プロジェクトいいえ。NVKP_16-1-2016-0017('ナショナル・ハート・プログラム')は、ハンガリーの国立研究開発・イノベーション基金から提供された支援を受けて実施され、NVKP_16資金制度の下で資金提供を受けています。この研究は、センメルワイス大学の治療開発とバイオイメージングテーマプログラムの枠組みの中で、ハンガリーのイノベーションと技術省のテーマ・エクセレンス・プログラム(2020-4.1.1.-TKP2020)によって資金提供されました。

この議定書は、センメルワイス大学の地域・学術研究倫理委員会のガイドラインに従っています。現在のプロトコルは、特定のベンダーに適用されます。いくつかのステップは、超音波機械と後処理ソフトウェアに関係なく有効なままですが、他のベンダーのソリューションを使用する場合、重要な違いが存在する可能性があります。
1. 技術的な要件
<ol><li>3Dイメージングが可能な心エコー検査機を利用する。</li>
	<li>3Dトランスソシック心エコー検査に対応したフェーズドアレイトランスデューサを接続します。</li>
	<li>超音波システムの内蔵3リードECGを適用して、システムが記録と分析を心臓周期に同期できるようにします。</li>
</ol>2. 3次元心エコー画像の取得
<ol><li>左横の褥瘡の位置に患者を配置する(患者は、左の側に横たわって、頭の上に左腕を伸ばした)。</li>
	<li>画面上の ECG トレースの品質が良好であることを確認します。 注:ソフトウェアは心電図信号に基づいて心周期のさまざまなポイントを検出するので、これは後処理のための前提条件です。</li>
	<li>画像を凍結解除し、トランスデューサで患者を検査し始めます。従来のアペカル4室ビューを視覚化します。</li>
	<li>セクタ幅をLVに調整し、深さを下げて左心房を切り詰め、わずかな過ゲインを使用して画質を最適化します。 注意:LVの全端を確認し、また心外膜面が見えるようにしてください。</li>
	<li>4Dボタンを押して3Dモードに切り替えます。 注: タッチスクリーンの マルチスライス ボタンを押すと、標準の短軸と長軸カットを使用して3Dデータセットの概要を表示する4つのオプション(5、7、8、12スライス)が利用可能になります。必要に応じて、トランスデューサの位置を補正して、円錐から僧帽弁レベルまでのLVの壁厚全体をピラミッド型3Dデータセットに確実に含めることができます。12 個のスライス (9 つの調整可能な短軸ビュー) の使用をお勧めします。</li>
	<li>マルチビートまたはシングルビートモードを使用して3D画像を取得します。<ol>
<li>マルチビートモードを使用して、2、3、4、または6の心拍サイクル(これは画面上で設定することができます)からデータセットを再構築する、より高い空間および時間分解能を達成するために使用してください - ステッチアーティファクトを最小限に抑えるために必要な患者と安定したトランスデューサ位置の終わりの息止め。 注: シングルビート の取得は、より低い空間と時間分解能です。しかし、ほとんどの最新のトランスデューサは高品質であるため、再構築せずに適切な3Dデータセットを取得してさらなる分析を行うことができます。一般的な推奨事項として、1 秒あたりのボリューム レートは、さらに分析するために推奨されます。</li>
		<li>フルボリュームがサブボリュームから再構築され、LV全体が表示されたら、イメージをフリーズします。 サイクル選択 と サイクル数ノブを 使用して、最適な取得した心臓サイクルを選択し、 イメージストアを押します。 注: ステッチアーティファクトは、空間的または一時的に位置がずれるサブボリューム同士です。LV 壁の大きなドロップアウトを持つデータセットや、ステッチアーティファクトを含むデータセットは、一般に、さらなる分析には適していません。すでに取得した 3D データセットの品質は、マルチスライス モードを使用してダブルチェックできます。</li>
	</ol>
</li>
</ol>3. LV形態と機能を定量化するための後処理
<ol><li>さらに解析に適した 3D データセットを選択します。 注:プロトコルのこの部分は、以前に取得し、良質の3D画像を保存する必要があり、超音波装置と別のワークステーション上で実行することができます。</li>
	<li>[測定]|をクリックします。ボリューム、4DオートLVQを選択します。</li>
	<li>クワッドスクリーン(4、2、3室のビュー、および1つの短軸ビューの3つの補助的なビュー)では、後者は長軸ビュー上の水平面で調整できます。</<c0>a0>必要に応じて、傾きと回転によって手動でアパイカルビューを修正し、対応する標準ビューを表示し、それによって短縮を排除します。長軸ビューでキャリパーをドラッグして移動することで、キャリパーを LV の長軸に合わせて傾斜を設定します。マシン上の対応するまたはすべての回転ノブまたは短軸イメージのキャリパーを調整することによって、回転を設定します。 注: ソフトウェアの推奨設定は 、[自動整列 ] ボタンを押してリセットできます。</li>
	<li>ビューの配置が完了したら、次のステップ EDV をクリックします。エンド拡張(ED)フレームは、ECG信号を使用して自動的に検出されますが、必要に応じて手動で修正することができます。</li>
	<li>LV内膜・外膜表面の半自動検出<ol><li>任意のアプカル ビューで 2 つのランドマーク ポイントを手動で選択します。まず、LV頂点を特定し、次にLVベースの中央(僧帽弁アヌラスレベル)を任意のアピックビューで識別します。アルゴリズムは、自動的に全体のLVの心内境界を輪郭を描きます。 注:2つのオプションがあります: 手動、つまり、2つの基礎と1つのアペカルランドマークは、すべてのアパイカルビューで設定する必要があることを意味し、 自動Initは、ユーザーの操作なしで自動的にLVを輪郭を描きます。</li>
		<li>3 つのアペカル ビュー、異なるレベルの 3 つの短軸ビュー、および第 4 のユーザー制御短軸で輪郭の信頼性を確認して、検出されたサーフェスの視覚的な検証を可能にします。輪郭補正は、手動でポイントを追加することで可能で、その後、コンター ラインに組み込まれます。 注: [元に戻す]を使用すると、以前に追加したポイントを削除できます。リセットボタンは、輪郭をリセットして、セクション全体を最初から開始します。輪郭の可視性を調整して、グレースケールの画像上の心内膜表面の鑑賞を可能にできます。心内膜および心筋輪郭は、正確かつ一貫した方法で行われるべきである。詳細な推奨事項については、以下の参照を確認してください。</li>
		<li>次のステップ( ESV)を選択します。</li>
		<li>前のポイントで説明したように、同じ手順(3.5.1~3.5.2)を繰り返して、エンドシストリックフレームの心内輪郭を特定して修正します。 メモ:エンドシストリック(ES)フレームは、ECG信号を使用して自動的に検出されますが、必要に応じて手動で修正することができます。エンド拡張期容積(EDV)、終期収縮期容積(ESV)、駆出率(EF)、心拍数(HR)、脳卒中量(SV)、心拍出量(CO)および球面指数(SpI)の値が既に画面に表示されています。</li>
		<li>次のステップで 音量波形 を押します。ソフトウェアは、心周期フレーム全体にわたって心内膜表面をトレースするLVと時間容積曲線の動的3Dモデルを表示します(図1)。 注:ここでは、任意のフレームで心内膜の境界線を編集する可能性があります。</li>
		<li>次のステップでは、 LV Mass を押します。ソフトウェアは自動的に末拡張期フレームのLV心外膜輪郭を輪郭を描き、LV質量(EDMass)を計算する。 注: 必要に応じて、短軸または長軸平面に含める点(前述の方法と同じ方法)を追加して、心外膜サーフェスの輪郭を編集します。調整する輪郭を選択できます: 内線、エピ、または Endo +エピ。</li>
		<li>次のステップに向けて 4D ひずみ ROI を押します。ソフトウェアは自動的に終部収縮期フレームのLV心外膜輪郭を輪郭し、LV末端収縮量質量(ESMass)を計算する。 注: 必要に応じて、短軸または長軸面に含める点(前述と同じ方法)を追加して、心外膜サーフェスの終点-収縮期の輪郭を編集します。ESMass は、EDMass と同様の値である必要があります。このステップは、スペックルトラッキングによって3Dひずみ値を計算するために不可欠です。</li>
		<li>次のステップで 4Dひずみ結果 を押します。ソフトウェアは、複数の短軸および長軸平面上の3D心筋追跡と、心臓サイクル全体の17の標準LVセグメントの対応する歪み値をフレームごとに視覚化します。時間ひずみ曲線と雄牛の目のプロットも表示されます。縦方向ひずみ、円周ひずみ、放射状ひずみ、面積ひずみ、回転、およびトーションのパラメータが計算され、実証できます。 注:特定のLVセグメントが画像の視覚的観察や時間ひずみ曲線に基づいて低いトラッキング品質を有すると考えられる場合は、解析から特定のLVセグメントを除外する可能性があります。ただし、ソフトウェアは、デフォルトでセグメントの承認または拒否を推奨します。色分け歪み値は、LVの動的3Dモデルで「レイアウト」を変更することで可視化できます。</li>
	</ol></li>
	<li>分析を終了するには、[ 承認と終了] を押します。</li>
</ol>

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Journal of the American Society of Echocardiography. 28 (1), 1-39 (2015).</li><li>Matyas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+speckle-tracking+echocardiography+with+invasive+hemodynamics+for+the+detection+of+characteristic+cardiac+dysfunction+in+type-1+and+type-2+diabetic+rat+models.">Comparison of speckle-tracking echocardiography with invasive hemodynamics for the detection of characteristic cardiac dysfunction in type-1 and type-2 diabetic rat models.</a> Cardiovascular Diabetology. 17 (1), 13 (2018).</li><li>Kovacs, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+hemodialysis,+left+ventricular+mass+and+FGF-23+on+myocardial+mechanics+in+end-stage+renal+disease:+a+three-dimensional+speckle+tracking+study.">Impact of hemodialysis, left ventricular mass and FGF-23 on myocardial mechanics in end-stage renal disease: a three-dimensional speckle tracking study.</a> International Journal of Cardiovascular Imaging. 30 (7), 1331-1337 (2014).</li><li>Muraru, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+analysis+of+left+ventricular+geometry+and+function+by+three-dimensional+echocardiography+in+healthy+adults.">Comprehensive analysis of left ventricular geometry and function by three-dimensional echocardiography in healthy adults.</a> Journal of the American Society of Echocardiography. 26 (6), 618-628 (2013).</li><li>Lakatos, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+Cardiac+Remodeling+and+Exercise+Capacity+in+Elite+Athletes:+Incremental+Value+of+Left+Atrial+Morphology+and+Function+Assessed+by+Three-Dimensional+Echocardiography.">Relationship between Cardiac Remodeling and Exercise Capacity in Elite Athletes: Incremental Value of Left Atrial Morphology and Function Assessed by Three-Dimensional Echocardiography.</a> Journal of the American Society of Echocardiography. 33 (1), 101-109 (2020).</li><li>Muraru, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervendor+Consistency+and+Accuracy+of+Left+Ventricular+Volume+Measurements+Using+Three-Dimensional+Echocardiography.">Intervendor Consistency and Accuracy of Left Ventricular Volume Measurements Using Three-Dimensional Echocardiography.</a> Journal of the American Society of Echocardiography. 31 (2), 158-168 (2018).</li><li>Kalam, K., Otahal, P., Marwick, T. 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LV形態学的および機能的測定は、心臓病の診断、管理、およびフォローアップの基礎を表します。さらに、それらは結果の強力な予測変数です。一般的に、LVの2D心エコーベースの評価は、現在の実践ガイドラインで推奨されています。しかし、3D心エコーは、LV shape7,8に関する幾何学的仮定から解放され、より正確であることが証明されています。?...

左心室(LV)形態および機能の評価は、心臓病学における一般的かつより具体的な調査の主な目的である1。情報の高密度を提供することができる広く利用可能で非侵襲的な胸部心エコー検査(TTE)は、便利で迅速で費用対効果の高い評価のための選択の方法です。
LV質量、体積、および後続の放出率の測定は、有意な診断および予後値2を保持します。与えられたメジャーが正確であればあるほど、その値は高くなります。金標準心臓磁気共鳴(CMR)の画像化の派生値とのよりよい相関関係は、心エコー技術のための継続的な追跡である。一般的に、臨床実習ガイドラインでは、LV容積および駆出率測定3のための複葉機シンプソンの方法をお勧めします。しかし、LVは不規則な形状を持つ3次元(3D)構造であるため、LVの形態や機能を正確に表現するためにいくつかの臨床シナリオでは、いくつかの断層面が失敗することは間違いありません。超音波ハードウェアおよびソフトウェア技術の最近の進歩は、心エコープロトコルを革命化するリアルタイム3Dイメージングの開発を可能にしました。
また、壁運動異常に関する定量的アプローチの必要性は、変形画像化4の台頭をもたらした。ひずみとひずみ率のパラメータは、標準のグレースケール画像を使用してスペックルトラッキングで計算できます。3D心エコー検査は、2次元歪み評価5のいくつかの欠点を克服することもできる。高価な科学的ツールから、3D心エコーは日常の臨床現場で使用される強力な技術になり始め、LVの定量化は確かにこのブレークスルーの最初の行にあります。
本稿は、ヒントやトリックを提示し、また、LVに関するこの重要な技術の広範だが技術的に健全な使用を容易にする潜在的な落とし穴を強調することによって、詳細な3Dプロトコルを通して読者を導くことを目的としています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3V-D/4V-D/4Vc-D</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>ultrasound probe</td>
		</tr>
		<tr>
			<td>4D Auto LVQ</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>software for analysis</td>
		</tr>
		<tr>
			<td>E9/E95</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>ultrasound machine</td>
		</tr>
		<tr>
			<td>EchoPac v203</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>software for analysis</td>
		</tr>
	</tbody>
</table>

evaluation of left ventricular structure and function using 3d echocardiography

左心室(LV)の3次元(3D)定量化は、様々な心臓障害における診断精度と正確なリスク階層化の点で有意な付加価値を提供する。最近では、3D心エコー検査は、日常的な心臓病の練習で利用可能になりました。ただし、高品質の画像取得とその後の解析には、学習曲線が急な値になります。本稿は、ヒントやトリックを提示し、また、LVに関するこの重要な技術の広範だが技術的に健全な使用を容易にする潜在的な落とし穴を強調することによって、詳細な3Dプロトコルを通して読者を導くことを目的としています。まず第一に、最適な空間と時間分解能を備えた高品質の3Dデータセットの取得を示します。次に、最も広く適用されている組み込みソフトウェアの1つを使用して、LVの詳細な定量化に向けた分析ステップを提示します。LV体積、球面性、質量、収縮期機能を、吐出分率と心筋変形(縦方向および経周歪み)を測定して定量化します。従来の心エコー手法から3Dベースの定量化への移行が強く推奨される重要なシナリオについて、臨床事例を紹介します。

LVの3D分析は、大多数の患者で実現可能である。ケース1は正常な心室容積と機能を有する健康なボランティアである(図1)。症例2(図2)は、拡張型心筋症を有する64歳の男性患者であり、左束枝ブロック形態の広いQRS複合体(160ms)である。ゴールドスタンダードCMR測定は以下の通り:末拡張期容積:243mL、終期収縮期容積:160mL、駆出...

Watch this Scientific Journal Video about Evaluation of Left Ventricular Structure and Function using 3D Echocardiography at JoVE.com

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

本稿では、3D心エコー検査による左心室の容積評価および分光追跡解析のためのステップバイステップの取得および分析プロトコルを提供し、特にこの技術の実現可能性を最大化する実用的な側面に焦点を当てる。

3D心エコーを用いた左心室構造と機能の評価

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk ...

evaluation-left-ventricular-structure-function-using-3d

Research

JoVE Journal

Bioengineering

3.8K ビュー.  Semmelweis University. 本稿では、3D心エコー検査による左心室の容積評価および分光追跡解析のためのステップバイステップの取得および分析プロトコルを提供し、特にこの技術の実現可能性を最大化する実用的な側面に焦点を当てる。

ビデオ: Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

後期胚と新生児マウスの計測左心室圧

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

生物工学

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

心筋の構造、機能の in vivo定量的評価では 、灌流および生存率は、心臓マイクロコンピュータ断層撮影法を使用しました

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

軟骨と皮膚組織類縁ミキシング ユニット Bioink Precellularization の小説のパッシブ バイオプリンティング

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

自動化された微生物叢システムを用いた中国ハムスター卵巣細胞からのモノクローナル抗体の精製と分析

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Four-Dimensional CT Analysis Using Sequential 3D-3D Registration

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data remains difficult because segmentation in all volumetric frames is time-consuming. We aimed to analyze joint kinematics using a sequential 3D-3D registration technique to provide the kinematics of the moving bone with respect to the fixed bone semiautomatically using 4DCT DICOM data and existing software. Surface data of the source bones are reconstructed from 3DCT. The trimmed surface data are respectively matched with surface data from the first frame in 4DCT. These trimmed surfaces are sequentially matched until the last frame. These processes provide positional information for target bones in all frames of the 4DCT. Once the coordinate systems of the target bones are decided, translation and rotation angles between any two bones can be calculated. This 4DCT analysis offers advantages in kinematic analyses of complex structures such as carpal or tarsal bones. However, fast or large-scale motions cannot be traced because of motion artifacts.

We analyzed joint kinematics from four-dimensional computed tomography data. The sequential 3D-3D registration method semiautomatically provides the kinematics of the moving bone with respect to the subject bone from four-dimensional computed tomography data.

シーケンシャル3D-3D登録を用いた4次元CT解析

Four-dimensional computed tomography (4DCT) provides a series of volume data and visualizes joint motions. However, numerical analysis of 4DCT data ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

単離マウス心室筋細胞の光学イメージング

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

音響セパレータを用いたCHO採取細胞培養液の一次解明

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

心臓血管磁気共鳴画像法によるマウス心左心室機能、心筋緊張、血行力の定量化

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

肺高血圧症と右心室不全の大型動物モデル:羊の左肺動脈結紮と進行性主肺動脈バンディング

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

搭載型ブタ大動脈弁の流体力学的特性の評価

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

著者スポットライト:大動物実験に着手する前に心臓手術試験を進める 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

実験的大動脈弁処置および新規医療機器の流体力学的試験のための ex vivo 豚モデル

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...