Towards 3D bioprinted spinal cord organoids

Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation

Vulnerability of Polarised Intestinal Porcine Epithelial Cells to Mycotoxin Deoxynivalenol Depends on the Route of Application

BACKGROUND AND AIMS: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. METHODS: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. RESULTS: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. CONCLUSIONS: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity

Mejora del dolor crónico en ratones con sobrepeso tras la administración de un prebiótico

Resumen trabajo presentado en el XII Workshop Sociedad Española de Microbiota, Probióticos y Prebióticos (SEMiPyP) y I Congreso Sociedad Iberoamericana de Microbiota, Probióticos y Prebióticos (SIAMPYP), celebrado de forma virtual del 15 al 18 de septiembre de 2021Introducción/Objetivo. El sobrepeso y la obesidad son problemas graves de salud pública, con alto riesgo de mortalidad y comorbilidades asociadas. El dolor crónico es más frecuente y severo en estos individuos, por lo que existe una necesidad de buscar terapias que ayuden a mitigarlo. La microbiota intestinal (MI) juega un papel crítico en la salud, y a través del eje intestino-cerebro ejerce funciones a nivel del sistema nervioso, por lo que se ha propuesto como una diana de actuación en trastornos metabólicos y/o neurológicos. Los prebióticos son conocidos por su efecto modulador de la MI. En este trabajo, el objetivo fue evaluar el efecto de un prebiótico en el desarrollo del dolor crónico inducido por el sobrepeso en un modelo animal. Metodología. Cuatro grupos de ratones macho C57Bl6/J (n= 10/grupo) recibieron dos tipos de dieta: 2 grupos dieta hipercalórica (HFD) y 2 grupos dieta control (CD). Un grupo de cada condición fue administrado con un prebiótico en el agua de bebida durante la duración del experimento (8 semanas). Se evaluó el desarrollo de alodinia mecánica e hiperalgesia térmica con diferentes pruebas in vivo, se realizaron análisis bioquímicos y se analizó la MI mediante secuenciación del gen ribosómico 16S. Resultados. La HFD indujo sobrepeso y desarrollo de dolor crónico en los ratones. La administración del prebiótico atenuó el peso corporal, tuvo efectos positivos sobre hormonas metabólicas y redujo el desarrollo del dolor inducido por el sobrepeso. La composición de la MI se vio alterada por la HFD y modulada por el prebiótico. Algunos grupos microbianos se asociaron con una menor respuesta al dolor. Conclusiones. Los resultados muestran el efecto positivo de los prebióticos en el desarrollo del dolor inducido por el sobrepeso, pudiendo actuar la MI como mediador. Este trabajo fortalece el papel de la MI como diana de actuación para mejorar trastornos metabólicos del eje intestino-cerebro

Targeting the Microbiota-Gut-Brain Axis for obesity-induced chronic pain

Trabajo presentado en la 13th International Scientific Conference on Probiotics, Prebiotics, Gut Microbiota and Health, celebrado en Praga (República Checa), del 17 al 20 de junio de 2019[Introduction] Obesity is a global epidemic and is associated with increased risk for mortality and several medical morbidities that create a substantial burden on both the aƿicted patients and society. Chronic pain is more frequent and severe among overweight patients and given the enormity of the problem and the lack of effective therapies, there is a pressing need to understand the mechanisms underlying pain development under these particular conditions. Multiple studies have now revealed that the microbiota-gut-brain axis plays a critical role in health and disease. In agreement, modulation of the gut microbiota via probiotic treatment is postulated to be an effective adjunctive therapy for metabolic disorders. In contrast, the role of prebiotics is less investigated. Prebiotic Ƽbres like short-chain fructo-oligosaccharides (FOS) are known to selectively modulate the composition of the intestinal microbiota and specially to stimulate proliferation of the lactobacilli and biƼdobacteria in the gut. [Methods] A model of overweight-induced exposure chronic pain was used in this study. Mice (40 C57Bl6/J male) were randomly assigned to the different diets for an entire period of 8 weeks: hypercaloric high-fat diet (5.21 kcal/g) and control diet (3.87 kcal/g). We tested whether the prebiotic FOS, which increases intrinsic enteric microbiota, affected obesity-induced chronic pain development. Mice were exposed to by exposing the animals to drinking water containing or not FOS during the entire experimental period. The development of allodynia and hyperalgesia were evaluated by using the von Frey and the plantar tests, respectively. Plasma leptin, adiponectin and insulin were measured. Cecum microbiota composition was determined. [Results] High fat diet induced overweight and the development of chronic pain in mice, revealed in the von Frey test. The administration of FOS attenuated body weight and epididymal fat gain and revealed reduced obesity-induced pain development. The prebiotic increased the weight of he cecum and had a positive effect on metabolic hormones such as leptin, insulin and adiponectin. Microbiota composition was also affected by diet and by FOS administration. High-fat diet reduced microbiota diversity and the presence of Roseburia was associated with reduced pain response. [Discussion] These data show that FOS produced a positive impact on obesity-induced pain development in mice. Collectively, our results suggest that prebiotics can ameliorate the overall metabolic proƼle of mice exposed to high-fat diet, partly by acting on the gut microbiota. Future studies in animal models of disease and in humans are now warranted. These Ƽndings strengthen the role of gut microbiota supplementation as prebiotic-based strategies for metabolic related brain-gut axis disorders opening new avenues in the Ƽeld of neurogastroenterology and nutritional neuroscience

Biocompatibility investigation of hybrid organometallic polymers for sub-micron 3D printing via laser two-photon polymerisation

Hybrid organometallic polymers are a class of functional materials which can be used to produce structures with sub-micron features via laser two-photon polymerisation. Previous studies demonstrated the relative biocompatibility of Al and Zr containing hybrid organometallic polymers in vitro. However, a deeper understanding of their effects on intracellular processes is needed if a tissue engineering strategy based on these materials is to be envisioned. Herein, primary rat myogenic cells were cultured on spin-coated Al and Zr containing polymer surfaces to investigate how each material affects the viability, adhesion strength, adhesion-associated protein expression, rate of cellular metabolism and collagen secretion. We found that the investigated surfaces supported cellular growth to full confluency. A subsequent MTT assay showed that glass and Zr surfaces led to higher rates of metabolism than did the Al surfaces. A viability assay revealed that all surfaces supported comparable levels of cell viability. Cellular adhesion strength assessment showed an insignificantly stronger relative adhesion after 4 h of culture than after 24 h. The largest amount of collagen was secreted by cells grown on the Al-containing surface. In conclusion, the materials were found to be biocompatible in vitro and have potential for bioengineering applications

3D microporous scaffolds manufactured via combination of fused filament fabrication and direct laser writing ablation

A 3D printing fused filament fabrication (FFF) approach has been implemented for the creation of microstructures having an internal 3D microstructure geometry. These objects were produced without any sacrificial structures or additional support materials, just by precisely tuning the nozzle heating, fan cooling and translation velocity parameters. The manufactured microporous structures out of polylactic acid (PLA) had fully controllable porosity (20%-60%) and consisted of desired volume pores (similar to 0.056 mu m(3)). The prepared scaffolds showed biocompatibility and were suitable for the primary stem cell growth. In addition, direct laser writing (DLW) ablation was employed to modify the surfaces of the PLA structures, drill holes, as well as shape the outer geometries of the created objects. The proposed combination of FFF printing with DLW offers successful fabrication of 3D microporous structures with functionalization capabilities, such as the modification of surfaces, the generation of grooves and microholes and cutting out precisely shaped structures (micro-arrows, micro-gears). The produced structures could serve as biomedical templates for cell culturing, as well as biodegradable implants for tissue engineering. The additional micro-architecture is important in connection with the cell types used for the intention of cell growing. Moreover, we show that surface roughness can be modified at the nanoscale by immersion into an acetone bath, thus increasing the hydrophilicity. The approach is not limited to biomedical applications, it could be employed for the manufacturing of bioresorbable 3D microfluidic and micromechanic structures