Name: technique to collect fungiform (taste) papillae from human tongue
Uploaded: 2010-09-18T17:00:00.000+00:00
Duration: 9 min 39 s
Description: Watch this Scientific Journal Video about Technique to Collect Fungiform Taste Papillae from Human Tongue at JoVE.com

Diese Arbeit wurde vom NIH R21 DC03969-01 (zu JGB) unterstützt. Wir danken Herrn Dr. Fritz Lischka für die Erfassung von einzelnen Geschmacksknospe Zellen, Dr. Tauf Huque für die Durchführung der RT-PCR in Abbildung 4B und Herr D. Bayley und Frau S. Alarcon für die technische Unterstützung gezeigt. M. Yanina Pepino ist ein Kerl von einem NIDAT32 DA07313 Washington University School of Medicine, St. Louis, MO (Cottler, LB PI) unterstützt.

<ol>
	<li>Breslin, P. A., Spector, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+taste+perception.">Mammalian taste perception.</a> Curr Biol. 26 (4), R148-R155 (2008).</li><li>Miller, I., Doty, R. I., Dekker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+peripheral+taste+system.">Anatomy of the peripheral taste system.</a> Handbook of Olfaction and Gustation. , 521-547 (1995).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Figure+12.67.">Figure 12.67.</a> Gray's Anatomy: The Anatomical Basis of Medicine and Surgery. , (1995).</li><li>Huque, T., Cowart, B. J., Dankulich-Nagrudny, L., Pribitkin, E. A., Bayley, D. L., Spielman, A., Feldman, I., S, R., Mackler, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sour+ageusia+in+two+individuals+implicates+ion+channels+of+the+ASIC+and+PKD+families+in+human+sour+taste+perception+at+the+anterior+tongue.">Sour ageusia in two individuals implicates ion channels of the ASIC and PKD families in human sour taste perception at the anterior tongue.</a> PLoS One. 4 (10), e7347-e7347 (2009).</li><li>MacDonald, J. F., Mody, I., Salter, M. W., Spielman, A. I., Spielman, A. I., Brand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamping+of+taste+cells+in+mouse.">Patch-clamping of taste cells in mouse.</a> Experimental Cell Biology of Taste and Olfaction. , 329-332 (1995).</li></ol>

Ein bemerkenswerter Vorteil der Verwendung von menschlichen Zellen ist ihre Spezifität. Das Verfahren hier im Detail beschrieben bietet Zunge Gewebe für die Isolierung des Geschmacks-Rezeptor-Zellen aus Mensch. Diese Geschmacks-Zellen können für molekulare Studien eingesetzt werden, suche, um Moleküle wichtig für die Transduktion des Geschmacks Informationen zu entdecken, und für die Kalzium-Imaging-Studien, die Geschmack-Zell-Aktivität zu überwachen, um Reize. Darüber hinaus kann die Zunge Gewebe durch dieses Verfahren mit verschiedenen Techniken wie Immunhistochemie, in situ Hybridisierung analysiert werden, und ermöglicht die Erfassung einzelner Zellen für molekulare Verfahren wie Polymerase Chain Reaction (PCR), einschließlich Real Time Quantitative Single Cell PCR. Da dieses Verfahren auf Personen, deren Sinn für Geschmack kann psychophysisch ausgewertet werden vor Biopsie durchgeführt werden kann, ist es möglich, ein klares Bild von den molekularen Details, die für Geschmack Anomalien (siehe z. B. Huque et al. 2009) erhalten. Diese Biopsie ermöglicht die Herstellung eines tragfähigen und metabolisch intakten Zelle Vorbereitung in Zellen von Geschmacksknospen angereichert und somit die Fähigkeit, Patch Clamp Messungen an diesen Zellen durchführen. Mit dieser Technik könnten künftige Studien nicht nur unser Verständnis des Geschmacks Transduktionsprozesse auf den Menschen beschränkt, sondern auch entschlüsseln molekularen Mechanismen Geschmack Anomalien in Krankheit oder Medikamenten-induzierte Zustände und entwickeln Therapien, um diese Anomalien zu behandeln. Schließlich werden die Ergebnisse unserer Beobachtungen deuten stark darauf hin, dass die Anwesenheit von &quot;Löchern&quot;, oft auf pilzförmigen Papillen in der Nähe von Verzweigungen zur Kenntnis genommen und allgemein als Geschmacksknospe Poren werden, sind in der Tat nicht gut Prädiktoren für die Existenz der Geschmacksnerven. Genau das, was die Natur dieser Löcher ist, oder welche Funktionen sie ausführen, wissen wir nicht. In 8 Jahren haben wir dieses Verfahren begann, haben wir immer wieder von mehreren Dutzend Freiwillige über 4000 Geschmack Papillen gesammelt. Zwei negative Inzidenz, sowohl mit der lokalen Anästhesie führten zu einer vorübergehenden Schwellung der Zunge, aber entschlossen innerhalb der nächsten 48 Stunden ohne Nebenwirkungen. Wenn richtig, unter sterilen Bedingungen durchgeführt und atraumatische, ist diese Methode sicher und wirksam.

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technique to collect fungiform (taste) papillae from human tongue

Der Geschmackssinn ist von entscheidender Bedeutung für das menschliche Leben. Er informiert den Körper über die Qualität von Lebensmitteln, die möglicherweise verschluckt werden und regt Stoffwechselprozesse, die den Verdauungstrakt Vorbereitung für die Verdauung. Stetige Fortschritte auf dem Weg zu Verständnis der frühen biochemischen und molekularen Ursachen Geschmack Transduktion (für eine Übersicht, Breslin und Spector, 2008 1) hergestellt. Allerdings hat die bisherigen Fortschritte im Wesentlichen aus Tiermodellen geführt. Doch, da deutliche Unterschiede in der Rezeptor-Spezifität und Rezeptor-Dichte zwischen den Arten variieren, wird die menschliche Geschmack Transduktion nur mit menschlichen Geschmack Gewebe verstanden werden. Hier beschreiben wir eine Biopsie-Technik für die menschliche pilzförmigen Papillen, sichtbar als runde rosa vorderen Strukturen, etwa 0,5 mm im Durchmesser zu sammeln, die Geschmacksknospen enthalten. Diese biopsiert Papillen sind für verschiedene Zwecke einschließlich der Trennung von lebensfähigen Geschmacksknospe Zellen in situ Hybridisierung, Immunhistochemie und durch Techniken der Molekularbiologie, die Identifizierung des Geschmacks-spezifische neuartige Proteine ​​verwendet.

Watch this Scientific Journal Video about Technique to Collect Fungiform Taste Papillae from Human Tongue at JoVE.com

Technique to Collect Fungiform Taste Papillae from Human Tongue

Die Kenntnis der zugrunde liegenden molekularen Mechanismen Geschmacks Transduktion hat kürzlich bedeutende Fortschritte genossen, vor allem aufgrund der Verwendung von Tiermodellen. Doch die große Vielfalt in Geschmack Sensitivität und Spezifität bei Säugetieren Optionsscheinen Studien in menschlichem Gewebe. Wir beschreiben eine Biopsie-Technik auf lebende Geschmack Zellen aus der Papillen auf die menschliche Zunge zu sammeln.

Technik zu pilzförmigen (Taste) Papillen aus menschlichen Zunge sammeln

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

technique-to-collect-fungiform-taste-papillae-from-human-tongue

Nanyang Technological University

Research

JoVE Journal

Medicine

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

28.9K Aufrufe.  College of Dentistry, New York University. Die Kenntnis der zugrunde liegenden molekularen Mechanismen Geschmacks Transduktion hat kürzlich bedeutende Fortschritte genossen, vor allem aufgrund der Verwendung von Tiermodellen. Doch die große Vielfalt in Geschmack Sensitivität und Spezifität bei Säugetieren Optionsscheinen Studien in menschlichem Gewebe. Wir beschreiben eine Biopsie-Technik auf lebende Geschmack Zellen aus der Papillen auf die menschliche Zunge zu sammeln.

Serie: Technique to Collect Fungiform Taste Papillae from Human Tongue

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce decellularized tongue extracellular matrix (TEM) which provides functional scaffolds for TSCC construction. TEM provides an in vitro niche for cell growth, differentiation, and cell migration. The microstructures of native extracellular matrix (ECM) and biochemical compositions retained in the decellularized matrix provide tissue-specific niches for anchoring cells. The fabrication of TEM can be realized by deoxyribonuclease (DNase) digestion accompanied with a serious of organic or inorganic pretreatment. This protocol is easy to operate and ensures high efficiency for the decellularization. The TEM showed favorable cytocompatibility for TSCC cells under static or stirred culture conditions, which enables the construction of the TSCC model. A self-made bioreactor was also used for the persistent stirred condition for cell culture. Reconstructed TSCC using TEM showed the characteristics and properties resembling clinical TSCC histopathology, suggesting the potential in TSCC research.

Fabrication of Tongue Extracellular Matrix and Reconstitution of Tongue Squamous Cell Carcinoma In Vitro

A method is shown here for the preparation of the tongue extracellular matrix (TEM) with efficient decellularization. The TEM can be used as functional scaffolds for the reconstruction of a tongue squamous cell carcinoma (TSCC) model under static or stirred culture conditions.

Herstellung von Zunge extrazellulären Matrix und Rekonstitution der Zunge Squamous Zelle Krebsgeschwür In-vitro-

In order to construct an effective and realistic model for tongue squamous cell carcinoma (TSCC) in vitro, the methods were created to produce ...

Forschung

Krebsforschung

Isolation and Culture of Human Fungiform Taste Papillae Cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8. We recently reported on a successful method for the extended culture of rodent taste cells9. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C &beta;2, (PLC-&beta;2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-&beta;2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation.
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 201110.

We aimed to develop a reproducible protocol for isolating and maintaining long-term cultures of human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability.

Isolierung und Kultivierung humaner Zellen pilzförmige Papillen Geschmack

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple ...

Neurowissenschaften

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

Mess Oral-Fettsäure-Schwellenwerte, Fett Wahrnehmung, Fatty Essen gefallen, und Papillen Dichte in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller &#38; Reedy&#8217;s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller &#38; Reedy&#8217;s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller &#38; Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Here we present a protocol to measure fungiform papilla density from digital photographs. This method builds prioritization and objective characteristic metrics into the original descriptive work on fungiform papillae by Miller &#38; Reedy (1990).

Denver Papillen Protokoll für objektive Analyse der pilzförmige Papillen

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

Geschmack-Prüfung: Eine kurze und validierten Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

Medizin

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

Färbung, Visualisierung und Analyse von Fungiform-, Circumvallate- und Gaumengeschmacksknospen

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

μTongue: Eine mikrofluidikbasierte funktionelle Bildgebungsplattform für die Zunge in vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

<meta charset="utf8"></head><body>Generierung und Charakterisierung von organoiden Kulturen der murinen Mundschleimhaut

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

Generierung und Charakterisierung von organoiden Kulturen der murinen Mundschleimhaut

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

Biologie

The Tongue and Taste Buds

The surface of the tongue is covered with various small bumps called papillae, which either distribute what has been ingested (filiform papillae) or contain the sensory taste (or gustatory) receptor cells (fungiform, circumvallate, and foliate papillae). Embedded within each taste-related papilla are the taste buds&mdash;clusters of 30 to 100 gustatory receptor cells.

Gustatory receptor cells extend finger-like projections called gustatory hairs (or microvilli) into a region known as the taste pore. Here, many of the cells contain receptors that detect different tastants&mdash;the molecules that can be tasted. The average number of taste buds varies significantly among individuals, with estimates ranging from 2,000-10,000 taste buds. Taste cells have a lifespan of about 10-14 days and are continually replaced. Thus, each taste bud contains taste cells at different stages of development.

<h4>Different Types of Taste Papillae</h4>

Aside from the filiform papillae, which do not contain taste buds, the mushroom-shaped fungiform papillae are the most numerous. Fungiform papillae are predominantly located on the anterior two-thirds of the tongue and contain between one and eight taste buds each.

In contrast, the other two types of papillae&mdash;circumvallate and foliate&mdash;contain more than 100 taste buds per papilla. Circumvallate papillae, the largest type, are located at the back of the tongue in &ldquo;V&rdquo;-like formation. Nearby, on the sides of the tongue, are the foliate papillae.

<h4>The Myth of the Tongue&rsquo;s Taste Map</h4>

The work of many scientists, including Collings, Yanagisawa, and colleagues, indicates that the five basic tastes can be perceived anywhere across the tongue. Thus, distinct tastes are not restricted to particular regions, as typically indicated on tongue maps.

Die Zunge und die Geschmacksknospen

The surface of the tongue is covered with various small bumps called papillae, which either distribute what has been ingested (filiform papillae) or ...

Technik zu pilzförmigen (Taste) Papillen aus menschlichen Zunge sammeln

Zusammenfassung

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