Building a tissue: Mesenchymal and epithelial cell spheroids mechanical properties at micro- and nanoscale.

Cell transitions between the epithelial and mesenchymal phenotypes provide the regulated morphogenesis and regeneration throughout the ontogenesis. The tissue mechanics and mechanotransduction play an essential role in these processes. Cell spheroids reproduce the cell density of native tissues and represent simple building blocks for the tissue engineering purposes. The mechanical properties of mesenchymal and epithelial cells have been extensively studied in 2D monolayer cultures, but have not been sufficiently compared in spheroids. Here, we have simultaneously applied several techniques to assess the mechanical parameters of such spheroids. The local surface mechanical properties were measured by AFM, and the bulk properties were analyzed with parallel-plate compression, as well as by observing cut opening after microdissection. The comparison of the collected data allowed us to apply the model of a solid body with surface tension, and estimate the parameters of this model. We found an expectedly higher surface tension in mesenchymal spheroids, as well as a higher bulk modulus and relaxation time. The two latter parameters agree with the bulk poroelastic behavior of spheroids, and with the higher cell density and extracellular matrix content in mesenchymal spheroids. The higher tension of the surface layer cells in mesenchymal cell spheroids was also confirmed by the viscoelastic AFM characterization. The cell phenotype affected the self-organization during the spheroid formation, as well as the structure, biomechanical properties, and spreading of spheroids. The obtained results will contribute to a more detailed description of spheroid and tissue biomechanics, and will help in controlling the tissue regeneration and morphogenesis. STATEMENT OF SIGNIFICANCE: Spheroids are widely used as building blocks for scaffold-based and scaffold-free strategies in tissue engineering. In most studies, either the concept of a solid body or a liquid with surface tension was used to describe the biomechanical behavior of spheroids. Here, we have used a model which combines both aspects, a solid body with surface tension. The "solid" aspect was described as a visco-poroelastic material, affected by the liquid redistribution through the cells and ECM at the scale of the whole spheroid. A higher surface tension was found for mesenchymal spheroids than that for epithelial spheroids, observed as a higher stiffness of the spheroid surface, as well as a larger spontaneous opening of the cut edges after microdissection.

Inhaled Placental Mesenchymal Stromal Cell Secretome from Two- and Three-Dimensional Cell Cultures Promotes Survival and Regeneration in Acute Lung Injury Model in Mice.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is a common clinical problem, leading to significant morbidity and mortality, and no effective pharmacotherapy exists. The problem of ARDS causing mortality became more apparent during the COVID-19 pandemic. Biotherapeutic products containing multipotent mesenchymal stromal cell (MMSC) secretome may provide a new therapeutic paradigm for human healthcare due to their immunomodulating and regenerative abilities. The content and regenerative capacity of the secretome depends on cell origin and type of cultivation (two- or three-dimensional (2D/3D)). In this study, we investigated the proteomic profile of the secretome from 2D- and 3D-cultured placental MMSC and lung fibroblasts (LFBs) and the effect of inhalation of freeze-dried secretome on survival, lung inflammation, lung tissue regeneration, fibrin deposition in a lethal ALI model in mice. We found that three inhaled administrations of freeze-dried secretome from 2D- and 3D-cultured placental MMSC and LFB protected mice from death, restored the histological structure of damaged lungs, and decreased fibrin deposition. At the same time, 3D MMSC secretome exhibited a more pronounced trend in lung recovery than 2D MMSC and LFB-derived secretome in some measures. Taking together, these studies show that inhalation of cell secretome may also be considered as a potential therapy for the management of ARDS in patients suffering from severe pneumonia, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, their effectiveness requires further investigation.

Proteomic and electron microscopy study of myogenic differentiation of alveolar mucosa multipotent mesenchymal stromal cells in three-dimensional culture.

Myocyte differentiation is featured by adaptation processes, including mitochondria repopulation and cytoskeleton re-organization. The difference between monolayer and spheroid cultured cells at the proteomic level is uncertain. We cultivated alveolar mucosa multipotent mesenchymal stromal cells in spheroids in a myogenic way for the proper conditioning of ECM architecture and cell morphology, which induced spontaneous myogenic differentiation of cells within spheroids. Electron microscopy analysis was used for the morphometry of mitochondria biogenesis, and proteomic was used complementary to unveil events underlying differences between two-dimensional/three-dimensional myoblasts differentiation. The prevalence of elongated mitochondria with an average area of 0.097 µm2 was attributed to monolayer cells 7 days after the passage. The population of small mitochondria with a round shape and area of 0.049 µm2 (p < 0.05) was observed in spheroid cells cultured under three-dimensional conditions. Cells in spheroids were quantitatively enriched in proteins of mitochondria biogenesis (DNM1L, IDH2, SSBP1), respiratory chain (ACO2, ATP5I, COX5A), extracellular proteins (COL12A1, COL6A1, COL6A2), and cytoskeleton (MYL6, MYL12B, MYH10). Most of the Rab-related transducers were inhibited in spheroid culture. The proteomic assay demonstrated delicate mechanisms of mitochondria autophagy and repopulation, cytoskeleton assembling, and biogenesis. Differences in the ultrastructure of mitochondria indicate active biogenesis under three-dimensional conditions.

Antimicrobial and Regenerative Effects of Placental Multipotent Mesenchymal Stromal Cell Secretome-Based Chitosan Gel on Infected Burns in Rats.

BACKGROUND: There is a need for better strategies to promote burn wound healing and prevent infection. The aim of our study was to develop an easy-to-use placental multipotent mesenchymal stromal cell (MMSC) secretome-based chitosan hydrogel (MSC-Ch-gel) and estimate its antimicrobial and regenerative activity in Staphylococcus aureus-infected burn wounds in rats. METHODS: Proteomic studies of the MMSC secretome revealed proteins involved in regeneration, angiogenesis, and defence responses. The MMSC secretome was collected from cultured cells and mixed with water-soluble chitosan to prepare the placental MSC-Ch-gel, which was stored in liquid phase at 4 °C. The wounds of rats with established II-IIIa-degree burns were then infected with S. aureus and externally covered with the MSC-Ch-gel. Three additional rat groups were treated with medical Vaseline oil, the antiseptic drug Miramistin®, or the drug Bepanthen® Plus. Skin wound samples were collected 4 and 8 days after burning for further microbiological and histological analysis. Blood samples were also collected for biochemical analysis. RESULTS: Application of the MSC-Ch-gel cleared the wound of microorganisms (S. aureus wasn't detected in the washings from the burned areas), decreased inflammation, enhanced re-epithelialisation, and promoted the formation of well-vascularised granulation tissue. CONCLUSIONS: MSC-Ch-gel effectively promotes infected wound healing in rats with third-degree burns. Gel preparation can be easily implemented into clinical practice.

Mechanical properties of cell sheets and spheroids: the link between single cells and complex tissues.

Cell aggregates, including sheets and spheroids, represent a simple yet powerful model system to study both biochemical and biophysical intercellular interactions. However, it is becoming evident that, although the mechanical properties and behavior of multicellular structures share some similarities with individual cells, yet distinct differences are observed in some principal aspects. The description of mechanical phenomena at the level of multicellular model systems is a necessary step for understanding tissue mechanics and its fundamental principles in health and disease. Both cell sheets and spheroids are used in tissue engineering, and the modulation of mechanical properties of cell constructs is a promising tool for regenerative medicine. Here, we review the data on mechanical characterization of cell sheets and spheroids, focusing both on advances in the measurement techniques and current understanding of the subject. The reviewed material suggest that interplay between the ECM, intercellular junctions, and cellular contractility determines the behavior and mechanical properties of the cell aggregates.

MAPK and Notch-Mediated Effects of Meso-Xanthin F199 Compounds on Proliferative Activity and Apoptosis of Human Melanocytes in Three-Dimensional Culture.

Meso-Xanthin (Meso-Xanthin F199™) is a highly active antiaging injection drug of the latest generation. The main acting compound is fucoxanthin, supplemented with several growth factors, vitamins, and hyaluronic acid. Previous examination of fucoxanthin on melanocytes showed its ability to inhibit skin pigmentation through different signaling pathways focused on suppression of melanogenic-stimulating receptors. In turn, the anticancer property of fucoxanthin is realized through MAPK and PI3K pathways. We aimed to evaluate the effect of fucoxanthin and supplemented growth factors on melanocyte growth and transformation at a proteomic level. The effect of fucoxanthin on melanocytes cultivated in three-dimensional (3D) condition was examined using high-throughput proteomic and system biology approaches to disclose key molecular events of the targeted action. Our results demonstrated significant inhibition of cell differentiation and ubiquitination processes. We found that the negative regulation of PSME1 and PTGIS largely determines the inhibition of NF-κB and MAPK2. Besides, fucoxanthin selectively inhibits cell differentiation via negative regulation of Raf signaling and the upstream activation of IL-1 signaling. It is assumed that inhibition of Raf influences the Notch-4 signaling and switches off the MAPK/MAPK2 cascade. Blockage of MAPK/MAPK2 is feasible due to suppression of Ras and NF-κB by the addressed action of IKKB, IKK2, and TRAF6. Suggestively, Meso-Xanthin F199™ can manage processes of proliferative activity and inhibition of apoptosis due to composition of fucoxanthin and growth-stimulating factors, which may increase the risk of skin cancer development under certain condition.

The Duo of Osteogenic and Angiogenic Differentiation in ADSC-Derived Spheroids.

Bone formation during embryogenesis is driven by interacting osteogenesis and angiogenesis with parallel endothelial differentiation. Thence, all in vitro bioengineering techniques are aimed at pre-vascularization of osteogenic bioequivalents to provide better regeneration outcomes upon transplantation. Due to appearance of cell-cell and cell-matrix interactions, 3D cultures of adipose-derived stromal cells (ADSCs) provide a favorable spatial context for the induction of different morphogenesis processes, including vasculo-, angio-, and osteogenesis and, therefore, allow modeling their communication in vitro. However, simultaneous induction of multidirectional cell differentiation in spheroids from multipotent mesenchymal stromal cells (MMSCs) was not considered earlier. Here we show that arranging ADSCs into spheroids allows rapid and spontaneous acquiring of markers of both osteo- and angiogenesis compared with 2D culture. We further showed that this multidirectional differentiation persists in time, but is not influenced by classical protocols for osteo- or angio-differentiation. At the same time, ADSC-spheroids retain similar morphology and microarchitecture in different culture conditions. These findings can contribute to a better understanding of the fundamental aspects of autonomous regulation of differentiation processes and their cross-talks in artificially created self-organizing multicellular structures. This, in turn, can find a wide range of applications in the field of tissue engineering and regeneration.

Engineering a Model to Study Viral Infections: Bioprinting, Microfluidics, and Organoids to Defeat Coronavirus Disease 2019 (COVID-19).

While the number of studies related to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is constantly growing, it is essential to provide a framework of modeling viral infections. Therefore, this review aims to describe the background presented by earlier used models for viral studies and an approach to design an "ideal" tissue model for SARS-CoV-2 infection. Due to the previous successful achievements in antiviral research and tissue engineering, combining the emerging techniques such as bioprinting, microfluidics, and organoid formation are considered to be one of the best approaches to form in vitro tissue models. The fabrication of an integrated multi-tissue bioprinted platform tailored for SARS-CoV-2 infection can be a great breakthrough that can help defeat coronavirus disease in 2019.

Cell spheroid fusion: beyond liquid drops model.

Biological self-assembly is crucial in the processes of development, tissue regeneration, and maturation of bioprinted tissue-engineered constructions. The cell aggregates-spheroids-have become widely used model objects in the study of this phenomenon. Existing approaches describe the fusion of cell aggregates by analogy with the coalescence of liquid droplets and ignore the complex structural properties of spheroids. Here, we analyzed the fusion process in connection with structure and mechanical properties of the spheroids from human somatic cells of different phenotypes: mesenchymal stem cells from the limbal eye stroma and epithelial cells from retinal pigment epithelium. A nanoindentation protocol was applied for the mechanical measurements. We found a discrepancy with the liquid drop fusion model: the fusion was faster for spheroids from epithelial cells with lower apparent surface tension than for mesenchymal spheroids with higher surface tension. This discrepancy might be caused by biophysical processes such as extracellular matrix remodeling in the case of mesenchymal spheroids and different modes of cell migration. The obtained results will contribute to the development of more realistic models for spheroid fusion that would further provide a helpful tool for constructing cell aggregates with required properties both for fundamental studies and tissue reparation.

Tissue engineering using a combined cell sheet technology and scaffolding approach.

Cell sheet technology has remained quite popular among tissue engineering techniques over the last several years. Meanwhile, there is an apparent trend in modern scientific research towards combining different approaches and strategies. Accordingly, a large body of work has arisen where cell sheets are used not as separate structures, but in combination with scaffolds as supporting constructions. The aim of this review is to analyze the intersection of these two vast areas of tissue engineering described in the literature mainly within the last five years. Some practical and technical details are emphasized to provide information that can be useful in research design and planning. The first part of the paper describes the general issues concerning the use of combined technology, its advantages and limitations in comparison with those of other tissue engineering approaches. Next, the detailed literature analysis of in vivo studies aimed at the regeneration of different tissues is performed. A significant part of this section concerns bone regeneration. In addition to that, other connective tissue structures, including articular cartilage and fibrocartilage, ligaments and tendons, and some soft tissues are discussed. STATEMENT OF SIGNIFICANCE: This paper describes the intersection of two technologies used in designing of tissue-engineered constructions for regenerative medicine: cell sheets as extracellular matrix-rich structures and supporting scaffolds as essentials in tissue engineering. A large number of reviews are devoted to each of these scientific problems. However, the solution of complex problems of tissue engineering requires an integrated approach that includes both three-dimensional scaffolds and cell sheets. This manuscript serves as a description of advantages and limitations of this method, its use in regeneration of bones, connective tissues and soft tissues and some other details.

Human Melanocyte-Derived Spheroids: A Precise Test System for Drug Screening and a Multicellular Unit for Tissue Engineering.

Pigmentation is the result of melanin synthesis, which takes place in melanocytes, and its further distribution. A dysregulation in melanocytes' functionality can result in the loss of pigmentation, the appearance of pigment spots and melanoma development. Tissue engineering and the screening of new skin-lightening drugs require the development of simple and reproducible in vitro models with maintained functional activity. The aim of the study was to obtain and characterize spheroids from normal human melanocytes as a three-dimensional multicellular structure and as a test system for skin-lightening drug screening. Melanocytes are known to lose their ability to synthesize melanin in monolayer culture. When transferred under non-adhesive conditions in agarose multi-well plates, melanocytes aggregated and formed spheroids. As a result, the amount of melanin elevated almost two times within seven days. MelanoDerm™ (MatTek) skin equivalents were used as a comparison system. Cells in spheroids expressed transcription factors that regulate melanogenesis: MITF and Sox10, the marker of developed melanosomes-gp100, as well as tyrosinase (TYR)-the melanogenesis enzyme and melanocortin receptor 1 (MC1R)-the main receptor regulating melanin synthesis. Expression was maintained during 3D culturing. Thus, it can be stated that spheroids maintain melanocytes' functional activity compared to that in the multi-layered MelanoDerm™ skin equivalents. Culturing both spheroids and MelanoDerm™ for seven days in the presence of the skin-lightening agent fucoxanthin resulted in a more significant lowering of melanin levels in spheroids. Significant down-regulation of gp100, MITF, and Sox10 transcription factors, as well as 10-fold down-regulation of TYR expression, was observed in spheroids by day 7 in the presence of fucoxanthin, thus inhibiting the maturation of melanosomes and the synthesis of melanin. MelanoDerm™ samples were characterized by significant down-regulation of only MITF, Sox10 indicating that spheroids formed a more sensitive system allowed for quantitative assays. Collectively, these data illustrate that normal melanocytes can assemble themselves into spheroids-the viable structures that are able to accumulate melanin and maintain the initial functional activity of melanocytes. These spheroids can be used as a more affordable and easy-to-use test system than commercial skin equivalents for drug screening.

Isolation and characterization of trophoblasts from enzymatic explants of human term placenta.

In the present study, we describe a new method of isolation and culture of human villous and extravillous trophoblasts from term placenta. The cultivation of trypsinized placental villous tissue explants, followed by the isolation of cells from outgrowth islets allows for obtaining a cytotrophoblast subpopulation that is free from contamination by other cell types. Compared to other methods, our protocol is mild, simple and effective, does not request costly reagents and provides isolation of the mononuclear cytotrophoblast cell populations free from contamination by other types of placental cells. The isolated cells proliferated and formed a pleomorphic monolayer, where cells fused into a small number of binuclear or polynuclear syncytiotrophoblasts. Isolated cytotrophoblast cells expressed the specific epithelial intermediate filament cytokeratin 7 (CK7), the epithelium-specific cell-cell adhesion molecule E-cadherin and were CD9-, CD45- and vimentin-negative. Cyto- and syncytiotrophoblasts obtained by this method can be used as a model or tool for the fundamental research of differentiation and function of human placental cells, and can provide a new understanding of drug distribution in placenta. Their combination with other in vitro cell models can be useful for studying a variety of other aspects concerning placental functions, which will provide new knowledge for understanding immunology, endocrinology and development of placenta.