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The general approach to industrial production of monoclonal antibodies is fed-batch culture using Chinese Hamster Ovary (CHO) cells. Perfusion culture is also attracting attention as a next-generation culture method. In these culture methods, optimization of amino acid and glucose concentration in the culture medium is essential, and influences cell proliferation, viability, productivity, and monoclonal antibody quality. Further, the maintenance of optimal nutrient levels – by avoiding both depletion and accumulation – is crucial. This study aimed to develop a dynamic feeding strategy based on specific indicators to maintain optimal amino acid and glucose concentrations. Multivariate correlation analysis confirmed a strong relationship between nutrient consumption and viable cell density (VCD). Regression analysis was used to establish a regression model to estimate amino acid and glucose consumption based on VCD. Using this model, the nutrient composition of feed media for both fed-batch and perfusion cultures was adjusted, and a dynamic feeding strategy guided by VCD was evaluated. The observed nutrient concentration trends closely matched the model’s predictions, confirming that VCD is a reliable indicator for implementing dynamic feeding. In both fed-batch and perfusion cultures, the VCD-guided dynamic feeding strategy enables the maintenance of multiple amino acids and glucose at target concentrations.

PurposePerfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra‐high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening.MethodsClonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors.ResultsThe selected clones achieved volumetric productivity (Pv) up to 5 g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60 g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline.ConclusionIn this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture.

Adenovirus is one of the most attractive viral vectors for therapeutic vaccines and gene therapy with the caveat that replication-competent adenoviruses (RCA) can be produced. To remediate this problem, engineered A-549 adenoviral vector complementing cells (SF-BMAdR cells) were previously generated by our organization for the production of E1-deleted adenoviral vectors without RCA. However, the production process remained to be improved for high titer production and scalability, as cost-effective and scalable biomanufacturing processes are critical for commercializing adenovirus-based vaccines and gene therapy. In this study, we first explored the potential of batch and fed-batch culture to increase maximum cell density and virus productivity by evaluating four different commercially available serum-free media and their combinations, and several feeds. A mixture (1:1) of two culture media improved the maximum cell density from 2.8 × 10 6 cells/mL obtained in the current batch culture to 4.2 × 10 6 cells/mL, and increased the virus productivity by 70% at a titer of 1.5 × 10 10 vp/mL. The fed-batch culture process, however, did not yield a significant improvement in either the maximum cell density or virus productivity. In contrast, batch culture with one medium replacement not only increased the cell growth but also resulted in an additional 70% improvement in the virus productivity at 2.6 × 10 10 vp/mL. The virus productivity was further increased to 6.3 × 10 10 vp/mL in a 3 L bioreactor perfusion culture infected at 7.0 × 10 6 cells/mL. This titer is 7.5 folds of the titer obtained in the current process. This study demonstrated the potential for a drastic improvement in the productivity of RCA-free adenovirus in the SF-BMAdR culture process. Furthermore, various processes developed fulfill different operational needs in manufacturing of RCA-free adenovirus to meet the increasing demands for therapeutic vaccines and gene therapy.

Continuous cultures of mammalian cells are complex systems displaying hallmark phenomena of nonlinear dynamics, such as multi-stability, hysteresis, as well as sharp transitions between different metabolic states. In this context mathematical models may suggest control strategies to steer the system towards desired states. Although even clonal populations are known to exhibit cell-to-cell variability, most of the currently studied models assume that the population is homogeneous. To overcome this limitation, we use the maximum entropy principle to model the phenotypic distribution of cells in a chemostat as a function of the dilution rate. We consider the coupling between cell metabolism and extracellular variables describing the state of the bioreactor and take into account the impact of toxic byproduct accumulation on cell viability. We present a formal solution for the stationary state of the chemostat and show how to apply it in two examples. First, a simplified model of cell metabolism where the exact solution is tractable, and then a genome-scale metabolic network of the Chinese hamster ovary (CHO) cell line. Along the way we discuss several consequences of heterogeneity, such as: qualitative changes in the dynamical landscape of the system, increasing concentrations of byproducts that vanish in the homogeneous case, and larger population sizes.

Background In recent years, gene therapy drugs have been widely marketed, and their effectiveness and potential have been confirmed. Thus, increasing their production on an industrial scale is critical. Recombinant adeno-associated viruses (rAAVs) are optimal vectors for gene therapy applications, and the baculovirus expression vector system (BEVS), which is based on Sf9 cell culture, is a common tool for rAAV production. Methods In this work, an Sf9 cell fed-batch process was developed using shake flasks. In the laboratory-scale bioreactor, four processes were selected as the key factors when carrying out the orthogonal experiment. On the basis of the equal P/V principle and considering the problem posed by air bubbles, a pilot-scale level bioreactor process was established. Results Here, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 10 6 cells/mL and the rAAV titre increasing to 20 × 10 11 VG/mL upon adding feed material. By resolving the problems associated with high-density cell culture and air bubbles, this process was successfully scaled to a 50 L pilot-scale level. Conclusions This successful experiment not only provides a technological basis for further scale-up but also guarantees product capacity. We hope that this development process can provide reference data for studying cell culture-based drug production.

3D cell culture has emerged as a relevant and promising alternative model for improving the preclinical phase of pharmaceutical development. It mimics cell-cell and cell-matrix interactions, as well as drug penetration and toxicity responses. However, there are currently no standardized methods that could lead to highly predictive treatment responses. In this context, the focus of this study was the adaptation of a liquid overlay technique, to generate a large and reproducible panel of six spheroid models, including melanoma, small cell lung cancer, non-small cell lung cancer, ovarian cancer, non-tumorigenic breast tissue and healthy kidney tissue. In this way, four cell concentrations (200 to 10,000 cells per well) with four matrix percentages (0 to 3%) were tested to determine the optimal combination based on their compaction, proliferation and viability. The surface characterization of each model was then assessed using scanning electron microscopy. Afterwards, the cytostatic and cytotoxic responses of these models to three targeted anti-PARP therapies, Olaparib, Rucaparib and Niraparib, were analyzed, revealing their sensitivity. These results demonstrated that our liquid overlay-based technique provides both a large cell culture panel, whatever the tissue type or pathological level, and an automated drug screening process that could lead to highly predictive efficacy results.

The adoption of three-dimensional (3D) cell culture systems represents a critical advancement in biomedical research, better mimicking complex 3D tissue environments than traditional two-dimensional (2D) models. However, variability in experimental outcomes has limited their reproducibility and clinical translation. Here, we systematically analyzed 32,000 spheroid images to identify key parameters influencing 3D model reliability. Our large-scale analysis revealed that oxygen levels significantly affect spheroid size and necrosis, while media composition (e.g., glucose and calcium concentrations) and serum levels (0–20%) critically regulate cell viability and structural integrity. For instance, spheroids cultured in 3% oxygen exhibited reduced dimensions and increased necrosis, whereas serum concentrations above 10% promoted dense spheroid formation with distinct necrotic and proliferative zones. By integrating single-cell RNA sequencing and automated image analysis, we uncovered dynamic gene expression patterns linked to spheroid maturation and hypoxia. These findings provide actionable guidelines for standardizing 3D culture protocols, addressing critical reproducibility challenges. Our work establishes a robust framework to enhance the reliability of 3D models in drug testing, personalized medicine, and tumor biology, facilitating their broader adoption in translational research.

Described herein is a protocol for producing a synthetic extracellular matrix that can be modified in situ during three-dimensional cell culture. The hydrogel platform is established using modular building blocks employing bio-orthogonal tetrazine (Tz) ligation with slow (norbornene, Nb) and fast ( trans -cyclooctene, TCO) dienophiles. A cell-laden gel construct is created via the slow, off-stoichiometric Tz/Nb reaction. After a few days of culture, matrix properties can be altered by supplementing the cell culture media with TCO-tagged molecules through the rapid reaction with the remaining Tz groups in the network at the gel–liquid interface. As the Tz/TCO reaction is faster than molecular diffusion, matrix properties can be modified in a spatiotemporal fashion simply by altering the identity of the diffusive species and the diffusion time/path. Our strategy does not interfere with native biochemical processes nor does it require external triggers or a second, independent chemistry. The biomimetic three-dimensional cultures can be analyzed by standard molecular and cellular techniques and visualized by confocal microscopy. We have previously used this method to demonstrate how in situ modulation of matrix properties induces epithelial-to-mesenchymal transition, elicits fibroblast transition from mesenchymal stem cells and regulates myofibroblast differentiation. Following the detailed procedures, individuals with a bachelor’s in science and engineering fields can successfully complete the protocol in 4–5 weeks. This protocol can be applied to model tissue morphogenesis and disease progression and it can also be used to establish engineered constructs with tissue-like anisotropy and tissue-specific functions. Key points This protocol describes a bio-orthogonal method for dynamically altering the adhesiveness or stiffness of the synthetic extracellular matrix during three-dimensional culture in a spatiotemporal manner to induce phenotypic changes and produce functional tissues. The method does not interfere with the native biological process, nor does it require external triggers or environmental changes. It thus more closely resembles the native extracellular environment and is straightforward to implement. This protocol describes a bio-orthogonal method for dynamically altering the adhesiveness or stiffness of the synthetic extracellular matrix during three-dimensional culture in a spatiotemporal manner to induce phenotypic changes and to produce functional tissues.

Three-dimensional spheroid models play a crucial role in cancer stem cell (CSC) research and drug screening. However, traditional methods often struggle with issues such as inconsistent shapes, difficulties in nutrient diffusion, and technical complexities. In this study, we introduce the SpheroidSync (SS) method, an innovative approach that combines conventional techniques to create uniform and size-adjustable MCF7 breast cancer spheroids at a very low cost, without the need for special growth factors or supplements. Our research involved culturing MCF7 cells using both the standard methods and the new SS method. We meticulously assessed various factors, including spheroid shape and cell viability, through fluorescent staining, colony formation assays, and gene expression analysis. The results revealed that the SS method produced spheroids that were not only uniform but also showed better structural integrity and maintained viability over time compared to traditional methods. Fluorescent viability tests indicated that SS spheroids consistently exhibited healthy cell viability as evidenced by sustained intracellular esterase activity throughout more extended culture periods. In contrast, spheroids generated through conventional methods exhibited declining viability, characterized by core deterioration and uneven staining. Furthermore, gene expression analysis showed a significant increase in CSC markers in SS spheroids, with CD44 levels rising over 40-fold, ALDH1 increasing more than threefold, CD24 decreasing, and HIF-1α elevating over 11-fold when compared to two-dimensional cultures. This establishes the typical breast CSC characteristics and confirms that a hypoxic environment was effectively created. Notably, as esterase activity declined, we observed an increase in stem cell populations, indicating a successful shift towards a quiescent, stem-like state. In summary, SpheroidSync represents a significant advancement in three-dimensional cancer modeling. It enables the production of uniform spheroids in a cost-effective manner, while ensuring long-term viability and enriching CSC populations.

Mesenchymal stromal/stem cells (MSCs) are promising candidates for regenerative medicine owing to their self-renewal properties, multilineage differentiation, immunomodulatory effects, and angiogenic potential. MSC spheroids fabricated by 3D culture have recently shown enhanced therapeutic potential. MSC spheroids create a specialized niche with tight cell-cell and cell-extracellular matrix interactions, optimizing their cellular function by mimicking the in vivo environment. Methods for 3D cultivation of MSCs can be classified into 2 main forms: static suspension culture and dynamic suspension culture. Numerous studies have reported the beneficial influence of these methods on MSCs, which is displayed by increased differentiation, angiogenic, immunomodulatory, and anti-apoptotic effects, and stemness of MSC spheroids. Particularly, recent studies highlighted the benefits of dynamic suspension cultures of the MSC spheroids in terms of faster and more compact spheroid formation and the long-term maintenance of stemness properties. However, only a few studies have compared the behavior of MSC spheroids formed using static and dynamic suspension cultures, considering the significant differences between their culture conditions. This review summarizes the differences between static and dynamic suspension culture methods and discusses the biological outcomes of MSC spheroids reported in the literature. In particular, we highlight the advantages of the dynamic suspension culture of MSC spheroids and contemplate its future applications for various diseases.