Cell Culture System Research Articles

Parameter Optimization and Capacitance-Based Monitoring of In Situ Cell Detachment in Microcarrier Cultures

This study delves into the scale-down optimization of the in situ cell detachment process for MA 104 cells cultivated on Cytodex 1 microcarriers (MCs). Through a systematic exploration, critical operational parameters—the agitation speed, incubation time, Trypsin–EDTA volume and corresponding activity, and washing steps—were identified as key factors influencing the efficiency and scalability of in situ cell detachment in microcarrier-based cell culture. Maintaining an appropriate agitation speed (1.25 × Njs, minimum agitation speed at which no microcarriers remain stationary for the signification period of 5 s), optimizing the Trypsinization incubation time (up to 60 min), and implementing multiple washing steps (two times) post-medium removal were found to be crucial for efficient cell detachment and subsequent growth. Our study demonstrates the feasibility of reducing the final Trypsin volume to 50 mL per gram of microcarrier while maintaining a Trypsin activity above 380 USP/mL. These conditions ensure complete cell dissociation and improve the cost effectiveness in large-scale productions. Additionally, we introduced real-time monitoring using a capacitance sensor during in situ cell detachment. This method has proven to be an effective process analytical technology (PAT) tool for tracking the cell detachment progress and efficiency. It allows for the prediction of cell detachment based on signals recorded between 3 and 7 min of Trypsinization, enabling rapid process decisions without the need for offline sampling, thereby enhancing the overall process control. This systematic approach not only optimizes in situ cell detachment processes but also has significant implications for the scalability and efficiency of microcarrier-based cell culture systems.

Norovirus-mediated translation repression promotes macrophage cell death.

Norovirus infection is characterised by a rapid onset of disease and the development of debilitating symptoms including projectile vomiting and diffuse diarrhoea. Vaccines and antivirals are sorely lacking and developments in these areas are hampered by the lack of an adequate cell culture system to investigate human norovirus replication and pathogenesis. Herein, we describe how the model norovirus, Mouse norovirus (MNV), produces a viral protein, NS3, with the functional capacity to attenuate host protein translation which invokes the activation of cell death via apoptosis. We show that this function of NS3 is conserved between human and mouse viruses and map the protein domain attributable to this function. Our study highlights a critical viral protein that mediates crucial activities during replication, potentially identifying NS3 as a worthy target for antiviral drug development.

Deciphering the physiopathology of neurodevelopmental disorders using brain organoids.

Neurodevelopmental disorders (NDD) encompass a range of conditions marked by abnormal brain development in conjunction with impaired cognitive, emotional, and behavioural functions. Transgenic animal models, mainly rodents, traditionally served as key tools for deciphering the molecular mechanisms driving NDD physiopathology, and significantly contributed to the development of pharmacological interventions aimed at treating these disorders. However, the efficacy of these treatments in humans has proven to be limited, due in part to the intrinsic constraint of animal models to recapitulate the complex development and structure of the human brain but also to the phenotypic heterogeneity found between affected individuals. Significant advancements in the field of induced pluripotent stem cells (iPSC) offer a promising avenue for overcoming these challenges. Indeed, the development of advanced differentiation protocols for generating iPSC-derived brain organoids gives the unprecedented opportunity to explore the human neurodevelopment. This review provides an overview of how 3D brain organoids have been used to investigate various NDD (i.e., Fragile X syndrome, Rett syndrome, Angelman syndrome, microlissencephaly, Prader-Willi syndrome, Timothy Syndrome, tuberous sclerosis syndrome), and elucidate their pathophysiology. We also discuss the benefits and limitations of employing such innovative 3D models compared to animal models and 2D cell culture systems, in the realm of personalized medicine.

Salidroside alleviates simulated microgravity-induced bone loss by activating the Nrf2/HO-1 pathway

BackgroundBone loss caused by microgravity exposure presents a serious threat to the health of astronauts, but existing treatment strategies have specific restrictions. This research aimed to investigate whether salidroside (SAL) can mitigate microgravity-induced bone loss and its underlying mechanism.MethodsIn this research, we used hindlimb unloading (HLU) and the Rotary Cell Culture System (RCCS) to imitate microgravity in vivo and in vitro.ResultsThe results showed that salidroside primarily enhances bone density, microstructure, and biomechanical properties by stimulating bone formation and suppressing bone resorption, thereby preserving bone mass in HLU rats. In MC3T3-E1 cells cultured under simulated microgravity in rotary wall vessel bioreactors, the expression of osteogenic genes significantly increased after salidroside administration, indicating that salidroside can promote osteoblast differentiation under microgravity conditions. Furthermore, the Nrf2 inhibitor ML385 diminished the therapeutic impact of salidroside on microgravity-induced bone loss. Overall, this research provides the first evidence that salidroside can mitigate bone loss induced by microgravity exposure through stimulating the Nrf2/HO-1 pathway.ConclusionThese findings indicate that salidroside has great potential for treating space-related bone loss in astronauts and suggest that Nrf2/HO-1 is a viable target for counteracting microgravity-induced bone damage.

An Evaluation of the Replacement of Animal-derived Biomaterials in Human Primary Cell Culture.

The likelihood that potential new drugs will successfully navigate the current translational pipeline is poor, with fewer than 10% of drug candidates making this transition successfully, even after their entry into clinical trials. Prior to this stage, candidate drugs are typically evaluated by using models of increasing complexity, beginning with basic in vitro cell culture studies and progressing through to animal studies, where many of these candidates are lost due to lack of efficacy or toxicology concerns. There are many reasons for this poor translation, but interspecies differences in functional and physiological parameters undoubtedly contribute to the problem. Improving the human-relevance of early preclinical in vitro models may help translatability, especially when targeting more nuanced species-specific cell processes. The aim of the current study was to define a set of guidelines for the effective transition of human primary cells of multiple lineages to more physiologically relevant, translatable, animal-free in vitro culture conditions. Animal-derived biomaterials (ADBs) were systematically replaced with non-animal-derived alternatives in the in vitro cell culture systems, and the impact of the substitutions subsequently assessed by comparing the kinetics and phenotypes of the cultured cells. ADBs were successfully eliminated from primary human dermal fibroblast, uterine fibroblast, pulmonary fibroblast, retinal endothelial cell and peripheral blood mononuclear cell culture systems, and the individual requirements of each cell subtype were defined to ensure the successful transition toward growth under animal-free culture conditions. We demonstrate that it is possible to transition ('humanise') a diverse set of human primary cell types by following a set of simple overarching principles that inform the selection, and guide the evaluation of new, improved, human-relevant in vitro culture conditions.

Drupacine as a potent SARS-CoV-2 replication inhibitor in vitro

Despite the availability of vaccines and antiviral treatments, the continued emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and breakthrough infections underscores the need for new, potent antiviral therapies. In a previous study, we established a transcription and replication-competent SARS-CoV-2 virus-like particle (trVLP) system that recapitulates the complete viral life cycle. In this study, we combined high-content screening (HCS) with the SARS-CoV-2 trVLP cell culture system, providing a powerful phenotype-oriented approach to assess the antiviral potential of compounds on a large scale. We screened a library of 3,200 natural compounds and identified drupacine as a potential candidate against SARS-CoV-2 infection. Furthermore, we utilized a SARS-CoV-2 replicon system to demonstrate that drupacine could inhibit viral genome transcription and replication. However, in vitro, enzymatic assays revealed that the inhibition could not be attributed to conventional antiviral targets, such as the viral non-structural proteins nsp5 (MPro) or nsp12 (RdRp). In conclusion, our findings position drupacine as a promising antiviral candidate against SARS-CoV-2, providing a novel scaffold for developing anti-coronavirus disease 2019 (COVID-19) therapeutics. Further investigation is required to pinpoint its precise target and mechanism of action.

Therapeutic potential of mesenchymal stem cell-derived extracellular vesicles in SARS-CoV-2 and H1N1 influenza-induced acute lung injury.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have shown anti-inflammatory potential in multiple inflammatory diseases. In the March 2022 issue of the Journal of Extracellular Vesicles, it was shown that EVs from human MSCs can suppress severe acute respiratory distress syndrome, coronavirus 2 (SARS-CoV-2) replication and can mitigate the production and release of infectious virions. We therefore hypothesized that MSC-EVs have an anti-viral effect in SARS-CoV-2 infection in vivo. We extended this question to ask whether also other respiratory viral infections could be treated by MSC-EVs. Adipose stem cell-derived EVs (ASC-EVs) were isolated using tangential flow filtration from conditioned media obtained from a multi-flask cell culture system. The effects of the ASC-EVs were tested in Vero E6 cells in vitro. ASC-EVs were also given i.v. to SARS-CoV-2 infected Syrian Hamsters, and H1N1 influenza virus infected mice. The ASC-EVs attenuated SARS-CoV-2 virus replication in Vero E6 cells and reduced body weight and signs of lung injury in infected Syrian hamsters. Furthermore, ASC-EVs increased the survival rate of influenza A-infected mice and attenuated signs of lung injury. In summary, this study suggests that ASC-EVs can have beneficial therapeutic effects in models of virus-infection-associated acute lung injury and may potentially be developed to treat lung injury in humans.

Development of a programmable automated cell culture system to study the lung pathophysiology of Cystic Fibrosis-related diabetes

The study of many diseases is limited by the in vitro systems available. Cystic Fibrosis-Related Diabetes (CFRD), the main co-morbidity of Cystic Fibrosis (CF), is a perfect example. Cells in vivo experience glucose fluctuations after meals. In contrast, cells cultured in vitro are initially exposed to high glucose media. Glucose gets progressively depleted until the next media change days later, which is not physiologically relevant and could negatively impact the results of research studies. To better study the mechanisms driving CFRD pathophysiology, we developed a programmable and automated cell culture system (PACCS) capable of mimicking acute hyperglycemic episodes experienced by CFRD patients after meals. We adapted a commercially available perfusion system and performed 3D modeling to develop this system. Results show that PACCS can be successfully used to culture airway epithelial cells, both immortalized and primary cells. Further, CF cells responded differently to meal-like conditioning when compared to controls, suggesting impaired adaptative responses in CF cells. Overall, PACCS will allow us to better study CFRD pathophysiology, and it could be used for a wide range of other applications.

Protein Immobilization on Bacterial Cellulose for Biomedical Application.

New carriers for protein immobilization are objects of interest in various fields of biomedicine. Immobilization is a technique used to stabilize and provide physical support for biological micro- and macromolecules and whole cells. Special efforts have been made to develop new materials for protein immobilization that are non-toxic to both the body and the environment, inexpensive, readily available, and easy to modify. Currently, biodegradable and non-toxic polymers, including cellulose, are widely used for protein immobilization. Bacterial cellulose (BC) is a natural polymer with excellent biocompatibility, purity, high porosity, high water uptake capacity, non-immunogenicity, and ease of production and modification. BC is composed of glucose units and does not contain lignin or hemicellulose, which is an advantage allowing the avoidance of the chemical purification step before use. Recently, BC-protein composites have been developed as wound dressings, tissue engineering scaffolds, three-dimensional (3D) cell culture systems, drug delivery systems, and enzyme immobilization matrices. Proteins or peptides are often added to polymeric scaffolds to improve their biocompatibility and biological, physical-chemical, and mechanical properties. To broaden BC applications, various ex situ and in situ modifications of native BC are used to improve its properties for a specific application. In vivo studies showed that several BC-protein composites exhibited excellent biocompatibility, demonstrated prolonged treatment time, and increased the survival of animals. Today, there are several patents and commercial BC-based composites for wounds and vascular grafts. Therefore, further research on BC-protein composites has great prospects. This review focuses on the major advances in protein immobilization on BC for biomedical applications.

PDE4B inhibition by nerandomilast: Effects on lung fibrosis and transcriptome in fibrotic rats and on biomarkers in human lung epithelial cells.

The PDE4 family is considered a prime target for therapeutic intervention in several fibro-inflammatory diseases. We have investigated the molecular mechanisms of nerandomilast (BI 1015550), a preferential PDE4B inhibitor. In addition to clinically relevant parameters of idiopathic pulmonary fibrosis (IPF; lung function measurement/high-resolution computed tomography scan/AI-Ashcroft score), whole-lung homogenates from a therapeutic male Wistar rat model of pulmonary fibrosis were analysed by next-generation sequencing (NGS). Data were matched with public domain data derived from human IPF samples to investigate how well the rat model reflected human IPF. We scored the top counter-regulated genes following treatment with nerandomilast in human single cells and validated disease markers discovered in the rat model using a human disease-relevant in vitro assay of IPF. Nerandomilast improved the decline of lung function parameters in bleomycin-treated animals. In the NGS study, most transcripts deregulated by bleomycin treatment were normalised by nerandomilast treatment. Most notably, a significant number of deregulated transcripts that were identified in human IPF disease were also found in the animal model and reversed by nerandomilast. Mapping to single-cell data revealed the strongest effects on mesenchymal, epithelial and endothelial cell populations. In a primary human epithelial cell culture system, several disease-related (bio)markers were inhibited by nerandomilast in a concentration-dependent manner. This study further supports the available knowledge about the anti-inflammatory/antifibrotic mechanisms of nerandomilast and provides novel insights into the mode of action and signalling pathways influenced by nerandomilast treatment of lung fibrosis.

Organ-On-A-Chip Devices: Technology Progress and Challenges.

Organ-On-a-Chip (OOC) is a multichannel 3D-microfluidic cell-culture system incorporated in a chip that simulates the behavior of an organ. This technology relies on a multidisciplinary science that benefits from and contributes in the progress of many fields including microbiology, microfluidics, biomaterials, and bioengineering. This review article summarizes the progress and achievements of various organ-on-chip technologies. It highlights the significant advantages of this technology in terms of reducing animal testing and providing personalized medical responses. In addition, this paper demonstrates how OOC is becoming a promising and powerful tool in pharmaceutical research to combat diseases. It predicts not only the effects of drugs on the target organs but also, using body-on-a-chip systems, it may provide insights into the side effects of the drug delivery on the other organs. Likewise, the models used for the construction of various organ-on-a-chip devices are investigated along with the design and materials of microfluidic devices. For each OOC, the integrated monitoring devices within the chips (e. g., sensors and biosensors) are discussed. We also discuss the evolution of FDA regulations and the potential in the near future for integrating OOCs into protocols that support and reduce the need for and the failure rates in preclinical and clinical studies.

Anti-SARS-CoV-2 gapmer antisense oligonucleotides targeting the main protease region of viral RNA

Given the worldwide risk for the outbreak of emerging/re-emerging respiratory viruses, establishment of new antiviral strategies is greatly demanded. In this study, we present a scheme to identify gapmer antisense oligonucleotides (ASOs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA that efficiently inhibit viral replication. We synthesized approximately 300 gapmer ASOs designed to target various SARS-CoV-2 RNA regions and evaluated their activity in cell-based assays. Through a multistep screening in cell culture systems, we identified that ASO#41, targeting the coding region for viral main protease, reduced SARS-CoV-2 RNA levels in infected cells and inhibited virus-induced cytopathic effects. Antiviral effect of ASO#41 was also observed in iPS cell-derived human lung organoids. ASO#41 depleted intracellular viral RNAs during genome replication in an endogenous RNaseH-dependent manner. ASO#41 showed a wide range of antiviral activity against SARS-CoV-2 variants of concern including Alpha, Delta, and Omicron. Intranasal administration to mice exhibited intracellular accumulation of ASO#41 in the lung and significantly reduced the viral infectious titer, with milder body weight loss due to SARS-CoV-2 infection. Further chemical modification with phosphoryl guanidine-containing backbone linkages provided an elevation of anti-SARS-CoV-2 activity, with 23.4 nM of 50% antiviral inhibitory concentration, one of the strongest anti-SARS-CoV-2 ASOs reported so far. Our study presents an approach to identify active ASOs against SARS-CoV-2, which is potentially useful for establishing an antiviral strategy by targeting genome RNA of respiratory viruses.

Human relevance of in vivo and in vitro skin irritation tests for hazard classification of pesticides

Background: Test methods to inform hazard characterization and labeling of pesticides to protect human health are typically conducted using laboratory animals, and for skin irritation/corrosion the rabbit Draize test is currently required by many regulatory agencies. Although the Draize test is generally regarded to provide protective classifications for human health, new approach methodologies (NAMs) have been developed that offer more human relevant models that circumvent the uncertainty associated with species differences that exist between rabbits and humans. Despite wide applicability and use of these test methods across a broad range of chemicals, they have not been widely adopted for testing pesticides and pesticidal formulations. One of the barriers to adoption of these methods in this sector is low concordance with results from the Draize rabbit test, particularly for chemicals within the mild to moderate irritation spectrum. Methods: This review compares and contrasts the extent to which available models used in skin irritation testing mimic the anatomy and physiology of human skin, and how each aligns with the known key events leading to chemically-induced adverse skin irritation and corrosion. Doing so fully characterizes the human relevance of each method. Results: As alternatives to the rabbit Draize test, several protocols using ex vivo, in chemico, and in vitro skin models are available as internationally harmonized test guidelines. These methods rely on a variety of models of human skin, including excised rodent skin, synthetic biochemical models of barrier function, cell culture systems, and reconstructed human tissue models. We find these models exhibit biological and mechanistic relevance aligned with human skin irritation responses. Further, recent retrospective analyses have shown that the reproducibility of the Draize test is less than 50% for mild and moderate responses, with many of the replicate predictions spanning more than one category (e.g., a moderate response reported in one study followed by a non-irritant response reported in another study). Conclusions: Based on this comparative evaluation, we recommend top-down and bottom-up testing strategies that use the most human relevant in vitro test methods for skin irritation and corrosion classification of pesticides and pesticide formulations. To further discriminate among mild and non-irritant formulations, optimization of a cytokine release protocol and subsequent analyses of reference formulation test results is recommended.

Cell-based assays and comparative genomics revealed the conserved and hidden effects of Wolbachia on insect sex determination.

It is advantageous for maternally transmitted endosymbionts to skew the sex ratio of their hosts toward females. Some endosymbiotic bacteria, such as Wolbachia, cause their insect hosts to exclusively produce female offspring through male killing (MK) or feminization. In some lepidopteran insects, MK is achieved by affecting the sex-determining process in males, and a unique mechanism of MK and its functional link with feminization have been implicated. However, comparative analysis of these phenotypes is often difficult because they have been analyzed in different host-symbiont systems, and transinfection of Wolbachia across different hosts is often challenging. In this study, we demonstrated the effects of nine Wolbachia strains on the splicing of sex-determining genes in Lepidoptera by fixing the host genetic background using a cell culture system. Cell transinfection assays confirmed that three MK-inducing Wolbachia strains and one feminization-inducing Wolbachia strain increased the female-type splicing products of the core sex-determining genes doublesex, masculinizer, and zinc finger protein 2. Regarding Wolbachia strains that do not induce MK/feminization, three had no effect on these sex-determining genes, whereas two strains induced female-type splicing of masculinizer and doublesex but not zinc finger protein 2. Comparative genomics confirmed that homologs of oscar, the Wolbachia gene responsible for MK in Ostrinia, were encoded by four MK/feminizing Wolbachia strains, but not by five non-MK/nonfeminizing strains. These results support the conserved effects underlying MK and feminization induced by oscar-bearing Wolbachia and suggested other potential mechanisms that Wolbachia might employ to manipulate host sex.

Genetic and antigenic characterization of two diarrhoeicdominant rotavirus A genotypes G3P[12] and G14P[12] circulating in the global equine population.

Equine rotavirus species A (ERVA) G3P[12] and G14P[12] are two dominant genotypes that cause foal diarrhoea with a significant economic impact on the global equine industry. ERVA can also serve as a source of novel (equine-like) rotavirus species A (RVA) reassortants with zoonotic potential as those identified previously in 2013-2019 when equine G3-like RVA was responsible for worldwide outbreaks of severe gastroenteritis and hospitalizations in children. One hurdle to ERVA research is that the standard cell culture system optimized for human rotavirus replication is not efficient for isolating ERVA. Here, using an engineered cell line defective in antiviral innate immunity, we showed that both equine G3P[12] and G14P[12] strains can be rapidly isolated from diarrhoeic foals. The genome sequence analysis revealed that both G3P[12] and G14P[12] strains share the identical genotypic constellation except for VP7 and VP6 segments in which G3P[12] possessed VP7 of genotype G3 and VP6 of genotype I6 and G14P[12] had the combination of VP7 of genotype G14 and VP6 of genotype I2. Further characterization demonstrated that two ERVA genotypes have a limited cross-neutralization. The lack of an in vitro broad cross-protection between both genotypes supported the increased recent diarrhoea outbreaks due to equine G14P[12] in foals born to dams immunized with the inactivated monovalent equine G3P[12] vaccine. Finally, using the structural modelling approach, we provided the genetic basis of the antigenic divergence between ERVA G3P[12] and G14P[12] strains. The results of this study will provide a framework for further investigation of infection biology, pathogenesis and cross-protection of equine rotaviruses.

Dynamic cell culture modulates colon cancer cell migration in a novel 3D cell culture system

The progression of cancer cell migration, invasion and subsequent metastasis is the main cause of mortality in cancer patients. Through creating more accurate cancer models, we can achieve more precise results, which will lead to a better understanding of the invasion process. This holds promise for more effective prevention and treatment strategies. Although numerous 2D and 3D cell culture systems have been developed, they poorly reflect the in vivo situation and many questions have remained unanswered. This work describes a novel dynamic 3D cell culture system aimed at advancing our comprehension of cancer cell migration. With the newly designed cultivation chamber, 3D tumor spheroids were cultivated within a collagen I matrix in the presence of fluid flow to study the migration of cancer cells from spheroids in the matrix. Using light sheet microscopy and histology, we demonstrated that the morphology of spheroids is influenced by dynamic culture and that, in contrast to static culture, spheroids in dynamic culture are characterized by the absence of a large necrotic core. Additionally, this influence extends to an increase in the size of migration area, coupled with an increase in expression of some genes related to epithelial-mesenchymal transition (EMT). The results here highlight the importance of dynamic culture in cancer research. Although the dynamic 3D cell culture system in this study was used to investigate migration of one cell type into a matrix, it has the potential to be further developed and used for more complex models consisting of different cell types or to analyze other steps of metastasis development such as transendothelial migration or extravasation.

Establishment of 3D cell culture systems with decellularized lung-derived extracellular matrix hydrogel scaffold

Decellularized tissue hydrogels, especially that mimic the native tissue, have a high potential for tissue engineering, three-dimensional (3D) cell culture, bioprinting, and therapeutic agent encapsulation due to their excellent biocompatibility and ability to facilitate the growth of cells. It is important to note that the decellularization process significantly affects the structural integrity and properties of the extracellular matrix, which in turn shapes the characteristics of the resulting hydrogels at the macromolecular level. Therefore, our study aims to identify an effective chemical decellularization method for sheep lung tissue, using a mixing/agitation technique with a range of detergents, including commonly [Sodium dodecyl sulfate (SDS), Triton X-100, and 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate] (CHAPS), and rarely used (sodium cholate hydrate, NP-40, and 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate) (ASB-14). After the effectiveness of the used detergents on decellularization was determined by histological and biochemical methods, lung derived decellularized extracellular matrix was converted into hydrogel. We investigated the interactions between lung cells and decellularized extracellular matrix using proliferation assay, scanning electron microscopy, and immunofluorescence microscopy methods on BEAS-2B cells in air-liquid interface. Notably, this study emphasizes the effectiveness of ASB-14 in the decellularization process, showcasing its crucial role in removing cellular components while preserving vital extracellular matrix biological macromolecules, including glycosaminoglycans, collagen, and elastin. The resulting hydrogels demonstrated favorable mechanical properties and are compatible with both cell-cell and cell-extracellular matrix interactions.

Organs on chips: fundamentals, bioengineering and applications.

Human body constitutes unique biological system containing specific fluid mechanics and biomechanics. Traditional cell culture techniques of 2D and 3D do not recapitulate these specific natures of the human system. In addition, they lack the spatiotemporal conditions of representing the cells. Moreover, they do not enable the study of cell-cell interactions in multiple cell culture platforms. Therefore, establishing biological system of dynamic cell culture was of great interest. Organs on chips systems were fabricated proving their concept to mimic specific organs functions. Therefore, it paves the way for validating new drugs and establishes mechanisms of emerging diseases. It has played a key role in validating suitable vaccines for Coronavirus disease (COVID-19). Herein, the concept of organs on chips, fabrication methodology and their applications are discussed.

A Review on the Development of Microcarriers for Cell Culture Applications

Microcarrier-based cell culture systems have gained significant attention and popularity in tissue engineering and regenerative medicine. In this culture system, tissue cells are grown as a monolayer on the surface of small solid particles called microcarriers (100 to 300 μm), kept suspended in the culture medium by stirring. This technology has paved the way for creating engineered tissues, one of the cutting-edge topics in tissue engineering and regenerative medicine. Microcarrier-based approaches have been proposed for three-dimensional (3D) cell culture in which cellular morphology and functions are maintained <i>in vivo</i>. This paper provides an overview of the optimal characteristics such as microcarriers’ size, shape, density and porosity. Various methods of preparation of microcarriers and surface modification techniques have been elaborated. Recent advances and applications of microcarriers in biotechnology fields, like the production of viral vaccines and recombinant proteins, culture and expansion of stem cells (SC), are described.

Analytical and computational studies predict negligible risk of cell death from eddy generation off flat surfaces in cell culture flow systems.

Cell-based therapies represent the current frontier of biomedical innovations, with the technologies required underpinning treatments as broad as CAR-T cell therapies, stem cell treatments, genetic therapies and mRNA manufacture. A key bottleneck in the manufacturing process for each of these lies in the expansion of cells within a bioreactor vessel, requiring by far the greatest share of time for what are often time-critical therapies. While various designs, culture feeding and mixing methods are employed in these bioreactors, a common concern among manufacturers and researchers lies in whether shear stresses generated by culture media flow will damage cells and inhibit expansion. This study develops an analytical tool to link macro-scale measures of flow to risk of cell death using relationships with eddy size and dissipation rates, from eddies generated off flat surfaces. This analytical tool was then employed using computational fluid dynamics (CFD) to replicate a range of generic bioreactor geometries and flow conditions. We found that no combination of flow condition or design parameter was predicted by the tool to cause cell death within eddies, indicating negligible risk of cell death due to eddy formation within cell culture systems. While this requires experimental validation, and does not apply when cells are expanded using microcarriers, this tool nonetheless provides reassurance and accessible prediction of bioreactor design parameters that could result in cell death. Finally, our findings show that bioreactor design can be tailored such that the shear stress stimulation of cells can be selectively altered through small changes in flow rate.