RESUMO
Indirect fundoscopy is challenging for novice learners, as patients are often intolerant of the procedure, impeding development of proficiency. To address this, we developed a canine ocular simulator that we hypothesized would improve student learning compared to live dogs. Six board-certified veterinary ophthalmologists and 19 second-year veterinary students (novices) performed an indirect fundic examination on the model and live dog. Prior to assessment, novices were introduced to the skill with a standardized teaching protocol and practiced (without feedback) with either the model (n = 10) or live dog (n = 9) for 30 minutes. All participants evaluated realism and usefulness of the model using a Likert-type scale. Performance on the live dog and model was evaluated in all participants using time to completion of task, performance of fundic examination using a checklist and global score, identification of objects in the fundus of the model, and evaluation of time spent looking at the fundus of the model using eye tracking. Novices (trained on simulator or live dogs) were compared in fundic examination performance on the live dog and identification of shapes in the model. In general, experts performed the fundic examination faster (p ≤ .0003) and more proficiently than the novices, although there were no differences in eye tracking behavior between groups (p ≥ .06). No differences were detected between training on simulator versus live dog in development of fundoscopy skills in novices (p ≥ .20). These findings suggest that this canine model may be an effective tool to train students to perform fundoscopy.
Assuntos
Educação em Veterinária , Animais , Competência Clínica , Simulação por Computador , Cães , Retroalimentação , Humanos , EstudantesRESUMO
AIMS/HYPOTHESIS: There are potential advantages to the low-temperature (-196 °C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years. METHODS: Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes. RESULTS: The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation. CONCLUSIONS/INTERPRETATION: We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.
Assuntos
Criopreservação , Ilhotas Pancreáticas/fisiologia , Bancos de Tecidos , Adulto , Animais , Diabetes Mellitus Experimental/terapia , Exocitose/fisiologia , Feminino , Proteínas de Homeodomínio/genética , Humanos , Insulina/sangue , Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Canais Iônicos/metabolismo , Transplante das Ilhotas Pancreáticas , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Transcriptoma/genéticaRESUMO
We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.
Assuntos
Técnicas de Cultura Celular por Lotes , Endoderma/citologia , Células-Tronco Pluripotentes/citologia , Engenharia Tecidual/métodos , Animais , Antígenos de Diferenciação/análise , Agregação Celular , Ciclo Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular/citologia , Linhagem Celular/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultura , Fibroblastos/metabolismo , Hepatócitos/citologia , Humanos , Camundongos , Pâncreas/citologia , SuspensõesRESUMO
We report an ultra-rapid prototyping technique for forming microchannel networks for lab-on-a-chip applications, called masters on-demand. Channels are produced by replica molding on masters formed by laser printing on flexible copper printed circuit board (PCB) substrates. Masters of various designs and dimensions can be individually or mass produced in less than 10 minutes. Using this technique, we have fabricated channels as narrow as 100 microm with heights ranging between 9 microm and 70 microm. Multi-depth channel fabrication is also reported, using a two-step printing process. The functionality of devices formed in this manner is verified by performing in-channel electrophoretic separations and culture and analysis of primary mammalian cells.
Assuntos
Microfluídica/instrumentação , Microfluídica/métodos , Microfluídica/economiaRESUMO
BACKGROUND: Human cells appear exquisitely sensitive to the levels of hTERT expression, the telomerase reverse transcriptase. In primary cells that do not express hTERT, telomeres erode with each successive cell division, leading to the eventual loss of telomere DNA, an induction of a telomere DNA damage response, and the onset of cellular senescence or crisis. In some instances, an average of less than one appropriately spliced hTERT transcript per cell appears sufficient to restore telomerase activity and telomere maintenance, and overcome finite replicative capacity. RESULTS: To underscore this sensitivity, we showed that a widely used system of transcriptional induction involving ecdysone (muristerone) led to sufficient expression of hTERT to immortalize human fibroblasts, even in the absence of induction. To permit tightly regulated expression of hTERT, or any other gene of interest, we developed a method of transcriptional control using an invertible expression cassette flanked by antiparallel loxP recombination sites. When introduced into human fibroblasts with the hTERT cDNA positioned in the opposite orientation relative to a constitutively active promoter, no telomerase activity was detected, and the cell population retained a mortal phenotype. Upon inversion of the hTERT cDNA to a transcriptionally competent orientation via the action of Cre recombinase, cells acquired telomerase activity, telomere DNA was replenished, and the population was immortalized. Further, using expression of a fluorescent protein marker, we demonstrated the ability to repeatedly invert specific transcripts between an active and inactive state in an otherwise isogenic cell background. CONCLUSION: This binary expression system thus provides a useful genetic means to strictly regulate the expression of a given gene, or to control the expression of at least two different genes in a mutually exclusive manner.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Integrases/genética , Engenharia de Proteínas/métodos , Telomerase/genética , Transfecção/métodos , Proteínas Virais/genética , Linhagem Celular , Inversão Cromossômica/genética , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Proteínas Recombinantes/metabolismo , Telomerase/metabolismo , Ativação Transcricional/fisiologiaRESUMO
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 , Alicerces Teciduais , 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/instrumentaçãoRESUMO
The formation of cells into more physiologically relevant three-dimensional multicellular aggregates is an important technique for the differentiation and manipulation of stem cells and their progeny. As industrial and clinical applications for these cells increase, it will be necessary to execute this procedure in a readily scalable format. We present here a method employing microwells to generate large numbers of human pluripotent stem cell aggregates and control their subsequent differentiation towards a cardiac fate.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Microtecnologia/métodos , Miocárdio/citologia , Células-Tronco Pluripotentes/citologia , Agregação Celular , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Miocárdio/metabolismo , Fatores de Tempo , Troponina T/metabolismoRESUMO
Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes.
Assuntos
Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Esferoides Celulares/metabolismo , Animais , Agregação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/ultraestrutura , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Fenótipo , Células-Tronco Pluripotentes/metabolismoRESUMO
BACKGROUND: Human embryonic stem cells (hESC) should enable novel insights into early human development and provide a renewable source of cells for regenerative medicine. However, because the three-dimensional hESC aggregates [embryoid bodies (hEB)] typically employed to reveal hESC developmental potential are heterogeneous and exhibit disorganized differentiation, progress in hESC technology development has been hindered. METHODOLOGY/PRINCIPAL FINDINGS: Using a centrifugal forced-aggregation strategy in combination with a novel centrifugal-extraction approach as a foundation, we demonstrated that hESC input composition and inductive environment could be manipulated to form large numbers of well-defined aggregates exhibiting multi-lineage differentiation and substantially improved self-organization from single-cell suspensions. These aggregates exhibited coordinated bi-domain structures including contiguous regions of extraembryonic endoderm- and epiblast-like tissue. A silicon wafer-based microfabrication technology was used to generate surfaces that permit the production of hundreds to thousands of hEB per cm(2). CONCLUSIONS/SIGNIFICANCE: The mechanisms of early human embryogenesis are poorly understood. We report an ultra high throughput (UHTP) approach for generating spatially and temporally synchronised hEB. Aggregates generated in this manner exhibited aspects of peri-implantation tissue-level morphogenesis. These results should advance fundamental studies into early human developmental processes, enable high-throughput screening strategies to identify conditions that specify hESC-derived cells and tissues, and accelerate the pre-clinical evaluation of hESC-derived cells.