Development of Good Manufacturing Practice-Compatible Isolation and Culture Methods for Human Olfactory Mucosa-Derived Mesenchymal Stromal Cells.

Demyelination in the central nervous system (CNS) resulting from injury or disease can cause loss of nerve function and paralysis. Cell therapies intended to promote remyelination of axons are a promising avenue of treatment, with mesenchymal stromal cells (MSCs) a prominent candidate. We have previously demonstrated that MSCs derived from human olfactory mucosa (hOM-MSCs) promote myelination to a greater extent than bone marrow-derived MSCs (hBM-MSCs). However, hOM-MSCs were developed using methods and materials that were not good manufacturing practice (GMP)-compliant. Before considering these cells for clinical use, it is necessary to develop a method for their isolation and expansion that is readily adaptable to a GMP-compliant environment. We demonstrate here that hOM-MSCs can be derived without enzymatic tissue digestion or cell sorting and without culture antibiotics. They grow readily in GMP-compliant media and express typical MSC surface markers. They robustly produce CXCL12 (a key secretory factor in promoting myelination) and are pro-myelinating in in vitro rodent CNS cultures. GMP-compliant hOM-MSCs are comparable in this respect to those grown in non-GMP conditions. However, when assessed in an in vivo model of demyelinating disease (experimental autoimmune encephalitis, EAE), they do not significantly improve disease scores compared with controls, indicating further pre-clinical evaluation is necessary before their advancement to clinical trials.

Specific Cationic Antimicrobial Peptides Enhance the Recovery of Low-Load Quiescent Mycobacterium tuberculosis in Routine Diagnostics.

The culture confirmation of Mycobacterium tuberculosis (MTB) remains the gold standard for the diagnosis of Tuberculosis (TB) with culture conversion representing proof of cure. However, over 40% of TB samples fail to isolate MTB even though many patients remain infectious due to the presence of viable non-culturable forms. Previously, we have shown that two short cationic peptides, T14D and TB08L, induce a hormetic response at low concentrations, leading to a stimulation of growth in MTB and the related animal pathogen Mycobacterium bovis (bTB). Here, we examine these peptides showing they can influence the mycobacterial membrane integrity and function through membrane potential reduction. We also show this disruption is associated with an abnormal reduction in transcriptomic signalling from specific mycobacterial membrane sensors that normally monitor the immediate cellular environment and maintain the non-growing phenotype. We observe that exposing MTB or bTB to these peptides at optimal concentrations rapidly represses signalling mechanisms maintaining dormancy phenotypes, which leads to the promotion of aerobic metabolism and conversion into a replicative phenotype. We further show a practical application of these peptides as reagents able to enhance conventional routine culture methods by stimulating mycobacterial growth. We evaluated the ability of a peptide-supplemented sample preparation and culture protocol to isolate the MTB against a gold standard routine method tested in parallel on 255 samples from 155 patients with suspected TB. The peptide enhancement increased the sample positivity rate by 46% and decreased the average time to sample positivity of respiratory/faecal sampling by seven days. The most significant improvements in isolation rates were from sputum smear-negative low-load samples and faeces. The peptide enhancement increased sampling test sensitivity by 19%, recovery in samples from patients with a previously culture-confirmed TB by 20%, and those empirically treated for TB by 21%. We conclude that sample decontamination and culture enhancement with D-enantiomer peptides offer good potential for the much-needed improvement of the culture confirmation of TB.

Crucial Roles of the Mesenchymal Androgen Receptor in Wolffian Duct Development.

Wolffian duct (WD) maintenance and differentiation is predominantly driven by the androgen action, which is mediated by the androgen receptor (AR). It is well established that the mesenchyme indicates the fate and differentiation of epithelial cells. However, in vivo developmental requirement of mesenchymal AR in WD development is still undefined. By designing a mesenchyme-specific Ar knockout (ARcKO), we discovered that the loss of mesenchymal Ar led to the bilateral or unilateral degeneration of caudal WDs and cystic formation at the cranial WDs. Ex vivo culture of ARcKO WDs invariably resulted in bilateral defects, suggesting that some factor(s) originating from surrounding tissues in vivo might promote WD survival and growth even in the absence of mesenchymal Ar. Mechanistically, we found cell proliferation was significantly reduced in both epithelial and mesenchymal compartments; but cell apoptosis was not affected. Transcriptomic analysis by RNA sequencing of E14.5 mesonephroi revealed 131 differentially expressed genes. Multiple downregulated genes (Top2a, Wnt9b, Lama2, and Lamc2) were associated with morphological and cellular changes in ARcKO male embryos (ie, reduced cell proliferation and decreased number of epithelial cells). Mesenchymal differentiation into smooth muscle cells that are critical for morphogenesis was also impaired in ARcKO male embryos. Taken together, our results demonstrate the crucial roles of the mesenchymal AR in WD maintenance and morphogenesis in mice.

Improved Culture Methods for Human Coronaviruses HCoV-OC43, HCoV-229E, and HCoV-NL63.

HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 are four of the seven known human coronaviruses (HCoVs) and, unlike the highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, these four so-called seasonal HCoVs generally cause mild upper-respiratory-tract illness. As Biosafety Level 2 (BSL-2) pathogens, the seasonal HCoVs are more accessible and can be used as surrogates for studying the highly pathogenic HCoVs. However, scientists have for many years found these difficult to study because of the lack of a universal culture system and the inability of typical culture methods to yield high-titer infectious stocks. We have developed assays to grow and quantify infectious virus and viral RNA for HCoV-OC43, -229E, and -NL63. We identified which immortalized cell lines should be used to optimize the replication of HCoV-OC43, -229E, and -NL63 in order to generate high titers (Vero E6, Huh-7, and LLC-MK2 cells, respectively). Here we present protocols for improved propagation and quantification of each seasonal HCoV. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Growth of HCoVs Basic Protocol 2: Quantification of HCoV by plaque assay Basic Protocol 3: Quantification of HCoV RNA products of replication Basic Protocol 4: Concentrating HCoVs via ultracentrifugation.

Isolation and Culture Techniques for Fetal Mouse Germ Cells.

The fetal gonad contains a great variety of differentiating cell populations, of which germ cells make up a relatively small percentage. In order to study germ cell-specific gene and protein expression, as well as determine direct effects of signaling molecules, it is necessary to prepare enriched populations of germ cells and maintain them in culture for several hours to multiple days. The protocols in this chapter are designed to provide a guide for the isolation or enrichment of primordial germ cells (from 9.5 days post coitum (dpc) to 18.5 dpc) by flow cytometry (Subheading 3.1) or magnetic sorting (Subheading 3.2), followed by feeder-free primary germ cell culture (Subheading 3.3).

Development of culture methods capable of culturing a wide range of predominant species of intestinal bacteria.

In recent years, with the development of non-cultivation approaches, it has become evident that intestinal bacteria have a significant impact on human health. However, because one-third of the genes cannot be annotated, it is difficult to elucidate the function of all intestinal bacteria by in silico analysis, and it is necessary to study the intestinal bacteria by culturing them. In addition, various media recommended for each individual bacterium have been used for culturing intestinal bacteria; however, the preparation of each medium is complex. To simultaneously culture many bacteria and compare bacterial phenotypes under the same conditions, a medium capable of culturing a wide range of bacteria is needed. In this study, we developed GAM + blood medium (GB medium), which consists of Gifu anaerobic medium containing 5% (v/v) horse blood; it is easy to prepare and it allowed the successful cultivation of 85% of the available predominant species in the human intestinal microbiota.

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Study of ex-ovo embryonic development of Gallus gallus domesticus / Estudio inicial del desarrollo embrionario ex-ovo de Gallus gallus domesticus

SUMMARY: The domestic chicken is a species of bird that has been extensively studied in regard to its biology and as a model organism for science. The reproduction of the species is by the laying of fertilized eggs, which in a period of 21 days will develop a chick inside. Several methods have been described to develop embryos ex-ovo, allowing the observation and manipulation of the organism. This work has the propose to standardize a method that allows the development of the embryos inside the artificial incubation system, which has a low cost and is easy to make. In this work, 100 chicken eggs were used to study the effects of humidity, mineral supplementation, and the preincubation time of the egg on the incubation ex-ovo of the embryos. Embryo development was documented through the different days. Pulverized eggshell was selected as an optimal source to provide calcium, magnesium, phosphorus, and other minerals to the developing embryo. By providing 900-1200 mg of pulverized eggshell, 40 mL of the 0.001 % solution of benzalkonium chloride, and a preincubation time of approximately 56 h, the embryos were able to develop until 19 days, and even though they did not reach hatching, the incubation conditions that allowed the survival and development of embryos until late stages were achieved. Thus, due to the conditions established for calcium, humidity and preincubation time, in the present work, the chicks reached 19 days of development.

El pollo doméstico es una especie de ave que ha sido ampliamente estudiada en cuanto a su biología y como organismo modelo para la ciencia. La reproducción de la especie es por la puesta de huevos fecundados, que en un período de 21 días desarrollarán un polluelo en su interior. Se han descrito varios métodos para desarrollar embriones ex-ovo, permitiendo la observación y manipulación del organismo. Este trabajo tuvo como objetivo estandarizar un método que permita el desarrollo de los embriones dentro del sistema de incubación artificial, el cual tiene un bajo costo y es fácil de realizar. En este trabajo se utilizaron 100 huevos de gallina para estudiar los efectos de la humedad, la suplementación mineral y el tiempo de preincubación del huevo sobre la incubación ex-ovo de los embriones. El desarrollo embrionario se documentó a través de los diferentes días. Se seleccionó la cáscara de huevo pulverizada como una fuente óptima para proporcionar calcio, magnesio, fósforo y otros minerales al embrión en desarrollo. Al suministrar 900-1200 mg de cáscara de huevo pulverizada, 40 mL de la solución de cloruro de benzalconio al 0.001 % y un tiempo de preincubación de aproximadamente 56 h, los embriones lograron desarrollarse hasta los 19 días, y aunque no llegaron a eclosionar, los embriones lograron desarrollarse hasta los 19 días. Se lograron condiciones de incubación que permitieron la supervivencia y desarrollo de los embriones hasta etapas tardías. Así, debido a las condiciones establecidas de calcio, humedad y tiempo de preincubación, en el presente trabajo los pollitos alcanzaron los 19 días de desarrollo.

Enrichment Culture but Not Metagenomic Sequencing Identified a Highly Prevalent Phage Infecting Lactiplantibacillus plantarum in Human Feces.

Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human diseases, but its phages in the human gut remain unexplored. Here, we report its first gut phage, Gut-P1, which we systematically screened using metagenomic sequencing, virus-like particle (VLP) sequencing, and enrichment culture from 35 fecal samples. Gut-P1 is virulent, belongs to the Douglaswolinvirus genus, and is highly prevalent in the gut (~11% prevalence); it has a genome of 79,928 bp consisting of 125 protein coding genes and displaying low sequence similarities to public L. plantarum phages. Physiochemical characterization shows that it has a short latent period and adapts to broad ranges of temperatures and pHs. Furthermore, Gut-P1 strongly inhibits the growth of L. plantarum strains at a multiplicity of infection (MOI) of 1e-6. Together, these results indicate that Gut-P1 can greatly impede the application of L. plantarum in humans. Strikingly, Gut-P1 was identified only in the enrichment culture, not in our metagenomic or VLP sequencing data nor in any public human phage databases, indicating the inefficiency of bulk sequencing in recovering low-abundance but highly prevalent phages and pointing to the unexplored hidden diversity of the human gut virome despite recent large-scale sequencing and bioinformatics efforts. IMPORTANCE As Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) is increasingly used as a probiotic to treat human gut-related diseases, its bacteriophages may pose a certain threat to their further application and should be identified and characterized more often from the human intestine. Here, we isolated and identified the first gut L. plantarum phage that is prevalent in a Chinese population. This phage, Gut-P1, is virulent and can strongly inhibit the growth of multiple L. plantarum strains at low MOIs. Our results also show that bulk sequencing is inefficient at recovering low-abundance but highly prevalent phages such as Gut-P1, suggesting that the hidden diversity of human enteroviruses has not yet been explored. Our results call for innovative approaches to isolate and identify intestinal phages from the human gut and to rethink our current understanding of the enterovirus, particularly its underestimated diversity and overestimated individual specificity.

Novel Antifungal and Antifeedant Metabolites from Penicillium chrysogenum Co-Cultured with Nemania primolutea and Aspergillus fumigatus.

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A-B (1-2), and penigenumin (3) from N. primolutea, penemin (4) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites (13-16) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A-B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites (13-16) were the culture driven. Compounds 4, 6, 8, 10, 11, 14, and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MICS of 1-8 µg/mL, and compounds 7, 9, and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.

Patient-derived three-dimensional culture techniques model tumor heterogeneity in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) outcomes remain stagnant, in part due to a poor understanding of HNSCC biology. The importance of tumor heterogeneity as an independent predictor of outcomes and treatment failure in HNSCC has recently come to light. With this understanding, 3D culture systems, including patient derived organoids (PDO) and organotypic culture (OTC), that capture this heterogeneity may allow for modeling and manipulation of critical subpopulations, such as p-EMT, as well as interactions between cancer cells and immune and stromal cells in the microenvironment. Here, we review work that has been done using PDO and OTC models of HNSCC, which demonstrates that these 3D culture models capture in vivo tumor heterogeneity and can be used to model tumor biology and treatment response in a way that faithfully recapitulates in vivo characteristics. As such, in vitro 3D culture models represent an important bridge between 2D monolayer culture and in vivo models such as patient derived xenografts.

Transcriptomics and metabolomics reveal changes in the regulatory mechanisms of osteosarcoma under different culture methods in vitro.

BACKGROUND: Recently, increasing attention has been drawn to the impact of the tumor microenvironment (TME) on the occurrence and progression of malignant tumors. A variety of 3D culture techniques have been used to simulate TME in vitro. The purpose of this study was to reveal the differences in transcriptional and metabolic levels between osteosarcoma (OS) 2D cells, 3D cells, 3D cell-printed tissue, isolated tissue, and transplanted tumor tissue in vivo. METHODS: We cultured the OS Saos-2 cell line under different culture methods as 2D cells, 3D cells, 3D cell-printed tissue and isolated tissue for 14 days and transplanted tumors in vivo as a control group. Through transcriptomic and metabonomic analyses, we determined the changes in gene expression and metabolites in OS tissues under different culture methods. RESULTS: At the transcriptional level, 166 differentially expressed genes were found, including the SMAD family, ID family, BMP family and other related genes, and they were enriched in the TGF-ß signaling pathway, complement and coagulation cascades, signaling pathways regulating pluripotency of stem cells, Hippo signaling pathway, ferroptosis, cGMP-PKG signaling pathway and other pathways. At the metabolic level, 362 metabolites were significantly changed and enriched in metabolic pathways such as the Fc Epsilon RI signaling pathway, histidine metabolism, primary bile acid biosynthesis, steroid biosynthesis, protein digestion and absorption, ferroptosis, and arachidonic acid metabolism. After integrating the transcriptome and metabolomics data, it was found that 44 metabolic pathways were changed, and the significantly enriched pathways were ferroptosis and pyrimidine metabolism. CONCLUSION: Different culture methods affect the gene expression and metabolite generation of OS Saos-2 cells. Moreover, the cell and tissue culture method in vitro cannot completely simulate TME in vivo, and the ferroptosis and pyrimidine metabolism pathways mediate the functional changes of OS Saos-2 cells in different microenvironments.

Effect of Human Amniotic Epithelial Stem Cell Transplantation on Preterm Premature Rupture of Fetal Membrane Using the Amniotic Pore Culture Technique in vitro.

OBJECTIVES: The objective of this study was to evaluate the efficacy of cell therapy using human amniotic epithelial stem cells (hAESCs) for the treatment of premature rupture of membranes (PROM) in vitro. DESIGN: Using the amniotic pore culture technique (APCT), we mimicked the environment of PROM in vitro, thus enabling the observation of the healing process of hAESC-treated amniotic membranes. MATERIALS: Amniotic membrane samples were collected from placentas of pregnant women who underwent elective cesarean sections. APCT model and isolated hAESCs were used in this study. All patients who participated in this study provided their written informed consent prior to the commencement of the study. SETTINGS: To create the APCT model in vitro, isolated amniotic membranes were punched to create 5 mm diameter circles and re-punched to form a 1-mm pore at the center. Membranes were cultured in α-minimal essential medium, and the hAESCs were collected and cultured as well. Subsequently, the APCT models were divided into two groups: hAESC treated and control. METHODS: Within the culture period, pore sizes were calculated to evaluate the degree of tissue regeneration in both groups. We then evaluated the histology, cell density, and epithelial thickness of the regenerated tissues. Statistical analyses were performed using SPSS software ver. 20.0 (IBM, Armonk, NY, USA) with repeated-measures one-way analysis of variance or paired samples t test. The significance level was set at p < 0.05. RESULTS: As per the evaluation of the APCT model in vitro, the pore size in the hAESC-treated group reduced by 62.2% on day 6 (62.2 ± 0.19, n = 24), whereas in the control group, it shrank by only 36.8% (p < 0.05) (36.8 ± 0.19, n = 24). Furthermore, the epithelial thickness in the amniotic epithelial stem cell-treated group (10.08 ± 1.26 µm, n = 8) was significantly higher than that in the control group (5.87 ± 0.94 µm, n = 8). Cell density in the regenerated tissue in the amniotic epithelial stem cell-treated group (57 ± 2.77, n = 8) was significantly higher than that in the control group (49 ± 2.23, n = 8). LIMITATIONS: In this study, we did not explore the molecular mechanisms by which hAESCs participate in membrane healing in the APCT model. Although our results showed a significant difference, this difference was not too obvious. Therefore, further research on the mechanisms of hAESCs is needed, with more amniotic tissues and APCT samples being tested. CONCLUSIONS: We developed an APCT model to investigate the PROM conditions in vitro. By implanting donor hAESCs in the pores of the APCT model, we observed that hAESCs seeding accelerated pore healing in vitro. Thus, hAESCs may be a valuable source of cells for cell therapies in regenerative medicine.

Selection of salt tolerant lines at cell level using gamma ray with callus and suspension culture techniques in black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.).

The main objective was to select salt tolerant lines at the cell level of Hatay region's black carrot (Daucus carota L. ssp. sativus var. atrorubens Alef.) using callus and suspension culture techniques combined with gamma rays. Hypocotyl explants of http://www.scialert.net/asci/result.php?searchin = Keywords&cat = &ascicat = ALL&Submit = Search&keyword = in+vitro (in vitro)">in vitro grown plantlets was used for callus induction. Effective mutation dose was determined by gamma radiation treatment at various doses (0, 5, 10, 20, 30, 40, 50, 60 Gy) to black carrot calli after in vitro optimization steps. According to regression analysis, the number of plants regenerated from calli was found 8.36 Gy as effective dose. In the ongoing study, calli with 7 Gy, 8 Gy and 9 Gy gamma rays were multiplied by subculture for 5 times. Shoot induction was achieved in medium containing 1 mg L-1 BAP concentration. Average plant height, root length and branching number parameters of plants regenerated from calli were determined. Salt stress was applied to the plants acclimatized from in vitro to the climate chambers. changes in the amount of peroxidase (POD) and superoxide dismutase (SOD) activities of antioxidant enzymes and the changes in lipid peroxidation were revealed in leaf samples taken from plants that continued to live in a salty environment after the 14 days of the treatment. At the end of the study, salt tolerance increased in mutant plants have the plant number of 8-21, 9-19, 7-9, 9-2 and 9-8 compared to the control, and these were determined as possible mutant plants.

Bacterial concentration and Campylobacter spp. quantification differ when fresh or ultra-frozen samples are analysed over time using molecular biology and culture-based methods.

The study aimed to delineate the robustness of the culture-based and molecular biology methods to assess the total bacterial concentration and Campylobacter jejuni (C. jejuni) quantification in caecal content, analysed as fresh or after being stored immediately at ultra-low (-80°C) temperature at different time points (for 3, 7, 14, 28 and 62 days post collection). The caecal content was collected from birds that were artificially colonised with C. jejuni (in-vivo), and quantification was performed using both colony-forming unit (CFU) and qPCR. The results showed that storage time affected the output of culture-based analyses but mostly did not alter concentration retrieved via qPCR. After an initial ~4.5 log10 reduction in CFU observed from fresh (day 0) to frozen samples, bacterial concentration retrieved with culture-based methods seemed to be constant in samples frozen for 3 to 62 days, indicating a possible threshold for C. jejuni loss of viability due to effect of storage temperature. Ranking order analyses, revealed that the molecular biology technique was able to attribute somewhat the same relative C. jejuni concentrations to the samples analysed via qPCR. However, day 0 measurements from culture-based methods were associated with the absence of or negatively weak correlations with the rest of the time points, but ranking order was maintained from day 3 onwards. On the other hand, ranking order correlations were less constant when measuring total bacterial concentration through qPCR. The study suggests that if biological samples can't be analysed as fresh (immediately after collection) and have to be stored prior to analysis, then storage at -80°C samples be recommended to avoid the temporal-dependent effects on C. jejuni concentrations. In addition, irrespective of the method of analysis, an initial loss of CFU must be factored in when interpreting the results obtained from frozen samples.

The Enrichment of Breast Cancer Stem Cells from MCF7 Breast Cancer Cell Line Using Spheroid Culture Technique.

Breast cancer is the most common malignancy worldwide in females, representing 29% of all cancer new cases and 14% of cancer deaths in the world. Amongst the reasons for the high mortality rate is resistance to chemotherapy resulting in therapeutic failure. Various studies have shown that the presence of cancer stem cells (CSCs) in breast tumors is responsible for chemotherapy resistance and tumor recurrence. This CSC population possesses the characteristics of normal stem cells, including their ability to self-renewal and give rise to other epithelial cells. One thing that unique to the CSC population is their ability to escape from chemotherapy drugs; this can make them resistant to therapy and able to repopulate the cancer. Isolation and enrichment of breast CSCs (BCSCs) is required in order to study their characteristics and the behavior that enables them to drive breast tumor development, in order to develop better therapies. This chapter describes a method for the isolation and enrichment of BCSCs from the MCF7 breast cancer cell line, which consists of a heterogeneous breast cancer cell population. This method depends on cancer stem cell behavior, specifically an ability to self-renew and form spheroids in harsh conditions that allow only cancer cells with stem cell characteristics to survive and form spheroids.

Using plant growth-promoting microorganisms (PGPMs) to improve plant development under in vitro culture conditions.

MAIN CONCLUSION: The use of beneficial microorganisms improves the performance of in vitro - cultured plants through the improvement of plant nutrition, the biological control of microbial pathogens or the production of phytohormones that promote plant growth and development. Plant in vitro culture techniques are highly useful to obtain significant amounts of true-to-type and disease-free plant materials. One of these techniques is clonal micropropagation which consists on the establishment of shoot tip cultures, shoot multiplication, in vitro rooting and acclimatization to ex vitro conditions. However, in some cases, the existence of recalcitrant genotypes, with a compromised multiplication and rooting ability, or the difficulties to overcome the overgrowth of endophytic contaminations might seriously limit its efficiency. In this sense, the establishment of beneficial interactions between plants and plant growth-promoting microorganisms (PGPMs) under in vitro culture conditions might represent a valuable approach to efficiently solve those restrictions. During the last years, significant evidence reporting the use of beneficial microorganisms to improve the yield of in vitro multiplication or rooting as well as their acclimatization to greenhouse or soil conditions have been provided. Most of these positive effects are strongly linked to the ability of these microorganisms to provide in vitro plants with nutrients such as nitrogen or phosphorous, to produce plant growth regulators, to control the growth of pathogens or to mitigate stress conditions. The culture of A. thaliana under aseptic conditions has provided high-quality knowledge on the root development signaling pathways, involving hormones, triggered in the presence of PGPMs. Overall, the present article offers a brief overview of the use of microorganisms to improve in vitro plant performance during the in vitro micropropagation stages, as well as the main mechanisms of plant growth promotion associated with these microorganisms.

Sars-Cov-2 Spike Protein-Induced Damage of hiPSC-Derived Cardiomyocytes.

Sars-Cov-2 may trigger molecular and functional alterations of cardiomyocytes (CMs) of the heart due to the presence of receptor angiotensin-converting enzyme 2 (ACE2) of the host cells. While the endocytic itinerary of the virus via cleavage of the spike protein of Sars-Cov-2 is well understood, the role of the remaining part of the spike protein subunit and ACE2 complex is still elusive. Herein, the possible effects of this complex are investigated by using synthetic spike proteins of Sars-Cov-2, human-induced pluripotent stem cells (hiPSC), and a culture device made of an arrayed monolayer of cross-linked nanofibers. hiPSCs are first differentiated into CMs that form cardiac tissue-like constructs with regular beating and expression of both ACE2 and gap junction protein Connexin 43. When incubated with the spike proteins, the hiPSC-CMs undergo a rhythmic fluctuation with overstretched sarcomere structures and dispersed gap junction proteins. When incubated with the spike proteins and supplementary angiotensin II, the damage of the spike protein on hiPSC-CMs is enhanced due to downregulated ACE2, chromatin margination, altered Connexin 43 expression, sarcomere disruption, and beating break. This discovery may imply latent effects of the spike proteins on the heart.

Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro.

Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and craniomaxillofacial implants. To screen biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity. International Organization for Standardization (ISO) standards have been widely utilized in the evaluation of biomaterials. However, most ISO standards were originally established to assess the cytotoxicity of nondegradable materials, thus providing limited value for screening biodegradable materials. This article introduces and discusses three different culture methods, namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials, including biodegradable polymers, ceramics, metals, and their composites, with different cell types. Research has shown that culture methods influence cell responses to biodegradable materials because their dynamic degradation induces spatiotemporal differences at the interface and in the local environment. Specifically, the direct culture method reveals the responses of cells seeded directly on the implants; the direct exposure culture method elucidates the responses of established host cells coming in contact with the implants; and the exposure culture method evaluates the established host cells that are not in direct contact with the implants but are influenced by the changes in the local environment due to implant degradation. This article provides examples of these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells (BMSCs). It also describes how to harvest, passage, culture, seed, fix, stain, characterize the cells, and analyze postculture media and materials. The in vitro methods described in this article mimic different scenarios of the in vivo environment, broadening the applicability and relevance of in vitro cytocompatibility testing of different biomaterials for various biomedical applications.