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      Actually Seeing What Is Going on – Intravital Microscopy in Tissue Engineering

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          Abstract

          Intravital microscopy (IVM) study approach offers several advantages over in vitro, ex vivo, and 3D models. IVM provides real-time imaging of cellular events, which provides us a comprehensive picture of dynamic processes. Rapid improvement in microscopy techniques has permitted deep tissue imaging at a higher resolution. Advances in fluorescence tagging methods enable tracking of specific cell types. Moreover, IVM can serve as an important tool to study different stages of tissue regeneration processes. Furthermore, the compatibility of different tissue engineered constructs can be analyzed. IVM is also a promising approach to investigate host reactions on implanted biomaterials. IVM can provide instant feedback for improvising tissue engineering strategies. In this review, we aim to provide an overview of the requirements and applications of different IVM approaches. First, we will discuss the history of IVM development, and then we will provide an overview of available optical modalities including the pros and cons. Later, we will summarize different fluorescence labeling methods. In the final section, we will discuss well-established chronic and acute IVM models for different organs.

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          Modeling Physiological Events in 2D vs. 3D Cell Culture

          Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research.
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            2D and 3D cell cultures – a comparison of different types of cancer cell cultures

            Cell culture is a widely used in vitro tool for improving our understanding of cell biology, tissue morphology, and mechanisms of diseases, drug action, protein production and the development of tissue engineering. Most research regarding cancer biology is based on experiments using two-dimensional (2D) cell cultures in vitro. However, 2D cultures have many limitations, such as the disturbance of interactions between the cellular and extracellular environments, changes in cell morphology, polarity, and method of division. These disadvantages led to the creation of models which are more closely able to mimic conditions in vivo. One such method is three-dimensional culture (3D). Optimisation of the culture conditions may allow for a better understanding of cancer biology and facilitate the study of biomarkers and targeting therapies. In this review, we compare 2D and 3D cultures in vitro as well as different versions of 3D cultures.
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              In vivo three-photon microscopy of subcortical structures within an intact mouse brain

              Two-photon fluorescence microscopy (2PM) 1 enables scientists in various fields including neuroscience 2,3 , embryology 4 , and oncology 5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of 2PM within the mouse brain to the cortical layer, and imaging subcortical structures currently requires the removal of overlying brain tissue 3 or the insertion of optical probes 6,7 . Here we demonstrate non-invasive, high resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy (3PM) at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein (RFP)-labeled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher order nonlinear excitation overcomes the limitations of 2PM, enabling biological investigations to take place at greater depth within tissue.

                Author and article information

                Contributors
                Journal
                Front Bioeng Biotechnol
                Front Bioeng Biotechnol
                Front. Bioeng. Biotechnol.
                Frontiers in Bioengineering and Biotechnology
                Frontiers Media S.A.
                2296-4185
                17 February 2021
                2021
                : 9
                : 627462
                Affiliations
                Department of Plastic and Hand Surgery, University Hospital of Erlangen, Friedrich–Alexander University Erlangen–Nürnberg (FAU) , Erlangen, Germany
                Author notes

                Edited by: Jetze Visser, Radboud University Medical Center, Netherlands

                Reviewed by: Roberto Weigert, National Institute of Dental and Craniofacial Research (NIDCR), United States; Lia Rimondini, University of Eastern Piedmont, Italy

                This article was submitted to Biomaterials, a section of the journal Frontiers in Bioengineering and Biotechnology

                Article
                10.3389/fbioe.2021.627462
                7925911
                33681162
                1193d441-53ae-499d-9851-4dcf22f41432
                Copyright © 2021 Vaghela, Arkudas, Horch and Hessenauer.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 09 November 2020
                : 26 January 2021
                Page count
                Figures: 5, Tables: 2, Equations: 0, References: 174, Pages: 18, Words: 0
                Categories
                Bioengineering and Biotechnology
                Review

                tissue engineering,intravital microscopy,leukocyte recruitment,biomaterial,fluorescence,in vivo

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