Microfluidic tools and high-content imaging for cell therapy bioprocessing

Culture parameters including type of matrix, feeding regimes and media formulations have to be fine-tuned in order to obtain the desired cell quality attributes that will be the basis of large scale bioprocess development. Costly and time consuming screenings, usually performed in multi-well plates, are undertaken to obtain preliminary results. In contrast, microfluidics offers the possibility to perform studies of different parameters in an integrated manner, with high-throughput analysis, lower consumption of reagents and reduction in time. This approach has the possibility to unveil valuable information in early stages of process development that can reduce the risks associated with scale-up. In one working example, a range of process parameters were screened for the development of a microcarrier-based cell culture of a commercial cell line. Two different media flow rates and four different microcarrier substrates were assessed for their capacity to sustain cell growth and known markers of different stages of cell differentiation. It was possible to define combinations of parameters that yield very distinct outcomes in terms of cell number and identity. In a second working example, modular production of bone-like tissue using mesenchymal stem cells, different implantable biomaterial scaffolds and different culture media feeds was assessed. In situ analysis identified optimal combinations of parameters that maintained multipotency in Mesenchymal Stem Cells (MSCs) and also combinations that promote MSCs maturation. Scaling up to 125 mL cultures enabled the production of large clusters of bone-like material

Xeno-free expansion of late-adherent human olfactory mucosa cells: Towards an allogeneic therapy for neural regeneration

Human olfactory mucosa cells (hOMCs) are anchorage dependent cells that have potential for treatment of spinal cord injury. However, current hOMC therapies relied on autologous transplantation and it is not feasible to prepare and characterize sufficient quantities of cells (in the order of 107 - 108 cells) within a timeframe to treat acute injury. Thus an allogeneic (universal) off-the-shelf approach would offer an alternative for this case. We incorporated the regulator-approved c-MycERTAM gene (ReNeuron) into primary late-adherent hOMCs to extend their ex vivo proliferation in the presence of the synthetic drug 4-hydroxytamoxifen (4-OHT). Polyclonal populations of hOMCs were generated and characterized, with an ultimate goal of developing a potential cell therapy product for application in spinal cord injury. Due to the lack of scalability, the availability of labour intensive manual processes and fetal bovine serum (FBS) supplementation, we aimed to develop a xeno-free process for the expansion of a these cells. An initial issue for the manufacture of hOMCs is that key bioprocess parameters have not been established. In this work, we performed cell growth characterization to provide information about their growth i.e. effect of initial cell seeding density, long-term culture, and metabolite profiles to ultimately define the expansion process window. Although widely used, FBS is a finite resource that raises concerns about the presence of adventitious agents. Alternative human-derived (xeno-free) or chemically-defined (serum-free) supplements were assessed for their ability to sustain cell growth. From these studies, human platelet lysate supplementation at 2-5% (% v/v) was found to be a viable xeno-free option to sustain growth of hOMCs with no adverse effects on their phenotype. Finally, we sought to replace the current manually intensive monolayer expansion process with a more flexible and scalable platform such as suspension culture on animal-free microcarriers. Successful expansion of c- MycERTAM-derived late-adherent hOMCs on plastic microcarriers at 80-mL scale was achieved to establish a suspension culture expansion platform for the translation of a potential candidate cell therapy for neural regeneration. In summary, we show a systematic approach to address main hOMC bioprocessing challenges for an allogeneic therapy to treat patients suffering from spinal cord injury

Microcarrier expansion of c-MycERTAM - modified human olfactory mucosa cells for neural regeneration

Human olfactory mucosa cells (hOMCs) have potential as a regenerative therapy for spinal cord injury. In our earlier work, we derived PA5 cells, a polyclonal population that retains functional attributes of primary human OMCs. Microcarrier suspension culture is an alternative to planar two-dimensinal culture to produce cells in quantities that can meet the needs of clinical development. This study aimed to screen the effects of 10 microcarriers on PA5 hOMCs yield and phenotype. Studies performed in well plates led to a 2.9-fold higher cell yield on plastic compared to plastic plus microcarriers with upregulation of neural markers β-III tubulin and nestin for both conditions. Microcarrier suspension culture resulted in concentrations of 1.4 × 10 5 cells/ml and 4.9 × 10 4 cells/ml for plastic and plastic plus, respectively, after 7 days. p75 NTR transcript was significantly upregulated for PA5 hOMCs grown on Plastic Plus compared to Plastic. Furthermore, coculture of PA5 hOMCs grown on Plastic Plus with a neuronal cell line (NG108-15) led to increased neurite outgrowth. This study shows successful expansion of PA5 cells using suspension culture on microcarriers, and it reveals competing effects of microcarriers on cell expansion versus functional attributes, showing that designing scalable bioprocesses should not only be driven by cell yields

Generation of c-MycERTAM-transduced human late-adherent olfactory mucosa cells for potential regenerative applications

Human olfactory mucosa cells (hOMCs) have been transplanted to the damaged spinal cord both pre-clinically and clinically. To date mainly autologous cells have been tested. However, inter-patient variability in cell recovery and quality, and the fact that the neuroprotective olfactory ensheathing cell (OEC) subset is difficult to isolate, means an allogeneic hOMC therapy would be an attractive “off-the-shelf” alternative. The aim of this study was to generate a candidate cell line from late-adherent hOMCs, thought to contain the OEC subset. Primary late-adherent hOMCs were transduced with a c-MycERTAM gene that enables cell proliferation in the presence of 4-hydroxytamoxifen (4-OHT). Two c-MycERTAM-derived polyclonal populations, PA5 and PA7, were generated and expanded. PA5 cells had a normal human karyotype (46, XY) and exhibited faster growth kinetics than PA7, and were therefore selected for further characterisation. PA5 hOMCs express glial markers (p75NTR, S100ß, GFAP and oligodendrocyte marker O4), neuronal markers (nestin and ß-III-tubulin) and fibroblast-associated markers (CD90/Thy1 and fibronectin). Co-culture of PA5 cells with a neuronal cell line (NG108-15) and with primary dorsal root ganglion (DRG) neurons resulted in significant neurite outgrowth after 5 days. Therefore, c-MycERTAM-derived PA5 hOMCs have potential as a regenerative therapy for neural cells

Terapias voltadas para o tratamento do transtorno dissociativo de identidade

O transtorno dissociativo de identidade compreende uma condição psicológica complexa provavelmente causada por inúmeros fatores, envolvendo trauma grave na primeira infância, como abuso sexual, físico ou emocional repetitivo e extremo e repetitivo. Este estudo teve como objetivo identificar as terapias voltadas para o tratamento do transtorno dissociativo de identidade. Para isso, foi realizada uma revisão integrativa de literatura, selecionando fontes a partir das bases de dados Medline e Lilacs. A partir da análise qualitativa de dados, concluiu-se que há vários tipos de terapias para o tratamento de pessoas transtorno dissociativo de identidade, devendo essas serem aplicadas conforme cada realidade. Nos estudos, foram identificados os modelos de tratamento psicanalítico relacional, fásico, psicoativo e psicotraumatológico. Em todos esses, foram registrados resultados satisfatórios, tais como a diminuição na dissociação e o aumento do funcionamento adaptativo do paciente, revelando a possibilidade de desconstruir crenças solidamente cultivadas e trazendo esperança aos pacientes no sentido de amenizar ou superar esse transtorno e garantir uma boa interação social

Alterações musculares e esqueléticas cervicais em mulheres disfônicas

Termo clínico, a disfonia envolve a todas as transformações e dificuldades durante a emissão vocal, as quais resultam no impedimento da produção normal da voz. Pacientes como esse problema, podem apresentar desequilíbrio da musculatura crâniocervical e laríngea e lesão orgânica subjacente. A disfonia resulta em modificações fonatórias, limitando atividades diárias relacionadas ao uso da voz, impactando na vida social e na qualidade de vida do indivíduo. Este estudo teve como objetivo analisar alterações musculares e esqueléticas cervicais em mulheres com disfonia, conforme identificado na literatura científica sobre o tema. Para isso, realizou-se uma revisão integrativa de literatura, selecionando estudos nas bases de dados Literatura Latino-americana e do Caribe em Ciências da Saúde (Lilacs) e Medical Literature Analysis and Retrieval System Online (Medline). A partir da análise qualitativa dos resultados, concluiu-se que dor intensa na região posterior do pescoço e na laringe se manifestam em mulheres disfônicas. Contribuem para isso a função prejudicada da articulação cervical e alterações da amplitude de movimento cervical. Com isso, compreende-se que o abuso vocal e o mau uso da voz como fatores mais comuns para a disfonia

Microscale processing tools as screening strategies for cell therapy manufacture

Allogeneic cell therapies require the manufacture of large amounts of cells in a reliable and robust manner. Microcarrier suspension cultures are scalable, providing a solution for this challenge. Screening the effects of raw materials on cell quantity and quality should be performed at early stages of process development to reduce the risks associated with scale-up. A microfuidic platform was investigated to measure quantity and phenotype of cells grown on microcarriers. This approach was tested using CTX0E03 cells and PA5 hOMCs, potential cell therapy candidates for the treatment of neurological diseases. In well plates Plastic L and Synthemax II LC microcarriers yielded 1.2 -1.5x10⁴ cells/cm². Expression of ß-III tubulin and nestin was maintained similar between the two conditions. For PA5 hOMCs, Plastic led to 3 times higher cell density compared to Plastic Plus microcarriers. The expression of b-III tubulin and nestin was 1.5 to 2-fold higher for both Plastic and Plastic Plus microcarriers compared to other tested conditions. In the second generation microfuidic platform, cell number and phenotype were investigated through an immunoassay and a tailored image processing tool. Results were comparable between platforms for cell quantity. CTX0E03 cells proliferated similarly on Plastic L and Synthemax II LC with cell densities ranging from 1.3-1.5x10⁴ cells/cm². For PA5 hOMCs, in the second generation microfuidic platform, the cell density on Plastic was 1.2 times higher compared to Plastic Plus. A study performed in stirred microcarrier cell culture showed 3 times higher cell density on Plastic microcarriers compared to Plastic Plus microcarriers. Furthermore, cells grown on Plastic Plus microcarriers led to 2-fold higher expression of p75NTR and signi cantly increased neurite outgrowth in a co-culture assay with NG108-15 neurons with 1.2 times higher average and maximum neurite length, and 1.7 times higher number of neurites per neuron

Ozone deposition modelling in a portuguese coastal zone

The deposition of pollutants from the atmosphere to ecosystems is the cause of some present environmental problems: acidification, eutrophication and, indirectly, ground level ozone. Related with these issues is the critical loads/levels concept, widely accepted in Europe as basis for pollution control strategies. The knowledge of atmospheric pollutants deposition on ecosystems is needed to setup this methodology. This paper presents the parameterisation done in the deposition module of the system of models MAR IV which is adequate to simulate the transport, dispersion and removal of pollutants, including ozone production for mesoscale application. The ozone surface resistance (Rc) pararneterisation was applied to Lisbon region and the modelk performance was assessed with ozone deposition experimental data observed in the study area during the fieldwork LisbEx 97. The influence of parameterisation became evident with the good fit between modelled results and measured data. Based on an empirical method described by the Stockholm Environmental Institute report a Critical Load map, for non-forested areas, was calculated for Pomgal. The methodology is based on the kind of soil and its buffer capacity to acid compounds, The results show that around 70°/0of the country is in sensitive areas reinforcing the need for the application of this concept.info:eu-repo/semantics/publishedVersio

Xylose fermentation to ethanol by new Galactomyces geotrichum and Candida akabanensis strains

The conversion of pentoses into ethanol remains a challenge and could increase the supply of second-generation biofuels. This study sought to isolate naturally occurring yeasts from plant biomass and determine their capabilities for transforming xylose into ethanol. Three yeast strains with the ability to ferment xylose were isolated from pepper, tomato and sugarcane bagasse. The strains selected were characterized by morphological and auxanographic assays, and they were identified by homology analysis of 5.8 S and 26 S ribosomal RNA gene sequences. The identities of two lineages of microrganism were associated with Galactomyces geotrichum, and the other was associated with Candida akabanensis. Fermentative processes were conducted with liquid media containing only xylose as the carbon source. YP/S values for the production of ethanol ranging between 0.29 and 0.35 g g−1 were observed under non-optimized conditions