Standard Culture Conditions Research Articles

Silicone rubber membrane devices permit islet culture at high density without adverse effects.

Conventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200IE/cm2. To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen. Oxygenation of individual islets under three culture conditions, standard low-density, non-GP high density, and GP high density, were first modeled with finite element simulations. Porcine islets from 30 preparations were cultured for 2days in devices with GP membrane bottoms or in paired cultures under conventional conditions. Islets were seeded at high density (HD, ∼4000IE/cm2, as measured by DNA) in both GP and non-GP devices. In simulations, individual islets under standard culture conditions and high density cultures on GP membranes were both well oxygenated whereas non-GP high density cultured islets were anoxic. Similarly, compared to the non-GP paired controls, islet viability and recovery were significantly increased in HD GP cultures. The diabetes reversal rate in nude diabetic mice was similar for HD GP devices and standard cultures but was minimal with non-GP HD cultures. Culturing islets in GP devices allows for a 20-fold increase of islet surface area density, greatly simplifying the culture process while maintaining islet viability and metabolism.

The sub-MIC selective window decreases along the digestive tract: determination of the minimal selective concentration of oxytetracycline in sterilised intestinal contents.

The administration of antibiotics can expose the digestive microbiota of humans and animals to sub-inhibitory concentrations, potentially favouring the selection of resistant bacteria. The minimal selective concentration (MSC) is a key indicator to understand this process. The MSC is defined as the lowest concentration of an antibiotic that promotes the growth of a resistant strain over a susceptible isogenic strain. It represents the lower limit of the sub-minimal inhibitory concentration (MIC) selective window, where resistant mutants can be selected. Previous studies focused on determining the MSC under standard culture conditions, whereas our research aimed to determine the MSC in a model that approximates in vivo conditions. We investigated the MSC of oxytetracycline (OTC) in Mueller-Hinton broth (MHB) and sterilised intestinal contents (SIC) from the jejunum, caecum and rectum (faeces) of pigs, using two isogenic strains of Escherichia coli (one susceptible and one resistant to OTC). Additionally, the MIC of OTC against the susceptible strain was determined to assess the upper limit of the sub-MIC selective window. Our study took a novel approach, and the results indicated that MIC and MSC values were lower in MHB than in SIC. In the latter, these values varied depending on the intestinal segment, with distal compartments exhibiting higher MIC and MSC values. Moreover, the sub-MIC selective window of OTC in SIC narrowed from the jejunum to the rectum, with a significantly closer MSC to MIC in faecal SIC. The results suggest that OTC binds to digestive contents, reducing the fraction of free OTC. However, binding alone does not fully explain our results, and interactions between bacteria and intestinal contents may play a role. Furthermore, our findings provide initial estimates of low concentrations facilitating resistance selection in the gut. Finally, this research enhances the understanding of antimicrobial resistance selection, emphasising the intricate interplay between antibiotics and intestinal content composition in assessing the risk of resistance development in the gut.

Striking differences in frost hardiness and inability to cold acclimate in two Mougeotia species (Zygnematophyceae) from alpine and lowland habitats

AbstractZygnematophyceae, a class of freshwater green algae, exhibit distinctive seasonal dynamics. The increasing frequency of cold snaps during the growing season might challenge the persistence of some populations. The present study explored the frost hardiness of two Mougeotia species isolated from different elevations and habitats. Additionally, a phylogenetic (rbcL sequence), ultrastructural and physiological characterization was performed. Both species, grown under standard culture conditions and cold acclimated cultures (+4°C), were exposed to freezing temperatures down to −9°C. Furthermore, ultrastructural‐, hydrogen peroxide (H2O2)‐ and photosynthetic pigment analysis were performed on cells exposed to −2°C, with and without induced ice nucleation. The alpine M. disjuncta showed a higher frost hardiness (LT50 = −5.8°C), whereas the lowland M. scalaris was susceptible to ice. However, frost hardiness did not improve after cold acclimation in either species but rather decreased significantly in M. disjuncta (LT50 = −4.7°C). Despite darkness, prolonged sub‐zero temperatures or freezing induced the activation of the xanthophyll (VAZ) cycle in M. scalaris. Our results demonstrate that frost hardiness varies within the genus Mougeotia and that the VAZ cycle can be activated in the dark under subzero temperature‐ and freezing stress but does not necessarily increase frost hardiness. As highly frost hardy cell types are usually formed at the end of the growing season, the ability of young cells to survive ice formation in the upper subzero temperature range represents a crucial survival strategy in populations exposed to late spring frosts.

Genome-based assessment of safety characteristics of Lacticaseibacillus paracasei NY1301 and genomic differences in closely related strains marketed as probiotics.

The probiotic attributes of Lacticaseibacillus paracasei NY1301 were comprehensively characterized, and a comparison between the closely related LcA (Actimel) and LcY (Yakult) probiotic strains was conducted using genomic tools. All strains exhibited high genetic similarity and likely shared a common ancestor; differences were primarily expressed as minor chromosomal re-arrangements, substitutions, insertions, and deletions. Compared with LcY, NY1301 exhibited 125 single-nucleotide polymorphisms. NY1301 lacked virulence factors, antibiotic resistance genes, and mutations associated with antibiotic resistance and had a 46-kbp prophage. This prophage is spontaneously induced at low levels and remains in a non-lytic state under standard culture conditions. The observed causal adaptive mutations were likely related to niche adaptation within the respective laboratory or manufacturing processes that occurred during the maintenance of the strains. However, the phenotypic effects of these genomic differences remain unclear. To validate the safety of NY1301, we conducted an open-label trial with healthy participants who consumed excessive amounts of NY1301 (3.0 × 1011 cfu) daily for 28 days. The results of this trial and those of other in vivo studies, coupled with the long history of human consumption without established risks to humans, provide strong evidence confirming the safety of NY1301.

CellShip: An Ambient Temperature Transport and Short-Term Storage Medium for Mammalian Cell Cultures.

Cell culture is a critical platform for numerous research and industrial processes. However, methods for transporting cells are largely limited to cryopreservation, which is logistically challenging, requires the use of potentially cytotoxic cryopreservatives, and can result in poor cell recovery. Development of a transport media that can be used at ambient temperatures would alleviate these issues. In this study, we describe a novel transportation medium for mammalian cells. Five commonly used cell lines, (HEK293, CHO, HepG2, K562, and Jurkat) were successfully shipped and stored for a minimum of 72 hours and up to 96 hours at ambient temperature, after which, cells were recovered into standard culture conditions. Viability (%) and cell numbers, were examined, before, following the transport/storage period and following the recovery period. In all experiments, cell numbers returned to pretransport/storage concentration within 24-48 hours recovery. Imaging data indicated that HepG2 cells were fully adherent and had established typical growth morphology following 48 hours recovery, which was not seen in cells recovered from cryopreservation. Following recovery, Jurkat cells that had been subjected to a 96 hours transport/storage period, demonstrated a 1.93-fold increase compared with the starting cell number with >95% cell viability. We conclude that CellShip® may represent a viable method for the transportation of mammalian cells for multiple downstream applications in the Life Sciences research sector.

Precise sensorimotor control impacts reproductive fitness of C. elegans in 3D environments.

The ability of animals to sense and navigate towards relevant cues in complex and elaborate habitats is paramount for their survival and reproductive success. The nematode Caenorhabditis elegans uses a simple and elegant sensorimotor program to track odors in its environments. Whether this allows the worm to effectively navigate a complex environment and increase its evolutionary success has not been tested yet. We designed an assay to test whether C. elegans can track odors in a complex 3D environment. We then used a previously established 3D cultivation system to test whether defect in tracking odors to find food in a complex environment affected their brood size. We found that wild-type worms can accurately migrate toward a variety of odors in 3D. However, mutants of the muscarinic acetylcholine receptor GAR-3 which have a sensorimotor integration defect that results in a subtle navigational defect steering towards attractive odors, display decreased chemotaxis to the odor butanone not seen in the traditional 2D assay. We also show that the decreased ability to locate appetitive stimuli in 3D leads to reduced brood size not observed in the standard 2D culture conditions. Our study shows that mutations in genes previously overlooked in 2D conditions can have a significant impact in the natural habitat, and highlights the importance of considering the evolutionary selective pressures that have shaped the behavior, as well as the underlying genes and neural circuits.

Development of Cryoconservation Protocol for In-vitro Shoot Bases of Allium ampeloprasum L. for Long-term Conservation

Critical factors for the development of cryo conservation protocol for Allium ampeloprasum L. conserved in the in-vitro Genebank (IVGB) at ICAR-NBPGR of India were investigated. Shoot bases (1.0 x 1.5 mm) excised from 8-wk-old shoot cultures grown on growth media (MS + 0.1 mg/l NAA + 0.02 mg/l 2iP + 0.3 M sucrose) and pregrown at 22/ 5ºC for 16/8 hours alternate temperature regime, precultured on MS medium supplemented with 0.3 M sucrose at standard culture conditions (SCC) for 16 hours followed by loading solution treatment for 60 minutes, PVS2 dehydration for 40 minutes were successfully cryoconserved using vitrification and droplet-vitrification technique. Droplet-vitrification technique improved post-thaw regrowth by ~58.33% as compared to vitrification. Thus, a standardized protocol was used for cryobanking of shoot bases of A. ampeloprasum

Influence of standard culture conditions and effect of oleoresin from the microalga Haematococcus pluvialis on splenic cells from healthy Balb/c mice — a pilot study

In this work, we used splenocytes from healthy mice to study the effects of the two most commonly used cell culture media (A, B) with different compositions of redox reagents. The incubation of cells for 24 h resulted in a significant decrease in viability and metabolic activity of splenocytes, and the negative effects of incubation in medium B were more pronounced. In standard conditions, oxidative stress in cells was manifested by reduced mitochondrial potential, and this effect correlated with the transition of 58.3% of cells to the early stage of apoptosis under reducing conditions of medium A and up to 66.1% of cells under super-reducing conditions in medium B, suggesting altered cell physiology. High levels of ROS/RNS activated transcription factor Nrf2, superoxide dismutase 1, and catalase. The higher mRNA levels of these genes were under the conditions of medium B, whose super-reducing environment in combination with the environment of conventional incubators proved to be less suitable for the cells compared to medium A. Treatment of the cells with a lower concentration (10 µg/ml) of oleoresin obtained from the microalga H. pluvialis partially eliminated the negative effects of cultivation. Higher concentration of oleoresin (40 µg/ml) was slightly cytotoxic, due to the significant antioxidant effect of astaxanthin, the main bioactive component of the extract, which eliminated most of the ROS/RNS acting as signalling molecules. This study shows that the standard culture conditions do not reflect the physiological in vivo cell conditions; therefore, they are not generally suitable for incubation of all cell types.

Long-term cold storage of clonal apple rootstocks on growth media with added jasmonic acid: effect and their subsequent development <I>in vitro</I>

The article examines the effect of jasmonic a cid used at concentrations of 0.1-1.0 mg/L in the longterm continuous in vitro cold storage of clonal apple rootstock explants (54-118, 57-490, 57-545, and MM 106) on their subsequent development on under standard cultivation conditions. A positive effect of jasmonic acid was noted on the preservation of the viable explants of clonal apple rootstocks at all used concentrations over a period of 48-54 months (+3-6 ℃) of continuous cold storage, as compared to the explants cultivated without its application. Different concentrations of jasmonic acid in the cold storage medium were found to have no significant effect on the net reproduction of explants that were subsequently cultivated without jasmonic acid under standard cultivation conditions (+20-22 ℃). However, a significant increase in the length of microshoots (by 1.4-1.6 times) and the average number of leaves per microshoot (by 1.2-1.6 times) was observed in all studied forms of clonal apple rootstocks on growth medium with a jasmonic acid concentration of 1.0 mg/L. Subsequently, these explants exhibited no delay in vegetative development in vitro as compared to the explants that did not undergo cold storage (control). Jasmonic acid addition to the growth medium for long-term continuous cold storage at can be recommended as a way to improve the preservation of explants when clonal apple rootstock collections are stored for long periods of time in in vitro culture.

Modeling the effect of multiple pests on ecosystem services provided by fruit crops: Application to apple

CONTEXTIn agroecosystems, crop pests have long been controlled by the intensive use of synthetic pesticides. To lower the dependency on pesticides, alternative crop protection strategies such as Integrated Pest Management (IPM) and Agroecological Crop Protection (ACP) have been proposed. In this context, it is crucial to consider multiple pests and their effects on host plant functioning and, ultimately, on system performance. OBJECTIVEWe aim to develop a pest-crop model to account for the effect of multiple pests on fruit tree functioning and the resulting ecosystem services provided by an orchard, using apple as a case study. METHODSFirst, we identified possible mechanisms of pest disturbance on apple tree for ten major pests: rosy and woolly apple aphid, codling, oriental and tortrix moths, red mite, powdery mildew, European canker, fire blight and apple scab. We classified them into seven functional groups: resource stealers, leaf area reducers, assimilation rate reducers, water transport reducers, assimilate sappers, fruit senescence accelerators and fruit marketability depreciators. Second, we defined coupling functions for each functional group. These describe the reduction of target variables in an existing fruit tree model (QualiTree), i.e. the variables to which the pest disturbances directly apply, as a function of pest abundances. Parameter values of the coupling functions were estimated based on the literature. Third, we used simulations in standard cultural conditions and a Monte Carlo Bayesian Sensitivity Analysis to test and hierarchize the effect of pests on the variation of nine indicators of four classes of ecosystem services (namely fruit production, climate regulation, soil nitrogen availability and water cycle maintenance and regulation).RESULTS AND CONCLUSIONSOnly four indicators were affected by the considered pests: refractometric index (measuring fruit sugar content), marketable yield, carbon sequestration and water drainage. The variations in refractometric index and in marketable yield were largely explained by the main effects of water transport reducers and fruit senescence accelerators, respectively. The latter also contributed the most to carbon sequestration variation. The variation in water drainage was largely explained by the main effect of mildew, an assimilation rate reducer. SIGNIFICANCEOur approach provides an operational tool to assess the impact of multiple pests on orchard ecosystem services. The model also opens up the possibility of studying the impact of pests in different ecological and technical contexts. For this, it must be coupled with models capable of predicting pest abundances as a function of cultivation practices and biotic and abiotic conditions.

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

Pathobiology and Targeting of CD38 in Cutaneous T-Cell Lymphoma

Introduction: Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature CD4+ T-cells that primarily affects the skin. Despite a wide variety of topical and systemic therapies for patients, CTCL remains difficult to treat. Because of its high likelihood of relapse and resistance, new therapeutic approaches are needed. We recently showed strong CD38 expression on neoplastic T-cells from patients with mature T-cell neoplasms, including CTCL. In this study, we evaluated i) the role of CD38 in CTCL pathogenesis ii) strategies to enhance CD38 expression in CTCL and iii) tested efficacy of combination therapy in vivo. Methods: Skin and peripheral blood samples from patients and healthy donors were evaluated for CD38 expression by immunohistochemistry (N=7), microarray (N=82), single-cell RNA-sequencing (N=11), and flow cytometry (N=14). To investigate the role of CD38 in CTCL, we generated CD38 knockout cell lines (H9, HH, and HuT-78) using CRISPR/Cas9-mediated genomic deletion. The CD38 wild-type (CD38 WT) and knockout (CD38 KO) cells were purified, cultured, and used for downstream functional analysis. We evaluated the lines for growth, metabolic capacities (XFe24 Seahorse assay), and engraftment potential in vivo. For in vivo therapeutic studies, we transduced firefly-luciferase gene into H9 CD38 WT and CD38 KO cells, and engrafted them intravenously into immunodeficient NOD Rag -/-γc -/- mice. Mice were treated with anti-CD38 antibody daratumumab or IgG control and disease progression was monitored over time using an in vivo imaging system. For combination studies, we evaluated CD38 expression by flow cytometry in H9 cells treated with increasing doses of HDAC inhibitor, panobinostat (5nM, 10nM, 25nM) for 24, 48, and 72 hours. Next, we tested co-treatment with panobinostat and daratumumab in vivo in mice engrafted with H9 CD38 WT cells with four treatment groups (vehicle/IgG, panobinostat/IgG, vehicle/daratumumab, and panobinostat/daratumumab). Results: Patient skin biopsies showed increased CD38 expression at both protein and RNA levels (log fold change 4.8; p<0.0001 by Mann-Whitney test). Across nine days of tracking under standard cell culture conditions, CD38 KO CTCL cells showed minimal growth differences compared to CD38 WT (p=0.22 by linear regression). However, CD38 KO CTCL cells showed significantly increased basal and maximum oxygen consumption rates (OCR) via seahorse assay when compared to CD38 WT cells (basal OCR 2.26 fold increase, p<0.0001; max OCR 1.83 fold increase, p<0.0001 by Mann-Whitney test). Using IVIS luminescent imaging, we found significantly increased in vivo growth of CD38 KO CTCL cells compared to CD38 WT cells (CD38 WT=2.2e8 photons/sec average total flux, N=7; CD38 KO=1e9 photons/sec, N=5; p=0.003 by Mann-Whitney test). These data suggest that the loss of CD38 in CTCL cells can accelerate the development of tumors in mice. In order to study the effectiveness of a targeted drug against CD38 in CTCL, we used anti-CD38 antibody daratumumab in our H9 xenograft model. Daratumumab treatment had a significant therapeutic effect on CD38 WT tumors compared to isotype control, reducing tumor burden by 84% (daratumumab average total flux=1.4e7 photons/sec, N=4; IgG average total flux=9.0e7 photons/sec, N=3; p=0.0002). Additionally, we explored methods to increase CD38 expression on CTCL cells. We found that panobinostat significantly increased CD38 expression on H9 CTCL cells in a dose dependent manner across multiple time points (max 85% increase in CD38 expression with 25nM panobinostat vs. DMSO at 72 hours; p<0.0001 by 2way ANOVA). In vivo testing demonstrated a statistically significant improved survival benefit in mice treated with the combination of panobinostat and daratumumab compared to mice that received daratumumab alone (vehicle median survival 23 days, N=4; daratumumab alone median survival 32 days N=4; panobinostat alone median survival 27.5 days, N=4; combination median survival 39 days, N=4; p=0.01 by log-rank test). The prolonged average survival benefit amounted to approximately one quarter of their entire overall lifespan. Conclusion: Our studies demonstrate strong evidence for further study into how CD38 regulates the growth and survival of CTCL cells. Our data also provide preliminary evidence for the clinical usefulness of combination therapies that increase CD38 expression to enhance tumor cell immunotherapeutic targeting.

Mesenchymal Cells Retain the Specificity of Embryonal Origin During Osteogenic Differentiation.

Mesenchymal stem cells (MSCs) are widely used in therapy, but the differences between MSCs of various origins and their ability to undergo osteogenic differentiation and produce extracellular matrix are not fully understood. To address this, we conducted a comparative analysis of mesenchymal cell primary cultures from 6 human sources, including osteoblast-like cells from the adult femur, adipose-derived stem cells, Wharton's jelly-derived mesenchymal cells, gingival fibroblasts, dental pulp stem cells, and periodontal ligament stem cells. We analyzed these cells' secretome, proteome, and transcriptome under standard and osteogenic cultivation conditions. Despite the overall similarity in osteogenic differentiation, the cells maintain their embryonic specificity after isolation and differentiation in vitro. Furthermore, we propose classifying mesenchymal cells into 3 groups: dental stem cells of neural crest origin, mesenchymal stem cells, and fetal stem cells. Specifically, fetal stem cells have the most promising secretome for various applications, while mesenchymal stem cells have a specialized secretome optimal for extracellular matrix production. Nevertheless, mesenchymal cells from all sources secreted core bone extracellular matrix-associated proteins. In conclusion, our study illuminates the distinctive characteristics of mesenchymal stem cells from various sources, providing insights into their potential applications in regenerative medicine and enhancing our understanding of the inherent diversity of mesenchymal cells in vivo.

Suspension-Induced Stem Cell Transition: A Non-Transgenic Method to Generate Adult Stem Cells from Mouse and Human Somatic Cells.

Adult stem cells (ASCs) can be cultured with difficulty from most tissues, often requiring chemical or transgenic modification to achieve adequate quantities. We show here that mouse primary fibroblasts, grown in suspension, change from the elongated and flattened morphology observed under standard adherent culture conditions of generating rounded cells with large nuclei and scant cytoplasm and expressing the mesenchymal stem cell (MSC) marker (Sca1; Ly6A) within 24 h. Based on this initial observation, we describe here a suspension culture method that, irrespective of the lineage used, mouse fibroblast or primary human somatic cells (fibroblasts, hepatocytes and keratinocytes), is capable of generating a high yield of cells in spheroid form which display the expression of ASC surface markers, circumventing the anoikis which often occurs at this stage. Moreover, mouse fibroblast-derived spheroids can be differentiated into adipogenic and osteogenic lineages. An analysis of single-cell RNA sequence data in mouse fibroblasts identified eight distinct cell clusters with one in particular comprising approximately 10% of the cells showing high levels of proliferative capacity expressing high levels of genes related to MSCs and self-renewal as well as the extracellular matrix (ECM). We believe the rapid, high-yield generation of proliferative, multi-potent ASC-like cells via the process we term suspension-induced stem cell transition (SIST) could have significant implications for regenerative medicine.

SARS-CoV-2 Infection of Human Primary Nasal Multiciliated Epithelial Cells Grown on Air-Liquid Interface Cultures.

Respiratory epithelial cells fail to exhibit natural phenotypic and morphological characteristics when grown in standard cell culture conditions. To better understanding respiratory pathogen host-cell interactions in the airways, one approach is to instead grow and differentiate these cells at an air-liquid interface (ALI). This chapter provides the working protocols used in our lab for producing ALI cultures, infecting them with SARS-CoV-2 and monitoring viral replication.

The Effect of Harmine on Dental Pulp Stem Cells Differentiation Into Neural Cells in Two-Dimensional and Three-Dimensional Cell Cultures

Background: As the repair capacity of the nervous system is low, stem cell therapy is a trend for replacement therapy. Dental pulp stem cells (DPSCs) have the potential to differentiate into many tissues, such as neurons. Harmine (7-methoxy-1methyl-9H-pyrido[3,4-b] indole) is an alkaloidal component of medicinal plants with a long history in traditional medicine. Alginate is a biocompatible hydrogel widely used as a biomaterial base in various scaffolds. Objectives: This study investigated whether harmine and encapsulation of cells in alginate hydrogel could improve DPSCs differentiation into neural cells. Methods: DPSCs were cultured under standard stem cell culture conditions, then encapsulated in alginate hydrogel, and treated with differentiation medium with and without harmine. After 14 days, cell proliferation and differentiation were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, real-time polymerase chain reaction (RT-PCR), and flow cytometry. Results: Harmine (5 and 10 μM) significantly increased the proliferation and viability of DPSCs compared to the control group in both two-dimensional and three-dimensional culture systems (P < 0.05). The expression levels of three neural cell markers (nestin, microtubule-associated protein [MAP-2], and β-tubulin III) in DPSCs-derived neural cells cultured in two-dimensional and three-dimensional culture systems were significantly increased in harmine-treated two-dimensional and three-dimensional culture systems compared to the control group (P < 0.05). Conclusions: Either harmine or alginate hydrogel had an accelerating effect on DPSCs differentiation into neural cells. Harmine also increased the proliferation of the cells.

Gut bacteria promote proliferation in benign S/RG/C2 colorectal tumour cells, and promote proliferation, migration and invasion in malignant HCT116 cells

Colorectal cancer (CRC) is a significant global health burden with a rising incidence worldwide. Distinct bacterial populations are associated with CRC development and progression, and it is thought that the relationship between CRC and associated gut bacteria changes during the progression from normal epithelium to benign adenoma and eventually malignant carcinoma and metastasis. This study compared the interaction of CRC-associated species Enterotoxigenic Bacteroides fragilis, Enterococcus faecalis and Fusobacterium nucleatum and one probiotic species, Escherichia coli Nissle 1917 with a colorectal adenoma (S/RG/C2) and a colorectal adenocarcinoma (HCT116) derived cell line. Gentamicin protection assays showed that all species displayed higher attachment to benign tumour monolayers when compared to malignant monolayers. However, invasion of 3/4 species was higher in the HCT116 cells than in the adenoma cells. All species were found to persist within tumour cell monolayers for a minimum of 48 h under standard aerobic cell culture conditions, with persistence significantly higher in HCT116 cells. Downstream assays were performed to analyse the behaviour of S/RG/C2 and HCT116 cells post-infection and revealed that all species increased the tumour cell yield of both cell lines. The migratory and invasive potential of HCT116 cells was increased after infection with F. nucleatum; however, no species significantly altered these characteristics in S/RG/C2 cells. These results add to the growing evidence for the involvement of microorganisms in CRC progression and suggest that these interactions may be dependent on tumour cell-specific characteristics.

Effects of ceramide C2 application on human laryngeal carcinoma cells: a cell culture study.

In the present study, we investigated the effects of Ceramide C2 application on human laryngeal carcinoma cells. Human larynx epidermoid carcinoma HEp-2 (ATCC® CCL-23™) cells were purchased from the American Type Culture Collection (ATCC, USA). Human larynx epidermoid carcinoma HEp-2 cells were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) (10%) and penicillin/streptomycin (1%) in a CO2 (5%) incubator under standard cell culture conditions. Ceramide C2 was prepared, and further dilutions ranging from 3.13 to 100 μM were prepared in a fresh culture medium. Cells on 96 well plates were exposed to the prepared concentrations of ceramide C2 for 24 and 48 hours. Cytotoxicity evaluation was performed by MTT. Apoptosis profiles of HEp-2 cells were detected by annexin-V analysis. The activated caspases 3/7 on HEp-2 cells after ceramide C2 exposure were evaluated with flow cytometric analysis. The morphological changes on HEp-2 cells caused by ceramide C2 were evaluated by staining with phalloidine and acridine orange via confocal microscopy. For the Wound Healing Assay, HEp-2 cells were cultured in 6 well-plates until they became confluent. MTT cytotoxicity test findings revealed that the viability of human laryngeal carcinoma cells decreased with the increased application of ceramide C2 for 24 hours compared to untreated (control) cells. The highest growth inhibition by ceramide C2 for short-term application for 24 hours was detected at the highest concentration of ceramide C2 (100 µM). Annexin-V findings showed that 98.97 of HEp-2 cells were alive, and 1.63% were detected as early apoptosis for the control group. The results showed that ceramide C2 triggered apoptosis on HEp-2 cells with a percentage of total apoptotic cells of 61,40 compared to untreated HEp-2 cells. Cysteine proteases (caspases) 3/7 activation percentages of HEp-2 cells exposed to ceramide C2 for 24 hours were compared to control cells, and the morphology of HEp-2 cells was changed with clear apoptotic signs that underlined the cytotoxicity and pro-apoptotic activity of ceramide C2. Scratch Assay assessed the migration capability of HEp-2 cells before and after the exposure to ceramide C2. It showed that ceramide C2 reduced human laryngeal carcinoma cells' migration capability and proliferation for 24 hours. Based on all study findings, it can be considered that short-chain ceramide C2 exerted cytotoxicity on human laryngeal carcinoma cells in a dose and time-dependent manner and reduced the viability via inducing caspase-dependent apoptosis. The overall effect might be derived from the elevated intracellular ceramide levels by the exogenous application of ceramide C2. Consequently, it was concluded that ceramide C2 has good potential to cause cytotoxicity and apoptosis in human laryngeal carcinoma cells and, after deeper in vitro and in vivo investigations, can be a good candidate for designing anti-cancer drugs with high efficiency.

PerTurboID, a targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria-causing parasites.

Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the Plasmodium falciparum-exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria-causing parasite, PfEMP1. We generated independent TurboID fusions of two proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

Engineered bone marrow as a clinically relevant ex vivo model for primary bone cancer research and drug screening

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. While numerous other cancers now have promising therapeutic advances, treatment options for OS have remained unchanged since the advent of standard chemotherapeutics and offer less than a 25% 5-y survival rate for those with metastatic disease. This dearth of clinical progress underscores a lack of understanding of OS progression and necessitates the study of this disease in an innovative system. Here, we adapt a previously described engineered bone marrow (eBM) construct for use as a three-dimensional platform to study how microenvironmental and immune factors affect OS tumor progression. We form eBM by implanting acellular bone-forming materials in mice and explanting the cellularized constructs after 8 wk for study. We interrogate the influence of the anatomical implantation site on eBM tissue quality, test ex vivo stability under normoxic (5% O2) and standard (21% O2) culture conditions, culture OS cells within these constructs, and compare them to human OS samples. We show that eBM stably recapitulates the composition of native bone marrow. OS cells exhibit differential behavior dependent on metastatic potential when cultured in eBM, thus mimicking in vivo conditions. Furthermore, we highlight the clinical applicability of eBM as a drug-screening platform through doxorubicin treatment and show that eBM confers a protective effect on OS cells that parallel clinical responses. Combined, this work presents eBM as a cellular construct that mimics the complex bone marrow environment that is useful for mechanistic bone cancer research and drug screening.