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1.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
2.
Sci Rep ; 11(1): 5118, 2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664329

RESUMO

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of "pieces" of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ-/- (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 µm achieving superior microtissue vascularisation relative to mesh with 36×36 µm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.

3.
PLoS One ; 13(8): e0202079, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30148832

RESUMO

A mathematical model was developed for mesenchymal stromal cell (MSC) growth in a packed bed bioreactor that improves oxygen availability by allowing oxygen diffusion through a gas-permeable wall. The governing equations for oxygen, glucose and lactate, the inhibitory waste product, were developed assuming Michaelis-Menten kinetics, together with an equation for the medium flow based on Darcy's Law. The conservation law for the cells includes the effects of inhibition as the cells reach confluence, nutrient and waste product concentrations, and the assumption that the cells can migrate on the scaffold. The equations were solved using the finite element package, COMSOL. Previous experimental results collected using a packed bed bioreactor with gas permeable walls to expand MSCs produced a lower cell yield than was obtained using a traditional cell culture flask. This mathematical model suggests that the main contributors to the observed low cell yield were a non-uniform initial cell seeding profile and a potential lag phase as cells recovered from the initial seeding procedure. Lactate build-up was predicted to have only a small effect at lower flow rates. Thus, the most important parameters to optimise cell expansion in the proliferation of MSCs in a bioreactor with gas permeable wall are the initial cell seeding protocol and the handling of the cells during the seeding process. The mathematical model was then used to identify and characterise potential enhancements to the bioreactor design, including incorporating a central gas permeable capillary to further enhance oxygen availability to the cells. Finally, to evaluate the issues and limitations that might be encountered scale-up of the bioreactor, the mathematical model was used to investigate modifications to the bioreactor design geometry and packing density.


Assuntos
Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Algoritmos , Proliferação de Células , Consumo de Oxigênio , Estresse Fisiológico
4.
Future Microbiol ; 12: 817-829, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28593794

RESUMO

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection worldwide and the leading cause of preventable blindness. Reports have emerged of treatment failure, suggesting a need to develop new antibiotics to battle Chlamydia infection. One possible candidate for a new treatment is the protease inhibitor JO146, which is an effective anti-Chlamydia agent that targets the CtHtrA protein. CtHtrA is a lynchpin on the chlamydial cell surface due to its essential and multifunctional roles in the bacteria's stress response, replicative phase of development, virulence and outer-membrane protein assembly. This review summarizes the current understanding of CtHtrA function and presents a mechanistic model that highlights CtHtrA as an effective target for anti-Chlamydia drug development.


Assuntos
Infecções por Chlamydia/tratamento farmacológico , Chlamydia trachomatis/enzimologia , Serina Endopeptidases/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidade , Dipeptídeos/uso terapêutico , Humanos , Modelos Biológicos , Modelos Moleculares , Organofosfonatos/uso terapêutico , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Serina Endopeptidases/química , Serina Endopeptidases/genética , Fatores de Virulência/química , Fatores de Virulência/metabolismo
5.
Tissue Eng Part C Methods ; 23(4): 200-218, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28406754

RESUMO

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25-400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38- cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38- cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38- cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.


Assuntos
Proliferação de Células , Dimetilpolisiloxanos/química , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Esferoides Celulares/metabolismo , Animais , Técnicas de Cocultura/métodos , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos NOD , Camundongos SCID , Esferoides Celulares/citologia
6.
J Vis Exp ; (112)2016 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-27340821

RESUMO

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.


Assuntos
Células-Tronco Mesenquimais , Placenta , Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Feminino , Feto , Humanos , Gravidez
7.
Sci Rep ; 6: 23886, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-27065210

RESUMO

Direct bone marrow (BM) injection has been proposed as a strategy to bypass homing inefficiencies associated with intravenous (IV) hematopoietic stem cell (HSC) transplantation. Despite physical delivery into the BM cavity, many donor cells are rapidly redistributed by vascular perfusion, perhaps compromising efficacy. Anchoring donor cells to 3-dimensional (3D) multicellular spheroids, formed from mesenchymal stem/stromal cells (MSC) might improve direct BM transplantation. To test this hypothesis, relevant combinations of human umbilical cord blood-derived CD34(+) cells and BM-derived MSC were transplanted into NOD/SCID gamma (NSG) mice using either IV or intrafemoral (IF) routes. IF transplantation resulted in higher human CD45(+) and CD34(+) cell engraftment within injected femurs relative to distal femurs regardless of cell combination, but did not improve overall CD45(+) engraftment at 8 weeks. Analysis within individual mice revealed that despite engraftment reaching near saturation within the injected femur, engraftment at distal hematopoietic sites including peripheral blood, spleen and non-injected femur, could be poor. Our data suggest that the retention of human HSC within the BM following direct BM injection enhances local chimerism at the expense of systemic chimerism in this xenogeneic model.


Assuntos
Medula Óssea/fisiologia , Quimerismo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Esferoides Celulares/fisiologia , Animais , Antígenos CD34/análise , Humanos , Antígenos Comuns de Leucócito/análise , Camundongos Endogâmicos NOD , Camundongos SCID , Resultado do Tratamento
8.
Lab Chip ; 16(8): 1473-83, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-27008339

RESUMO

Polydimethylsiloxane (PDMS) is the most commonly used material in the manufacture of customized cell culture devices. While there is concern that uncured PDMS oligomers may leach into culture medium and/or hydrophobic molecules may be absorbed into PDMS structures, there is no consensus on how or if PDMS influences cell behaviour. We observed that human umbilical cord blood (CB)-derived CD34(+) cells expanded in standard culture medium on PDMS exhibit reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. Through a series of experiments we demonstrated that ATRA-mediated CD38 expression was attenuated when cultures were maintained on PDMS. Medium pre-incubated on PDMS for extended durations resulted in a time-dependant reduction of ATRA in the medium and increasingly attenuated CD38 expression. This indicated a time-dependent absorption of ATRA into the PDMS. To better understand how PDMS might generally influence cell behaviour, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34(+) haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithelial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a). IPA predicted that the most likely common upstream regulator of perturbed pathways was ATRA. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced expression of CD38 on the cell surface of CB-derived CD34(+) cells.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Absorção Fisico-Química , Dimetilpolisiloxanos/farmacologia , Retinoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Tretinoína/metabolismo , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Meios de Cultura/química , Humanos , Poliestirenos/farmacologia , Tretinoína/farmacologia
9.
Nanomedicine ; 12(3): 633-641, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26656628

RESUMO

UNLABELLED: Isolating, purifying, and identifying proteins in complex biological matrices are often difficult, time consuming, and unreliable. Herein we describe a rapid screening technique for proteins in biological matrices that combines selective protein isolation with direct surface enhanced Raman spectroscopy (SERS) detection. Magnetic core gold nanoparticles were synthesized, characterized, and subsequently functionalized with recombinant human erythropoietin (rHuEPO)-specific antibody. The functionalized nanoparticles were used to capture rHuEPO from horse blood plasma within 15 min. The selective binding between the protein and the functionalized nanoparticles was monitored by SERS. The purified protein was then released from the nanoparticles' surface and directly spectroscopically identified on a commercial nanopillar SERS substrate. ELISA independently confirmed the SERS identification and quantified the released rHuEPO. Finally, the direct SERS detection of the extracted protein was successfully demonstrated for in-field screening by a handheld Raman spectrometer within 1 min sample measurement time. FROM THE CLINICAL EDITOR: The rapid detection of recombinant human erythropoietin (rHuEPO) is important in competitive sports to screen for doping offences. In this article, the authors reported their technique of direct surface enhanced Raman spectroscopy (SERS) detection using magnetic core gold nanoparticles functionalized with recombinant human erythropoietin-specific antibody. The findings should open a new way for future detection of other proteins.


Assuntos
Eritropoetina/sangue , Eritropoetina/isolamento & purificação , Ouro/química , Nanopartículas de Magnetita/química , Imãs/química , Análise Espectral Raman/métodos , Animais , Anticorpos Imobilizados/química , Cavalos , Humanos , Detecção do Abuso de Substâncias/métodos
10.
PLoS One ; 10(12): e0144941, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26660475

RESUMO

Large numbers of Mesenchymal stem/stromal cells (MSCs) are required for clinical relevant doses to treat a number of diseases. To economically manufacture these MSCs, an automated bioreactor system will be required. Herein we describe the development of a scalable closed-system, packed bed bioreactor suitable for large-scale MSCs expansion. The packed bed was formed from fused polystyrene pellets that were air plasma treated to endow them with a surface chemistry similar to traditional tissue culture plastic. The packed bed was encased within a gas permeable shell to decouple the medium nutrient supply and gas exchange. This enabled a significant reduction in medium flow rates, thus reducing shear and even facilitating single pass medium exchange. The system was optimised in a small-scale bioreactor format (160 cm2) with murine-derived green fluorescent protein-expressing MSCs, and then scaled-up to a 2800 cm2 format. We demonstrated that placental derived MSCs could be isolated directly within the bioreactor and subsequently expanded. Our results demonstrate that the closed system large-scale packed bed bioreactor is an effective and scalable tool for large-scale isolation and expansion of MSCs.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Células-Tronco Mesenquimais/citologia , Placenta/citologia , Adipogenia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Condrogênese , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteogênese , Oxigênio/química , Oxigênio/metabolismo , Poliestirenos/química , Gravidez
11.
PLoS One ; 10(11): e0142473, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26566128

RESUMO

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009-0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.


Assuntos
Vírus da Dengue/genética , Dengue/virologia , Variação Genética , Filogenia , Genoma Viral , Humanos , RNA Viral/genética , RNA Viral/isolamento & purificação , Análise de Sequência de RNA , Proteínas do Envelope Viral/genética
12.
BMC Microbiol ; 15: 194, 2015 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-26424482

RESUMO

BACKGROUND: Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism's intracellular developmental cycle. HtrA is a critical stress response serine protease and chaperone for many bacteria and in C. trachomatis has been previously shown to be important for heat stress and the replicative phase of development using a chemical inhibitor of the CtHtrA activity. In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized. METHODS: SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis. The C. trachomatis strains containing the SNVs with marked reductions in proteolysis were investigated in cell culture to identify phenotypes that could be linked to CtHtrA function. RESULTS: The strain harboring the SNV with the most marked impact on proteolysis (cthtrA P370L) was detected to have a significant reduction in the production of infectious elementary bodies. CONCLUSIONS: This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.


Assuntos
Substituição de Aminoácidos , Chlamydia trachomatis/metabolismo , Corpos de Inclusão/microbiologia , Proteínas Mutantes/metabolismo , Serina Proteases/metabolismo , Fatores de Virulência/metabolismo , Linhagem Celular , Chlamydia trachomatis/genética , Análise Mutacional de DNA , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/genética , Proteólise , Serina Proteases/genética , Fatores de Virulência/genética
13.
Cell Tissue Res ; 361(3): 755-68, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25924853

RESUMO

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.


Assuntos
Osso e Ossos/citologia , Cartilagem/citologia , Diferenciação Celular/fisiologia , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual , Técnicas de Cultura de Células , Células Cultivadas , Condrogênese/fisiologia , Humanos , Osteogênese/fisiologia
14.
PLoS One ; 10(5): e0122250, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26020956

RESUMO

We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100-500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells alone, however when replicating matrix-rich tissues such as cartilage it would be ideal if matrix or biomaterials supplements could be incorporated directly into the micropellet during the manufacturing process. Herein we describe a method to efficiently incorporate donor cartilage matrix into tissue engineered cartilage micropellets. We lyophilized bovine cartilage matrix, and then shattered it into microscopic pieces having average dimensions < 10 µm diameter; we termed this microscopic donor matrix "cartilage dust (CD)". Using a microwell platform, we show that ~0.83 µg CD can be rapidly and efficiently incorporated into single multicellular aggregates formed from 180 bone marrow mesenchymal stem/stromal cells (MSC) each. The microwell platform enabled the rapid manufacture of thousands of replica composite micropellets, with each micropellet having a material/CD core and a cellular surface. This micropellet organization enabled the rapid bulking up of the micropellet core matrix content, and left an adhesive cellular outer surface. This morphological organization enabled the ready assembly of the composite micropellets into macroscopic tissues. Generically, this is a versatile method that enables the rapid and uniform integration of biomaterials into multicellular micropellets that can then be used as tissue building blocks. In this study, the addition of CD resulted in an approximate 8-fold volume increase in the micropellets, with the donor matrix functioning to contribute to an increase in total cartilage matrix content. Composite micropellets were readily assembled into macroscopic cartilage tissues; the incorporation of CD enhanced tissue size and matrix content, but did not enhance chondrogenic gene expression.


Assuntos
Materiais Biocompatíveis/metabolismo , Cartilagem Articular/citologia , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Animais , Cartilagem Articular/crescimento & desenvolvimento , Bovinos , Técnicas de Cultura de Células , Células Cultivadas , Condrogênese , Humanos , Tamanho da Partícula
15.
Biomaterials ; 62: 1-12, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26010218

RESUMO

Microwell platforms are frequently described for the efficient and uniform manufacture of 3-dimensional (3D) multicellular microtissues. Multiple partial or complete medium exchanges can displace microtissues from discrete microwells, and this can result in either the loss of microtissues from culture, or microtissue amalgamation when displaced microtissues fall into common microwells. Herein we describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells; the microwell-mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells. To demonstrate the utility of this platform, we used the microwell-mesh to manufacture hundreds of cartilage microtissues, each formed from 5 × 10(3) bone marrow-derived mesenchymal stem/stromal cells (MSC). The microwell-mesh enabled reliable microtissue retention over 21-day cultures that included multiple full medium exchanges. Cartilage-like matrix formation was more rapid and homogeneous in microtissues than in conventional large diameter control cartilage pellets formed from 2 × 10(5) MSC each. The microwell-mesh platform offers an elegant mechanism to retain microtissues in microwells, and we believe that this improvement will make this platform useful in 3D culture protocols that require multiple medium exchanges, such as those that mimic specific developmental processes or complex sequential drug exposures.


Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Tecidos Suporte , Diferenciação Celular/fisiologia , Separação Celular/instrumentação , Células Cultivadas , Condrócitos/fisiologia , Condrogênese/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Células-Tronco Mesenquimais/fisiologia , Miniaturização , Engenharia Tecidual/instrumentação , Ultrafiltração/instrumentação
16.
PLoS One ; 9(9): e107264, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25222471

RESUMO

Genetically diverse RNA viruses like dengue viruses (DENVs) segregate into multiple, genetically distinct, lineages that temporally arise and disappear on a regular basis. Lineage turnover may occur through multiple processes such as, stochastic or due to variations in fitness. To determine the variation of fitness, we measured the distribution of fitness within DENV populations and correlated it with lineage extinction and replacement. The fitness of most members within a population proved lower than the aggregate fitness of populations from which they were drawn, but lineage replacement events were not associated with changes in the distribution of fitness. These data provide insights into variations in fitness of DENV populations, extending our understanding of the complexity between members of individual populations.


Assuntos
Vírus da Dengue/genética , Vírus da Dengue/classificação , Evolução Molecular , Variação Genética/genética , Filogenia , RNA Viral/genética
17.
Talanta ; 130: 20-5, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25159374

RESUMO

We present a proof of concept for a novel nanosensor for the detection of ultra-trace amounts of bio-active molecules in complex matrices. The nanosensor is comprised of gold nanoparticles with an ultra-thin silica shell and antibody surface attachment, which allows for the immobilization and direct detection of bio-active molecules by surface enhanced Raman spectroscopy (SERS) without requiring a Raman label. The ultra-thin passive layer (~1.3 nm thickness) prevents competing molecules from binding non-selectively to the gold surface without compromising the signal enhancement. The antibodies attached on the surface of the nanoparticles selectively bind to the target molecule with high affinity. The interaction between the nanosensor and the target analyte result in conformational rearrangements of the antibody binding sites, leading to significant changes in the surface enhanced Raman spectra of the nanoparticles when compared to the spectra of the un-reacted nanoparticles. Nanosensors of this design targeting the bio-active compounds erythropoietin and caffeine were able to detect ultra-trace amounts the analyte to the lower quantification limits of 3.5 × 10(-13)M and 1 × 10(-9)M, respectively.


Assuntos
Cafeína/análise , Eritropoetina/análise , Ouro/química , Nanopartículas Metálicas/química , Dióxido de Silício/química , Análise Espectral Raman/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Técnicas Biossensoriais , Ouro/metabolismo , Humanos , Camundongos , Dióxido de Silício/metabolismo , Propriedades de Superfície
18.
Trends Biochem Sci ; 39(4): 151-3, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24703407

RESUMO

Multimedia communication capabilities are rapidly expanding, and visual information is easily shared electronically, yet funding bodies still rely on paper grant proposal submissions. Incorporating modern technologies will streamline the granting process by increasing the fidelity of grant communication, improving the efficiency of review, and reducing the cost of the process.


Assuntos
Multimídia , Revisão da Pesquisa por Pares/métodos , Apoio à Pesquisa como Assunto/métodos , Papel
19.
Trends Mol Med ; 20(3): 137-42, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24485902

RESUMO

Type 2 diabetes is the epidemic of our generation, and diabetic foot ulcers (DFUs) are a major complication. Although DFU formation itself can indicate disease progression, the failure to effectively treat ulcers contributes further to a decay in patient quality of life and increased mortality. Herein we discuss the development of next-generation DFU therapies including: (i) topical growth factors, (ii) scaffolds, and (iii) cellular therapies. Individually these therapies have yielded measurable but modest improvements in DFU repair. Because DFUs arise as a result of multiple biochemical deficiencies, a singular treatment modality is unlikely to be effective. Next-generation DFU technologies must be combined to address effectively the complex underlying pathology and enable reliable DFU repair.


Assuntos
Diabetes Mellitus Tipo 2/complicações , Pé Diabético/terapia , Cicatrização , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Células-Tronco Mesenquimais/citologia , Tecidos Suporte
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