저자는 친절하게 자신의 도움말 및 기술 전문 지식에 대한 수산나 Gräfe과 아르노 WIEHE 특별 감사와 함께, 리포좀 mTHPC 제형으로 우리를 제공 biolitec 연구 GmbH의 (예나, 독일) 감사의 말씀을 전합니다. 우리는 또한 결과의 많은 부분을 생성 및 출판 (11)에 대해 coresponsible했다 케르 스틴 Reidy을, 감사의 말씀을 전합니다. 이 작품은 Schweizerischer Verein에 Balgrist, 취리히 대학, Krebsliga 취리히뿐만 아니라 월터 L.와 요한 울프 재단, 취리히, 스위스에서 부여 및 EuroNanoMed ERA-NET / SNF 스위스에서 부여로에서 교부금에 의해 지원되었다 국립 과학 재단 (National Science Foundation) 31NM30-1분의 131,004. 이 작품은 또한 HSM Kanton 취리히의 근골격계 종양학 (고도로 전문화 된 의학) 프로그램에 의해 지원되었다.

1. 각각의 로우 mTHPC의 통풍과 높은 전이성 HOS와 143B OS 세포주의 비교 <ol><li> DMEM, 햄 F12 및 비율 4.5:4.5:1에서 열 불 활성화 된 태아 혈청을 포함하는 세포 배양 매체를 준비합니다. 주 : PS mTHPC의 리포솜 제형은 1.5 ㎎ / ㎖ mTHPC의 최종 농도로 물에 용해시켰다. </li><li> 플레이트 0.2 × 10 6 HOS와 143B 세포 / 잘 6 - 웰 플레이트 (각 조건에서 삼중)과 세포가 하룻밤을 준수 할 수 있습니다. 참고 번호가 개별 세포주로 조정되어야한다. 80~90%의 포화 상태는 실험시에 최적이다. </li><li> 다음 날에, 다른 기간 (시간 의존적 흡수)에 대한 mTHPC의 고정 농도로 세포를 배양, 또는 고정 된 시간주기 (농도 의존적​​ 흡수) 중에 mTHPC의 농도 증가. </li><li> 특정 시간 P에 mTHPC와 세포를 품어oints 또는 mTHPC의 농도. 주 : 본 연구에서는 HOS와 143B 세포주 모두가 0.6 ㎍ / ㎖의 mTHPC와 함께 배양 0, 2.5, 5 또는 10 시간, 5 시간 동안 0, 0.6, 2.5, 10 ㎍ / ㎖의 mTHPC와. 적용 기간 및 투여 량은 사용되는 세포주에 따라 다를 수 있습니다. </li><li> 항상 세포의 직사광선 노출을 방지하기 위해 어둠 속에서 또는 흐리게 조명 조건에서 작동해야합니다. </li><li> 지정된 조건에서 세포를 배양 한 후, 배지를 흡인하고, PBS로 세포를 세 번 씻는다. 이어서, 0.5 ml의 트립신 / EDTA로 세포를 분리하고 세포를 센다. </li><li> 박리 한 후, 5 분 동안 400 XG에서 세포를 원심 분리하여 배지를 흡인하고 200,000 세포 / ml의 최종 농도로 PBS에 세포를 재현 탁. </li><li> 얻어진 세포 현탁액 피펫 100 μL를 96 웰 플레이트 및 형광 분광 광도계에서 이러한 세포의 형광을 측정한다 (즉, 20,000 세포). 설정mTHPC 형광 측정의는 다음과 같습니다 방출의 여기 및 652 nm의 417 nm의. </li><li> 셀당 내면화 mTHPC의 양을 계산은 다음과 같이 세포 수와 세포 부피에 대한 상대적 형광 단위 (RFU)를 정상화하기 위해 : </li><li> 0-4 ㎍ / ㎖ (사용 세중의 측정)에서 PBS에 mTHPC의 서로 다른 농도를 사용하여 표준 곡선을 준비합니다. 96 - 웰 플레이트에 각 희석 100 μl를 피펫 단계 1.6에 설명 된 설정을 사용하여 우물의 형광을 측정합니다. </li><li> 우물에서 (㎍ / ml 중) mTHPC의 농도를 제공 mTHPC의 선형 표준 곡선의 기울기가 (20,000 세포 얻은) RFU를 나눈다. </li><li> (세포 현탁액 100 μl를 나타내는 μg에서) 하나의 잘 mTHPC의 총량을하고 20,000 세포의 양이 값을 나누는 10이 mTHPC 농도를 나눈다. </li><li> X (4 / 3 개의 x π) 구에 대한 공식으로 볼륨을 계산R 3. 현미경 하에서 20 트립신 세포의 직경을 측정하고, (2)에 의해이 값을 나눔으로써 반경 (r)을 구하는. </li><li> 마지막으로, 10 배양 (농도 의존적​​ 흡수)의 시간 또는 100 %로 설정 한 10 ㎍ / ㎖의 mTHPC (시간 의존 흡수), 치료 후 HOS 세포를 얻은 것과 모든 값을 정상화. </li></ol> 2. PS 체외의 광독성 측정 <ol><li> 96 - 웰 플레이트 (mTHPC의 각 농도에 대한 삼중 사용)에 3,000 세포 / 웰 씨와 그 하룻밤을 준수 할 수 있습니다. 마찬가지로 추가로 96 - 웰 플레이트를 준비하고 아래에 설명 된대로 세포를 치료하지만 위상 콘트라스트 이미지 따로 넣어. 주 : 숫자는 각각의 세포주에 따라 조정되어야한다. 실험시의 50 ~ 60 %의 컨 플루가 이상적이다. <ol><li> mTHPC 농도의 각 셀의 해당 어두운 독성 컨트롤을 추가하기 위해 염두에 두십시오.SE는 mTHPC의 선택 농도의 부재 또는 존재 배양 할 세포하지만 조명되지 않습니다. </li></ol></li><li> 다음 날, 세포주에 따라 mTHPC (0, 0.0001, 0.001, 0.01, 0.03, 0.075, 0.15, 0.3, 0.6, 1.25, 2.5, 5 및 10 ㎍ / ㎖)을 다양한 농도로 세포를 배양하고 5 시간 동안 어둠에 보관하십시오. <ol><li> 1.3.2 절을 참조하십시오 주 : 배양 시간을 조명 세포주에 사용 된 PS에 따라 달라집니다 전에, 따라서 별도의 실험에서 서로 다른 농도와 서로 다른 시간의 포인트를 테스트하는 것이 좋습니다. </li></ol></li><li> 지정된 시간 동안 배양 한 다음, PBS로 두 번 세포를 씻어 mTHPC없이 새로운 신선한 매체를 추가 할 수 있습니다. </li><li> mTHPC 흡수 스펙트럼에 대한 구체적인 652 nm의 다이오드 레이저 (도 1 참조)와 함께 세포를 밝히는. <ol><li> 13.5 cm의 dista의 높이에 21.88 ㎿가 / cm 2 전원에 레이저를 설정5 J / cm 2의 에너지 량을 얻기 위하여 세포 NCE. 230 초 동안 세포를 조명. (함께하고 mTHPC없이) 어두운 독성 제어 세포를 조명하지 마십시오. 참고 : 항상 PS의 여기에 사용되는 레이저 빛으로부터 눈을 보호하는 안경을 착용. </li></ol></li><li> 조명 한 후, 24 시간 동안 37 º C에서 어둠 속에서 세포를 유지. 그 후, 수용성 테트라 졸 (WST-1) 시약 (매체의 10 μl/100 μL)를 추가합니다. </li><li> 세 시간 WST-1 시약을 첨가 후, 415 nm에서 96 - 웰 플레이트의 각각의 웰의 흡광도를 측정한다. </li><li> 생존 세포의 비율을 계산하기 위해서, (즉 mTHPC없이 mTHPC와 nonilluminated없이 조명) 100 %의 각 조건에 대해 mTHPC 비 처리 된 세포에 대해 얻어진 값의 평균치를 설정한다. </li></ol> 3. 셀 계산하여 세포 수의 추정 <ol><li> 종자 20,000 세포 / 우리24 웰 플레이트에 LL하고 하룻밤을 준수 할 수 있습니다. </li><li> mTHPC과 5 시간 (0.001, 0.003, 0.15, 0.6, 1.25 ㎍ / ml)에 대한 세포를 품어 전술 한 바와 같이 그들을 조명. </li><li> 매체를 수집, 5 분 400 XG에서 원심 분리하여 PBS로 한 번 세포를 씻어 그들을를 Trypsinize 그들을 수집합니다. </li><li> 원심 분리 후, 배지를 흡인하고, 신선한 배지 200 μL에 세포를 재현 탁. </li><li> 네우 바우 실에서 세포를 계산합니다. </li></ol>

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저자는 더 경쟁 재정적 이익 또는 이해 관계의 충돌 선언하지 않습니다.

PDT에 응답하여 최적의 세포 독성을 달성하기 위해, 오른쪽 레이저 광 설정과 배양 시간을 선택하는 것이 중요하다. 여기에 설명 된 절차는 PS의 이해를 확인하고 체외에서 PDT 유도 된 세포 독성을 정량화하기 위해 일관되고 효율적입니다. PS mTHPC의 특정 흡수 파장을 사용하여, 셀룰러 PS 흡수는 직접적으로 측정 될 수 있고, PS는 세포 독성 활성 산소 종을 생성하기 위해 활성화 될 수있다. ...

골육종 (OS)의 예술 치료의 오늘의 상태는 기본 뼈 종양, 신 보조 항암 화학 요법 및 수술의 조합을 포함한다. 이 치료 요법은 화학 요법의 사용 전에 약 20 % 내지 국소 질환 환자의 생존율 증가를 밝혀, 현재까지 04 세 사이 60~70%. 그러나 지난 수십 년 동안, 현지 질환 OS 환자의 전체 생존 기간은 5 4 plateaued했다. 또한, 이러한 환자 30 ~ 40 %의 진단 후 3 년 이내에 재발 및 전이성 질환을 가진 환자는 20 ~ 30 % 4의 생존율, 6, 7을 계속합니다. 이들 환자의 결과를 개선하기 위해, 새로운 치료 전략을 개발해야한다. 광 역학 요법 (PDT), 오히려 신규 항암 치료는 photosensi의 여기를 위해 특정 파장의 광을 사용혈류로의 주입 후 종양 세포에 축적 tizer (PS). PS의 레이저 광을 여기에는 종양 세포 및 세포 죽음에서 세포 독성 반응을 유도하는 산소의 존재 하에서 산소 라디칼을 생성한다. PDT는 종양 경색으로 이어지는, 혈관 수축 및 종양 미세 혈관의 혈전 형성을 일으키는 원인이되고, 종양 내부 결과적으로 지역의 저산소증과 산소 결핍이 기본 메커니즘 외에 두 개의 추가 PDT는 생물 학적 과정이 감소 된 종양의 성장에 기여 유발. 마지막으로, PDT 부상 죽어가는 종양 세포는 로컬 면역 반응, PDT의 다소 독특한 기능을 트리거합니다. 이것은 보체 시스템 및 수지상 세포 8 항원 제시의 활성화를 수반한다. 따라서, 조건은 종양 특정 면역에 이르는, 림프 세포의 후속 활성화와 종양 항원의 프레 젠 테이션을위한 생성됩니다. 지금까지, PDT 연조직 종양 및 hyperpl 여러 종류의 치료를 위해 사용되었다아시아의, 같은 광선 각화증, 바렛 식도, 기관지 종양, 방광 암, 기저 세포 암, 머리와 목 암 2의 완화 치료로. 치료는 단지 적은 부작용과 로컬 대규모 괴사를 유도하는 것으로 알려져, 따라서 선택적으로 종양 조직을 근절 할 수있는 잠재력을 가지고있다. 이러한 장점에도 불구하고, PDT의 응용 프로그램은 화학 요법 약물의 투여보다 기술적으로 더 까다로운 남아있다. 최대 효능, PS 농도, 노광 시간 및 전체 광 에너지 전달을 달성하기 위해 최적화 될 필요가있다. 이는 생체 내 실험에서 수행 될 수 있지만, 인해 최적화 될 필요가 파라미터의 상대적 다수, 그것은 초기 시험 관내에서 최적 조건을 결정하는 것이 더 효율적이다. 후술하는 실험에서는, PS 5,10,15,20 - 테트라 키스 (메타 - 하이드 록시 페닐) CHL를 사용하여 PDT의 효능을 시험 관내 시험오린은 mTHPC (그림 1A)를 단축. mTHPC는 현재 두 경부암의 완화 치료를위한 병원에서 사용되는 의약품 Foscan있는 활성 물질이다. 그것은 낮은 농도에서 이미 대규모 세포의 손상을 유도하는 가장 유력한 PS 중 하나이며, 이는 조직 침투 (9, 10)의 측면에서 다른 PS 우수한 것으로 입증되었다. 의 광 흡수 스펙트럼 (그림 1B)는 각각 PS를 축적 조직 현지화 및 PDT 유도에 사용되는 두 개의 눈에 띄는 봉우리, 417 nm에서 한 652 nm에서 두 번째를 보여줍니다. 현재 mTHPC의 리포솜 제형은 개발 중입니다. 여기서, 우리는이 리포솜 제형의 흡수를 정량화하고,이 OS 인간 세포주에서 PDT를 수행하는 절차를 설명; 낮은 전이성 HOS 및 높은 전이성 143B 세포. 여기에 제시된 데이터 중 일부는 11 이전에보고 된 바있다. 일여기에 설명 된 전자의 방법은 PDT의 효과에 전이성 표현형의 효과를 공부하기 위해 수 있습니다. orthotopically 면역 결핍 SCID 마우스의 뒷다리에 주입 143B 세포는 intratibial을 전이 차 종양, 밀접하게 인간을 전이 질환을 흉내 낸 모델을 일으킬 수 있습니다. 따라서, 제안 된 시험 관내 실험은 최적 PDT 설정하기 생체 내 실험에 사용되는 평가할에게도 적합하다.

<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>
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cytotoxic efficacy of photodynamic therapy in osteosarcoma cells in vitro

최근, 적은 전신 부작용과 암에 대한보다 효과적인 치료법을 찾는데 어려움이있어왔다. 따라서 광 역학 치료보다 종양 선택적 치료에 대한 새로운 접근 방식입니다. 산소의 존재 하에서 특정 파장의 빛을 활성화시에, 세포 독성 반응을 유도 한 산소 라디칼을 생성하고, 비 독성 감광제 (PS)을 이용한다 광 역학 치료 (PDT). FDA에 의해 거의 20 년 전 승인에도 불구하고, PDT가 요즘에만 암 종류 (피부, 방광) 및 nononcological 질환 (건선, 광선 각화증) 2 제한된 수를 치료하는 데 사용됩니다. PDT의 사용의 중요한 이점은 전신적인 부작용을 방지 지방 치료를 수행 할 수있는 능력이다. 또한, (신경 또는 혈관 주위 예) 민감한 부위에서의 종양의 치료를 허용한다. 여기서, intraoperatPDT의 필자의 응용 프로그램은 골육종 (OS)에서 기본 종양 위성을 대상으로 뼈의 종양이 외과 종양 절제술 후 조직을 둘러싼 종양에 남아 간주됩니다. 치료 재발의 수를 감소시 및 (수술후) 전이에 대한 위험을 감소시키는 것을 목표로한다. 본 연구에서 우리는 잘 확립 intratibial OS 마우스 모델에서 인간의 질병을 재현하기 위해 사용되는 널리 사용되는 OS 세포주의 효과적인 치료를위한 최적의 PDT 설정을 확립하는 체외 PDT 절차에서 제시한다. PS mTHPC의 흡수 분광 광도계 및 광독성는 WST-1 분석으로 살아남은 세포의 계산에 의해 평가 된 세포의 죽음을 유도하기 위해 652 nm에서 mTHPC의 레이저 광 자극으로 유발 된으로 조사 하였다. 기존 기술은 동물 모델에서 미래 연구를위한 최적의 PDT 설정을 정의 할 수있게. 그들은 PDT의 효과의 평가를위한 쉽고 빠른 도구입니다 <em&gt; 체외에서 생체 내에서 응용 프로그램 전.

여기에보고 된 기술로, 우리는 인간의 OS 세포에서 mTHPC 기반 PDT를 조사 하였다. 먼저, mTHPC의 시간 및 농도 의존적​​ 흡수는 낮은 전이성 HOS 및 고도로 전이성 143B OS 세포주에서 조사 하였다. mTHPC 흡수는 형광 분광 광도계 mTHPC의 형광 (Reidy 등. (11)로부터 허가를 재현 그림 2) 측정에 의해 평가 될 수있다. 그림 2A는 시간 의존적으로 mTHPC의 이해를 보여줍니다...

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

여기에보고 된 프로토콜은 수 광역 세포주의 치료 (PDT) 및 동물 모델에서 치료를 적용하기 전에 PDT 설정의 최적화의 효과의 평가.

골육종 세포의 광 역학 치료의 세포 독성 효능<em&gt; 체외</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 조회수.  Balgrist University Hospital, Zurich, Switzerland.  여기에보고 된 프로토콜은 수 광역 세포주의 치료 (PDT) 및 동물 모델에서 치료를 적용하기 전에 PDT 설정의 최적화의 효과의 평가. 

비디오: 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.

마우스 눈에 유전자 치료 벡터와 줄기 세포의 Subretinal 주입

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 ...

연구

의학

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.

난소 암 종양 시작 세포의 생체 농축에

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.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

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.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

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.

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.

준비 및<em&gt; 체외</em&gt; 자화 특성의 내피 세포를 미르 변성

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.

가속도계를 사용하여 일상 생활에서 상지 성능을 정량화하는 방법

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.

Photodynamic 치료 체 외에서 접근

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.

구슈강(GSK) 생체내 및 체외 실험을 위한 과립 준비

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.