In vitro vascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture

The bioengineering of artificial tissue constructs requires special attention to their fast vascularization to provide cells with sufficient nutrients and oxygen. We addressed the challenge of in vitro vascularization by employing a combined approach of cell sheet engineering, 3D printing, and cellular self-organization in dynamic maturation culture. A confluent cell sheet of human umbilical vein endothelial cells (HUVECs) was detached from a thermoresponsive cell culture substrate and transferred onto a 3D-printed, perfusable tubular scaffold using a custom-made cell sheet rolling device. Under indirect co-culture conditions with human dermal fibroblasts (HDFs), the cell sheet-covered vessel mimic embedded in a collagen gel together with additional singularized HUVECs started sprouting into the surrounding gel, while the suspended cells around the tube self-organized and formed a dense lumen-containing 3D vascular network throughout the gel. The HDFs cultured below the HUVEC-containing cell culture insert provided angiogenic support to the HUVECs via molecular crosstalk without competing for space with the HUVECs or inducing rapid collagen matrix remodeling. The resulting vascular network remained viable under these conditions throughout the 3 week cell culture period. This static indirect co-culture setup was further transferred to dynamic flow conditions, where the medium perfusion was enabled via two independently addressable perfusion circuits equipped with two different cell culture chambers, one hosting the HDFs and the other hosting the HUVEC-laden collagen gel. Using this system, we successfully connected the collagen-embedded HUVEC culture to a dynamic medium flow, and within 1 week of the dynamic cell culture, we detected angiogenic sprouting and dense microvascular network formation via HUVEC self-organization in the hydrogel. Our approach of combining a 3D-printed and cell sheet-covered vascular precursor that retained its sprouting capacity together with the self-assembling HUVECs in a dynamic perfusion culture resulted in a vascular-like 3D network, which is a critical step toward the long-term vascularization of bioengineered in vitro tissue constructs

Th2 and metabolic responses to nematodes are independent of prolonged host microbiota abrogation

Antibiotic treatment can lead to elimination of both pathogenic bacteria and beneficial commensals, as well as to altered host immune responses. Here, we investigated the influence of prolonged antibiotic treatment (Abx) on effector, memory and recall Th2 immune responses during the primary infection, memory phase and secondary infection with the small intestinal nematode Heligmosomoides polygyrus. Abx treatment significantly reduced gut bacterial loads, but neither worm burdens, nor worm fecundity in primary infection were affected, only worm burdens in secondary infection were elevated in Abx treated mice. Abx mice displayed trends for elevated effector and memory Th2 responses during primary infection, but overall frequencies of Th2 cells in the siLP, PEC, mLN and in the spleen were similar between Abx treated and untreated groups. Gata3+ effector and memory Th2 cytokine responses also remained unimpaired by prolonged Abx treatment. Similarly, the energy production and defence mechanisms of the host tissue and the parasite depicted by NAD(P)H fluorescence lifetime imaging (FLIM) did not change by the prolonged use of antibiotics. We show evidence that the host Th2 response to intestinal nematodes, as well as host and parasite metabolic pathways are robust and remain unimpaired by host microbiota abrogation

NAD(P)H fluorescence lifetime imaging of live intestinal nematodes reveals metabolic crosstalk between parasite and host

Infections with intestinal nematodes have an equivocal impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. One basic mechanism governing parasite and host cellular and tissue functions, metabolism, has largely been neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic activity and possible metabolic pathways in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via metabolic pathways resembling oxidative phosphorylation/aerobic glycolysis features. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis-like and effective oxidative phosphorylation-like metabolic pathways, towards mainly anaerobic glycolysis-like pathways, back to oxidative phosphorylation/aerobic glycolysis-like pathways during their different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of NAD(P)H-FLIM in live tissues constitutes a unique tool to study possible shifts between metabolic pathways in host-parasite crosstalk, in various parasitic intestinal infections

Application to the Analysis of Germinal Center Reactions In Vivo

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method

Analyzing nicotinamide adenine dinucleotide phosphate oxidase activation in aging and vascular amyloid pathology

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive O−2, which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age- dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APPE693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein

Vektoranalytischer Fluoreszenz-Lebensdauer-Mikroskopie-Ansatz: neue Einblicke in den Zellstoffwechsel und die Kalzium-Signalübertragung, in vitro und in vivo

The ability to dynamically observe functional processes at the (sub-) cellular level holds a high potential to answer open questions in medical biology, e.g. immunology. The ubiquitous co-enzymes nicotinamide adenine dinucleotide (NADH) and its phosphorized variant (NADPH) play a major role in metabolic processes, for instance in the production of the energy carrier ATP, or in the generation of reactive oxygen species (ROS) for cellular defense against invading pathogens. Since these two co-enzymes are autofluorescent, they provide an endogenous sensor to study those basic mechanisms label-free in vitro and in vivo using multi-photon microscopy. When NADH/NADPH bind to another enzyme to catalyze one of these processes, the molecular structure of the assembled enzyme compartment changes and thus (in contrast to their emission wavelengths) their fluorescence lifetime, meaning the time the molecule remains in the excited state before it emits light. However, the evaluation and interpretation of NAD(P)H fluorescence lifetime images (FLIM) in real biological environments are challenging. Here, the method of phasor analysis has been adopted and applied to stimulated/non-stimulated ROS-producing lymphocytes. This way it was possible to directly image the temporal dynamics of NADPH oxidase activation and its requirement for triggering NETosis in phagocyting neutrophils. In order to meet the growing demand for a systematic interpretation of NAD(P)H-FLIM measurements, especially to reflect the complex distribution and activity of NAD(P)H-dependent enzymes, the phasor approach was extended by the analysis of the phase vectors involved. For this purpose, a priori knowledge about the specific fluorescence lifetimes of the most abundant enzymes in tissue was created by measuring NADH or NADPH with the respective enzyme in solution. Applied to 3T3-L1 cell line or the data of phagocyting neutrophils, this method reveals insights into the enzyme composition and its enzymatic activity, which was shown to be even more complex than the pure FLIM images suggest. In addition to NAD(P)H-FLIM, the phasor approach and the vector analysis application to extract further information from the FLIM images was applied to Förster resonance energy transfer (FRET). The CD19+ lymphocytes of the reporter mouse line YellowCaB, carry the genetically encoded calcium-sensitive FRET-construct TN-XXL. In the presence of calcium ions, a second messenger of signal transduction, this construct is folded and the donor quenched. The fluorescence lifetime of the donor is shortened proportionally to the cytosolic Ca2+ concentration. Using vector analysis in the phase domain, maps of the absolute calcium ion concentration of the different B-cell populations in the germinal center of the lymph node were generated and their cell-to-cell interaction visualized. Together, both NAD(P)H-FLIM and donor-FRET-FLIM, hold the power to map mechanisms in cellular metabolism, defense, and intercellular communication, providing new insights into their interaction.Funktionelle Prozesse auf (sub-) zellulärer Ebene dynamisch beobachten können, birgt ein hohes Potential ausstehende biomedizinische Fragestellungen z.B. der Immunologie beantworten zu können. Die ubiquitären Co-Enzyme Nicotinamid-Adenin-Dinucleotid (NADH) und seine phosphorisierte Variante (NADPH) spielen eine wichtige Rolle bei Stoffwechselprozessen, zum Beispiel bei der Produktion des Energieträgers ATP oder bei der Bildung reaktiver Sauerstoffspezies (ROS) zur zellulären Abwehr eindringender Krankheitserreger. Da diese beiden Co-Enzyme autofluoreszierend sind, liefern sie einen endogenen Sensor, um diese grundlegenden Mechanismen markierungsfrei in vitro und in vivo mittels Multiphotonenmikroskopie zu untersuchen. Wenn sich diese Co-Enzyme an ein weiteres Enzym binden, um einen dieser Prozesse zu katalysieren, verändert sich der molekulare Aufbau des Gesamtkonstrukts und damit (im Gegensatz zu ihrer Emissionswellenlänge) die Fluoreszenzlebensdauer, also die Zeit, die das Moleküle im angeregten Zustand verweilt, bevor es fluoresziert. Allerdings ist seit langem bekannt, dass sowohl die Auswertung als auch die Interpretation einer solchen Lebenszeitmessung in einer realen biologischen Umgebung eine Herausforderung ist. In der vorliegenden Arbeit wurde die Methode der phasor-Analyse übernommen und auf stimulierte/ nicht-stimulierte ROS-produzierende Lymphozyten angewendet. Auf diese Weise war es möglich, die zeitliche Dynamik der NADPH-Oxidase-Aktivierung und ihre Voraussetzung für die Auslösung der NETosis in phagozytierenden Neutrophilen direkt abbilden. Um den wachsenden Bedarf einer systematischen Interpretation von NAD(P)H-Fluoreszenzlebenszeitaufnahmen zu begegnen, speziell um die komplexe Verteilung und Aktivität der NAD(P)H-abhängigen Enzymen wiedergeben zu können, wurde die phasor-Anwendung um eine Analyse der beteiligen Phasenvektoren erweitert. Zu diesem Zweck wurde durch die Messung von NADH oder NADPH mit dem jeweiligen Enzym in Lösung a priori Wissen über die spezifischen Fluoreszenzlebensdauern der am häufigsten im Gewebe vorkommenden Enzyme geschaffen. Angewendet auf die 3T3-L1 Zelllinie oder die Daten von phagozytierenden Neutrophilen, offenbart diese Methode Einblicke in die Enzymzusammensetzung und enzymatische Aktivität, die noch komplexer ist als die reinen FLIM-Bilder vermuten lassen. Neben NAD(P)H-FLIM kann die phasor-Anwendung und die Herangehensweise der Vektoranalyse zum Extrahieren weiterer Informationen aus den FLIM-Aufnahmen auch auf den Förster-Resonanzenergietransfer (FRET) angewendet werden. Die CD19+ Lymphozyten der Reporter Mauslinie YellowCaB tragen genetisch kodiert das calciumsensitive FRET-Konstrukt TN-XXL. In Anwesenheit von Kalziumionen, einem sekundären Botenstoff der Signaltransduktion, wird das Konstrukt gefaltet und der donor gequenched, dessen Fluoreszenzlebensdauer sich proportional zur zytosolischen Ca2+ Konzentration verkürzt. Mit Hilfe der Vektoranalyse in der Phasenebene wurden hier Karten der absoluten Kalziumionenkonzentration verschiedener B-Zellpopulationen im Keimzentrum des Lymphknotens erstellt und auf diese Weise deren Zell-zu-Zell-Kommunikation sichtbar gemacht. Zusammen sind beide, NAD(P)H-FLIM und donor-FRET-FLIM, in der Lage, Mechanismen des Zellstoffwechsels, der zellulären Abwehr und der interzellulären Kommunikation abzubilden und eröffnen so neue Einblicke in deren Zusammenspiel

Two-Photon Excitation Spectra of Various Fluorescent Proteins within a Broad Excitation Range

Two-photon excitation fluorescence laser-scanning microscopy is the preferred method for studying dynamic processes in living organ models or even in living organisms. Thanks to near-infrared and infrared excitation, it is possible to penetrate deep into the tissue, reaching areas of interest relevant to life sciences and biomedicine. In those imaging experiments, two-photon excitation spectra are needed to select the optimal laser wavelength to excite as many fluorophores as possible simultaneously in the sample under consideration. The more fluorophores that can be excited, and the more cell populations that can be studied, the better access to their arrangement and interaction can be reached in complex systems such as immunological organs. However, for many fluorophores, the two-photon excitation properties are poorly predicted from the single-photon spectra and are not yet available, in the literature or databases. Here, we present the broad excitation range (760 nm to 1300 nm) of photon-flux-normalized two-photon spectra of several fluorescent proteins in their cellular environment. This includes the following fluorescent proteins spanning from the cyan to the infrared part of the spectrum: mCerulean3, mTurquoise2, mT-Sapphire, Clover, mKusabiraOrange2, mOrange2, LSS-mOrange, mRuby2, mBeRFP, mCardinal, iRFP670, NirFP, and iRFP720

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions