Die Entwicklung dieses Protokoll wurde von der California Institute for Regenerative Medicine (CIRM, TR1-01246) und Parkinson Allianz finanziert.

Die Haut Stanzbiopsie unter Verwendung Standardverfahren 3 (zB mit 4mm lang Visipunch Instrument erhalten) sollten in komplettem DMEM 20% FBS Medien auf Eis gehalten werden. Nachdem die Probe im Labor angekommen, verarbeiten die Biopsie so schnell wie möglich. 1. Vorbereitung der Haut Stanzbiopsie STEPS 1,1-1,3 SIND IN EINEM Biosicherheitswerkbank DURCHZUFÜHRENDEN <ol><li> Bereiten Sie im Voraus: In 1 ml 0,1% Gelatine in jede Vertiefung einer 6-Well-Platte. Stellen Sie den Teller beiseite für 30-60 min. Saugen Sie die Gelatine-Lösung und fügen Sie 800 ul komplette DMEM/20% FBS Medien in jede Vertiefung. Sicherstellen, dass die gesamte Oberfläche des Bohrlochs mit Medien abgedeckt wird. </li><li> Kehren Sie die Deckel eines sterilen 10 cm Gewebekulturschale und fügen Sie 1,5 ml DMEM / 20% FBS Medien auf die Mitte des Deckels und breitete die Medien Tropfen mit der Spitze des serologischen Pipette. </li><li> Mit einer sterilen Pinzette Platz ter Hautbiopsie Stück in den Medien auf dem Teller. </li></ol> 2. Dissektion der Haut Stanzbiopsie STEPS 9 2.1-2.2 SIND IN EINEM HORIZONTAL Laminarströmungshaube DURCHZUFÜHRENDEN <ol><li> Setzen Sie den unteren Teil 10 cm Schale auf umgekehrten Deckel, und übertragen Sie die Haut an den Binokular im Laminarströmungshaube Biopsie. </li><li> Präparieren Sie ein 4-mm rund Hautbiopsie in 12-15 gleich große Stücke mit scharfen Kanten durch Schneiden Stücke in gleiche Hälften mit einem Skalpell, um die Biopsie in Position zu halten und die zweite Skalpell mit einer rollenden Bewegung in eine Richtung geschnitten. Stücke mit ausgefranste Kanten tragen zur schlechten Befestigung / Zelle Auswuchs. </li></ol> 3. Übertragung von Dissected Hautbiopsie Pieces in Zellkulturplatten STEPS 3.1-3.6 SIND IN EINEM Biosicherheitswerkbank DURCHZUFÜHRENDEN <ol><li> Platzieren des Bodenabschnitts des 10 cm Zellkulturschalenauf der Oberseite der umgedrehten Deckel und Schüssel Transfer zurück in die Biosicherheitswerkbank. </li><li> Mit einem spitzen Pinzette, Platz 2-3 Biopsie Stücke in jede Vertiefung der vorbereiteten 6-Well-Platte mit 800 ul und nicht auf trockenen Brunnen. Verwenden Sie tippen oder gleitende Bewegung um die Stücke auf den Grund des Brunnens zu befestigen. Ein Skalpell ist nützlich, um keine Biopsie Stücke aus der Zange entfernen. </li><li> Legen Sie die 6-Well-Platte in der 37 ° C-Inkubator. Überwachen Sie täglich, um sicherzustellen, es ist ein Film von Medien Beschichtung der Boden der gut für die erste Woche, fügen ~ 200 ul alle 2 Tage, um alle verdampft Medien zu ersetzen. </li><li> Nach einer Woche erhöhen Anzahl der Medien bis 2 ml komplettem DMEM/20% FBS und wechseln Sie das Medium alle 2-3 Tage. </li><li> Sobald Fibroblasten sind in jeder Vertiefung konfluenten bis zu dem Punkt, wo die Fibroblasten Erreichen der Kanten der Bohrung, trypsinize und Kanal 6-Well-Platte in 2X T75-Flaschen (Kanal 1). Die Gewebestücke werden mit übertragen werden. Sie werden nicht an und gewaschen werden out bei der nächsten Änderung Medien. </li><li> Sobald Fibroblasten konfluent sind, übertragen Sie sie auf 3X T175-Flaschen (Durchgang 2), frieren in komplettem DMEM Medium plus 10% DMSO bei 1x10 6 Zellen / ml pro Fläschchen. </li></ol> 4. Charakterisierung der Fibroblasten durch Immunfärbung <ol><li> Kultur Fibroblasten in 8 gut Kammerobjektträgers mit Gelatine beschichtet. </li><li> Wenn die Zellen bei 80% Konfluenz sind, Saugen Sie das Medium aus jedem Well. </li><li> Fix Zellen in 4% Paraformaldehyd für zehn Minuten bei Raumtemperatur gerührt. </li><li> Waschen Sie die Wells 3-mal mit 1X PBS. </li><li> Permeabilisierung der Zellen mit 150 ul 0,3% Triton X-100 (in PBS) für 5 min bei Raumtemperatur gerührt. </li><li> Waschen Sie die Wells 3-mal mit 1X PBS. </li><li> Jeweils 200 ul Blockierungslösung (PBS + 5% normalem Ziegenserum (Vector, S-1000)) für 1 Stunde bei Raumtemperatur. </li><li> Bereiten Sie eine Verdünnung von 1:250 Anti-Rilit-1 (Anti-Kaninchen, mAB, Sigma, S5950-200 ul) in Blocking-Lösung. </li><li> Aspirate Blocking-Lösung, und fügen Sie 150 ul der 1:250 Verdünnung AntiSERPINH-1. Inkubation bei 1-2 Stunden bei Raumtemperatur oder bei 4 ° C über Nacht. </li><li> Waschen Sie die Wells 3-mal mit 1X PBS. </li><li> Bereiten Sie eine 1:200 Ziege-Anti-Kaninchen-Antikörper (Alexa Fluor Ziege Anti-Kaninchen-IgG (H + L), Invitrogen, A11008) in Blocking-Lösung. </li><li> In 150 ul 1:200 Ziege-Anti-Kaninchen monoklonaler Antikörper in jede Vertiefung. Inkubation für 1 h in der Dunkelheit bei Raumtemperatur. </li><li> Waschen Vertiefungen einmal mit 1xPBS. </li><li> Saugen Sie die PBS, und heben Sie die Kammern aus, um nur die Folie zu offenbaren. </li><li> Befestigen Sie die Folie mit einem 2-3 Tropfen Eindeckmedium enthält DAPI (Vector, H-1200). </li><li> Analysieren der Objektträger unter einem Fluoreszenzmikroskop. </li></ol>

<ol>
	<li>Nguyen, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress.">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li><li>Mak, S. K., Tewari, D., Tetrud, J. W., Langston, J. W., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+skin+fibroblasts+from+a+Parkinson's+disease+patient+with+an+alpha-synuclein+triplication.">Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.</a> Journal of Parkinson's Disease. 1, 175-183 (2011).</li><li>Punch Zuber, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=biopsy+of+the+skin.">biopsy of the skin.</a> Am. Fam. Physician. 65, 1155-1164 (2002).</li><li>Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+fibroblast+cultures.">Establishment of fibroblast cultures.</a> Curr. Protoc. Cell Biol. Chapter 2, Unit 2, 1 (2001).</li><li>Kuroda, K., Tajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP47+is+a+useful+marker+for+skin+fibroblasts+in+formalin-fixed,+paraffin+embedded+tissue+specimens.">HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin embedded tissue specimens.</a> J. Cutan. Pathol. , 241-246 (2004).</li></ol>

Die Autoren haben nichts zu offenbaren.

Mit diesem Protokoll kann relativ reine Kulturen von Fibroblasten gewonnen werden. Die Fibroblasten weisen charakteristische morphologische Merkmale von langgestreckten, spindelförmigen Zellkörper, runde bis ovale Zellkerne und die Fibroblasten wachsen ausgerichtet und in Bündeln, wenn konfluent. Das Medium unterstützt das Wachstum von Fibroblasten während andere Zellpopulationen zB Keratinozyten benötigen Zusatzgebühren und Wachstumsfaktoren oder sind mitotisch weniger aktiv, ermöglicht dies eine relat...

<table border="1">
<tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica</td>
 <td>S6E</td>
 </tr>
 <tr>
 <td>10 cm Tissue Culture Petri dish</td>
 <td>VWR</td>
 <td>25382-166</td>
 </tr>
 <tr>
 <td>6-well Tissue Culture plate </td>
 <td>VWR</td>
 <td>73520-906</td>
 </tr>
 <tr>
 <td>Stainless steel disposable sterile scalpels (2), blade No#15, Miltex</td>
 <td>VWR</td>
 <td>21909-660</td>
 </tr>
 <tr>
 <td>Sterile pointed-tip forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Conical tubes</td>
 <td>VWR</td>
 <td>21008-940</td>
 </tr>
 <tr>
 <td>2 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-894</td>
 </tr>
 <tr>
 <td>5 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-896</td>
 </tr>
 <tr>
 <td>10 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-898</td>
 </tr>
 <tr>
 <td>Pasteur Pipettes</td>
 <td>VWR</td>
 <td>14672-380</td>
 </tr>
 </tbody>
</table>
Table A. Table of specific reagents and equipments.
 <table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company </td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1X DMEM: High Glucose</td>
 <td>Invitrogen/Gibco</td>
 <td>11960-069</td>
 </tr>
 <tr>
 <td>20% Fetal Bovine Serum</td>
 <td>Invitrogen/Gibco</td>
 <td>26140-079</td>
 </tr>
 <tr>
 <td>1X L-Glutamine</td>
 <td>Invitrogen/Gibco</td>
 <td>25030-164</td>
 </tr>
 <tr>
 <td>1X MEM Non-essential Amino Acids </td>
 <td>Invitrogen/Gibco</td>
 <td>11140-076</td>
 </tr>
 <tr>
 <td>1X Penicillin/Streptomycin</td>
 <td>Invitrogen/Gibco</td>
 <td>15140-163</td>
 </tr>
 </tbody>
 </table>
Table B. Reagents.

skin punch biopsy explant culture for derivation of primary human fibroblasts

Geweben und Zelllinien von einem Individuum mit der Krankheit abgeleitet sind eine ideale Quelle zu krankheitsbedingten zellulären Phänotypen zu studieren. Patienten stammenden Fibroblasten in diesem Protokoll wurden erfolgreich in der Ableitung von induzierten pluripotenten Stammzellen zu modellieren Krankheit 1 verwendet. Frühen Passagen dieser Fibroblasten können auch zellbasierten funktionellen Assays verwendet werden, um spezifische Krankheitsbahnen Mechanismen 2 und nachfolgende Wirkstoff-Screening-Ansätzen zu studieren. Der Vorteil der vorgestellten Protokoll über enzymatische Verfahren sind 1) die Reproduzierbarkeit der Technik von kleinen Mengen von Gewebe von älteren Patienten, zB Patienten mit Parkinson-Krankheit, 2 betroffen) der technisch einfachen Ansatz über schwierigere Methoden mittels enzymatischer Behandlungen abgeleitet und 3 ) die Zeit berücksichtigt: Dieses Protokoll dauert 15-20 min und kann sofort nach der Ankunft Biopsie durchgeführt werden. Enzymatische Behandlungen kann bis zu 4 Stunden und haben die Probleme deroverdigestion, Verminderung der Lebensfähigkeit der Zellen und die anschließende Befestigung der Zellen, wenn sie nicht richtig gehandhabt. Dieses Protokoll beschreibt die Präparation und Herstellung einer 4-mm menschlichen Hautbiopsie zur Ableitung eines Fibroblasten-Kultur und hat eine sehr hohe Erfolgsquote, die wichtig ist, wenn man mit einem Patienten abgeleitete Gewebeproben. In dieser Kultur, Migration Keratinozyten aus der Biopsie Gewebe innerhalb der ersten Woche nach der Zubereitung. Fibroblasten erscheinen 7-10 Tage nach der ersten Folge von Keratinozyten. DMEM high glucose Medium mit 20% FBS begünstigt das Wachstum von Fibroblasten in Keratinozyten und Fibroblasten werden die Keratinozyten überwuchern. Nach 2 Passagen Keratinozyten wurden durchgeführt, was zu relativ homogenen Fibroblastenkulturen die die Fibroblasten-Marker SERPINH1 (HSP-47) drückt verdünnt. Mit diesem Ansatz können 15-20 Millionen Fibroblasten in 4-8 Wochen für die Zell-Banking abgeleitet werden. Die Haut Präparation dauert etwa 15-20 min werden die Zellen dann überwacht einmal täglichunter dem Mikroskop und Medien alle 2-3 Tage nach der Befestigung und Auswachsen der Zellen verändert.

Keratinozyten wachsen aus der Biopsie Stücke, sobald 48 Stunden nach der Sektion. Erste Fibroblasten Auswuchs kann etwa eine Woche nach der Verarbeitung zu beachten. Sobald die Brunnen Konfluenz erreicht haben, sind Fibroblasten für zwei weitere Passagen passagiert zu drei 150 cm Kolben (T150) zu erreichen und die Zellen werden kryokonserviert. Wir erzeugen mit dieser Methode 15-20 Millionen Zellen. Die Fibroblasten sind für anti-SERPINH1 (auch als HSP-47 bekannt) (Abbildung 1), die ein Kollagen-spez...

Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Die Fibroblasten Explantatkultur Protokoll aus der menschlichen Haut Stanzbiopsien ist eine technisch robuste und einfache Weise abzuleiten Hautzellen innerhalb von 4-8 Wochen für Banken von etwa 15-20 Millionen Zellen auf einem niedrigen Durchgang Nummer.

Haut Stanzbiopsie Explantatkultur für die Ableitung des primären humanen Fibroblasten

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

skin-punch-biopsy-explant-culture-for-derivation-primary-human

Research

JoVE Journal

Medicine

40.8K Aufrufe.  The Parkinson's Institute. Die Fibroblasten Explantatkultur Protokoll aus der menschlichen Haut Stanzbiopsien ist eine technisch robuste und einfache Weise abzuleiten Hautzellen innerhalb von 4-8 Wochen für Banken von etwa 15-20 Millionen Zellen auf einem niedrigen Durchgang Nummer.

Serie: Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Experimentelle Erzeugung von Karzinom-assoziierte Fibroblasten (CAF) von menschlichen Brustkrebszellen Fibroblasten

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Forschung

Medizin

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Die Verwendung von primären humanen Fibroblasten zur Überwachung Mitochondriale Phänotypen auf dem Gebiet der Parkinson-Krankheit

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In-vitro-Kultur von Organoide Primäre Maus Kolontumoren

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Eine dreidimensionale Zellkulturmodell zu primären humanen Knochenmark und seine Malignome Studieren

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Primäre Kultur der Menschen vestibuläre Schwannome

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Menschlichen Skelettmuskelbiopsie Verfahren unter Verwendung der modifizierten Technik Bergström

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Gründung des Menschen Epithelial Enteroids und Colonoids von Whole Gewebe und Biopsie

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Mensch<em&gt; Vastus lateralis</em&gt; Skeletal Muscle Biopsy die Weil-Blakesley Conchotom Verwendung

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Gewebecharakterisierung nach einer neuen Disaggregation Methode für Haut Micro-Grafts-Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Quantitative Visualisierung von Leukozyten-Infiltrat in einem Murin-Modell der Fulminant-Myokarditis durch Licht-Blatt-Mikroskopie

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...