The accurate study of cellular microenvironments is limited by the lack of technologies that can manipulate cells in 3D at a sufficiently small length scale. The ability to build and manipulate multicellular microscopic structures will facilitate a more detailed understanding of cellular function in fields such as developmental and stem cell biology. We present a holographic optical tweezers based technology to accurately generate bespoke cellular micro-architectures. Using embryonic stem cells, 3D structures of varying geometries were created and stabilized using hydrogels and cell-cell adhesion methods. Control of chemical microenvironments was achieved by the temporal release of specific factors from polymer microparticles positioned within these constructs. Complex co-culture micro-environmental analogues were also generated to reproduce structures found within adult stem cell niches. The application of holographic optical tweezers-based micromanipulation will enable novel insights into biological microenvironments by allowing researchers to form complex architectures with sub-micron precision of cells, matrices and molecules.
Introduction: The benefits of endoscopic fetal surgery are deteriorated by the high risk of iatrogenic preterm prelabor rupture of fetal membranes (iPPROM). While previous studies have reported good sealing candidates to prevent membrane rupture, the delivery of these materials to the location of membrane puncture remains unsolved. Materials and Methods: We describe an approach to apply sealing materials onto the amnion through the fetoscopy port. We developed a device composed of an umbrella-shaped polyester coated nitinol mesh and an applicator. The spontaneously unfolding umbrella is pushed through the port, pulled against the amnion, and glued onto the amnion defect site. We tested the adhesion strength of multiple glues and tested the feasibility and reproducibility of this fetal membrane sealing approach in an ex vivo model. Results: The umbrella unfolded and was well positioned in all tests (n = 18). When applied via the fetoscopy port, umbrellas were successfully glued onto the fetal membrane, and all of them completely covered the defect (n = 5). The mean time needed for the whole procedure was 3 min. Discussion: This study is a proof of concept presenting a potential future solution for the precise local application of bioadhesives for the prevention of iPPROM.
The perivascular niche is a complex microenvironment containing mesenchymal stem cells (MSCs), among other perivascular cells, as well as temporally organized biochemical and biophysical gradients. Due to a lack of conclusive phenotypic markers, MSCs' identity, heterogeneity and function within their native niche remain poorly understood. The in vitro reconstruction of an artificial three-dimensional (3D) perivascular niche would offer a powerful alternative to study MSC behavior under more defined conditions. To this end, we here present a poly(ethylene glycol)-based in vitro model that begins to mimic the spatiotemporally controlled presentation of biological cues within the in vivo perivascular niche, namely a stably localized platelet-derived growth factor B (PDGF-BB) gradient. We show that 3D-encapsulated MSCs respond to soluble PDGF-BB by proliferation, spreading, and migration in a dose-dependent manner. In contrast, the exposure of MSCs to 3D matrix-tethered PDGF-BB gradients resulted in locally restricted morphogenetic responses, much as would be expected in a native perivascular niche. Thus, the herein presented artificial perivascular niche model provides an important first step towards modeling the role of MSCs during tissue homeostasis and regeneration.
Cells modulate the functional properties of their environment by depositing extracellular matrix (ECM) proteins during biological processes in vivo and in vitro. Despite the ECMs central role in tissue formation, its quantification in hydrogels like Matrigel, which have a complex materials‐inherent biopolymer composition is exceptionally challenging. Here, the use of protein‐free, synthetic poly(ethylene glycol) hydrogels enables the analysis of deposited human bone marrow mesenchymal stromal cells ECM directly harvested from fresh 3D cell cultures by a tandem mass spectrometry (LC‐MS/MS) method. In this study, it is proved that a label‐free LC‐MS/MS quantification method can selectively identify proteins deposited in 3D synthetic hydrogels following different growth factor (GF) treatments. Furthermore, it is shown that the sequence in which GFs are administered and the choice of stimuli significantly influences the number and abundance of ECM proteins. Therefore, this provides a versatile method to optimize GF treatments in synthetic hydrogel‐based regenerative medicine and tissue engineering approaches.
<b><i>Introduction:</i></b> Iatrogenic preterm premature rupture of the membrane remains the Achille’s heel of fetoscopy. The aim of this study was to show in vivo feasibility of fetal membrane (FM) defect sealing by the application of tissue glues with umbrella-shaped receptors. <b><i>Methods:</i></b> First, we adapted our previously described ex vivo strategy and evaluated the adhesion strength of different tissue glues, Histoacryl® and Glubran2®, by bonding polytetrafluoroethylene or silicone encapsulated nitinol glue receptor to human FM. Then, we exposed pregnant sheep uterus through a laparotomy and placed a 10-French trocar into the amniotic cavity through which the umbrella-shaped glue receptor (<i>n</i> = 9) was inserted and fixated onto the FM with the tissue glues (<i>n</i> = 8). The tightness of the sealed defects was assessed 4 h post-surgery. <b><i>Results:</i></b> Both tissue glues tested resulted in adhesion of the glue receptors to the FM ex vivo. In vivo, all glue receptors opened in the amniotic cavity (<i>n</i> = 9) and all successfully placed glue receptors sealed the FM defect (<i>n</i> = 8). Four hours post-surgery, 2 treatment sites showed minimal leakage whereas the negative control without glue (<i>n</i> = 1) showed substantial leakage. <b><i>Discussion:</i></b> This in vivo study confirms that fetoscopically induced FM defects can be sealed by the application of tissue adhesives.
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