Abstract
Esophageal engineering aims to create replacement solutions by generating hollow organs using a combination of cells, scaffolds, and regeneration-stimulating factors. Currently, the fate of cells on tissue-engineered grafts is generally determined retrospectively by histological analyses. Unfortunately, quality-controlled cell seeding protocols for application in human patients are not standard practice. As such, the field requires simple, fast, and reliable techniques for non-invasive, highly specific cell tracking. Here, we show that bioluminescence imaging (BLI) is a suitable method to track human mesoangioblast seeding of an esophageal tubular construct at every stage of the preclinical bioengineering pipeline. In particular, validation of BLI as longitudinal quantitative assessment of cell density, proliferation, seeding efficiency, bioreactor culture, and cell survival upon implantation in vivo was performed against standard methods in 2D cultures and in 3D decellularized esophageal scaffolds. The technique is simple, non-invasive, and provides information on mesoangioblast distribution over entire scaffolds. Bioluminescence is an invaluable tool in the development of complex bioartificial organs and can assist in the development of standardized cell seeding protocols, with the ability to track cells from bioreactor through to implantation.Impact StatementMethodologies for incorporation of cells into tissue-engineered grafts, particularly at the later preclinical stages, are suboptimal and non-validated, and monitoring cell fate within scaffolds cultured in bioreactors and in vivo is challenging. In this study, we demonstrate how bioluminescence imaging (BLI) can overcome these difficulties and allow quantitative cell tracking at multiple stages of the bioengineering preclinical pipeline. Our robust bioluminescence-based approach allowed reproducible longitudinal monitoring of mesoangioblast localization and survival in 2D/3D tissue culture, in organ-scale bioreactors, and in vivo. Our findings will encourage the use of BLI in tissue engineering studies, improving the overall quality of cell–scaffold interaction research.
Highlights
Esophageal tissue engineering aims to create a suitable replacement by following the principle of engrafting cells onto a tubular scaffold, creating a functional tissue or organ.[1]
Primary human mesoangioblasts (hMABs) were transduced with a lentivirus carrying the pHIV-Luc-ZsGreen vector to express both ZsGreen fluorescent protein and luciferase
To avoid contamination with nontransduced cells, transduced hMABs were fluorescence-activated cell sorting (FACS)-sorted based on the expression of ZsGreen (Fig. 1C) and further expanded in culture before seeding
Summary
Esophageal tissue engineering aims to create a suitable replacement by following the principle of engrafting cells onto a tubular scaffold, creating a functional tissue or organ.[1] A common aim in tissue engineering endeavors is the development of robust, controlled protocols for cell seeding and 3D culturing.[2,3] While technology to seed cells onto scaffolds in controlled environments, or bioreactors, has developed rapidly, methods to monitor the fate of seeded cells have not kept pace.[4,5] As such, tracking the engraftment, survival, and proliferation of cells in esophageal constructs from organ-scale bioreactor studies through to in vivo implantation represents a challenge for the field, where validation is necessary for clinical translation.[6,7]. The mainstay methods to image and/or quantify cells on tissue-engineered esophageal scaffolds include scanning electron microscopy, metabolic activity assays, DNA quantification assays, flow cytometry, confocal microscopy, and histochemistry.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
