Human intrinsic choroidal neurons do not alter the expression of intrinsic markers in response to pressure

BackgroundThe choroid is densely innervated by all parts of the autonomic nervous system and further harbours a network of local nerve cells, the intrinsic choroidal neurons (ICN). Their function in ocular control is currently unknown. While morphological data assume a role in intraocular pressure regulation, we here test if increased pressure on isolated choroids may activate ICN.MethodsDonor tissue was transferred into a pressurisable tissue culture chamber, and nasal and temporal choroid halves incubated for 1 or 4 hours, with pressures set to 15 or 50 mm Hg, followed by qRT-PCR expression analysis of the ICN-specific markers VIP, UCN, NOS1, UCH-L1. POL2-normalised data in the different pressure settings, incubation times and localisations were statistically analysed.ResultsThe presence of the ICN-specific markers VIP, UCN, NOS1, UCH-L1 was confirmed using immunohistochemistry, and mRNA of all markers was detected in all experimental conditions. Marker analysis revealed no significant changes of mRNA expression levels between 15 and 50 mm Hg in the different incubation times. When comparing all samples over all experimental conditions, a significant increase of VIP and NOS1 mRNA was detected in temporal versus nasal choroids.ConclusionIn this functional analysis of human ICN in vitro, higher amounts of VIP and NOS1 mRNA were detected in the temporal choroid, that is, the choroidal site with ICN accumulation. Further, our data indicate that elevated pressure is apparently not able to trigger ICN responses via the investigated markers. Alternative markers and stimuli need to be investigated in upcoming studies in order to unravel ICN function.

A programmable culture platform for stimulation and in situ sensing of lung epithelial cells

A programmable dynamic cell culture chamber compatible with a standard multi-well plate was designed and characterized. The system is integrated with an array of OECT biosensors, in view of an in-situ monitoring of the dynamic cultures.

Crosstalk between astrocytes and microglia results in increased degradation of α-synuclein and amyloid-β aggregates

Background Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aβ) and alpha-synuclein (αSYN), respectively. In order to develop effective therapies, it is crucial to understand how the Aβ/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear. Methods The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aβ pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aβ and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized “close-culture” chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space. Results Our data show that intracellular deposits of αSYN and Aβ are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the “close-culture” chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte. Conclusions Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aβ/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.

Designing Hydrogel-Based Bone-On-Chips for Personalized Medicine

The recent development of bone-on-chips (BOCs) holds the main advantage of requiring a low quantity of cells and material, compared to traditional In Vitro models. By incorporating hydrogels within BOCs, the culture system moved to a three dimensional culture environment for cells which is more representative of bone tissue matrix and function. The fundamental components of hydrogel-based BOCs, namely the cellular sources, the hydrogel and the culture chamber, have been tuned to mimic the hematopoietic niche in the bone aspirate marrow, cancer bone metastasis and osteo/chondrogenic differentiation. In this review, we examine the entire process of developing hydrogel-based BOCs to model In Vitro a patient specific situation. First, we provide bone biological understanding for BOCs design and then how hydrogel structural and mechanical properties can be tuned to meet those requirements. This is followed by a review on hydrogel-based BOCs, developed in the last 10 years, in terms of culture chamber design, hydrogel and cell source used. Finally, we provide guidelines for the definition of personalized pathological and physiological bone microenvironments. This review covers the information on bone, hydrogel and BOC that are required to develop personalized therapies for bone disease, by recreating clinically relevant scenarii in miniaturized devices.

Propagation of phytosanitary clones by in vitro culture

This article presents the process of accelerated reproduction of healthy clones of grapes, including the following stages: growing young shoots of the original plants of clones, introducing tops into in vitro culture, microclonal cuttings, adaptation of microplants to ex vitro culture, transplanting into cassettes with a soil substrate, transferring plants to a greenhouse for growing to the condition of vegetative seedlings and planting in a pre-propagation mother stock. For the successful implementation of each of the above operations in the SPIHVFT, a Cultural Complex has been equipped, consisting of a number of interconnected premises: a sterile box, a culture chamber and a vegetation chamber. The use of this Complex allows multiplying the required number of plants during the year, to grow vegetative seedlings by the spring of the next year and plant them in the pre-propagation “Pre-base” mother plant. After a year, grafted vegetative seedlings grown from the vines of the mother plant were used for laying the mother stock.

Physiological shear stress enhances differentiation and mucus-formation of intestinal epithelial cells in vitro.

The gastrointestinal (GI) mucus layer plays a pivotal role in tissue homoeostasis and functionality of the gut. However, due to the shortage of affordable, realistic in vitro mucus models, studies with deeper insights into its structure and characteristics are rare. To obtain an improved mucus model, we developed a reusable culture chamber facilitating the application of physiologically relevant GI shear stresses (0.002-0.08 dyn/cm) to cells in a bioreactor system. Differentiation of a confluent monolayer of human mucus-producing epithelial HT29-MTX cells was monitored under dynamic and static culture conditions. Cells under flow remained highly proliferative and analysis via confocal microscopy revealed superior reorganization into 3-dimensional villi-like structures compared to static culture (up to 120 vs. 80 µm in height). Additionally, the median mucus thickness was significantly increased under dynamic conditions compared to static culture (41±14 vs. 29±14 µm) with a simultaneous drastic reduction of culture time from three to two weeks for sufficient maturation into goblet-like cells. We demonstrated the impact of culture conditions on the differentiation of HT29-MTX cells, revealing outstanding in vivo like reorganization of cells and the production of thick adherent mucus networks when cultured under physiological shear stress using our newly designed culture chamber.

Bioelectronics-on-a-chip for cardio myoblast proliferation enhancement using electric field stimulation

Background Cardio myoblast generation from conventional approaches is laborious and time-consuming. We present a bioelectronics on-a-chip for stimulating cells cardio myoblast proliferation during culture. Method The bioelectronics chip fabrication methodology involves two different process. In the first step, an aluminum layer of 200 nm is deposited over a soda-lime glass substrate using physical vapor deposition and selectively removed using a Q-switched Nd:YVO4 laser to create the electric tracks. To perform the experiments, we developed a biochip composed of a cell culture chamber fabricated with polydimethylsiloxane (PDMS) with a glass coverslip or a cell culture dish placed over the electric circuit tracks. By using such a glass cover slip or cell culture dish we avoid any toxic reactions caused by electrodes in the culture or may be degraded by electrochemical reactions with the cell medium, which is crucial to determine the effective cell-device coupling. Results The chip was used to study the effect of electric field stimulation of Rat ventricular cardiomyoblasts cells (H9c2). Results shows a remarkable increase in the number of H9c2 cells for the stimulated samples, where after 72 h the cell density double the cell density of control samples. Conclusions Cell proliferation of Rat ventricular cardiomyoblasts cells (H9c2) using the bioelectronics-on-a-chip was enhanced upon the electrical stimulation. The dependence on the geometrical characteristics of the electric circuit on the peak value and homogeneity of the electric field generated are analyzed and proper parameters to ensure a homogeneous electric field at the cell culture chamber are obtained. It can also be observed a high dependence of the electric field on the geometry of the electrostimulator circuit tracks and envisage the potential applications on electrophysiology studies, monitoring and modulate cellular behavior through the application of electric fields.

Bioelectronics-on-a-chip for cardio myoblast proliferation enhancement using electric field stimulation.

Background: Cardio myoblast generation from conventional approaches is laborious and time-consuming. We present a bioelectronics on-a-chip for stimulating cells cardio myoblast proliferation during culture. Method: The bioelectronics chip fabrication methodology involves two different process. In the first step, an aluminum layer of 200 nm is deposited over a soda-lime glass substrate using physical vapor deposition and selectively removed using a Q-switched Nd:YVO4 laser to create the electric tracks. To perform the experiments, we developed a biochip composed of a cell culture chamber fabricated with polydimethylsiloxane (PDMS) with a glass coverslip or a cell culture dish placed over the electric circuit tracks. By using such a glass cover slip or cell culture dish we avoid any toxic reactions caused by electrodes in the culture or may be degraded by electrochemical reactions with the cell medium, which is crucial to determine the effective cell-device coupling. Results: The chip was used to study the effect of electric field stimulation of Rat ventricular cardiomyoblasts cells (H9c2). Results shows a remarkable increase in the number of H9c2 cells for the stimulated samples, where after 72 hours the cell density double the cell density of control samples. Conclusions: Cell proliferation of Rat ventricular cardiomyoblasts cells (H9c2) using the bioelectronics-on-a-chip was enhanced upon the electrical stimulation. The dependence on the geometrical characteristics of the electric circuit on the peak value and homogeneity of the electric field generated are analyzed and proper parameters to ensure a homogeneous electric field at the cell culture chamber are obtained. It can also be observed a high dependence of the electric field on the geometry of the electrostimulator circuit tracks and envisage the potential applications on electrophysiology studies, monitoring and modulate cellular behavior through the application of electric fields

Bioelectronics-on-a-chip for Cardiomyoblast Proliferation Enhancement Using Electric Field Stimulation

Background:Cardiomyoblast generation from conventional approaches is laborious and time-consuming. We present a bioelectronics on-a-chip for stimulating cells cardio myoblast proliferation during culture. Method:The bioelectronics chip fabrication methodology involves two different process. In the first step, an aluminum layer of 200 nm is deposited over a soda-lime glass substrate using physical vapor deposition and selectively removed using a Q-switched Nd:YVO4laser to create the electric tracks. To perform the experiments, we developed a biochip composed of a cell culture chamber fabricated withpolydimethylsiloxane (PDMS) with a glass coverslip placed over the electric circuit tracks.By using such a glass coverslip we avoid any toxic reactions caused by electrodes in the culture or may be degraded by electrochemical reactions with the cell medium, which is crucial to determine the effective cell-device coupling. Results:The chip was used to study the effect of electricfield stimulation of Rat ventricular cardiomyoblasts cells (H9c2). Results shows a remarkable increase in the number of H9c2 cells for the stimulated samples, where after 72 hours the cell density double the cell density of control samples. Conclusions:Cell proliferation of Rat ventricular cardiomyoblasts cells (H9c2) using the bioelectronics-on-a-chip was enhanced upon the electrical stimulation. The dependence on the geometrical characteristics of the electric circuit on the peak value and homogeneity of the electric field generated are analyzed and proper parameters to ensure a homogeneous electric field at the cell culture chamber are obtained. It can also be observed a high dependence of the electric field on the geometry of the electrostimulator circuit tracks and envisage the potential applications on electrophysiology studies, monitoring and modulate cellular behavior through the application of electric fields