recCD47 1. 수정 고분자 표면 NOTE :. 프로토콜은도 1에 개략적으로 요약은도 1a는 티올 반응성 중합체 표면의 생성을 도시 한도 1b는 티올 반응성 recCD47의 생성을 도시한다.. <ol><li> 1 일 <ol><li> 멸균 수에 PDT-BzPh의 용액 (1 ㎎ / ㎖)과 탄산 수소 칼륨 (KHCO 3) (0.7 ㎎ / ㎖)을 준비한다. 4 ° C (빛으로부터 보호)에서 밤새 교반한다. </li></ol></li><li> 2 일 <ol><li> (회전 바퀴 주위에 맞게 충분히) 40cm 길이의 조각으로 고분자 튜브를 잘라. </li><li> 통이나 챈들러 루프 장치에 실온에서 90 분 동안 hexacylpyridinium의 0.1 % 수용액에 튜브를 만끽. 90 분 담근 후 멸균 수의 3 배와 튜브를 씻어. </li><li> (15 % 수성 칼륨 인산을 첨가하여 1 일부터 PDT-BzPh의 용액을 산성화 KH 2 </suB&gt; PO 4). 흐린 미셀 솔루션을 형성 PDT-BzPh의 ML 당 50 μL KH 2 PO 4를 추가합니다. </li><li> 진탕 또는 장치 상에 실온에서 40 분 동안 산성화 PDT-BzPh 용액 튜브를 담근다. </li><li> 한 번 : 묽은 초산 (1000 일)와 튜브를 씻어. </li><li> 60 분의 전체 지속 시간 동안 UV 조사에 튜브를 노출. 전체 면적을 조사하기 위해 15 분마다 차례 ¼ 튜브를 회전합니다. </li><li> 쉐이커 또는 장치에 실온에서 20 분 동안 20 ㎎ / ㎖의 탄산 칼륨의 수용액 (KHCO 3)에서 튜브를 담근다. </li><li> 멸균 수에 4 ° C에서 멸균의 3 배 및 저장소와 튜브를 씻어. </li></ol></li><li> 3 일 <ol><li> (: 흄 후드에서 수행주의) 200 ㎕의 디메틸 포름 아미드 (DMF)에 5 mg의 설포-SMCC을 녹인다. </li><li> recCD47 폴리 라이신 용액을 0.1 ㎎ / ㎖에 1 단계에서 제조 된 설포-SMCC 용액 50 μl를 추가하고, RO에서 60 분 동안 교반톰 온도. </li><li> 60 분 교반하는 동안, 가스 제거는 둘 베코 인산염 완충 생리 식염수 (DPBS)와 따로 멸균. </li><li> 정화 설포-SMCC는 제조업체의 지침에 따라 7K 분자량 컷 - 오프 스핀 탈염 컬럼을 사용하여 recCD47을 반응. 최종 관류 (고품질 티올 반응성 recCD47)를 모아서 코트 DPBS와 관의 내부에 필요한 볼륨으로 희석한다. </li><li> 멸균과 일이에서 튜브를 씻어 DPBS 배는 탈기. </li><li> 이하 2 분간 탈기 DPBS 20 ㎎ / ㎖와 트리스 (2 - 카르복시 에틸) 포스 핀 히드로 클로라이드 (TCEP)의 용액으로 튜브의 표면 개질 반응. 멸균 탈기 DPBS 배와 튜브를 씻어. </li><li> 튜브에 설포-SMCC-반응 recCD47를 추가하고 통 또는 장치에 4 ° C에서 하룻밤 배양한다. </li></ol></li></ol> 수정 된 표면에 recCD47의 2 면역 정량 <ol><li> 수정 된 튜브를 씻어하는 3 일 기지에서 정량시간 DPBS 배. 저장 튜브는 4 ° C에서 DPBS에 정량을 사용하지. </li><li> 96 웰 플레이트의 우물에서 생검 펀치를 중복 튜브 샘플을 만들 수 및 배치 4mm 생검 펀치를 사용합니다. </li><li> 검출 항체로 처리하지 변성 샘플 튜브로 이루어지는 음성 대조군을 준비한다. 검출 항체 처리 변성 샘플 튜브로 이루어진 항체 제어를 준비한다. 검출 항체 처리 변성 튜브 시험 샘플을 준비한다. </li><li> 진탕 기에서 실온에서 60 분 동안 DPBS에서 0.4 % 소 혈청 알부민 (BSA)으로 차단 샘플. </li><li> 차단 대기음 BSA 트리스 완충 식염수 플러스 1 % 트윈 20 (TBST) 3 배, 통에 10 분 각 씻어 후. </li><li> 면역에 대한 제조업체의 권장 희석에 따라 0.4 %의 BSA에 항체의 희석 작업을 준비합니다. 인간 CD47 항체 B6H12-FITC 일을 희석 : (100) 0.4 %의 BSA에. </li><li> 적절한 우물에 항체 희석 200 μl를 추가합니다. Incuba0.4 %의 BSA 만과 테 부정적인 제어합니다. 쉐이커에 60 분 (빛으로부터 보호)을 실온에서 품어. </li><li> TBST 배, 10 분 쉐이커 각 씻어. 마지막 TBST 린스를 대기음 및 튜브 샘플 웰에 200 μL의 DPBS를 추가합니다. </li><li> 최종 관류 고품질 티올 반응성 재조합 CD47이다 모아서 코트 DPBS와 튜브의 내부에 필요한 양으로 희석한다. </li><li> FITC 여기 (485 나노 미터) 및 배출 (538 나노 미터) 설정을 사용하여 마이크로 플레이트 리더를 사용하여 FITC 신호 강도를 읽으십시오. </li><li> 계산 recCD47 자동 형광 및 비특이적 항체는 대조 샘플에서 바인딩 차지 표준 값을 기초로 중합체 표면에 결합. </li></ol> 3 챈들러 루프 장치 프로토콜 혈액 수집 프로토콜의 임상 시험 심사위원회 (IRB) 승인 및 동의서 서류 이전에 인간의 혈액 샘플 수집을 시작하기를 추구합니다. 정보를 얻rmed 인간의 혈액 기증자의 동의. 참고 : 장치를 묘사 한 그림이 그림 2에 나타나있다. <ol><li> 약 2 직경 바퀴의 때까지 증류수로 수조를 작성하는 침수된다. 10 % 표백제 솔루션을 만들기 위해 물을 욕조에 충분히 표백제를 추가합니다. 설정 물 37 ° C에 욕조와 온도가 평형을 할 수 있습니다. </li><li> 금속 (도 1b에 도시) 어댑터, 미 변성 및 변성 튜브 튜브를 수집하고,도 1c에 도시 된 바와 같이, 장치를 바퀴 주위에 조립한다. 그 튜브가 제자리에 금속 어댑터 바퀴 snuggly 주위에 적합해야합니다. </li><li> 구연산이나 다른 항응고제의 2 ㎖로 채워져 유리 병에, IRB 승인 프로토콜을 사용하여 30 ml의 혈액 샘플을 확보하고, 시료가 수집 된 후 응고 방지하기 위해 반전으로 섞는다. </li><li> 튜브에 약간의 공기를 떠나, 금속 밸브와 주사기를 사용하여 각 튜브에 혈액의 약 10 ML을 추가합니다. 밸브 캡과 연구를 고정3 시간 동안 otate. </li><li> 10 % 표백제 용액으로 처리 폐기물 비커에 혈액을 배출 (또는 기관 정책 필요). </li><li> 부드럽게 혈액의 모든 흔적을 제거하는 DPBS와 튜브 내부를 씻어. 흐름을 통해 폐기물 비커를 수집합니다. 기관의 정책에 따라 혈액 폐기하십시오. </li><li> 장치 및 표면 10 % 표백제 용액을 사용하거나 기관 방침에 따라 접촉하는 혈액 소독. </li><li> 형광 현미경 또는 주사 전자 현미경을위한 프로세스 샘플 후술. </li></ol> 4 형광 현미경과 세포 계수 <ol><li> 4 % 파라 포름 알데히드 (PFA) 용액 (주의 : 흄 후드에서 수행)을 준비. </li><li> 4 ° C에서 하룻밤 4 % PFA 용액에 튜브를 품어. </li><li> 하룻밤 배양 후, 4 % PFA를 제거하고 DPBS 매우 부드럽게 영화를 씻어. </li><li> 염색에 대한 루멘 표면을 노출하는 섹션으로 튜브를 잘라. </li><li> 얼룩실온에서 30 분 동안 4 &#39;, 6-diamidino-2-페닐 인돌 (DAPI)로 미디어를 장착 몇 방울의 튜브 섹션 (광으로부터 보호). 염색 후, DAPI 배경 신호를 감소시키기 DPBS로 매우 조심스럽게 튜브를 헹군다. </li><li> DAPI 필터 및 디지털 카메라가 장착 된 형광 현미경을 사용하는 이미지 200X 배율 하에서 뷰 9 맹목적 선택된 분야에서 세포의 수를 계산한다. </li><li> 기록 셀은 시야 당 계산 및 결과의 통계적 유의성을 결정하기 위해 적절한 통계 분석을 수행합니다. </li></ol> (5) 주사 전자 현미경 <ol><li> 2 % 글루 타르 알데히드 용액 (주의 : 흄 후드에서 수행) 준비합니다. </li><li> 밤새 4 ° C에서 2 % 글루 타르 알데히드 용액 튜브를 품어. </li><li> 하룻밤 배양 후, 부드럽게 DPBS와 함께 호스의 3 배를 씻어. </li><li> 산화 오스뮴의 1 % 용액을 준비합니다 (주의 : 심각한 흡입 위험! ) 흄 후드에서 사용합니다. </li><li> 실온에서 15 분 동안 산화 오스뮴의 1 % 용액에서 배양 튜브. </li><li> 부드럽게 DPBS와 함께 호스의 3 배를 씻어. </li><li> 에탄올 농도의 시리즈 (25 %, 50 %, 75 %, 95 %, 100 % 에탄올)을 준비한다. </li><li> 에탄올 농도의 일련의 인큐베이션 튜브 탈수. 25 %, 50 %, 75 %, 20 분마다 95 % 에탄올에서 배양한다. 30 분 동안 100 % 에탄올에서 배양한다. </li><li> 초 임계 점은 모든 수분을 제거하고 45 분 동안 CO 2와 함께 호스를 건조. </li><li> 페이스트 또는 흑연 표본 스텁에 마운트 튜브 섹션. </li><li> 금 - 팔라듐의 12.5 nm의 코트 튜브 섹션. </li><li> 주사 전자 현미경으로 조사한다. </li></ol>

<ol>
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F., de Hart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+modified+chandler,+roller+pump,+and+ball+valve+circulation+models+for+in+vitro+testing+in+high+blood+flow+conditions:+application+in+thrombogenicity+testing+of+different+materials+for+vascular+applications.">Comparison of modified chandler, roller pump, and ball valve circulation models for in vitro testing in high blood flow conditions: application in thrombogenicity testing of different materials for vascular applications.</a> Int J Biomater. 2012, (2012).</li></ol>

이 책에서보고 된 연구는 국가의 수상 번호 T32 HL007915 (JBS와 RJL)에서 수상 번호 R21 EB015612 (SJS) 및 국립 심장, 폐, 혈액 연구소에서 바이오 메디컬 이미징 및 생물의 국립 연구소에 의해 지원되었다 건강 연구소.

(그림 1에 요약) photoactivation의 화학에 대한 PDT-BzPh 첨부 및 후속 UV 조사 용이하게하기 위해 충분한 탄화수소가 거의 모든 고분자 표면의 수정을 허용 PDT-BzPh을 사진을 활성화. 반응성 티올 그룹과 고분자 표면을 기능화하는 것은 관심의 검증 가능한 분자의 범위의 후속 첨부 할 수 있습니다. 우리의 특정 연구에서 우리는 재조합 CD47 11 ~ 13을 선택했다. 우리는 이관 능성 가교 링...

그러한 체외 순환 및 신장 투석과 같은 많은 임상 절차, 중합체 혈액 도관의 사용을 필요로하고 종종 시술 후 합병증 1과 연관된다. 혈액 관류 할 때,이 폴리머는 혈액 단백질 및 혈소판, 단핵구 / 대식 세포 접착의 흡착 결과, 이물 반응 (FBR)를 이끌어 낼, 그리고 할 시술 후 합병증에 기여 모두 염증성 사이토 카인의 방출 / 또는 장치 고장 2,3. 따라서, 이러한 문제를 해결하기위한 전략은 생체 재료 연구의 중요한 영역 및 지속적인 유지. 연구자들은 생리 활성 또는 생체 불활성 분자 4-6 혈액 접촉면을 수정하여이 문제를 해결하려고했습니다. 우리의 실험실에서 연구는 FBR을 완화하고 이들 물질의 효능을 증가시키기위한 전략으로 고분자 생체 재료에 재조합 CD47 (recCD47를) 추가에 초점을 맞추고있다. CD47는 재하 표현 transmembr입니다세포 7-10와 쇼를 표현하는시의 면역 회피의 알려진 역할 부여 &quot;자기&quot;상태 메탄 단백질은 고분자 표면에 11 ~ 13에 추가 할 때 생체 적합성을 부여에 약속드립니다. 신호 규제 단백질 알파 (SIRPα), CD47에 대한 동족 수용체 및 횡단 단백질의 가족을 함유 면역 수용체 티로신 기반의 억제 모티프 (ITIM)의 회원은 골수 기원 (14)의 세포에 표현된다. 우리는 이전에 CD47가 SIRPα 매개 세포 신호를 통해, 생체 외, 생체 ​​폴리 우레탄 (PU)와 폴리 염화 비닐 (PVC)에 대한 면역 반응을 하향 조정하고 생체 내 모델 11-13에 있음을 증명하고있다. 우리의 조사에 중앙 화학적으로 반응성 티올 그룹이 공유 다기능 고분자 (PDT-의 BZ와 튜브를 반응시켜 고분자 튜브에 추가되는 여기에 설명 된 비교적 새로운 photoactivati​​on 화학은있다2 - 피리 딜 디티 오 (PDT)로 이루어지는) 액, 광 반응성 페논 (BzPh) 및 카르복시 변성 폴리 알릴 11-13. 트리스 함께 공유 첨부 PDT기를 환원 (2 - 카르복시 에틸) 포스 핀 히드로 클로라이드 (TCEP)는 11 일 이후 일 수 티올 표면은 치료 잔기와 반응하여 산출한다. 본원 이전 상세한 12,13, 상기 C-말단 폴리 - 리신 12,13 꼬리의 첨가로 개질 recCD47는, Sulfosuccinimidyl - 4 - [N -maleimidomethyl] 시클로 헥산 카르 복실 레이트 1과 반응 (술포-SMCC) 1 시간 동안 튜브 (11) 사이 및 recCD47 우람 모노 설파이드 결합 형성을 허용 티올 반응성기를 생성한다. CD47의 표면 기능화 항염증제 용량은 원래 응고 혈전 (15)의 시험 관내 모델로서 1958 년에 설명 된 전체 인간 혈액과 챈들러 루프 장치를 이용하여, 전 비브 O, 시험 하였다. 장치에 의존밀폐 된 튜브 시스템은 부분적으로 공기 튜브 (15)를 통해 혈액을 순환하는 회전 모터 가득합니다. 이 실험 모델 수정 및 표면 개질시 혈액 노출의 효과뿐만 아니라, 혈액 세포의 생리학에 따라 그 표면 변형의 영향을 조사 할 수있는 기회를 제공한다. recCD47이 photoactivation 화학을 사용하여 중합체의 다양한 표면에 추가 될 수 있고, 그것의 항 염증 능력은 중합체 표면 (11, 12)를 통해 혈류를 흉내 임상 적으로 중요한 생체 모델을 이용하여 평가 될 수있다. 장치에서 인간의 혈액에 노출되었을 때 변성 중합체에 비해 recCD47 변성 임상 등급의 혈액 도관은 상당히 적은 혈소판과 염증 세포 부착을 보여준다. 본 변형 과정의 단계별 설명은 아래에 자세히 설명된다.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:107px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">16% Paraformaldehyde (PFA)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">58906</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25% Glutaraldehyde</td>
			<td style="width:175px;">VWR</td>
			<td style="width:107px;">AAA17876-AP&#160;</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">2-pyridyldithio,benzophenone (PDT-BzPH)</td>
			<td style="width:175px;">Synthesized in lab</td>
			<td style="width:107px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">A3059-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Citrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">S5770-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Digital Camera</td>
			<td style="width:175px;">Leica</td>
			<td style="width:107px;">DC500</td>
			<td>Out of production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dimethylformamide (DMF)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">270547-100ML</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:175px;">Gibco/Life Technologies</td>
			<td style="width:107px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fluorescent Microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:107px;">TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glacial Acetic Acid</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">A38-212</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Human CD47 (B6H12) &#8211; FITC Antibody</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:107px;">SC-12730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Osmium Tetroxide</td>
			<td style="width:175px;">Acros Organics</td>
			<td style="width:107px;">197450050</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Bicarbonate (KHCO3)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">237205-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PVC Tubing (Cardiovascular Procedure Kit)</td>
			<td style="width:175px;">Terumo Cardiovascular Systems</td>
			<td style="width:107px;">60050</td>
			<td>Most clinical-grade tubing will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>JSM-T330A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Sodium Chloride (NaCl)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:269px;">Microplate Reader</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:107px;">Spectramax Gemini EM&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sulfo-SMCC</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">M6035-10MG</td>
			<td>Moisture Sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">tris (2-carboxyethyl) phosphine (TCEP-HCl)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">20491</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tris Base</td>
			<td style="width:175px;">Sigma</td>
			<td>T1503-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tween-20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Vectashield with DAPI</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">H-1200</td>
			<td>Light sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Zeba Spin Desalt Columns &#8211; 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">89891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

합성 체 표면에 도입 될 때 이물 반응이 일어난다. 이 시술 후 합병증으로 이어지는, 혈액 단백질의 흡착 및 혈소판의 후속 부착 및 활성화, 단핵구 / 대 식세포 접착 및 염증 세포의 신호 이벤트에 의해 특징입니다. 챈들러 루프 장치 연구자들은 혈액의 많은 양이 고분자 도관을 통해 관류 때 발생하는 분자 및 세포의 상호 작용을 연구 할 수있는 실험 시스템입니다. 이를 위해, 본 장치는 다양한 중합체 표면 변형 항염의 평가를 허용하는 생체 외 모델로 사용되고있다. 우리 연구실은 공유 재조합 CD47와 photoactivati​​on 화학을 통해 수정 된 혈액 도관, 고분자 표면에 생체 적합성을 부여 할 수 있습니다 것으로 나타났습니다. 고분자 표면에 CD47를 추가하는 것은 중합체 혈액 도관 효능을 촉진 할 수있는 효과적인 수단이 될 수있다. 그녀의EIN은 CD47 변성 및 제어 도관과 혈액의 상호 작용을 조사하는 임상 적으로 중요한 중합체 혈액 도관 및 생체 외 실험 모델로서 챈들러 루프의 사용 재조합 CD47을 추가하는 데 사용 photoactivation 화학 디테일 방법론이다.

SMCC를 사용하여 티올 - 반응성 recCD47 폴리 리신과 함께 PDT-BzPh 및 TCEP를 사용하여 티올 - 반응성 중합체 표면을 생성하는 것은 중합체 recCD47 표면에의 부착을 허용한다. 개질 방법은도 1에 개략적으로 요약된다. 본 변형 공정의 편의성이 단백질을 가정 할 아민 - 함유 리신과 같은 충분한 화학적 반응성기로 변성 될 수 있으며, 다양한 단백질 및 다양한 중합체 표면에인가 될 수 있다는 ?...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

사용<em&gt; 전의 VIVO</em&gt; 챈들러 루프 장치 수정 된 고분자 혈액 도관의 생체 적합성을 평가하기 위해

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

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Universidad de Cartagena UDEC

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Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

11.5K 조회수.  The Children's Hospital of Philadelphia. Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. 

비디오: The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypic 콜라겐 I 분석 : 3 차원 컨텍스트에서 셀 행동을 평가하기 위해 가단 플랫폼

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

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생체공학

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

태반의 맞은 편에 생체이 물과 나노 물질의 전송 속도의 결정을 사용하여<em&gt; 생체</em&gt; 인간 태반 관류 모델

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

방법 혈액 - 뇌 장벽의 인간 세포 기반의 연락 모델의 준비를 위해 개선

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

유 전적으로 변형 된 세포 이식을위한 미세 패턴 배양 플레이트에 회전 타원체 세포를 향해 유전자 형질

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

높은 처리량의 사용<em&gt; 체외</em&gt; 미세 유체 시스템 구강 다중 종의 바이오 필름을 개발하는

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

내 시경 Submucosal 주입 재료 성능 평가 대 한 새로운 Ex Vivo 모델

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

교란 성 포유류 심장의 경부 흡광도 분광학을 사용하여 세포 대사를 모니터링하기위한 심장 내 측 발사 라이트 카테터

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

CD47 유래 펩타이드 고정화를 통해 금속 임플란트에 대한 혈액 매개 세포 부착 완화

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

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