저자는 체외 재관류 챔버의 설계 잭 Milwid 감사하고 싶습니다. 이 작품은 KU, 미국 NSF KU 및 BEU 어린이를위한 이너스 병원 CBET - 0853569을​​ 내, R00DK080942에 미국 NIH, R01DK59766 및 R01DK084053에서 부여 (부여 안돼. 8503)에 의해 지원되었다. 우리는 또한 지원과 어린이를위한 이너스 병원 인정합니다.

1. 간 Decellularization <ol><li> 포털 정맥 사용 cannulation 및 18 게이지 카테터와 쥐의 간을 수확. 열등하고 우수한 베나 카바를 열어 둡니다. 인산염의 기관은 수산화 유지 10 cm 배양 접시에있는 호수 (PBS)가 버퍼. </li><li> 8 리터 저수지, 연동 펌프와 버블 트랩으로 구성되어 있습니다 재관류 시스템을 설정합니다. </li><li> 인산염과 재관류 시스템을 작성하는 것은 생리 버퍼링하고 10 분 동안 계속 실행. 10 cm 배양 접시에 PBS를 입력하고 인산의 흐름 속도는 1 ML / 분에 생리 버퍼 줄일 수 있습니다. </li><li> 조심스럽게 인산염에 수확 간 전송하면 생리 채워진 10 cm 배양 접시를 버퍼. </li><li> 하룻밤 PBS의 재관류를 계속합니다. </li><li> 5 분 증류수에 0.01 % (W / V) 나트륨 dodecyl 황산 (SDS)와 재관류를 시작합니다. </li><li> 1 시간 PBS로 Perfuse. </li><li> 매번 10, 15 및 20 분 SDS 재관류 시간을 증가 단계 1.6 및 1.7 이상을 세 번 반복합니다. </li><li> 24 시간 동안 0.01 % (W / V) SDS와 재관류를 계속합니다. </li><li> 24 시간 동안 0.1 % (W / V) SDS와 재관류를 계속합니다. </li><li> 3 시간 동안 0.2 % (W / V) SDS와 Perfuse. </li><li> 3 시간 동안 0.5 % (W / V) SDS와 Perfuse. </li><li> 15 분 동안 증류수로 Perfuse. </li><li> 어떤 바운드 핵산을 제거하는 데 30 분의 1 % (W / V) 증류수의 트리톤 X - 100 Perfuse. </li><li> decellularized 간 매트릭스 (DLM), 2 시간 동안 PBS로 perfuse을 씻어. </li><li> 옵션 : 중간 엽을 제외한 모든 엽 (叶)을 잘라 내다. </li><li> (그림 1)를 사용하여 준비까지 4 O C에서 PBS에 담가 깨끗하고 봉인 페트리 접시에서 DLM을 저장합니다. </li><li> DLM를 검사하려면 : 1) 멸균 PBS 4 O C.에서 0.1 % (V / V) peracetic 산성과 4 % (V / V) 3 시간 동안 에탄올과 부화를 포함하는으로 DLM을 플러쉬 2) 멸균 PBS로 2 번 씻으십시오. 3) 멸균 PBS가 2% 페니실린 - 스트렙토 마이신, 10ug / ML gentamicin, 및 2.5 UG / ML amphotericin B. 스토어 recellularization 실험에 사용할 준비까지 4 O C에서 동일한 솔루션에 decellularized 간을 포함로 씻으십시오. </li></ol> 2. Decellularized 간 매트릭스 Recellularization <ol><li> 재관류 챔버, 무균 조건 하에서 연동 펌프와 버블 트랩으로 구성되어 있습니다 재관류 시스템을 설정합니다. 문화 매체, 예를 들어, 높은 혈당 DMEM (시그마) 10 % 태아 소 혈청 (Hyclone), 100 U ML -1 페니실린, 100 μg ML -1 스트렙토 마이신 (Invitrogen) 200 ML과 재관류 시스템을 채우십시오. </li><li> 재관류 챔버에서 decellularized 간 매트릭스를 놓고 펌프 어떤 공기 거품의 형성을 피하기 5 ML / 분 실행되는 동안 포털 정맥 정맥을 통해 관류 시스템에 DLM를 연결합니다. </li><li> 30 분 DLM을 통해 매체의 재관류를 허용합니다. </li><li> 적어도 90 % 생존과 성인 쥐의 기본 hepatocytes을 분리. </li><li> 재관류 시스템의 흐름을 중지하고 천천히 거품 트랩을 통해 관류 시스템에 50,000,000 hepatocytes를 (문화 매체 1-3 ML)을 주입. </li><li> 10 ML / 분 흐름을 시작하고 10 분 동안 매체를 recirculate. </li><li> 200000000 세포의 총이 DLM (그림 2) 6에 도입되기 전까지 단계 2.5 2.6 반복합니다. </li><li> 일단 모든 세포가 DLM에 perfused 있으며, 10 분 동안 600 RPM에서 4 50 ML 원심 분리기 튜브와 원심 분리기에 perfusate를 수집합니다. supernatants를 버리고 하나의 튜브로 알약을 수집합니다. 시딩 효율을 결정하는 trypan 파랑 배제를 통해 세포 생존의 수를 결정합니다. </li></ol> 3. Recellularized 간 이식의 체외 문화 <ol><li> 재관류 챔버, 멸균 조건 (그림 3)에 따라 연동 펌프, oxygenator 및 버블 트랩으로 구성되어 있습니다 재관류 시스템을 설정합니다. 문화 매체 50 ML과 재관류 시스템을 채우기 예, 윌리엄스 &#39;E (시그마) 5 % 태아 소 혈청 (Hyclone가), 0.5 U ML -1 인슐린 (엘리 릴리), 20 NG ML -1 EGF (Invitrogen) 14 NG ML -1 글루카곤 (베드퍼드 연구소), 7.5 μg ML -1 하이드로코티손 (Pharmacia), 100 U ML -1 페니실린, 100 μg ML -1 스트렙토 마이신 (Invitrogen). </li><li> 재관류 챔버에서 recellularized 간 이식을 놓고 펌프 어떤 공기 거품의 형성을 피하기 5 ML / 분 실행되는 동안 포털 정맥 정맥을 통해 관류 시스템에 DLM를 연결합니다. </li><li> 무균 재관류 챔버를 닫고 문화 동안 누출을 방지하기 위해 단단히 밀봉합니다. </li><li> 37 O C이며 10 %의 CO가 인큐베이터에 재관류 시스템을 전송 2 15 ML / 분에 재관류 흐름 속도를 향상시킬 수 있습니다. </li><li> 95 % O 2 5% CO 2 가스 혼합물 탱크에 oxygenator를 연결하고 가스 유량 0.5 리터 / 분으로 설정합니다. 이것은 approxima의 산소 분압을 달성한다tely 400 mmHg. </li><li> 문화 문화 매체의 매일 변화에 최대 10 일 계속 사용할 수 있습니다. 문화 매체는 알부민, 요소 및 총 담즙산의 분비 등 그래프트의 간 기능의 모니터링을 위해 매일 맛볼 수 있습니다. 문화 기간의 끝에, recellularized 간 이식은 분자와 histological 분석을 위해 샘플 수 있습니다. </li></ol> 4. 대표 결과 : 쥐 간 전체 decellularization는 설명 프로토콜을 사용하는 방법에 대한 72 시간 정도 소요됩니다. 결과 매트릭스는 원섬유의의 콜라겐의 100 %, glycosaminoglycans의 50 %와 원시 간 DNA (표 1) 6 단 5 %를 유지합니다. 모체의 혈관 구조로 부식은 주조 및 전자 현미경 분석 (그림 4) 6을 검색하여 입증 보존되어 있습니다. DLM 내에서 혈관 마이크로 아키텍처의 존재는 96 % 효율성과 체외 문화에 대한 이후의 재관류와 세포와의 repopulation을 용이하게합니다. recellularized 간 이식은 체외에서 10 일까지 교양과 같은 알부민, 요소 및 총 담즙산의 분비 (그림 5) 6를 통해 확인 적절한 간 기능을 표시 수 있습니다. <img src="/files/ftp_upload/2394/2394fig1.jpg" alt="그림 1" /> 그림 1. decellularization 과정의 끝에 간 매트릭스를 Decellularized. 절제술 후 (A) 전체 간은 (B) 평균 엽. <img src="/files/ftp_upload/2394/2394fig2.jpg" alt="그림 2" /> 그림 2. DLM의 recellularization의 도식 표현. <img src="/files/ftp_upload/2394/2394fig3.jpg" alt="그림 3" /> 그림 3. recellularized 간 이식의 체외 문화에 대한 재관류 시스템 설정. <img src="/files/ftp_upload/2394/2394fig4.jpg" alt="그림 4" /> 그림 4. microvascular 구조는 decellularized 간 매트릭스에 유지됩니다. A) 정상 간 B) decellularized 간, 포털 (적색)과 정맥 (파란색) vasculature의 부식 캐스트 이미지. DLM C의 전자 현미경 이미지) 선박, D) 담즙 덕트와 같은 소형 선박 (화살표), 스케일 바 (A, B) 5mm (C, D) 20 μm의를 특징으로 섹션을 검사합니다. <img src="/files/ftp_upload/2394/2394fig5.jpg" alt="그림 5" /> 그림 5. 체외 재관류 문화에있는 동안 recellularized 간 이식의 간 특정 기능을 수행합니다. A) 알부민 분비 (P = 0.5249가), B) 우레아 생산 (P = 0.5271가)와 C) 총 담즙산의 분비 (P = 0.0114가). 실험과 통제 사이의 차이의 통계 분석은 = 0.01에서 프리드먼의 시험에 의해 10 D 문화에 걸쳐 이루어졌다. 오차 막대는 SEM (N = 3)을 나타냅니다. <table border="1"><tbody><tr><td></td><td> 신선한 livera </td><td> Decellularized 간 매트릭스 </td><td rowspan="2"> P - 값 </td><td rowspan="2"> 신선한 간 비율 (%) </td></tr><tr><td></td><td> N = 4 </td><td> N = 8 </td></tr><tr><td> 콜라겐 </td><td rowspan="2"> 0.07 ± 0.01 </td><td rowspan="2"> 0.08 ± 0.03 </td><td rowspan="2"> 0.56 </td><td rowspan="2"> 1백14퍼센트 </td></tr><tr><td> (g 간 당 MG) </td></tr><tr><td> Glycosaminoglycans </td><td rowspan="2"> 73.1 ± 6.7 </td><td rowspan="2"> 34.2 ± 2.9 </td><td rowspan="2"> 0.004 </td><td rowspan="2"> 47% </td></tr><tr><td> (g 간 당 MG) </td></tr><tr><td> DNA </td><td rowspan="2"> 14.9 ± 5.6 </td><td rowspan="2"> 0.44 ± 0.08 </td><td rowspan="2"> 3.3 10 -5 </td><td rowspan="2"> 2.9 % </td></tr><tr><td> (g 간 당 MG) </td></tr></tbody></table> 표 1. decellularized 간 매트릭스의 생화 학적 조성은 원래 간 비교. 값은 ± SEM 의미로 표시됩니다

<ol>
	<li>Kulig, K. M., Vacanti, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+tissue+engineering.">Hepatic tissue engineering.</a> Transpl Immunol. 12, 303-310 (2004).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature biotechnology. 23, 47-55 (2005).</li><li>Dahl, S. L., Koh, J., Prabhakar, V., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+native+and+engineered+arterial+scaffolds+for+transplantation.">Decellularized native and engineered arterial scaffolds for transplantation.</a> Cell Transplant. 12, 659-666 (2003).</li><li>Schechner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engraftment+of+a+vascularized+human+skin+equivalent.">Engraftment of a vascularized human skin equivalent.</a> FASEB J. 17, 2250-2256 (2003).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F., F, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3683 (2006).</li><li>Uygun, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat Med. , (2010).</li></ol>

관심 없음 충돌 선언하지 않습니다.

여기에 설명된 재관류의 decellularization 방법은 동일한 총 구조와 기본 간 혈관 마이크로 아키텍처를 가지고 전체 간을 발판을 생산하고 있습니다. 발판은 원래 간장과 비슷한 세포외 기질 구성을하고 있습니다. recellularization 방법은 높은 효율성과 세포에서 세포 검사 체외 문화 시대의 동안 가능한 및 기능 유지와 비계의 repopulation을 구현합니다. recellularized 간 이식에 nonparenchymal 세포의 추가와 함께, 우리는 recellularized 간 이식이 같은 약물 대사, 재생 및 개발 등 다양한 간 기능을 연구하기위한 플랫폼으로 사용될 수있는 구상. 그것이 잠재적으로 그라 프트 6 혈관 구조의 존재에받는 동물 감사의 혈액 순환에 연결을 통해 이식 수 있으므로 또한, recellularized 수술이, 임상 응용 프로그램을 찾을 수 있습니다.

A correction was made to <a href="https://www-jove-com.remotexs.ntu.edu.sg/Details.php?ID=2394">Decellularization and Recellularization of Whole Livers</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Nima Saeidi
instead of:
Nima Saedi.

Erratum: Decellularization and Recellularization of Whole Livers

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sodium dodecyl sulfate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L4390</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Masterflex L/S Digital Drive</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW-07523-80</td>
<td>&nbsp;</td>
<tr>
<td>Masterflex L/S Standard pump head</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW- 07013-81</td>
<td>&nbsp;</td>
<tr>
<td>Bubble trap</td>
<td>&nbsp;</td>
<td>Radnoti</td>
<td>130149</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

decellularization and recellularization of whole livers

간은 1 생존하기 위해서는 영양분과 산소와 폐기물의 제거 전달 지속적인 살포가 필요 복잡한 기관이다. 조직 공학 및 microfabrication 기술을 사용하여 접근까지 경내로 간 미세 구조를 재현하거나 모방하는 노력은 지금까지이 디자인 도전으로 인해 성공적으로되지 않았습니다. 또한, 간 조직 공학 응용 프로그램에 대한 공사장 공중 발판을 만드는 데 사용되는 합성 biomaterials 인해 적절한 세포 기능 2 유도 것이 특정 세포 바인딩 작품의 부족으로 많은 부분에서 조직 재생 및 수리를 유도 제한되었습니다. 반면에 Decellularized 기본 tissues 같은 혈관 3 피부에 4 조직 공학에 많은 응용 프로그램을 발견하고 도전 중 일부에 대한 실용적인 솔루션을 제공하고 있습니다. decellularized 기본 행렬의 장점은 따라서 세포 부착 및 개편 5 강화, 정도, 원래 구성 및 미세로 유지한다는 것입니다. 이 작품에서 우리는 방법이 주요 혈관의 구조를 유지 손상 간 bioscaffold가 얻은 것입니다 그러한 간의 재관류 - decellularization을 수행하기 위해 설명합니다. 또한, 우리는 체외에서 기능을하는 간 이식을 만드는, 성인 주요 hepatocytes 이러한 bioscaffolds을 recellularize하는 방법을 설명하고, 생체내에서 이식에 필요한 선박 액세스할 수 있습니다.

Watch this Scientific Journal Video about Decellularization and Recellularization of Whole Livers at JoVE.com

Decellularization and Recellularization of Whole Livers

재관류의 decellularization는 기관의 세포외 기질 구성과 마이크로 아키텍처를 유지 전체 간 공사장 공중 발판을 생산하는 새로운 기술입니다. 여기에, hepatocytes과 재관류의 decellularization 이후 repopulation를 사용하여 전체 오르간 공사장 공중 발판을 준비하는 방법이 설명되어 있습니다. 기능 및 transplantable 간 이식이 기술을 사용하여 생성할 수 있습니다.

전체 간은의 Decellularization 및 Recellularization

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

decellularization-and-recellularization-of-whole-livers

Nanyang Technological University

Research

JoVE Journal

Bioengineering

21.9K 조회수.  Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children. 재관류의 decellularization는 기관의 세포외 기질 구성과 마이크로 아키텍처를 유지 전체 간 공사장 공중 발판을 생산하는 새로운 기술입니다. 여기에, hepatocytes과 재관류의 decellularization 이후 repopulation를 사용하여 전체 오르간 공사장 공중 발판을 준비하는 방법이 설명되어 있습니다. 기능 및 transplantable 간 이식이 기술을 사용하여 생성할 수 있습니다.

비디오: Decellularization and Recellularization of Whole Livers

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

퇴행성 관상 재관류에 의한 돼지 심장의 Decellularization 절차

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

연구

생체공학

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

자연 무 세포 창자 매트릭스의 생산을위한 탈세 포화 방법론

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

처리되지 않은 전체 혈액에서 호중구 주 화성을 측정하기위한 미세 유체 플랫폼

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

전체 담배 연기에의 Organotypic 3D 기관지와 코 조직 문화 모델의 반복 노출의 영향 평가

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

진동 압 관류 장치에서 쥐 간을의 탈세 포화 절차

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

높은 강도의 실시간 모니터링의 초음파 (HIFU) 절제를 집중<em&gt; 체외</em&gt; 집중 초음파에 대한 조화 운동 이미징을 사용하여 개 간을 (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

시험관 연구에 대 한 3D 같은 플랫폼으로 태아와 성인 심장 조직 Explants의 비교 Decellularization

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Decellularization 식물 조직의 조직 공학 응용 프로그램에 대 한 두 가지 방법

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Decellularization 및 Recellularization 방법론을 인간의 Saphenous 정 맥에 대 한

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...