Name: establishment of a primary culture of patient-derived soft tissue sarcoma
Uploaded: 2018-04-11T13:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

著者は、編集者の支援のため Gráinne ティアニーを感謝したいです。

STS 細胞質量手術 STS 患者から摘出した腫瘍から分離されます。このプロトコルは、ローカル倫理委員会によって承認されている、よい臨床練習のガイドラインおよびヘルシンキ宣言に従って実行されます。すべての患者は、研究に参加する書面によるインフォームド コンセントを与えた。摘出標本は経験豊富な肉腫の病理学者によって解析し、手術後 3 時間以内に処理されます。
1. 腫瘍標本収集と処理:
<ol><li>パラフィン フィルムで密封 DMEM 低グルコース培地 50 mL で 100 mL 滅菌尿容器に肉腫の病理学者の助けを借りて、腫瘍標本を収集します。</li>
	<li>腫瘍切除後 3 h の最大 4 ° C でサンプルを格納します。</li>
	<li>滅菌のティッシュ文化フードの下のすべての次の手順を実行します。<ol>
<li>70% エタノールでフードを消毒し、滅菌手袋を着用します。</li>
		<li>70% のエタノール (滅菌ガーゼを使用) と鋼ステンレス ピンセットを消毒します。</li>
		<li>尿容器からパラフィン フィルムを削除し、破棄します。</li>
		<li>70% エタノールで尿容器の外装を洗ってください。</li>
		<li>角型シャーレを開きます。</li>
		<li>尿容器を開き、滅菌鋼ステンレス ピンセットを使用して角型シャーレに腫瘍の標本を転送します。</li>
	</ol>
</li>
	<li>PBS での腫瘍の標本を 2 回洗います。</li>
	<li>無菌手術のメスを開きます。<ol>
<li>1-2 mm3個の腫瘍の標本細かくシュレッドします。</li>
		<li>2 mL チューブに総サンプルの 4 分の 1 を収集します。</li>
		<li>(購入) チューブを trizol 試薬 1 mL を追加し、-80 ° C (サンプルは STS 遺伝子発現評価ダウン ストリームの解析に使用されます) ですぐにフリーズします。</li>
	</ol>
</li>
	<li>15 mL チューブに精巧に寸断された腫瘍材料の残りの部分を収集します。<ol>
<li>コラゲナーゼの 4 つの mL を追加私にソリューション (4 μ G/ml の濃度で、PBS に溶解しているコラゲナーゼ タイプ) を入力します。</li>
		<li>コラゲナーゼを希釈 4 ml DMEM 高文化メディアのソリューション型 (最終濃度コラゲナーゼ型の私は 2 μ G/ml)。</li>
		<li>15 mL チューブを閉じてパラフィン フィルムでシールします。</li>
		<li>15 mL チューブを消化させる 15 分攪拌条件で 37 ° C でインキュベーターでローリング シェーカーに入れてください。</li>
		<li>15 分後に、一晩室温で撹拌条件下でローリング シェーカーにチューブを格納します。</li>
	</ol>
</li>
	<li>次の日は、滅菌のフードの下で 15 mL のチューブを置きます。</li>
	<li>軽く 50 mL チューブの上部に接続されている 100 μ m の滅菌フィルターを介して細胞懸濁液をフィルターします。</li>
	<li>1% ペニシリン/ストレプトマイシン、グルタミン 1%, 10% 牛胎児血清を添加した DMEM 高文化メディアでフィルターを洗浄します。</li>
	<li>100 μ m の滅菌フィルターを捨てて、50 mL のチューブを閉じ、室温で 5 分間 225 × g で遠心します。</li>
	<li>チューブを反転して上清を破棄します。</li>
	<li>1-2 mL DMEM 高文化メディアの 1% ペニシリン/ストレプトマイシン、グルタミン 1%, 10% 牛胎児血清を添加した細胞再中断します。 注:ボリュームは、腫瘍サンプルの処理量に依存します。</li>
	<li>ノイバウアー室を持つセルをカウントします。<ol>
<li>トリパン ブルー色素のサンプル 1:2 を希釈し、希釈の 10 μ L を使用してセルをカウントします。</li>
		<li>少なくとも 2 回カウントし、収集したセルの合計の平均数を知っている複製の平均を計算します。</li>
	</ol>
</li>
	<li>80,000 のセル/cm2の密度で単層培養細胞。 注:常に実験でコントロールを含めます。各条件の少なくとも 3 の技術的なレプリケートを実行します。少なくとも 2 の生物的複製を実行します。臨床薬理学、遺伝子発現、タンパク質発現解析の実験実施されなければならない文化通路の数が少ないと細胞が癌細胞の表現型を失うことを避けるために播種の 30-45 日以内。</li>
	<li>細胞試料。<ol>
<li>200 μ L 1% ペニシリン/ストレプトマイシン、グルタミン 1%, 10% 牛胎児血清を添加した DMEM 高文化メディアに 100,000 細胞を収集します。</li>
		<li>ピペット使い捨て漏斗にソリューション (200 μ L ピペット) フィルター、ガラス スライドを搭載します。</li>
		<li>収集を開き、サンプルを挿入します。</li>
		<li>20 分間 18.6 x g で、収集を実行します。</li>
		<li>目標到達プロセス、フィルター搭載を破棄します。</li>
		<li>10 分 5 分クロロホルムによって続いてのアセトンのスライドを修正します。</li>
		<li>-20 ° C でスライドを保存します。 注:雪解けに汚れる前にサンプルをメタンハイド レートし、染色の製造元の指示に従ってを実行します。少なくとも 3 技術的複製を準備します。</li>
	</ol>
</li>
</ol>2. STS 細胞培養:
<ol><li>週に一度 (DMEM 高文化メディア 10% 牛胎児血清、グルタミン 1% 1% ペニシリン/ストレプトマイシンと補われる) メディアを変更します。<ol>
<li>200-1,000 μ L ピペットを使用して培養液を破棄します。</li>
		<li>200-1,000 μ L ピペットを使用して新しい新鮮な培地を追加します。</li>
		<li>セル通過:<ol>
<li>合流点が約 90% のセルをデタッチします。 注:STS 細胞培養増幅は、培養条件に基づいて週に一度実行必要があります。</li>
			<li>媒体を廃棄し、PBS で洗浄、トリプシン 1 2 3 mL を追加し PBS で x 注:トリプシンの使用量は、得られた培養細胞の量に依存します。</li>
			<li>37 ° C で 3-5 分のための細胞を孵化させなさい</li>
			<li>200-1,000 μ L ピペットを使用してプレートから剥離細胞を収集します。</li>
			<li>15 mL チューブに剥離細胞をピペット、1% ペニシリン/ストレプトマイシン酵素反応を停止する、1% のグルタミン, 10% 牛胎児血清を添加した DMEM 高培地 4 mL を追加します。</li>
			<li>室温で 5 分間 225 × g で遠心分離機します。</li>
			<li>上清を捨てます。</li>
			<li>再 1% ペニシリン/ストレプトマイシン、グルタミン 1%, 10% 牛胎児血清を添加した DMEM 高培地 1 mL を追加することによって細胞のペレットを中断します。</li>
			<li>新しい培養プレートまたは製造元の指示に従って最終巻とフラスコのセルをシードします。 注:1:3 以上の細胞を分割しないでください。</li>
		</ol>
</li>
	</ol>
</li>
</ol>3. 下流解析:
注:さまざまなさまざまな実験は、このモデル (下記参照) を使用して実施することができます。したがって、どの解析が実行されるは、十分な数のサンプルと複製を準備することができますので、研究の設計段階の間に決定することが重要です。
<ol><li>MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium ブロマイドなど増殖アッセイ</li>
	<li>ターミナルトランスフェラーゼ (TdT) などアポトーシス アッセイ ニック端標識 (tunel)</li>
	<li>免疫組織化学的解析</li>
	<li>遺伝子発現解析</li>
	<li>西部のしみの分析</li>
	<li>ELISA の試金</li>
	<li>蛍光活性化セル (FACS) 分析を並べ替え</li>
	<li>
異種移植モデル生体内で</li>
</ol>

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著者はある利益相反を開示します。

よく定義された臨床前モデルは、腫瘍の分子の背景を解明、予後不良を予測して癌に対する新しい治療戦略の開発に必要です。これは特に STS などのまれな腫瘍の重要な形態の面でその高い異質性、攻撃的な潜在的なおよび臨床的行動課題 STS 生物学および患者管理21の私達の理解。さらに、利用可能ないくつかの商業肉腫細胞株は、間葉系腫瘍18のこのグ?...

軟部組織肉腫 (STS) は、間葉系由来会計すべて固形腫瘍1,2, 1% の異機種混在病変のスペクトルと新しい世界保健機関の分類は 50 以上の存在を認識しサブタイプが異なる3。これらのうち、最も一般的な histotypes、脂肪肉腫や脂肪肉腫、平滑筋肉腫、15% とすべてアダルト STS、それぞれ4、5の 11% を占めます。STS は、体のさまざまな部分で成長できる、四肢と後腹膜が 60% とそれぞれ6の場合の 20% で発生する、最も一般的なサイトです。
ローカライズされた疾患の治療の基礎は広範囲の外科的切除、術前・術後の化学療法の利点がまだ明確でないと選択された場合は7にのみと見なされます。転移性の設定では、化学療法の標準治療が限られた結果8に示します。STS の分子生物学のよりよい理解は、現在の差動診断を改善、利用可能な治療の最適化、および療法のための新しいターゲットを識別に役立つでしょう。
このコンテキスト内で不死化細胞ライン9を使用して長年にわたって癌の研究を行った。これらの実験モデルは通常標準的な単分子膜サポートで培養または、生体内で異種移植モデル10を確立する免疫不全齧歯動物に注入します。不死化細胞株は、研究実験の十分な数を行うことができます管理およびストア簡単材料を表します。彼らは、実際には、少なくとも高価な最も広く臨床研究11でツールを使用します。
ただし、セルライン基づく癌モデルもいくつかの制限があります、例えば延長通路数の増加とともに単分子膜における文化誘導表現型、遺伝的浮動作る細胞ほどキーとなる癌を要約するには機能。さらに、ライン培養細胞は、細胞間相互作用やシグナル伝達の分子クロストーク腫瘍の生物学的挙動を模倣し、がん微小環境の特徴を再現する失敗します。これらの問題は、既存の前臨床および臨床結果12の間のギャップの基礎を形成します。
上記を考えると、患者由来の主な文化への興味は13に増加しています。確かに、癌と正常組織の切除は癌細胞の表現型とより腫瘍微小環境の代表は、原料をご提供の不均一性を失うことのリスクを軽減します。また、外科的切除を受けた患者から収穫される新鮮な腫瘍の標本の使用は、単一の腫瘍を調べると患者の体14の同じ部分から異なる病変を比較する私たちできます。上記の理由から、組織由来細胞培養腫瘍病態と薬理学15,16,17, 特にどの市販肉腫細胞株における STS の研究に貴重な資料を提供します。限定18です。我々 はこのため手術時摘出腫瘍組織13,19,20から始まって患者由来 STS 一次電池文化を確立する方法を最適化しました。本手法は、腫瘍標本を単一細胞懸濁液を取得する一晩の酵素組織解離に続いて小さな組織片に分解ので構成されます。次の日は新鮮な培地を追加することで酵素消化を停止し、得られた懸濁液を抽出し、組織片、細胞凝集塊と余分なマトリックス材料やゴミを取り除きます。最後に、孤立した腫瘍細胞の割合はスライド ガラス固定し、細胞形態学的、免疫組織化学、分子細胞遺伝学的評価による STS の主な文化の確立を目的と下流の分析のため保存に cytospinned(例えば魚における MDM2 増幅は高分化型脂肪肉腫と dedifferentiated の脂肪肉腫の標準的な鑑別診断を表します)4します。 残りの細胞は遺伝子発現プロファイリングと薬理学的研究のための標準的な単層培養に播かれます。低い使用して、実験を行いすべては通路の初代培養特異的ながんのサブクローンの選択を避けるために、経験豊富な肉腫の病理学者によって分析しました。このように完全に人間 STS ex vivoモデル13,19,20を作成しました。この汎用性の高い、簡単にハンドル モデルは異なったタイプの臨床研究など診断と予後のための新しい分子マーカーを識別するかのメカニズムと活動をより深く理解するための便利なツールを表すことができます。STS の管理で使用される標準および新しい薬剤のアクション。

<table><tbody><tr><td>DMEM High Glucose</td><td>EuroClone</td><td>ECB7501L</td><td></td></tr><tr><td>Fetal bovine serum</td><td>EuroClone</td><td>ECS0180D</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Paraformaldehyde 4% aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>157-4-100</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Triazol reagent</td><td>Ambion Life-Technologies</td><td>15596018</td><td></td></tr><tr><td>Trypsin</td><td>EuroClone</td><td>ECB3052D</td><td></td></tr></tbody></table>

establishment of a primary culture of patient-derived soft tissue sarcoma

軟部組織肉腫 (STS) は、異種悪性腫瘍診断が困難、分類、および管理とのスペクトルを表します。日には、これらのまれな腫瘍の 50 以上の組織学的サブタイプが同定されています。したがって、彼らの驚くべき多様性と発生率が低いため、これらの腫瘍の生物学の私達の理解はまだ限られています。患者由来の文化は、STS の病態と薬理学の研究に理想的なプラットフォームを表します。このように外科的切除を受けた患者から収穫される腫瘍の標本から STS の人間前臨床モデルを作成しました。患者由来 STS 細胞培養は、コラゲナーゼの消化力によって摘出標本から得られ、濾過によって分離します。セルがカウント、シードしの標準的な単層培養 14 日間左し、下流の分析によって処理されます。細胞機能と、利用可能な場合の評価を通じて確認した STS 初代培養の確立医薬や分子の解析を実行する前に免疫組織化学的マーカー。このメソッドは、これらの不十分な検討悪性腫瘍の自然史を勉強するには 1) と 2) の作用メカニズム詳細については努力でさまざまな薬物の効果をテストするための便利なツールを表します。

患者由来の軟部肉腫の主な文化の確立を取得し、結果を得られた 1 つの特定の STS histotype13の例をここで報告する簡単な方法を考案しました。プロトコルは高分化脂肪肉腫を含む別の STS histotypes の初代培養の確立のために使用された、脱分化型脂肪肉腫、粘液型脂肪肉腫、多形型脂肪肉腫、未分化 myxofibrosarcoma多形細胞肉腫、GIST、デスモイド線維腫?...

Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

次のプロトコルは、患者由来の軟部肉腫 (STS) の主要な文化の確立に焦点を当てください。このモデルでは、分子の背景、これらのまれな悪性腫瘍の薬理学的プロファイルを理解する私たちを助けることができる、STS 管理の改善に向けたさらなる研究のための開始点を表すことができます。

患者由来の軟部肉腫の主な文化の確立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

establishment-primary-culture-patient-derived-soft-tissue

Research

JoVE Journal

Cancer Research

14.2K ビュー.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 次のプロトコルは、患者由来の軟部肉腫 (STS) の主要な文化の確立に焦点を当てください。このモデルでは、分子の背景、これらのまれな悪性腫瘍の薬理学的プロファイルを理解する私たちを助けることができる、STS 管理の改善に向けたさらなる研究のための開始点を表すことができます。

ビデオ: Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

がんの確立は、ヒト従来の骨肉腫からの幹細胞培養物

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

がん研究

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

肉腫患者由来異種移植片からの腫瘍始発細胞の単離と特徴付け

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

胃癌患者由来異種移植片モデルおよび原発細胞株の確立

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

外科的組織からの初代ヒト結腸腫瘍細胞の単離およびトラクションサイトメトリーのためのソフト弾性基板上にそれらを直接培養

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

生物工学

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人間の前庭神経鞘腫の初代培養

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

医学

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

患者由来の腫瘍サンプルから3次元スフェロイドを樹立し、薬物に対する感受性を評価

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

患者由来乳房オルガノイドの確立と培養

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

新鮮組織からの膵臓癌由来腫瘍オルガノイドおよび線維芽細胞

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

著者スポットライト:新鮮組織からの膵臓がん由来腫瘍オルガノイドと線維芽細胞の確立  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

膵臓癌由来腫瘍オルガノイドおよび線維芽細胞の新鮮組織からの樹立

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

患者由来の軟部肉腫の主な文化の確立

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がんの確立は、ヒト従来の骨肉腫からの幹細胞培養物

肉腫患者由来異種移植片からの腫瘍始発細胞の単離と特徴付け

胃癌患者由来異種移植片モデルおよび原発細胞株の確立

外科的組織からの初代ヒト結腸腫瘍細胞の単離およびトラクションサイトメトリーのためのソフト弾性基板上にそれらを直接培養

人間の前庭神経鞘腫の初代培養

患者由来の腫瘍サンプルから3次元スフェロイドを樹立し、薬物に対する感受性を評価

患者由来乳房オルガノイドの確立と培養

膵臓癌由来腫瘍オルガノイドおよび線維芽細胞の新鮮組織からの樹立

膵臓癌由来腫瘍オルガノイドおよび線維芽細胞の新鮮組織からの樹立

膵臓癌由来腫瘍オルガノイドおよび線維芽細胞の新鮮組織からの樹立