Evaluation of loop-mediated isothermal amplification for detecting COVID-19.

The emergence of SARS-CoV-2 has caused worldwide pandemic of COVID-19. Infection is difficult to diagnose early as some patients remain asymptomatic and may carry this virus to other people. Currently, qRT-PCR is the widely accepted mode for detection. However, the need for sophisticated instrument and trained personnel may hinder its application, especially in remote and facility-lacking areas. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) may serve as a potential approach for detection of SARS-CoV-2 as the resources needed in its application is far less complex than those of qRT-PCR. Herein, we evaluated RT-LAMP based analytical method (COVIDNow), relative to qRT-PCR, in detecting SARS-CoV-2 by using 63 clinical respiratory samples. Based on our finding, COVIDNow exhibited sensitivity and specificity values of 87.5% and 80.6%, respectively. Taken together, RT-LAMP based detection of SARS-CoV-2 by utilizing COVIDNow might serves as a valuable diagnostic tool in the management of global COVID-19 pandemic condition.

Novel ECM Patch Combines Poly(vinyl alcohol), Human Fibroblast-Derived Matrix, and Mesenchymal Stem Cells for Advanced Wound Healing.

Decellularized extracellular matrix (ECM)-based scaffold has been a very useful resource for effective tissue regeneration. In this study, we report a novel ECM patch that physically combines human fibroblast-derived matrix (hFDM) and poly(vinyl alcohol) (PVA) hydrogel. hFDM was obtained after decellularization of in vitro cultured human fibroblasts. We investigated the basic characteristics of hFDM alone using immunofluorescence (fibronectin, collagen type I) and angiogenesis-related factor analysis. Successful incorporation of hFDM with PVA produced an hFDM/PVA patch, which showed excellent cytocompatibility with human mesenchymal stem cells (hMSCs), as assessed via cell adhesion, viability, and proliferation. Moreover, in vitro scratch assay using human dermal fibroblasts showed a significant improvement of cell migration when treated with the paracrine factors originated from the hMSC-incorporated hFDM. To evaluate the therapeutic effect on wound healing, hMSCs were seeded on the hFDM/PVA patch and they were then transplanted into a mouse full-thickness wound model. Among four experimental groups (control, PVA, hFDM/PVA, hMSC/hFDM/PVA), we found that hMSC/hFDM/PVA patch accelerated the wound closure with time. More notably, histology and immunofluorescence demonstrated that compared to the other interventions tested, hMSC/hFDM/PVA patch could lead to significantly advanced tissue regeneration, as confirmed via nearly normal epidermis thickness, skin adnexa regeneration (hair follicle), mature collagen deposition, and neovascularization. Additionally, cell tracking of prelabeled hMSCs suggests the in vivo retention of transplanted cells in the wound region after the transplantation of hMSC/hFDM/PVA patch. Taken together, our engineered ECM patch supports a strong regenerative potential toward advanced wound healing.

A Fibrous Hybrid Patch Couples Cell-Derived Matrix and Poly(l-lactide-co-caprolactone) for Endothelial Cells Delivery and Skin Wound Repair.

Skin wound healing is an intricate orchestration that involves different cell types, an extracellular matrix (ECM), cytokines, and growth factors. On the basis of the great benefits of cell-derived ECM in regenerative applications, here we propose an electrospun fibrous membrane that combines poly(l-lactide-co-caprolactone) (PLCL) and human fibroblast-derived ECM (hFDM). hFDM-deposited PLCL (hFDM-PLCL) was obtained via decellularization of a confluent layer of fibroblasts cultivated on PLCL. An organized assembly of fibrillar structure on hFDM-PLCL was notable via immunostaining. As human umbilical vein endothelial cells (HUVECs) were seeded on the hFDM-PLCL, they proliferated faster and exhibited more elongated, capillary-like morphology than those on PLCL or fibronectin-coated PLCL (Fn-PLCL). HUVECs have a relatively large aspect ratio, spreading area, and vinculin-positive area per cell on the fibrillary structure of hFDM-PLCL. In addition, transwell cell migration assay showed the chemoattractant effect of hFDM for HUVECs and human dermal fibroblasts. Furthermore, HUVECs-loaded hFDM-PLCL membranes showed the most promising therapeutic effects on a mouse skin wound model as proved via the wound closure rate, neovascularization effect, regenerated epidermis, and skin appendage. This study shows that biodegradable PLCL fibers not only support the weak mechanical properties of hFDM but also allow hFDM to reserve ECM macromolecules and to maintain structural integrity. Current results also demonstrate the critical role of hFDM with biochemical and biophysical cues on HUVECs adhesion, proliferation, and vascular morphogenesis in vitro and even on the wound healing process in vivo. Taken together, hFDM-functionalized PLCL patch should be a promising platform for cell delivery and regenerative applications.

Novel skin patch combining human fibroblast-derived matrix and ciprofloxacin for infected wound healing.

Skin injuries are frequently encountered in daily life, but deep wounds often poorly self-heal and do not recover completely. In this study, we propose a novel skin patch that combines antibiotic, cell-derived extracellular matrix (ECM) and biocompatible polyvinyl alcohol (PVA) hydrogel. Methods: Decellularized human lung fibroblast-derived matrix (hFDM) was prepared on tissue culture plate (TCP) and PVA solution was then poured onto it. After a freeze-thaw process, PVA was peeled off from TCP along with hFDM tightly anchored to PVA. Subsequently, ciprofloxacin (Cipro)-incorporated PVA/hFDM (PVA/Cipro/hFDM) was fabricated via diffusion-based drug loading. Results: In vitro analyses of PVA/Cipro/hFDM show little cytotoxicity of ciprofloxacin, stability of hFDM, rich fibronectin in hFDM, and good cell attachment, respectively. In addition, hFDM proved to be beneficial in promoting cell migration of dermal fibroblasts and human umbilical vein endothelial cells (HUVECs) using transwell inserts. The antibacterial drug Cipro was very effective in suppressing colony growth of gram-negative and -positive bacteria as identified via an inhibition zone assay. For animal study, infected wound models in BALB/c mice were prepared and four test groups (control, PVA, PVA/Cipro, PVA/Cipro/hFDM) were administered separately and their effect on wound healing was examined for up to 21 days. The results support that Cipro successfully reduced bacterial infection and thus encouraged faster wound closure. Further analysis using histology and immunofluorescence revealed that the most advanced skin regeneration was achieved with PVA/Cipro/hFDM, as assessed via re-epithelialization, collagen texture and distribution in the epidermis, and skin adnexa (i.e., glands and hair follicles) regeneration in the dermis. Conclusion: This work demonstrates that our skin patch successfully consolidates the regenerative potential of ECM and the antibacterial activity of Cipro for advanced wound healing.

Human lung fibroblast-derived matrix facilitates vascular morphogenesis in 3D environment and enhances skin wound healing.

Extracellular matrix (ECM) is crucial to many aspects of vascular morphogenesis and maintenance of vasculature function. Currently the recapitulation of angiogenic ECM microenvironment is still challenging, due mainly to its diverse components and complex organization. Here we investigate the angiogenic potential of human lung fibroblast-derived matrix (hFDM) in creating a three-dimensional (3D) vascular construct. hFDM was obtained via decellularization of in vitro cultured human lung fibroblasts and analyzed via immunofluorescence staining and ELISA, which detect multiple ECM macromolecules and angiogenic growth factors (GFs). Human umbilical vein endothelial cells (HUVECs) morphology was more elongated and better proliferative on hFDM than on gelatin-coated substrate. To prepare 3D construct, hFDM is collected, quantitatively analyzed, and incorporated in collagen hydrogel (Col) with HUVECs. Capillary-like structure (CLS) formation at 7day was significantly better with the groups containing higher doses of hFDM compared to the Col group (control). Moreover, the group (Col/hFDM/GFs) with both hFDM and angiogenic GFs (VEGF, bFGF, SDF-1) showed the synergistic activity on CLS formation and found much larger capillary lumen diameters with time. Further analysis of hFDM via angiogenesis antibody array kit reveals abundant biochemical cues, such as angiogenesis-related cytokines, GFs, and proteolytic enzymes. Significantly up-regulated expression of VE-cadherin and ECM-specific integrin subunits was also noticed in Col/hFDM/GFs. In addition, transplantation of Col/hFMD/GFs with HUVECs in skin wound model presents more effective re-epithelialization, many regenerated hair follicles, better transplanted cells viability, and advanced neovascularization. We believe that current system is a very promising platform for 3D vasculature construction in vitro and for cell delivery toward therapeutic applications in vivo. STATEMENT OF SIGNIFICANCE: Functional 3D vasculature construction in vitro is still challenging due to the difficulty of recapitulating the complex angiogenic extracellular matrix (ECM) environment. Herein, we present a simple and practical method to create an angiogenic 3D environment via incorporation of human lung fibroblast-derived matrix (hFDM) into collagen hydrogel. We found that hFDM offers a significantly improved angiogenic microenvironment for HUVECs on 2D substrates and in 3D construct. A synergistic effect of hFDM and angiogenic growth factors has been well confirmed in 3D condition. The prevascularized 3D collagen constructs also facilitate skin wound healing. We believe that current system should be a convenient and powerful platform in engineering 3D vasculature in vitro, and in delivering cells for therapeutic purposes in vivo.

Novel Platform of Cardiomyocyte Culture and Coculture via Fibroblast-Derived Matrix-Coupled Aligned Electrospun Nanofiber.

For cardiac tissue engineering, much attention has been given to the artificial cardiac microenvironment in which anisotropic design of scaffold and extracellular matrix (ECM) are the major cues. Here we propose poly(l-lactide-co-caprolactone) and fibroblast-derived ECM (PLCL/FDM), a hybrid scaffold that combines aligned electrospun PLCL fibers and FDM. Fibroblasts were grown on the PLCL fibers for 5-7 days and subsequently decellularized to produce PLCL/FDM. Various analyses confirmed aligned, FDM-deposited PLCL fibers. Compared to fibronectin (FN)-coated electrospun PLCL fibers (control), H9c2 cardiomyoblast differentiation was significantly effective, and neonatal rat cardiomyocyte (CM) phenotype and maturation was improved on PLCL/FDM. Moreover, a coculture platform was created using multilayer PLCL/FDM in which two different cells make indirect or direct cell-cell contacts. Such coculture platforms demonstrate their feasibility in terms of higher cell viability, efficiency of target cell harvest (>95% in noncontact; 85% in contact mode), and molecular diffusion through the PLCL/FDM layer. Coculture of primary CMs and fibroblasts exhibited much better CM phenotype and improvement of CM maturity upon either direct or indirect interactions, compared to the conventional coculture systems (transwell insert and tissue culture plate (TCP)). Taken together, our platform should be very useful and have significant contributions in investigating some scientific or practical issues of crosstalks between multiple cell types.

Tunable Crosslinked Cell-Derived Extracellular Matrix Guides Cell Fate.

Extracellular matrix (ECM), comprised of multiple cues (chemical, physiomechanical), provides a niche for cell attachment, migration, and differentiation. Given that different cells give rise to distinct physiological milieus, the role of such microenvironmental cues on various cells has been well-studied. Particularly, the effect of various physiomechanical factors on stem cell lineage has been resolved into individual variables via ECM protein-coated polymeric systems. Such platforms, while providing a reductionist approach as a means to remove any confounding factors, unfortunately fall short of capturing the full biophysical scope of the natural microenvironment. Herein, the use of a cell-derived ECM platform is reported in which its crosslinking density is tunable; varying concentrations (0, 0.5, 1, 2% w/v) of genipin (GN), a naturally derived crosslinker with low toxicity, are used to form inter- and intrafibril crosslinks. ECM crosslinking produces GN concentration-dependent changes in ECM stiffness (<0.1-9.4 kPa), roughness (96-280 nm), and chemical composition (100-60% amine content). The effect of the various crosslinked ECM profiles on human mesenchymal stem cell differentiation, vascular morphogenesis, and cardiomyogenesis are then evaluated. Taken together, this study demonstrates that tunable crosslinked cell-derived ECM platform is capable of providing a comprehensive physiological platform, and envisions its use in future tissue engineering applications.

Erratum to: Investigation of the changes of biophysical/mechanical characteristics of differentiating preosteoblasts in vitro.

[This corrects the article DOI: 10.1186/s40824-015-0046-y.].

Elasticity Modulation of Fibroblast-Derived Matrix for Endothelial Cell Vascular Morphogenesis and Mesenchymal Stem Cell Differentiation.

Biophysical properties of the microenvironment, including matrix elasticity and topography, are known to affect various cell behaviors; however, the specific role of each factor is unclear. In this study, fibroblast-derived matrix (FDM) was used as cell culture substrate and physically modified to investigate the influence of its biophysical property changes on human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) behavior in vitro. These FDMs were physically modified by simply storing them at different temperatures: the one stored at 4°C, maintained its original properties, was considered natural FDM, whereas the ones stored at -20°C or -80°C, exhibited a distinct surface morphology, were considered physically modified FDM. Physical modification induced matrix fiber rearrangement in FDM, forming different microstructures on the surface as characterized by focused ion beam (FIB)-cryoSEM. A significant increase of matrix elasticity was found with physically modified FDMs as determined by atomic force microscopy. HUVEC and hMSC behaviors on these natural and physically modified FDMs were observed and compared with each other and with gelatin-coated coverslips. HUVECs showed a similar adhesion level on these substrates at 3 h, but exhibited different proliferation rates and morphologies at 24 h; HUVECs on natural FDM proliferated relatively slower and assembled to capillary-like structures (CLSs). It is observed that HUVECs assembled to CLSs on natural FDMs are independent on the exogenous growth factors and yet dependent on nonmuscle myosin II activity. This result indicates the important role of matrix mechanical properties in regulating HUVECs vascular morphogenesis. As for hMSCs multilineage differentiation, adipogenesis is improved on natural FDM that with lower matrix elasticity, while osteogenesis is accelerated on physically modified FDMs that with higher matrix elasticity, these results further confirm the crucial role of matrix elasticity on cell fate determination.

Investigation of the changes of biophysical/mechanical characteristics of differentiating preosteoblasts in vitro.

BACKGROUND: Topography, stiffness, and composition of biomaterials play a crucial role in cell behaviors. In this study, we have investigated biochemical (gene markers), biophysical (roughness), and biomechanical (stiffness) changes during the osteogenic differentiation of preosteoblasts on gelatin matrices. RESULTS: Our results demonstrate that gelatin matrices offer a favorable microenvironment for preosteoblasts as determined by focal adhesion and filopodia formation. The osteogenic differentiation potential of preosteoblasts on gelatin matrices is confirmed by qualitative (Alizarin red, von kossa staining, immunofluorescence, and gene expression) and quantitative analyses (alkaline phosphatase activity and calcium content). The biomechanical and biophysical properties of differentiating preosteoblasts are analyzed using atomic force microscopy (AFM) and micro indentation. The results show sequential and significant increases in preosteoblasts roughness and stiffness during osteogenic differentiation, both of which are directly proportional to the progress of osteogenesis. Cell proliferation, height, and spreading area seem to have no direct correlation with differentiation; however, they may be indirectly related to osteogenesis. CONCLUSIONS: The increased stiffness and roughness is attributed to the mineralized bone matrix and enhanced osteogenic extracellular matrix protein. This report indicates that biophysical and biomechanical aspects during in vitro cellular/extracellular changes can be used as biomarkers for the analysis of cell differentiation.