我々は、フローサイトメトリーの支援についてはマルセロ·トロに感謝したい。本研究では、スウェーデンのハートと肺財団、欧州委員会FP7 &quot;CardioCell&quot;、スウェーデンの研究評議会、AFA保険やALFによってサポートされていました。 OBは、ドイツ学術振興によってサポートされていました。

1。心臓核の単離<ol><li> 1％BSA / PBSコーティング溶液10mlとコート遠心管（Beckmanの遠心チューブ＃363664）。チューブにフタをし、それらがチューブローテーターで30分間回転させましょう。コー​​ティング溶液を除去し、遠心チューブの空気乾燥した（マウスの心臓ごとに管が異なる種からの単一のマウスの心臓、交互に最大5つのマウスの心臓や心臓の組織の1グラムの分析のために必要とされるみましょう（例えば、ヒト）処理することができます1チューブで）。 </li><li>すべての次の手順は氷の上で実行する必要があります。メスで新鮮な、またはスナップインを凍結したマウスの心臓から左心室を解剖。 （注）このプロトコルは、マウスの心臓のために最適化されていますが、また、ラットやヒトの心臓に適合させることができます。また、異なる種から心臓組織の1グラムまで使用しています。 </li><li>メスの小さな個室に試料をトリミングします。 </li><li>溶解緩衝液15ミリリットルを充填した50mlファルコンチューブに組織片を転送します。 </li><li>ホモジナイズ10秒24,000 rpmでT-25ウルトラタラックスプローブホモジナイザー（IKA）の心臓組織。 </li><li> 30ミリリットルに溶解用緩衝液と等量のホモジネートを希釈する。 </li><li>さらに組織と自由核を均一にガラスdouncer（40ミリリットル）を使用します。大クリアランス乳棒で8打を実行します。 </li><li>連続して、100μmと70μmのナイロンメッシュセルストレーナー（BD Biosciences社）を介して分離し、粗核を渡します。 </li><li>原油核スピンダウンし、10分間700×gで冷却遠心（4°C）に分離します。 </li><li>反転管によって注意深く上清を除去し、ペーパータオルで管の内側を拭いてください。核ペレットを乱さないように注意してください。 </li><li>原油核が上下にソリューションを数回ピペッティングしてショ糖緩衝液の5ミリリットルで分離溶解する。溶解したペレットに、ショ糖バッファのさらに25ミリリットルを追加します。 </li><li>コー​​ティングされた遠心チューブ（ステップ1.1参照）に新たに調製し、ショ糖緩衝液10mlを追加します。&lt;/ LI&gt; <li>慎重にステップ1.9からショ糖緩衝液に溶解し再懸濁した核ペレットを蔗糖バッファーの追加10ミリリットルを重ねる。 </li><li> JS13.1フリースイングローターにそれらを配置する前に、遠心チューブのバランスをとり、高速遠心機（ベックマンアヴァンティS-25）にローターを配置します。 </li><li> 60分間、4℃で13000×gで核サンプルをスピン。 </li><li>スピンが完了したとき、ロータから慎重にチューブを取り外し、反転チューブの上清を捨て、ペーパータオルのチューブの内側から、残りの破片を拭く。 </li><li>核格納バッファの1ミリリットル（NSBプラスバッファ）で核ペレットを溶解する。注：NSBに加えて、DNAの安定剤として1.5mMのスペルミンが含まれています。 </li><li>心筋細胞核の免疫染色は、ステップ2.1に進みます。 </li></ol> 2。フローサイトメトリーのために免疫染色<ol><li>免疫染色のために、ネガティブコントロールを用意します。核サンプルの20μlのアリコートを取り出し、NSB PlusバッファのDD 980μL。 </li><li> immunolabel心筋細胞核に1:500の希釈で核サンプルに抗中心体周辺物質1抗体（ウサギ抗-PCM-1、アトラス抗体）を追加します。ステップ2.1で調製したネガティブコントロールに抗-PCM-1抗体と同じ希釈でアイソタイプの抗体を追加します。 </li><li> 4℃で一晩、ネガティブコントロールとサンプルチューブをインキュベートします。 </li><li> NSBプラスバッファ（10分間700×gで冷却遠心（4℃）にチューブをスピンダウンし、上清を捨て、NSB Plusバッファの1ミリリットルで核ペレットを溶解）で少なくとも1回はネガティブコントロールとサンプルを洗浄します。 </li><li> 1:1000の希釈で、ネガティブコントロールとサンプルチューブに抗ウサギ蛍光二次抗体（FITCまたはAPC）を追加します。 </li><li> 1時間4℃で、ネガティブコントロールとサンプルチューブをインキュベートします。 </li><li> NSBプラスバッファ（10分間700×gで冷却遠心（4℃）にチューブをスピンダウンして、少なくとも一度はネガティブコントロールとサンプルを洗う。上清を破棄して）NSB Plusバッファの1ミリリットルで核ペレットを溶解する。 </li><li>フローサイトメトリー分析およびソートを続行します。 </li></ol> 3。フローサイトメトリー<ol><li>手順1.1で説明したようにソートし、フローサイトメトリーを開始する前に、1％BSA / PBS溶液で被覆核のコレクションチューブ（ファルコン15ミリリットル）。 </li><li> 30μmのセルストレーナーを通してサンプルとネガティブコントロールをフィルタリングし、フローサイトメーター（BD流入）に最初の負の制御をロードします。前方散乱光（FSC）、前方散乱パルス幅（FSパルス幅）と側方散乱（SSC）（ 図2AおよびB）に基づいて、核とシングレット（単核）を定義する第1および第2のゲートを定義します。サンプルにDNA染色（DRAQ5（1:500））を追加すると、最初に核の集団を識別するのに役立ちます。 </li><li>免疫標識サンプルをロードし、非心筋細胞核（PCM-1陰性）から心筋細胞核（PCM-1高い正）を分離する第3のゲートを定義します。ソートを開始（ 図2cおよびd）。 </li></ol> オプション：核DNA量（倍数性）を分析し、核への染色適切なDNA（例えば、ヘキスト33342またはDRAQ5）（ 図2e）を追加し 、細胞周期の解析を実行するために。 <ol start="4"><li>フローサイトメトリー選別した後、氷上に核を配置し、選別純度（ 図3AおよびB）を決定するために再分析します。 </li><li> 15分間冷却遠心機で1500×gでコレクションチューブ内のソート済み核スピンダウンする。 </li><li>下流のアプリケーションと互換性の緩衝液中で核ペレットを溶解する。 </li></ol> 4。代表的な結果核の形態との整合性は、DNA染色により評価し、顕微鏡（ 図1）によって可視化することができます。成功したPCM-1標識は、落射蛍光顕微鏡により、フローサイトメトリー（ 図1、図2CおよびD）で評価することができる。 PCM-1-positiveと負の集団がよく（ 図2cおよびd）互いに分離する必要があります。マウスの左心室にすべての核の約30％が心筋細胞核（ 図2D）でなければなりません。純度のソートは、再分析ソート核（ 図3a及びb）によって評価することができる。両方の原子核の集団は95％を超えて選別純度を持っている必要があります。 <img alt="図1" src="/files/ftp_upload/4205/4205fig1.jpg" /> 図1。 PCM-1は心筋細胞の核を識別します。心臓核（）は、成体マウスの心臓にPCM-1（b）に及びNkx2.5（C）に対する抗体で染色されています。 （d）のPCM-1-標識心筋細胞の核細胞質に囲まれた（ミオシン重鎖（MHC））と転写因子Nkx2.5を発現し、PCM-1染色（スケールバーは20μmと10心筋細胞核の正確な識別を文書化されているμmの（D、挿入図））。 （e）のDNAを用いて可視化心臓核分離株はDRAQ5を染色する。 （FおよびG）Cardiomyocyte核は、PCM-1（スケールバーは10μm）に対する抗体で標識されています。 （注）、組織切片の筋細胞核内および単離された核（矢印）のPCM-1のepinuclear染色パターン。 <img alt="図2" src="/files/ftp_upload/4205/4205fig2.jpg" /> 図2。心筋細胞核のフローサイトメトリー選別（）心臓核は前方散乱光（FSC）と側方散乱（SSC）によって識別されます。 （b）は第二のゲートは、FSCとFSパルス幅5で、単一の核を識別します。 （c、d）の蛍光ゲーティングは、心臓組織から心筋細胞核の分離（PCM-1陽性）と非心筋細胞（PCM-1陰性）が核になります。マウスの心筋細胞はほとんど（&gt; 80％）二倍体（2n）である（e）は、唯一の小さなサブセットの（4n）6倍体である。 （注）、人間の心筋細胞は倍数性核（&gt; 2n）の7,8の高い周波数が含まれています。 <img alt="図3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <stroNG&gt;図3。ソートされた心筋細胞と非心筋細胞核の純度分析。ソートされた非心筋細胞（）と心筋細胞核（b）の再解析。両集団は99％を超えるソート純度を示しています。

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利害の衝突が宣言されません。

心筋細胞核の正確な識別は、心筋2,3の再生過程の分析のために重要である。新鮮な組織から心筋細胞を分離する従来の手法は、主に細胞外マトリックスタンパク質の酵素消化と低速遠心分離により間質細胞からの後続の精製に基づいています。胚性幹細胞（ESC）から生きている心筋細胞のさらなる精製は、そのようなSIRPA 9またはミトコンドリアの染料10などの表面マ?...

<table border="1"><tbody><tr><td> 1。溶解バッファー </td></tr><tr><td> 試薬の名前 </td></tr><tr><td> 0.32Mスクロース</td></tr><tr><td> 10mMトリス-HCl液（pH = 8） </td></tr><tr><td> 5mMのCaCl 2を </td></tr><tr><td> 5mMの酢酸マグネシウム</td></tr><tr><td> 2.0mMのEDTA </td></tr><tr><td> 0.5mMのEGTA </td></tr><tr><td> 1mMのDTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2。ショ糖バッファ </td></tr><tr><td> 試薬の名前 </td></tr><tr><td> 2.1 Mのショ糖</td></tr><tr><td> 10mMトリス-HCl液（pH = 8） </td></tr><tr><td> 5mMの酢酸マグネシウム</td></tr><tr><td> 1mMのDTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3。核貯蔵緩衝液（NSBプラス） </td></tr><tR&gt; <td> 試薬の名前 </td></tr><tr><td> 0.44 Mショ糖</td></tr><tr><td> 10mMトリス-HCl液（pH = 7.2） </td></tr><tr><td> 70mMのKClを</td></tr><tr><td> 10mMのMgCl 2 </td></tr><tr><td> 1.5 mMのスペルミン</td></tr></tbody></table><table border="1"><tbody><tr><td> 試薬および器具 </td><td> 会社 </td></tr><tr><td>アイソタイプウサギIgG-ChIP用グレード、＃ab37415 </td><td>アブカム</td></tr><tr><td>ウサギ抗-PCM-1抗体、＃HPA023374 </td><td>アトラス抗体</td></tr><tr><td>ロバ秒。抗体、抗ウサギアレクサ488弗素、＃-21206または同等秒。蛍光抗体</td><td>ライフテクノロジーズ</td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td>セルストレーナーは30μm、70μmと100μm </td><td> BDバイオサイエンス</td></tr><tr><td>ガラスdouncer（40ml）および&quot;L&quot;を乳棒</td><td> VWR（ウィートン工業株式会社） </td></tr><tr><td> T-25ウルトラタラックスホモジナイザー</td><td> IKAドイツ</td></tr><tr><td>分散ツールS25 N-18 G </td><td> IKAドイツ</td></tr><tr><td>ベックマン·アヴァンティ遠心</td><td>ベックマン·コールター</td></tr><tr><td>ファルコンチューブ15ミリリットルと50ミリリットル</td><td> VWR </td></tr><tr><td>ベックマン遠心チューブ＃363664 </td><td>ベックマン·コールター</td></tr><tr><td> JS13.1フリースイングローター</td><td>ベックマン·コールター</td></tr><tr><td>流入メーター</td><td>ベックマン·コールター</td></tr><tr><td>チューブローテーター</td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

ほとんどの戦略は、唯一の細胞マーカータンパク質1に依存しているとして、心筋細胞の核の同定は、組織切片に挑戦されています。そのような増殖およびアポトーシスとして心筋細胞では稀なイベントは、心筋細胞核の正確な同定は、恒常性と病理学的状態2の細胞の更新を分析する必要があります。ここでは、中心体周辺物質1（PCM-1）およびそれ以降のフローサイトメトリー選別に対する抗体を用いた密度の堆積作用と免疫標識によって死後組織から心筋細胞核を単離する方法を提供します。この戦略は、新鮮な組織と凍結アーカイブ材料にも同様の作業の利点を有するハイスループット解析と分離することができます。これにより、既にバイオバンクで収集された材料を研究することができます。この手法は適用可能で、種の広い範囲でテストし、そのような炭素14年代測定3、セル-CYなどの複数のダウンストリームアプリケーションに適していますCLE分析4、チミジン類似体の可視化（例えば、BrdUとIDU）4、トランスクリプトームとエピジェネティクス解析。

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

心臓核密度沈降を介して分離し、フローサイトメトリーにより心筋細胞核を識別し、ソートするための中心体周辺物質1（PCM-1）に対する抗体で免疫標識されています。

事後組織から心筋細胞核の分離

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

isolation-of-cardiomyocyte-nuclei-from-post-mortem-tissue

Research

JoVE Journal

Medicine

16.9K ビュー.  University of Lund. 心臓核密度沈降を介して分離し、フローサイトメトリーにより心筋細胞核を識別し、ソートするための中心体周辺物質1（PCM-1）に対する抗体で免疫標識されています。

ビデオ: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

部分的に膵臓を摘出された患者からのヒト膵島の単離

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

医学

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

ヒト前立腺癌サンプルからがん幹細胞の単離

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

成体マウスからの心房筋細胞の単離

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

光遺伝学的変調型心筋細胞活性の電気機械評価

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

hiPSC由来心筋細胞を用いた虚血性心疾患のモデルと心筋細胞収縮の映像的比較

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

RNA解析およびマクロファージ表現型解析に適したヒト内臓脂肪組織からの生存脂肪細胞および間質血管画分の単離

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

心筋梗塞マウスにおける超音波ガイド下多能性幹細胞由来心筋細胞移植術

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

心筋梗塞後の成体マウス心臓からのシングルセルRNAシーケンシングのための非心筋細胞懸濁液の調製

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

ヒト異種異系フリーのヒト治療法によるヒト脂肪由来幹細胞の単離と培養

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

ヒトケロイド組織からの初代皮膚線維芽細胞の単離、培養、および特性評価

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...