We would like to thank the American Cancer Society, Illinois Division (282802) and the National Science Foundation CBET (1512598) for funding support. We also would like to thank Dr. Dianwen Zhang from the University of Illinois Beckman Institute for microscopy training. Finally, we would like to thank Jared Weddell, Stacie Chen, and Spencer Mamer for insightful discussions.

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The authors have nothing to disclose.

細胞分離技術の改善は、血管生物学19で細胞の再生生物学でのプログラミング、および血管新生シグナル伝達幹、神経科学18における構造と機能の関係に科学的研究をfurthers。実際、初代細胞培養物20（ 例えば 、HUVECを）血管生物学のは、主として細胞分離技術を使用して行われます。細胞の単離は、最近細胞膜受容体3,14,15,19,21の定量的な流れ（qFlow）フローサイトメトリー分析に使用しました。しかしながら、既存の細胞単離方法は、細胞表面受容体のレベルに影響し、人員および試薬の両方に高価です。我々は、これらの欠点を満たす細胞タイプの混合物からの単一細胞型の再現可能な捕獲の穏やかなシステムの構築を可能にする表面機能17に新たな方法を進めてきました。この技術は、cの可能性を可能にする、反復システムに統合することができ特異的抗体を使用して段階的に異なる細胞型をapturing。さらに手順を明確にするために、トラブルシューティングの質問と回答の数は、以下に提供されています： 官能の最適化：官能化プロセスが改善された均一性のために最適化され、MCF7gfp細胞のプルダウンされています。このプロトコルは、代替的に、任意の細胞型および抗体コンフィギュレーションのために最適化し、カスタマイズすることができます。これは、捕捉表面、またはタイプまたは抗体の濃度を変化させることにより、ベース成分の濃度を変化させることによって達成することができます。ほとんどの抗体の種類は、上記のプロトコルを用いてビオチン化することができます。ビオチン化されると、それらは次にプルダウンのための理想的な濃度を見つけるために（ 図1）に滴定することができます。細胞濃度は、捕捉する細胞の理想的な濃度を見つけるために（ 図2）を滴定することができます。このキャップと特定の細胞タイプを捕捉する可能性を使用してチャー面を確認することができます。例えば、目的の細胞型が理想的である官能化表面を使用して、その範囲内の細胞の濃度を捕捉する、百万個の細胞あたり100個の細胞の規模である場合。滴定中に、表面は、その低濃度の細胞を捕捉することができない場合には、表面または抗体の最適化は、その濃度範囲内のセルを除外することができるようにするために必要です。 これは、細胞の種類は、このプロトコルで使用されていますか？どのように誰かが異なる細胞型を使用するには、このプロトコルを変更することができますか？ 現在、このプロトコルは、MCF7-GFP細胞を使用し、RAW 264.7マクロファージは、多くの場合、デュアル細胞混合物中の細胞型を引き出す能力を示すために含まれています。彼らは、マウス異種移植モデル（マウスの環境内でヒト腫瘍）に最も関連の細胞型二たように、これらの細胞を選択しました。他の細胞の様々なプロセスを較正するために、抗体SPecificityが最も重要です。抗体22を選択するために利用可能ないくつかのオンラインリソースがあります。 この方法のいくつかの利点は何ですか？ ジェントルアプローチ：力の欠如は、このアプローチは穏やかになります。確かに、私たちの計算されたせん断力は、最大以下の応力の値ながら2.09 Paの（314 pMの2のセル面積を想定し656 PN）の流体力学的応力が壊死を誘発するので、バイオマーカーに大きな混乱を引き起こしてはならない24×10 -6 pNで17であり、 0.59 Paの（314 pMの2のセル面積を想定し185 PN）ない23を行います。この穏やかなアプローチが原因タンパク質発現パターンおよび形態25を変更することができる600 Gの周りの値で急加速にピークせん断応力の0.78 Paの25まで発揮するような遠心分離ベース24に接近するなど、いくつかの商業的に利用可能なオプション、オーバーが有利で ​​す、26 </suP&gt;、さらには細胞壊死23を誘導します 。こうして遠心用途の量を減らすことができる方法は、細胞が、精製を通じて経験し、最終的に多くの生理学的データを保証する応力の量を減少させるであろう。我々の現在のプロトコルは、精製およびキャプチャする前に細胞濃度を再構成するために遠心分離機を使用して、しかし、この製造プロセスは、磁気ビーズ分離27,28のような多くの精製技術において標準であり、フローサイトメトリー29を流れ、FACS 30（ 図5）。我々のアプローチは、他の技術は、準備工程に加えて、使用するすべての下流の遠心分離工程を減らすことができます。 ビオチン-アビジンのアプローチ：一般的に使用される生体材料（DSB-SAVおよびビオチン-SAV）のアプリケーションでは、このアプローチの有利31,32をレンダリングします。アビジンファミリータンパク質は、ビオチンファミリータンパク質に非常に選択的に結合し、scientの範囲に使用されていますIFICを含む医療用途：抗体-フルオロフォアアタッチメント33、定量的Qドット-ポリスチレンビーズアタッチメント34、およびカプセル化された薬剤32を解放するために、環境に刺激に応答するヒドロゲルの作成 ​​。さらに、ビオチン化抗体を取得する相対的な容易さは、このアプローチが広くアクセス可能で、カスタマイズ可能になります。 ビーズ無しデスチオビオチンアプローチ：捕獲表面は、細胞を放出するための厳しい試薬および力を使用せずにデスチオビオチンの使用を実装することができます。 DSBは、DSB-抗体および磁気ビーズ35と連動して他のシステムによって可逆的細胞接着および放出のために使用されています。しかし、分離技術の過酷な影響だけでなく、差動受容体およびケモカインの発現28,36内のサンプル27,28の結果を準備するに関連付けられている大規模な携帯損失。このアプローチは、緩和することを目的とそして、はるかに穏やかな洗浄および解放の方法論を作成するために、磁石とビーズの両方の使用を排除することによって、これらの制限を克服します。 このプロトコルでの重要なステップは何ですか？どのような変動を引き起こす可能性がありますか？その変動はどのように制御することができますか？ このプロセスは、捕捉表面の効率低下をもたらすことができる4つの重要なステップを有します。最初の重要なステップは、自己組織面の破壊をもたらすであろうAPTES官能ステップ、中にAPTES表面への水の予防です。これは、後に水溶液から加水分解を減少させるためにオーブンにAPTESを、溶媒としてエタノールを用いて焼成することにより改善されます。二層の架橋を可能にする。第二の重要なステップは、EDCがAPTESでDSBの反応を触媒するEDC反応工程です。 EDCは除外または十分に追加されていない場合、DSBはwhic、床を添付することはできません<html> hは放出機構を危うくします。 SAV層を維持することが重要であるように、第3の重要なステップは、SAV官能中にあります。捕捉効率の低下で、ストレプトアビジン層の結果の不均一。捕獲表面への抗体の結合は、捕獲表面と抗体のビオチン - ストレプトアビジン相互作用を介して促進されるように第四の重要なステップは、抗体のビオチン化です。抗体が非ビオチン化である場合には、ストレプトアビジン層は、それを捕捉し、捕捉表面は無用作り、それをプルダウンすることができません。表面捕獲の変動と矛盾は濃度非規則性だけでなく、層の添付ファイルの偶然性に起因することができます。この変化は、表面に較正層成分の正確な濃度および捕捉の意図されたターゲットを使用することによって制御することができます。この設計では、濃度は、MCF7-GFP細胞の捕捉に特異的に較正されます。 e_content &quot;&gt; この方法のいくつかの欠点は何ですか？ 現在、APTESの官能化は、最大2時間の焼成工程に続いて55分間、表面の液相の浸漬を使用して行われます。これは、表面に結合するAPTESシランの完全な層を可能にするが、より均一なプロセスは、気相シラン37です。これにより、研究者の時間を節約するだけでなく、均一性を増加させる、APTESの接触時間を減少させます。さらに、改善されたプルダウンSAVの一晩のインキュベーションを使用している17観察されており、現在、表面官能化は、全体的なプロセスは、三日かかる作り、2つの一晩のインキュベーションを必要とします。だから、化学的性質を最適化することは研究者にとって大きな時間の節約を提供するであろう。 役に立つかもしれいくつかの警告または注意事項は何ですか？ 表面を官能とき、それはイムです適切なケアは、呼吸器損傷の危険に取られていることをperative。両方APTESとメルカプトエタノールは、このように、化学ヒュームとの相互作用を低減するために、化学フード内で官能化プロセスを行う必要があり、肺および関連臓器に非常に危険なことができます。メルカプトエタノールは、匂いで特に刺激的であるチオールです。メルカプトエタノールを使用する場合は、処分前に一日か二日のためにフード内に座って汚染廃棄物を可能にします。 この表面の将来の目標やアプリケーションは何ですか？ 私たちの未来は、血管新生関連疾患に関連する種々の細胞型で動作するように、この面をカスタマイズ伴うことを目指しています。特に、我々は、血液サンプルを使用して、そこから目的の細胞を分離するセグエするために、ヒト臍帯静脈内皮細胞に注力していきます。さらに、我々はそのような官能surfacの設計へのアプタマーとして分離様式を統合しますeはさらに、当社のキャプチャとリリース割合を増加させます。

現在のベンチトップ細胞分離手法（ 例えば 、蛍光活性化細胞は1ソート、レーザーキャプチャーマイクロダイセクション2、免疫磁気ビーズ分離1）が準備し、ソートの数時間かかることがあります。これらの大きな時間スケールは、生理学的応答3の代表ではない分析の結果、生理学的応答および発現レベルに影響を与えることができます。システムは、迅速かつ効率的な生物医学的用途のための細胞の単離及び濃縮を改善するために、細胞表面受容体レベルを中断することなく、特定の細胞型を分離することができる必要とされています。したがって、我々のアプローチの理論的根拠は、細胞単離のための穏やかなアプローチを開発することです。 「ラボオンチップ」概念が速く（時間・ツー・分）細胞単離を桁違いの約束を提供し、最も頻繁に表面上に細胞を捕捉し、細胞や細胞内のコンテを解放することを含みます4,5物理的または化学的方法6を通じてNTS。これらのアプローチは、このようなRNA発現9-11、あるいはin vitro培養12,13のための細胞を提供する識別、タンパク質7,8式を識別するなど、いくつかの利点を提供していますが、これらの技術の多くは、このような原因で細胞受容体プロファイリングなどの診断に翻 ​​訳することはできませんそれらの非生理的な環境です。このようなコラゲナーゼなどの酵素リフティング剤もこれらのリフティングエージェントが正確な生理学的データを生成しません使用する細胞受容体の定量化技術を意味し、これらの受容体の量14,15に影響を与えることができます。細胞溶解は、天然の表面受容体との間の差別化を防止し、あらかじめ16を内在したもの。このプロトコルは、細胞単離のための高速かつ穏やかなアプローチを説明しています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(3-Aminopropyl) triethoxysilane (APTES)</td>
			<td>Acros Organics</td>
			<td>919-30-2</td>
			<td>Used to make 2% APTES solution</td>
		</tr>
		<tr>
			<td>Plasma Cleaner Pico</td>
			<td>Diener</td>
			<td>Model 1</td>
			<td>Cleans surfaces and allows for bonding of PDMS to glass</td>
		</tr>
		<tr>
			<td>d-Desthiobiotin (DSB)</td>
			<td>Sigma</td>
			<td>D20655</td>
			<td>Used as the releasing mechanism in the cellular capture surface.</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>British Drug Houses (BDH)</td>
			<td>BDH1115-1LP</td>
			<td>Dissolves the DSB into solution</td>
		</tr>
		<tr>
			<td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Thermo-Scientific</td>
			<td>5g: 22980 
			25g: 22981</td>
			<td>Activates Carboxylic Acids and allows binding of proteins to glass surface.</td>
		</tr>
		<tr>
			<td>uncoated 8-well culture slide</td>
			<td>BD Falcon</td>
			<td>Case of 24: 354118 
			Case of 96: 354108</td>
			<td>Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments</td>
		</tr>
		<tr>
			<td>Glass bottom 24-well plates</td>
			<td>MatTek</td>
			<td>P24G-0-13-F</td>
			<td>Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments</td>
		</tr>
		<tr>
			<td>Mercaptoethanol</td>
			<td>Science Lab</td>
			<td>60-24-2</td>
			<td>Used to quench reaction between EDC and DSB</td>
		</tr>
		<tr>
			<td>4-Morpholinoethanesulfonic acid hydrate 
			(MES Hydrate 99%)</td>
			<td>Fisher Scientific</td>
			<td>AC172590250</td>
			<td>Used to make 0.1 M MES Buffer for use in EDC reaction</td>
		</tr>
		<tr>
			<td>Precision Oven</td>
			<td>Thermo Scientific</td>
			<td>11-475-153</td>
			<td>Used in curing of PDMS and APTES layer.</td>
		</tr>
		<tr>
			<td>Titramax 1000 Shaker</td>
			<td>Heidolph</td>
			<td>13-889-420</td>
			<td>Used to ensure even distribution of APTES on surfaces.</td>
		</tr>
		<tr>
			<td>1X Streptavidin 5mg 
			[e7105-5mg]</td>
			<td>Proteo Chem</td>
			<td>9013-20-1</td>
			<td>Biotin-binding protein 
			May cause irritation</td>
		</tr>
		<tr>
			<td>5 cm Glass Dish</td>
			<td>Fisher Scientific</td>
			<td>08748A</td>
			<td>Used in HUVEC studies as well as future profiling studies.</td>
		</tr>
		<tr>
			<td>14 cm Petri Dish with Cover</td>
			<td>Sigma-Aldrich</td>
			<td>Z717231</td>
			<td>Used to hold samples being functionalized and transport them.</td>
		</tr>
		<tr>
			<td>MCF7-GFP cells</td>
			<td>Cell Biolabs</td>
			<td>AKR211</td>
			<td>Stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>RAW264.7 
			mouse macrophages</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td>Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Life Technologies</td>
			<td>12605036</td>
			<td>Stored in 100mL at room temperature</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium</td>
			<td>Cell Media Facility at School of Chemical Sciences at UIUC</td>
			<td>50003PC</td>
			<td>Supplier: Corning</td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Cell Media Facility at School of Chemical Sciences at UIUC</td>
			<td>25-025-CI</td>
			<td>Already added into DMEM by facility. 
			Supplier: Corning</td>
		</tr>
		<tr>
			<td>10% fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>03-600-511</td>
			<td>Stored in 500mL at &#60; -10&#8304;C</td>
		</tr>
		<tr>
			<td>1% Penicillin&#8211;Streptomycin</td>
			<td>Life Sciences Storeroom at UIUC</td>
			<td>17602e</td>
			<td>Supplier: VWR 
			Stored in 100 ml at 4&#8304;C</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>12-565-58</td>
			<td>Small 23cm 50 pack</td>
		</tr>
		<tr>
			<td>Cell Dissociation Solution</td>
			<td>Corning</td>
			<td>MT-25-056CI</td>
			<td>Used to lift cells non-enzymatically for the use in cell experiments</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td>Used to count cells for quantification of cell solutions and capture and release effectivity.</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Amresco</td>
			<td>58-85-5</td>
			<td>Used to release cells from surface.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Created from Recipe</td>
			<td>N/A</td>
			<td>Used to keep cells alive in suspension as well as wash surfaces of non-specific binding. (Adapted from Cold Spring Harbor Protocols): In 500 mL, use 4 g NaCl, .2 g KCl, .0402 g Na2PO4*7H2O, .03 g KH2PO4 and .5 g Glucose. Add DI water to get to 500 mL, filter, and then refrigerate.</td>
		</tr>
		<tr>
			<td>HLA-ABC Antibody</td>
			<td>BioLegend</td>
			<td>311402</td>
			<td>Antibody used to capture MCF7gfp cells</td>
		</tr>
		<tr>
			<td>hIgG Antibody</td>
			<td>BioLegend</td>
			<td>HP6017</td>
			<td>Antibody used to capture MCF7gfp cells</td>
		</tr>
		<tr>
			<td>MCF7 GFP cells</td>
			<td>Cell Biolabs</td>
			<td>AKR-211</td>
			<td>Luminal Breast Cancer line that has been transfected with green fluorescent protein.</td>
		</tr>
		<tr>
			<td>Assorted Conicals</td>
			<td>Thermo-Scientific</td>
			<td>15mL: 12-565-268</td>
			<td>50/15 mL plastic conicals for storing solutions and aliquots.</td>
		</tr>
		<tr>
			<td>Mini-Tube Rotators (End over End Mixer)</td>
			<td>Fisher Scientific</td>
			<td>05-450-127</td>
			<td>Used to incubate antibody and mix other cellular solutions in order to mix</td>
		</tr>
		<tr>
			<td>Axiovert 200M (Fluorescence Microscope)</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Zeiss Axiovert 200 M inverted florescence microscope.</td>
		</tr>
		<tr>
			<td>Zeba Desalting columns</td>
			<td>Thermo-Scientific</td>
			<td>PI-87770</td>
			<td>Used to purify newly biotinylated antibodies after the use of the Biotinylation Kit. Instructions provided at: http://www.funakoshi.co.jp/data/datasheet/PCC/89894.pdf</td>
		</tr>
		<tr>
			<td>EZ Link Sulfo NHS Low Weight Biotinylation Kit</td>
			<td>Thermo- Scientific</td>
			<td></td>
			<td>Used to biotinylate antibodies to allow them to integrate with the capture surface</td>
		</tr>
		<tr>
			<td>Plate Reader</td>
			<td>BioTek</td>
			<td>Synergy HTX Multimode Reader</td>
			<td>Used to quantitatively measure fluorescent intensity in the titration experiments.</td>
		</tr>
	</tbody>
</table>

a method of targeted cell isolation via glass surface functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

このプロトコルを使用して、我々は、細胞捕捉（ 図3A）とMCF7GFP細胞ならびに生細胞対照（ 図4）の細胞の放出（ 図3C）を示しています。 60％と80％が放出されたとして、我々は（ 図3C）細胞捕獲を定量化しました。私たちは、RAW 264.7マクロファージおよびMCF7GFP細胞の混合物にこのアプローチを拡張すると、RAWマクロファージの50％が捕獲された（ 図3D）およびRAWマクロファージの80％は、20 mMのビオチン（ 図3B）でリリースしました。過剰な抗体は、細胞捕獲を減少させることができるので、我々はHLA抗体の0〜10,000 nMのを滴定を経由して、最適化、および理想的な抗体濃度は100〜1000 mMの抗体（ 図1）の間にあることを確認します。同様に、我々は、細胞の数の下に、値が背景17より低いので、捕捉することができる細胞の理想的な濃度は、1×10 5〜1×10 6であることを決定します（ 図2）。各ウェルからの蛍光データは、下記のように処理されます。 <img alt="式（1）" src="/files/ftp_upload/54315/54315eq1.jpg" />ここでは、3反復の平均はない抗体と試料（ブランク）から採取し、各試料から得られた蛍光から差し引かれます。この値は、平均最大蛍光に対して正規化です。この処理は、研究者が明らかに携帯低い検出限界を観察することができます。 <img alt="図1" src="/files/ftp_upload/54315/54315fig1.jpg" /> 図1の 正規化Blankedを抗体滴定ヒト HLA-ABCはMCF7GFP細胞（ウェルあたり125,000細胞）の一定量を横切って滴定され、捕捉された細胞の蛍光を、プレートリーダーを用いて定量しました。前の官能化表面に露出し、細胞および抗体が非特異的な抗体の付着を除去するために遠心分離しました。この遠心分離することなく、過剰な抗体は、遠心分離は、表面への結合より少ない細胞を生じ、表面の抗体過飽和を防止するのに十分ではなかった万ng / mlでの濃度で示されるように、表面を飽和させ、細胞のプルダウンを防止します。灰色の線は、コントロールを表します。エラーバーは平均の標準誤差を表す。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54315/54315fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54315/54315fig2.jpg" /> 図2 正規Blankedを細胞の滴定。抗体滴定から最適化された抗体濃度を用いて、MCF7GFP細胞KEながら、最適化された捕捉濃度を見つけるために滴定しました一定の官能基化表面及び抗体濃度をeping。これは、異なるアプリケーションのための細胞捕獲を較正するために使用することができます。行は、携帯捕獲のためのアイデアの範囲を見つけるために、細胞の濃度を変更しながら、この数字の列は、複製されています。灰色の線は、コントロールを表し、エラーバーは平均の標準誤差を表す。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54315/54315fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54315/54315fig3.jpg" /> 図3 キャプチャ及び実験を放出は単一細胞混合物は、HLA-ABC抗体を用いMCF7GFP細胞を捕捉する、官能化表面（A）に曝露しました。 HBSS（B）を用いて洗浄した場合に、細胞が捕捉されたままであったが、20mMのビオチン（C）の溶液に曝されたとき、細胞はRELEましたASED。 MCF7GFP細胞およびRAW 264.7マクロファージの細胞混合物は、官能化表面に露出しmCD11b抗体（D）を用いて捕捉しました。非特異的MCF7GFP細胞は緑色で標識されています。表面は、それらをターゲットにしなかったことを意味し、中性洗浄（E）にさらされたときMCF7GFP細胞の蛍光強度は減少し、それらが非特異的に取り付けられています。ビオチン洗浄（F）にさらされたときに、対象となるRAWマクロファージは、表面から放出されました。スケールバーは250μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54315/54315fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54315/54315fig4.jpg" /> デュアル細胞分離図 4. 正と負のセル制御。ポジティブコントロールRAWマクロファージはOをイメージしています全く蛍光活動だけでなく、細胞の相対的なサイズがないことを示す、蛍光顕微鏡をナ。 GFPの励起下で、マージされた画像（C）に示されてない蛍光（B）が存在しないながら明RAWマクロファージは、細胞のサイズ（A）を示します。陰性対照MCF7GFP細胞を蛍光大型蛍光活性を示し、顕微鏡、ならびにMCF7GFP細胞の相対的大きさに結像されます。細胞は、サイズを示し、明視野（D）上に結像され、GFPの励起下ながら（E）が明らかにマージされた画像（F）で示すことができる大型の蛍光を示します。スケールバーは250μmである。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54315/54315fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図5" src="/files/ftp_upload/54315/54315fig5.jpg" /> F標準的な精製技術の遠心分離機の使用 igure 5 比較分析前の遠心分離工程は、サンプル調製に保存されています。磁気ビーズ分離だけでなく、FACSは、細胞にストレスを導入し、発現レベルを変化させることができるいくつかの追加の遠心分離工程を伴う。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54315/54315fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 補足コードファイル。計算。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54315/Supplemental_Code_File.docx" target="_blank">このファイルをダウンロードするにはこちらをクリックしてください。</a>

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A Method of Targeted Cell Isolation via Glass Surface Functionalization

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

ガラスの表面機能を介して、標的細胞の単離方法

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

a-method-targeted-cell-isolation-via-glass-surface

Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.3K ビュー.  University of Illinois at Urbana-Champaign. This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process. 

ビデオ: A Method of Targeted Cell Isolation via Glass Surface Functionalization

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

濃縮細胞集団を生成し、細胞間コミュニケーションを調べるための簡単​​な方法：腫瘍微小環境のミミック

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

がん研究

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

肺癌標的化ナノ粒子と光線力学療法の抗がん効能

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

世代と複雑な染色体と細胞周期逮捕細胞の分離

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

表面増強共鳴ラマン葉酸受容体を介して顕微鏡による卵巣癌の検出のためのナノプローブ比を散乱によるターゲット設定

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

腫瘍標的免疫のパラミクソ ウイルス: 設計と評価 Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

3次元スフェロイド培養からの幹様細胞の分離

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

細胞および組織サンプルからのヒト乳がん幹細胞の単離と機能評価

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

HiBiT CRISPR細胞株を用いた標的タンパク質分解化合物のハイスループット細胞プロファイリング(英語)

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

レプトメニンゲレス病の直接標的療法を研究するマウス・オンマヤ異種移植片モデル

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...