This work was supported by the Ronald Peter Griggs Memorial Fellowship in ALS Research (to KP) and a Neuromuscular Research Partnership Program grant from the CIHR, the ALS Society of Canada, and Muscular Dystrophy Canada (JNM-69682) to CK and FMR. Additional support was also provided by the ALS Society of America (57969). We would like to thank Dr. R. Keith Humphries from the Terry Fox Laboratory/Department of Pathology, BC Cancer Agency, Vancouver, Canada and the staff at the Simon Fraser University Animal Care Unit.

HINWEIS: Ethikerklärung: Dieses Protokoll wurde überprüft und von der Animal Care Committee der Simon Fraser University zugelassen (UACC; erlauben Zahlen 1037K-12 und 1060K-03) und ist in Übereinstimmung mit dem Canadian Council on Animal Care, der NIH Leitfaden für die Pflege und Verwendung von Labortieren und der EWG Richtlinie. 1. Spezielle Überlegungen HINWEIS: Busulfan ist zytotoxische. Behandeln und entsorgen Sie nach dem Sicherheitsdatenblatt und institutionellen Richtlinien. <ol><li> Führen Sie alle Techniken aseptisch in einer Sterilbank. </li><li> Sterilisieren von Instrumenten vor der durch Wickeln in einer geeigneten Verpackung, beispielsweise einer abziehbaren Verpackung und Autoklavieren zu verwenden. </li><li> Während das Risiko für eine Infektion mit niedriger Busulfan Anlage gegenüber Bestrahlung, Griff Tieren nur in einer laminaren Strömungshaube für die ersten 2 Wochen nach der Transplantation. </li></ol> 2. KonditionierungEmpfänger Mäuse <ol><li> Verdünne Busulfan bis 3 mg / ml mit steriler phosphatgepufferter Salzlösung (PBS) ml. HINWEIS: Es ist wichtig, um eine frische Verdünnung von Busulfan kurz vor jeder Tag der Injektion verwendet werden. </li><li> Verwalten Sie 20 mg / kg des verwässerten Busulfan auf Empfängermäuse über IP-Injektion täglich. </li><li> Täglich wiederholen IP-Injektionen von 20 mg / kg Busulfan, bis zu einer Gesamtdosis von 60-100 mg / kg abgegeben wurde (dh für eine 80 mg / kg Gesamtdosis verabreichen 20 mg / kg Busulfan für 4 aufeinanderfolgende Tage). </li></ol> 3. Isolierung von Spenderknochenmarkzellen HINWEIS: Dieses Protokoll wurde erfolgreich zur Isolierung und Vorbereitung BM-Zellen von bis zu 5 Spendermäusen eingesetzt. Typischerweise wird die Zellausbeute pro Maus etwa 30 bis 40.000.000 BMDCs, die ausreicht, um 12 bis 16 Empfängermäuse verpflanzen ist. Wenn mehr Donormäusen benötigt das Protokoll müssen entsprechend angepasst werden. <ol><li> Nach dem letzten Tag der Busulfan Anlage EuthAL TEN ein GFP Spendermaus (1-6 Monate) mit CO 2 (oder von anderen Euthanasie-Verfahren bei Hochschule angenommen). Zur Vermeidung von Transplantat Komplikationen verwenden syngenen Spendern, die das gleiche Geschlecht wie die Empfänger sind. </li><li> Spray-Maus mit 70% Ethanol. Fahrstuhl Haut an den Bauch und mit chirurgische Scheren einen Einschnitt durch die Haut aus der Bauchhöhle bis das Bein auf die Knöchel. Halten Sie den Fuß fest ziehen Sie die Haut vom Knöchel zur Hüfte Freilegung der Beingewebe. </li><li> Schneiden Sie Muskel- und Fettgewebe aus dem Oberschenkelknochen, das Hüftgelenk aus. </li><li> Langsam nach unten auf den Fuß, das Bein zu verlängern, drücken Sie die Schere gegen das Hüftgelenk. Schneiden Sie gerade über dem Kopf des Oberschenkelknochens kümmert sich nicht um den Oberschenkelknochen selbst geschnitten. </li><li> Zur Aufrechterhaltung der Sterilität, halten Sie das Bein mit dem Fuß, und reinigen Sie alle verbleibenden Gewebe von den Knochen durch Reiben der Knochenoberfläche mit autoklaviert Gewebe. </li><li> Trennen Sie die Oberschenkel und Schienbein durch Schneiden durch das Kniegelenk undlegen Sie das Femur in einer Kulturschale mit steriler PBS. Auf Eis inkubieren. </li><li> Entfernen und entsorgen Sie das Wadenbein, indem an den Stellen, wo das Wadenbein verbindet sich mit dem Schienbein. Legen Sie die Tibia in der Kulturschale mit dem Oberschenkelknochen und auf Eis inkubieren. </li><li> Die Schritte 3,2-3,7 für das andere Bein, und gegebenenfalls weitere Spendermäusen. Nach der Entfernung der Knochen, sterilisieren chirurgische Instrumente mit einem heißen Perle Sterilisator oder verwenden Sie eine neue Reihe von sterilen Instrumente für die weiteren Schritte. </li><li> Für den Oberschenkelknochen, halten Sie die Oberschenkel mit einer Pinzette und mit chirurgische Scheren sorgfältig &quot;Rasur&quot; die distalen Enden der Knochen. So wenig von den Knochen wie nötig, um die BM Höhle freizulegen. </li><li> Füllen Sie eine Spritze mit 3 ml sterilem PBS und fügen Sie eine 23 G Nadel. Vorsichtig trug die Nadel in die BM Höhle und spülen Sie das BM in eine sterile Petrischale. Achten Sie darauf, die Markhöhle mit der Nadelspitze zu kratzen, um die Entfernung aller gewünschten Zellen zu gewährleisten. Nach der Extraktionsicherzustellen, dass der rote BM nicht mehr sichtbar ist und der Knochen wird jetzt weiß. </li><li> Wiederholen Sie die Schritte 3,9-3,10 für nachfolgende Oberschenkelknochen, die Bündelung all der BM in der gleichen Kulturschale. </li><li> Für die Schienbeine, halten Sie die Tibia mit einer Pinzette vorsichtig &quot;Rasur&quot; das Ende, wo das Schienbein bis zum Knie, um den BM Höhle aussetzen befestigt. Machen Sie einen zweiten Schnitt entlang der Knochen, wo die sichtbaren roten BM endet. </li><li> Füllen Sie eine Spritze mit 3 ml sterilem PBS und fügen Sie eine 25 G Nadel. Schieben Sie die Nadel in die BM Hohlraum (vom Ende, die bis zu den Knien befestigt war) und spülen Sie das BM in der Kulturschale mit dem BM aus den Oberschenkelknochen. Kratzen die Wände des Hohlraums BM mit der Nadel, um alle Zellen zu entfernen. Nach der Extraktion zu gewährleisten, dass die rote BM nicht mehr sichtbar ist und der Knochen wird jetzt weiß. </li><li> Wiederholen Sie die Schritte von 3,12 bis 3,13 für nachfolgende tibiae, die Bündelung all der BM in der gleichen Kulturschale. </li><li> Sanft verreiben die BM mit einer 1 ml Pipettenspitze, um die c distanzierenEllen. </li><li> Passieren die Zellsuspension durch ein 40 &amp; mgr; M Korbfilter in ein steriles 50 ml Zentrifugenröhrchen. Spülen Sie die Schale mit PBS, um alle verbleibenden Zellen und übertrage diese durch den Filter in dem Zentrifugenröhrchen. </li><li> Zentrifuge 5 min bei 450 × g bei 4 ° C. Entfernen Sie den Überstand. </li><li> Re-suspendieren des Pellets in 3 ml Erythrozyten Lysepuffer. Inkubieren auf Eis für 8,5 min. </li><li> Hinzufügen ~ 30 ml sterilem PBS, die Lysepuffer quenchen. </li><li> Zentrifuge 5 min bei 450 × g bei 4 ° C. Entfernen Sie den Überstand. </li><li> Resuspendieren des Pellets in 1 ml sterilem PBS. Halten Sie auf dem Eis. </li><li> Nehmen ein kleines Aliquot des Zellhomogenats mit PBS verdünnt (1: 100 für 1 Maus, 1: 200 für die 2-Mäuse, etc.), um die Anzahl der Zellen zu quantifizieren, unter Verwendung eines Hämocytometers. </li><li> Verdünne die Zellsuspension auf 8 x 10 6 Zellen / ml und Inkubation auf Eis bis zur Injektion. </li></ol> 4. Knochenmarktransplantation <ol><li> Fill ein 0,5-ml-Spritze (mit einer 28 G Nadel Insulin) mit 300 ul verdünnten Zellsuspension für jede Maus injiziert werden. Seien Sie sicher, dass alle in der Spritze Luftblasen zu entfernen. </li><li> Platzieren Sie die Maus Käfig mit den konditionierten Empfängermäuse auf einem Heizkissen und lassen Sie die Schwanzvenen zu erweitern. </li><li> Nehmen Sie eine Empfängermaus und in die Rückhalteeinrichtung. Wischen Sie den Schwanz mit chirurgischen Gaze mit 70% Ethanol eingeweicht. </li><li> Halten Sie das Heck und mit der Fase nach oben, sanft die Nadel in eine der seitlichen Schwanzvenen und injizieren Sie die Zellsuspension. </li><li> Halten chirurgischer Gaze auf die Injektionsstelle, bis die Blutung aufhört vor dem Einführen der Maus in einen neuen sauberen Käfig. </li></ol> 5. Schätzung der Umfang der BM Chimärismus HINWEIS: Chimärismus Ebenen sind in der Regel variable im peripheren Blut nach 1 Woche nach der KMT, und als solche Chimärismus Ebenen werden in der Regel geschätzt mindestens 2 Wochen nach der KMT. Ebenen der chimerism sollte&gt; 80% GFP + Zellen im peripheren Blut in 3-4 Wochen erreichen nach der KMT. <ol><li> Bereiten FACS Puffer (2 mM EDTA + 2% fötalem Rinderserum in PBS). HINWEIS: FACS-Puffer bei 4 ° C für mehrere Wochen gelagert werden. </li><li> Platzieren Sie die Maus in einem Halterohr und rasieren Sie das Fell auf dem hinteren Bein, um die laterale Vena saphena aus. </li><li> Dünn Mantel das Bein mit Vaseline und durchbohren die Vena saphena mit einer 25 G-Nadel. </li><li> Sammeln etwa 50 ul Blut mit Heparin beschichteten Kapillarröhrchen. Füge das Blut in einem Mikrozentrifugenröhrchen mit 1 ml FACS-Puffer. Mischen Sie das Rohr durch Umdrehen und Lagerung auf Eis. </li><li> Halten chirurgischer Gaze auf die Injektionsstelle bis die Blutung aufhört, bevor er Maus wieder in den Käfig. </li><li> Zentrifugieren der Blutproben für 5 min bei 900 x g. Entfernen Sie den Überstand. </li><li> Re-suspendieren des Pellets in 500 ul Erythrocyten-Lysepuffer. Inkubieren auf Eis für 8,5 min. </li><li> 1 ml FACS-Puffer, um die Lysepuffers quenchen. </li><li> Zentrifugiere die Proben für 5 min bei 900 x g. Entfernen Sie den Überstand. </li><li> Führen Sie eine zweite Lyseschritt in 500 ul Erythrozyten Lysepuffer. Inkubieren auf Eis für 8,5 min. </li><li> 1 ml FACS-Puffer, um die Lysepuffers quenchen. </li><li> Zentrifugiere die Proben für 5 min bei 900 x g. Entfernen Sie den Überstand. Überprüfen Sie, ob das Pellet ist jetzt weiß. HINWEIS: Wenn das Pellet noch rot angezeigt wird, kann ein zusätzlicher Lyse, bevor Sie mit dem nächsten Schritt benötigt. </li><li> Resuspendieren des Pellets in 1 ml FACS-Puffer. Zentrifugiere die Proben für 5 min bei 900 x g. Entfernen Sie den Überstand. </li><li> Re-suspendieren des Pellets in 300 ul FACS-Puffer und Quantifizierung GFP + -Zellen mittels Durchflusscytometrie (ANMERKUNG: An diesem Punkt jede gewünschte Immunfärbung der Zellen kann durchgeführt werden unter Verwendung von Standardtechniken vor der Durchflusszytometrie-Analyse). </li></ol>

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The authors have nothing to disclose.

Busulfan Anlage ermöglicht die Erzeugung von High-Level BM Chimärismus in Mäusen, die mit hämatopoetischen Zellen, die leicht nachweisbar sind. Diese Konditionierung kann ohne die Notwendigkeit für Bestrahlungsanlagen durchgeführt werden. Außerdem ist die Myelosuppression mit den Dosen von Busulfan in diesem Protokoll umrissen gut verträglich Minimierung toxischer Nebenwirkungen von myeloablativen Dosis der Bestrahlung verursacht werden, und somit kann eine weitere geeignete Technik zur BM Chimärismus in junge,...

Recently, there has been considerable interest in the roles of microglia and bone marrow (BM)-derived cells (BMDCs) in the central nervous system (CNS), both during disease as well as with normal aging. Microglia, the resident immune cells of the CNS, are now known to develop in the CNS following the entry of primitive myeloid progenitors during embryogenesis1. Microglia retain aspects of their myelomonocytic lineage well into adult life. While evidence suggests that the contribution of BMDCs to the microglial pool is minimal in healthy adult animals1, the role BMDCs play in the progression of various neurodegenerative diseases remains unclear. This uncertainty is compounded by the fact that it is difficult to distinguish BMDCs that accumulate within the CNS from endogenous resident microglia, as no universally accepted discriminating immunohistochemical marker has been identified. In order to monitor BMDCs in vivo, we employ a BM transplantation (BMT) protocol whereby endogenous BM cells of a recipient mouse are replaced with those of a donor mouse ubiquitously expressing green fluorescent protein (GFP) under control of the &#946;-actin promoter. This protocol permits the determination of both the localization and morphology of GFP+ BMDCs within the recipient mouse using immunohistochemistry, and facilitates further characterization of these cells using fluorescence-activated cell sorting (FACS) followed by subsequent biochemical assays. 
For a successful BMT, recipient BM cells need to be ablated (termed myeloablation) in order to generate niche space within the recipient BM to allow for donor cell engraftment. Often, myeloablation is achieved using total body gamma irradiation which induces double strand breaks in DNA that leads to cell death, particularly in actively dividing cells such as hematopoietic progenitor cells2,3. The irradiation protocols are done to induce sufficient BM cell death such that animal lethality results if the recipient animal does not achieve adequate donor cell engraftment from BMT (so called &#8216;lethal irradiation&#8217;). However, irradiation requires a specialized facility and equipment, along with veterinary and animal husbandry resources not available to researchers at all institutions. Furthermore, myeloablative irradiation can cause potentially lethal damage to other tissues, and due to immunosuppresion, irradiated animals are more susceptible to secondary infections3. As such, reduced intensity conditioning (so called &#8216;RIC&#8217; protocols) regimens have been developed that are intended to minimize potentially toxic side effects of the conditioning protocol in patients, particularly for use in at risk populations such as children and the elderly4. 
Some RIC protocols rely upon chemotherapeutics such as busulfan in order to condition the BM compartment. Busulfan is a bifunctional DNA alkylating agent often used clinically as an alternative to irradiation5,6. Notably, busulfan can be safely administered to mice by intraperitoneal (IP) injection and does not require the specialized facilities and equipment necessary to irradiate mice. Busulfan conditioning has been used extensively in our lab7, as well as in several other recent publications8&#8211;11. When doses of 60-100 mg/kg are employed, high degrees of stable chimerism (>80% GFP+ cells) can be established in the peripheral blood and BM7. Importantly, at these doses myeloablation is not complete and as such mice are able to survive without receiving support BM cells (K. Peake, J. Manning, C. Lewis, and C. Krieger, unpublished observations). Moreover, with myelosuppressive busulfan conditioning there is rapid reconstitution of peripheral blood myelomonocytic cells by donor cells. However, the replacement of peripheral blood lymphocytes by donor cells is slower, highlighting the lack of immunosuppression that occurs with 60-100 mg/kg doses of busulfan compared to total body irradiation7. 
We have successfully used busulfan-induced chimerism to investigate BMDCs in wild-type mice, as well as in mouse models of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS)7 and Alzheimer disease. It has been observed that under certain conditions significant numbers of GFP+ BMDCs accumulate within the CNS7,12. Importantly, like myeloablative conditioning with irradiation, busulfan conditioning followed by BMT is sufficient to allow for GFP+ BMDC accumulation within the CNS in both wild-type mice, and mice with neurological disorders7&#8211;11. As a major obstacle in treating neurodegenerative disorders is the ability to get therapeutic molecules from the circulation into the CNS where they can exert beneficial effects, this raises the possibility that BMDCs could be engineered to express therapeutic molecules, and subsequently used as a vehicle to deliver therapeutics to the CNS of busulfan conditioned recipients, a mechanism we are actively investigating. Although we have found that myeloablative conditioning with irradiation leads to greater accumulation of GFP+ BMDCs in the CNS compared to busulfan conditioning7, irradiation may have considerable toxicity in patients with neurodegenerative disorders and in murine models of CNS disease.
While we, and many others, have used BMT models to study BMDCs in neurodegenerative disorders, the ability to largely replace the BM compartment of a recipient mouse with a distinctly identifiable population of BM cells is an invaluable tool for studying various aspects of the hematopoietic system. This includes a wide array of research topics such as hematopoietic lineage development, leukemia, organ transplantation, graft-versus-host disease, and immunobiology.

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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:224px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Busulfex</td>
			<td style="width:224px;">Otsuka Canada Pharmaceutical Inc.</td>
			<td style="width:119px;">DIN 02240602</td>
			<td>Dilute busulfan to 3 mg/mL with sterile PBS just prior to use. Busulfan is cytotoxic. Handle and dispose according to the MSDS and institutional guidelines.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ACK Lysing Buffer</td>
			<td style="width:224px;">Life Technologies</td>
			<td style="width:119px;">A10492-01</td>
			<td>Other lysis buffer could be substituted but incubation times may need to be adjusted accordingly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:224px;">Life Technologies</td>
			<td>12483-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

busulfan as a myelosuppressive agent for generating stable high-level bone marrow chimerism in mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

Die Verabreichung von 60-100 mg / kg Busulfan im allgemeinen gut von Mäusen gut vertragen. Doch in der Regel Tiere, Gewicht zu verlieren während der Konditionierungsphase (~ 5-10%) und damit die Nahrung muss unter Umständen mit Leckereien wie bestrahlt Sonnenblumenkerne zu essen zu fördern ergänzt werden. Ebenen der Chimärismus von&gt; 80% GFP + Zellen im peripheren Blut und BM konsequent erreichbar mit 60 bis 100 mg / kg Busulfan 7 sein. Unser Labor hat erfolgreich dieses Protokoll, mit dem über 100 c...

Watch this Scientific Journal Video about Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice at JoVE.com

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Busulfan als myelosuppressiven Agenten zur Erwirtschaftung stabiler Hochrangige Knochenmark Chimärismus bei Mäusen

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

busulfan-as-myelosuppressive-agent-for-generating-stable-high-level

Research

JoVE Journal

Medicine

13.1K Aufrufe.  Simon Fraser University. We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Serie: Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Präklinische Evaluierung von Tyrosinkinase-Inhibitoren für die Behandlung von akuter Leukämie

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Forschung

Medizin

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 ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Harnblase Aufdehnung evozierte viszeromotorische Antworten als Modell für Bladder Pain in Mäuse

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Oberschenkelknochenmark-Aspiration im Live-Mäuse

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Halsschlagader Infusionen für pharmakokinetischen und pharmakodynamischen Analyse von Taxanen in Mäusen

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Ein<em&gt; In-vitro-</em&gt; Winterruhe Model of Östrogen-sensitiven Brustkrebs im Knochenmark: Ein Werkzeug für Molecular Mechanism Studien und Hypothesengenerierung

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

In-vivo-Imaging und Verfolgung von Technetium-99m markierten Knochenmark mesenchymale Stammzellen in Equine Tendinopathie

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

Datenbank-geführte Durchflusszytometrie zur Auswertung von Knochenmark myeloische Zellen Reifung

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Quantitative Mapping of Specific Ventilation in the Human Lung using Proton Magnetic Resonance Imaging and Oxygen as a Contrast Agent

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of the fresh gas entering a lung region divided by the region&#8217;s end-expiratory volume &#8213; in the human lung, using only inhaled oxygen as a contrast agent. Regional quantification of specific ventilation has the potential to help identify areas of pathologic lung function. Oxygen in solution in tissue shortens the tissue&#8217;s longitudinal relaxation time (T1), and thus a change in tissue oxygenation can be detected as a change in T1-weighted signal with an inversion recovery acquired image. Following an abrupt change between two concentrations of inspired oxygen, the rate at which lung tissue within a voxel equilibrates to a new steady-state reflects the rate at which resident gas is being replaced by inhaled gas. This rate is determined by specific ventilation. To elicit this sudden change in oxygenation, subjects alternately breathe 20-breath blocks of air (21% oxygen) and 100% oxygen while in the MRI scanner. A stepwise change in inspired oxygen fraction is achieved through use of a custom three-dimensional (3D)-printed flow bypass system with a manual switch during a short end-expiratory breath hold. To detect the corresponding change in T1, a global inversion pulse followed by a single shot fast spin echo sequence was used to acquire two-dimensional T1-weighted images in a 1.5&#160;T MRI scanner, using an eight-element torso coil. Both single slice and multi-slice imaging are possible, with slightly different imaging parameters. Quantification of specific ventilation is achieved by correlating the time-course of signal intensity for each lung voxel with a library of simulated responses to the air/oxygen stimulus. SVI estimations of specific ventilation heterogeneity have been validated against multiple breath washout and proved to accurately determine the heterogeneity of the specific ventilation distribution.

Specific ventilation imaging is a functional magnetic resonance imaging technique that allows for quantification of regional specific ventilation in the human lung, using inhaled oxygen as a contrast agent. Here, we present a protocol to collect and analyze specific ventilation imaging data.

Quantitative Mapping spezieller Belüftung in der menschlichen Lung mit Proton-Magnetresonanztomographie und Sauerstoff als Kontrastmittel

Specific ventilation imaging (SVI) is a functional magnetic resonance imaging technique capable of quantifying specific ventilation &#8213; the ratio of ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Durchflusszytometrie Analyse von Immunzell-Subsets innerhalb der Murinenmilen, Knochenmark, Lymphknoten und Synovialgewebe in einem Osteoarthritis-Modell