Cells Tissues Organs最新文献

Until September 2021, the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2; COVID-19) pandemic caused over 217 million infections and over 4.5 million deaths. In pregnant women, the risk factors for the need of intensive care treatment are generally the same as in the overall population. Of note, COVID-19-positive women deliver earlier than COVID-19-negative women, and the risk for severe neonatal and perinatal morbidity and mortality is significantly higher. The probability and pathways of vertical transmission of the virus from the pregnant woman to the fetus are highly controversial. Recent data have shown that 54 (13%) of 416 neonates born to COVID-19-positive women were infected. Here, we investigated term placentas collected before the SARS-CoV-2 pandemic and studied the main COVID-19 receptors angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine subtype 2 (TMPRSS2), as well as neuropilin 1 (NRP1). We performed real-time PCR and immunofluorescence on cryosections in combination with markers for syncytiotrophoblast, endothelial cells, macrophages and stromal cells. The PCR studies showed expression of both the truncated delta form of ACE2, which does not bind the COVID-19 spike protein, and the long form. The ACE2 antibody used does not distinguish between the two forms. We did not observe expression of the canonical SARS-CoV-2 entry machinery on syncytio- and cytotrophoblast. ACE2 and TMPRSS2 are co-expressed in a subpopulation of stromal cells, which in part are CD68-positive macrophages. NRP1 is localized to endothelial cells. In sum, the term placenta is not an organ that directly favors vertical transmission of COVID-19; however, microtraumas and placentitis may weaken its barrier function.

The production of a cellularized silk fibroin scaffold is very difficult because it is actually impossible to differentiate cells into a well-organized cardiac tissue. Without vascularization, not only do cell masses fail to grow, but they may also exhibit an area of necrosis, indicating a lack of oxygen and nutrients. In the present study, we used the so-called tyrosine protein kinase kit (c-Kit)-positive cardiac progenitor cells (CPCs) to generate cardiac cellularized silk fibroin scaffolds, multipotent cells isolated from the adult heart to date that can show some degree of differentiation toward the cardiac phenotype. To test their ability to differentiate into the cardiac phenotype in vivo as well, CPC and collagen organoid-like masses were implanted into nude mice and their behavior observed. Since the 3-dimensional structure of cardiac tissue can be preserved by scaffolds, we prepared in parallel different silk fibroin scaffolds with 3 different geometries and tested their behavior in 3 different models of immunosuppressed animals. Unfortunately, CPC cellularized silk fibroin scaffolds cannot be used in vivo. CPCs implanted alone or in collagen type I gel were destroyed by CD3+ lymphocyte aggregates, whereas the porous and partially oriented scaffolds elicited a consistent foreign body response characterized by giant cells. Only the electrospun meshes were resistant to the foreign body reaction. In conclusion, c-Kit-positive CPCs, although expressing a good level of cardiac differentiation markers in vitro with or without fibroin meshes, are not suitable for an in vivo model of cardiac organoids because they are degraded by a T-cell-mediated immune response. Even scaffolds which may preserve the survival of these cells in vivo also induced a host response. However, among the tested scaffolds, the electrospun meshes (F-scaffold) induced a lower response compared to all the other tested structures.

Over the past 50 years, several different types of extracellular vesicles have been discovered including exosomes, microvesicles, and matrix vesicles. These vesicles are secreted by cells for specific purposes and contain cargo such as microRNA, cytokines, and lipids. A novel extracellular vesicle, the matrix-bound nanovesicle (MBV), has been recently discovered. The MBV is similar to the microvesicle, however, it is attached to the extracellular matrix, instead of being secreted. This review compares MBVs to other types of extracellular vesicles to try and better understand their origin and function. Further, this review will explain various extracellular vesicle isolation methods and how these can be used for MBVs and summarize characterization of MBV cargo such as microRNA, proteins, and lipids. Lastly, we will summarize the effects of MBVs on cells. MBVs are a novel class of extracellular vesicles that hold great promise as a platform for delivery of targeted gene and drug therapeutics.

Genetic engineering of farm animals is commonly carried out via cell-mediated transfection followed by somatic cell nuclear transfer. However, efficient transfer of exogenous DNA into ovine embryonic fibroblast (EF) cells without compromising cell viability has remained a challenging issue. Here, we aimed to develop a protocol for electrotransfection of sheep EF cells. First, we optimized the pulsing condition using an OptiMEM-GlutaMAX medium as the electroporation buffer and found 2 pulses of 270 V, each for 10 ms and 10 s interval, is the most efficient condition to have a high rate of transfection and cell survival. Moreover, supplementing 3% dimethyl sulfoxide (DMSO) into the electroporation medium considerably improved the cell viability after the electroporation process. The electroporation procedure resulted in >98% transfection efficiency and >97% cell survival rate using reporter plasmids. Finally, using CRISPR/Cas9-encoding vectors, we targeted BMP15 and GDF9 genes in sheep EF cells. The electroporated cells are associated with a 52% indels rate using single gRNAs as well as a highly efficient target deletion using 2 gRNAs. In conclusion, we have developed an electrotransfection protocol using the OptiMEM-GlutaMAX medium supplemented with 3% DMSO for sheep EF cells. The electroporation method can be used for cell-mediated gene-editing in sheep.

Cell-derived matrices are useful tools for studying the extracellular matrix (ECM) of different cell types and testing the effects on cell migration or wound repair. These matrices typically are generated using extended culture with ascorbic acid to boost ECM production. Applying this technique to cancer cell cultures could advance the study of cancer ECM and its effects on recruitment and training of the tumor microenvironment, but ascorbic acid is potently cytotoxic to cancer cells. Macromolecular crowding (MMC) agents can also be added to increase matrix deposition based on the excluded volume principle. We report the use of MMC alone as an effective strategy to generate brain cancer cell-derived matrices for downstream analyses and cell migration studies. We cultured the mouse glioblastoma cell line GL261 for 1 week in the presence of three previously reported MMC agents (carrageenan, Ficoll 70/400, and hyaluronic acid). We measured the resulting deposition of collagens and sulfated glycosaminoglycans using quantitative assays, as well as other matrix components by immunostaining. Both carrageenan and Ficoll promoted significantly more accumulation of total collagen content, sulfated glycosaminoglycan content, and fibronectin staining. Only Ficoll, however, also demonstrated a significant increase in collagen I staining. The results were more variable in 3D spheroid culture. We focused on Ficoll MMC matrices, which were isolated using the small molecule Raptinal to induce cancer cell apoptosis and matrix decellularization. The cancer cell-derived matrix promoted significantly faster migration of human astrocytes in a scratch wound assay, which may be explained by focal adhesion morphology and an increase in cellular metabolic activity. Ultimately, these data show MMC culture is a useful technique to generate cancer cell-derived matrices and study the effects on stromal cell migration related to wound repair.

Peristalsis is a nuanced mechanical stimulus comprised of multi-axial strain (radial and axial strain) and shear stress. Forces associated with peristalsis regulate diverse biological functions including digestion, reproductive function, and urine dynamics. Given the central role peristalsis plays in physiology and pathophysiology, we were motivated to design a bioreactor capable of holistically mimicking peristalsis. We engineered a novel rotating screw-drive based design combined with a peristaltic pump, in order to deliver multi-axial strain and concurrent shear stress to a biocompatible polydimethylsiloxane (PDMS) membrane "wall." Radial indentation and rotation of the screw drive against the wall demonstrated multi-axial strain evaluated via finite element modeling. Experimental measurements of strain using piezoelectric strain resistors were in close alignment with model-predicted values (15.9 ± 4.2% vs. 15.2% predicted). Modeling of shear stress on the "wall" indicated a uniform velocity profile and a moderate shear stress of 0.4 Pa. Human mesenchymal stem cells (hMSCs) seeded on the PDMS "wall" and stimulated with peristalsis demonstrated dramatic changes in actin filament alignment, proliferation, and nuclear morphology compared to static controls, perfusion, or strain, indicating that hMSCs sensed and responded to peristalsis uniquely. Lastly, significant differences were observed in gene expression patterns of calponin, caldesmon, smooth muscle actin, and transgelin, corroborating the propensity of hMSCs toward myogenic differentiation in response to peristalsis. Collectively, our data suggest that the peristalsis bioreactor is capable of generating concurrent multi-axial strain and shear stress on a "wall." hMSCs experience peristalsis differently than perfusion or strain, resulting in changes in proliferation, actin fiber organization, smooth muscle actin expression, and genetic markers of differentiation. The peristalsis bioreactor device has broad utility in the study of development and disease in several organ systems.

The heart is a dynamic organ, and the cardiac tissue experiences changes in pressure and stretch during the cardiac cycle. Existing cell culture and animal models are limited in their capacity to decouple and tune specific hemodynamic stresses implicated in the development of physiological and pathophysiological cardiac tissue remodeling. This study focused on creating a system to subject engineered cardiac tissue to either pressure or stretch stimuli in isolation and the subsequent evaluation of acute tissue remodeling. We developed a cardiac tissue chip containing three-dimensional (3-D) cell-laden hydrogel constructs and cultured them within systems where we could expose them to either pressure changes or volume changes as seen in the left ventricle. Acute cellular remodeling with each condition was qualitatively and quantitatively assessed using histology, immunohistochemistry, gene expression studies, and soluble factor analysis. Using our unique model system, we isolated the effects of pressure and stretch on engineered cardiac tissue. Our results confirm that both pressure and stretch mediate acute stress responses in the engineered cardiac tissue. However, both experimental conditions elicited a similar acute phase injury response within this timeframe. This study demonstrates our ability to subject engineered cardiac tissue to either pressure or stretch stimuli in isolation, both of which elicited acute tissue remodeling responses.

The field of tissue engineering has benefited greatly from the broad development of natural and synthetic polymers. Extensive work in neural engineering has demonstrated the value of conductive materials to improve spontaneous neuron activity as well as lowering the necessary field parameters for exogenous electrical stimulation. Further, cell fate is directly coupled to the mechanical properties of the cell culture substrate. Increasing the conductivity of hydrogel materials often necessitates the addition of dopant materials that facilitate electron mobility. However, very little electron transfer is observed in native cell signaling and most of these materials are opaque, severely limiting microscopy applications commonly employed to assess cell culture morphology and function. To overcome these shortcomings, the inclusion of an ionic liquid, choline acrylate, into the backbone of a modified collagen polymer increases the bulk conductivity 5-fold at a 1:1 ratio while maintaining optical transmission of visible light. Here, we explore how the inclusion of choline acrylate influences bulk material properties including the mechanical, swelling, and optical properties of our hydrogels, referred to as Gel-Amin hydrogels, as a material for tissue culture. Despite an increase in swelling over traditional GelMA materials, the conductive hydrogels support whole dorsal root ganglia encapsulation and outgrowth. Our results indicate that our Gel-Amin system holds potential for neural engineering applications and lowering the required charge injection for the application of exogenous electrical stimulation. This is this first time an ionic liquid-hydrogel system has been used to culture and support primary neurons in vitro.

Autologous fat transplantation is plagued by an unpredictable and often significant degree of graft loss. AdE4+ endothelial cells (ECs) are human endothelial cells that have been transduced with the E4ORF1 region of human adenovirus type 5, resulting in long-term preservation of EC proliferation and angiogenic capability without immortalization. We hypothesized that AdE4+ EC-enriched fat grafts would demonstrate improved volume retention secondary to enhanced angiogenesis. Three experimental groups were prepared by admixing 400 µL of patient lipoaspirate with 100 µL of AdE4+ EC suspensions (high AdE4+ EC concentration-enriched [5 × 106/mL], low AdE4+ EC concentration-enriched [1.25 × 106/mL], or PBS) and injected subcutaneously into the bilateral dorsa of nude mice. Fat transplants were explanted at 90 and 180 days for volumetric and histologic analyses. After both 90 and 180 days, AdE4+ EC-enriched fat grafts showed greater mean volume preservation compared to control grafts (p < 0.05). Regions of focal necrosis were only noticed in low AdE4+ EC concentration-enriched and control groups after 180 days. Histologic analysis demonstrated the presence of healthy adipocytes in all AdE4+ EC-enriched fat grafts in which both human and host ECs were evident after 90 and 180 days. AdE4+ EC enrichment improved fat graft volume preservation and vascularization in this murine xenograft model. Though further study is warranted, AdE4+ ECs demonstrated to be promising as a potential off-the-shelf adjunct for improving the volume, quality, and consistency of fat engraftment.

Despite their critical roles in tissue repair and pathological processes such as fibrosis, tumor invasion, and metastasis, the origins of mesenchymal cells remain poorly understood. Among the likely routes, epithelial-to-mesenchymal transitions (EMTs) emerge as important source of these cells. EMTs manifest themselves as a phenotypic transition in terminally differentiated epithelial cells into mesenchymal cells which are closely related to embryogenesis and organ development as well as in chronically inflamed tissues and neoplasia. There exists a potential for successful engineering of biomimetic environments that closely reflects and reciprocates the dynamic changes in the cellular microenvironment during EMT and relies on integrating the mechanical sensing mechanisms found in the native tissues into the synthetic scaffolds to understand cellular plasticity. Extracellular matrix (ECM) has complex structures composed of a collection of extracellular molecules including fibrous proteins and glycoproteins in a hydrated mixture of glycosaminoglycans and proteoglycans. Therefore, fibrous materials have been increasingly applied in tissue engineering applications since biomaterials need to restore ECM structures to provide physical, biochemical, and biomechanical signals to define cellular behaviors and tissue functions. This review summarizes materials used for fibrous scaffolds including natural and synthetic materials, highlights recent development of fabrication techniques, characteristic architectures, and properties and different applications of fibrous scaffolds in tissue engineering. The prospects and challenges about fibrous materials in tissue engineering applications are also discussed. Finally, we summarized relevant bioengineering approaches to modulate each type of EMT as potential avenues to consider toward future biomaterials design.