synthase (ARG1) and argininosuccinate lyase (ARG4) was identified as a key node driving fumarate-mediated TCA cycle anaplerosis and ATP supply. This foundational analysis led to the identification of ARG4 as a key metabolic engineering target. Overexpression of ARG4 significantly improved SAM production, achieving a 56.46% increase in shake flasks and 9.06 g/L SAM (a 22.10% improvement) in 500 mL fermenters compared to control strains. Integrated physiological and metabolic flux analysis (MFA) demonstrated that ARG4 overexpression redirects metabolic flow, channeling energy substrates toward SAM synthesis, effectively maintaining cellular respiratory metabolism and alleviating energy limitations in the late fermentation phase. This work establishes a novel strategy for enhancing product synthesis by restructuring energy allocation rather than merely increasing overall energy supply.

Tyrosine sulfation is a post-translational modification that has been reported to occur infrequently on recombinant monoclonal antibodies (mAbs). We recently demonstrated that tyrosine sulfation occurred on a bispecific antibody (bsAb) produced in Chinese hamster ovary (CHO) cells, using a multi-enzymatic approach in combination with intact mass and peptide-based mass spectrometry analysis supplemented with the use of synthetic peptides. Tyrosine sulfation needs to be controlled during the manufacturing process due to potential undesired effects, such as impact on potency and immunogenicity. Here, we report that tyrosine sulfation was not significantly inhibited by the addition of chemical inhibitors, such as sodium chlorate. Individual knockout and double knockout (DKO) of two key genes in the tyrosine sulfation pathway were carried out sequentially. Tyrosyl protein sulfotransferase 1/2 (TPST1/2) DKO by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease)-mediated gene editing eliminated tyrosine sulfation while maintaining cell growth, antibody production, and overall product quality.

The recent spread of human monkeypox (mpox) beyond Africa has attracted global attention. Although mpox is a DNA virus with relatively lower pandemic potential, and smallpox vaccines and therapeutics may offer protection, the distinct clinical features observed in the current outbreak highlight the need for novel antiviral drugs. Here, we developed a drug repurposing workflow based on interspecies protein analysis and advanced virtual screening, integrating pre- and post-docking processes. After screening 6,074 approved or clinical-stage drugs against viral DNA topoisomerase, we expanded the analysis to viral DNA polymerase, increasing the compound library to 11,484 molecules, including approved or clinical-stage drugs and DNA polymerase inhibitors from ChEMBL. To assess dynamic stability, we performed molecular dynamics (MD) simulations and quantified protein-ligand contact occupancy, distinguishing stably bound candidates from transient docking poses. The MD and convolutional neural-network (GNINA) scoring analyses converged on vindesine for DNA topoisomerase and LY2090314 for DNA polymerase. Importantly, the workflow was applied to targets with distinct prior information, including a structurally characterized DNA polymerase supported by homologue inhibitor data and a less-characterized DNA topoisomerase requiring homology modelling. These results illustrate an adaptable repurposing pipeline for prioritizing therapeutic candidates against emerging infectious diseases, although experimental validation remains required.

Nucleases hydrolyze phosphodiester bonds and participate in numerous cellular and metabolic processes. Intracellular nucleases repair nonfunctional or damaged DNA using DNA base excision repair (BER), mismatch repair (MMR), and homologous recombination (HR). Apoptotic nucleases systematically degrade cellular DNA during programmed cell death (PCD). Non-apoptotic nucleases support DNA repair and replication. Small noncoding RNAs (sncRNAs) degrade the RNA of viral particles. Extracellular and membrane-associated nucleases replenish nucleotides, especially in biofilms where cells rely on additional carbon, phosphorus, and energy. Restriction endonucleases (REs) are indispensable in recombinant DNA technology. Some genetic disorders and cancers have been treated by changing the genetic code of host cells using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system. Nucleases are also used in vaccine development. Heterologous expression of nucleases remains challenging, largely due to cytotoxicity and product instability. Some successes have been reported using the T7 promoter-based system. However, due to the formation of inclusion bodies (IBs), the nucleases were insoluble and of low activity. Refolding misfolded nucleases from IBs, tight control (sequestration) of periplasmic secretion, and coexpression with natural inhibitor proteins increased yield, purity, and biological activity. This review addresses the significance of nucleases, heterologous expression, gene regulation, activity inhibition, and product yield.

Stable expression of regulatory non-coding RNAs enables targeted cell engineering in Chinese hamster ovary (CHO) cells but commonly relies on random integration, resulting in variable expression. Here, we establish a transposase-mediated platform for stable microRNA (miRNA) expression in CHO cells and demonstrate the first application of a DNA transposon system for long-term miRNA genome integration. Using miR-3096b-5p, we compared piggyBac-mediated integration with conventional random integration in antibody-producing CHO cells. Transposase-mediated integration enabled rapid generation of stable cell pools with higher fractions of GFP-expressing cells. Both integration strategies supported comparable growth, viability, and antibody production in batch cultures, indicating that transposase-mediated miRNA integration does not compromise bioprocess performance. At the molecular level, transposase-mediated integration resulted in significantly higher transgene copy numbers and markedly increased miRNA expression, translating into stronger and more consistent regulation of the target genes Fuk and Gmds. By combining high integration efficiency and expression stability with the regulatory potential of miRNAs, this approach expands the applicability of transposase systems beyond protein-coding transgenes and provides a robust platform for precise and durable post-transcriptional gene regulation in CHO cell engineering.

Methanol is a promising renewable C1 feedstock for sustainable single-cell protein (SCP) production. However, its inherent cytotoxicity and metabolic trade-offs between cell growth and protein synthesis remain significant bottlenecks. Here, we established an "evolutionary-rational" dual-driven paradigm to construct a high-yield Pichia pastoris chassis. Through UV mutagenesis and adaptive laboratory evolution, we developed a highly tolerant strain A40, capable of growing in 70 g/L methanol. Notably, at 30 g/L methanol, A40 achieved a 3.4-fold higher maximum biomass than the wild-type. Whole-genome resequencing and reverse genetics revealed that this superior performance stems from a multi-gene synergistic network rather than a single dominant mutation. To further optimize SCP production, we rationally co-overexpressed nitrogen assimilation genes (GLN1, GDH1) and a translation elongation factor (PpeEF3) in the A40 background. This targeted metabolic engineering effectively redirected carbon flux toward protein biosynthesis. The engineered strain A40-2Ge3 achieved a peak intracellular crude protein content of 67.9% and a 51.3 g/L total titer in a 5-L bioreactor, representing a 23.1% increase over the wild-type strain. Collectively, this study provides deep insights into the synergistic mechanisms of methanol adaptation and establishes an efficient, scalable strategy for sustainable SCP production from C1 feedstocks.