Characterization of cellular responses and cell lysis to elevated hydrodynamic stress from benchtop perfusion bioreactors.

The effective design of perfusion cell culture is currently challenging regarding balancing the operating parameters associated with the hydrodynamic conditions due to increased system complexity. To address this issue, cellular responses of an industrial CHO cell line to different types of hydrodynamic stress in benchtop perfusion bioreactors originating from agitation, sparging, and hollow fibers (HF) in the cell retention devices were systematically investigated here with the analysis of cell lysis. It was found that cell lysis was very common and most associated with the sparging stress, followed by the HF and lastly the agitation, consequently heavily impacting the estimation of process descriptors related to biomass. The results indicated that the agitation stress led to a reduced cell growth with a shift toward a more productive phenotype, suggesting an energy redirection from biomass formation to product synthesis, whereas the sparging stress had a small impact on the intracellular metabolic flux distribution but increased the cell death rate drastically. For HF stress, a similar cell maintenance profile was found as the sparging while the activity of glycolysis and the TCA cycle was significantly impeded, potentially leading to the lack of energy and thus a substantial decrease in cell-specific productivity. Moreover, a novel concept of volume average shear stress was developed to further understand the relations of different types of stress and the observed responses for an improved insight for the perfusion cell culture.

Using a micro-device with a deformable ceiling to probe stiffness heterogeneities within 3D cell aggregates.

Recent advances in the field of mechanobiology have led to the development of methods to characterise single-cell or monolayer mechanical properties and link them to their functional behaviour. However, there remains a strong need to establish this link for three-dimensional (3D) multicellular aggregates, which better mimic tissue function. Here we present a platform to actuate and observe many such aggregates within one deformable micro-device. The platform consists of a single polydimethylsiloxane piece cast on a 3D-printed mould and bonded to a glass slide or coverslip. It consists of a chamber containing cell spheroids, which is adjacent to air cavities that are fluidically independent. Controlling the air pressure in these air cavities leads to a vertical displacement of the chamber's ceiling. The device can be used in static or dynamic modes over time scales of seconds to hours, with displacement amplitudes from a fewµm to several tens of microns. Further, we show how the compression protocols can be used to obtain measurements of stiffness heterogeneities within individual co-culture spheroids, by comparing image correlations of spheroids at different levels of compression with finite element simulations. The labelling of the cells and their cytoskeleton is combined with image correlation methods to relate the structure of the co-culture spheroid with its mechanical properties at different locations. The device is compatible with various microscopy techniques, including confocal microscopy, which can be used to observe the displacements and rearrangements of single cells and neighbourhoods within the aggregate. The complete experimental and imaging platform can now be used to provide multi-scale measurements that link single-cell behaviour with the global mechanical response of the aggregates.

Bioreactor Culture to Create Adipose Tissue from Human Mesenchymal Stromal Cells.

Adipose tissue is a complex and multifaceted endocrine organ located throughout the body. The dysfunction of adipose tissue is known to induce a wide variety of comorbidities that can negatively impact one's health and quality of life. In addition to behavioral changes, drugs that target dysfunctional adipose tissue to treat associated diseases are clinically needed. Regarding drug-testing platforms, animal models are the most popular models, limited by known differences from humans in genetics and physiology. Two-dimensional and static three-dimensional (3D) cell cultures are also used. Still, these in vitro models with static culture fail to recapitulate the phenotype and function of adipocytes seen in vivo. To combat this, our lab has developed an adipose tissue microphysiological system. A perfusion bioreactor with dual-flow chambers is 3D printed, which enables individualized top and bottom medium flows after adipose tissues are inserted as a barrier. Human progenitor cells, such as human mesenchymal stem cells, are embedded within a gelatin scaffold and in situ adipogenic differentiation within the bioreactor. Medium flow is established via a syringe pump system, allowing in vivo-like conditions to be maintained. The novel bioreactor-cultured adipose tissues represent a versatile disease modeling and drug-testing system. Here, we present the step-by-step methods to generate the bioreactors and adipose tissues. We also show the process of collecting and analyzing samples. In addition, we highlight the critical steps that require particular attention in notes.

Advantages of Using 3D Spheroid Culture Systems in Toxicological and Pharmacological Assessment for Osteogenesis Research.

Bone differentiation is crucial for skeletal development and maintenance. Its dysfunction can cause various pathological conditions such as rickets, osteoporosis, osteogenesis imperfecta, or Paget's disease. Although traditional two-dimensional cell culture systems have contributed significantly to our understanding of bone biology, they fail to replicate the intricate biotic environment of bone tissue. Three-dimensional (3D) spheroid cell cultures have gained widespread popularity for addressing bone defects. This review highlights the advantages of employing 3D culture systems to investigate bone differentiation. It highlights their capacity to mimic the complex in vivo environment and crucial cellular interactions pivotal to bone homeostasis. The exploration of 3D culture models in bone research offers enhanced physiological relevance, improved predictive capabilities, and reduced reliance on animal models, which have contributed to the advancement of safer and more effective strategies for drug development. Studies have highlighted the transformative potential of 3D culture systems for expanding our understanding of bone biology and developing targeted therapeutic interventions for bone-related disorders. This review explores how 3D culture systems have demonstrated promise in unraveling the intricate mechanisms governing bone homeostasis and responses to pharmacological agents.

Tracking Tau in Neurons: How to Grow, Fix, and Stain Primary Neurons for the Investigation of Tau in All Developmental Stages.

Primary murine neurons are a well-established tool for investigating Tau in the context of neuronal development and neurodegeneration. However, culturing primary neurons is usually time-consuming and requires multiple feeding steps, media exchanges, proprietary media supplements, and/or preparation of complex media. Here, we describe (i) a relatively cheap and easy cell culture procedure for the cultivation of forebrain neurons from embryonic mice (E13.5) based on a commercially available neuronal supplement (NS21), (ii) a protocol for the cultivation of hippocampal and cortical neurons from postnatal (P0-P3) animals, and (iii) basic fixation and immunofluorescence techniques for the staining of neuronal markers and endogenous Tau. We demonstrate a staining technique, which minimizes antibody consumption and allows for fast and convenient processing of samples for immunofluorescence microscopy of endogenous Tau in primary neurons. We also provide a protocol that enables cryopreservation of fixed cells for years without measurable loss of Tau signal. In sum, we provide reliable protocols enabling microscopy-based studies of Tau in primary murine neurons.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation.

BACKGROUND: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. METHODS AND RESULTS: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. CONCLUSIONS: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

The generation of glioma organoids and the comparison of two culture methods.

BACKGROUND: The intra- and inter-tumoral heterogeneity of gliomas and the complex tumor microenvironment make accurate treatment of gliomas challenging. At present, research on gliomas mainly relies on cell lines, stem cell tumor spheres, and xenotransplantation models. The similarity between traditional tumor models and patients with glioma is very low. AIMS: In this study, we aimed to address the limitations of traditional tumor models by generating patient-derived glioma organoids using two methods that summarized the cell diversity, histological features, gene expression, and mutant profiles of their respective parent tumors and assess the feasibility of organoids for personalized treatment. MATERIALS AND METHODS: We compared the organoids generated using two methods through growth analysis, immunohistological analysis, genetic testing, and the establishment of xenograft models. RESULTS: Both types of organoids exhibited rapid infiltration when transplanted into the brains of adult immunodeficient mice. However, organoids formed using the microtumor method demonstrated more similar cellular characteristics and tissue structures to the parent tumors. Furthermore, the microtumor method allowed for faster culture times and more convenient operational procedures compared to the Matrigel method. DISCUSSION: Patient-derived glioma organoids, especially those generated through the microtumor method, present a promising avenue for personalized treatment strategies. Their capacity to faithfully mimic the cellular and molecular characteristics of gliomas provides a valuable platform for elucidating tumor biology and evaluating therapeutic modalities. CONCLUSION: The success rates of the Matrigel and microtumor methods were 45.5% and 60.5%, respectively. The microtumor method had a higher success rate, shorter establishment time, more convenient passage and cryopreservation methods, better simulation of the cellular and histological characteristics of the parent tumor, and a high genetic guarantee.

Emerging models for studying adipose tissue metabolism.

Understanding adipose metabolism is essential for addressing obesity and related health concerns. However, the ethical and scientific pressure to animal testing, aligning with the 3Rs, has triggered the implementation of diverse alternative models for analysing anomalies in adipose metabolism. In this review, we will address this issue from various perspectives. Traditional adipocyte cell cultures, whether animal or human-derived, offer a fundamental starting point. These systems have their merits but may not fully replicate in vivo complexity. Established cell lines are valuable for high-throughput screening but may lack the authenticity of primary-derived adipocytes, which closely mimic native tissue. To enhance model sophistication, spheroids have been introduced. These three-dimensional cultures better mimicking the in vivo microenvironment, enabling the study of intricate cell-cell interactions, gene expression, and metabolic pathways. Organ-on-a-chip (OoC) platforms take this further by integrating multiple cell types into microfluidic devices, simulating tissue-level functions. Adipose-OoC (AOoC) provides dynamic environments with applications spanning drug testing to personalized medicine and nutrition. Beyond in vitro models, genetically amenable organisms (Caenorhabditis elegans, Drosophila melanogaster, and zebrafish larvae) have become powerful tools for investigating fundamental molecular mechanisms that govern adipose tissue functions. Their genetic tractability allows for efficient manipulation and high-throughput studies. In conclusion, a diverse array of research models is crucial for deciphering adipose metabolism. By leveraging traditional adipocyte cell cultures, primary-derived cells, spheroids, AOoCs, and lower organism models, we bridge the gap between animal testing and a more ethical, scientifically robust, and human-relevant approach, advancing our understanding of adipose tissue metabolism and its impact on health.

Application of Multivariate Data Analysis on Historical Recombinant Adenovirus Zoster Vaccine Production Data for Upstream Process Improvements.

In recent years, multivariate data analysis (MVDA) has been widely used for process characterization and fault diagnosis in the biopharmaceutical industry. This study aims to investigate the feasibility of using MVDA for the development and scale-up of a perfusion process for HEK293 cell-based recombinant adenovirus zoster vaccine (Ad-HER) production. The Principal Component Analysis (PCA) results suggested comparable performance among the ATF, PATFP, and BFP perfusion systems in benchtop-scale stirred-tank bioreactor (STR). Then a Batch Evolution Model (BEM) was built using representative data from 10 L STR with a BFP system to assess the Ad-HER perfusion process performance at pilot-scale bioreactor (50 L STR and 50 L wave bioreactor). Furthermore, another BEM model and Batch Level Model (BLM) were built to monitor process parameters over time and predict the final adenovirus titer in 50 L wave bioreactor. The loading plot revealed that lactate dehydrogenase activity, viable cell diameter, and base-added during the virus production phase could be used as preliminary indicators of adenovirus yield. Finally, an adenovirus titer of 2.0±0.3×1010 IFU/mL was achieved in the 50 L wave bioreactor with BFP system, highlighting the robustness of the Ad-HER perfusion process at pilot-scale. Overall, this study emphasizes the effectiveness of MVDA as a tool for advancing the understanding of recombinant adenovirus vaccine perfusion production process development and scale-up.

Comparison of permeable cell culture inserts for use in culture of a human in vitro air-liquid interface model system.

In this study, we compared 12 mm cell culture inserts with permeable polyester membranes (0.4 µm pores) from two different manufacturers: CELLTREAT® and Corning®. Physical dimensions and masses of the inserts were found to be very similar between the two brands, with CELLTREAT® inserts having a slightly smaller diameter and growth area (11.91 mm; 1.11 cm2 ) compared to Corning® Transwells® (12 mm; 1.13 cm2 ). We compared cell differentiation outcomes of human nasal epithelial cells (HNECs) at air-liquid interface grown on inserts from the two different manufacturers, including trans-epithelial electrical resistance, ciliary beat frequency, ciliated area, and gene expression. HNECs from three male donors were used for all endpoints. No statistically significant differences were observed between paired cultures grown on different brands of insert. In conclusion, these inserts are comparable for use with airway epithelial cell model systems and likely do not impact cellular differentiation or cell culture quality.

Vascularised cardiac spheroids-on-a-chip for testing the toxicity of therapeutics.

Microfabricated organ-on-a-chips are rapidly becoming the gold standard for the testing of safety and efficacy of therapeutics. A broad range of designs has emerged, but recreating microvascularised tissue models remains difficult in many cases. This is particularly relevant to mimic the systemic delivery of therapeutics, to capture the complex multi-step processes associated with trans-endothelial transport or diffusion, uptake by targeted tissues and associated metabolic response. In this report, we describe the formation of microvascularised cardiac spheroids embedded in microfluidic chips. Different protocols used for embedding spheroids within vascularised multi-compartment microfluidic chips were investigated first to identify the importance of the spheroid processing, and co-culture with pericytes on the integration of the spheroid within the microvascular networks formed. The architecture of the resulting models, the expression of cardiac and endothelial markers and the perfusion of the system was then investigated. This confirmed the excellent stability of the vascular networks formed, as well as the persistent expression of cardiomyocyte markers such as cTNT and the assembly of striated F-actin, myosin and α-actinin cytoskeletal networks typically associated with contractility and beating. The ability to retain beating over prolonged periods of time was quantified, over 25 days, demonstrating not only perfusability but also functional performance of the tissue model. Finally, as a proof-of-concept of therapeutic testing, the toxicity of one therapeutic associated with cardiac disfunction was evaluated, identifying differences between direct in vitro testing on suspended spheroids and vascularised models.

3D Cell Culture: Techniques For and Beyond Organoid Applications.

In the rapidly evolving landscape of cell biology and biomedical research, three-dimensional (3D) cell culture has contributed not only to the diversification of experimental tools available but also to their improvement toward greater physiological relevance. 3D cell culture has emerged as a revolutionary technique that bridges the long-standing gap between traditional two-dimensional (2D) cell culture and the complex microenvironments found in living organisms. By providing conditions for establishing critical features of in vivo environment, such as cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper tissue-like architecture and differentiated function of cells. Since the early days of 3D cell culture in the 1970s, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. One particular 3D cell culture technique has caught the attention of many scientists and has experienced an unprecedented boom and enthusiastic application in both basic and translational research over the past decade - the organoid technology. This book chapter provides an introduction to the fundamental concepts of 3D cell culture including organoids, an overview of 3D cell culture techniques, and an overview of methodological- and protocol-oriented chapters in the book 3D Cell Culture.

The potential of Rhodobacter sphaeroides extract as an alternative supplement for cell culture systems.

It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.

High throughput generating stable spheroids with tip-refill wafer.

Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210 rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60 rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1% ± 3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.

3D modeling of normal skin and cutaneous squamous cell carcinoma. A comparative study in 2D cultures, spheroids, and 3D bioprinted systems.

The current cancer research and drug testing are primarily based on 2D cell cultures and animal models. However, these methods have limitations and yield distinct drug response patterns. This study addressed this gap by developing an innovativein vitrohuman three-dimensional (3D) normal skin model and a multicellular model of human cutaneous squamous cell carcinoma (cSCC) using 3D bioprinting technology. Comparative analyzes were performed between bioprinted 3D-cSCC model, consisting of HaCaT keratinocytes, primary normal human dermal fibroblasts and A431 cancer cells (tricellular), bioprinted 3D-A431 model composed of A431 cancer cells only (monocellular), A431 cancer cell spheroids, and conventional 2D models. The models were structurally characterized by light microscopy, immunofluorescence (LIVE/DEAD assay, confocal microscopy) and immunohistochemistry (hematoxylin/eosin, p63, vimentin, Ki67, epidermal growth factor receptor stainings). The spatial arrangement of the 3D models was analyzed using the ARIVIS scientific image analysis platform. All models were also functionally assessed by cetuximab (CTX) response testing with the MTS assay. 3D-cSCC models were maintained for up to 16 weeks. Morphological and histological examinations confirmed the presence of skin-like layers in the bioprinted 3D models of normal skin, and the intricate and diverse features of the bioprinted skin cancer model, replicating the criticalin vivocharacteristics. In both mono- and tricellular bioprinted tumor constructs, there was a gradual formation and continuous growth of spheroid-like clusters of cancer cells, significantly influencing the morphology of the models. Cancer cells in the 3D bioprinted constructs showed reduced sensitivity to CTX compared to spheroids and 2D cultures. This study underscores the potential of 3D multicellular models in elucidating drug responses and gaining a better understanding the intricate interplay of cellular components within the tumor microenvironment. Developing the multicellular 3D tumor model paves the way for new research critical to advancing fundamental cancer research and future clinical applications, particularly drug response testing.

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration.

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification. Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 µm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2-5 µm) allowed not only to achieve high viable cell concentrations (VCC, 16.4-20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL). Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained. KEY POINTS: • Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield • Use of a TFDF system allowed continuous virus harvesting and clarification • TFDF perfusion system has great potential towards the establishment of an intensified vector production.

A strategy of novel molecular hydrogen-producing antioxidative auxiliary system improves virus production in cell bioreactor.

In the increasing demand for virus vaccines, large-scale production of safe, efficient, and economical viral antigens has become a significant challenge. High-cell-density manufacturing processes are the most commonly used to produce vaccine antigens and protein drugs. However, the cellular stress response in large-scale cell culture may directly affect host cell growth and metabolism, reducing antigen production and increasing production costs. This study provided a novel strategy of the antioxidant auxiliary system (AAS) to supply molecular hydrogen (H2) into the cell culture media via proton exchange membrane (PEM) electrolysis. Integrated with a high-density cell bioreactor, the AAS aims to alleviate cellular stress response and increase viral vaccine production. In the results, the AAS stably maintained H2 concentration in media even in the high-air exposure tiding cell bioreactor. H2 treatment was shown safe to cell culture and effectively alleviated oxidative stress. In two established virus cultures models, bovine epidemic fever virus (BEFV) and porcine circovirus virus type 2 (PCV-2), were employed to verify the efficacy of AAS. The virus yield was increased by 3.7 and 2.5 folds in BEFV and PCV-2 respectively. In conclusion, the AAS-connected bioreactor effectively alleviated cellular oxidative stress and enhanced virus production in high-density cell culture.

Investigation of cell infiltration and colonization in 3D porous scaffolds via integrated experimental and computational strategies.

This study aimed to integrate experimental and computational methods to systematically investigate cell infiltration and colonization within porous scaffolds. Poly(lactic acid) discs (Diameter: 6 mm; Thickness: 500 µm) with open pores (Diameter: 400-1100 µm), corners (Angle: 30-120°) and gaps (Distance: 100-500 µm), and cellulosic scaffolds with irregular pores (Diameter: 50-300 µm) were situated in tissue culture plates and cultured with human dermal fibroblasts (HDFs). Both phase contrast and scanning electron microscopy revealed that HDFs initially proliferated on scaffold surfaces, then infiltrated into the porous structures via cell bridging and stacking strategies, which was affected by the initial cell seeding densities, porous structures and culture times. Based on the density-dependent cell growths in two-dimensional cell cultures, power law models were developed to quantitatively simulate cell growths on scaffold surfaces. Model analysis predicted the effect of cell seeding efficiency on cell infiltrations into the porous scaffolds, which was further validated via series cell seeding experiments. The novelty of this research lies in the incorporation of multiple experimental and computational strategies, which enables the mechanistic insights of cell invasion and colonization in porous scaffolds, also facilitates the development of suitable bioprocesses for cell seeding and tissue manufacturing in Tissue Engineering and Regenerative Medicine.

Facilitating long-term cell examinations and time-lapse recordings in cell biology research with CO2 mini-incubators.

In recent years, microscopy has revolutionized the study of dynamic living cells. However, performing long-term live cell imaging requires stable environmental conditions such as temperature, pH, and humidity. While standard incubators have traditionally provided these conditions, other solutions, like stagetop incubators are available. To further enhance the accessibility of stable cell culture environments for live cell imaging, we developed a portable CO2 cell culture mini-incubator that can be easily adapted to any x-y inverted microscope stage, enabling long-term live cell imaging. This mini-incubator provides and maintains stable environmental conditions and supports cell viability comparable to standard incubators. Moreover, it allows for parallel experiments in the same environment, saving both time and resources. To demonstrate its functionality, different cell lines (VERO and MDA-MB-231) were cultured and evaluated using various assays, including crystal violet staining, MTT, and flow cytometry tests to assess cell adhesion, viability, and apoptosis, respectively. Time-lapse imaging was performed over an 85-h period with MDA-MB-231 cells cultured in the mini-incubator. The results indicate that this device is a viable solution for long-term imaging and can be applied in developmental biology, cell biology, and cancer biology research where long-term time-lapse recording is required.