作者は親切に彼らの助けのためのスザンナグレーフェとアルノWieheに感謝し、技術的な専門知識と、リポソームmTHPC製剤を私たちに提供するためbiolitecリサーチ社（イエナ、ドイツ）に感謝したいと思います。また、結果の大部分を生成し、その出版11 coresponsibleたカースティンレイディを、感謝したいと思います。 この作品は、Schweizerischerの法令に基づく連合組織体Balgrist、チューリッヒ大学、チューリッヒKrebsligaだけでなく、ウォルター·L.とヨハンナウルフ財団、チューリッヒ、スイス、EuroNanoMed ERA-NET / SNFスイスからの助成金からの助成金によってからの補助金によって支えられて国立科学財団31NM30-1分の131004。この作品はまた、カントンチューリッヒの筋骨格系の腫瘍学のためのHSM（高度専門医療）プログラムによってサポートされていました。

1。それぞれの低および高転移性HOSおよび143B、OSの細胞株におけるmTHPCの取り込みを比較する<ol><li> DMEM、ハムF12、及び比4.5:4.5:1の熱不活性化ウシ胎児血清を含む細胞培養培地を調製する。 注：PS mTHPCのリポソーム製剤を1.5 mg / mlのmTHPCの最終濃度で水に溶解した。 </li><li>プレート0.2×10 6 HOSおよび143B細胞/ 6ウェルプレート（各条件について三重）での、細胞を一晩接着してみましょう。 注：番号は個々の細胞株に調整しなければならない。 80から90までパーセントの集密度は、実験時に最適です。 </li><li>次の日に、異なる期間（時間依存性の取り込み）用、又は一定時間（用量依存性の取り込み）の間mTHPCの濃度を増加させてmTHPCの固定濃度で細胞をインキュベートする。 </li><li>特定の時間PのためmTHPCで細胞を培養するointsまたはmTHPCの濃度。 注：本研究では、HOSおよび143B細胞株の両方が5時間0.6μg/ mlの0のためmTHPC、2.5、5、または10時間、または0で、0.6、2.5、および10μg/ mlのmTHPCとインキュベートした。印加される期間及び投与量は、使用される細胞株に依存して変化し得る。 </li><li>常に細胞の直接露光を防ぐために、暗闇の中、または淡色表示光の状態で動作するように覚えている。 </li><li>指示された条件での細胞のインキュベーション後、培地を吸引し、PBSで細胞を3回洗浄する。次に、0.5ミリリットルのトリプシン/ EDTAで細胞を剥離し、細胞をカウントします。 </li><li>剥離した後、5分間400×gで細胞を遠心分離培地を吸引し、200,000細胞/ mlの最終濃度にPBSで細胞を再懸濁。 </li><li>得られた細胞懸濁液をピペットで100μlを96ウェルプレート中、蛍光分光光度計におけるこれらの細胞の蛍光を測定する（ すなわち、20,000細胞）。設定mTHPC蛍光測定用のSは、次のとおりです。励起用417 nmおよび発光のための652 nmの。 </li><li>次のように細胞あたり内部移行mTHPCの量を算出するために、細胞数および細胞容積相対蛍光単位（RFU）を正規化する。 </li><li> 0-4μg/ mlの（使用3回の測定）からPBSにmTHPCの異なる濃度を用いて標準曲線を作成。 96ウェルプレートの各希釈液100μlをピペットステップ1.6で説明した設定を使用して、ウェルの蛍光を測定する。 </li><li>ウェル内（μg/ ​​mlで）mTHPCの濃度を与えmTHPC、線形標準曲線の傾きによって（20,000細胞のために取得した）RFUを分割。 </li><li> 1ウェル内mTHPCの総量（μgの中で、細胞懸濁液100μlを表す）を取得し、20,000細胞の体積で、この値を分割するために10でこのmTHPC濃度を分割します。 </li><li> X（4/3×π）：球の計算式で体積を計算するR 3。顕微鏡下で20トリプシン処理した細胞の直径を測定し、この値を2で割ることにより、半径rを得る。 </li><li>最後に、インキュベーションの10時間後（用量依存性の取り込み）の後HOS細胞について得られた又は100％に設定した10μg/ mlのmTHPC（時間依存性の取り込み）で処理したものにすべての値を正規化する。 </li></ol> 2。 in vitroでの PSの光毒性の測定<ol><li> 96ウェルプレート中の種子3,000細胞/ウェル（mTHPCの各濃度三重が使用される）、それらを一晩接着してみましょう。同様に、付加的な96ウェルプレートを調製し、下記のように細胞を処理するが、その後、位相コントラスト撮影のために取ってそれらを置く。 注：数は、各々のセルラインについて調整しなければならない。実験時の百分の50から60までの集密度が理想的です。 <ol><li> mTHPC濃度ごとに、セルの対応する暗い毒性コントロールを追加するために覚えておいてください。ザ·SEはmTHPCの選択された濃度の存在下または非存在下でインキュベートされる細胞であるが、点灯しません。 </li></ol></li><li>翌日、細胞株に応じてmTHPC（0、0.0001、0.001、0.01、0.03、0.075、0.15、0.3、0.6、1.25、2.5、5、および10μg/ ml）を異なる濃度で細胞をインキュベートし5時間暗所に保管してください。 <ol><li>第1.3.2項を参照してください。 注意：インキュベーション時間の照明が細胞株および使用されるPSに依存する前に、したがって、それは別々の実験において、異なる濃度および異なる時点を試験することが望ましい。 </li></ol></li><li>指示された時間の間インキュベートした後、PBSで2回細胞を洗浄し、mTHPCずに新しい新鮮な培地を追加します。 </li><li> mTHPC吸収スペクトルに特異的な652 nmのダイオードレーザ（ 図1参照）で細胞を照らす。 <ol><li> 13.5センチメートルのDISTAの高さ21.88 MW / cm 2とパワーにレーザーを設定5 J / cm 2のエネルギー量を得るために細胞からのNCE。 230秒のセルを照らす。 （ととmTHPCなしで）ダーク毒性対照細胞に照射しないでください。 注意：必ずPSの励起に用いられるレーザ光 ​​から目を保護するメガネを着用してください。 </li></ol></li><li>照明の後、24時間37℃の暗所で細胞を維持する。その後、水溶性のテトラゾリウム（WST-1）試薬（培地10μl/100μL）を追加します。 </li><li>三時間WST-1試薬の添加後、415nmで96 - ウェルプレートの各ウェルの吸光度を測定する。 </li><li>生存細胞のパーセンテージを計算するために、（ すなわち mTHPCことなく、mTHPCとnonilluminatedことなく、照明）を100％に各条件についてmTHPC非処理細胞について得られた値の平均値を設定する。 </li></ol> 3。細胞計数による細胞数の推定<ol><li>シード20,000細胞/ WE24ウェルプレート中のLL、彼らが一晩を密着させ。 </li><li> mTHPC（0.001、0.003、0.15、0.6、及び/ mlの1.25μgの）で5時間細胞をインキュベートし、上記のように、それらを照らす。 </li><li>メディアを集め、5分間400×gで遠心分離することにより、PBSで細胞を1回洗浄してトリプシン処理し、それらを収集します。 </li><li>遠心分離後、培地を吸引し、新鮮な培地200μl中に細胞を懸濁します。 </li><li>ノイバウアー室で細胞を数える。 </li></ol>

<ol>
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著者らは、競合する経済的利益や利益相反を宣言していません。

PDTに応じて最適な細胞毒性を達成するためには、右のレーザ光設定およびインキュベーション時間を選択することが重要である。ここで説明する手順は、PSの取り込みを決定し、in vitroで 、PDT誘導される細胞毒性を定量化するために一貫性があり、効率的です。 PS mTHPCの特定の吸収波長を用いて、PSの細胞取り込みは、直接的に決定することができ、PSは、細胞傷害性活性酸素種を生成...

骨肉腫の芸術治療の現在の状態（OS）、原発性骨腫瘍は、ネオアジュバント化学療法と手術との組み合わせを包含する。この治療計画は、現在、60〜70％3、4との間に、化学療法の使用前に約20％からローカライズされた疾患を有する患者の生存率の増加を明らかにした。しかし、過去20年間、地元の疾患を持つOSの患者の全生存期間は4、5を頭打ちしている。さらに、これらの患者の30〜40％は、診断後3年以内に再発および転移性疾患の患者は、20から30パーセント4、6、7の貧しい人々の生存を持ち続ける。これらの患者の転帰を改善するために、新たな治療戦略を開発する必要がある。 光線力学療法（PDT）、むしろ新規な抗癌治療は、photosensiの励起のために特定の波長の光を使用する血流への注入後に腫瘍細胞中に蓄積tizer（PS）。 PSのレーザー光励起は、腫瘍細胞および細胞死を細胞毒性反応を誘発し、酸素の存在下で酸素ラジカルを生成する。この主要なメカニズムに加えて、二つの追加PDTは生物学的プロセスが減少し、腫瘍の成長に寄与する誘発：PDTは、腫瘍の微小血管の血管収縮および血栓形成を引き起こすと、腫瘍内部の結果として、地元の低酸素および無酸素、腫瘍梗塞につながる。最後に、PDTは負傷し、瀕死の腫瘍細胞が局所的免疫応答、PDTのかなりユニークな機能をトリガします。これは、補体系及び樹状細胞の抗原提示8の活性化が関与している。従って、条件は、腫瘍特異的免疫をもたらす、リンパ系細胞のその後の活性化による腫瘍抗原の提示のために作成される。 今のところ、PDTは、軟組織腫瘍及びhyperplのいくつかのタイプを治療するために使用されているアジアの、例えば光線性角化症、バレット食道、気管支内腫瘍、膀胱癌、基底細胞癌、及び頭頸部癌2の姑息的治療として。治療は少しだけ副作用が局所大規模壊死を誘導することが知られ、したがって、選択的に腫瘍組織を根絶する可能性を有している。これらの利点にもかかわらず、PDTの適用は、化学療法薬の投与よりも技術的により厳しいままである。最大効果を達成するために、PS濃度、露光時間、および総光エネルギー伝達を最適化する必要がある。これは、最適化が必要なパラメータの相対的な多数の、それが最初にインビトロで最適な条件を決定する方が効率的であり、in vivo実験で行うことができるが。 下記の実験では、PS 5,10,15,20 -テトラキス（メタ-ヒドロキシフェニル）クロロフィルを用いたPDT のインビトロ効力を試験しおりん、mTHPC（ 図1A）は省略。 mTHPCは現在頭頸部癌の緩和治療のために臨床で使用される医薬品Foscan、活性物質である。これは、低濃度ですでに大量の細胞損傷を誘導し、最も強力なPSの一つであり、それは組織浸透9,10の点で他のPSよりも優れていることが実証された。その光吸収スペクトル（ 図1B）は、それぞれPSを蓄積する組織局在およびPDT誘導のために使用される2つの顕著なピーク、417 nmでのオンおよび652 nmでの第二を示す。 現在、mTHPCためのリポソーム製剤を開発中である。ここでは、このリポソーム製剤の取り込みを定量化するための手順を記述し、2人のOSの細胞株でPDTを行うために、低転移HOSと高転移性の143B細胞。ここに提示されたデータの中には、11以前に報告されている。目ここで説明するEアプローチは、PDTの有効性に転移性表現型の効果を研究することを可能にします。同所免疫不全SCIDマウスの後肢に注入された143B細胞は、intratibial転移原発腫瘍、密接に人間の転移性疾患を模倣したモデルを引き起こす。したがって、提案されたin vitro実験は、最適なPDT設定が保存インビボ実験で使用するための完全に評価するのに適している。

<table border="0" cellpadding="0" cellspacing="0" width="955">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:251px;">PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-883</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HAM F12</td>
			<td>PAA GmbH, Freiburg, Germany</td>
			<td style="width:127px;">E15-817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">heat-inactivated fetal calf serum</td>
			<td>GIBCO, Basel, Switzerland</td>
			<td style="width:127px;">10500-064</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:291px;">mTHPC</td>
			<td style="width:251px;">biolitec research GmbH, Jena, Germany</td>
			<td style="width:127px;"></td>
			<td style="width:287px;">As this liposomal formulation is originating from R &#38; D 
			no catalogue number is available at the moment. Stock: 1.5mg/ml; provided in a 9:1 mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG; &#62;99% purity)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectramax Gemini XS plate reader</td>
			<td style="width:251px;">Molecular Devices, Sunnyvale, CA</td>
			<td style="width:127px;">ID# 861</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Microscope Zeiss Observer.Z1</td>
			<td style="width:251px;">Axio Observer, Axio Vision Release 4.6.3 SP1, Jena Germany</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Ceralas PDT 652 nm Laser</td>
			<td style="width:251px;">Biolitec AG, Jena, Germany</td>
			<td style="width:127px;">LD652nm2W400u</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Water-soluble tetrazolium (WST) reagent</td>
			<td style="width:251px;">Roche Diagnostics AG, Rotkreuz, Switzerland</td>
			<td style="width:127px;">1644807</td>
			<td></td>
		</tr>
	</tbody>
</table>

cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

近年では、より少ない全身性副作用、癌に対してより効果的な治療法を見つけることが困難であった。したがって光線力学療法は、それ以上の腫瘍選択的治療のための新規なアプローチである。 酸素の存在下で特定の波長の光で活性化されると、細胞傷害性応答を誘発する1酸素ラジカルを生成し、非毒性の光増感剤（PS）を使用する光線力学療法（PDT）。 FDAによる、ほ ​​ぼ20年前の承認にもかかわらず、PDTは、最近だけのタイプの癌（皮膚、膀胱）とnononcological疾患（乾癬、光線性角化症）2限られた数の治療に使用されます。 PDTの使用の主な利点は、全身性の副作用を防止する局所治療を行うことができることである。また、（神経や血管の周囲などで ）繊細な部位における腫瘍の治療を可能にする。ここでは、intraoperatPDTのIVEアプリケーションは、骨肉腫（OS）で、原発腫瘍の衛星を対象とする骨の腫瘍は、外科的腫瘍切除後に周囲の組織の腫瘍に残さ考えられている。治療は、再発の数を減少時と（術後の）転移のリスクを低減することを目的とする。 本研究では、十分に確立されintratibial OSマウスモデルにおいてヒトの疾患を再現するために使用される広く使用されているOS細胞株の効果的な治療のためのPDTの最適な設定を確立するためにインビトロ PDT処置で提示する。分光光度計や光毒性は、WST-1アッセイを用いて、および生存細胞の計数によって評価し、細胞死を誘導するために652 nmでのmTHPCのレーザー光励起により引き起こされたとPS mTHPCの取り込みを調べた。確立された技術は、動物モデルでの今後の研究のために最適な、PDTの設定を定義するために私達を可能にする。彼らは、PDTの有効性を評価するための簡単​​かつ迅速なツールです<em&gt; 生体内での適用前にin vitroで。

ここで報告された技術では、我々人間のOS細胞内mTHPCベースのPDTを調査した。まず、mTHPCの時間および用量依存的取り込みは、低転移HOSおよび高転移143B OS細胞株で調べた。 mTHPC取り込みは、蛍光分光光度計でmTHPCの蛍光を測定することによって評価することができる（レイディらからの許可を得て複製、 図2、 図11）。 図2Aは、時間依存的?...

Watch this Scientific Journal Video about Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro at JoVE.com

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

ここで報告されたプロトコルは、細胞株における光線力学療法（PDT）の有効性および動物モデルにおいて治療を適用する前に、PDTの設定の最適化の評価を可能にする。

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 ...

cytotoxic-efficacy-photodynamic-therapy-osteosarcoma-cells

Research

JoVE Journal

Medicine

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

12.7K ビュー.  Balgrist University Hospital, Zurich, Switzerland. ここで報告されたプロトコルは、細胞株における光線力学療法（PDT）の有効性および動物モデルにおいて治療を適用する前に、PDTの設定の最適化の評価を可能にする。 

ビデオ: Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

マウスの眼の中に遺伝子治療ベクターと幹細胞の網膜下注入

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

医学

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

卵巣がんの腫瘍開始細胞の体外富化

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

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. ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

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 ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

インビボおよびインビトロ実験用グシュカン(GSK)顆粒の調製

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Large Animal Model for Evaluating the Efficacy of the Gene Therapy in Ischemic Heart

Coronary artery disease is one of the significant causes of mortality and morbidity worldwide. Despite the progression of current therapeutics, a considerable proportion of coronary artery disease patients remain symptomatic. Gene therapy-mediated therapeutic angiogenesis offers a novel therapeutic method for improving myocardial perfusion and relieving symptoms. Gene therapy with different angiogenic factors has been studied in few clinical trials. Due to the novelty of the method, the progress of myocardial gene therapy is a continuous path from bench to bedside. Therefore, large animal models are needed for evaluating the safety and efficacy. The more the large animal model identifies the original disease and the endpoints used in clinics, the more predictable outcomes are from clinical trials. Here, we introduce a large animal model for evaluating the efficacy of the gene therapy in the ischemic porcine heart. We use clinically relevant imaging methods such as ultrasound imaging and 15H2O-PET. For targeting the gene transfers into the desired area, electroanatomical mapping is used. The aim of this method is: (1) to mimic chronic coronary artery disease, (2) to induce therapeutic angiogenesis at hypoxic areas of the heart, and (3) to evaluate the safety and efficacy of the gene therapy by using relevant endpoints.

Myocardial gene therapy for ischemic heart disease holds great promise for future therapeutics. Here, we introduce a large animal model for evaluating the efficacy of gene therapy in the ischemic heart.