We gratefully acknowledge the open-source sharing of MATLAB particle-tracking code provided by Maria Kilfoil (<a href="http://people.umass.edu/kilfoil/">http://people.umass.edu/kilfoil/</a>), along with the earlier IDL code and extensive documentation provided by John C. Crocker and Eric R. Weeks. This work was made possible by funding from the National Cancer Institute (NCI/NIH), K99CA155045 and R00CA155045 (PI: JPC).

1。培養腫瘍球<ol><li> 1％アガロース溶液を得るために、アガロース0.1gの細胞培養グレードの水10mlを混合する。 </li><li> 96ウェルプレートにウェルにアガロース溶液40μlを小分けする前に、70℃以上（標準電子レンジでまたは加熱板を用いておよそ14秒）への熱アガロース溶液。 </li><li>標準的な技術を用いて細胞を採取しながら、少なくとも1時間、37℃でプレートをインキュベートする。 </li><li>懸濁液中の細胞の濃度を決定するために、血球計を使用する。 </li><li>千個/ mlに細胞懸濁液を希釈し、十分に硬化したアガロース床を含む、この希釈液100μlを加える。 </li><li> 37℃、5％CO 2に設定したインキュベーター内で一晩振とう機上でサンプルを置きます。 </li><li>シェーカーからサンプルを取り出して、細胞培養培地を100μlを加える。 </li><li>所望の直径に到達するまでスフェロイドをインキュベートする。例えば、9日後に、SPheroidは、直径約450ミクロンとなります。この時間の間、新鮮な増殖培地をウェルに添加することができるが、これは回転楕円体の除去をもたらすので、吸引は避けるべきである。 </li></ol> 2。ECMに埋め込まれた3D腫瘍球の準備<ol><li>層流フード内に必要な材料とワークステーションを準備します。 </li><li> 25部の滅菌水に2部株価プローブ（2％固体）を添加することにより、カルボン酸で修飾された直径1μm蛍光トレーサープローブの希釈液を調製する。 </li><li>冷蔵庫から3.1 mg / mlのウシコラーゲンのボトルを取り出して、氷の上に置きます。 </li><li>空の2ミリリットルバイアルにアリコートコラーゲンの125μlの。 </li><li>プローブを配布するために簡単にコラーゲンと渦を含むバイアルに希釈したトレーサープローブ溶液50μlを追加します。 </li><li> 41の総容積のために（黄色に変わります）フェノールレッドを含む適切な細胞培養培地の235μl加え205ミリリットル（ハーフ総量）を除去し、新たな2ミリリットルバイアル中に配置する前に簡単に0ミリリットルと渦。バイアル1は、腫瘍スフェロイドが含まれていますし、バイアル2は、対照混合物となります。 </li><li>中性pH（フェノールレッドを含む培地が赤に戻らなければならない）に戻す解決策を持って来るために1をバイアルに1 M NaOHでの〜2を添加する。これは混合物は、それが氷上に保持されていない場合は、硬化を開始させることに注意してください。簡単に、すぐに氷棚に戻り、その後、混合する渦。 注：回転楕円体は、文化の9日後に肉眼ではほとんど見えないし、アガロース床からの除去がデリケートな作業である。解剖顕微鏡の使用は、一部のユーザーのために有用であり得る。転送中に回転楕円体の完全性を維持することが最重要である。 </li><li>広口ピペットチップを使用して、静かによく腫瘍スフェロイドを含むからメディアを40μlを削除します。次のステップを行いながら、この40μlを保管しておいてください。 </li><li>回転楕円体かどうかを確認するために十分に確認してください前のステップで除去した。それがあった場合は、1をバイアルに回転楕円体を含む40μlを加える。ていない場合は、バックウェルに40μLを配置し、前の手順を繰り返します。 </li><li>静かに96ウェルプレートの3つの別々のウェルに60μlの部分に物を転送する前に（ボルテックスを危険にさらすしないでください、それは回転楕円体に損傷を与える場合があります）バイアル1をかき混ぜる。うまく回転楕円体が含まれているかを決定するために混合物を添加した後に顕微鏡で各ウェルを検査します。 </li><li>コントロールとして96ウェルプレートとラベルの空のウェルにこの混合物の60μlのを等分する前にバイアル2と渦に〜2μlの1 M NaOHおよび細胞培養培地40μlを追加します。 </li><li>少なくとも1時間、硬化させるために37℃のインキュベーターでプレートを置きます。 </li></ol> 3。サンプル点のグリッドを構築し、各ポイントで動画を撮影<ol><li>顕微鏡のステージにインキュベーターからサンプルプレートを転送します。サンプルのための10分EQUILすることができます加熱されたステージが利用できない場合、室温でibrate。 </li><li>必ずそれはそのままとイメージングのための準備ができているように、低パワーの対物レンズで試料を観察する。ウェル内の腫瘍の位置を決定する。 </li><li>取る何サンプル点を決める。一般的には、回転楕円体の周囲に同心円状に分布して各ウェル中の20のサンプル点は、十分に詳細な結果が生成されます。 </li><li>各目的の位置にステージを移動し、（ソフトウェアのインタフェースが使用できない場合は、手動で座標またはレコード）は、x座標とy座標を記録するために顕微鏡のインタフェースソフトウェアを使用しています。 </li><li>ハイパワーの対物レンズ（通常は100倍）に顕微鏡を切り替え、トレーサープローブの励起波長のための適切なフィルターキューブを選択します。グリッドの最初のポイントに移動するために、ステップ3.4で作成された点のリストを使用してください。 </li><li>視野を見つける上に移動して、ウェルの底を見つけることでピントを合わせる（FOV）は、いくつかの焦点の合ったTracを含むERプローブ。 </li><li>輝度ヒストグラムを観察し、画像が飽和しないようにしながら可能な最大のダイナミックレンジを与えるために露光強度や時間を調整します。 </li><li>フレーム当たり20〜30ミリ秒のフレームレートでビデオシーケンスを取得する（約800フレームまたは長さが16〜24秒では、各空間格子点における捕捉時間を最小化する必要性とのバランスがロバストMSD計算のための十分な統計を提供することをお勧めします）そして、適切な慣例で保存します。録音中は、顕微鏡やテーブルに触れないでください。 </li><li>繰り返しは、グリッド内の各サンプル点のために3.9〜3.6を繰り返します。 </li><li>繰り返し実験で各ウェルについて3.10に3.3を繰り返します。 </li></ol> 4。各サンプル点でのレオロジー特性を計算するためにビデオデータの分析<ol><li> （おそらく別のコンピュータ上の）分析フォルダに、すべてのビデオ·データをコピーします。 </li><li> MATLABにインポートされた画像データや他の解析ソフトウェア。 NOTEは：ここで採用したMATLABの粒子追跡ルーチンのセットに関する詳細なドキュメントは次の場所にあります<a href="http://people.umass.edu/kilfoil/">http://people.umass.edu/kilfoil</a> 。異なる位置に複数のファイルの処理を自動化するためのカスタム呼び出し元の関数の使用が推奨されます。 </li><li>適宜、プローブの中心位置を識別するための選択および拒絶パラメータ（サイズ、帯域など）を決定するために、ビデオのいくつかのフレームを分析することによってソフトウェアを較正する。これらの重要なステップのための広範囲な背景はクロッカーとグリアに記載されている。 </li><li>自動的にすべてのビデオフレームのための各プローブの位置を決定し、軌道にこれらの位置をリンクするためにソフトウェアを使用する。 </li><li>など具体的な対物レンズ、任意のピクセルビニングのために適切な空間較正係数（ピクセルあたりμm）を適用してくださいという、遅れ時間の関数としての平均二乗変位（MSD）を計算するために軌跡データを使用します（通常は行わメタデータ）。 </li><li>特定のサンプル24,28,43用GSERの適用可能性の限界は、また、ユーザは単にべき乗スケーリング、プラトーを比較することにより、MSDから直接ECMのプロパティを解釈したい場合を考慮し一般化されたストークス·アインシュタインの関係を用いて、G *は（計算異質性やその他のパラメータの値、指標。 </li><li>繰り返し実験で、各FOVのために4.6を通じて4.2を繰り返します。 </li><li>関心のある特定の周波数での粘弾性moduliiでステップ3.4からの位置データを共同登録してください。使用される顕微鏡の製造業者によっては、このステップは、顕微鏡メタデータまたは位置データを手動で表にすることができる読み出すカスタムルーチンによって促進することができる。 </li><li> ECMの空間的なレオロジーマップを生成する3次元の補間関数を使用します。 </li></ol>

The authors declare that they have no competing financial interests.

In this protocol we introduce a robust and widely applicable strategy for longitudinally tracking local changes in ECM rigidity in 3D tumor models. We envision that this methodology could be adopted by cancer biologists and biophysicists interested in mechanosensitive behavior implicated in matrix remodeling during tumor growth and invasion processes. Precise quantification of matrix degradation kinetics could be particularly valuable to those studying the activity of matrix metalloproteases, lysyl oxidase or other relevant biochemical species in 3D tumor models that are directly linked to matrix remodeling. Beyond the application to 3D tumor models described here, one could further imagine the implementation of this approach in conjunction with other disease model systems in which modification of matrix rheology over time plays important roles46,47. The utility of this method continues to be enhanced through software improvements that further automate the process and allow for increased throughput studies. This will result in increasingly detailed snapshots of the physical landscape of many samples, simultaneously, for use in multi-well formats.
Here we specifically describe the implementation of passive particle-tracking microrheology in 3D tumor models, which is well suited for measurement of linear viscoelastic response in the range of ~tens of Pascal typical of commercially available extracellular matrix products (bovine collagen, EHS-tumor extracts or commercial nanofiber scaffolds). The observation of stochastic, thermally driven motion, even with high numerical aperture optics to quantify small probe displacements, is inherently limited to the linear regime of these relatively soft ECM environments, but investigators desiring to probe more rigid materials may be able to adapt this protocol for use with active manipulations via optical48 or magnetic trapping49 of probes. Since the optical or magnetic tweezers probe a single point within a sample and do not affect the integrity of the sample, these techniques could possibly be incorporated into the above protocol to obtain longitudinal measurements of more rigid samples.
It is important to observe that other cell generated forces may also contribute to tracer probe movements at various time scales. For example, during cell migration occurring over the course of hours and days in culture, clusters of probes will be pushed and pulled due to cell movements and it is indeed this motion that is utilized in traction force microscopy50,51. The thermally driven probe movements which are analyzed to obtain microrheology data take place on the order of seconds, a clearly separable time scale. While it is also true that the slower dynamics of stresses and strains associated with traction forces can contribute to local changes in rheology36, a strength of this imaging-based approach lies in the ability to correlate visual and rheological changes in the sample. For example in the representative results in Figure 3, an obvious matrix invasion and cell migration event is correlated with a change in G&#8242; of nearly four orders of magnitude during the time this migration takes place.
The protocol described here assumes that users have access to a microscope with a motorized stage that provides reproducible positioning of the sample. Labs that do not have this instrumentation could still make point measurements using marks or other visual cues in the sample or sample carrier to allow repeated measurements at the same point, though it would likely be difficult to achieve the same reproducibility in positioning as shown in the representative longitudinal measurements here. For the purposes of these representative results above, positioning on the z-axis was kept approximately constant. However, the protocol could also be amended to include additional measurements along the z-axis in order to create a 3D rheological map of the sample. The method could also be modified to utilize a scanning laser confocal or a multiphoton techniques for 3D sectioning though scan speeds would impose a limitation on the upper frequency limits accessible that may or may not be problematic for a given application.
In this method, a significant time constraint is imposed by the necessity of returning live cells to the incubator. Without a time-lapse weather station enclosure to control for temperature, humidity, and CO2 levels, image acquisition time must be minimized. Even with the proper equipment, data collection takes up large amounts of time and digital memory, which are both subject to practical limitations. For instance, with microscope and camera used in this study, it would take roughly 35 hr&#160;to take the roughly 4,000 videos necessary to map a single well on a 96-well plate using a 100X&#160;objective lens with no space between fields of view. These factors impose constraints on the number of data points that can realistically be collected. The level of detail is, therefore subject to the practical constraints imposed by time available for data collection, data storage space and the availability of an environmental housing.
The ability to quantify changes in ECM rheology could be further integrated alongside imaging-based methods for quantification of therapeutic response in 3D tumor models17,18,52,53. The multi-well 3D model culture system adopted in both methods suggests a parallel implementation providing correlation between cytotoxic response and modification of the mechanical environment. This could facilitate development of further strategies that explicitly target the mechanical phenotype or elucidate the impact of existing therapeutics in this capacity. Such insight could be directly relevant to approaches to enhance drug delivery through dense collagen-rich tumor stroma. For example, in the context of the representative illustration of this protocol presented here with in vitro pancreatic tumor models, the evaluation of matrix degradation following interventions could be used in screening stromal depletion regimens, an increasingly important therapeutic paradigm for pancreatic cancer54.

それは、癌細胞が、非悪性の哺乳動物の上皮細胞と同様に、周囲の細胞外マトリックス（ECM）の機械的および生物物理学的特性および他の微小環境の成分1-9に非常に敏感であることが文献において多くの証拠から明らかである。エレガントなメカニズムの研究は、悪性増殖挙動や形態形成2,3,10,11を規制する複雑な機械受容シグナル伝達パートナーのような細胞外剛性の役割への洞察を提供してきました。この研究は、生物学的に関連する組織の構造を復元し、調整可能な力学と足場材料中で増殖させ、光学顕微鏡12-19によって画像化することができるインビトロ腫瘍モデルにおける 3Dの開発によって、特に、促進された。しかし、今度は癌細胞がその周囲のレオロジーを改変を通して、腫瘍微小環境との間のこの対話mechanoregulatory、他方の側は、多少残る勉強するのがより困難。例えば、侵入プロセスの間、腫瘍の周囲の細胞は、順番にの機械受容増殖挙動に影響を与えるECM 20-22の局所的な劣化を引き起こす間葉移行（EMT）に上皮を受け、マトリックスメタロプロテアーゼ（MMP）の発現を増加させることができる他の近位の腫瘍細胞。生化学的プロセスのさまざまな、癌細胞は、継続的に異なる時間に異なるプロセスに合わせて上下にその環境の局部剛性をダイヤルします。ここに記載された方法論は、3D腫瘍モデルと統合され、文化を終了せずに生化学的および表現型の変化を縦方向に相関させることができる成長中の剛性とECMの遵守の局所的な変化を報告分析ツールの必要性が動機とされている。 これに関連して実施するための適切な技術を求めて、粒子追跡マイクロレオロジー（PTM）が有力な候補として浮上。この方法は、メイソンとワイツ23,24によって元々の先駆者、ミクロンの長さスケールでの周波数依存性複合体の粘弾性せん断弾性率G *（ω）を報告するために、複雑な流体中に埋め込 ​​まれたトレーサプローブの動きを使用しています。この一般的なアプローチは、ソフト凝縮物質、コロイド、生物物理学、高分子物理学25-31で別の用途に適した複数のバリエーションで展開してきた。局所粘弾性の読み出しを培養調製時に組み込まれ、成長の長期間にわたって定位置に残留する生化学的に不活性トレーサープローブの非破壊ビデオイメージングにより提供されているのでPTMは、他の方法に比べて特定の利点を有している。これは必ずしも文化の終了を必要とし、複雑な3D腫瘍microenvir内のポイントの測定値ではなく、サンプルのバルク巨視的レオロジーを報告する振動剪断バルクレオメーター、とゴールドスタンダードの測定とは対照的であるふりかかる。実際、多くの研究は、細胞遊走32、拡大回転楕円体33によって誘発される機械的ストレス、細胞内レオロジー34,35に関連付けられた変形を測定するために、癌または非癌細胞または周りトレーサープローブの動きの測定値を解釈するの有用性を例示している機械的応力とひずみエンジニアリング組織36内、および細孔径および浸潤速度の37との関係をマッピングする。例えば、原子間力顕微鏡（AFM）などのマイクロレオロジーのに適した他の技術は、実現することができるが、主に、試料表面の点をプロービングするための長手方向の測定値38を複雑培養無菌性の問題を引き起こすことがある。 ここでは、確実に空間をマッピングするためのビデオの粒子追跡および分析方法のために埋め込まれた蛍光プローブを用いたECMへの転送に適した3D腫瘍スフェロイドの成長のための方法を網羅する総合的なプロトコルを記述文化の中で時間をかけてマイクロレオロジーの変化。現在の実装では、3次元腫瘍モデルはこのフォーマットが助長している他の伝統的なアッセイ（ 例えば 、細胞毒性）とマイクロレオロジー測定を組み込むことを視野に、マルチウェル形式で増殖させる。この方法論のこの代表的な実例では、本明細書に記載のPANC-1細胞を用いてインビトロ 3Dスフェロイド培養 、スフェロイド39を形成することが知られている確立された膵臓癌細胞株が、すべての測定は、様々な細胞株を用いて、固形腫瘍を研究するために広範に適用可能である3D培養に適しています。この方法は、本質的に画像化ベースであるためには、理想的に細胞の増殖、移動および表現型の変化をレポートビューの大透過光のフィールドを持つ高解像度のマイクロレオロジーデータの共同登録のために適しています。このように、透過光顕微鏡と統合され、PTMの実装では、顕微鏡ステージWHの再現可能なポジショニングを前提としていICHは電動商用広視野落射蛍光生物顕微鏡に一般的に使用可能です。下の開発されたプロトコルは、いかなる合理的に装備し、自動化蛍光生物顕微鏡を用いて実施することができる。これはオフライン処理のためのデジタルビデオ顕微鏡ギガバイトのデータの取得が必要で、本質的にデータ集約型の方法である。 以下のプロトコルにおいて、プロトコル1アガロース上でオーバーレイを使用してここに記載されているが、このような懸滴40、又は41回転培養技術などの他の様々な方法で置換することができる腫瘍スフェロイドの最初の調製に関する。プロトコル2は、代替的に、 インビトロの3D腫瘍はECM 12,15で再懸濁された細胞ではなく、単一の予め形成された非接着性スフェロイドの封入又は包埋することによって成長させることができたもののコラーゲン足場でスフェロイドを埋め込 ​​む方法が記載されている。その後のプロトコルは、Oのための手順について説明それぞれ、ビデオ顕微鏡データを取得し、処理することにより、時間分解マイクロレオロジー測定をbtaining。データ処理は、もともとも広範囲に異なるソフトウェア·プラットフォーム用に開発されているクロッカーとグリア42で説明したアルゴリズムに基づいて構築さPTMのためのオープン·ソース·ルーチンを利用して、MATLABを使用して記述されている（参照http://www.physics.emory.edu/ 〜週/ IDL /）。

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:177px;">Bovine type 1 collagen</td>
			<td style="width:235px;">BD Biosciences, San Jose, California</td>
			<td style="width:100px;">354231</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PANC-1</td>
			<td>American Type Cell Culture, Manassas, VA</td>
			<td>CRL1469</td>
			<td>or other appropriate cell type</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescent Microspheres</td>
			<td>Life Technologies, Carlsbad, California</td>
			<td>906906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>BD Biosciences, Bedford MA</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Fisher Bioreagents, Waltham, MA</td>
			<td>C12H18O9</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH</td>
			<td>Fisher Bioreagents, Waltham, MA</td>
			<td>NC0480985</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well Imaging plates</td>
			<td>Corning Inc., Corning, NY</td>
			<td>3904</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Hyclone, Waltham, MA</td>
			<td>SH30243.01</td>
			<td>or appropriate&#160; cell culture media</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Zeiss AxioObsever Microscope</td>
			<td>Zeiss, Oberkochen, Germany</td>
			<td></td>
			<td>includes high-speed camera and imaging software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB software</td>
			<td>The Mathworks, Natick, MA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal measurement of extracellular matrix rigidity in 3d tumor models using particle-tracking microrheology

機械的な微小環境は、それ自体が複雑な、双方向の機械受容相互作用のセットの一部として改造および修飾された腫瘍増殖挙動およびシグナル伝達の重要な調節因子として作用することが示されている。生物学的に関連する3D腫瘍モデルの開発は、腫瘍成長に対するマトリックスレオロジーの影響に関する機構研究を促進してきたが、腫瘍によって誘発される機械的環境におけるマッピングの変更の逆問題は、依然として厳しい。ここでは、縦方向にその場で 、腫瘍微小環境内の物理的変化を監視するための堅牢かつ実行可能なアプローチの一環として、膵臓癌の3Dモデルと連携して、粒子追跡マイクロレオロジー（PTM）の実装について説明します。ここに記載した方法では、蛍光標識プローブが均一に経口投与するための分散モデル細胞外マトリックスI型コラーゲンの（ECM）足場内に埋め込 ​​まインビトロ 3Dモデルを製造するシステムを統合試料全体sitionおよび時間依存性マイクロレオロジー測定。 インビトロ腫瘍はマルチウェルイメージングプレートを用いた並列状態でプレーティングし、プローブする。確立された方法で描画、トレーサプローブの動きのビデオは複雑な周波数依存粘弾性せん断弾性率G *（ω）を報告するために一般化ストークスアインシュタインの関係（GSER）を介して変換されます。この方法は画像ベースであるため、機械的特性評価も同時に3Dの腫瘍の大きさと表現の質的変化を報告するために大規模な透過光の空間のフィールドにマッピングされます。ローカライズされた侵入に誘導されるマトリックス分解だけでなく、システムのキャリブレーションに関連するサブ地域での機械的応答を対比示す代表的な結果は、検証データを提示する。一般的な実験誤差およびこれらの問題のトラブルシューティングから望ましくない結果も提示されています。このプロトコルで実装された96ウェルの3D培養メッキ形式はc治療スクリーニングアッセイまたは機械的微小環境に対する治療的または生化学的な刺激の影響に新たな洞察を得るための分子イメージングとマイクロレオロジー測定の相関onducive。

To verify the validity of G*(&#969;) measurements at localized positions within a complex model tumor microenvironment, two initial validation experiments were conducted. First, we sought to validate our measurements against the "gold standard" of bulk oscillatory shear rheometry. We prepared identical samples of collagen matrix (without cells) at a concentration of 1.0 mg/ml collagen. These samples were probed with a bulk rheometer (TA Instruments AR-G2, using 40 mm parallel plate geometry) and by PTM (using the same parameters as throughout this protocol) and the resulting measurements of G'(&#969;) and G" (&#969;) were compared (Figure 2A). Excellent agreement was found between the two measurements. Secondly, to establish the ability of this protocol to measure point changes in ECM rheology we prepared a 1.0 mg/ml collagen matrix and embedded tracer beads. The sample was then treated with a centralized injection of 5 &#956;l dispase (Figure 2B), which rapidly digests collagen. After incubating for 3 hr to allow localized collagen digestion to occur, video data was taken at varying distances from the injection site, and measurements of G'(&#969;) and G"(&#969;) were made in each case. The magnitude of G"(&#969; = 1 rad/sec) was found to be larger than that of G'(&#969; = 1 rad/sec) at the injection site. Measurements taken at increasing distances from the injection site were found to have an increasing magnitude of G*, as well as an increasing ratio of G' to G" (Figure 2C). The size of the highly degraded region of ~2 mm is consistent with an estimated distance of diffusion of a protein with hydrodynamic radius RH = 1 nm (the molecular weight of dispase is 32.5 kDa) through digested collagen with viscosity of &#951; = 10-3 Pa&#183;sec at 37 &#176;C: <img alt="Equation 1" fo:content-width="1.5in" src="/files/ftp_upload/51302/51302eq1.jpg" />, where <img alt="Equation 2" fo:content-width=".7in" src="/files/ftp_upload/51302/51302eq2.jpg" />. Therefore the results agree with the expected softening of the matrix near the injection site, and demonstrate our ability to make accurate, localized measurements of matrix rheology.
Representative data from a 3D tumor model prepared according to this protocol is shown in Figure 3. Video data was collected at 19 fixed locations within the collagen scaffold, forming a rough grid around the tumor (Figure 3A). This coordinate data was combined with calculated values of G'(&#969;) to create a spatial map of ECM stiffness (Figure 3B). This spatial map clearly reveals a zone of heightened rigidity immediately proximal to the tumor spheroid at day 2, which could be correlated with the deposition of collagen IV, laminin V and other basement membrane components deposited by the spheroid itself17,18,44,45, or local compression of the ECM by the expanding spheroid.
Tumor growth and matrix invasion events were monitored over 4 days. Two regions were identified as having either no evidence of invasive cells at the tumor periphery, or pronounced evidence of invading cells (Figure 3C). PTM measurements were taken at regions 1 and 2 over 4 days. By day 4, there was a significant difference in G' (reported at 1 rad/sec) between the two regions (Figure 3D). Frequency-dependent measurements of G' and G" for regions 1 and 2 on day 4 show significant softening of the ECM at region 2, the invasive region (Figure 3E). On day 4 region two exhibits a liquid-like viscoelastic response, evident by viscous scaling of the G" plot (Figure 3E).
Representative sub-optimal results from common experimental and analysis errors are shown in Figure 4. Perhaps the most obvious source of error lies in the stability of the setup on which video microscopy is performed. If the lab bench is displaced or if there is some other external source of vibration, bead trajectories can be artificially influenced, which results in a drift in the data. Drift causes the MSD to increase dramatically at high lag times (Figure 4A) which also causes the G'(&#969;) and G"(&#969;) calculated values to increase sharply at low frequencies (Figure 4B). Drift can be reduced through the use of a pneumatic, air cushioned lab bench, and by simply taking care to not bump the setup while videos are being recorded. Additionally, drift may be removed during post processing using software routines. These routines are suitable for removing drift which is approximately uniform over a known time interval. Erratic drift (perhaps caused by bumping the stage during data acquisition) is more problematic. Therefore it is important to make sure that the sample remains mechanically isolated from the environment during data collection.
Another potential source of unclear results comes from improper interpretation of sample heterogeneity. Although the ability to observe and quantify spatial heterogeneity in a sample is indeed one of the noted strengths of PTM26 (and something that is lost in traditional bulk rheology measurements), improper grouping of clusters of probe trajectories can lead to incorrect conclusions. Given that changes in ECM rigidity reported by this methodology are inherently heterogeneous, as is the material itself, in any given field of view, there could be a few tracer probes that are not elastically confined by collagen fibers and thus free to stochastically explore larger water-filled pores (Figure 4C). These "loose" probes exhibit mobility approximately consistent with a purely viscous environment. Since data for all probes is averaged before viscoelastic moduli are calculated, having a few loose probes in a given sampling region (field of view) can produce frequency dependent plots of G*(&#969;) that vary wildly (Figure 4D).
Improper software calibration can lead to errors during video collection and analysis. During video data collection, it is important to observe the intensity histogram and adjust the exposure time to give the greatest dynamic range possible while ensuring that the image does not become saturated. The analysis computes refined center positions for each tracer probe by integrating the intensity value of each pixel. If the image is saturated, this integration will be less accurate due to a 'flat top' section of the intensity profile. Additionally, the feature finding calibration is an extremely critical step in ensuring accurate results. Proper choice of selection parameters is paramount. This process has been described extensively by Crocker and Grier42, Savin and Doyle43, and others.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51302/51302fig1highres.jpg" width="500" /> 
	Figure 1. Schematic of Experimental Procedure. (A) Tumor spheroids are formed on agarose beds, and then transferred into a 3D matrix containing embedded tracer probes. (B) Video data is taken at several points within the sample well, both adjacent to and far away from the tumor spheroid. (C) The stochastic motion of the probes in each video is analyzed in order to determine the local rheological properties of the matrix at each sample point. This data is compiled into a spatial mapping of ECM stiffness throughout the well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51302/51302fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51302/51302fig2highres.jpg" width="500" /> 
	Figure 2. Verification of Measurements.(A) The components of G*(&#969;) are shown here. The calculated values of G'(&#969;) and G"(&#969;) using PTM are very similar to those obtained via a bulk rheometer. (B) 5 &#956;l of dispase was added to the center of a well with radius r = 8.5 mm containing 1.0 mg/ml collagen to measure spatial changes in G*(&#969;). (C) Calculated values of G'(&#969;) and G"(&#969;) at the site of the dispase injection, 1.5 mm from the site, and 3 mm from the site. Additionally, a control measurement was made in a well containing the same collagen gel but with no dispase treatment. Near the treatment site G'(&#969;) is smaller than G"(&#969;). As the gel is sampled further from the treatment site the values of G'(&#969;) become larger than G"(&#969;), and the magnitude of G*(&#969;) increases. Measurements were taken 2.5 hr after dispase injection. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51302/51302fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51302/51302fig3highres.jpg" width="500" /> 
	Figure 3. Quantifying changes in ECM rheology concomitant with onset of local matrix invasion. (A) The well containing a PANC-1 3D spheroid is probed at multiple locations throughout the collagen ECM surrounding the tumor. (B) Coordinate data is combined with calculated G'(&#969;) for each location to produce a spatial ECM rigidity map. (C) Tumor growth and invasion behavior were monitored over 4 days. Two regions were identified as having either no interaction with invading cells (region 1, blue asterisk) or heavy interaction with invading cells which spontaneously branched out from the large primary multicellular spheroid mass (region 2, red asterisk). (D) Rheological measurements were taken at regions 1 and 2 over 4 days. By the fourth day, there was a significant difference in G' (reported at 1 rad/sec) between the two regions. (E) Frequency dependent measurements of G'(&#969;) and G"(&#969;) for regions 1 and 2 on day 4 show significant softening of the ECM at region 2, precisely correlated with the presence of invasive populations. G'(&#969;) and G"(&#969;) values calculated using a power law fit to MSD data are shown as filled and unfilled point respectively. On day 4, region two exhibits a liquid-like viscoelastic response as is evident by viscous scaling of the G"(&#969;) plot and the absence of the fit line for G'(&#969;). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51302/51302fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51302/51302fig4highres.jpg" width="500" /> 
	Figure 4. Data from sub-optimal results due to drift and incorrect interpretation of ECM heterogeneity. (A) The MSD of a tracer probe confined in a complex material follows power law scaling &#60;&#916;r2(&#964;)&#62; ~ &#964;&#945; (0 &#8804;&#945; &#8804; 1). Accordingly, MSD values should approach a plateau at long lag times. Drift in the sample can artificially eliminate this behavior, leading to escalating MSD values at long lag times. (B) The long lag time drift in the MSD will cause the values of G'(&#969;) and G"(&#969;) to become significantly erroneous at short frequencies. Drift can be eliminated or reduced either by ensuring that the sample is mechanically isolated during data collection or by using software routines to remove drift during the analysis. (C) Even in a small field of view, two distinct varieties of probe trajectories may be present. In this example, most of the probes are confined in the matrix, but a few are 'loose' - their trajectories are much less constrained. (D) Attempting to interpret MSD data from both constrained and unconstrained probes at the same time without proper interpretation of sample heterogeneity will confound measurements of viscoelastic moduli. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51302/51302fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology at JoVE.com

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

粒子追跡マイクロレオロジーを用いて3次元腫瘍モデルにおける細胞外マトリックス剛性の縦測定

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

longitudinal-measurement-extracellular-matrix-rigidity-3d-tumor

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.4K ビュー.  University of Massachusetts Boston. Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

ビデオ: Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

ラマン分光法を用いた細胞と細胞外マトリックスの非接触、ラベルフリーの監視

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

生物工学

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

グライディングアッセイを用いて生体高分子の曲げ剛性の測定

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

粒子追跡マイクロレオロジーによりバイオフィルムの機械的特性のその場でのマッピング

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

2Dと3Dの肌の改善<em&gt;インビトロ</em分子クラウディングの使用&gt;モデル

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

リアルタイム 3 D 単一粒子追跡のためのプロトコル

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

腎臓皮質細胞外マトリックス由来ハイドロゲルを製造

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

3D細胞-細胞-膵組織コンストラクトを印刷するための膵組織由来細胞外マトリックスバイオインク

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

マトリックス細胞代謝クロストークを研究するためのヒト3D細胞外マトリックス-脂肪細胞培養モデル

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

ハイドロゲルアレイにより、3D腫瘍モデルにおけるマトリックス成分および治療薬の効果をスクリーニングするためのスループットの向上が可能

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...