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1.
Proc Natl Acad Sci U S A ; 117(32): 19033-19044, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709748

RESUMO

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).


Assuntos
Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/citologia , Microgéis/química , Neovascularização Fisiológica , Animais , Proliferação de Células , Células Imobilizadas/química , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Membro Posterior/cirurgia , Humanos , Integrinas/genética , Integrinas/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus
2.
Chemosphere ; 258: 127148, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32535434

RESUMO

Tetrahydrofuran (THF) is a common highly toxic cyclic aliphatic ether that frequently exists in waste gases. Removal of gaseous THF is a serious issue with important environmental ramifications. A novel three-phase airlift bioreactor (TPAB) loaded with immobilized cells was developed for efficient THF removal from gas streams. An effective THF-degrading transformant, Pseudomonas oleovorans GDT4, which contains the pTn-Mod-OTc-gfp plasmid and was tagged with a green fluorescent protein (GFP), was constructed. Continuous treatment of THF-containing waste gases was succeeded by the GFP-labelled cells immobilized with calcium alginate and activated carbon fiber in the TPAB for 60 days with >90% removal efficiency. The number of fluorescent cells in the beads reached 1.7 × 1011 cells·g-1 of bead on day 10, accounting for 83.3% of the total number of cells. The amount further increased to 3.0 × 1011 cells·g-1 of bead on day 40. However, it decreased to 2.5 × 1011 cells·g-1 of bead with a substantial increase in biomass in the liquid because of cell leakage and hydraulic shock. PCR-DGGE revealed that P. oleovorans was the dominant microorganism throughout the entire operation. The maximum elimination capacity was affected by empty bed residence time (EBRT). The capacity was only 25.9 g m-3·h-1 at EBRT of 80 s, whereas it reached 37.8 g m-3·h-1 at EBRT of 140 s. This work provides an alternative method for full-scale removal of gaseous THF and presents a useful tool for determining the biomass of a specific degrader in immobilized beads.


Assuntos
Reatores Biológicos/microbiologia , Furanos/metabolismo , Pseudomonas oleovorans/metabolismo , Gerenciamento de Resíduos/métodos , Alginatos/química , Biodegradação Ambiental , Biomassa , Fibra de Carbono , Células Imobilizadas/metabolismo , Carvão Vegetal , Desenho de Equipamento , Gases , Proteínas de Fluorescência Verde/genética , Microbiota , Microrganismos Geneticamente Modificados , Pseudomonas oleovorans/citologia , Pseudomonas oleovorans/genética , Gerenciamento de Resíduos/instrumentação
3.
J Biosci Bioeng ; 130(3): 260-264, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32456985

RESUMO

Vanillin is a well-known fragrant, flavoring compound. Previously, we established a method of coenzyme-independent vanillin production via an oxygenase from Caulobacter segnis ATCC 21756, called Cso2, that converts 4-vinylguaiacol to vanillin and formaldehyde using oxygen. In this study, we found that reactive oxygen species inhibited the catalytic activity of Cso2, and the addition of catalase increased vanillin production. Since Escherichia coli harbors catalases, we used E. coli cells expressing Cso2 to produce vanillin. Cell immobilization in calcium alginate enabled the long-term use of the E. coli cells for vanillin production. Thus, we demonstrate the possibility of using immobilized E. coli cells for both continuous and repeated batch vanillin production without any coenzymes.


Assuntos
Benzaldeídos/metabolismo , Células Imobilizadas/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Guaiacol/análogos & derivados , Oxigenases/genética , Biotecnologia , Caulobacter/enzimologia , Expressão Gênica , Guaiacol/metabolismo
4.
Enzyme Microb Technol ; 135: 109495, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146929

RESUMO

E. coli has been engineered to produce xylitol, but the production faces bottlenecks in terms of production yield and cell viability. In this study, recombinant E. coli (rE. coli) was immobilized on untreated and treated multiwalled carbon nanotubes (MWCNTs) for xylitol production. The immobilized rE. coli on untreated MWCNTs gave the highest xylitol production (5.47 g L-1) and a productivity of 0.22 g L-1 h-1. The doubling time for the immobilized cells increased up to 20.40 h and was higher than that of free cells (3.67 h). Cell lysis of the immobilized cells was reduced by up to 73 %, and plasmid stability improved by up to 17 % compared to those of free cells. Xylitol production using the optimum parameters (pH 7.4, 0.005 mM and 29 °C) achieved a xylitol production and productivity of 6.33 g L-1 and 0.26 g L-1 h-1, respectively. A seven-cycle repeated batch fermentation was carried out for up to 168 h, which showed maximum xylitol production of 7.36 g L-1 during the third cycle. Hence, this new adsorption immobilization system using MWCNTs is an alternative to improve the production of xylitol.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Nanotubos de Carbono/química , Xilitol/metabolismo , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Escherichia coli/química , Fermentação
5.
Molecules ; 25(4)2020 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-32093025

RESUMO

The demand for natural lactone gamma-decalactone (GDL) has increased in the fields of food and cosmetic products. However, low productivity during bioprocessing limits its industrial production. In this study, a novel composite porous cell carrier, bacterial cellulose-alginate (BC-ALG), was used for long-term biotransformation and production of GDL. The effects of this carrier on biotransformation and related mechanisms were investigated. BC-ALG carriers showed improved mechanical strength over ALG carriers, with their internal embedded cell pattern changed to an interconnected porous structure. In five repeated-batch biotransformation experiments, the maximum concentration of GDL obtained in culture with BC-ALG carriers was 8.37 g/L, approximately 3.7 times higher than that from the medium with an ALG carrier alone. The result indicated that multiple hydrogen bonding interactions at the interface between BC and ALG contributed to the compatibility and stability of BC-ALG carriers. On the basis of the above results, the BC-ALG composite carrier can be considered ideal for immobilisation of cells for the production of GDL on an industrial scale, and has the potential to be utilised in other biological processes.


Assuntos
Alginatos/química , Células Imobilizadas/metabolismo , Celulose/química , Lactonas/metabolismo , Yarrowia/metabolismo
6.
Bioengineered ; 11(1): 141-153, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-31994978

RESUMO

Cell immobilization on the magnetic nanoparticles (MNPs) and magnetic harvesting is a novel approach for microalgal cells separation. To date, the effect of these nanoparticles on microalgal cells was only studied over a short period of time. More studies are hence needed for a better understanding of the magnetic harvesting proposes or environmental concerns relating to long-term exposure to nanoparticles. In this study, the impact of various concentrations of MNPs on the microalgal cells growth and their metabolic status was investigated over 12 days. More than 60% reduction in mitochondrial activity and pigments (chlorophyll a, chlorophyll b, and carotenoids) content occurred during the first 6 days of exposure to ≥50 µg/mL nanoparticles. However, more than 50% growth inhibitory effect was seen at concentrations higher than 400 µg/mL. Exposure to MNPs gradually induced cellular adaptation and after about 6 days of exposure to stress generating concentrations (˂400 µg/mL) of IONs, microalgae could overcome the imposed damages. This work provides a better understanding regarding the environmental impact of MNPs and appropriate concentrations of these particles for future algal cells magnetic immobilization and harvesting.


Assuntos
Chlorella vulgaris/química , Nanopartículas/química , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Chlorella vulgaris/crescimento & desenvolvimento , Chlorella vulgaris/metabolismo , Clorofila/análise , Clorofila/metabolismo , Clorofila A/análise , Clorofila A/metabolismo , Fenômenos Magnéticos , Microalgas/química , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo
7.
Talanta ; 206: 120192, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514850

RESUMO

In biosensors fabrication, entrapment in polymeric matrices allows efficient immobilization of the biorecognition elements without compromising their structure and activity. When considering living cells, the biocompatibility of both the matrix and the polymerization procedure are additional critical factors. Bio-polymeric gels (e.g. alginate) are biocompatible and polymerize under mild conditions, but they have poor stability. Most synthetic polymers (e.g. PVA), on the other hand, present improved stability at the expense of complex protocols involving chemical/physical treatments that decrease their biological compatibility. In an attempt to explore new solutions to this problem we have developed a procedure for the immobilization of bacterial cells in polyethersulfone (PES) using phase separation. The technology has been tested successfully in the construction of a bacterial biosensor for toxicity assessment. Biosensors were coated with a 300  µm bacteria-containing PES membrane, using non-solvent induced phase separation (membrane thickness ≈ 300 µm). With this method, up to 2.3 × 106 cells were immobilized in the electrode surface with an entrapment efficiency of 8.2%, without compromising cell integrity or viability. Biosensing was performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. PES biosensors showed good stability and reusability during dry frozen storage for up to 1 month. The analytical performance of the sensors was assessed carrying out a toxicity assay in which 3,5-dichlorophenol (DCP) was used as a model toxic compound. The biosensor provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 9.2 ppm, well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive bacterial biosensors.


Assuntos
Técnicas Biossensoriais/métodos , Clorofenóis/toxicidade , Escherichia coli/isolamento & purificação , Polímeros/química , Sulfonas/química , Testes de Toxicidade/métodos , Sobrevivência Celular/efeitos dos fármacos , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/metabolismo , Técnicas Eletroquímicas/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ferricianetos/química , Ferricianetos/metabolismo , Glucose/metabolismo , Membranas Artificiais , Oxirredução , Reprodutibilidade dos Testes
8.
Lett Appl Microbiol ; 70(4): 263-273, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31879967

RESUMO

Bioaugmentation is an effective treatment method to reduce nitrogenous pollutants from wastewater. A strain of DYTN-1, which could effectively remove TN from sewage, was isolated from the sludge of a wastewater treatment plant and was identified as Paracoccus denitrificans. The TN in wastewater reduced to <20 mg l-1 within 12 h under optimal conditions by free cells of P. denitrificans DYTN-1. To enhance the removal of TN, P. denitrificans DYTN-1 cells were immobilized in sodium alginate (SA) using different divalent metal ions as cross-linking agents. It was found that the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 to below 20 mg l-1 within 8 h. After the optimization of an orthogonal experiment, the immobilized P. denitrificans DYTN-1 cells could reduce the TN concentration from 100 mg l-1 to below 20 mg l-1 within 1 h and significantly reduce the fermentation cycle. These findings would provide an economical and effective method for the removal of total nitrogen in wastewater by immobilized cells of P. denitrificans DYTN-1. SIGNIFICANCE AND IMPACT OF THE STUDY: We identified a new Paracoccus denitrificans strain (DYTN-1) for removal of the total nitrogen in wastewater. The total nitrogen could be removed effectively by P. denitrificans DYTN-1 within 12 h in wastewater. Using sodium alginate as the carrier and Ba2+ as cross-linking agent, the immobilized P. denitrificans DYTN-1 cells could improve the removal efficiency of total nitrogen in wastewater and significantly reduce the fermentation cycle. The assay has provided an economical and effective method for the removal of total nitrogen in wastewater by immobilized cell.


Assuntos
Nitrogênio/metabolismo , Paracoccus denitrificans/metabolismo , Águas Residuárias/microbiologia , Purificação da Água/métodos , Biodegradação Ambiental , Reatores Biológicos/microbiologia , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Desnitrificação , Fermentação , Paracoccus denitrificans/química , Paracoccus denitrificans/genética , Paracoccus denitrificans/isolamento & purificação , Esgotos/microbiologia , Purificação da Água/instrumentação
9.
Huan Jing Ke Xue ; 40(7): 3126-3134, 2019 Jul 08.
Artigo em Chinês | MEDLINE | ID: mdl-31854710

RESUMO

Application of microalgae in wastewater treatment is regarded as a potential green technology. However, its engineering application has been largely hindered because of the difficulty of biomass separation and harvesting. This study aimed to identify the key parameters influencing the process of microalgae immobilization. To do this, the study focused on immobilization technology and Scenedesmus obliquus, and employed the response surface methodology (RSM) and the Box-Behnken design (BBD). In an evaluation of the performance of microalgae beads, the fixing agent concentration, the cross-linking agent concentration, and the cross-linking time were selected as the independent variables, and the mechanical strength, the mass transfer rate, and the growth rate of immobilized microalgae beads were the response values. The optimal conditions and the uptake potential of the microalgae beads with respect to ammonia nitrogen (NH4+-N) were further explored and analyzed. The results showed that the optimal parameters for the preparation of immobilized microalgae beads were 5%, 2%, and 16 h, and the maximum removal capacity was obtained using mixotrophic cultivation with an embedding density of 1×106 cells·mL-1 and an organic matter concentration of 300 mg·L-1. In addition, the removal capacity of immobilized microalgae with respect to high concentrations of NH4+-N was significantly higher than for free-living microalgae. When the initial concentrations of NH4+-N were approximately 50 and 70 mg·L-1, NH4+-N was removed by the immobilized microalgae (after a 5-day mixotrophic cultivation) at a rate of (96.6±0.1)% and (65.2±4.5)%, respectively. With an initial NH4+-N concentration of 30 mg·L-1, the dominance of free-living microalgae was clear, with a removal rate of (97.8±0.6)% after a 3-day cultivation. However, under heterotrophic cultivation, the removal rate of NH4+-N by immobilized microalgae was generally low and gradually decreased with increasing concentrations. When the initial concentration was approximately 30 mg·L-1, the removal rate was only (49.0±3.1)%. This study provides new prospects for sustainable urban wastewater treatment, a new approach for resource recycling, and a strong theoretical foundation for the popularization and application of microalgae in wastewater treatment.


Assuntos
Amônia/isolamento & purificação , Microalgas/metabolismo , Nitrogênio/isolamento & purificação , Águas Residuárias , Purificação da Água/métodos , Biomassa , Células Imobilizadas/metabolismo
10.
Biosensors (Basel) ; 9(4)2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739597

RESUMO

Cancer cell lines are important tools for anticancer drug research and assessment. Impedance measurements can provide valuable information about cell viability in real time. This work presents the proof-of-concept development of a bioelectrical, impedance-based analysis technique applied to four adherent mammalian cancer cells lines immobilized in a three-dimensional (3D) calcium alginate hydrogel matrix, thus mimicking in vivo tissue conditions. Cells were treated with cytostatic agent5-fluoruracil (5-FU). The cell lines used in this study were SK-N-SH, HEK293, HeLa, and MCF-7. For each cell culture, three cell population densities were chosen (50,000, 100,000, and 200,000 cells/100 µL). The aim of this study was the extraction of mean impedance values at various frequencies for the assessment of the different behavior of various cancer cells when 5-FU was applied. For comparison purposes, impedance measurements were implemented on untreated immobilized cell lines. The results demonstrated not only the dependence of each cell line impedance value on the frequency, but also the relation of the impedance level to the cell population density for every individual cell line. By establishing a cell line-specific bioelectrical behavior, it is possible to obtain a unique fingerprint for each cancer cell line reaction to a selected anticancer agent.


Assuntos
Técnicas Biossensoriais , Células Imobilizadas/metabolismo , Impedância Elétrica , Técnicas Eletroquímicas , Impressão Tridimensional , Alginatos/química , Técnicas Biossensoriais/instrumentação , Sobrevivência Celular , Células Cultivadas , Técnicas Eletroquímicas/instrumentação , Células HEK293 , Humanos
11.
Biosens Bioelectron ; 146: 111710, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31600628

RESUMO

Farming, industry and urbanization lead to increases in the concentrations of potentially harmful compounds in waste, surface and drinking waters. One example of such pollution are estrogens, the steroidal female reproductive hormones. Already at a few nanograms per litre, these hormones can trigger endocrine disruption and cause acute and chronic health problems in humans and wildlife. Here, we present a Saccharomyces cerevisiae estrogen biosensor capable of detecting estradiol, as well as ethinylestradiol, at concentrations of 1 nM. After an initial characterization of the sensor strain performance in an optimal laboratory setting, we focused on developing a biosensor device. We addressed current limitations of biosensors, such as the requirement of the cells for a liquid growth matrix, controlled storage conditions required to preserve cell viability, and the usually required bulky, as well as expensive, laboratory equipment. Our study provides significant new insights into the field of applied biosensors. The system presented in this work takes microorganism-based analytics one step closer to field application in decentralized locations.


Assuntos
Técnicas Biossensoriais/instrumentação , Disruptores Endócrinos/análise , Estradiol/análise , Saccharomyces cerevisiae/efeitos dos fármacos , Poluentes Químicos da Água/análise , Células Imobilizadas/efeitos dos fármacos , Células Imobilizadas/metabolismo , Disruptores Endócrinos/metabolismo , Desenho de Equipamento , Estradiol/metabolismo , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Smartphone , Poluentes Químicos da Água/metabolismo
12.
Mater Sci Eng C Mater Biol Appl ; 105: 110071, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546377

RESUMO

In the current study, in vitro analysis of the osteogenic potential of different scaffolds based on strontium-substituted ß-TCP (Sr-TCP) and bioactive glass (BG) ceramics was conducted using rabbit bone marrow-derived mesenchymal stem cells (rBMSCs) and the osteogenic ability of the prepared Sr-TCP and BG scaffold was evaluated through alkaline phosphatase activity, mineral deposition by Alizarin red staining, and osteoblastic gene expression experiments. The obtained in vitro results revealed that among experimental Sr-TCP/BG nanocomposite scaffold samples with the composition of Sr-TCP/BG: 100/0, 50/50, 75/25, and 25/75, the 50Sr-TCP/50BG sample presented better osteoinductive properties. Therefore, the optimized 50Sr-TCP/50BG nanocomposite scaffold was chosen for further in vivo experiments. In vivo implantation of 50Sr-TCP/50BG scaffold and hydroxyapatite (HA)/TCP granules in a rabbit calvarial defect model showed slow degradation of 50Sr-TCP/50BG scaffold and high resorption rate of HA/TCP granules at 5 months' post-surgery. However, the 50Sr-TCP/50BG scaffolds loaded by mesenchymal stem cells (MSCs) were mainly replaced with new bone even at 2 months post-implantation. Based on the obtained engineering and biological results, 50Sr-TCP/50BG nanocomposite scaffold containing MSCs could be considered as a promising alternative substitute even for load-bearing bone tissue engineering applications.


Assuntos
Células da Medula Óssea/metabolismo , Fosfatos de Cálcio/química , Células Imobilizadas , Cerâmica/química , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Nanocompostos/química , Estrôncio/química , Tecidos Suporte/química , Animais , Células da Medula Óssea/patologia , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Células-Tronco Mesenquimais/patologia , Porosidade , Coelhos
13.
ACS Appl Mater Interfaces ; 11(41): 38232-38239, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31550878

RESUMO

Currently, there are limited approaches to tailor 3D scaffolds cross-linked with a stable covalent C-C bond that does not require any catalysts or initiators. We present here the first hydrogels employing aldol condensation chemistry that exhibit exceptional physicochemical properties. We investigated the aldol-cross-linking chemistry using two types of aldehyde-modified hyaluronic acid (HA) derivatives, namely, an enolizable HA-aldehyde (HA-Eal) and a non-enolizable HA-aldehyde (HA-Nal). Hydrogels formed using HA-Eal demonstrate inferior cross-linking efficiency (due to intramolecular loop formation), when compared with hydrogels formed by mixing HA-Eal and HA-NaI leading to a cross-aldol product. The change in mechanical properties as a result of cross-linking at different pH values is determined using rheological measurements and is interpreted in terms of molecular weight between cross-links (Mc). The novel HA cross-aldol hydrogel demonstrate excellent hydrolytic stability and favorable mechanical properties but allow hyaluronidase-mediated enzymatic degradation. Interestingly, residual aldehyde functionality within the aldol product rendered the tissue-adhesive properties by bonding two bone tissues. The aldehyde functionality also facilitated facile post-synthetic modifications with nucleophilic reagents. Finally, we demonstrate that the novel hydrogel is biocompatible with encapsulated stem cells that show a linear rate of expansion in our 3-6 days of study.


Assuntos
Células Imobilizadas/metabolismo , Ácido Hialurônico , Hidrogéis , Teste de Materiais , Células-Tronco Mesenquimais/metabolismo , Adesivos Teciduais , Células Imobilizadas/citologia , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Concentração de Íons de Hidrogênio , Células-Tronco Mesenquimais/citologia , Adesivos Teciduais/química , Adesivos Teciduais/farmacologia
14.
Appl Microbiol Biotechnol ; 103(21-22): 8923-8935, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520132

RESUMO

UV and gamma irradiation mutagenesis was applied on Aspergillus fumigatus and Alternaria tenuissima in order to improve their producing ability of paclitaxel. Among the screened mutants, two stable strains (designated TXD105-GM6 and TER995-GM3) showed the maximum paclitaxel production. Paclitaxel titers of the two respective mutants were dramatically intensified to 1.22- and 1.24-fold, as compared by their respective parents. Immobilization using five different entrapment carriers of calcium alginate, agar-agar, Na-CMC, gelatin, and Arabic gum was successfully applied for production enhancement of paclitaxel by the two mutants. The immobilized cultures were superior to free-cell cultures and paclitaxel production by the immobilized mycelia was much higher than that of the immobilized spores using all the tried carriers. Moreover, calcium alginate gel beads were found the most conductive and proper entrapment carrier for maximum production of paclitaxel. The feasibility of the paclitaxel production by the immobilized mycelia as affected by incubation period, medium volume, and number of beads per flask was adopted. Under the favorable immobilization conditions, the paclitaxel titers were significantly intensified to 1.31- and 1.88-fold by the respective mutants, as compared by their free cultures. The obtained paclitaxel titers by the immobilized mycelia of the respective mutants (694.67 and 388.65 µg L-1) were found promising in terms of fungal production of paclitaxel. Hence, these findings indicate the future possibility to reduce the cost of producing paclitaxel and suggest application of the immobilization technique for the biotechnological production of paclitaxel at an industrial scale.


Assuntos
Alternaria/metabolismo , Antineoplásicos/metabolismo , Aspergillus fumigatus/metabolismo , Paclitaxel/biossíntese , Alginatos/química , Alternaria/química , Alternaria/genética , Aspergillus fumigatus/química , Aspergillus fumigatus/genética , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Fermentação , Microbiologia Industrial , Micélio/química , Micélio/genética , Micélio/metabolismo
15.
Regen Med ; 14(10): 915-923, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31556342

RESUMO

Aim: The major symptom of many closed spinal dysraphism patients is that the laminas or arches of vertebra are not fused well. To date, the bone repair of spina bifida for young children is a significant challenge in clinical practice. Materials & methods: Bovine bone collagen particle (BBCP) scaffolds combined with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were implanted in the defect area. X-ray analysis was performed after 3 months. Tissues were harvested for gross observation, and histological and immunohistochemical staining. Results: The BBCP supported hUC-MSCs adhesion and growth. Implanted BBCP combined with hUC-MSCs also promoted bone regeneration in the vertebral lamina and arch defect area. Conclusion: This method represents a new strategy for vertebral lamina and arch reconstruction in children.


Assuntos
Regeneração Óssea , Células Imobilizadas , Colágeno/química , Vértebras Lombares , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Cordão Umbilical , Animais , Bovinos , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Humanos , Vértebras Lombares/lesões , Vértebras Lombares/metabolismo , Vértebras Lombares/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Coelhos , Cordão Umbilical/metabolismo , Cordão Umbilical/patologia
16.
Biosens Bioelectron ; 142: 111571, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445395

RESUMO

The extracellular electron transfer of Shewanella oneidensis MR-1 (MR-1) has been extensively studied due to the importance of the biosensors and energy applications of bioelectrochemical systems. However, the oxidation of metal compounds by MR-1, which represents the inward extracellular electron transfer from extracellular electron donors into the microbe, is barely understood. In this study, MR-1 immobilized on an electrode electrocatalyzes the oxidation of [Fe(CN)6]4- to [Fe(CN)6]3- efficiently and selectively. The selectivity depends on midpoint potential and overall charge(s) of redox molecules. Among 12 investigated redox molecules, the negatively charged molecules with high midpoint potentials, i.e., [Ru(CN)6]4- and [Fe(CN)6]4-, show strong electrocatalysis. Neither reference bacteria (Escherichia coli K-12 nor Streptococcus mutans) electrocatalyze the oxidation of [Fe(CN)6]4-. The electrocatalysis decays when MR-1 is covered with palladium nanoparticles presumptively involved with cytochromes c. However, cytochromes c MtrC and OmcA on MR-1 do not play an essential role in this process. The results support a model that [Fe(CN)6]4- donor electrons to MR-1 by interacting with undiscovered active sites and the electrons are subsequently transferred to the electrode through the mediating effect of [Fe(CN)6]4-/3-. The selective electron uptake by MR-1 provides valuable and fundamental insights of the applications of bioelectrochemical systems and the detection of specific redox molecules.


Assuntos
Ferrocianetos/metabolismo , Metais/metabolismo , Shewanella/metabolismo , Técnicas Biossensoriais , Catálise , Células Imobilizadas/metabolismo , Técnicas Eletroquímicas , Eletrodos , Transporte de Elétrons , Elétrons , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Oxirredução , Paládio/metabolismo
17.
Int J Biol Macromol ; 141: 51-59, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31442504

RESUMO

Hydrogels have shown to be advantageous in supporting damaged cartilage because of its analogous to the extracellular matrix (ECM) of cartilage tissue. However, problems such as infection and inflammation are still a challenge to be solved. In terms of tissue engineering, natural materials are more advantageous than synthetic materials in biocompatibility and biodegradability status. Herein, physically blended nature-derived gellan gum (GG) hydrogel and hyaluronic acid (HA) hydrogel is suggested as a one of solution for cartilage tissue engineering material. The purpose of this study is to determine the effect of GG/HA hydrogel in vitro and in vivo. The chemical and mechanical properties were measured to confirm the compatibility of hydrogels for cartilage tissue engineering. The viability, proliferation, morphology, and gene expression of chondrocytes encapsulated in hydrogels were examined in vitro. Furthermore, the beneficial effect of the blended hydrogel was confirmed by performing the in vivo experiment. The chemical properties of hydrogels confirmed the well physically blended hydrogels. The mechanical studies of hydrogels displayed that as the content of HA increases, the swelling ratio was higher, compressive strength decreased and degradation was faster. Therefore, to use the hydrogel of GG and HA network, the proper amount must be blended. The in vitro study of chondrocytes encapsulated GG/HA hydrogel showed that the proper amount of HA enhanced the cell growth, attachment, and gene expression. The in vivo examination verified the advantageous effect of GG/HA hydrogel. Overall results demonstrate that GG/HA hydrogel is suitable for culturing chondrocyte and can be further applied for the treatment of cartilage defects.


Assuntos
Cartilagem , Células Imobilizadas , Condrócitos , Ácido Hialurônico/química , Hidrogéis/química , Polissacarídeos Bacterianos/química , Regeneração , Animais , Cartilagem/lesões , Cartilagem/patologia , Cartilagem/fisiologia , Células Imobilizadas/metabolismo , Células Imobilizadas/patologia , Células Imobilizadas/transplante , Condrócitos/metabolismo , Condrócitos/patologia , Condrócitos/transplante , Coelhos
18.
Colloids Surf B Biointerfaces ; 181: 989-993, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382350

RESUMO

Saccharomyces cerevisiae immobilized in electrospun Pluronic F127 dimethacrylate (FDMA) was successfully employed for the production of ethanol in an immobilized cell reactor. Yeast cells were immobilized into fibers formed through the process of electrospinning and cross-linking. The threads had an average diameter of 0.88 µm and were used in continuous-flow immobilized cell reactors. The immobilized cell reactors were able to maintain a high ethanol yield of >90% from day 4 through to the end of the time course at day 14. The reactor was able to achieve a maximum ethanol yield of 94.3%. This study shows that the use of electrospinning is a promising method for continuous ethanol production through immobilized cell-based continuous flow reactors.


Assuntos
Reatores Biológicos , Células Imobilizadas/metabolismo , Etanol/metabolismo , Metacrilatos/metabolismo , Poloxâmero/metabolismo , Saccharomyces cerevisiae/metabolismo , Etanol/química , Fermentação , Metacrilatos/química , Tamanho da Partícula , Poloxâmero/química , Propriedades de Superfície
19.
ACS Appl Mater Interfaces ; 11(34): 30585-30595, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31378063

RESUMO

One of the challenges for extrusion bioprinting using low-viscosity bioinks is the fast gravity-driven sedimentation of cells. Cells in a hydrogel bioink that features low viscosity tend to settle to the bottom of the bioink reservoir, and as such, their bioprintability is hindered by association with the inhomogeneous cellularized structures that are deposited. This is particularly true in cases where longer periods are required to print complex or larger tissue constructs. Increasing the bioink's viscosity efficiently retards sedimentation but gives rise to cell membranolysis or functional disruption due to increased shear stress on the cells during the extrusion process. Inspired by the rainbow cocktail, we report the development of a multilayered modification strategy for gelatin methacryloyl (GelMA) bioink to manipulate multiple liquid interfaces, providing interfacial retention to retard cell sedimentation in the bioink reservoir. Indeed, the interfacial tension in our layer-by-layer bioink system, characterized by the pendant drop method, was found to be exponentially higher than the sedimental pull (ΔGravity-Buoyancy = ∼10-9 N) of cells, indicating that the interfacial retention is crucial for preventing cell sedimentation across the adjacent layers. It was demonstrated that the encapsulated cells displayed better dispersibility in constructs bioprinted using the multilayered GelMA bioink system than that of pristine GelMA where the index of homogeneity of the cell distribution in the multilayered bioink was 4 times that of the latter.


Assuntos
Células Imobilizadas/metabolismo , Hidrogéis/química , Tinta , Animais , Bioimpressão , Células Imobilizadas/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Células NIH 3T3
20.
Nat Commun ; 10(1): 3656, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409793

RESUMO

In this work we model the glomerular filtration barrier, the structure responsible for filtering the blood and preventing the loss of proteins, using human podocytes and glomerular endothelial cells seeded into microfluidic chips. In long-term cultures, cells maintain their morphology, form capillary-like structures and express slit diaphragm proteins. This system recapitulates functions and structure of the glomerulus, including permselectivity. When exposed to sera from patients with anti-podocyte autoantibodies, the chips show albuminuria proportional to patients' proteinuria, phenomenon not observed with sera from healthy controls or individuals with primary podocyte defects. We also show its applicability for renal disease modeling and drug testing. A total of 2000 independent chips were analyzed, supporting high reproducibility and validation of the system for high-throughput screening of therapeutic compounds. The study of the patho-physiology of the glomerulus and identification of therapeutic targets are also feasible using this chip.


Assuntos
Glomérulos Renais/metabolismo , Dispositivos Lab-On-A-Chip , Nefrite Hereditária/metabolismo , Albuminas/metabolismo , Albuminúria/tratamento farmacológico , Albuminúria/metabolismo , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Células Endoteliais/química , Células Endoteliais/metabolismo , Humanos , Glomérulos Renais/química , Glomérulos Renais/efeitos dos fármacos , Masculino , Nefrite Hereditária/tratamento farmacológico , Podócitos/química , Podócitos/metabolismo
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