Digital holographic microscopy: a noninvasive method to analyze the formation of spheroids

Digital holographic (DH) microscopy is a unique noninvasive method to analyze living cells. With DH microscopy, in vitro cell cultures can be imaged in 2D and pseudo-3D and measurements of size and morphology of the cells are provided. Here, a description of a novel methodology utilizing DH microscopy for the analysis of spheroids is presented. A cell culture protocol is introduced and morphological parameters of cell spheroids as measured by DH microscopy are presented. The study confirms the use of DH microscopy for the analysis of cell spheroids. In the future, organoids can be analyzed with DH microscopy, and it can also be used for drug response and cell death analyses.

IVTech: The next generation of in vitro models

In vitro cell cultures are often proposed as “Alternatives” to animal models, but they are still inadequate to reproduce the human pathophysiology. This is mainly due to the technological limitations of the standard equipment used in cell culture laboratories, such as the lack of a 3D micro-architecture, the static environment and the absence of cross talk between different tissues. These limitations cause the poor predictivity of a standard in-vitro model, if compared with the human reality.

Towards reliable in vitro models – New bi-compartmental platforms for replicating tissue barriers

In vitro cell cultures represent a widespread methodology for pathophysiology studies with a high benefit/cost ratio. We have developed and are validating TToP, a novel, versatile, and scalable culture system allowing compartmentalized cell/tissue cultures with the aim of mimicking the human pathophysiological microenvironment of tissue barriers.

Shape fidelity and sterility assessment of 3D printed polycaprolactone and hydroxyapatite scaffolds

Polycaprolactone (PCL) and hydroxyapatite (HA) composite are widely used in tissue engineering (TE). They are fit to being processed with three-dimensional (3D) printing technique to create scaffolds with verifiable porosity. The current challenge is to guarantee the reliability and reproducibility of 3D printed scaffolds and to create sterile scaffolds which can be used for in vitro cell cultures. In this context it is important for successful cell culture, to have a protocol in order to evaluate the sterility of the printed scaffolds. We proposed a systematic approach to sterilise 90%PCL-10%HA pellets using a 3D bioprinter before starting the printing process. We evaluated the printability of PCL-HA composite and the shape fidelity of scaffolds printed with and without sterilised pellets varying infill pattern, and the sterility of 3D printed scaffolds following the method established by the United States Pharmacopoeia. Finally, the thermal analyses supported by the Fourier Transform Infrared Spectroscopy were useful to verify the stability of the sterilisation process in the PCL solid state with and without HA. The results show that the use of the 3D printer, according to the proposed protocol, allows to obtain sterile 3D PCL-HA scaffolds suitable for TE applications such as bone or cartilage repair.

Biotic and Abiotic Elicitors of Stilbenes Production in Vitis vinifera L. Cell Culture

The in vitro cell cultures derived from the grapevine (Vitis vinifera L.) have been used for the production of stilbenes treated with different biotic and abiotic elicitors. The red-grape cultivar Váh has been elicited by natural cellulose from Trichoderma viride, the cell wall homogenate from Fusarium oxysporum and synthetic jasmonates. The sodium-orthovanadate, known as an inhibitor of hypersensitive necrotic response in treated plant cells able to enhance production and release of secondary metabolite into the cultivation medium, was used as an abiotic elicitor. Growth of cells and the content of phenolic compounds trans-resveratrol, trans-piceid, δ-viniferin, and ɛ-viniferin, were analyzed in grapevine cells treated by individual elicitors. The highest accumulation of analyzed individual stilbenes, except of trans-piceid has been observed after treatment with the cell wall homogenate from F. oxysporum. Maximum production of trans-resveratrol, δ- and ɛ-viniferins was triggered by treatment with cellulase from T. viride. The accumulation of trans-piceid in cell cultures elicited by this cellulase revealed exactly the opposite effect, with almost three times higher production of trans-resveratrol than that of trans-piceid. This study suggested that both used fungal elicitors can enhance production more effectively than commonly used jasmonates.

A Review on the Adaption of Alginate-Gelatin Hydrogels for 3D Cultures and Bioprinting

Sustaining the vital functions of cells outside the organism requires strictly defined parameters. In order to ensure their optimal growth and development, it is necessary to provide a range of nutrients and regulators. Hydrogels are excellent materials for 3D in vitro cell cultures. Their ability to retain large amounts of liquid, as well as their biocompatibility, soft structures, and mechanical properties similar to these of living tissues, provide appropriate microenvironments that mimic extracellular matrix functions. The wide range of natural and synthetic polymeric materials, as well as the simplicity of their physico-chemical modification, allow the mechanical properties to be adjusted for different requirements. Sodium alginate-based hydrogel is a frequently used material for cell culture. The lack of cell-interactive properties makes this polysaccharide the most often applied in combination with other materials, including gelatin. The combination of both materials increases their biological activity and improves their material properties, making this combination a frequently used material in 3D printing technology. The use of hydrogels as inks in 3D printing allows the accurate manufacturing of scaffolds with complex shapes and geometries. The aim of this paper is to provide an overview of the materials used for 3D cell cultures, which are mainly alginate–gelatin hydrogels, including their properties and potential applications.

Asthma-derived fibroblast to myofibroblast transition is enhanced in comparison to fibroblasts derived from non-asthmatic patients in 3D in vitro culture due to Smad2/3 signalling

The basic hallmarks of bronchial asthma, one of the most common chronic diseases occurring in the world, are chronic inflammation, remodelling of the bronchial wall and its hyperresponsiveness to environmental stimuli. It was found out that the fibroblast to myofibroblast transition (FMT), a key phenomenon in subepithelial fibrosis of the bronchial wall, was crucial for the development of asthma. Our previous studies showed that HBFs derived from asthmatic patients cultured in vitro display some inherent features which facilitate their TGF-b-induced FMT. Although usefulness of standard ‘2D’ cultures is invaluable, they have many limitations. As HBFs interact with extracellular matrix proteins in the connective tissue, which can affect the FMT potential, we have decided to expand our ‘2D’ model to in vitro cell cultures in 3D using collagen gels. Our results showed that 1.5 mg/ml concentration of collagen is suitable for HBFs growth, motility, and phenotypic shifts. Moreover, we demonstrated that in the TGF-β1-activated HBF populations derived from asthmatics, the expression of fibrosis-related genes (ACTA2, TAGLN, SERPINE1, COL1A1, FN1 and CCN2) was significantly increased in comparison to the non-asthmatic ones. We also confirmed that it is related to the TGF-β/Smad2/3 profibrotic pathway intensification. In summary, the results of our study undoubtedly demonstrate that HBFs from asthmatics have unique intrinsic features which predispose them, regardless the culture conditions, to the increased FMT under the influence of TGF-β1.