The authors have no acknowledgements.

이 프로토콜은 인간의 샘플에 대한 연구에 대한 기관 윤리 지침을 다음과 동의서가 포함 된 모든 기증자로부터 얻은 것입니다. 여기에 설명 된 실험에 대한 승인은 앤트워프 대학 병원의 임상 시험 심사 보드에서 얻었다. 주 : 지정하지 않는 온도 표시는 항상 실온이다. 1. 준비 유량 용기 설치 <ol><li> 준비 레인, 튜브와 핀 <ol><li> 격렬하게 소용돌이 콜라겐 현탁액를 50㎍ / ㎖의 최종 농도 제공자에 의해 공급되는 등장 글루코스 용액 1/20 희석. 참고 : 우리는 주로 유형 I 피 브릴의 구성, 말 건 콜라겐을 사용합니다. 말의 콜라겐 타입 I은 종종 &quot;HORM&quot;콜라겐로하고, 과거뿐만 아니라 생물학적 분석 이유 모두 9 이러한 유형의 황금 표준이다. 인간 타입 III 콜라겐도 사용되지만, 일 수있다E는 덜 코트 브릴 및 혈소판 반응은 강하지 않다. 기타 코팅 표면은 예 폰 빌레 브란트 인자 (VWF), 피브리노겐, 피브로넥틴, 라미닌, 비트로 넥틴, 트롬 보스 폰딘 -1 (16) 또는 이들의 조합을 위해 사용될 수있다. </li><li> 공급자의 컨테이너에서 새로운 일회용 바이오칩을 가져 가라. 여기에 사용되는 바이오칩의 크기는 mm 3 0.4W X 0.1H의 X 20L입니다. </li><li> 피펫 출구로 칩과 마크의 한쪽 끝에서 미세 유체 바이오 칩의 레인 (들)에 0.8 μL. 차선은 1.1.1에서 제조 된 코팅 용액을 포함하는 콜라겐과 5 / 6를 충전되어 있는지 확인합니다. 기포가 없는지 확인합니다. 주 : 채널은 부분적으로 (논의를 참조)의 채널의 입구에서 콜라겐 섬유의 축적을 방지하기 위해 도포된다. </li><li> 가습 및 밀폐 용기에 4 시간 또는 밤새 4 ° C에서 품어. </li><li> / v)의 소 혈청은 알부민 승 (버퍼를 차단 (1.0 % 피펫 팅으로 코팅 된 채널을 차단타단 마크에서, D는 0.1 % (w / v) 글루코오스 된 10 mM 4- (2- 히드 록시 에틸) -1- 피페 라진 에탄 술폰산 (HEPES) 완충 된 염수 (/ v)의 염화나트륨, pH 7.4의 0.9 % w (HBS)에서 입구로. 차선이 완전히 공기 방울을 방지 차단 버퍼 충전되어 있는지 확인합니다. </li><li> 동일한 길이 (12cm)에서 튜브를 잘라. 레인 당 하나를 사용하여 핀 각 튜브를 연결합니다. 예를 들어, 중복에서 실행되는 두 가지 조건을 비교 실험는 4 개의 튜브 뻗어과 4 개의 핀을 준비 할 필요합니다. </li><li> 주사기 및 26 G 바늘 또는 첨부 커넥터를 사용하여 증류수로 튜브를 씻어. </li><li> 버퍼를 차단와 튜브를 포화. 1 시간의 최소 폐쇄 및 가습 용기에 보관할 것. </li></ol></li><li> 펌프 및 매니 폴드 준비 <ol><li> 기포를 제거 펌프 린스 증류수 매니 폴드. </li><li> 출구에서의 고정 된 10 ㎕의 팁을 사용하여 바이오칩 레인 (들)에서 차단 완충액 대기음. 의 표면을 청소정밀 무료로 닦아 먼지와 변성 알코올로 바이오 칩은 지문과 먼지를 제거합니다. </li><li> 자동화 된 현미경 무대에 바이오칩을 수정합니다. 하나 이상의 레인 하나의 실행에서 동시에 사용되는 경우, 바이오칩 콘센트 8 레인 매니 스플리터를 연결한다. 참고 : 8 레인 매니 폴드 스플리터는 하드웨어의 조각 (그림 S1)는 펌프와 바이오칩에 연결. 그것은 바이오칩 또는 운영자 정의 첨부 된 소프트웨어에있을 수 레인의 조합에 사용 가능한 모든 (팔) 레인의 작업을 할 수 있습니다. </li><li> 바이오칩 입구에서 그들을 해결하기 위해 튜브에 핀을 사용합니다. HBS 가득 1.5 ML 원뿔 테스트 튜브 (핀없이) 튜브의 다른 쪽 끝을 놓습니다. 나머지는 버퍼를 차단하고 제대로 콜라겐을 준수 제거하기 위해, 모든 튜브 1 ML의 HBS는 펌프를 사용하여와의 연결 차선을 씻어. </li></ol></li></ol> 혈액 샘플 2. 준비 <ol><li> 수집 및 분리건강한 지원자에서 신선한 전혈. (17) <ol><li> 항응고제로 에틸렌 디아민 산 (EDTA)를 포함하는 진공 튜브에 혈액의 첫 번째 밀리리터를 수집하고 독점적으로 자동 혈액 분석기 전체 혈액 카운트 (CBC)이 샘플을 사용합니다. </li><li> 대피 튜브를 사용하여 적절한 항 응고 혈액의 부피를 수집합니다. 피브린을 형성하는 연구 프로토콜과는 구연산 나트륨의 일부가 아닌 경우 유동 챔버 실험 표준 항응고제 헤파린 또는 히 루딘이다. 주 : 헤파린 결과에 기재된 모든 실험에서 항응고제로 사용되었다. 주 : 혈액의 양이 수행되는 실험의 수에 의존한다. 약 3 차선 1 튜브 (7 ㎖). </li><li> 회 전자 출원중인 혈액 재구성에 튜브를 놓습니다. 참고 : 분석은 정맥 절개의 3 시간 이내에 완료해야합니다. </li><li> 혈소판 풍부 혈장을 제조 250 g에서 15 분 동안 원심 분리기 (PR피). 느슨하게 포장 된 펠릿의 교란을 방지하기 위해 원심 브레이크를 사용하지 마십시오. <ol><li> 하나 이상의 튜브가 수집 될 때, 하나의 원추형 원심 분리 튜브 내의 혈액 풀링. 참고 : 원심 분리가 PRP 수율 및 차등 셀 &quot;오염&quot;에 따라, 더 느리게 이하 긴 수행 할 수 있습니다 바람직하다. </li></ol></li><li> 제거하고 몇 혈소판 가득 적혈구를 맺는 PRP과 버피 코트를 폐기합니다. 우리의 손에 평균 μL 당 13 ± 5 × 10 (3) 포장 붉은 세포 분획의 혈소판이 (SD ± 의미, N = 12) : 참고. </li></ol></li><li> 혈액에서 재구성 <ol><li> 5 분 4 ml의 당 20 초 동안 37 ° C에서 해동 혈액형 AB (히말라야 D 음) 플라즈마. </li><li> 자동 혈액 분석기를 사용 2.1.5에서 제조 한 포장 적혈구 용적률을 결정한다. </li><li> 혈액 은행에서 혈소판 농도를 결정 혈소판 농축 그쪽 제조t 위 적혈구 분획을 재구성하는데 사용된다. </li><li> 1 mL의 샘플을 40 %의 적혈구 용적 및 250 × 10³ 혈소판 / μl를 산출 할 것이다 충전 적혈구 및 혈소판 농축 물의 양을 계산한다. 주의 : 다른 타겟 셀의 역가가 연구 프로토콜에 따라 임의로 설정할 수있다. </li><li> 전송은 클리핑 된 피펫 팁을 사용하고, 1 mL의 샘플 볼륨에 도달 할 때까지 혈소판 농축 물을 추가 새로운 튜브로 적혈구와 혈장을 포장. 주 : 플라즈마 혈전 형성 비율에 큰 영향이 있기 때문에 변수 연구에 따라, 혈장 분획 모든 재구성 된 샘플에서 동일해야한다. 예를 들어, 결과에 영향을 미칠 수있는 다른 도너 또는 다른 항응고제 촬영 동결 해동 또는 플라즈마를 반복했다. </li><li> 부드럽게 반전에 의해 재구성 된 혈액을 혼합하고 CBC를 수행합니다. </li><li> 혈소판 부분의 볼륨이 교체되는 &quot;빈&quot;제어 샘플을 준비0.9 % 물 (m / v)의 염화나트륨 같은 볼륨하여 CBC를 사용하여 재구성 된 혈액 내인성 혈소판의 농도 (즉, 비 - 혈액 은행 혈소판)을 결정한다. </li></ol></li><li> 라벨링 <ol><li> 1 mL를 정확하게 취하여 1 ㎕의 5 mM의 칼 세인 AM (5 μM 최종 농도)를 함유하는 시험관에 혈액을 재구성. 주의 : 다른 세포 염료 (14)를 사용할 수있다. </li><li> 반전에 의해 부드럽게 섞는다. </li><li> 사용하기 전에 37 ℃에서 5 분 동안 인큐베이션. </li></ol></li></ol> 3. 관류 분석 <ol><li> 차선의 하단에 부착 된 콜라겐 섬유에 대물 초점. 이상적으로,이 집중 전략 위상차 또는 차등 간섭 대비 (DIC) 설정을 사용합니다. 디지털 선택된 Z-위치를 해결하기 위해 실험 소프트웨어 &#39;를 선택 타일 영역에 대한 설정 현재 Z&#39;를 선택합니다. </li><li> 제 m의 실험 소프트웨어 차선 (XY)에서의 ROI를 선택실험 기간 동안 기록됩니다 icroscope. 참고 : ROI가 임의로 관류 차선 내에서 선택하는 표면적이 될 수 있습니다. 너무이 비교적 작은 경우에도, 그 영역에서 가변 유동 프로파일의 부작용을 방지하기 위해 근접 차선의 유입구들 및 배출구 혈전 형성을 분석 할 수없는 것이 바람직하다. 관심 영역의 표면적은 혈소판의 상당수를 포함하거나, 신호의 수평 있도록 혈전한다. 이 프로토콜에서, ROI는 2 ㎝ 길이 차선의 중앙에 0.62 mm 2의 결과로 동일한 크기의 세 개의 나란히 이미지의 디지털 스티치 집합체이다. </li><li> 부드럽게 반전 샘플을 혼합하고 자동화 된 단계에있는 바이오 칩에이 옆에 위치. </li><li> 재구성 된 혈액 샘플을 함유하는 시험관에 바이오칩의 입구와 연결되어 튜브를 놓는다. </li><li> 튜브를 테스트하기 위해 연결하는 채널 (필요에 따라 또는 다른 전단 응력) 50 다인 / ㎝ 2에서 펌프를 실행펌프의 소프트웨어를 사용하여 재구성 된 혈액 시료를 포함. 참고 : 다른 전단 응력 사용할 수 있습니다. </li><li> 이미지를 기록 현미경의 수집 및 실험 소프트웨어를 사용하여 실시간으로 5 분 동안 매 15 초. 주의 : 다른 시계열 실험적 셋업에 따라 사용될 수있다. 참고 : 우리는 일반적으로 100 배의 배율 (10 배 대물 렌즈 10 배)를 사용하지만, 더 높은 (또는 낮은) 배율 쉽게 대체 할 수있다. </li></ol> 4. 워시 아웃 <ol><li> 차아 염소산 나트륨 (표백제) 0.5 %이어서 모든 튜브 출구에 부착 된 증류수를 사용하여 멀티 매니 폴드 또는 펌프에 연결을 세척 (v / v)의 물에 마지막으로 0.1 M의 NaOH. 유해 폐기물로 바이오 칩 입구에 고정 튜브를 폐기하십시오. </li></ol> 5. 데이터 분석 <ol><li> 이미지 분석 소프트웨어 혈전 성장 반응 속도를 결정한다. 다음 명령은 ZEN2012에 대한 구체적인이다. <ol&gt; <li> 혈소판의 표면 적용 범위를 결정하기 위해 플러그인 이미지 분석을 엽니 다. </li><li> ㄱ 긍정적 인 신호와 상관 픽셀 강도를 정의하는 대화 형 분석 탭, 즉 부착 식 혈소판 또는 부착 혈소판의 형광 임계 값을 설정합니다. </li><li> 자동으로 선택한 임계 값 사이의 신호로 픽셀을 포함하는 &quot;객체&quot;의 (μm의 2) 별도의 표면 영역을 포함하는 스프레드 시트를 생성 할 테이블을 작성합니다. 이는 각각의 시점에 대해 수행된다. 참고 : 형광 임계 값이 선택되고 나면, 분석 소프트웨어가 자동으로 기준을 충족보기 필드에서 &#39;개체&#39;를 검색합니다. 이러한 개체는 혈전 작은 혈소판 응집 또는 단일 혈소판되며 다수의 픽셀을 커버한다. 모든 개체는 별도 스프레드 시트에 나열됩니다. </li><li> XML 형식으로 이러한 스프레드 시트를 저장하고 스프레드 시트 프로그램에서 열더 계산. </li><li> 합산하여 선택된 객체의 전체 표면적과 측정 영역 (μm의 2)의 합계 면적 결과를 나눈다. 이것은 상대적 흡착율 (%)을 수득한다. 각 시점에 대해 그렇게. </li><li> 재관류 시간 함수 이러한 표면 커버리지를 플롯 및 특정 조건의 혈전 성장 동역학을 수득 선형 회귀에 의해 기울기를 계산한다. </li></ol></li></ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+of+blood+platelets+by+adenosine+diphosphate+and+its+reversal.">Aggregation of blood platelets by adenosine diphosphate and its reversal.</a> Nature. 194, 927-929 (1962).</li><li>Cattaneo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+the+Standardization+of+Light+Transmission+Aggregometry:+A+Consensus+of+the+Working+Party+from+the+Platelet+Physiology+Subcommittee+of+SSC/ISTH.">Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.</a> J Thromb Haemost. 11, 1183-1189 (2013).</li><li>Deckmyn, H., Feys, H. 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The authors have nothing to disclose.

미세 유체 흐름 챔버 실험은 혈액을 흐르는 혈소판 기능을 조사하는 훌륭한 도구입니다 실험 상황 변화에 체외에서 지혈을 평가하는 데 사용됩니다. 가난한 실험실 간 표준화 (9)에도 불구하고, 우리는 우리의 실험실에서 실험 변동이 허용되는 것을 보여줍니다. 이것은 확실하게 주어진 연구에서 (쌍) 샘플을 비교할 수 있습니다. 이것은 혈액 은행 조건 11 혈소판 저장 해로?...

지혈은 제한된 시공간 상황 (1) 세포, 단백질, 이온 및 조직의 결합과 규제 활동을 필요로한다. 제어되지 않은 활동은 혈액 응고 관련 질환의 스펙트럼에 출혈이나 혈전증 및 이환율이나 사망률의 원인이됩니다. 미세 유체 흐름 챔버 실험은 시험관 내에서 지혈을 모방 도전적인 기술이다. 이 방법은 혈소판에 대한 선도적 인 역할과 지혈에 참여 프로세스의 복잡한 상호 작용의 조사를 할 수 있습니다. 혈관 손상에 따라, 혈소판은 혈액 손실을 방지하기 위해 피하 노출 매트릭스 (글리코) 단백질에 부착. 접착 후, 혈소판 활성화 및 답변 집합체 자동 - 최종적 혈소판 네트워크의 형성에 의해 안정화 피브린 및 회사의 결과에 이르게 분비 시그널링 혈전이 밀봉 권취. 대부분 혈소판 기능 검사, 체험관 달리흐름 챔버와 사항이 고려 혈류의 물리적 파라미터 따라서 멀티플 세포 및 생체에 3,4- 레올의 영향을. 흐름 챔버 실험은 접착제 매트릭스, 유동성 및 흐름 프로필, 세포 성분, 독성 물질이나 약물, 이온 강도 및 더 많은 존재 포함되는 프로세스 지혈 (하위)에 영향을 미치는 주요 매개 변수를 변화시켜 지혈과 혈전의 랜드 마크 통찰력을 생성했다. 지난 20 년에 큰 샘플 볼륨 (10 ~ 100 ml)에 요구되는 낮은 처리량 흐름 챔버 실험은 미세 유체 챔버 종종 작은 평행 판 챔버로 구성된 제어 벽 전단 조건 5에서 전체 혈액 관류를 위해 현대 기술을 포함하여 진화했다. 하드웨어 설치가 간단하고 이하 (혈액) 볼륨이 필요한 대부분 때문에 Microscaling 크게 실험이 더 접근하고 versati 렌더링, 분석 처리량을 증가르. 예를 들어, 작은 실험실 동물에서 혈액 해주기 동물을 희생하지 않고도 사용될 수있다. 유전자 변형 생쥐의 혈액 샘플 따라서 지혈을 촉진 또는 억제 키 분자의 식별과 새로운 기본적인 통찰력 6 도움을했습니다. 전문 연구소들은 여전히 소프트웨어에 의해 청사진을 할 수 석판 금형에 중합 폴리 디메틸 실록산 (PDMS) 7에서 예를 들어 사용자 정의 만든 흐름 챔버를 사용합니다. 그 결과 챔버는 저렴한 일회용하고 쉽게 사후 분석을 위해 분해 할 수 있습니다. 또한, 기본적으로 선박을 포함 분기점 또는 날카로운 회전의 디자인은 명령에 구축 할 수 있습니다. 이러한 장점은 표준화가 이미 흐름 챔버 실험과 기본 문제 이후의 단점이며, PDMS 사용자 정의 챔버이 도움하지 않은했다. 이 특정 문제의 위에, 코팅 (조건), 형광 프로브, 항응고제, 온도에샘플링과 분석 사이 erature와 시간이 모두 제대로 팔을 표준화하고 있습니다. 이러한 변수의 표준화에 도전하지만, 그럼에도 불구하고 실험실 간 결과의 비교를 가능하게 할 필요가 있습니다. 이 항목에서는 Biorheology 9,10에 과학 및 표준화 소위원회에서 혈전증 및 지혈에 관한 국제 사회의 주요 주제이다. 혈소판 농축액 (PC)는 혈소판 감소증 및 / 또는 출혈의 원인이 다양한 질병을 앓고있는 환자에 수혈된다. 그러나 PC 혈소판 특히 저장 시간 (11)의 기능에 둔감하도록 공지되어 열화 과정 노화 일반적 혈소판 기억 장애라고 링크. 때때로 혈소판 번 (12) 수혈 혈액 순환에 복원 주장하지만, 이것에 대한 증거는 부족하다. 또한, PC를 구성하는 혈소판의 기능은 통상적으로 시험되지 않기 때문에 이러한 분석법과의 관계치료 또는 예방 효과는 불분명 (13)이다. 미세 유동 챔버 수집 및 발의 조작 사이의 사슬을 최적화하는 PC에서 혈소판 기능을 조사하기위한 수단을 제공한다. 우리가 이전에 14,15을 발표하고 여기에 설명 된 바와 같이 PC의 직접 (쌍) 비교를위한 강력한 연구 도구입니다.

<table border="0" cellpadding="0" cellspacing="0" width="1113">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer tube with EDTA&#160;</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368856</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Heparin</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368480</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Sodium Citrate</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">366575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hirudin Blood tube</td>
			<td style="width:220px;">Roche</td>
			<td style="width:268px;">6675751 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer Eclipse</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368650</td>
			<td>Blood collection needle with preattached holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 100-1000</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">740290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 2-200</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">739280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1-10</td>
			<td style="width:220px;">Eppendorf</td>
			<td style="width:268px;">A08928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tube 5mL</td>
			<td style="width:220px;">Simport</td>
			<td style="width:268px;">11691380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 15mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">1888271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 50mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:268px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision wipes</td>
			<td style="width:220px;">Kimtech</td>
			<td style="width:268px;">5511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Fluoro+ Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">188V8CF-400-100-02P10</td>
			<td>Named in figure S1 A as &#39;Biochip&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Tubing</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">TUBING-TYGON-B1IC-B1OC-ROLL 100F</td>
			<td>Named in figure S1 B as &#39;Disposable tubing&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Needles</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">SS-P-B1IC-B1OC-PACK200</td>
			<td>Named in figure S1 B as &#39;Pin&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Connectors for single inlet cables of biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">CONNECTORS-B1IC-PACK100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multiflow8 connect</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">MF8-CONNECT-BIC3-N-THROMBOSIS</td>
			<td>Named in figure S1 B as &#39;Reusable tubing&#39; and &#39;Splitter&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Humidified box</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">HUMID-BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microfluidic pump</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">N/A</td>
			<td>Venaflux Assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horm Collagen</td>
			<td style="width:220px;">Takeda/Nycomed</td>
			<td style="width:268px;">1130630</td>
			<td style="width:409px;">Native equine tendon collagen (type I) 
			Isotonic glucose solution to dilute collagen is supplemented</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES buffered saline (HBS)</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blocking buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein AM</td>
			<td style="width:220px;">Molecular probes</td>
			<td style="width:268px;">C1430</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach 10%</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1M NaOH</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturated alcohol</td>
			<td style="width:220px;">Fiers</td>
			<td style="width:268px;">T0011.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mirus Evo Nanopump</td>
			<td>Cellix</td>
			<td>188-MIRUS-PUMP-EVO</td>
			<td>with Multiflow8. Named in figure S1 A as &#39;Pump&#39; and &#39;Manifold&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z1</td>
			<td>equipped with a colibri-LED and high resolution CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:268px;">N/A</td>
			<td>ZEN 2012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematology analyzer</td>
			<td>Sysmex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge</td>
			<td>Eppendorf</td>
			<td>521-0095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Platelet incubater</td>
			<td style="width:220px;">Helmer</td>
			<td style="width:268px;">PF-48i</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubation water bath</td>
			<td>GFL</td>
			<td>1013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette</td>
			<td>Brand</td>
			<td>A03429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Roller</td>
			<td>Ratek</td>
			<td>BTR5-12V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile docking device</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">TSCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing Sealer</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">AC-155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:220px;">VWR</td>
			<td style="width:268px;">58816-121</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

내부 분석 변화를 설명하기 위해, 3 개의 동일한 복원 된 전혈 시료를 콜라겐 코팅 된 표면 (도 1)를 통해 동시에 관류 하였다. 이는 8.7 %의 변동 계수가 발생했습니다. 이 통계는 관련 샘플 간의 신뢰성있는 비교를 허용 허용 내 분석 및 intralaboratory 변화를 의미한다. 우리는 여기 설명 상업적 유동 챔버의 입구는 상...

Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

미세 유체 흐름 챔버 지혈 및 혈소판 수혈을 모델링하기 위해 재구성 된 혈액을 사용하여<em&gt; 체외</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

microfluidic-flow-chambers-using-reconstituted-blood-to-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

9.3K 조회수.  Belgium Red Cross-Flanders. Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro. 

비디오: Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

종양 Microenvironment를 재현을위한 마이크로 유체 장치<em&gt; 체외에서</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

연구

생체공학

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

멀티 채널 Microfluidic 장치에서 분리 비즈와 세포 Dielectrophoresis와 층류를 사용하여

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

의 체계적인 분석<em&gt; 체외</em멀티 웰 플레이트 미세 유체 시스템을 사용하여&gt; 셀 롤링

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

혈관 세포 리모델링의 조사에 생리의 체외 모델 및 응용 프로그램과 함께 병리학 혈액 흐름

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

혈소판 수혈 및 지혈을위한 미세 유체 유량 용기 모델은 실시간으로 혈소판 증착 및 섬유소 형성을 측정합니다

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

<em&gt; 체외</em쥐의 추간판의&gt; 장기 문화 모델

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

가 여러 명의 병렬 접시 흐름 챔버를 사용 하 여 스트레스에 노출 된 내 피 세포의 유전자 표정 분석

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

다중 스펙트럼 화상 진찰 교류 세포측정을 사용하여 시험관 내 미세핵 분석기를 능력을 발휘하는 자동화된 방법

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

미세 유체 흐름 챔버 지혈 및 혈소판 수혈을 모델링하기 위해 재구성 된 혈액을 사용하여

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