Menschliche embryonale Stammzellen-Studien in der Teitell Labor werden von einem California Institute for Regenerative Medicine (CIRM) Saatgut gewähren RS1-00313 unterstützt. Wir danken Mitglieder der Broad Center of Regenerative Medicine and Stem Cell Research an der UCLA, vor allem Dr. Amander Clark, Dr. Jerome Zack, und die Mitglieder des UCLA Broad Institute Stem Cell Core Facility für ihre Unterstützung unserer Studien.

Splitting hESCs von Matrigel auf Matrigel Normalerweise wird eine konfluente 6-Well-Platte von hESCs auf Matrigel können split von 1.03 Uhr bis 01.05 Uhr zu einem anderen Matrigel Platte werden, mit dem Brunnen zu konfluenten wieder 4-5 Tage nach der Trennung. <ol><li> CM und Matrigel Platten werden wie oben beschrieben vor der Spaltung. </li><li> Am Tag der Aufspaltung, waschen jede Vertiefung für die Spaltung mit 1 x PBS, pH 7,4, 1ml von 1mg/ml Dispase (verdünnte 5mg/ml Stammlösung mit DMEM/F12 Medien) in jede Vertiefung, und bei 37 ° C für 3 min. </li><li> Wash jedes Well dreimal mit 1 × PBS, pH 7,4, 1ml von ES Medien ohne bFGF, verwenden Sie einen Zellschaber Kolonien aus dem Boden des Brunnens zu kratzen, sammeln die suspendierten hESCs in einem 50ml Falcon-Röhrchen und Pellet durch Zentrifugation bei 200g für 5 min bei Raumtemperatur. </li><li> Zum erneuten Platte der hESCs auf Matrigel Platten ersten Waschen die Matrigel-Platten mit DMEM/F12 und fügen 2,5 ml CM mit 10ng/ml bFGF pro Vertiefung ergänzt. Resuspendieren hESC Pellet in einem geeigneten Volumen von CM mit 10ng/ml bFGF, Pipette die Zellsuspension nach oben und unten drei Mal ergänzt. (Hinweis: Wenn hESCs zur Aufspaltung von Matrigel Platten kommen, sind die Klumpen sehr leicht zu brechen, deshalb drei Runden Pipettieren ist genug und mehr Pipettieren kann over-stören den Kolonien und Einzel-Zellen.) Aliquot von 0,5 ml hESC Klumpen Suspension pro auch der 6-well Matrigel Platte 3ml pro Vertiefung zu erreichen, wie ein Endvolumen. Die Platte wird in einem 37 ° C Inkubator. </li></ol> Nachweis der Pluripotenz Marker durch Immunfluoreszenz-Mikroskopie Immunfluoreszenzmikroskopie wird verwendet, um die Expression von humanen embryonalen Stammzellen Pluripotenz Marker Oct-4 und SSEA-4 während der Kultivierung zu untersuchen. <ol><li> Saugen Sie Medien aus einer gut und waschen mit 1 x PBS, pH 7,4. Add 2 ml 3,7% Formaldehyd zum Brunnen, um für 10 Minuten zu beheben. </li><li> Spülen Sie mit 1xPBS, pH 7,4, fügen 2ml Methanol zum Brunnen und bei -20 ° C für 2 min. </li><li> Spülen Sie mit 1xPBS, pH 7,4, und Block mit 1 ml 0,2% Rinder serume Albumin (BSA) für 5 min. </li><li> 1ml von Oct-4 oder anti-h/mSSEA-4 Antikörper (1:200 Verdünnung in 0,2% BSA) und Inkubation bei Raumtemperatur für 1 Stunde oder bei 4 ° C über Nacht. (Hinweis: für SSEA-4-Färbung, gehen Sie direkt zu Schritt 6) </li><li> Spülen Sie dreimal mit 1xPBS, pH 7,4, fügen FITC-konjugierten Kaninchen-IgG (1:500 Verdünnung in 0,2% BSA) und Inkubation bei Raumtemperatur für 1 Stunde. </li><li> Spülen Sie 3-mal mit 1xPBS, pH 7,4, und geben Sie einen Tropfen der Montage-Lösung (kann DAPI enthalten, wenn nukleare Visualisierung gewünscht wird) in der Mitte des Brunnens. Put Deckglas oben auf die Zellen, das Siegel mit Nagellack und evaluieren durch Immunfluoreszenz-Mikroskopie. </li></ol> Menschliche embryonale Stammzellen Einnahmen ES Medien (embryonale Stammzell-Medien): DMEM/F12 - 400ml Knockout Serum Replacer - 100ml Non-Essential Amino Acids - 5ml 200 mM Glutamax / BME-Lösung - 5ml Penicillin / Streptomycin - 5ml Gut durchmischen und endgültige hESC Nährmedien in 500ml-Flaschen bei 4 ° C. Gut für maximal zwei Wochen. MEF Culture Media: DMEM - 1000ml FBS - 100ml Non-Essential Amino Acids - 10ml Glutamin - 10ml Penicllin / Streptomycin - 10ml Gut durchmischen und endgültige MEF Nährmedien bei 4 ° C. 200 mM Glutamax / BME-Lösung: Glutamax - 100ml β-Mercaptoethanol (Stock-Lösung bei 14,3 Mio.) - 140 μ1 Mix beiden Lösungen gut und dann aliquoten 10ml Portionen. Keep Aliquots bei -20 ° C. bFGF-Lösung (Endkonzentration Lager: 10 Mikrogramm / ml): bFGF (FGF2) - 50 ug 0,1% BSA in 1xPBS, pH 7,4 - 5ml Lösen Sie 50 Mikrogramm bFGF in 5ml 0,1% BSA auf eine 10 ug / ml Aktie zu machen. Aliquot in 500 ul Portionen. Lagerung bei -80 ° C. Collagenase IV-Lösung (1mg/ml): Collagenase IV - 50mg DMEM/F12 Media - 50ml Fügen Sie die Kollagenase IV Pulver zu einer 50ml Falcon-Röhrchen. In der Motorhaube, in 50 ml sterile DMEM/F12 Media-Mix. Vortex die Lösung für &lt;1 min. um sicherzustellen, dass das Pulver aufgelöst ist. In der Motorhaube, durch ein 0,22 um-Filter in eine neue sterile 50ml Falcon-Röhrchen passieren. Diese Lösung ist gut für maximal 2 Wochen bei 4 ° C gelagert Dispase-Lösung (1mg/ml): Dispase 5mg/ml - 2ml DMEM/F12 Media - 8ml Aliquot kommerziellen 5mg/ml Dispase-Lösung und bei -20 ° C. Verdünnen Dispase zu 1mg/ml mit DMEM/F12. Diese Lösung kann für 1 Woche bei 4 ° C aufbewahrt werden

<ol>
	<li>Thomson, J. a. m. e. s. A., Itskovitz-Eldor, J. o. s. e. p. h., Shapiro, S. a. n. d. e. r. S., Waknitz, M. i. c. h. e. l. l. e. A., Swiergiel, J. e. n. n. i. f. e. r. J., Marshall, V. i. v. i. e. n. n. e. S., Jones, J. e. f. f. r. e. y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts+.">Embryonic Stem Cell Lines Derived from Human Blastocysts .</a> Science. 282, 1145-1147 (1998).</li><li>Xu, C. h. u. n. h. u. i., Inokuma, M. a. r. g. a. r. e. t. S., Denham, J. e. r. r. o. d., Golds, K. a. t. h. a. l. e. e. n., Kundu, P. r. a. t. i. m. a., Gold, J. o. s. e. p. h. D., Carpenter, M. e. l. i. s. s. a. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells+Nature+Biotechnology+19."> Feeder-free growth of undifferentiated human embryonic stem cells Nature Biotechnology 19.</a> Nature Biotechnology. 19, 971-974 (2001).</li></ol>

Diese Serie von 3 Videos demonstriert, wie humanen embryonalen Stammzellen (hES) am embryonalen Maus-Fibroblasten wachsen (MEF) Feeder-Zellen (Video 1), wie die Passage ihnen Feeder zellfreien Platten (Video 2) Matrigel, und wie halten hESCs durch Passagieren in Matrigel Feeder zellfreien Bedingungen (Video 3). Zahlreiche frühere Studien zeigten, dass die Wartung von lebensfähigen, undifferenzierte hES erfordert Kultur auf inaktivierten MEF Feeder-Zellen. Doch für viele Experimente, ist eine reine Population von hESCs frei von Feeder-Zel...

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<td>Knockout Serum Replacer (KSR)</td>
<td>Reagent</td>
<td>Gibco</td>
<td>10828-028</td>
<td>&nbsp;</td>
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<td>DMEM/F12</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11330-057</td>
<td>&nbsp;</td>
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<td> Non-essential Amino Acids</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11140-050</td>
<td>&nbsp;</td>
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<td>GlutaMax </td>
<td>Reagent</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
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<td>DMEM</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11995-065</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Clontech</td>
<td>631107</td>
<td>&nbsp;</td>
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<td>L-glutamine</td>
<td>Reagent</td>
<td>Gibco</td>
<td>25030-081</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>Fisher</td>
<td>BP176-100</td>
<td>&nbsp;</td>
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<td>Reagent</td>
<td>R&D Systems</td>
<td>233-FB-025</td>
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<td>Reagent</td>
<td>Gibco</td>
<td>17104-019</td>
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<td>Reagent</td>
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<td>Penicillin / Streptomycin </td>
<td>Reagent</td>
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<td>Reagent</td>
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<td>Matrigel</td>
<td>Reagent</td>
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<td>Oct-4 antibody</td>
<td>Reagent</td>
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<td>SC-9081</td>
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<td>anti-h/mSSEA-4 Phycoerythrin Conjugated Mouse IgG3</td>
<td>Reagent</td>
<td>R&D Systems</td>
<td>FAB1435P</td>
<td>&nbsp;</td>
<tr>
<td>FITCI-conjugated antirabbit IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Laboratories. Inc.</td>
<td>715-095-152</td>
<td>&nbsp;</td>		</tbody>
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from mefs to matrigel 3: passaging hescs from matrigel onto matrigel

Dieses Video zeigt, wie das Wachstum von humanen embryonalen Stammzellen (hES) in Feeder-Zell-freien Bedingungen und wie kontinuierlich Durchgang hESCs in Feeder-Zell-freien Bedingungen aufrecht zu erhalten. Bestätigung der hESC Pluripotenz gewachsen in Feeder-Zell-freien Bedingungen durch Immunfluoreszenz-Mikroskopie ist auch bewiesen.

Watch this Scientific Journal Video about From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel at JoVE.com

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

Von MEFs auf Matrigel 3: Die Passage hESCs von Matrigel auf Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

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Research

JoVE Journal

Biology

13.7K Aufrufe.  University of California, Los Angeles. Dieses Video zeigt, wie das Wachstum von humanen embryonalen Stammzellen (hES) in Feeder-Zell-freien Bedingungen und wie kontinuierlich Durchgang hESCs in Feeder-Zell-freien Bedingungen aufrecht zu erhalten. Bestätigung der hESC Pluripotenz gewachsen in Feeder-Zell-freien Bedingungen durch Immunfluoreszenz-Mikroskopie ist auch bewiesen.

Serie: From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Passagierung humaner neuraler Stammzellen

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Forschung

Biologie

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

Von MEFs auf Matrigel I: Die Passage hESCs in Gegenwart von MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Generieren von iPS-Zellen aus MEFS durch Forced Expression von Sox-2, Oct-4, c-Myc und Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

Von MEFs auf Matrigel 2: Splitting hESCs aus MEFs auf Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Frachtladesysteme auf Kinesin Powered Molekulare Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

An Introduction to Worm Lab: von Kultivierung Worms zu Mutagenese

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Ein Matrigel-Based Rohr Formation Assay zur vaskulären Aktivität von Tumorzellen Beurteilen

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Effizienten Herstellung und Reinigung von rekombinantem murinem Kindlin-3 von Insektenzellen für biophysikalische Studien

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generierung von Myospheres Von hESCs durch epigenetische Reprogrammierung

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Magnetische aktiviert Zelle Sortieren Strategien zu isolieren und reinigen Synovial Flüssigkeit abgeleitet mesenchymalen Stammzellen aus einem Kaninchen-Modell

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...