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.

hESC Kultur tägliche Wartung <ol><li> hESC Kulturmedien müssen alle 24 Stunden gewechselt werden. Von der ES Medien Vorratsflasche bei 4 ° C gelagert, nehmen Sie die Menge der Lösung benötigt werden (~ 20ml pro 6-well-Platte), in einem 50ml Falcon-Röhrchen und warm bis 37 ° C in einem Wasserbad. Nachdem die Medien wird erwärmt, fügen bFGF Lagerung bei 4 ° C bis zu einer Endkonzentration von 10ng/ml. Legen Sie die bFGF Aktie wieder bei 4 ° C sofort nach Gebrauch! </li><li> Entfernen Sie aus dem 6-Well-Platten alle, aber ~ 500μl der Medien. Vergewissern Sie sich, wirbeln die Kulturschale zu Schutt zu entfernen auszusetzen. Sicherstellen, dass die bFGF in der hESC Kulturmedien wird gründlich gemischt und fügen 3 ml frisches Medium zurück in jede Vertiefung einer 6-Well-Platte. Legen Sie die Platte wieder in den Inkubator. </li></ol> Splitting hESCs aus MEFs auf Matrigel Normalerweise wird eine konfluente 6-Well-Platte von hESCs auf MEFs können von 1.02 Uhr bis 01.03 Uhr aufgeteilt werden auf Matrigel 6-well Platten mit den Vertiefungen zu konfluenten wieder 4-5 Tage nach der Trennung. <ol><li> Bei der Aufteilung hESCs auf Matrigel, MEF-konditionierte Medien (CM) wird verwendet, um zu halten Pluripotenz. MEF-konditionierten Medien erfolgt im Voraus. Am ersten Tag werden 1,5 × 10 7 γ-bestrahlten MEFs in eine T75 ausgesät. Am zweiten Tag sind MEFs einmal mit Raumtemperatur 1 × PBS, pH 7,4 gewaschen und anschließend 15 ml ES-Medien mit 5ng/ml bFGF ergänzt wird, um den Kolben gegeben. (HINWEIS: MEFs kann in kleineren oder größeren Kolben beschichtet werden, sondern Verhältnis zwischen Zelldichte und das Volumen der ES Medien sollten konstant sein.) Der Kolben bei 37 ° C wird für weitere 24 Stunden inkubiert. Am dritten Tag ist der CM gesammelt und mit 15 ml frischem ES-Medien mit 5ng/ml bFGF ergänzt ersetzt. Die gesammelten CM ist bei 4 ° C gelagert MEF-konditionierten Medien von einer Beschichtung von MEFs sind über sieben aufeinander folgenden Tagen geerntet bis 15 x 7 = 105ml generieren. Nach sieben Tagen sind alle CM Fraktionen vereinigt, sterilfiltriert, aliquotiert und bei -20 ° C. CM Media hergestellt, wie können gespeichert beschrieben und verwendet werden für 1 Monat. </li><li> Einen Tag vor hESC Splitting, legte Matrigel Aliquots in Eppendorf-Röhrchen bei 4 ° C auftauen über Nacht (ein Aliquot enthält 76.2mg von Matrigel, und dies ist der Betrag für eine 6-Well-Platte erforderlich). Am Tag der Aufspaltung, nehmen Sie eine Flasche des aufgetauten Matrigel für eine 6-Well-Platte, legen Sie die Flasche und 6ml der kalten DMEM/F12 Medien auf Eis. Übertragen Sie die Matrigel in die DMEM/F12 Medien und gut mischen. 1ml der Lösung in jede Vertiefung einer 6-Well-Platte. Schwenken Sie die Platte auf Matrigel auf der Oberfläche verteilen und inkubieren Sie die Matrigel-bedeckten Platte bei Raumtemperatur für mindestens 1 Stunde vor der Spaltung der hESCs. </li><li> hESCs auf MEFs werden einmal mit 1 × PBS, pH 7,4 gewaschen. Anschließend 1ml warmen Kollagenase IV (1mg/ml) in jede Vertiefung der 6-well-Platte gegeben und dann wird die Platte bei 37 ° C für 5-10 min. </li><li> 1ml von ES Medien ohne bFGF in jede Vertiefung. Verwenden Sie eine 1ml Pipette auf die Stammzellen von der Platte zu blasen. Sammeln Sie alle Medien mit Klumpen von hES und tote MEFs in ein 50ml Falcon-Röhrchen. Lassen Sie große Klumpen von Zellen für 5-10 Minuten absetzen, so dass die hESC Klumpen ein Pellet Formular am Ende der Röhre, während die MEFs im Überstand suspendiert bleiben. Überstand verwerfen, und waschen durch Resuspendieren des hESC Pellet mit ES Medien fehlt bFGF, durch Zentrifugation bei Raumtemperatur bei 200g folgte für 5 min. </li><li> Um Platte die Stammzellen auf Matrigel Platten ersten Waschen die Matrigel-Platten mit Raumtemperatur DMEM/F12 (1 ml pro well) und das Hinzufügen von 2,5 ml CM mit 10ng/ml bFGF pro Vertiefung ergänzt. Das Pellet in einem geeigneten Volumen von CM mit 10ng/ml bFGF, Pipette die Zellsuspension nach oben und unten mehrfach ergänzt, bis die Klumpen einheitlichen und klein geworden. Aliquot der Suspension bei 0,5 ml pro Vertiefung auf dem Matrigel Platte. Beachten Sie, dass die Kolonie Dichte auf Matrigel höher als die Dichte der Kolonien, die von üblichen Aufteilung auf MEFs ist. Die Platte wird in einem 37 ° C Gewebekultur-Inkubator. </li><li> Zur Aufrechterhaltung der hESCs auf Matrigel ist CM-Medien mit 10ng/ml bFGF ergänzt alle 24 Stunden für bis zu 4-5 Tage gewechselt. </li></ol>

<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.">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nature Biotechnology. 19, 971-974 (2001).</li></ol>

Dieses Video zeigt, wie die Passage hESCs von MEF Platten Feeder zellfreien Matrigel Platten. Beachten Sie, dass die Kolonie Dichte auf Matrigel höher als die Dichte der Kolonien, die von üblichen Aufteilung auf MEFs ist. Immunfluoreszenzfärbung und Mikroskopie oder Durchflusszytometrie für hESC Pluripotenz Marker, wie Oct-4 und SSEA-4, sind nötig, um Wartung von Zellkulturen in einem undifferenzierten Zustand in Feeder-freien Kulturbedingungen zu bestätigen.

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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>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>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>
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<td>Reagent</td>
<td>R&D Systems</td>
<td>233-FB-025</td>
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<td>Collagenase IV</td>
<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>Reagent</td>
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<td>354277</td>
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<td>Oct-4 antibody</td>
<td>Reagent</td>
<td>Santa Cruz Biotechnology</td>
<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 2: splitting hescs from mefs onto matrigel

Dieses Video zeigt, wie menschliche embryonale Stammzellen (hES) am embryonalen Maus-Fibroblasten (MEF) Feeder-Zellen, wie die Passage hESCs von MEF Platten Feeder zellfreien Matrigel Platten zu wachsen.

Watch this Scientific Journal Video about From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel at JoVE.com

From MEFs to Matrigel 2: Splitting hESCs from MEFs 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.

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 ...

from-mefs-to-matrigel-2-splitting-hescs-from-mefs-onto-matrigel

Research

JoVE Journal

Biology

11.4K 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 2: Splitting hESCs from MEFs onto Matrigel

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.
...

Forschung

Biologie

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 3: Passaging hESCs from Matrigel onto 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 hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

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 3 of 3.

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 ...

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 ...

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 ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis als Modell zur Übersetzung Impairment Identifizieren

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Verwenden von Caco-2-Zellen, die Lipidtransport Studie der Darm

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

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 ...