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

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

Comorbidities in a sample of adults with HIV in Puerto Rico: an exploratory study.

Background: Puerto Rico is among the areas with the highest estimated rates of people living with HIV in the United States. Despite the epidemiologic data available, there is limited real-world information that can help understand the comorbidities of people with HIV. In this study, we describe common comorbidities among adults with HIV attending treatment clinics in Puerto Rico. Methods: An exploratory, retrospective, cross-sectional study was conducted at five HIV clinics in Puerto Rico. A random sample of medical records was reviewed. Descriptive statistics were used to summarize patient demographics, morbidity, and clinical characteristics. Multivariate analyses were conducted to explore comorbidities by age and sex. Results: A total of 250 (179 men; 71 women) medical records were reviewed. Participants\u27 mean age was 47.9 years and on average they had been living with HIV for 9 years. Most (97.6%) had at least one comorbidity. The most common comorbidities were dyslipidemia and hypertension. Men were more likely to have been diagnosed with alcohol misuse while women were more likely to have been diagnosed with obesity, human papillomavirus (HPV), hypothyroidism, and osteoporosis. Participants younger than 50 years of age were more likely to have history of alcohol misuse while older individuals (50 years and old) were more likely to have been diagnosed with dyslipidemia, hypertension, and diabetes. Adjusting by sex and age, women were more likely to have been diagnosed with obesity and depression and those older than 50 years were more likely to have had a diagnosis of dyslipidemia, hypertension, HPV, and diabetes. Conclusions: This is one of the few studies assessing comorbidities among adults with HIV in Puerto Rico, among Latino/Hispanics within the United States, and Latin America. Consistent with other studies, cardiovascular diseases are common among adults with HIV in Puerto Rico. Findings support the need for awareness and real-world evidence about comorbidities among people with HIV when implementing screenings and prescribing drugs

Large-scale manufacturing of base-edited chimeric antigen receptor T cells

Base editing is a revolutionary gene-editing technique enabling the introduction of point mutations into the genome without generating detrimental DNA double-stranded breaks. Base-editing enzymes are commonly delivered in the form of modified linear messenger RNA (mRNA) that is costly to produce. Here, we address this problem by developing a simple protocol for manufacturing base-edited cells using circular RNA (circRNA), which is less expensive to synthesize. Compared with linear mRNA, higher editing efficiencies were achieved with circRNA, enabling an 8-fold reduction in the amount of RNA required. We used this protocol to manufacture a clinical dose (1 × 108 cells) of base-edited chimeric antigen receptor (CAR) T cells lacking expression of the inhibitory receptor, PD-1. Editing efficiencies of up to 86% were obtained using 0.25 μg circRNA/1 × 106 cells. Increased editing efficiencies with circRNA were attributed to more efficient translation. These results suggest that circRNA, which is less expensive to produce than linear mRNA, is a viable option for reducing the cost of manufacturing base-edited cells at scale

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

Development of a xeno-free cell culture platform for novel candidate human cell therapies for neural regeneration

The olfactory mucosa is a source of cell types that are associated with neural regeneration such as olfactory ensheathing cells (OECs), mesenchymal stromal cells (MSCs), and neural stem cells (NSCs). Although cells from this region have been used for autologous transplantation onto damaged spinal cord injury, there is a gap for an allogeneic cell therapy approach. The aim of this thesis was to establish key bioprocessing parameters for the expansion of c-MycERTAM-derived populations of human late-adherent olfactory mucosa cells (hOMCs). First, growth kinetics, identity and potency were characterised in both monolayer and suspension culture platforms. Profiles of cell growth kinetics were obtained for seeding densities ranging from 3,000 cells/cm² to 12,000 cells/cm² by manual counting and confluence measurements. PA5 hOMCs achieved between 35-41 population doublings in culture when seeded at 6,000 cells/cm² in long term culture. Moreover, removal of 4-hydroxytamoxifen (4-OHT) did not have an effect on cell growth kinetics. Replicative senescence was observed due to undesired silencing of c-MycERTAM. Next, alternative media compositions were examined as a means of optimising the process and transitioning to xeno-free culture. hOMCs showed a high dependence on fetal bovine serum (FBS) supplementation, and substitution with commercially available supplements did not sustain growth in serum-free conditions. However, human platelet lysate (hPL) was found to be a comparable to FBS when used to supplement basal media at concentrations of 2% and 5% (v/v). In this xeno-free media, hOMCs showed comparable cell growth kinetics to FBS supplemented media. Additionally, identity and potency marker expression in hOMCs was comparable between the FBS and the xeno-free hPL conditions. Finally, to progress towards scalable production of hOMCs, microcarrier screening and subsequent cell expansion on microcarriers in spinner flasks was undertaken. PA5 hOMCs were successfully expanded as a suspension culture on microcarriers at 80-mL scale in spinner flasks. A total of 8.40 ± 0.54 ×10⁶ viable cells were harvested when grown in medium supplemented with 5% hPL, and a similar number of 6.70 ± 1.05 ×10⁶ cells when grown in 10% FBS using Plastic microcarriers. PA5 hOMCs expanded on microcarriers conserved markers of identity post-harvest. In conclusion, by addressing bioprocessing fundamentals, advances towards scalable production of hOMCs using xeno-free culture reagents have been achieved

Fish collagen stabilization by phenolic compounds and enzymes for cosmetic applications

Fish industry generates a large biomass of waste that is generally discarded in spite of the potential added value of its components. Enzyme-aided and biomechanical modification steps were explored for fish gelatin in order to obtain a biomaterial suitable for exploitation in skin care applications. The experimental strategies were focused on the use of biocatalysts such as laccase, tyrosinase and transglutaminase as promoters of gelatin cross-linking and stabilization for cosmetic uses. Natural phenolic compounds such as gallic acid, tannic acid and ferulic acid were enzymatically grafted on gelatin for improved structural stability, anti-oxidant and antimicrobial activity. In parallel, chemical cross-linkers 1-ethyl3-(3-dimethylaminopropyl) carbodiimide (EDAC) and N-hydroxysuccinimide (NHS) were applied for gelatin modification. Due to the low molecular weight and the high degree of hydrolysis of the gelatin impeding its transformation into tridimensional solid matrices, a novel approach combining ultrasound, enzymes and natural phenolics was used for the generation of fish gelatin nanoparticles. The simultaneous enzymatic-sonochemical approach provided nanospheres of acceptable particle size distribution and low polidispersity. The selection of a suitable oil phase, cross-linking enzyme and the addition of antioxidant phenolics during sonication renders the fish gelatin nanoparticles potential bioactive carriers in skin care formulations

Survey of mosquito-borne flaviviruses in the Cuitzmala River Basin, Mexico: do they circulate in rodents and bats?

Abstract Background RNA viruses commonly infect bats and rodents, including mosquito-borne flaviviruses (MBFV) that affect human and animal health. Serological evidence suggests past interactions between these two mammalian orders with dengue viruses (DENV), West Nile virus (WNV), and yellow fever virus (YFV). Although in Mexico there are reports of these viruses in both host groups, we know little about their endemic cycles or persistence in time and space. Methods Rodents and bats were captured at the Cuitzmala River Basin on the Pacific coast of Jalisco state, Mexico, where MBFV, such as DENV, have been reported in both humans and bats. Samples were taken during January, June, and October 2014, at locations adjacent to the river. Tissue samples were collected from both bats and rodents and serum samples from rodents only. Highly sensitive serological and molecular assays were used to search for current and past evidence of viral circulation. Results One thousand nine hundred forty-eight individuals were captured belonging to 21 bat and 14 rodent species. Seven hundred sixty-nine liver and 764 spleen samples were analysed by means of a specific molecular protocol used to detect flaviviruses. Additionally, 708 serum samples from rodents were examined in order to demonstrate previous exposure to dengue virus serotype 2 (which circulates in the region). There were no positive results with any diagnostic test. Discussion To our knowledge, this is the first survey of rodents and only the second survey of bats from the Pacific Coast of Mexico in a search for MBFV. We obtained negative results from all samples. We validated our laboratory tests with negative and positive controls. Our findings are consistent with other empirical and experimental studies in which these mammalian hosts may not replicate mosquito-borne flaviviruses or present low prevalence. Conclusions True-negative results are essential for the construction of distribution models and are necessary to identify potential areas at risk. Negative results should not be interpreted as the local absence of MBFV in the region. On the contrary, we need to establish a long-term surveillance programme to find MBFV presence in the mosquito trophic networks, identifying the potential role of rodents and bats in viral dynamics

Antropologías hechas en Ecuador. El quehacer antropológico (Volumen IV)

Al igual que en otros países, en Ecuador la antropología no es solo una disciplina, son varias genealogías que obedecen a temas diversos con enfoques interdisciplinarios y que cambian de acuerdo al contexto social, económico y político; pero a diferencia de la región, registra pocas escuelas de antropología y centros de formación de profesionales en el área. Esta recopilación de textos muestra la diversidad y las múltiples facetas de las antropologías ecuatorianas. La antropología ecuatoriana no se agota en estas historiografías y resalta aquellas genealogías del pensamiento ecuatoriano, nutrido por reflexiones desde las escuelas clásicas de la antropología, que dialogan fuertemente con el contexto nacional y que, particularmente, tienen la capacidad de recrearse a la luz de las necesidades reales de la gente con quienes se co-construye el conocimiento