Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

Generation of human neuronal networks by three-dimensional (3D) bioprinting is promising for drug testing and hopefully will allow for the understanding of cellular mechanisms in brain tissue. The application of neural cells derived from human induced-pluripotent stem cells (hiPSCs) is an obvious choice, since hiPSCs provide access to cells unlimited in number and cell types that could be generated by differentiation. The questions in this regard include which neuronal differentiation stage is optimal for printing of such networks, and to what extent the addition of other cell types, especially astrocytes, supports network formation. These aspects are the focus of the present study, in which we applied a laser-based bioprinting technique and compared hiPSC-derived neural stem cells (NSCs) with neuronal differentiated NSCs, with and without the inclusion of co-printed astrocytes. In this study, we investigated in detail the effects of cell types, printed droplet size, and duration of differentiation before and after printing on viability, as well as proliferation, stemness, differentiation potential, formation of dendritic extensions and synapses, and functionality of the generated neuronal networks. We found a significant dependence of cell viability after dissociation on differentiation stage, but no impact of the printing process. Moreover, we observed a dependence of the abundance of neuronal dendrites on droplet size, a marked difference between printed cells and normal cell culture in terms of further differentiation of the cells, especially differentiation into astrocytes, as well as neuronal network formation and activity. Notably, there was a clear effect of admixed astrocytes on NSCs but not on neurons

Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

Generation of human neuronal networks by three-dimensional (3D) bioprinting is promising for drug testing and hopefully will allow for the understanding of cellular mechanisms in brain tissue. The application of neural cells derived from human induced-pluripotent stem cells (hiPSCs) is an obvious choice, since hiPSCs provide access to cells unlimited in number and cell types that could be generated by differentiation. The questions in this regard include which neuronal differentiation stage is optimal for printing of such networks, and to what extent the addition of other cell types, especially astrocytes, supports network formation. These aspects are the focus of the present study, in which we applied a laser-based bioprinting technique and compared hiPSC-derived neural stem cells (NSCs) with neuronal differentiated NSCs, with and without the inclusion of co-printed astrocytes. In this study, we investigated in detail the effects of cell types, printed droplet size, and duration of differentiation before and after printing on viability, as well as proliferation, stemness, differentiation potential, formation of dendritic extensions and synapses, and functionality of the generated neuronal networks. We found a significant dependence of cell viability after dissociation on differentiation stage, but no impact of the printing process. Moreover, we observed a dependence of the abundance of neuronal dendrites on droplet size, a marked difference between printed cells and normal cell culture in terms of further differentiation of the cells, especially differentiation into astrocytes, as well as neuronal network formation and activity. Notably, there was a clear effect of admixed astrocytes on NSCs but not on neurons

Dispensing pico to nanolitre of a natural hydrogel by laser-assisted bioprinting

<p>Abstract</p> <p>Background</p> <p>Laser-assisted bioprinting of multi-cellular replicates in accordance with CAD blueprint may substantially improve our understandings of fundamental aspects of 3 D cell-cell and cell-matrix interactions <it>in vitro</it>. For predictable printing results, a profound knowledge about effects of different processing parameters is essential for realisation of 3 D cell models with well-defined cell densities.</p> <p>Methods</p> <p>Time-resolved imaging of the hydrogel jet dynamics and quantitative assessment of the dependence of printed droplet diameter on the process characteristics were conducted.</p> <p>Results</p> <p>The existence of a counterjet was visualised, proving the bubble collapsing theory for the jet formation. Furthermore, by adjusting the viscosity and height of the applied hydrogel layer in combination with different laser pulse energies, the printing of volumes in the range of 10 to 7000 picolitres was demonstrated. Additionally, the relationship between the viscosity and the layer thickness at different laser pulse energies on the printed droplet volume was identified.</p> <p>Conclusions</p> <p>These findings are essential for the advancement of laser-assisted bioprinting by enabling predictable printing results and the integration of computational methods in the generation of 3 D multi-cellular constructs.</p

Cell type-specific adhesion and migration on laser-structured opaque surfaces

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell–implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Hydrogel-based microfluidics for vascular tissue engineering

In this work, we have explored 3-D co-culture of vasculogenic cells within a synthetically modified fibrin hydrogel. Fibrinogen was covalently linked with PEG-NHS in order to improve its degradability resistance and physico-optical properties. We have studied influences of the degree of protein PEGylation and the concentration of enzyme thrombin used for the gel preparation on cellular responses. Scanning electron microscopy analysis of prepared gels revealed that the degree of PEGylation and the concentration of thrombin strongly influenced microstructural characteristics of the protein hydrogel. Human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem cells (hASCs), used as vasculogenic co-culture, could grow in 5:1 PEGylated fibrin gels prepared using 1:0.2 protein to thrombin ratio. This gel formulation supported hASCs and HUVECs spreading and the formation of cell extensions and cell-to-cell contacts. Expression of specific ECM proteins and vasculogenic process inherent cellular enzymatic activity were investigated by immunofluorescent staining, gelatin zymography, western blot and RT-PCR analysis. After evaluation of the optimal gel composition and PEGylation ratio, the hydrogel was utilized for investigation of vascular tube formation within a perfusable microfluidic system. The morphological development of this co-culture within a perfused hydrogel over 12 days led to the formation of interconnected HUVEC-hASC network. The demonstrated PEGylated fibrin microfluidic approach can be used for incorporating other cell types, thus representing a unique experimental platform for basic vascular tissue engineering and drug screening applications. © 2016 by De Gruyter

In Vitro Development of Human iPSC-Derived Functional Neuronal Networks on Laser-Fabricated 3D Scaffolds

Neural progenitor cells generated from human induced pluripotent stem cells (hiPSCs) are the forefront of ″brain-on-chip″ investigations. Viable and functional hiPSC-derived neuronal networks are shaping powerful in vitro models for evaluating the normal and abnormal formation of cortical circuits, understanding the underlying disease mechanisms, and investigating the response to drugs. They therefore represent a desirable instrument for both the scientific community and the pharmacological industry. However, culture conditions required for the full functional maturation of individual neurons and networks are still unidentified. It has been recognized that three-dimensional (3D) culture conditions can better emulate in vivo neuronal tissue development compared to 2D cultures and thus provide a more desirable in vitro approach. In this paper, we present the design and implementation of a 3D scaffold platform that supports and promotes intricate neuronal network development. 3D scaffolds were produced through direct laser writing by two-photon polymerization (2PP), a high-resolution 3D laser microstructuring technology, using the biocompatible and nondegradable photoreactive resin Dental LT Clear (DClear). Neurons developed and interconnected on a 3D environment shaped by vertically stacked scaffold layers. The developed networks could support different cell types. Starting at the day 50 of 3D culture, neuronal progenitor cells could develop into cortical projection neurons (CNPs) of all six layers, different types of inhibitory neurons, and glia. Additionally and in contrast to 2D conditions, 3D scaffolds supported the long-term culturing of neuronal networks over the course of 120 days. Network health and functionality were probed through calcium imaging, which revealed a strong spontaneous neuronal activity that combined individual and collective events. Taken together, our results highlight advanced microstructured 3D scaffolds as a reliable platform for the 3D in vitro modeling of neuronal functions.publishedVersio

Capillary-like Formations of Endothelial Cells in Defined Patterns Generated by Laser Bioprinting

Bioprinting is seen as a promising technique for tissue engineering, with hopes of one day being able to produce whole organs. However, thick tissue requires a functional vascular network, which naturally contains vessels of various sizes, down to capillaries of ~10 &micro;m in diameter, often spaced less than 200 &micro;m apart. If such thick tissues are to be printed, the vasculature would likely need to be printed at the same time, including the capillaries. While there are many approaches in tissue engineering to produce larger vessels in a defined manner, the small capillaries usually arise only in random patterns by sprouting from the larger vessels or from randomly distributed endothelial cells. Here, we investigated whether the small capillaries could also be printed in predefined patterns. For this purpose, we used a laser-based bioprinting technique that allows for the combination of high resolution and high cell density. Our aim was to achieve the formation of closed tubular structures with lumina by laser-printed endothelial cells along the printed patterns on a surface and in bioprinted tissue. This study shows that such capillaries are directly printable; however, persistence of the printed tubular structures was achieved only in tissue with external stimulation by other cell types

Laser-assisted bioprinting at different wavelengths and pulse durations with a metal dynamic release layer: A parametric study

For more than a decade, living cells and biomaterials (typically hydrogels) are printed via laser-assisted bioprinting. Often, a thin metal layer is applied as laser-absorbing material called dynamic release layer (DRL). This layer is vaporized by focused laser pulses generating vapor pressure that propels forward a coated biomaterial. Different lasers with laser wavelengths from 193 to 1064 nanometer have been used. As a metal DRL gold, silver, or titanium layers have been used. The applied laser pulse durations were usually in the nanosecond range from 1 to 30 ns. In addition, some studies with femtosecond lasers have been published. However, there are no studies on the effect of all these lasers parameters on bioprinting with a metal DRL, and on comparing different wavelengths and pulse durations except one study comparing 500 femtosecond pulses with 15 ns pulses. In this paper, the effects of laser wavelength (355, 532, and 1064 nm) and laser pulse duration (in the range of 8 to 200 ns) are investigated. Furthermore, the effects of laser pulse energy, intensity, and focal spot size are studied. The printed droplet volume, hydrogel jet velocity, and cell viability are analyzed

Капитальный ремонт подводного перехода магистрального газопровода "Парабель–Кузбасс " через реку Обь методом санации

Наша страна покрыта обширной сетью рек, по дну которых проложены тысячи километров дюкерных переходов трубопроводов различного назначения (газовые, нефтяные и др.). Однако их объединяет то, что изготовлены они в основном из стали и срок их эксплуатации превысил все возможные пределы. Некоторые дюкеры построены еще в 70–80-х годах прошлого века и нуждаются в срочном ремонте или замене.Our country is covered with a wide water network, including thousand kilometers of underwater pipelines of different purposes (sewerage system, water-pipe, gas-pipeline etc). However they have one thing in common, they are made from steel and their operation life exceeds all possible limits. Some underwater pipelines were built as early as the 40’s – 60’s of the last century and it is necessary to reconstruct them or substitute them