Three-Dimensional Rotary Culture to Model Mycobacterial Biofilms in Low-Shear Detergent-Free Liquid Suspension.

In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.

Preventing biofouling in microalgal photobioreactors.

Photobioreactors (PBRs) are used to grow the light-requiring microalgae in diverse commercial processes. Often, they are operated as continuous culture over months period. However, with time, biofouling layer develops on the inner surfaces of their walls. The fouling layer formation deteriorates the PBR performance as foulants reduce light penetration in it. Light is essential for photosynthetic cultures, and a deterioration in lighting adversely impacts algae growth and biomass productivity. Fouling requires a frequent shutdown to clean the PBR and add to the environmental impact of the operation by generating many wastewaters contaminated with the cleaning chemicals. Antibiofouling coatings could be used to modify the surfaces of existing and future PBRs. Therefore, transparent and non-toxic fouling-release coatings, produced using hydrogel technology, could transform the existing PBRs into efficient and enduring microalgae culture systems, requiring only the application of the coating to the inner walls, without additional investments in new PBRs.

Establishment of an in vitro choroid complex system for vascular response screening.

The choroid, a vascularized tissue situated between the retina and the sclera, plays a crucial role in maintaining ocular homeostasis. Despite its significance, research on choroidal abnormalities and the establishment of effective in vitro models have been limited. In this study, we developed an in vitro choroid model through the co-culture of human induced pluripotent stem cells (hiPSC)-derived endothelial cells (ECs) and mouse choroidal fibroblasts (msCFs) with hiPSC-derived retinal pigment epithelial (RPE) cells via a permeable membrane. This model, inclusive of ECs, CFs, and RPE cells, exhibited similarities with in vivo choroidal vessels, as confirmed through immunohistochemistry of extracellular matrix markers and vascular-related markers, as well as choroid angiogenesis sprouting assay analysis. The effectiveness of our in vitro model was demonstrated in assessing vascular changes induced by drugs targeting vasoregulation. Our model offers a valuable tool for gaining insights into the pathological mechanisms underlying choroid development and the progression of choroidal vascular diseases.

Insight on cytotoxic NHC gold(I) halide complexes evaluated in multifaceted culture systems.

Gold complexes can be a useful system in the fight against cancer. Although many studies have been carried out on in vitro 2D cell culture models embryotoxic assays are particularly lacking. Embryotoxicity and DNA damage are critical concerns in drug development. In this study, the effects of a new N-Heterocyclic carbene (NHC)-Au compound (Bromo[1,3-di-4-methoxybenzyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]gold(I)) at different concentrations were explored using multifaceted approach, encompassing 2D cancer cell cultures, in vivo zebrafish and in vitro bovine models, and compared with a consolidated similar complex (Bromo[1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]gold(I)). The results obtained from 2D cancer cell cultures revealed concentration-dependent effects of the gold compounds by estimating the cytotoxicity with MTT assay and cellular damage as indicated by LDH release. Selected concentrations of gold complexes demonstrated no adverse effects on zebrafish embryo development. However, in bovine embryos, these same concentrations led to significant impairments in the early developmental stages, triggering cell apoptosis and reducing blastocyst competence. These findings underscore the importance of evaluating drug effects across different model systems to comprehensively assess their safety and potential impact on embryonic development.

3D layered co-culture model enhances Trastuzumab Deruxtecan sensitivity and reveals the combined effect with G007-LK in HER2-positive non-small cell lung cancer.

Human epidermal growth factor receptor 2 (HER2) aberrations are observed in various cancers. In non-small cell lung cancer, genetic alterations activating HER2, mostly exon 20 insertion mutations, occur in approximately 2-4% of cases. Trastuzumab deruxtecan (T-DXd), a HER2-targeted antibody-drug conjugate has been approved as the first HER2-targeted drug for HER2-mutant lung cancer. However, some cases are not responsive to T-DXd and the primary resistant mechanism remains unclear. In this study, we assessed sensitivity to T-DXd in JFCR-007, a patient-derived HER2-mutant lung cancer cell line. Although JFCR-007 was sensitive to HER2 tyrosine kinase inhibitors, it showed resistance to T-DXd in attachment or spheroid conditions. Accordingly, we established a three-dimensional (3D) layered co-culture model of JFCR-007, where it exhibited a lumen-like structure and became sensitive to T-DXd. In addition, an in-house inhibitor library screening revealed that G007-LK, a tankyrase inhibitor, was effective when combined with T-DXd. G007-LK increased the cytotoxicity of topoisomerase-I inhibitor, DXd, a payload of T-DXd and SN-38. This combined effect was also observed in H2170, an HER2-amplified lung cancer cell line. These results suggest that the proposed 3D co-culture system may help in evaluating the efficacy of T-DXd and may recapitulate the tumor microenvironment.

Guide for generating single-cell-derived knockout clones in mammalian cell lines using the CRISPR/Cas9 system.

Genome editing has developed rapidly in various research fields for targeted genome modifications in many organisms, including cells, plants, viruses, and animals. The clustered regularly interspaced short palindromic repeats-associated protein 9 system stands as a potent tool in gene editing for generating cells and animal models with high precision. The clinical potential of clustered regularly interspaced short palindromic repeats-associated protein 9 has been extensively reported, with applications in genetic disease correction, inhibition of viral replication, and personalized or targeted therapeutics for various cancers. In this study, we provide a guide on single-guide RNA design, cloning single-guide RNA into plasmid vectors, single-cell isolation via transfection, and identification of knockout clones using next-generation sequencing. In addition, by providing the results of insertion into mammalian cell lines through next-generation sequencing, we offer useful information to those conducting research on human and animal cell lines.

Current status and challenges for cell-cultured milk technology: a systematic review.

Cellular agriculture is an innovative technology for manufacturing sustainable agricultural products as an alternative to traditional agriculture. While most cellular agriculture is predominantly centered on the production of cultured meat, there is a growing demand for an understanding of the production techniques involved in dairy products within cellular agriculture. This review focuses on the current status of cellular agriculture in the dairy sector and technical challenges for cell-cultured milk production. Cellular agriculture technology in the dairy sector has been classified into fermentation-based and animal cell culture-based cellular agriculture. Currently, various companies synthesize milk components through precision fermentation technology. Nevertheless, several startup companies are pursuing animal cell-based technology, driven by public concerns regarding genetically modified organisms in precision fermentation technology. Hence, this review offers an up-to-date exploration of animal cell-based cellular agriculture to produce milk components, specifically emphasizing the structural, functional, and productive aspects of mammary epithelial cells, providing new information for industry and academia.

Enhancing high-efficiency breeding and microbial microdroplet cultivation techniques for Ganoderma lucidum.

Ganoderma lucidum is known for its bioactive compounds, such as polysaccharides and triterpenoids, which are crucial in food and medicine. However, liquid fermentation encounters challenges in terms of strain differentiation and stability. In this research, we employed atmospheric room temperature plasma mutation and a microbial microdroplet culture system to identify strains with enhanced biomass and triterpenoid production. The three mutant strains, YB05, YB09, and YB18, exhibited accelerated growth rates and antagonized the initial strain G0023 more effectively than the controls. Notably, YB18 displayed the fastest growth, with a 17.25% increase in colony radius. Shake flask cultivation demonstrated that, compared with the initial strain, YB05 and YB18 had 26.33% and 17.85% greater biomass, respectively. Moreover, the triterpenoid production of YB05 and YB18 surpassed that of the control by 32.10% and 15.72%, respectively, as confirmed by colorimetric detection. Importantly, these mutant strains remained stable for five generations. This study revealed a comprehensive screening system utilizing atmospheric pressure, room temperature plasma mutation technology and microbial droplet cultivation. This innovative approach offers a promising pathway for obtaining advantageous Ganoderma strains for liquid fermentation. The methodology of atmospheric room temperature plasma mutation and microbial microdroplet culture systems is detailed for better comprehension.

Gel microspheres enhance the stemness of ADSCs by regulating cell-ECM interaction.

The gel microsphere culture system (GMCS) showed various advantages for mesenchymal stem cell (MSC) expansion and delivery, such as high specific surface area, small and regular shape, extensive adjustability, and biomimetic properties. Although various technologies and materials have been developed to promote the development of gel microspheres, the differences in the biological status of MSCs between the GMCS and the traditional Petri dish culture system (PDCS) are still unknown, hindering gel microspheres from becoming a culture system as widely used as petri dishes. In the previous study, an excellent "all-in-one" GMCS has been established for the expansion of human adipose-derived MSCs (hADSCs), which showed convenient cell culture operation. Here, we performed transcriptome and proteome sequencing on hADSCs cultured on the "all-in-one" GMCS and the PDCS. We found that hADSCs cultured in the GMCS kept in an undifferentiation status with a high stemness index, whose transcriptome profile is closer to the adipose progenitor cells (APCs) in vivo than those cultured in the PDCS. Further, the high stemness status of hADSCs in the GMCS was maintained through regulating cell-ECM interaction. For application, bilayer scaffolds were constructed by osteo- and chondro-differentiation of hADSCs cultured in the GMCS and the PDCS. The effect of osteochondral regeneration of the bilayer scaffolds in the GMCS group was better than that in the PDCS group. This study revealed the high stemness and excellent functionality of MSCs cultured in the GMCS, which promoted the application of gel microspheres in cell culture and tissue regeneration.

Angiotensin II promotes erythroid proliferation in a three-stage erythroid culture system.

At present, the numbers of cultured erythroid cells obtained from culture systems are not on a scale that can be used for therapeutics since the cultured erythroid cells have limited proliferation capacity. Stromal cells are believed to play important roles during erythropoiesis. Our previous study shows that factors secreted by stromal cells enhance the proliferation capacity of adult erythroid cells in the culture system. Among the identified factors, angiotensinogen is one of the most abundant proteins secreted by the stromal cells. This study aims to investigate the effect of angiotensin II, an angiotensinogen derivative, on the proliferation of erythroid cells. •The receptor for angiotensin II was first checked by PCR analysis. It was expressed in erythroblasts at all stages during differentiation.•To study the effect of angiotensin II, CD34+ hematopoietic stem cells were cultured in a 3-stage erythroid culture system with and without angiotensin II. The addition of angiotensin II to the culture media, from day 0 to 8, significantly increases the numbers of cultured erythroid cells, whereas no difference in enucleation is observed.

Impaired astrocytic synaptic function by peripheral cholesterol metabolite 27-hydroxycholesterol.

Astrocytes represent the most abundant cell type in the brain, where they play critical roles in synaptic transmission, cognition, and behavior. Recent discoveries show astrocytes are involved in synaptic dysfunction during Alzheimer's disease (AD). AD patients have imbalanced cholesterol metabolism, demonstrated by high levels of side-chain oxidized cholesterol known as 27-hydroxycholesterol (27-OH). Evidence from our laboratory has shown that elevated 27-OH can abolish synaptic connectivity during neuromaturation, but its effect on astrocyte function is currently unclear. Our results suggest that elevated 27-OH decreases the astrocyte function in vivo in Cyp27Tg, a mouse model of brain oxysterol imbalance. Here, we report a downregulation of glutamate transporters in the hippocampus of CYP27Tg mice together with increased GFAP. GLT-1 downregulation was also observed when WT mice were fed with high-cholesterol diets. To study the relationship between astrocytes and neurons, we have developed a 3D co-culture system that allows all the cell types from mice embryos to differentiate in vitro. We report that our 3D co-cultures reproduce the effects of 27-OH observed in 2D neurons and in vivo. Moreover, we found novel degenerative effects in astrocytes that do not appear in 2D cultures, together with the downregulation of glutamate transporters GLT-1 and GLAST. We propose that this transporter dysregulation leads to neuronal hyperexcitability and synaptic dysfunction based on the effects of 27-OH on astrocytes. Taken together, these results report a new mechanism linking oxysterol imbalance in the brain and synaptic dysfunction through effects on astrocyte function.

CRISPR-Cas9-Induced Gene Editing in Primary Human Monocytes.

Monocytes play important and diverse roles in both homeostatic and inflammatory immune responses. The CRISPR-Cas9 system in lentiviral vectors has been widely used to manipulate specific genes of immortal monocyte cell lines to study monocyte functions. However, human primary monocytes are refractory to this method with low gene knockout (KO) efficiency. In this chapter, we developed an in vitro gene-editing procedure for primary human monocytes with a consistent and high-gene KO efficiency via a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single-guide RNA (sgRNA). This method can be adapted to study the functions of targeted signaling molecules involved in modulating monocyte polarization in primary human monocytes.

3D culture applied to reproduction in females: possibilities and perspectives.

In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction.

A sustainable and efficient strategy for bioconverting naringin to L-rhamnose, 2R-naringenin, and kaempferol.

The development of a sustainable and efficient bioconversion strategy is crucial for the full-component utilization of naringin. In this study, an engineering Pichia pastoris co-culture system was developed to produce L-rhamnose and 2S/2R-naringenin. By optimizing transformation conditions, the co-culture system could completely convert naringin while fully consuming glucose. The production of 2S/2R-naringenin reached 59.5 mM with a molar conversion of 99.2%, and L-rhamnose reached 59.1 mM with a molar conversion of 98.5%. In addition, an engineering Escherichia coli co-culture system was developed to produce 2R-naringenin and kaempferol from 2S/2R-naringenin. Maximal kaempferol production reached 1050 mg/L with a corresponding molar conversion of 99.0%, and 996 mg/L 2R-naringenin was accumulated. Finally, a total of 17.4 g 2R-naringenin, 18.0 g kaempferol, and 26.1 g L-rhamnose were prepared from 100 g naringin. Thus, this study provides a novel strategy for the production of value-added compounds from naringin with an environmentally safe process.

In vitro characterization of 3D culture-based differentiation of human liver stem cells.

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system, guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study, we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR, immunofluorescence, and Western blot analysis. Additionally, we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes, including CYP450, urea cycle enzymes, and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes, evident as early as 1 day post-differentiation. Interestingly, HLSCs transiently upregulated stem cell markers during differentiation, followed by downregulation after 7 days. Furthermore, differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h, with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system, its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications.

A growth factor-reduced culture system for colorectal cancer organoids.

Although organoids derived from tumor tissues have been widely used in cancer research, it is a great challenge for cultured organoids to retain the characteristics of the original tumor tissues due to their heterogeneity. In this study, we explore organoid culture recipes to capture tumor features of colorectal cancers. We find that the activation of Wnt and EGF signaling and inhibition of BMP signaling are non-essential for the survival of most colorectal cancer organoids (CRCOs). We design a growth factor-reduced culture medium containing FGF10, A83-01 (TGF-ß type I receptor inhibitor), SB202190 (p38 MAPK inhibitor), gastrin, and nicotinamide. Using this medium, we can maintain tumor features in long-term CRCO cultivation, as evidenced by histopathology, genetic stability, tumorigenicity, and response of clinical treatments. Our findings offer a reliable and economical strategy for CRCO culture, facilitating the utilization of organoids in colorectal cancer research and treatment.

The Role of Light on the Microalgae Biotechnology: Fundamentals, Technological Approaches, and Sustainability Issues.

Light energy directly affects microalgae growth and productivity. Microalgae in natural environments receive light through solar fluxes, and their duration and distribution are highly variable over time. Consequently, microalgae must adjust their photosynthetic processes to avoid photo limitation and photoinhibition and maximize yield. Considering these circumstances, adjusting light capture through artificial lighting in the main culture systems benefits microalgae growth and induces the production of commercially important compounds. In this sense, this review provides a comprehensive study of the role of light in microalgae biotechnology. For this, we present the main fundamentals and reactions of metabolism and metabolic alternatives to regulate photosynthetic conversion in microalgae cells. Light conversions based on natural and artificial systems are compared, mainly demonstrating the impact of solar radiation on natural systems and lighting devices, spectral compositions, periodic modulations, and light fluxes when using artificial lighting systems. The most commonly used photobioreactor design and performance are shown herein, in addition to a more detailed discussion of light-dependent approaches in these photobioreactors. In addition, we present the principal advances in photobioreactor projects, focusing on lighting, through a patent-based analysis to map technological trends. Lastly, sustainability and economic issues in commercializing microalgae products were presented.

Canine induced pluripotent stem cells can be successfully maintained in weekend-free culture systems.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.

Development and Optimization of a Multilayer Rat Purkinje Neuron Culture.

Elucidation of the mechanisms involved in neurodegenerative diseases of the cerebellum has been hampered by the lack of robust single cell models to study Purkinje neurons and replicate at the same time in vivo features. Cerebellar Purkinje neurons are difficult to grow in dispersed cell culture, and only limited work has been done using rat cells. We developed a refined protocol for growing rat Purkinje neurons from embryonic and postnatal tissue ex vivo that results in well-developed, mature, functional, and synaptically active neurons. The rat Purkinje neurons generated are responsive to paracrine factors and genetic manipulation, allowing great experimental flexibility at the single-cell level. This ex vivo model can be used to investigate disease mechanisms that disturb Purkinje neuron morphology, function, and communication in high- and low-throughput screening formats.

Use of Epigenetic Cues and Mechanical Stimuli to Generate Blastocyst-Like Structures from Mammalian Skin Dermal Fibroblasts.

Mammalian embryogenesis is characterized by complex interactions between embryonic and extra-embryonic tissues that coordinate morphogenesis, coupling bio-mechanical and bio-chemical cues, to regulate gene expression and influence cell fate. Deciphering such mechanisms is essential to understand early embryogenesis, as well as to harness differentiation disorders. Currently, several early developmental events remain unclear, mainly due to ethical and technical limitations related to the use of natural embryos.Here, we describe a three-step approach to generate 3D spherical structures, arbitrarily defined "epiBlastoids," whose phenotype is remarkably similar to natural embryos. In the first step, adult dermal fibroblasts are converted into trophoblast-like cells, combining the use of 5-azacytidine, to erase the original cell phenotype, with an ad hoc induction protocol, to drive erased cells into the trophoblast lineage. In the second step, once again epigenetic erasing is applied, in combination with mechanosensing-related cues, to generate inner cell mass (ICM)-like spheroids. More specifically, erased cells are encapsulated in micro-bioreactors to promote 3D cell rearrangement and boost pluripotency. In the third step, chemically induced trophoblast-like cells and ICM-like spheroids are co-cultured in the same micro-bioreactors. The newly generated embryoids are then transferred to microwells, to encourage further differentiation and favor epiBlastoid formation. The procedure here described is a novel strategy for in vitro generation of 3D spherical structures, phenotypically similar to natural embryos. The use of easily accessible dermal fibroblasts and the lack of retroviral gene transfection make this protocol a promising strategy to study early embryogenesis as well as embryo disorders.