Failure of Y-27632 to improve the culture of adult human adipose-derived stem cells

Y-27632 is a well-known inhibitor of the Rho-associated coiled kinase (ROCK) and has been shown to significantly improve the culture of a variety of multipotent stem cell types. However, the effects of Y-27632 on the expansion of adult human adipose-derived stem cell (hADSC) cultures remain to be established. Here, we aimed to characterize the effects of Y-27632 on the culture of hADSCs. Adult hADSCs were isolated from subjects submitted to elective plastic surgery procedures and cultivated in vitro under optimized conditions. Our results show that the continuous supplementation of hADSC cultures with Y-27632 led to decreased numbers of cells and decreased global metabolic viability of hADSC cultures when compared with control conditions. This effect appeared to be dependent on the continuous presence of the drug and was shown to be concentration-dependent and significant for 10 muM and 20 muM of Y-27632. Moreover, the Y-27632-induced decrease in hADSC numbers was not linked to a block in global cell proliferation, as cell numbers consistently increased from the moment of plating until passaging. In addition, Y-27632 was not able to increase the number of hADSCs present in culture 24 hours after passaging. Taken together, our results suggest that, in contrast to other stem cell types, Y-27632 supplementation is not a suitable strategy to enhance hADSC culture expansion.We thank Jeffrey M Gimble (Center for Stem Cell Research and Regenerative Medicine, Tulane University and LaCell LLC, New Orleans, LA, USA) for kindly isolating, characterizing, and sharing the cellular lines of hADSCs used in the present study and for critical input on the manuscript. We also would very much like to thank Laurent Roybon (Stem Cell Laboratory for CNS Disease Modeling, Department of Experimental Medical Science, Lund University, Lund, Sweden) for the utilization of Metamorph NX software for automated cell quantification. We are grateful to Miguel Carvalho, Ana Pires, Eduardo Gomes, Fabio Teixeira and Nuno Silva for technical assistance. This work was supported by the Portuguese Foundation for Science and Technology (predoctoral fellowship to NJL [SFRH/BD/33421/2008]; FCT Investigator Program to AJS) and the Luso-American Development Foundation

Impacto da doença crónica na qualidade de vida: Comparação entre indivíduos da população geral e doentes com tumor do aparelho locomoto

O diagnóstico de cancro, a própria doença e os seus tratamentos têm associadas implicações físicas, psicológicas e sociais que acarretam uma redução significativa da qualidade de vida dos doentes. Este estudo exploratório teve como objectivo avaliar o impacto de uma doença crónica específica – a patologia tumoral do aparelho locomotor – na qualidade de vida. Para o efeito, o WHOQOL-100 foi administrado a uma amostra constituída por 30 doentes com tumor do aparelho locomotor e a um grupo de controlo de 30 indivíduos saudáveis. Os resultados obtidos revelam a existência de diferenças estatisticamente significativas entre o grupo clínico e o grupo de controlo, sugerindo o impacto negativo da patologia tumoral do aparelho locomotor na qualidade de vida global (t=2,38; p<0,05), nos domínios físico (t=2,06; p<.0,5) e nível de independência (t=6,51; p<0,001) da qualidade de vida, e alertando para a necessidade de delinear intervenções multidisciplinares que ajudem a pessoa a lidar com esta doença e com os seus tratamentos, promovendo o seu bem-estar

Animal model for chronic massive rotator cuff tear: behavioural and histologic analysis

Purpose Massive rotator cuff tears (MRCT) are usually chronic lesions that present associated degenerative changes of the myotendinous unit that have been implicated in limitations for surgical repair. In order to develop effective therapies, it is important to establish animal models that mimic the hallmarks of the injury itself. Therefore, in the present work, we aimed to (1) optimize a rodent animal model of MRCT that closely reproduces the fatty infiltration of the cuff muscles seen in humans and (2) describe the effects of unilateral or bilateral lesion in terms of histology and behaviour. Methods Massive tear was defined as two rotator cuff tendons—supraspinatus and infraspinatus—section. Twenty-one Wistar rats were randomly assigned to four groups: bilateral lesion (five animals), right-sided unilateral lesion (five animals), left-sided unilateral lesion (five animals) and control (six animals). Behaviour was analyzed with open field and staircase test, 16 weeks after lesion. After that, animals were killed, and the supraspinatus and infraspinatus muscles were processed. Results Histologic analysis revealed adipocytes, fatty infiltration and atrophy in the injured side with a greater consistency of these degenerative changes in the bilateral lesion group. Behaviour analysis revealed a significant functional impairment of the fine motor control of the forepaw analyzed in staircase test where the number of eaten pellets was significantly higher in sham animals (sham = 7 ± 5.0; left unilateral = 2.6 ± 3.0; right unilateral = 0 ± 0; and bilateral = 0 ± 0, p left unilateral = 2 ± 2.1 > right unilateral = 0.8 ± 1.3 > bilateral = 0.8 ± 1.1). Conclusions The present study has been able to establish an animal model that disclosed the hallmarks of MRCT. This can now be used as a valuable, cost-effective, pre-clinical instrument to assist in the development of advanced tissue engineered strategies. Moreover, this animal model overcomes some of the limitations of those that have been reported so far and thus represents a more reliable source for the assessment of future therapeutic strategies with potential clinical relevance.Portuguese Foundation for Science and Technology (FCT)Programa Operacional Regional do Norte (ON.2—O Novo Norte), ao abrigo do Quadro de Referência Estratégico Nacional (QREN), através do Fundo Europeu de Desenvolvimento Regional (FEDER

In vitro evaluation of cell-seeded chitosan films for peripheral nerve tissue engineering

Natural biomaterials have attracted an increasing interest in the field of tissue-engineered nerve grafts, representing a possible alternative to autologous nerve transplantation. With the prospect of developing a novel entubulation strategy for transected nerves with cell-seeded chitosan films, we examined the biocompatibility of such films in vitro. Different types of rat Schwann cells (SCs)-immortalized, neonatal, and adult-as well as rat bone-marrow-derived mesenchymal stromal cells (BMSCs) were analyzed with regard to their cell metabolic activity, proliferation profiles, and cell morphology after different time points of mono-and cocultures on the chitosan films. Overall the results demonstrate a good cytocompatibility of the chitosan substrate. Both cell types were viable on the biomaterial and showed different metabolic activities and proliferation behavior, indicating cell-type-specific cell-biomaterial interaction. Moreover, the cell types also displayed their typical morphology. In cocultures adult SCs used the BMSCs as a feeder layer and no negative interactions between both cell types were detected. Further, the chitosan films allow neurite outgrowth from dissociated sensory neurons, which is additionally supported on film preseeded with SC-BMSC cocultures. The presented chitosan films therefore demonstrate high potential for their use in tissue-engineered nerve grafts.This work was supported by the European Community's Seventh Framework Programme (FP7-HEALTH-2011) under grant agreement No. 278612. This work was also co-funded by Programa Operacional Regional do Norte (ON.2-O Novo Norte), ao abrigo do Quadro de Referencia Estrategico Nacional (QREN), and atraves do Fundo Europeu de Desenvolvimento Regional (FEDER). The authors gratefully acknowledge the delivery of the chitosan raw material by Altakitin S.A., Portugal, and the fabrication of chitosan films by Medovent GmbH, Germany

Bioprocess integration for human mesenchymal stem cells: from up to downstream processing scale-up to cell proteome characterization

Human mesenchymal stem cells (hMSC) are relevant cell-based products for autologous and allogeneic therapies. To deliver the required cell numbers and doses to therapy, scaling up production and purification processes (at least to the liter-scale) while ensuring high purity, viability and maintaining cells’ critical quality attributes (CQA) and functionality is essential [1]. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissues (AT-MSC). BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2 L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50 x 106 cell.L-1.day-1), expansion factors (14 - 16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30 - 50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (\u3e 95%) hMSC could be recovered without compromising cell’s CQA of viability, identity and differentiation potential. “Omic” tools in combination with standard analytical assays allow for a better cell characterization, increasing product and process understanding [2] and are thus fundamental for process development. Thus, alongside the standard quality assays for evaluating hMSC’s CQA, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC’ CQA. Overall, through sensitivity, robustness and throughput, this type of workflow provided the identification of specific signatures of the final product. Therefore, it proves to be essential to understand the cells’ final quality as well as to evaluate the impact of manufacturing at different stages of processing. References: [1] Pattasseril J et al, BioProcess Int. 2013, 3, 38–46. [2] Campbell A et al, Stem Cells Transl. Med. 2015, 4, 1155–1163. The authors acknowledge UniMS – Mass Spectrometry Unit team (ITQB-NOVA/iBET, Oeiras, Portugal), iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), and Fundação para a Ciência e Tecnologia (FCT, Portugal) for funding the project CARDIOSTEM (MITP-TB/ECE/0013/2013), and the grants SFRH/BD/51940/2012 (MIT-Portugal), SFRH/BD/52302/2013, SFRH/BD/52481/2014, SFRH/BPD/86513/201

Engineering scalable manufacturing of high-quality human MSC for cell therapy: From up to downstream processing integration to cell proteome characterization

Human mesenchymal stem cells (hMSC) are relevant cell therapy products for autologous and allogeneic therapies. To deliver the required cell numbers and doses to therapy, scaling up production and purification processes (at least to the liter-scale) while ensuring high purity, viability and maintaining cells’ critical quality attributes (CQA) and functionality is essential. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting, volume reduction and washing unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissues (AT-MSC). Single-use technologies were adopted at different steps of the manufacturing workflow to support process integration and scale-up. BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2 L single-use stirred tank bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50 x 106 cell.L-1.day-1), expansion factors (14 - 16 fold) and cell recovery yields (\u3e80%). For the volume reduction and washing steps, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30 - 50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC performed better allowing recovering 18% more cells after a volume reduction factor of 50 without compromising cell’s CQA of viability, identity and differentiation potential. “Omic” tools in combination with standard analytical assays allow for a better cell characterization, increasing product and process understanding and are thus fundamental for process development. Thus, alongside the standard quality assays for evaluating hMSC’s CQA, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC’ CQA. Overall, through sensitivity, robustness and throughput, this type of workflow provided the identification of specific signatures of the final product. Therefore, it proves to be essential to understand the cells’ final quality as well as to evaluate the impact of manufacturing at different stages of processing. The authors acknowledge UniMS – Mass Spectrometry Unit team (ITQB-NOVA/iBET, Oeiras, Portugal), iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344), and Fundação para a Ciência e Tecnologia (FCT, Portugal) for funding the project CARDIOSTEM (MITP-TB/ECE/0013/2013), and the grants SFRH/BD/51940/2012 (MIT-Portugal), SFRH/BD/52302/2013, SFRH/BD/52481/2014, SFRH/BPD/86513/2012

Online holistic program to foster health amongst students: a pilot study in a Portuguese university during COVID-19 pandemic

During the COVID-19 pandemic, several preventive mental health interventions took place to increase the psychological well-being of university students due to the high levels of stress, anxiety and negative emotions experienced in that period. This context reinforced the role of universities in supporting students and preventing the mental health risk factors they faced. In this context a multidisciplinary team of professionals (psychologists, nurses, nutritionists, and artists) in the Portuguese Catholic University, gathered efforts and developed an holistic intervention program for university students based on a mind and body integrated approach. This program of 8 online sessions aims to improve students’ resilience to the psychosocial consequences of COVID-19 pandemic and promote their wellbeing. The twenty university students that participated in this pilot study reported that this intervention improved their emotional self-awareness, their ability to apply self-care strategies, as well as they believed it promoted healthier lifestyle changes. These findings suggest that this program consists in an innovative approach with the potential to promote the psychological health and well-being of university students in adverse circumstances.info:eu-repo/semantics/publishedVersio

Combinatorial activity of flavonoids with antibiotics against drug resistant Staphylococcus aureus

The use of resistance-modifying agents is a potential strategy that is used to prolong the effective life of antibiotics in the face of increasing antibiotic resistance. Since certain flavonoids are potent bacterial efflux pump inhibitors, we assessed morin, rutin, quercetin, hesperidin, and (+)-catechin for their combined activity with the antibiotics ciprofloxacin, tetracycline, erythromycin, oxacillin, and ampicillin against drug-resistant strains of Staphylococcus aureus, including methicillin-resistant S. aureus. Four established methods were used to determine the combined efficacy of each combination: microdilution checkerboard assays, time-kill determinations, the Etest, and dual disc-diffusion methods. The cytotoxicity of the flavonoids was additionally evaluated in a mouse fibroblast cell line. Quercetin and its isomer morin decreased by 3- to 16-fold the minimal inhibitory concentration of ciprofloxacin, tetracycline, and erythromycin against some S. aureus strains. Rutin, hesperidin, and (+)-catechin did not promote any potentiation of antibiotics. Despite the potential cytotoxicity of these phytochemicals at a high concentration (fibroblast IC50 of 41.8 and 67.5mg/L, respectively), quercetin is commonly used as a supplement for several therapeutic purposes. All the methods, with exception of the time-kill assay, presented a high degree of congruence without any apparent strain specificity.This work was supported by Operational Program for Competitiveness Factors—COMPETE, FCT/MEC (PIDDAC), and FEDER through Projects Bioresist—PTDC/EBB-EBI/ 105085/2008; Phytodisinfectants—PTDC/DTP-SAP/1078/ 2012 (COMPETE: FCOMP-01-0124-FEDER-028765) and the PhD grants awarded to Ana Abreu (SFRH/BD/84393/ 2012) and Anabela Borges (SFRH/BD/63398/2009). The authors are very grateful to Professor Simon Gibbons (De- partment of Pharmaceutical and Biological Chemistry, The School of Pharmacy, UCL School of Pharmacy, London) for providing the bacterial strains.info:eu-repo/semantics/publishedVersio

Diseño y desarrollo de un modelo de portafolio para la evaluación de competencias en el Grado de Estudios Portugueses y Brasileños

Memoria ID-0205. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2014-2015

Proyecto Escritiva

Memoria ID-0132. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2015-2016