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.

注：倫理声明：このプロトコルは、見直し、サイモン·フレーザー大学の動物実験委員会によって承認されている（UACC、許可証番号1037K-12と1060K-03）とアニマルケアに関するカナダの協議会に準拠している、のためのNIHガイド実験動物の管理と使用、およびEEC理事会指令。 1.特別な考慮事項注：ブスルファンは、細胞傷害性である。ハンドルと安全データシートと制度ガイドラインに従って廃棄してください。 <ol><li>層流フード内で無菌的にすべての技術を実行する。 </li><li>このような剥離パッケージ、およびオートクレーブのような適切な包装材料で包むことによって、使用前に器具を滅菌する。 </li><li>感染のリスクは、照射に比べブスルファン付きの下ですが、唯一の移植後最初の2週間は、層流フードで動物を扱う。 </li></ol> 2.コンディショニングレシピエントマウス<ol><li>滅菌リン酸緩衝生理食塩水（PBS）を用いて溶液を3mg /にブスルファンを希釈する。 注：注射の毎日を使用直前にブスルファンの新鮮な希釈することが重要である。 </li><li>毎日のIP注射を経由して、レシピエントマウスに希釈しブスルファンの20mg / kgのを管理します。 </li><li> 60-100 mgの総用量まで20mg / kgのブスルファンの毎日のIP注射を繰り返し/ kgの（ すなわち、80 mg / kgを総用量のために、4日間連続ブスルファンを20mg / kgで投与）送達された。 </li></ol>ドナー骨髄細胞の3.絶縁注意：このプロトコルは正常に最大5ドナーマウスからのBM細胞を単離および調製するために使用されてきた。通常、マウス当たり細胞収量は、12-16レシピエントマウスに移植するのに十分である約30から40000000 BMDCを、である。以上のドナーマウスが必要な場合は、プロトコルに応じて調整する必要があるかもしれない。 <ol><li>ブスルファンコンディショニングの最後の日に続いてeuthCO 2を使用した（一から六ヶ月）のGFPドナーマウスをanize（または機関に受け入れられ、他の安楽死法による）。移植の合併症を回避するために、受信者と同じ性別である同系ドナーを使用しています。 </li><li> 70％エタノールでマウススプレー。リフト腹部の皮膚と外科用ハサミを使用するには、足首に向かって腹腔まで足から皮膚を介して切開する。足を持ち、しっかりと脚組織を暴露腰に向かって足首から肌を引き出します。 </li><li>股関節を露出するために大腿骨から筋肉及び脂肪組織を切り取る。 </li><li>優しく足を拡張するために、足を引っ張っている間、股関節に対してハサミを押してください。ちょうど大腿骨自体をカットしないように注意しながら、大腿骨の頭の上にカット。 </li><li> 、無菌性を維持するのに役立ち足で足を保持し、オートクレーブ組織と骨表面をこすることにより骨から残りの組織をクリーニングするには。 </li><li>膝関節を切断することにより、大腿骨と脛骨を分離滅菌PBSを含む培養皿に大腿骨を置く。氷上でインキュベートする。 </li><li>腓骨は脛骨に接続している箇所で切断することにより、腓骨を取り外し、廃棄します。大腿骨との培養皿で脛骨を置き、氷上でインキュベートする。 </li><li>繰り返して、他の足のために3.2から3.7を段階的に説明し、必要に応じて、追加のドナーマウス。骨を除去した後、ホットビーズ滅菌器で手術器具を滅菌するか、その後の工程のための無菌の新しいツールセットを使用します。 </li><li>大腿骨の場合は、鉗子で大腿骨を保持し、慎重に手術用ハサミを使用して「剃る」は遠位骨をオフに終了します。 BM空洞を公開するために、必要に応じて骨のような少しを削除します。 </li><li>滅菌PBS 3mlで注射器を記入し、23 Gの針を取り付けます。慎重にBM空洞に針を産んと無菌培養皿にBMをフラッシュする。すべての所望の細胞の除去を確実にするために、ニードルポイントで髄空洞をこすりしてください。抽出に続いて、赤いBMは表示されなくなりと骨が今白く見えることを確認してください。 </li><li>繰り返しは、同じ培養皿にBMのすべてをプール、後続の大腿骨用3.9から3.10を繰り返します。 </li><li>脛骨のために脛骨がBM空洞が露出するように膝に取り付けた最後を、鉗子で脛骨を保持し、慎重に「剃る」。可視赤色BMが終わる骨に沿って第2のカットを行います。 </li><li>滅菌PBS 3mlで注射器を記入し、25 Gの針を取り付けます。静かに（膝に取り付けた端から）BM空洞の中に針を挿入し、大腿骨からBMを含む培養皿にBMをフラッシュする。すべての細胞を除去するために針でBMキャビティの壁をこすり。抽出に続いて、赤のBMが見えなくなると骨が今白表示されていることを確認していない。 </li><li>繰り返しは、同じ培養皿にBMのすべてをプール、後続の脛骨のため3.12から3.13を繰り返します。 </li><li>穏やかcと解離を1 mlピペットチップでBMを粉砕するのエル。 </li><li>滅菌した50mLの遠心分離管に40μMバスケットフィルターを通して細胞懸濁液を渡す。残りの細胞を取得し、遠心管にフィルターを介してこれを転送するためにPBSでディッシュをすすぐ。 </li><li> 4℃で450×gで5分間遠心。上清を除去し、廃棄する。 </li><li>再懸濁赤血球溶解緩衝液3ml中にペレットを再。 8.5分間氷上でインキュベートする。 </li><li>溶解バッファをクエンチするの無菌PBS〜30ミリリットルを追加します。 </li><li> 4℃で450×gで5分間遠心。上清を除去し、廃棄する。 </li><li>滅菌PBSの1ミリリットル中にペレットを再懸濁。氷の上に保管してください。 </li><li>細胞ホモジネートの小アリコートを取り、PBSで希釈し（1：1マウス100; 1：2匹のマウス200等）の血球計数器を用いて細胞数を定量する。 </li><li> 8×10 6細胞/ mlの細胞懸濁液を希釈し、注射まで氷上でインキュベートする。 </li></ol> 4.骨髄移植<ol><li> Fiのllの各マウスに希釈した細胞懸濁液を300μlと（28 Gイン​​スリン針付き）が0.5 mlシリンジに注入する。注射器中に存在する任意の気泡を除去するようにしてください。 </li><li>加熱パッドを条件とレシピエントマウスを含むマウスケージを置き、尾静脈を拡張することができます。 </li><li>拘束装置において、レシピエントマウスと起こる。 70％エタノールを浸した外科ガーゼでテールを拭きます。 </li><li>しっかりと尾を保持し、ベベルを上にして、軽く外側尾静脈の1に針を挿入し、細胞懸濁液を注入する。 </li><li>出血が新しいクリーンケージにマウスを導入する前に停止するまで注射部位に外科ガーゼを持ってください。 </li></ol> 5. BMキメラ現象の程度を推定 NOTE：キメラ化レベルが1週間後BMTにおける末梢血中で一般的に可変であり、そのようキメリズムのレベルは、通常、少なくとも2週間後BMT推定される。チムのレベルerismはBMT後3〜4週間以内に末梢血中の&gt; 80％のGFP +細胞に到達する必要があります。 <ol><li> FACS緩衝液（PBS中の2mM EDTA + 2％ウシ胎児血清）を準備。 NOTE：FACSバッファは数週間4℃で保存することができる。 </li><li>拘束チューブにマウスを置き、横伏在静脈を露出させるために後部脚に毛を剃る。 </li><li>薄くコートワセリンとピアス25 G針で伏在静脈と足。 </li><li>ヘパリンコーティングされた毛細管血液の約50μlのを収集します。 FACS緩衝液1mlを含むマイクロ遠心チューブに血液を加える。反転によってチューブを混合し、氷上で保存する。 </li><li>戻ってケージにマウスを返す前に停止し、出血するまで、注射部位に外科ガーゼを保持します。 </li><li>遠心分離機900×gで5分間の血液サンプルは上清を除去し、廃棄する。 </li><li>赤血球溶解緩衝液500μl中にペレットを再懸濁。 8.5分間氷上でインキュベートする。 </li><li>溶解緩衝液をクエンチし、FACS緩衝液1mlを加える。 </li><li> 900×gで5分間、サンプルを遠心する。上清を除去し、廃棄する。 </li><li>赤血球溶解緩衝液500μl中に第二溶解ステップを実行します。 8.5分間氷上でインキュベートする。 </li><li>溶解緩衝液をクエンチし、FACS緩衝液1mlを加える。 </li><li> 900×gで5分間、サンプルを遠心する。上清を除去し、廃棄する。ペレットは今白であることを確認してください。 注：ペレットがまだ赤い表示される場合は、追加の溶解工程は、次のステップに進む前に必要になる場合があります。 </li><li>再懸濁FACS緩衝液1ml中でペレット。 900×gで5分間、サンプルを遠心する。上清を除去し、廃棄する。 </li><li> FACS緩衝液300μl中にペレットを再懸濁し、フローサイトメトリーを用いて、GFP +細胞を定量化する（注：この時点で、細胞の任意の免疫染色は、標準的な技術を使用して、行うことができる前にフローサイトメトリー分析する）。 </li></ol>

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The authors have nothing to disclose.

ブスルファン調整は容易に検出された造血細胞を有するマウスにおける高レベルのBMキメラの生成を可能にする。この調整は、照射設備を必要とせずに行うことができる。また、このプロトコルで概説ブスルファンの用量の骨髄抑制はよく照射骨髄破壊用量によって引き起こされる有害な副作用を最小限に許容され、従って、よりあり、若い古い、または罹患したマウスにおけるBMキメラを生...

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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:224px;">Life Technologies</td>
			<td>12483-020</td>
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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.

ブスルファンの60〜10mg / kgの投与は、一般的にマウスが許容される。しかし、動物は、一般的に、コンディショニング相（〜5から10までパーセント）の間に体重を減らすため、食事は、そのような食べることを奨励するために照射ヒマワリの種のような扱いで補充する必要があるかもしれません。末梢血中の&gt; 80％のGFP +細胞のキメラ現象とBMのレベルは60〜100 mg / kgをブスルファン7を</sup...

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.

マウスにおける安定な高レベルの骨髄キメラ現象を生成するための骨髄抑制剤としてブスルファン

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 ビュー.  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.

ビデオ: 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.

急性白血病の治療のためのチロシンキナーゼ阻害剤の前臨床評価

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

医学

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.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

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.

マウスの膀胱痛に対するモデルとしての膀胱膨満誘発内臓運動応答

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.

生きたマウスにおける大腿骨髄吸引

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.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

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.

アン<em&gt;インビトロ</em&gt;骨髄中のエストロゲン感受性乳癌の休眠モデル：分子機構の研究と仮説生成のためのツール

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.

馬腱でテクネチウム-99m標識骨髄間葉系幹細胞のin vivoイメージングと追跡中

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.

データベース ガイド-フローサイトメトリー評価骨髄骨髄細胞の成熟のため

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.

造影剤としてのプロトン磁気共鳴イメージングと酸素を用いたヒト肺における特定の換気の定量的マッピング

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.