This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (26461185 and 25460137).

注：この研究は、適切な倫理委員会によって承認された。 ヒト肺線維芽細胞の1。初代培養<ol><li>肺組織のコレクション： <ol><li>直接の外科手術室からのヒト肺組織のサンプルを得る。できるだけ離れ腫瘍から収集した非癌性サンプルと、癌性及び非癌性肺組織から約1cm 3ブロックを収集します。 </li><li> 100単位/ mlペニシリン、100μg/ mlのストレプトマイシンおよび0.25 / mlのアンホテリシンB（無血清培地）を補充したダルベッコ改変イーグル培地（DMEM）中でサンプルを懸濁する。無菌50mlチューブ中の浮遊サンプルを維持し、実験室に移す。 NOTE：線維芽細胞は、上皮細胞、内皮細胞および平滑筋細胞などの他の細胞型と比較して高度回遊あり、増殖性。成長方法は、線維芽細胞のこれらの機能を活用し、かつoutgro組織切片から翼細胞は、線維芽細胞に豊富に存在する。いくつかの細胞継代後に、他の細胞型は、精製された線維芽細胞培養物中で得られる、生存し、10％ウシ胎児血清（FBS）を含むDMEM中で伝播することができない。細胞は、初代​​培養後3~7通路を使用することができる。 </li></ol></li><li>細胞成長のための移植片培養およびコンディショニング： <ol><li>修正5と以前に報告されたプロトコルを使用して、線維芽細胞の初代培養を行います。 </li><li>研究室では、培地なし10cmの組織培養皿に組織サンプルを配置し、小さな切片に切断〜滅菌ピンセットとメスを用いて大きさ2〜3mmの。このプロセスの間、部分乾燥を避ける、そうでなければ細胞伸長の効率が損なわれる。 </li></ol></li><li>上皮細胞層および結合組織の分離。 <ol><li> 4℃で16時間培養2000 PU / mlのディスパーゼIを含む培地と培養中の切断組織切片を浸し。&lt;/ LI&gt; <li>皿に組織切片を転送し、隣接する結合組織から上皮細胞層を分離する。微生物の汚染を避けるために、クリーンベンチ内でセクションを処理する。 注：上皮細胞の初代培養が必要な場合は、ステップ1.3を行う。唯一の線維芽細胞を単離することがある場合は、その後の移植片培養し、細胞の継代は、精製線維芽細胞集団をもたらす上皮細胞、のために不利であるため、ステップ1.3を省略します。 </li></ol></li><li>組織切片の添付ファイル： <ol><li> 2メスを使用して1ミリメートル片に組織をミンチ。片が十分に小さくない場合は、皿からより容易に切り離して、細胞をディッシュ表面に脱却することができないであろう。 </li><li>各ピースを囲む空の領域を維持するために、互いに離間して小肺組織切片を置く。組織切片の取り付けを容易にするために、手術用メスの刃を用いて組織培養皿の表面に傷を作る。 注：スクラッチまた、細胞が移動することができ、それに沿ってトラックを提供し、細胞成長を促進するために表示されます。 <ol><li>代替的に、個別に6ウェルプレートの各ウェルに細分化した組織片を置き、カバースリップでそれらをカバー。シリコーングリースを使用したプレートの表面にカバースリップを取り付けます。 </li><li>無菌のピンを使用して、各ウェルに1.5センチメートル離れた2点でのシリコーングリースを配置します。組織片のカバレッジは、いくつかの継代後の細胞成長および細胞増殖の効力を増強する。 </li></ol></li></ol></li><li>その後の移植片培養： <ol><li>サンプルが完全に乾く場合は、効率的な細胞成長は大部分が失われているように、組織切片は、完全に乾く前に静かに皿に培養液を追加します。あまりにも急速に組織切片を切り離すと同じようにメディアをかけないでください。 </li><li> 10％のFBSを含むDMEM中で培養細胞。追加の浮力から脱離を促進するようにそれは、浮動可能にすることをあまりにも多くのちょうど組織をカバーするために十分なメディアを使用してではなく、一品。 </li></ol></li><li>細胞継代： <ol><li>培養皿の繰り返し運動が組織切片の剥離につながることができように、常に注意して培養液を処理します。 </li><li> 5-7日間、37℃のインキュベーターで培養皿を置きます。一日おきに培地を更新し、培地交換の際に最小限の培養皿を扱う。線維芽細胞は、今後数週間にわたって組織切片の端から脱却する。 NOTE：〜2-3週間後、外部増殖線維芽細胞は、ほとんどの表面積の量に依存して、コンフルエンスに達する。 </li><li> 1×PBSで細胞を洗浄し、私はプレートにトリプシンの1ミリリットルを追加します。 </li><li>トリプシン処理後、10％FBSを補充したDMEMの9ミリリットルを用いて細胞を切り離す。組織切片と一緒に、10ミリリットルチューブ内の培地に懸濁した細胞を収集します。細胞ペレットを形成するために遠心分離した後、新しい培地で得られたペレットを再懸濁する。 </li><li>新しいCUL上に細胞や組織切片をシードトゥーレの料理は、その時点で組織は、細胞が付着し、成長しながら、皿に​​取り付けることができない。翌日、吸引により、浮遊組織片を除去し、培地を変更してください。添付線維芽細胞は、その後の文化に伝播する。 </li></ol></li></ol>ヒト肺癌線維芽細胞と癌細胞の2三次元共培養<ol><li>細胞および試薬の調製<ol><li>約2×10 5上皮細胞およびウェルあたり2.5×10 5線維芽細胞を使用しています。コラーゲンタイプIA（3 mg / mlの、pHが3）、FBS（100％）、再構成緩衝液（50mMのNaOH、260ミリモルのNaHCO 3、200mMのHEPES）、5×DMEMを調製し、DMEMは、線維芽細胞培養10％FBSを補充した3次元共培養培地（1：癌細胞に対する線維芽細胞培養培地および培地の1：1混合物）。 </li><li>培養A549肺腺癌細胞、線維芽細胞およびDMEM中でA549の三次元共培養は、10％FBSを補充した。 </li></ol></li><li>コラーゲンゲルfはormation： <ol><li> 1×PBSで10cmディッシュ上で培養した線維芽細胞を洗浄し、プレートにトリプシンの1ミリリットルに私を追加します。 37℃で約5分間トリプシン処理した後、10％のFBSを添加した9ミリリットルのDMEMを用いて細胞を切り離す。 10ミリリットルチューブに培地に懸濁した細胞を収集し、細胞ペレットを形成するために、200〜300×gで、それらを遠心する。 </li><li> 5×10 5 / mlの密度で、100％FBS中で得られたペレットを再懸濁する。 </li><li>氷上のコラーゲンゲルを準備します。次の手順の前に冷蔵庫での試薬、ピペット、チューブクール。偶数氷上で、混合物をある程度固化し、全量を全成分の合計よりも小さい。 </li><li>各ウェルに、（2.5×10 5細胞）、線維芽細胞懸濁液を0.5mlとを混合してコラーゲンゲルを調製FBS、2.3ミリリットルタイプIAコラーゲン、670μlの5×DMEM、および330μlの再構成緩衝液中で。ゲルは、室温で容易に設定することができます。 <ol><li>激しく均質マイルを確実にするためにピペット固定装置。ペッティングプロセス中に気泡を作成しないでください。 </li></ol></li><li> 6ウェルプレートの各ウェルに、混合物（3 ml）を添加し、30〜60分間妨害することなく、37℃のインキュベーター内でゲル化することを可能にする。 </li></ol></li><li>コラーゲンゲル上の癌細胞培養： <ol><li> 6変更を加えて、以前に報告されたプロトコルを使用して、3次元共培養を行います。 </li><li> 3D共培養培地に再懸濁癌細胞（またはA549細胞の場合は10％FBSを含むDMEM中で）1×10 5 / mlの密度で。 </li><li>各ゲルの表面上に再懸濁した細胞溶液2ml（2×10 5細胞）を注ぐ。実験条件に依存して、共培養中の癌細胞または線維芽細胞の細胞数を変更する。 </li></ol></li><li>ゲル収縮： <ol><li>癌細胞がコラーゲンゲルに付着することができるように、37℃で一晩ゲルをインキュベートする。各ゲルを切り離し、およびOを各ウェルに「浮遊培養 &#39;を生成6ウェルプレートfは。 NOTE：浮遊培養を使用することにより、ゲルの大きさの様々な変更は、共培養癌細胞と線維芽細胞との間の細胞の相互作用によって媒介される機械的張力およびコラーゲン分解によって誘導される。 </li><li>ウェルの端から各ゲルを分離するために角度の付いた21 G針や小さなへらを使用してください。 2〜3日おきに、3次元共培養培地2mlでウェルをリフレッシュ。 5日間毎日各ゲルの大きさを測定します。 </li></ol></li><li>コラーゲンゲルの分析： <ol><li>このようSircolアッセイコラーゲン定量法を用いて、各ゲルのコラーゲン含有量を測定する。ゲル5からRNAを収集した後、トランスクリプトーム解析または定量的RT-PCRを行います。 </li></ol></li></ol> 3.気液界面文化と浸潤アッセイ<ol><li> 5日間37℃でコラーゲンゲルを培養した後、新たな6-メッシュ（70μmの孔サイズ）に配置して空気に収縮したゲルを暴露ウェルプレート。フローサイトメトリーのために使用される市販の細胞ストレーナーからメッシュを作成します。 </li><li>静かに3D共培養培地の11ミリリットルで、各ウェルを埋める。 </li><li>無菌のスプーンを使用してメッシュ上にゲルを置き、ゲルから、残りの培地を除去。空気にゲルの上面を露出しながら、この状態では、媒体中のゲルを水没。気液界面、この培養条件を定義する。 注：インキュベーター中、37℃で気液界面培養を維持5-7日後に、浸潤性癌細胞がゲル中に移動する。細胞型および細胞の相互作用の結果として依存して、癌細胞の細胞形態変化の可変度は、気液界面培養によって誘導される。 </li><li>組織学的評価のために、室温で一晩ホルマリン溶液中でゲルを固定し、パラフィンに埋め込む。ヘマトキシリン​​およびエオシン（H＆E）で垂直セクション（4μm）を染色する。免疫組織化学的分析のOを実行nはセクション4。 </li></ol>

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The authors declare that they have no competing financial interests.

CAFSは、癌細胞の周囲のECMの主要な成分を形成し、腫瘍のための足場を提供するだけでなく、積極的に腫瘍の発達7に参加するだけでなく。証拠を蓄積すると、CAF媒介腫瘍進行8の重要な役割を強調し、最終的な予後上のCAFSまたはその関連分子の影響を解く。 以前の研究では、肺CAFS 4を単離するための成長方法を用いた。この実験では、皿の表面上...

Cancer tissue can be perceived as a type of organ, which evolves through close interactions between the cancer and the tumor stromal microenvironment, composed of cancer-associated fibroblasts (CAFs), immune cells, tumor vessels and the extracellular matrix (ECM). CAFs are the major source of soluble factors (cytokines, growth factors and chemokines) that exert mitogenic, pro-migratory and pro-invasive effects on cancer cells. They also stimulate tumor vessel formation and recruit precursor cells, such as bone marrow-derived cells (BMDC). Activated CAFs are involved in the production and remodeling of the ECM, thereby promoting the growth and spread of cancer cells1. CAFs also provide a niche that facilitates tumor cell colonization and metastasis and are capable of conferring stem cell phenotypes onto neighboring cancer cells. Pathological observations suggest that stromal reactions or fibrotic changes in cancer tissues are indicative of a poor prognosis. Recent studies have also demonstrated that tumor stromal features, such as the gene signature, can predict patient prognosis. Furthermore, CAF-derived factors can modulate sensitivity to chemotherapy, highlighting the role of CAFs in determining drug sensitivity and resistance2.
As CAFs play a multifaceted role in the promotion of tumor progression through signaling pathways that mediate interactions between CAFs and different cell types within the tumor microenvironment, they have attracted increasing attention as novel targets for cancer therapies. The heterogeneity of the cell populations within the cancer microenvironment presents an obstacle for targeting CAFs. Several markers for CAFs have been proposed, such as &#945;-smooth muscle actin (&#945;-SMA), fibroblast activation protein (FAP), and fibroblast specific proten-1 (FSP-1: also called S100A4); however, these molecular markers are not specific for distinguishing CAFs from other cells present in non-cancerous tissues3. Therefore, further studies are needed to obtain more knowledge about the specific properties of CAFs. To this end, it is informative to characterize primary cultured CAFs compared with patient-matched normal fibroblasts.
Recently, analyses on patient-derived CAFs have been reported in several cancer types, revealing unique gene expression patterns and cell behaviors compared with fibroblasts derived from non-cancerous tissues. Using isolated CAFs from human lung cancer tissues, we developed a three-dimensional co-culture method, enabling us to evaluate the properties of lung CAFs. In this model, we investigated the effects of the CAFs on lung cancer cell invasion, proliferation and collagen gel contraction, which experimentally recapitulated the tumor-promoting roles of lung CAFs4.

<table border="0" cellpadding="0" cellspacing="0" width="1035">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:267px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:471px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DMEM</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:471px;">D5796</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FBS</td>
			<td style="width:297px;">GIBCO</td>
			<td style="width:471px;">10437</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Collagen type IA</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;">CELL-1A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Reconstitution buffer</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cover slip</td>
			<td style="width:297px;">NUNC</td>
			<td style="width:471px;">174934</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Silicone grease</td>
			<td style="width:297px;">Dow Corning Toray</td>
			<td style="width:471px;">High vacuum grease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dispase I</td>
			<td style="width:297px;">WAKO</td>
			<td style="width:471px;">386-02271</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">6-well plate</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">353046</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell strainer (70 &#956;m)</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">352350</td>
		</tr>
	</tbody>
</table>

three-dimensional co-culture model for tumor-stromal interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

この共培養法は、腫瘍微小環境を模倣し、コラーゲンゲルに埋め込まれた癌細胞と線維芽細胞との間の相互作用を研究するための有用なツールである。コラーゲンゲル収縮、癌細胞浸潤および形態学的変化：以前の研究では、3つのパラメータは、この実験モデルで評価した。癌細胞の増殖はKi67の免疫染色4を用いて推定した。肺の組織サンプルを切除肺葉（ 図1A）の癌性...

Watch this Scientific Journal Video about Three-dimensional Co-culture Model for Tumor-stromal Interaction at JoVE.com

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

腫瘍間質相互作用のための三次元共培養モデル

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

three-dimensional-co-culture-model-for-tumor-stromal-interaction

Research

JoVE Journal

Medicine

17.3K ビュー.  The University of Tokyo. Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

ビデオ: Three-dimensional Co-culture Model for Tumor-stromal Interaction

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

ヒト乳腺線維芽細胞からの癌関連線維芽細胞の実験的生成（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

医学

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人間の神経内分泌腫瘍療法の研究のための三次元モデルとしての人間の神経内分泌腫瘍細胞株

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3次元（3D）モデルを用いた乳癌細胞浸潤の定量化

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

ヒト下垂体腺腫細胞の三次元アルギン酸ビーズ文化

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

複雑な大動脈の異常の三次元印刷

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

3次元印刷ガイドテンプレートアシスト経皮椎形成術(PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

マウス後肢壊疽モデルにおける足蹠血管系の高解像度3次元イメージング

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Guided Endodontics: Three-Dimensional Planning and Template-Aided Preparation of Endodontic Access Cavities

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, pulpitis or apical periodontitis may develop in the long term. Root canal treatment of teeth with severe PCO and pulpal or periapical pathosis is challenging for general practitioners and even for well-equipped endodontic specialists. To ensure detection of the calcified root canal and avoid excessive loss of tooth structure or root perforation, static navigation using templates ("Guided Endodontics") was introduced a few years ago. The general workflow includes three-dimensional imaging using cone-beam computed tomography (CBCT), a digital surface scan, and superimposition of both in a planning software. This is followed by virtual planning of the access cavity and the design of a template that will guide the drill to the desired target point. To do this, a true-to-scale virtual image of the drill must be placed in a way that the tip of the drill reaches the orifice of the calcified root canal. Once the template has been fabricated using computer-aided design and computer-aided manufacturing (CAD/CAM) or a 3D printer, guided preparation of the access cavity can be performed clinically. For research purposes, a postoperative CBCT image can be used to quantify the accuracy of the access cavity performed. This work aims to present the technique of static guided endodontics from imaging to clinical implementation.

Guided endodontics describes a template-aided approach for access cavity preparation. The procedure requires cone-beam computed tomography and a surface scan to produce a template. An incorporated sleeve guides the drill to the target point. This allows the preparation of minimally invasive endodontic access cavities in calcified teeth.

ガイド付き歯内療法:歯内アクセス虫歯の3次元計画とテンプレート支援準備

Pulp canal obliterations (PCO) are often a consequence of dental trauma, such as luxation injuries. Even though dentin apposition is a sign of vital pulp, ...