A dynamic flow fetal membrane organ-on-a-chip system for modeling the effects of amniotic fluid motion.

Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

Caspase-9 suppresses metastatic behavior of MDA-MB-231 cells in an adaptive organoid model.

Caspase-9, a cysteine-aspartate protease traditionally associated with intrinsic apoptosis, has recently emerged as having non-apoptotic roles, including influencing cell migration-an aspect that has received limited attention in existing studies. In our investigation, we aimed to explore the impact of caspase-9 on the migration and invasion behaviors of MDA-MB-231, a triple-negative breast cancer (TNBC) cell line known for its metastatic properties. We established a stable cell line expressing an inducible caspase-9 (iC9) in MDA-MB-231 and assessed their metastatic behavior using both monolayer and the 3D organotypic model in co-culture with human Foreskin fibroblasts (HFF). Our findings revealed that caspase-9 had an inhibitory effect on migration and invasion in both models. In monolayer culture, caspase-9 effectively suppressed the migration and invasion of MDA-MB-231 cells, comparable to the anti-metastatic agent panitumumab (Pan). Notably, the combination of caspase-9 and Pan exhibited a significant additional effect in reducing metastatic behavior. Interestingly, caspase-9 demonstrated superior efficacy compared to Pan in the organotypic model. Molecular analysis showed down regulation of epithelial-mesenchymal transition and migratory markers, in caspase-9 activated cells. Additionally, flow cytometry analysis indicated a cell cycle arrest. Moreover, pre-treatment with activated caspase-9 sensitized cells to the chemotherapy of doxorubicin, thereby enhancing its effectiveness. In conclusion, the anti-metastatic potential of caspase-9 presents avenues for the development of novel therapeutic approaches for TNBC/metastatic breast cancer. Although more studies need to figure out the exact involving mechanisms behind this behavior.

Hepatitis C virus-induced differential transcriptional traits in host cells after persistent infection elimination by direct-acting antivirals in cell culture.

Chronic hepatitis C virus infection (HCV) causes liver inflammation and fibrosis, leading to the development of severe liver disease, such as cirrhosis or hepatocellular carcinoma (HCC). Approval of direct-acting antiviral drug combinations has revolutionized chronic HCV therapy, with virus eradication in >98% of the treated patients. The efficacy of these treatments is such that it is formally possible for cured patients to carry formerly infected cells that display irreversible transcriptional alterations directly caused by chronic HCV Infection. Combining differential transcriptomes from two different persistent infection models, we observed a major reversion of infection-related transcripts after complete infection elimination. However, a small number of transcripts were abnormally expressed in formerly infected cells. Comparison of the results obtained in proliferating and growth-arrested cell culture models suggest that permanent transcriptional alterations may be established by several mechanisms. Interestingly, some of these alterations were also observed in the liver biopsies of virologically cured patients. Overall, our data suggest a direct and permanent impact of persistent HCV infection on the host cell transcriptome even after virus elimination, possibly contributing to the development of HCC.

Multifaceted interactions between cancer cells and glial cells in brain metastasis.

Cancer brain metastasis has a poor prognosis, is commonly observed in clinical practice, and the number of cases is increasing as overall cancer survival improves. However, experiments in mouse models have shown that brain metastasis itself is an inefficient process. One reason for this inefficiency is the brain microenvironment, which differs significantly from that of other organs, making it difficult for cancer cells to adapt. The brain microenvironment consists of unique resident cell types such as neurons, oligodendrocytes, astrocytes, and microglia. Accumulating evidence over the past decades suggests that the interactions between cancer cells and glial cells can positively or negatively influence the development of brain metastasis. Nevertheless, elucidating the complex interactions between cancer cells and glial cells remains challenging, in part due to the limitations of existing experimental models for glial cell culture. In this review, we first provide an overview of glial cell culture methods and then examine recent discoveries regarding the interactions between brain metastatic cancer cells and the surrounding glial cells, with a special focus on astrocytes and microglia. Finally, we discuss future perspectives for understanding the multifaceted interactions between cancer cells and glial cells for the treatment of metastatic brain tumors.

Development and qualification of 3 L scale-down model for large scale vaccine process on vero cell culture using microcarriers.

Scale-down models (SDM) are pivotal tools for process understanding and improvement to accelerate the development of vaccines from laboratory research to global commercialization. In this study, a 3 L SDM representing a 50 L scale Vero cell culture process of a live-attenuated virus vaccine using microcarriers was developed and qualified based on the constant impeller power per volume principle. Both multivariate data analysis (MVDA) and the traditional univariate data analysis showed comparable and equivalent cell growth, metabolic activity, and product quality results across scales. Computational fluid dynamics simulation further confirmed similar hydrodynamic stress between the two scales.

Establishing Three-Dimensional Explant Culture of Human Dental Pulp Tissue.

Mesenchymal stem cells in the dental tissue indicate a disposition for differentiation into diverse dental lineages and contain enormous potential as the important means for regenerative medicine in dentistry. Among various dental tissues, the dental pulp contains stem cells, progenitor cells and odontoblasts for maintaining dentin homeostasis. The conventional culture of stem cells holds a limit as the living tissue constitutes the three-dimensional (3D) structure. Recent development in the organoid cultures have successfully recapitulated 3D structure and advanced to the assembling of different types. In the current study, the protocol for 3D explant culture of the human dental pulp tissue has been established by adopting the organoid culture. After isolating dental pulp from human tooth, the intact tissue was placed between two layers for Matrigel with addition of the culture medium. The reticular outgrowth of pre-odontoblast layer continued for a month and the random accumulation of dentin was observed near the end. Electron microscopy showed the cellular organization and in situ development of dentin, and immunohistochemistry exhibited the expression of odontoblast and stem cell markers in the outgrowth area. Three-dimensional explant culture of human dental pulp will provide a novel platform for understanding stem cell biology inside the tooth and developing the regenerative medicine.

Downregulation of ATP8B2 to Assess Plasmalogen Distribution and Far1 Expression in Primary Trabecular Meshwork Cells.

The trabecular meshwork (TM) from primary open-angle glaucoma (POAG) cases has been found to contain decreased levels of intracellular plasmalogens. Plasmalogens are a subset of lipids involved in diverse cellular processes such as intracellular signaling, membrane asymmetry, and protein regulation. Proper plasmalogen biosynthesis is regulated by rate-limiting enzyme fatty acyl-CoA reductase (Far1). ATPase phospholipid transporting 8B2 (ATP8B2) is a type IV P-type ATPase responsible for the asymmetric distribution of plasmalogens between the intracellular and extracellular leaflets of the plasma membranes. Here we describe the methodology for extraction and culturing of TM cells from corneal tissue and subsequent downregulation of ATP8B2 using siRNA transfection. Further quantification and downstream effects of ATP8B2 gene knockdown will be analyzed utilizing immunoblotting techniques.

Improving assay feasibility and biocompatibility of 3D cyclic olefin copolymer microwells by superhydrophilic modification via ultrasonic spray deposition of polyvinyl alcohol.

Sample partitioning is a crucial step towards digitization of biological assays on polymer microfluidic platforms. However, effective liquid filling into microwells and long-term hydrophilicity remain a challenge in polymeric microfluidic devices, impeding the applicability in diagnostic and cell culture studies. To overcome this, a method to produce permanent superhydrophilic 3-dimensional microwells using cyclic olefin copolymer (COC) microfluidic chips is presented. The COC substrate is oxidized using UV treatment followed by ultrasonic spray coating of polyvinyl alcohol solution, offering uniform and long-term coating of high-aspect ratio microfeatures. The coated COC surfaces are UV-cured before bonding with a hydrophobic pressure-sensitive adhesive to drive selective filling into the wells. The surface hydrophilicity achieved using this method remains unchanged (water contact angle of 9°) for up to 6 months and the modified surface is characterized for physical (contact angle & surface energy, morphology, integrity of microfeatures and roughness), chemical composition (FTIR, Raman spectroscopy) and coating stability (pH, temperature, time). To establish the feasibility of the modified surface in biological applications, PVA-coated COC microfluidic chips are tested for DNA sensing (digital LAMP detection of CMV), and biocompatibility through protein adsorption and cell culture studies (cell adhesion, viability, and metabolic activity). Kidney and breast cells remained viable for the duration of testing (7 days) on this modified surface, and the coating did not affect the protein content, morphology or quality of the cultured cells. The ultrasonic spray coated system, coating with 0.25 % PVA for 15 cycles with 0.12 A current after UV oxidation, increased the surface energy of the COC (naturally hydrophobic) from 22.04 to 112.89 mJ/m2 and improved the filling efficiency from 40 % (native untreated COC) to 94 % in the microwells without interfering with the biocompatibility of the surface, proving to be an efficient, high-throughput and scalable method of microfluidic surface treatment for diagnostic and cell growth applications.

Test accuracy of rapid diagnostic tests and reverse-transcription polymerase chain reaction against virus isolation in cell culture for assessing SARS-CoV-2 infectivity: Systematic review and meta-analysis.

We aimed to assess the performance of Ag-RDT and RT-qPCR with regard to detecting infectious SARS-CoV-2 in cell cultures, as their diagnostic test accuracy (DTA) compared to virus isolation remains largely unknown. We searched three databases up to 15 December 2021 for DTA studies. The bivariate model was used to synthesise the estimates. Risk of bias was assessed using QUADAS-2/C. Twenty studies (2605 respiratory samples) using cell culture and at least one molecular test were identified. All studies were at high or unclear risk of bias in at least one domain. Three comparative DTA studies reported results on Ag-RDT and RT-qPCR against cell culture. Two studies evaluated RT-qPCR against cell culture only. Fifteen studies evaluated Ag-RDT against cell culture as reference standard in RT-qPCR-positive samples. For Ag-RDT, summary sensitivity was 93% (95% CI 78; 98%) and specificity 87% (95% CI 70; 95%). For RT-qPCR, summary sensitivity (continuity-corrected) was 98% (95% CI 95; 99%) and specificity 45% (95% CI 28; 63%). In studies relying on RT-qPCR-positive subsamples (n = 15), the summary sensitivity of Ag-RDT was 93% (95% CI 92; 93%) and specificity 63% (95% CI 63; 63%). Ag-RDT show moderately high sensitivity, detecting most but not all samples demonstrated to be infectious based on virus isolation. Although RT-qPCR exhibits high sensitivity across studies, its low specificity to indicate infectivity raises the question of its general superiority in all clinical settings. Study findings should be interpreted with caution due to the risk of bias, heterogeneity and the imperfect reference standard for infectivity.

Upstream Process Protocol for MSCs Isolated from Different Human-Based Tissue Origins.

This chapter introduces the increasing significance of mesenchymal stromal/stem cell (MSC) production in regenerative medicine and cellular therapeutics, outlines the growing interest in MSCs for various medical applications, and highlights their potential in advanced therapy medicinal products (ATMPs) and the advancements in cell culture technologies that have facilitated large-scale MSC production under Good Manufacturing Practices (GMP), ensuring safety and efficacy. This chapter describes an optimized upstream protocol for laboratory-scale MSC production from different tissue sources. This protocol, conducted in flasks, controls critical parameters and lays the foundation for downstream processing to generate ATMPs. This comprehensive approach underscores the potential of MSCs in clinical applications and the importance of tailored production processes.

Hydrogel-Integrated Millifluidic Systems: Advancing the Fabrication of Mucus-Producing Human Intestinal Models.

The luminal surface of the intestinal epithelium is protected by a vital mucus layer, which is essential for lubrication, hydration, and fostering symbiotic bacterial relationships. Replicating and studying this complex mucus structure in vitro presents considerable challenges. To address this, we developed a hydrogel-integrated millifluidic tissue chamber capable of applying precise apical shear stress to intestinal models cultured on flat or 3D structured hydrogel scaffolds with adjustable stiffness. The chamber is designed to accommodate nine hydrogel scaffolds, 3D-printed as flat disks with a storage modulus matching the physiological range of intestinal tissue stiffness (~3.7 kPa) from bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA). Computational fluid dynamics simulations were conducted to confirm a laminar flow profile for both flat and 3D villi-comprising scaffolds in the physiologically relevant regime. The system was initially validated with HT29-MTX seeded hydrogel scaffolds, demonstrating accelerated differentiation, increased mucus production, and enhanced 3D organization under shear stress. These characteristic intestinal tissue features are essential for advanced in vitro models as they critically contribute to a functional barrier. Subsequently, the chamber was challenged with human intestinal stem cells (ISCs) from the terminal ileum. Our findings indicate that biomimicking hydrogel scaffolds, in combination with physiological shear stress, promote multi-lineage differentiation, as evidenced by a gene and protein expression analysis of basic markers and the 3D structural organization of ISCs in the absence of chemical differentiation triggers. The quantitative analysis of the alkaline phosphatase (ALP) activity and secreted mucus demonstrates the functional differentiation of the cells into enterocyte and goblet cell lineages. The millifluidic system, which has been developed and optimized for performance and cost efficiency, enables the creation and modulation of advanced intestinal models under biomimicking conditions, including tunable matrix stiffness and varying fluid shear stresses. Moreover, the readily accessible and scalable mucus-producing cellular tissue models permit comprehensive mucus analysis and the investigation of pathogen interactions and penetration, thereby offering the potential to advance our understanding of intestinal mucus in health and disease.

Constructing Stiffness Tunable DNA Hydrogels Based on DNA Modules with Adjustable Rigidity.

DNA hydrogel represents a potent material for crafting biological scaffolds, but the toolbox to systematically regulate the mechanical property is still limited. Herein, we have provided a strategy to tune the stiffness of DNA hydrogel through manipulating the rigidity of DNA modules. By introducing building blocks with higher molecular rigidity and proper connecting fashion, DNA hydrogel stiffness could be systematically elevated. These hydrogels showed excellent dynamic properties and biocompatibility, thus exhibiting great potential in three-dimensional (3D) cell culture. This study has offered a systematic method to explore the structure-property relationship, which may contribute to the development of more intelligent and personalized biomedical platforms.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Protein Production at the 100L Scale.

The Baculovirus Expression Vector System (BEVS) has revolutionized the field of recombinant protein expression by enabling efficient and high yield production. The platform offers many advantages including manufacturing speed, flexible design, and scalability. In this chapter, we describe the methods including strategies and considerations to successfully optimize and scale-up using BEVS as a tool for production (Fig. 1). As an illustrative case study, we present an example focused on the production of a viral glycoprotein.

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Conductive extracellular matrix derived/chitosan methacrylate/ graphene oxide-pegylated hybrid hydrogel for cell expansion.

Electrical stimulation has emerged as a cornerstone technique in the rapidly evolving field of biomedical engineering, particularly within the realms of tissue engineering and regenerative medicine. It facilitates cell growth, proliferation, and differentiation, thereby advancing the development of accurate tissue models and enhancing drug-testing methodologies. Conductive hydrogels, which enable the conduction of microcurrents in 3D in vitro cultures, are central to this advancement. The integration of high-electroconductive nanomaterials, such as graphene oxide (GO), into hydrogels has revolutionized their mechanical and conductivity properties. Here, we introduce a novel electrostimulation assay utilizing a hybrid hydrogel composed of methacryloyl-modified small intestine submucosa (SIS) dECM (SISMA), chitosan methacrylate (ChiMA), and GO-polyethylene glycol (GO-PEG) in a 3D in vitro culture within a hypoxic environment of umbilical cord blood cells (UCBCs). Results not only demonstrate significant cell proliferation within 3D constructs exposed to microcurrents and early growth factors but also highlight the hybrid hydrogel's physiochemical prowess through comprehensive rheological, morphological, and conductivity analyses. Further experiments will focus on identifying the regulatory pathways of cells subjected to electrical stimulation.

Long-term culture of patient-derived mammary organoids in non-biogenic electrospun scaffolds for identifying metalloprotein and motor protein activities in aging and senescence.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotype in tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactone scaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids and patient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

Protocol for establishing knockout cell clones by deletion of a large gene fragment using CRISPR-Cas9 with multiple guide RNAs.

Genome editing is a powerful tool for establishing gene knockout or mutant cell lines. Here, we present a protocol for establishing knockout cell clones by deletion of large gene fragments using CRISPR-Cas9 with multiple guide RNAs. We describe steps for designing guide RNAs, cloning them into CRISPR-Cas9 vectors, cell seeding, transfection into cultured cells, clonal selection, and screening assays. This protocol can delete gene regions over 100 kbp, including GC-rich domains, and is applicable to various cell lines. For complete details on the use and execution of this protocol, please refer to Saito et al.,1 Saito and Endo et al.,2 and Higashi et al.3.