著者の謝辞があります。

1. 細胞の調製
<ol><li>バイオ セーフティ キャビネットの無菌技術を使用して 6 ウェル プレートの各ウェルに培地 2 mL に 20,000 の細胞をプレートします。</li>
	<li>6 ウェル プレートをプレートに付着する細胞をできるように 24 時間加湿インキュベーター (37 ° C、5% CO2) に配置します。</li>
</ol>2. 光増感剤の細胞の治療のための準備
<ol><li>培地の 0.5、1、および 2 mM 5 ALA ソリューションを準備します。ウシ胎児血清 (FBS) は、培地の成分が場合を準備し、細胞培養液 0.1% 溶液中 5 ALA の治療の孵化の FBS。11,12この場合、SCC 13 セルは必要ありません FBS、5 ALA は無血清培地ウシ脳下垂体抽出物および表皮成長因子を添加したケラチノ サイトに直接追加されたので。</li>
	<li>培養培地を吸引し、少なくとも 2 ml のリン酸緩衝生理食塩水 (PBS) の細胞を洗ってください。</li>
	<li>PBS を吸引し、個別の刷版や井戸に 0、0.5、1、または 2 mM 5 ALA ソリューションの 2 mL を追加します。</li>
	<li>0、0.5、1、セルを孵化させなさいまたは暖房の 30 分のブロック 36、37 ° C で 2 mM 5 ALA はアルミ箔を使用して潜伏中に露光量から細胞を保護します。</li>
	<li>0、0.5、1、および 2 mM 5 ALA ソリューションを吸引し、2 ml の PBS のセルを洗ってください。</li>
	<li>PBS を吸引し、青色光照射用培養液 2 mL を追加します。</li>
</ol>3. ブルーライト光線
<ol><li>青い光のデバイス (例えば、発光ダイオード、蛍光灯、またはハロゲン ライト) オンにセルを照射する前に、1 つのサイクルを実行 (1,000 s) デバイスを暖める。青い光デバイス必要があります 5 +/-417 の出力波長 nm。治療の前に 1 つのサイクルを実行するデバイスを許可する青い光の波長や放射照度の光位相で一貫していることを保証します。</li>
	<li>光度計を使用して、セル表面の放射照度を測定します。細胞表面の青い光の照度は、10 mW/cm2をする必要があります。光とセル間の距離は、光源の強さに応じて 10 mW/cm2の照度を達成するために調整する必要があります。</li>
	<li>青い光源下で黒の表面に 0、0.5、1、2 mM の ALA の治療グループを配置します。1,000 の青い光で細胞を照射秒 (16 分と 40 s) 10 J/cm2の合計フルエンスの。青い光光次セル分析の準備が整いました。</li>
</ol>4. コレクションと染色
<ol><li>製造元のガイドライン (表の材料を参照) によるとフロー バッファーを準備します。</li>
	<li>吸引培養培地、2 ml の PBS のセルを洗浄します。</li>
	<li>0.25% トリプシン-EDTA の 1 つの mL を追加し、約 3 〜 5 分のデタッチするセルします。細胞は、細胞の剥離を確認を顕微鏡下で調べることができます。</li>
	<li>10% 牛胎児血清トリプシンを非アクティブ化すると培養液 (PBS) の 1 つの mL を追加します。</li>
	<li>ラベル付きの 5 mL のフロー チューブに細胞懸濁液を収集します。</li>
	<li>細胞ペレットを削除せず 201 x gで 5 分吸引ソリューション間の遠心で 5 mL のフロー チューブをスピンします。</li>
	<li>共役アネキシン V 抗体 (フロー バッファーの 39 μ L あたりアネキシン v 1 μ) フロー バッファーの 200 μ L を各サンプルに追加します。</li>
	<li>細胞を再懸濁します、インキュベーター (37 ° C、5% CO2) 結合する annexin V を許可する 20 分間置きなさい。</li>
	<li>7 AAD の 3 μ L を各サンプルに追加します。室温で 5 分間インキュベートします。</li>
</ol>5 フローサイトメトリーによる解析
<ol><li>セットアップの流れの cytometer メーカー ガイドラインに従う (材料の表を参照してください)。</li>
	<li>収集し、フローサイトメトリーによるサンプルを分析します。13
</li>
	<li>治療法と分散分析 (ANOVA)14を使用してコントロール グループの統計的な比較を実行します。Dunnett のテスト投稿するアドホック分析とコントロールの意味する治療グループの平均を比較します。</li>
</ol>

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DUSA/太陽製薬の場合は、5 ALA とブル U の光デバイスが用意されています。この原稿は、博士国の残留によって支えられた資金。内容は、米国退役軍人局やアメリカ合衆国政府の見解を表していません。

SCC 13 アポートシス 0.5、1、および 2 mM 5-ALA 1,000 続いて 30 分孵化の大幅な増加があった青い光の s。これらの結果は 30 分青色光活性化によって続いての 5 翼の孵化を受けてアポトーシス12、線維芽細胞の割合で用量に依存して増加につながることを示す私たちの以前に発行された研究に一貫して 14。
勉強 PDT へのこの実験的アプローチには利点と制限。説明の方法は市販の PS として臨床に準拠し、光源が使用されました。研究者は、異なる種類の細胞、PSs、光源、照射パラメーターとメソッドをカスタマイズ可能性があります。ただし、このプロトコルは付着性細胞の単層培養向けにこの方法に制限があります。臨床において組織のアーキテクチャまたは病気の病理学 (すなわち、過角化症や線維症) の違いが低下する PS 吸収や浸透15光します。その結果、治療用量との実験結果が直接に対応しない臨床実習。回転楕円体腫瘍モデルとマイクロ流体チップより密接に腫瘍微小16,17,18をレプリケートする方法として研究されています。回転楕円体、感光を組み込むかもしれない差動異種腫瘍アーキテクチャを表す内側と外側の細胞ニッチがあります。他の研究者は、画面処理条件、光増感剤の血管配信にマイクロ流体チップを使用しています。ただし、マイクロ流体チップを開発する高価なまたは技術に馴染みのない研究者の実装することは困難があります。さらに、回転楕円体とマイクロ流体チップで光増感剤が血管普及せず腫瘍表面に直接適用される皮膚癌の臨床治療反映されません。研究者は異なった病気のシステムで勉強 PDT の単層培養法、回転楕円体腫瘍モデルおよびマイクロ流体チップの賛否両論を評価する必要があります。免疫細胞や細胞外マトリックスがない、ない PDT が複雑な細胞間相互作用に与える影響を判断することが可能です。研究者は動物モデルや臨床試験を用いた実験結果体外を確認する必要があります。高の間のテストの妥当性を達成するために体外実験を行う際に一貫性のあるインキュベーションと光源のパラメーターを維持するために重要です。
温度は、PDT19の効果を変更する知られています。1 つの研究は、細胞死、5 ALA 吸収, PP IX 形成およびサイトカインのリリースは、44 ° C19を温 5 ALA の細胞を培養によって強化される可能性があります示した。孵化温度 44 ° C の熱誘導細胞死につながる可能性があり、孵卵温度 20 ° C の下の重要な 5 ALA の吸収と蓄積19につながらない場合があります。研究者が熱の影響を潜在的交絡コントロールに一定した温度で温度調節可能な暖房のブロックで PS の孵化を実行することをお勧めします。また、5 ALA の PP IX への変換を許可するのに十分な時間、光増感剤をインキュベートすることが重要です。このプロトコルでは、正常に細胞のアポトーシスを誘発 30 分間 5 ALA に SCC 13 細胞培養。我々 は以前 5 ALA の 10 分インキュベーションが大幅無処理線維芽細胞11,14と比較して線維芽細胞のアポトーシスを増加しなかったことを示しています。PP IX は、5 ALA は 37 ° C で 6 分間インキュベートした後でマウス皮膚における蓄積開始します。したがって、5 ALA インキュベーションの 10 分後があります PP-IX20細胞アポトーシスを誘導するための十分な蓄積。私たちの以前の研究11,14に基づいて「ALA の 20 から 30 分の最小潜伏期間をお勧めします。研究者は、潜伏期間の設定、関心、セル型、および臨床徴候の光増感剤研究室に基づくを最適化する必要があります。製造業者の指針を使用して最良の結果のため、抗体の滴定および最適化の実験が行われます。興味の他のアッセイは、ROS の dihydroethidium フロー フローサイトメトリー法による定量化を含む青い光の photoactivation を次実行されるかもしれない。14
配布された表面積 (すなわち、放射照度) の単位あたりの光量変化と ROS 形成と細胞アポトーシス21総照射線量 (すなわちフルエンス) 光フェーズがあります。フルエンス、照度と時間との関係は以下の式で記述できます。
フルエンス (J/cm2) = 照度 (W/cm2) × 時間 (s)
フルエンスは時間に依存して、延長または短縮 photoactivation の段階は治療効果を変更できます。照度は光源と標的組織の間直交距離に比例します。その結果、光があまりにも遠くにある場合ができない場合があります十分な光エネルギーを刺激する対象の組織に配信される、PS の周囲の表面に反射する光の放射照度を増加可能性がありますまた。そのため、光の反射を防ぐために黒の表面に置かれた細胞培養皿で光照射を実行することをお勧めします。研究者が取得する商業的利用または FDA 承認された青色発光ダイオードと PDT の実験で使用する蛍光のデバイスが別の電源出力と光の電界均一性にこれらのデバイスがあります。特定の波長光度計は細胞表面と一貫性のある結果を得るのためのすべての実験の前に光のフィールドの均一性で照度を測定するために使わなければなりません。必要に応じて、電界均一性を強化する市販のディフューザーを使用する可能性があります。
要約すると、理論、実験方法、制限、潜在的な落とし穴と最適化のための推奨事項の詳細で PDT を調査する生体外でアプローチを説明しました。PDT は有用な臨床プロシージャおよび生体外での研究、新規 PSs、プロトコル、および PDT の新しい適応の最適化の開発できるようにします。

光線力学療法 (PDT) は、細胞のアポトーシスを誘導するために目に見える光した後ジベレリン光増感剤 (PS) の孵化を含む医療処置です。中央政府薬剤の管理 (FDA) が日光角化症 (AK) の治療の PDT を承認、臨床ガイドラインは、特定の非黒色腫皮膚癌とにきび尋常性1,2のための治療として PDT をお勧めします。PDT 用途新興非皮膚科にはには、消化器、婦人科、前立腺癌3治療が含まれます。PDT は、それは低コストで非侵襲的かつ最小限の副作用と瘢痕2に関連付けられて有利な治療法です。
PDT の最初のステップで PS は適用、2を細胞内に蓄積すること。アメリカ合衆国外用 5-アミノレブリン酸 (5-ALA)、AKs を治療するために使用されます。孵化の間に相、5 ALA が組み込まれた細胞3ヘム生合成経路を介してプロトポルフィリン IX (PP IX) に変換されます。がんや前がん細胞はプロトポルフィリン IX (PP IX)、最終製品に変換、鉄付加酵素の活動を減少しているヘム。その結果、がん細胞は、PP IX 細胞の4、5と比較して蓄積されます。がんや前がん細胞周囲の組織に対する PP IX のこの蓄積 PDT 最小限の有害事象に対象を絞ったアプローチをすることができます。光照射は、酸素が PP IX から励起電子を受け入れるとき活性酸素種 (ROS) の生成に します。PP IX は青と赤の光2を含む、目に見える光のスペクトル全体の小さいピークを持つ紫外スペクトルのピークの吸収です。ROS の生成は最終的に細胞のアポトーシス、膜の中断、ミトコンドリア障害、免疫調節、表皮細胞の増殖とコラーゲン売り上げ高6,7,8,9につながる可能性があります。,10。
PDT は 1970 年代に開発された、進化の治療3です。AKs の処置のための FDA 承認されたプロトコルは、14-18 h、5 ALA 皮膚培養をお勧めしますが、1 ~ 2 時間の孵化は臨床実習2でよく使用されます。体外では、我々 は短い 15 分 5 ALA 孵化が未処理の線維芽細胞11,12と比較して線維芽細胞のアポトーシスを高める可能性がありますを示しています。さらに、新規クロリン、フタロシアニン、およびナノ粒子ベースの PSs が PDT 効果3を改善するために研究されています。ここで、細胞付着性の扁平上皮癌における PDT に体外手法を示す.このプロトコルでは SCC 13 セルは 0、0.5、1.0、および 2 mM 5-翼 30 min とセンサーの 1,000 の 417 nm の青い光と孵化させる s。主要転帰指標はアポトーシスと壊死、アネキシン V 7 aminoactinomycin D (7 AAD) フローサイトメトリーによって測定されます。この治療プロトコル PDT をシミュレートするように設計および他の細胞、光増感剤、培養温度や光の波長で PDT の使用を勉強に調整する可能性があります。追加の管理グループの準備必要、含む無染色、単一分子蛍光染色、フローサイトメトリー用、正および負のコントロールがあります。理論、プロトコル、実験的なデザイン、およびアポトーシス/壊死フローサイトメトリー用ゲートの包括的な見直しは13他の場所で見つけることができます。

<table border="0" cellpadding="0" cellspacing="0" width="1259">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Keratinocyte serum free medium&#160;</td>
			<td style="width:211px;">Thermofisher</td>
			<td style="width:204px;">17005042</td>
			<td style="width:623px;">Culture medium for SSC-13 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM low glucose glutamax</td>
			<td>Thermofisher</td>
			<td>10567022</td>
			<td>Possible culture medium for other cell types</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">6-well culture plates</td>
			<td>Thermofisher</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Thermofisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA</td>
			<td>Thermofisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Thermofisher</td>
			<td>15250061</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLU-U light&#160;</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td>Other blue LED, flourescent, or halogen lights may used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-ALA</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta Biologicals</td>
			<td>C17032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowCellect Annexin Red Kit</td>
			<td>Millipore Sigma&#160;</td>
			<td>FCCH100108&#160;</td>
			<td>Propidium iodide may replace 7-AAD. Comes with annexin-V, 7-AAD, and flow buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCanto II</td>
			<td>BD</td>
			<td>Request from manufacturer</td>
			<td>Any flow cytometer may be used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Counting Chamber</td>
			<td>Hausser</td>
			<td>3120</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo</td>
			<td>FlowJo</td>
			<td>Request from manufacturer</td>
			<td>Flow cytometry analysis software</td>
		</tr>
	</tbody>
</table>

an in vitro approach to photodynamic therapy

光線力学療法 (PDT) は、細胞のアポトーシスを誘導するために目に見える光した後ジベレリン光増感剤 (PS) の孵化を含む医療処置です。連邦麻薬局が日光角化症の治療の PDT を承認、臨床ガイドラインは、特定の非黒色腫皮膚癌と尋常性ざ瘡の治療薬として PDT をお勧めします。PDT は、それは低コスト、非侵襲的、最小限の副作用に関連付けられてギョッとさせる有利な治療です。PDT の最初のステップで PS は適用、細胞内に蓄積すること。その後の光照射細胞のアポトーシス、膜の中断、ミトコンドリア障害、免疫調節、表皮細胞の増殖とコラーゲン売り上げ高につながる可能性があります最終的に活性酸素種の形成を誘導します。ここに、付着性のセル行における PDT に体外手法を示す.この治療プロトコルは PDT をシミュレートするように設計し、様々 な細胞、光増感剤、培養温度や光の波長で PDT の使用を勉強に調整することがあります。扁平上皮癌細胞培養し, 0、0.5、1.0、および 2 mM 5-アミノレブリン酸 (5-ALA) 30 min と 1,000 の 417 nm の青色光でセンサー s。主要転帰指標はアポトーシスと壊死、アネキシン V 7 aminoactinomycin D フローサイトメトリーによって測定されます。5 アラバマ州の 30 分間インキュベーション アポトーシスの線量依存性増加があった高の間のテストの妥当性を達成するために体外実験を行う際に一貫性のあるインキュベーションと光源のパラメーターを維持するために重要です。PDT は有用な臨床プロシージャおよび生体外での研究、新規 PSs、プロトコル、および PDT の新しい適応の最適化の開発できるようにします。

SCC 13 後セル 0、0.5、1、および 2 mM 5 ALA で 30 分インキュベートし、照射 1,000 s 青の光は、細胞のアポトーシスの線量依存性増加があったの。合計アポトーシス (平均の平均 ± 標準誤差) は 3.94 ± 0.34、7.90 ± 0.52、23.86 ± 0.52 と 38.33 ± 1.81 0、0.5、1、および 2 mM 5-ALA と孵化の 30 分後にそれぞれ、および青の 1000 秒の光 (図 1)。0、0.5、1、および分散分析を使用して 2 mM 5 ALA 培養グループの中でアポトーシス細胞の平均の割合を比較しました。1 と 2 mM 5 翼細胞 0 mM 5 ALA 処理細胞と比較して細胞のアポトーシスの重要な増加していた。図 2 aを示しています代表的なフロー フローサイト メーターは、インキュベートを 0、0.5、1、および 2 mM 5 アラバマ SCC 13 セルの側方散乱 (SSC) 対前方散乱 (FSC) のゲートSCC 13 セル人口を囲むサークル ゲートします。この実験のため 7500 イベントが収集され、SCC 13 セル人口ゲートのイベントの少なくとも 90% を捕獲しました。図 2 bは、x 軸と y 軸にアネキシン V 7 AAD の代表的なプロットを示しています。細胞集団は、4 つの象限にゲート (第 4 四半期に Q1) アポトーシス細胞を区別します。第 1 四半期 (高アネキシン v、低 7 AAD) を表すセル早期アポトーシスを受けます。Q2 (高アネキシン v、高 7 AAD) を表すセルを受けて後半 apoptosis か壊死します。第 3 四半期 (低アネキシン v、高 7 AAD) 表す細胞壊死を受けています。Q4 (低アネキシン v、低 7 AAD) 表す細胞 apoptosis か壊死を受けていません。アポトーシス細胞の割合を計算するには、我々 は第 1 四半期と第 2 四半期のセルの割合を追加しました。5 ALA 潜伏期間の長さ、孵化の温度、または光照射線量の矛盾は、PDT 効果を変更できます。図 3代表アネキシン V と 7 AAD 流れフローサイトメトリーによるプロットを示しますは、孵化の温度が変化するとき (すなわち27、36、または 42 ° C)。アポトーシスの温度依存増加があった。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58190/58190fig1.jpg"> 図 1: 5 アラバマ州の 30 分間インキュベーション後のアポトーシスの線量依存性に増加36 ° c の 1,000 に続いて 30 分インキュベート 5 ALA SCC 13 セルにおける青色光の s。バーは、それぞれの治療とコントロール群の平均パーセントのアネキシン V 陽性の細胞を表します。技術 3 通で実験を行った。統計的有意性はp 、分散分析によって決定された < 0.05 アスタリスク (*) で示されます。誤差範囲は、平均の平均 ± 標準誤差を表しています。<a href="https://www.jove.com/files/ftp_upload/58190/58190fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58190/58190fig2.jpg"> 図 2: 代表フローサイトメトリー プロットします。(A) 代表的な前方散乱対側フローサイトメトリー散布任意単位 (AU) の x 軸と y 軸の 0、0.5、1、および 2 mM 5 ALA 培養細胞の。この実験の 7500 のイベントが収集され、SCC 13 セル人口ゲートがイベントの 90% を獲得します。(B)対代表アネキシン V 7 AAD フローサイトメトリー プロット AU の x 軸と y 軸の 0、0.5、1、および 2 mM 5 ALA 培養細胞の。Q1: 早期アポトーシス、Q2: 第 3 四半期後半アポトーシス/壊死細胞: 壊死細胞および第 4 四半期: 実行可能なセル。<a href="https://www.jove.com/files/ftp_upload/58190/58190fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58190/58190fig3.jpg"> 図 3: PDT 効果を変更可能性があります異なる温度で 5 ALA をインキュベートします。7 AAD フローサイトメトリー対代表アネキシン V プロット AU での x 軸と y 軸 0.5 mM 5-ALA 27、36、42 ° c. で孵化 SCC 13 セルのQ1: 早期アポトーシス、Q2: 第 3 四半期後半アポトーシス/壊死細胞: 壊死細胞および第 4 四半期: 実行可能なセル。<a href="https://www.jove.com/files/ftp_upload/58190/58190fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about An In Vitro Approach to Photodynamic Therapy at JoVE.com

An In Vitro Approach to Photodynamic Therapy

光線力学療法 (PDT) は、ジベレリン光増感剤 (PS) 目に見える光した後アポトーシス誘導へのインキュベーションを含む医療処置です。PS 培養と光治療パラメーターの差異を検討するために使用可能性があります PDT をシミュレートするように設計の in vitro PDT プロトコルを提案します。

体外光線力学的治療へのアプローチ

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

an-in-vitro-approach-to-photodynamic-therapy

Research

JoVE Journal

Medicine

An In Vitro Approach to Photodynamic Therapy

8.6K ビュー.  University of California, Davis. 光線力学療法 (PDT) は、ジベレリン光増感剤 (PS) 目に見える光した後アポトーシス誘導へのインキュベーションを含む医療処置です。PS 培養と光治療パラメーターの差異を検討するために使用可能性があります PDT をシミュレートするように設計の in vitro PDT プロトコルを提案します。

ビデオ: An In Vitro Approach to Photodynamic Therapy

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

アンインビトロ</em転移性増殖に腫瘍の休眠とスイッチを研究する>システム

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

医学

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

骨肉腫細胞における光線力学療法の細胞毒性有効性<em&gt;体外</em

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

A Multi-Modal Approach to Assessing Recovery in Youth Athletes Following Concussion

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can experience a range of short and long term neurobehavioral symptoms (somatic, cognitive and emotional/behavioral) that can have a significant impact on one&#8217;s participation in daily activities and pursuits of interest (e.g., school, sports, work, family/social life, etc.). Despite this, there remains a paucity in clinically driven research aimed specifically at exploring concussion within the youth sport population, and more specifically, multi-modal approaches to measuring recovery. This article provides an overview of a novel and multi-modal approach to measuring recovery amongst youth athletes following concussion. The presented approach involves the use of both pre-injury/baseline testing and post-injury/follow-up testing to assess performance across a wide variety of domains (post-concussion symptoms, cognition, balance, strength, agility/motor skills and resting state heart rate variability). The goal of this research is to gain a more objective and accurate understanding of recovery following concussion in youth athletes (ages 10-18 years). Findings from this research can help to inform the development and use of improved approaches to concussion management and rehabilitation specific to the youth sport community.

This article provides an overview of a multi-modal approach to assessing recovery following concussion in youth athletes. The described protocol uses pre- and post-concussion assessment of performance across a wide variety of domains and can inform the development of improved concussion rehabilitation protocols specific to the youth sport community.

脳震盪に続いてユース選手での回復を評価するマルチモーダルアプローチ

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can ...

A Neuroscientific Approach to the Examination of Concussions in Student-Athletes

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement &#8220;may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an &#8216;impulsive&#8217; force transmitted to the head.&#8221; Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual&#8217;s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.

There is great variability in an individual&#8217;s risk for concussion and their corresponding recovery. A multifaceted approach to concussion evaluation is warranted; including baseline testing of athletes before participation in sport and timely evaluation post injury. The goal of this protocol is to provide an appropriate multifaceted approach to examine concussions.

学生·アスリートで脳震盪の検討に神経科学のアプローチ

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

加速度計を使用して日常生活に上肢のパフォーマンスを定量化する方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生児マウス蝸牛移植法<em&gt;インビトロ</em&gt;聴覚研究のスクリーニングツール

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

社内の取り組みを用いた上顎再建の CAD/CAM サージカル ガイドの設計

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...