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1.
Food Microbiol ; 109: 104136, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36309439

RESUMO

Under stressful conditions, bacteria can enter viable but non-culturable (VBNC) state to survive. VBNC cells lost ability to grow on routine culture medium but are still alive and may revive in suitable conditions. The revived cells can consume nutrients or produce toxins, leading to food spoilage or human illness, posing great risk to food safety and public health. Previously, we have reported that high pressure carbon dioxide (HPCD), an environment-friendly sterilization technology, can induce VBNC formation. However, the underlying mechanism is unclear. By performing a comprehensive transcriptomic analysis, we revealed that HPCD initiated high expression of asr, encoding an acid shock protein, could promote VBNC formation of E. coli O157:H7. Quantitative reverse transcription PCR analysis suggested that high expression of asr (i) inhibited acid resistance (AR) systems, resulting in endogenous proton accumulation; (ii) inhibited hchA expression, a protein stabilizing factor. The two effects resulted in endogenous protein aggregation, which was highly correlated to VBNC formation. Accordingly, HPCD-stressed cells exhibited decreased efficiency of electron transfer chain and ATP production, which was also contributory to cytoplasmic protein aggregation. Taken together, HPCD-initiated high expression of Asr coupled with decreased ATP production led to protein aggregation, finally promoted the cells to enter VBNC state.


Assuntos
Escherichia coli O157 , Humanos , Dióxido de Carbono/farmacologia , Agregados Proteicos , Meios de Cultura/farmacologia , Trifosfato de Adenosina
2.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232777

RESUMO

The intrinsic healing following tendon injury is ideal, in which tendon progenitor cells proliferate and migrate to the injury site to directly bridge or regenerate tendon tissue. However, the mechanism determining why and how those cells are attracted to the injury site for tendon healing is not understood. Since the tenocytes near the injury site go through apoptosis or necrosis following injury, we hypothesized that secretions from injured tenocytes might have biological effects on cell proliferation and migration to enhance tendon healing. Tenocyte apoptosis was induced by 24 h cell starvation. Apoptotic body-rich media (T-ABRM) and apoptotic body-depleted media (T-ABDM) were collected from culture media after centrifuging. Tenocytes and bone marrow-derived stem cells (BMDSCs) were isolated and cultured with the following four media: (1) T-ABRM, (2) T-ABDM, (3) GDF-5, or (4) basal medium with 2% fetal calf serum (FCS). The cell activities and functions were evaluated. Both T-ABRM and T-ABDM treatments significantly stimulated the cell proliferation, migration, and extracellular matrix synthesis for both tenocytes and BMDSCs compared to the control groups (GDF-5 and basal medium). However, cell proliferation, migration, and extracellular matrix production of T-ABRM-treated cells were significantly higher than the T-ABDM, which indicates the apoptotic bodies are critical for cell activities. Our study revealed the possible mechanism of the intrinsic healing of the tendon in which apoptotic bodies, in the process of apoptosis, following tendon injury promote tenocyte and stromal cell proliferation, migration, and production. Future studies should analyze the components of the apoptotic bodies that play this role, and, thus, the targeting of therapeutics can be developed.


Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Traumatismos dos Tendões , Proliferação de Células , Células Cultivadas , Meios de Cultura/farmacologia , Fator 5 de Diferenciação de Crescimento/farmacologia , Humanos , Células-Tronco Mesenquimais/fisiologia , Soroalbumina Bovina/farmacologia , Traumatismos dos Tendões/terapia , Tenócitos
3.
Reprod Fertil Dev ; 34(15): 980-990, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36075881

RESUMO

AIMS: The main objective of this work is to elucidate whether Quercetin (Qc) and 4-Hidroxistradiol (4OHE2 ) decrease the level of reactive oxygen species (ROS) in in vitro obtained embryos and to analyse which genes are activated under the treatments that could explain this improvement. METHODS: Oxidative stress was induced during embryo culture by H2 O2 treatment and ROS production was measured and compared with embryos treated with Qc or 4OHE2 . Gene expression was analysed by Q-PCR in control embryos obtained in utero (IU) or by IVF and compared with the levels found in embryos cultured with Qc or 4OHE2 to determine the effect of these compounds. KEY RESULTS: Qc strongly reduces ROS levels in embryos after a treatment of 4h. On the contrary, 4OHE2 had no effect in reducing ROS levels in embryos. The addition of these molecules to the culture media upregulate several hypoxia-related genes when Qc is added to the culture media, and implantation-related genes when 4OHE2 is used. CONCLUSIONS: Qc is a very strong antioxidant molecule that when used for short periods of time during culture can reduce ROS levels and improve embryo quality by activating antioxidant enzymes. 4OHE2 supplementation, despite having no effects in reducing ROS levels, acts directly in the molecular signalling implicated in the implantation process and could be also considered as a supplement for embryo culture during IVF. IMPLICATIONS: Proper supplementation of the culture media could greatly improve the quality of embryos cultured in vitro , resulting in better results in IVF clinics.


Assuntos
Técnicas de Cultura Embrionária , Quercetina , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Blastocisto/metabolismo , Meios de Cultura/farmacologia , Desenvolvimento Embrionário , Fertilização In Vitro/métodos , Expressão Gênica , Camundongos , Quercetina/farmacologia , Espécies Reativas de Oxigênio/metabolismo
4.
Altern Lab Anim ; 50(5): 339-348, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36062749

RESUMO

Vero cells are one of the most frequently used cell types in virology. They can be used not only as a vehicle for the replication of viruses, but also as a model for investigating viral infectivity, cytopathology and vaccine production. There is increasing awareness of the need to limit the use of animal-derived components in cell culture media for a number of reasons, which include reducing the risk of contamination and decreasing costs related to the downstream processing of commercial products obtained via cell culture. The current study evaluates the use of protein hydrolysates (PHLs), also known as peptones, as partial substitutes for fetal bovine serum (FBS) in Vero cell culture. Eleven plant-based, two yeast-based, and three casein-based peptones were assessed, with different batches evaluated in the study. We tested the effects of three concentration ratios of FBS and peptone on Vero cell proliferation, four days after the initial cell seeding. Some of the tested peptones, when in combination with a minimal 1% level of FBS, supported cell proliferation rates equivalent to those achieved with 10% FBS. Collectively, our findings showed that plant-based peptones could represent promising options for the successful formulation of serum-reduced cell culture media for vaccine production. This is especially relevant in the context of the current COVID-19 pandemic, in view of the urgent need for SARS-CoV-2 virus production for certain types of vaccine. The current study contributes to the Three Rs principle of reduction, as well as addressing animal ethics concerns associated with FBS, by repurposing PHLs for use in cell culture.


Assuntos
COVID-19 , Peptonas , Animais , Caseínas , Técnicas de Cultura de Células , Chlorocebus aethiops , Meios de Cultura/farmacologia , Humanos , Pandemias , Peptonas/metabolismo , Peptonas/farmacologia , Hidrolisados de Proteína , SARS-CoV-2 , Soroalbumina Bovina , Células Vero
5.
Mol Biol Rep ; 49(11): 10327-10338, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36097112

RESUMO

BACKGROUND: Chlorogenic acid (CGA) is one of the well-known polyphenol compounds possessing several important biological and therapeutic functions. In order to optimize a culture system to achieve complete development of follicles, we focused on the effects of CGA supplementation during in vitro culture (IVC) on follicular development, oxidative stress, antioxidant capacity, developmental gene expression, and functional potential in cultured mouse ovarian tissue. METHODS AND RESULTS: The collected whole murine ovaries were randomly divided into four groups: (1) non-cultured group (control 1) with 7-day-old mouse ovaries, (2) non-cultured group (control 2) with 14-day-old mouse ovaries, (3) cultured group (experimental 1) with the culture plates containing only the basic culture medium, (4) cultured group (experimental 2) with the culture plates containing basic culture medium + CGA (50, 100 and 200 µmol/L CGA). Afterward, histological evaluation, biochemical analyses, the expression assessment of genes related to follicular development and apoptosis as well as the analysis of 17-ß-estradiol were performed. The results showed that supplementation of ovarian tissue with the basic culture media using CGA (100 µmol/l) significantly increased the survival, developmental and functional potential of follicles in whole mouse ovarian tissues after 7 days of culture. Furthermore, CGA (100 µmol/L) attenuated oxidative damage and enhanced the concentration of antioxidant capacity along with developmental gene expression. CONCLUSION: It seems that supplementation of ovarian tissue with culture media using CGA could optimize follicular growth and development in the culture system.


Assuntos
Ácido Clorogênico , Folículo Ovariano , Feminino , Camundongos , Animais , Ácido Clorogênico/farmacologia , Ácido Clorogênico/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Ovário , Meios de Cultura/farmacologia , Meios de Cultura/metabolismo
6.
Braz J Biol ; 82: e263282, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36043661

RESUMO

The growth of Haematococcus pluvialis in two alternative culture media NPK (10:10:10) and ME (macrophyte extract), under mixotrophic conditions using sugarcane molasses as a carbon source were evaluated for 28 days. The molasses was used in two different ways, in a native form (untreated) and a hydrolyzed (pretreated). Cell density of Haematococcus pluvialis in mixotrophic cultivation was higher in pretreated molasses. Growth rate was higher when pretreated molasses were employed in mixotrophic cultivation with NPK culture medium (k=0.5 7th growth day). Biomass, chlorophyll-a, conductivity and total inorganic nitrogen were not significantly different (p>0.05) during the experimental period for two mixotrophic cultivation and culture media. Protein contents of H. pluvialis biomass were higher in NPK culture medium with pretreated molasses (50% dry biomass). Annual biomass production was 520 kg-1 dry biomass with untreated molasses for two culture media, and 650 and 520 kg-1 dry biomass with pretreated molasses for NPK and ME culture media, respectively. The use of NPK and ME culture media in mixotrophic cultivation may be a new protocol for H. pluvialis cultivation due to the low cost and similar annual production.


Assuntos
Clorófitas , Microalgas , Saccharum , Biomassa , Meios de Cultura/farmacologia , Fertilizantes , Melaço , Extratos Vegetais
7.
Protein Expr Purif ; 199: 106154, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35970490

RESUMO

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 ± 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 × 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium Eden™-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes.


Assuntos
Interleucinas , Mamíferos , Animais , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Meios de Cultura/farmacologia , Humanos , Interleucinas/genética
8.
Int J Mol Sci ; 23(15)2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35955667

RESUMO

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicusfur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicusfur protein was able to complement an E. colifur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus.


Assuntos
Gluconacetobacter , Ferro , Proteínas de Bactérias/metabolismo , Meios de Cultura/farmacologia , Ferro/metabolismo , Transcriptoma
9.
Front Immunol ; 13: 915277, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35795685

RESUMO

Introduction: To study human physiological and pathological bone remodeling while addressing the principle of replacement, reduction and refinement of animal experiments (3Rs), human in vitro bone remodeling models are being developed. Despite increasing safety-, scientific-, and ethical concerns, fetal bovine serum (FBS), a nutritional medium supplement, is still routinely used in these models. To comply with the 3Rs and to improve the reproducibility of such in vitro models, xenogeneic-free medium supplements should be investigated. Human platelet lysate (hPL) might be a good alternative as it has been shown to accelerate osteogenic differentiation of mesenchymal stromal cells (MSCs) and improve subsequent mineralization. However, for a human in vitro bone model, hPL should also be able to adequately support osteoclastic differentiation and subsequent bone resorption. In addition, optimizing co-culture medium conditions in mono-cultures might lead to unequal stimulation of co-cultured cells. Methods: We compared supplementation with 10% FBS vs. 10%, 5%, and 2.5% hPL for osteoclast formation and resorption by human monocytes (MCs) in mono-culture and in co-culture with (osteogenically stimulated) human MSCs. Results and Discussion: Supplementation of hPL can lead to a less donor-dependent and more homogeneous osteoclastic differentiation of MCs when compared to supplementation with 10% FBS. In co-cultures, osteoclastic differentiation and resorption in the 10% FBS group was almost completely inhibited by MSCs, while the supplementation with hPL still allowed for resorption, mostly at low concentrations. The addition of hPL to osteogenically stimulated MSC mono- and MC-MSC co-cultures resulted in osteogenic differentiation and bone-like matrix formation, mostly at high concentrations. Conclusion: We conclude that hPL could support both osteoclastic differentiation of human MCs and osteogenic differentiation of human MSCs in mono- and in co-culture, and that this can be balanced by the hPL concentration. Thus, the use of hPL could limit the need for FBS, which is currently commonly accepted for in vitro bone remodeling models.


Assuntos
Reabsorção Óssea , Soroalbumina Bovina , Animais , Plaquetas , Células Cultivadas , Meios de Cultura/farmacologia , Humanos , Osteogênese , Reprodutibilidade dos Testes
10.
Trials ; 23(1): 558, 2022 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-35804457

RESUMO

BACKGROUND: Minimally invasive dentistry is a highly convenient and efficient method of managing caries in pediatric patients. Silver diamine fluoride (SDF) is commonly used to arrest active caries lesions. However, the associated black stain, possibility of soft tissue injury, and unpleasant taste often limit its use. Recently, nanosilver fluoride (NSF) emerged as a promising topical fluoride agent with potent cariostatic and antibacterial potentials. This novel anticaries agent has gained attention as an alternative to overcome the drawbacks of SDF in caries arrest. OBJECTIVES: To assess the antibacterial effect of NSF in relation to caries activity in dentin caries lesions, as well as to investigate the change in saliva bacterial levels in primary teeth in comparison to SDF after 1 and 3 months. MATERIALS AND METHODS: Fifty children aged 4 to 6 years old with active dentin caries lesions (score 5 according to International Detection and Assessment System (ICDAS II) criteria) will be enrolled in the study. They will be equally and randomly allocated into 2 groups: a group receiving NSF and a control group receiving SDF treatment. Microbiological samples will be collected from the carious lesions and from unstimulated saliva at the baseline and at the 1 and 3 months' follow-up appointments. Bacterial counts will be assessed using Mitis Salivarius agar (selective culture media for S. mutans) and Rogosa agar (selective culture media for lactobacilli), and the results will be expressed in colony-forming units. Data regarding the children's oral health will be collected and their dmf index will be scored. The arrest of active carious lesions will be measured at the follow-up appointments according to ICDAS II criteria. RESULTS: The relation between bacterial colony counts and lesion activity for both groups will be assessed, as well as the change in salivary bacterial counts. The collected data will be statistically evaluated and tabulated. This clinical trial has been registered on ClinicalTrials.gov in January 2022 (original version) with ID: NCT05221749.


Assuntos
Cárie Dentária , Fluoretos Tópicos , Compostos de Amônio Quaternário , Ágar/farmacologia , Antibacterianos/uso terapêutico , Criança , Pré-Escolar , Meios de Cultura/farmacologia , Cárie Dentária/tratamento farmacológico , Fluoretos Tópicos/uso terapêutico , Humanos , Compostos de Amônio Quaternário/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Dente Decíduo
11.
PLoS One ; 17(7): e0259482, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35857759

RESUMO

Serum-containing medium is widely used to support cell attachment, stable growth and serial passaging of various cancer cell lines. However, the presence of cholesterols and lipids in serum greatly hinders the analysis of the effects of cholesterol depletion on cells in culture. In this study, we developed a defined serum-free culture condition accessible to a variety of different types of adherent cancer cells. We tested different factors that are considered essential for cell culture and various extracellular matrix for plate coating, and found cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) basal media supplemented with Albumin (BSA) and insulin-transferrin-selenium-ethanolamine (ITS-X) on fibronectin-precoated plate (called as "DA-X condition") showed comparable proliferation and survival to those in a serum-containing medium. Interestingly, we observed that DA-X condition could be adapted to a wide variety of adherent cancer cell lines, which enabled the analysis of how cholesterol depletion affected cancer cells in culture. Mechanistically, we found the beneficial effects of the DA-X condition in part can be attributed to the appropriate level of membrane cholesterol, and fibronectin-mediated signaling plays an important role in the suppression of cholesterol production.


Assuntos
Colesterol , Fibronectinas , Adesão Celular , Proliferação de Células , Células Cultivadas , Colesterol/farmacologia , Meios de Cultura/farmacologia , Fibronectinas/farmacologia
12.
Cell Death Dis ; 13(7): 650, 2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35882857

RESUMO

Arterial calcification is highly prevalent, particularly in patients with end-stage renal disease (ESRD). The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is the critical process for the development of arterial calcification. However, the detailed mechanism of VSMCs calcification remains to be elucidated. Here, we investigated the role of exosomes (Exos) derived from endothelial cells (ECs) in arterial calcification and its potential mechanisms in ESRD. Accelerated VSMCs calcification was observed when VSMCs were exposed to ECs culture media stimulated by uremic serum or high concentration of inorganic phosphate (3.5 mM Pi). and the pro-calcification effect of the ECs culture media was attenuated by exosome depletion. Exosomes derived from high concentrations of inorganic phosphate-induced ECs (ECsHPi-Exos) could be uptaken by VSMCs and promoted VSMCs calcification. Microarray analysis showed that miR-670-3p was dramatically increased in ECsHPi-Exos compared with exosomes derived from normal concentrations of inorganic phosphate (0.9 mM Pi) induced ECs (ECsNPi-Exos). Mechanistically, insulin-like growth factor 1 (IGF-1) was identified as the downstream target of miR-670-3p in regulating VSMCs calcification. Notably, ECs-specific knock-in of miR-670-3p of the 5/6 nephrectomy with a high-phosphate diet (miR-670-3pEC-KI + NTP) mice that upregulated the level of miR-670-3p in artery tissues and significantly increased artery calcification. Finally, we validated that the level of circulation of plasma exosomal miR-670-3p was much higher in patients with ESRD compared with healthy controls. Elevated levels of plasma exosomal miR-670-3p were associated with a decline in IGF-1 and more severe artery calcification in patients with ESRD. Collectively, these findings suggested that ECs-derived exosomal miR-670-3p could promote arterial calcification by targeting IGF-1, which may serve as a potential therapeutic target for arterial calcification in ESRD patients.


Assuntos
Exossomos , Falência Renal Crônica , MicroRNAs , Calcificação Vascular , Animais , Meios de Cultura/farmacologia , Células Endoteliais/metabolismo , Exossomos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Falência Renal Crônica/metabolismo , Camundongos , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Osteogênese , Fosfatos/metabolismo , Fósforo/metabolismo , Fósforo/farmacologia , Calcificação Vascular/metabolismo
13.
Stem Cell Res Ther ; 13(1): 223, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35658933

RESUMO

BACKGROUND: Human-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost. METHODS: In this study, we developed a xeno-free ON2/AscleStem PSC medium (ON2) and cultured 253G1 hiPSCs on different matrices, including iMatrix-511 and gelatin nanofiber (GNF) in ON2. Over 20 passages, we evaluated cell proliferation by doubling times; pluripotency by flow cytometry, immunofluorescence staining and qRT-PCR; and differentiation ability by three germ layer differentiation in vitro and teratoma formation in severe combined immunodeficiency mice, followed by histological analysis. In addition, we compared the maintenance effect of ON2 on hiPSCs with StemFit® AK02 (AK02N) and Essential 8™ (E8). Besides 253G1 hiPSCs, we cultivated different hiPSC lines, including Ff-l01 hiPSCs, ATCC® ACS-1020™ hiPSCs, and Down's syndrome patient-specific ATCC® ACS-1003™ hiPSCs in ON2. RESULTS: We found that 253G1 hiPSCs in ON2 demonstrated normal morphology and karyotype and high self-renewal and differentiation abilities on the tested matrices for over 20 passages. Moreover, 253G1 hiPSCs kept on GNF showed higher growth and stemness, as verified by the shorter doubling time and higher expression levels of pluripotent markers. Compared to AK02N and E8 media, 253G1 hiPSCs grown in ON2 showed higher pluripotency, as demonstrated by the increased expression level of pluripotent factors. In addition, all hiPSC lines cultivated in ON2 were able to grow for at least 10 passages with compact clonal morphology and were positive for all detected pluripotent markers. CONCLUSIONS: Our xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.


Assuntos
Células-Tronco Pluripotentes Induzidas , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Medicina Regenerativa
14.
Int J Mol Sci ; 23(11)2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35682966

RESUMO

The osteogenic differentiation of mesenchymal stem cells is now a standard procedure in modern bone tissue engineering. As this is a promising field for future clinical applications, many cell culture media exist to promote osteogenic differentiation. Prior to differentiation, cells must be expanded to obtain sufficient numbers for experiments. Little evidence is available regarding the optimal media combination for expansion and differentiation to maximize the osteogenic response. Therefore, human BM-MSCs (n = 6) were expanded in parallel in DMEM (Dulbecco's Modified Eagle Medium) LG (Low Glucose) and α-MEM (Minimum Essential Media alpha-modification), followed by simultaneous monolayer differentiation toward the osteogenic lineage in: 1. DMEM LG (Low Glucose), 2. DMEM HG (High Glucose), 3. α-MEM, 4. "Bernese medium", and 5. "Verfaillie medium", with a corresponding negative control (total 20 groups). As a marker for osteogenic differentiation, hydroxyapatite was accessed using radioactive 99mTc-HDP labeling and quantitative alizarin red staining. The results indicate that all media except "Bernese medium" are suitable for osteogenic differentiation, while there was evidence that DMEM LG is partly superior when used for expansion and differentiation of BM-hMSCs. Using "Verfaillie medium" after DMEM LG expansion led to the highest grade of osteogenic differentiation. Nevertheless, the difference was not significant. Therefore, we recommend using DMEM LG for robust osteogenic differentiation, as it is highly suitable for that purpose, economical compared to other media, and requires little preparation time.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células/fisiologia , Células Cultivadas , Meios de Cultura/farmacologia , Glucose/farmacologia , Humanos
15.
Stem Cell Res Ther ; 13(1): 268, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729640

RESUMO

BACKGROUND: Mesenchymal stem cells (MSCs) have been suggested as an appropriate source for diabetes cell-based therapies. The high proliferation and differentiation capacity of fetal MSCs and the role of fetal pancreatic-derived MSCs (FPMSCs) in islet generation make them good candidates for diabetes treatment. To manufacture clinical-grade MSCs, animal-free culture protocols are preferred. The current study aimed to establish a xeno-free/GMP-compliant protocol for FPMSCs manufacturing. The focus was on the effects of fetal bovine serum (FBS) replacement with pooled human serum (HS). MATERIAL AND METHODS: FPMSCs were isolated and expanded from the pancreas of legally aborted fetuses with few modifications in our previously established protocol. The cells were expanded in two different culture media, including DMEM supplemented with 10% FBS or 10% pooled HS. A side-by-side comparison was made to evaluate the effect of each serum on proliferation rate, cell cycle, senescence, multi-lineage differentiation capacity, immunophenotype, and tumorigenesis of FPMSCs. RESULTS: Flow cytometry analysis and three-lineage differentiation ability demonstrated that fibroblast-like cells obtained from primary culture had MSCs' characteristics. The FPMSCs displayed similar morphology and CD markers expression in both sera. HS had a higher proliferative effect on FPMSCs than FBS. In FBS, the cells reached senescence earlier. In addition to normal karyotypes and anchorage-dependent growth, in vivo tumor formation was not seen. CONCLUSION: Our results demonstrated that HS was a better serum alternative than FBS for in vitro expansion of FPMSCs. Compared with FBS, HS increased FPMSCs' proliferation rate and decreased their senescence. In conclusion, HS can effectively replace FBS for clinical-grade FPMSCs manufacturing.


Assuntos
Células-Tronco Mesenquimais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultura/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Pâncreas , Soro/metabolismo
16.
Lett Appl Microbiol ; 75(4): 951-956, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35699349

RESUMO

Cisplatin was originally discovered through its antibacterial action and subsequently has found use as a potent broad-spectrum anticancer agent. This study determines the effect of growth media and solvent on the antibacterial activity of cisplatin and its analogue, oxaliplatin. Escherichia coli MG1655 or MG1655 ΔtolC was treated with the platinum compounds under different conditions and susceptibility was determined. Our results showed that DMSO reduced the activity of cisplatin by fourfold (MIC 12·5 mg l-1 ) compared with 0·9% NaCl-solubilized cisplatin (MIC 3·12 mg l-1 ) when tested in MOPS. Surprisingly, complete loss of activity was observed in Mueller-Hinton Broth II (MHB II). By supplementing MOPS with individual components of MHB II such as the sulphur-containing amino acids, l-cysteine and l-methionine, individually or in combination reduced activity by ≥8-fold (MIC ≥25 mg l-1 ). Oxaliplatin was less active against E. coli (MIC 100 mg l-1 ) but exhibited similar inactivation in the presence of DMSO, MHBII or MOPS spiked with l-cysteine and l-methionine (MIC ≥400 mg l-1 ). Our data suggest that the antibacterial activity of cisplatin and oxaliplatin is modulated by both choice of solvent and composition of growth media. We demonstrate that this is primarily due to sulphur-containing amino acids cysteine and methionine, an essential component of the recommended media for testing antimicrobial susceptibility, MHBII.


Assuntos
Antineoplásicos , Cisplatino , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Cisplatino/química , Cisplatino/farmacologia , Meios de Cultura/farmacologia , Cisteína , Dimetil Sulfóxido/farmacologia , Escherichia coli , Metionina/química , Metionina/farmacologia , Testes de Sensibilidade Microbiana , Oxaliplatina/farmacologia , Solução Salina/farmacologia , Solventes , Enxofre
17.
Braz Dent J ; 33(3): 8-17, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35766720

RESUMO

This study evaluated the effect of photodynamic therapy (PDT) on infected root canals. Twenty-one human teeth were selected, and 18 were infected by E. faecalis for 60 days. The antimicrobial strategies tested were: G1. Root canal preparation (RCP) using Niquel-Titanium (NiTi) rotary instruments, 2.5% NaOCl, and final irrigation with 17% EDTA, followed by PDT with methylene blue photosensitizer and laser diode low power; G2. RCP using stainless steel files and the same irrigation and PDT protocols as G1; G3. Same RCP protocol as G1 without PDT; G4. Only irrigation with 2.5% NaOCl; G5. Same PDT protocol as G1 without RCP; G6. Negative control; G7. Positive control. Samples for microbiological tests were collected initially (S1), after RCP (S2), and after PDT (S3). Subsequently, the roots were sectioned and prepared for Scanning Electron Microscopy (SEM) analysis. Bacterial growth was analyzed according to the turbidity of the culture medium, followed by spectrophotometric optical density (nm). The effect of PDT on the dentinal structure was evaluated at magnifications 1,600X and 5,000X and described qualitatively. The Wilcoxon test was used for the comparisons from the same specimens, and the Mann-Whitney test was used to compare groups ((=5%). Bacteria were found in all experimental groups' microbiological samples (S1, S2 and S3). The optical density of culture media was lower in S2 than in S1 of G1, 2, 3, and 4 (p> 0.05). After PDT (S3) in G1 and 2, there was an additional reduction in optical density of the culture medium, respectively (p>0.05). In Group 5, the analysis of culture media at S2 revealed an increase in optical density compared to S1(p>0.05). In SEM images of G1, 2, and 5, dentin with melting and recrystallization areas were evidenced. After preparation of the root canal with the rotary system or manually associated with 2.5% NaOCl, PDT was not able to completely eliminate E. faecalis present in the root canal.


Assuntos
Cavidade Pulpar , Lasers Semicondutores , Meios de Cultura/farmacologia , Cavidade Pulpar/microbiologia , Enterococcus faecalis , Humanos , Lasers Semicondutores/uso terapêutico , Irrigantes do Canal Radicular/farmacologia , Preparo de Canal Radicular/métodos , Hipoclorito de Sódio/farmacologia , Hipoclorito de Sódio/uso terapêutico
18.
Reprod Domest Anim ; 57(10): 1277-1279, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35694897

RESUMO

Selenium is commonly used as an antioxidant in a serum-free culture medium setting. However, lycopene has emerged as a potent antioxidant being twice as efficient as ß-carotene and 10 times as efficient as α-tocopherol with beneficial effects when supplemented in a serum-free maturation medium. Here, we aimed to evaluate the effect of lycopene supplementation in a serum-free culture medium on blastocyst development and quality. After in vitro maturation and fertilization, presumed zygotes were cultured in groups of 25 in 50 µl droplets of synthetic oviductal fluid. Culture medium supplementation was done using four experimental groups: insulin, transferrin, selenium (ITS, control); ITS + DMSO (diluent control); ITS + DMSO-lycopene 0.1 µM (ITSL); and IT + DMSO-lycopene 0.1 µM (ITL). DMSO was used as a diluent for lycopene. Blastocyst development among experimental groups was fitted in mixed-effects models, and blastocyst quality parameters (assessed via differential apoptotic staining) were evaluated in mixed linear regression models. The cleavage (85.3 ± 2.4, 82.6 ± 2.7, 86 ± 2.3 and 86.4 ± 2.3% for control, diluent control, ITSL and ITL, respectively) and day 8 blastocyst rates (37.4 ± 3.3, 36.9 ± 3.4, 39.7 ± 3.3 and 46.2 ± 3.4% for control, diluent control, ITSL and ITL, respectively) were not different (p > .1) among experimental groups. Embryos produced in the ITL group resulted in blastocysts with higher total cell numbers (TCN; 141 ± 19.2), inner cell mass (ICM; 65.3 ± 11.6) and trophectoderm cells (TE; 75.2 ± 8.8) compared with the control (129 ± 19.2, 56.3 ± 11.6 and 72.7 ± 8.8, for TCN, ICM and TE; p < .01, respectively). Lycopene-supplemented groups (ITSL and ITL) resulted in blastocysts with similar TCN, ICM and TE (p > .2). The number of apoptotic cells was not different among experimental groups (p > .1). Lycopene supplementation to the culture medium only produced a numerical increase in the blastocyst rate but replacing selenium with lycopene in a serum-free culture medium resulted in blastocysts with more cells.


Assuntos
Insulinas , Selênio , Animais , Antioxidantes/farmacologia , Blastocisto , Bovinos , Meios de Cultura/farmacologia , Suplementos Nutricionais , Dimetil Sulfóxido/farmacologia , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Fertilização In Vitro/veterinária , Insulinas/farmacologia , Licopeno/farmacologia , Selênio/farmacologia , Transferrinas/farmacologia , alfa-Tocoferol/farmacologia , beta Caroteno/farmacologia
19.
Ann Lab Med ; 42(6): 638-649, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35765872

RESUMO

Background: Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality. Methods: UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells. Results: The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity. Conclusions: Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.


Assuntos
Células Matadoras Naturais , Proliferação de Células , Meios de Cultura/farmacologia , Humanos
20.
BMC Microbiol ; 22(1): 131, 2022 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-35568814

RESUMO

BACKROUND: During the last decades, outbreaks of foodborne illnesses have increasingly been linked to fresh and/or minimally processed fruit and vegetables. Enterohemorrhagic Escherichia coli was the causal agent for major outbreaks in Europe with leafy green vegetables and sprouts. To improve food safety, microbial antagonism has received attention during recent years and could be one of the solution to prevent contamination of food borne pathogens on fresh produce. Here we investigate the antagonistic effect of three bacterial strains (Pseudomonas orientalis, P. flavescens and Rhodococcus sp.) isolated from spinach leaves against E. coli O157:H7gfp + under laboratory and greenhouse conditions. RESULTS: Our results shows that significantly less culturable E.coli O157:H7gfp + were retrieved from the spinach canopy subjected to antagonist seed treatment than canopy inoculation. Seeds inoculated with Rhodococcus sp. significantly reduced growth of E. coli O157:H7gfp + compared with the other antagonists. The result from the in vitro study shows a significant reduction of growth of E. coli O157:H7gfp+, but only after 44 h when E. coli O157:H7gfp + was propagated in a mixture of spent media from all three antagonists. CONCLUSIONS: The antagonistic effect on phyllospheric E.coli O157:H7gfp + observed after seed inoculation with Rhodococcus sp. might be an indication of induced resistance mechanism in the crop. In addition, there was a small reduction of culturable E.coli O157:H7gfp + when propagated in spent media from all three antagonists. Nutritional conditions rather than metabolites formed by the three chosen organisms appear to be critical for controlling E. coli O157:H7gfp+.


Assuntos
Escherichia coli O157 , Bactérias , Contagem de Colônia Microbiana , Meios de Cultura/farmacologia , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Folhas de Planta/microbiologia , Sementes , Spinacia oleracea/microbiologia
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