저자는 우수한 기술 지원을 위한 리사 피 칠을 감사합니다.

<ol>
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저자는 공개 없다.

돼지 동물 모델에서 biobank 샘플 컬렉션의 대표, 중복 조직 샘플의 광범위 한 범위에 적합의 재현할 수 세대의 기관/조직, 결정에 대 한 강력한 기술 및 프로토콜 필요 다른 분석 방법, 그리고 랜덤 양적 stereological 분석에 대 한 샘플 섹션의 방향에 대 한. 현재 문서에서 설명 하는 방법 돼지 장기와 조직의 크기에 적응 하 고 효과적으로 이러한 요구2,7을 ?...

변환 의학, 돼지는 점점 사용을 위해 일반적으로 큰 동물 모델1,2,3,,45는 돼지 사이의 여러 유리한 유사성 때문과 인간 해부학 및 생리학, 및 설립된 분자 생물학적 방법의 세대에 대 한 허용 여부 맞춤, 유전자 변형 돼지 질병 조건1,4의 넓은 범위에 대 한 모델.
그러나, 설치류 모델에 비해, 동물 실험에 대 한 언제 든 지 제공 될 수 있다 각각 돼지 모델의 수가 제한 됩니다. 이것은 약 1 년, 그리고 돼지 모델 및 축산의 생성에 필요한 금융 및 시간 집중적인 노력 돼지 세대 간격. 따라서,이 돼지에서 생성 될 수 있는 샘플으로 서 돼지 모델의 개별 동물 들은 매우 귀중 한 유전자 변형 돼지 모델 및 장기 실험 문제 (예를 들면, 늦은 합병증의 경우에 특히 만성 질환) 세 개인2,,67에 검사 합니다.
모든 연구 과정에서 연구의 초기 실험 설계에 예정 하지 했다 추가 분석의 성능 수 나중으로 판명 관련, 예를 들어, 이전에서 발생 하는 별개 질문 발견 주소 예기치 않은 결과. 이러한 추가 실험에 대 한 적합 한 샘플을 사용할 수 없는 경우 disproportionally 높은 비용과 시간이 많이 소요 비용 추가 돼지와 조직 샘플을 생성 하는 데 필요한 수 있습니다. 그런 우발적 사건을 위해 준비 되, 다른 기관, 조직, 또는 바이오-액체, 양적 및 질적으로 후속 분석의 광범위 한 적합의 보존 백업 샘플 biobank 컬렉션의 중요 한 여겨진다 접근2,,67. 돼지 동물 모델에서 최적의 혜택을 파생, 적절 한 biobank 샘플의 가용성 또한 가능성을 제공 합니다 독특한 멀티 기관 수준에 동일한 샘플 자료에 광범위 한 다양 한 분석 방법 수행 하는 매우 동일한 개별 동물, 예를 들어, 다른 작업 그룹의 과학자 샘플의 유통 조직 연구 네트워크2,,67. 또한, biobanking에 '앞 ' 샘플링 전략 연구에 필요한 동물의 수의 감소에도 기여 한다. 돼지 모델 biobanking의 장점은 최근 다 기관, multiomics 연구, 검사 기관에서에서 입증 되었습니다에서 뮌헨 MIDY 돼지 Biobank 표본을 사용 하 여 장기 당뇨병의 유전자 변형된 돼지 모델에서 크로스 토크 2.
일반적으로 안정성과 이후에 수행된 분석의 결과의 판독을 준수 해야 합니다 biobank 샘플 몇 가지 필수 요구 사항이 있습니다. 샘플 reproducibly, 생성 해야 합니다 그리고 그들은 대표 해야 합니다, 즉, 적절 하 게 반영 하는 샘플7에서 찍은 조직/기관의 관심이 형태학 및 분자 기능. 다운스트림 분석 종류의 넓은 범위에 적합 될, 샘플 되어야 충분 한 수량에 촬영 하며 설명 포함 하 여 다른 분석 방법의 요구 사항 (시간 및 온도 조건 등)에 따라 처리 histopathological 분석, cryohistology, 파라핀 및 플라스틱 조직학, immunohistochemistry, 제자리에서 교 잡, ultrastructural 전자 현미경 분석, 임상 실험실 뿐만 아니라 분자 진단 분석 등 DNA, RNA, 단백질, 및 대사 산물의 분석.
수 있도록 다양 한 숫자, 볼륨, 길이, 또는 독특한 조직 구조의 표면 영역 같은 뚜렷한 양적 형태학 매개 변수 평가 대 한 양적 stereological 분석에 의해, 무작위로 섹션의 비행기는 각각 장기/조직의 조직학 샘플7,,89,,1011준비를 해야 합니다. 양적 형태학 연구, 조직, 기관, 또는 기관 구획의 총 볼륨의 정확한 결심에에서 샘플 (즉, 참조 공간)에서 찍은 결정적으로 중요 한7,9 , 12 각 기관, 조직, 또는 유기 체 내에서 관심이 매개 변수의 절대 수량 계산 하. 결국, 조직학 단면도의 준비 하는 동안 포함-관련 조직 수축 효과 결정 하 여 계정13에 있다. 따라서, 이전 연구에서 보관 된 샘플 (고정 조직 샘플, 임베디드 조직 블록, 조직학 단면도, 등등)의 특히 양적 stereological 분석은 때때로 심각 하 게 제한 하거나 심지어 불가능 한12, 특히 각 기관/조직의 volumetry 수행 되지 않았습니다, 아니 적절 한 샘플링 디자인 영장 대표 샘플을 숫자와 양의 사용 가능한 개별 샘플 충분 하지 않은 경우에 적용 하는 경우 또는 경우의 처리는 샘플의 양적 형태학 매개 변수 추정과 호환 되지 않습니다. 매니폴드 가능한 영향을 미치는 요인으로 인해 뚜렷한 양적 형태학 매개 변수 분석에 대 한 아카이브 샘플 자료의 적합성 명백 하 게 답을 수 없는, 그러나 각 개인적인 케이스의 신중한 평가에 따라 달라 집니다.
따라서, 위치, 크기, 숫자, 처리, 트리밍, 방향과 샘플의 후속 분석의 결과 영향을 줄 것 이다, 이러한 요인 매우 중요 하 고 어떤 연구의 실험 설계에 고려 되어야 한다. 이러한 측면 및 돼지 해부학, 돼지에 적응된 동물 모델 설립 되었습니다 최근, 포괄적인, 상세한, 대규모 샘플링 지침의 특수 기능 표준화, 재현성에 대 한 강력한 참조를 제공 하 그리고 50 개 이상의 다른 돼지 장기와 조직6,7에서 중복, 적절 하 게 처리, 높은-품질의 효율적인 생성.
방법론 설명 및 현재 문서에 표시 된 비디오 튜토리얼 제공, 설명, 이해할 수 있는, 단계-의해 단계를 지시 volumetry를 위한 기술의 다양 한의 실제 성능에 대 한 돼지 직물의 샘플링 및 장기, 고 다른 다운스트림 분석 방법에 대 한 조직 샘플의 처리. 관련 조직의 3 차원 수축 량의 크기의 결정을 포함 하 여 추천된 기술 기관/조직 볼륨 및 Archimedes 및 Cavalieri9의 원칙에 따라 밀도의 결정에 대 한 메서드를 포함 합니다 조직학 검사에 대 한 처리 하는 동안 다른 포함 미디어14 에 포함, 실용적인 볼륨 가중치 체계적인 무작위 샘플링의 응용 프로그램, 샘플된 조직 표본에 대 한 처리 접근 다른 후속 분석7,8,,915및 적절 하 게 지향 및 잠재적인 양적 stereological 분석7,8, 샘플 처리 9,,1011. 돼지 biobank 프로젝트에 응용 프로그램, 옆 시연된 메서드는 일반적으로 장기/조직의 양적 조직 형태학 속성을 검사 하는 모든 연구에 대 한 적절 한. 또한, 체계적인 무작위 샘플링 디자인은 분자 분석 방법을 사용 하 여 풍부한 변경의, 예를 들면, RNAs, 단백질, 또는에서 대사 산물을 검출 하는 실험에서 대표 샘플의 세대에 대 한 특히 유용 각종 기관 및 조직입니다.
다음 단락에서는 그들의 실질적인 성능을 프로토콜 섹션에서 설명 하는 동안 이러한 방법에 대 한 간략 한 소개를 제공 합니다.
기관/조직 볼륨의 결정 장기 무게와 볼륨의 결정은 여러 실험 설정에서 중요 한으로 이러한 요소를 나타낼 수 있습니다 변경, 잠재적으로 관련 된 실험적으로 시험 관심의 요인. 기관/조직의 총 볼륨 또한 일반적으로 절대 양적 매개 변수 (예:, 전체 휴대폰 번호), stereologically 예상된 숫자 볼륨 밀도 (즉, 볼륨 단위 당 세포의 수에서 계산 하는 데 필요한 조직)의7,12. 컴퓨터 단층 촬영, 같은 복잡 한 기술적인 장비를 사용 하 여 기술을 제외 하 고는 기본적으로 일반적으로 기관 또는 조직의 절대 볼륨을 결정 하는 데 사용 하는 세 가지 실용적인 방법. 장기의 볼륨 "직접 체적 측정" Archimedes, 즉의 원칙에 따라, 물 또는 염 분 완전히 빠져들 때 구조에 의해 난민의 볼륨을 측정 하 여 확인할 수 있습니다. 그러나, comparably 대형 돼지 장기에 대 한 이러한 접근은 허무 하 고 경향이 부정확성, 이후 그들은 매우 큰 체적/측정 플라스 크를 필요로. 더 편리 하 게,의 기관/조직 볼륨의 무게와 밀도7,,1216, 효율적으로 "침수 방법"7,12 사용 하 여 결정 될 수 있는 산출 될 수 있다 ,16 (프로토콜 단계 1.1.). 기관/조직 볼륨 (1598-1647) "Cavalieri의 원칙"에 따라 volumetry 접근을 사용 하 여 추정 또한 수 있습니다. 간단한 기간에서는, Cavalieri 원칙 상태, 두 개체는 접지 평면에 평행한 평면에 구분 하 고 섹션의 프로필에 해당에 두 개체를 극복 하는 경우 지 면에서 거리는 동일한 지역, 두 개체 동일한 볼륨이 있다. 따라서, 임의로 모양의 개체의 볼륨 병렬, 동등 하 게 먼 섹션 평면 및 단면 평면 사이의 거리에 그들의 섹션 프로필 분야의 제품으로 추정 될 수 있습니다. 이것은 다음과 같은 비유를 이해할 수: 동일한 동전 같은 수로 구성 된 두 개의 스택을 병행 하 여, 서로 양보는 동전, 스택과 다른 원통 모양 모양 스택 동전 질서와 하나의 스택 배치 됩니다 고려 오프 센터 코인의 스택 위치 동전 (그림 3A). 두 동전 더미의 형태는 다른, 그들의 볼륨 동일 합니다, 이후 두 스택의 해당 수준에서 동전의 영역 (즉, 병렬 섹션의 프로필 영역에서 동등한 거리에서 두 동전 스택을 통해 잘라는 접지) 같습니다. 돼지 장기 및 조직 Cavalieri 원리7,,1215 를 사용 하 여 볼륨의 추정 단계 1.2에서에서 설명 되어 있습니다.
조직학 포함 관련 조직 수축의 범위 결정 조직학 직물 단면도에서 측정 된 여러 양적 형태학 매개 변수 분석, 포함-관련 조직 수축 조직 조직학에 대 한 처리 중에 발생의 효과 결정 하 고 고려 한다. 포함-관련 조직 수축 범위 변수를 있을 수 있습니다 그리고 모두 처리, 조직과 포함 중간8,,1317,,1819에 따라 달라 집니다. 포함-관련 변경 사항 (즉, 주로 수축) 조직 샘플의 볼륨의 공간, 및, 따라서, 양적 stereological 분석8에 의해 모든 차원 매개 변수 추정의 모든 3 개 차원에서 발생 하는 일반적으로, . 기본적으로, 단계 1.3에서에서와 같이 포함-관련 조직 수축, 선형 조직 수축 계수 (fS)로 표현 된 범위 추정 수 있습니다. 그리고 (수축 구분) 양적 형태학 매개 변수14의 수정에 대 한 사용.
기관/조직의 체계적인 무작위 샘플링 볼륨 가중치 돼지 장기/조직 샘플의 biobank 컬렉션의 설립에 대 한 볼륨 가중치 체계적인 무작위 샘플링 방법과 같은 2 단계에서 설명한 입증 실용적, 시간-절약 및 효율적인 기술 대표의 생성에 대 한 다목적 조직7,8,,915샘플링합니다.
Stereological 정량 분석에 대 한 임의의 균일 한 등방성 섹션 및 임의의 유니폼 수직 섹션의 세대 Biobank 조직 샘플 매개 변수를 적절 하 게 준비 된 표본 없이 확인할 수 없습니다 그의 최대의 견적에 대 한 다른 양적 stereological 분석 방법의 넓은 범위에 적합 해야 합니다. 거의 모든 양적 stereological 매개 변수, "등방성 (독립) 획 일 한 무작위 (IUR) 섹션"8,9를 사용 하 여 결정 수 있습니다. IUR 섹션에서 조직 샘플의 섹션 평면 3 차원 방향 무작위입니다. "Isector" 방법11 (3.1 단계 프로토콜), 또는 단면 평면을 기준의 방향의 랜덤 적용이 섹션 비행기의 위치를 기준으로 조직 샘플의 위치는 랜덤에 의해 달성 될 수 있다는 "Orientator" 방법10 (프로토콜 단계 3.2)에서 조직 샘플 피부 또는 점 막 견본 표시는 또는 정의 자연스럽 게 존재 하 고 제대로 식별 수직 축, "수직 획 일 한 무작위 (VUR) 섹션"의 준비 등 조직 샘플에서 (프로토콜 단계 3.3.)의 평면 내에서 엄격 하 게 구분 그들의 수직 축은 유리8,20입니다. IUR/VUR 샘플링의 이론적 기초 및 잠재적인 다운스트림 양적 stereological 분석에 대 한 포괄적인 논의의 완전 한 담 론에 대 한 관심이 있는 독자 양적 stereology 인생에서의 교과서 라고 과학8,9.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Carl Roth GmbH, Germany</td>
			<td>Agar (powder), Cat.: 5210.3</td>
			<td>Dissolve approximately 1 g of agar in 10 ml cold water in a glass or plastic beaker, heat in microwave-oven at 700 W, boil the solution twice with rigorous stirring. Cast into mold while still warm and let solidify. Caution: While handling with hot liquid agar, wear protective goggles and gloves.</td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Hornbach Baumarkt GmbH, Bornheim, Germany</td>
			<td>Schieblehre Chrom/Vernickelt 120 mm Cat.: 3664902</td>
			<td>Any kind of caliper (mechanical or electronic) will do as well.</td>
		</tr>
		<tr>
			<td>Casting molds (metal)</td>
			<td>Engelbrecht Medizin &#38; Labortechnik, Ederm&#252;nde, Germany</td>
			<td>Einbettsch&#228;lchen aus Edelstahl, 14 x 24 x 5 mm, Cat.: 14302b</td>
			<td>Any other kind of metal casting mold used for paraffin-embedding will do as well.</td>
		</tr>
		<tr>
			<td>Copy templates of cross grids (5mm - 6 cm)</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Copy templates of cross grids (5mm - 6 cm) are provided in the supplemental data file of Albl et al.&#160; Toxicol Pathol. 44, 414-420, doi: 10.1177/0192623316631023 (2016)</td>
		</tr>
		<tr>
			<td>Copy templates of equiangular and cosine-weighted circles</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Copy templates of equiangular and cosine-weighted circles are provided in Nyengaard &#38; Gundersen. Eur Respir Rev. 15, 107-114, doi: 10.1183/09059180.00010101 (2006) and in Gundersen et al. Stereological Principles and Sampling Procedures for Toxicologic Pathologists. In: Haschek and Rousseaux&#180;s Handbook of Toxicologic Pathology. 3rd ed, 215-286, ISBN: 9780124157590 (2013).</td>
		</tr>
		<tr>
			<td>Foldback clamps (YIHAI binder clips, 15 mm and 19 mm)</td>
			<td>Ningbo Tianhong Stationery Co ltd., China</td>
			<td>Y10006 and Y10005</td>
			<td>Any other type of standard office foldback clamps will do as well.</td>
		</tr>
		<tr>
			<td>Forceps (anatomical)</td>
			<td>NeoLab Migge GmbH, Heidelberg, Germany</td>
			<td>neoLab Standard -Pinzette 130 mm, anatomisch, rund, Cat.: 1-1811</td>
			<td>Any type of anatomical forceps will do.</td>
		</tr>
		<tr>
			<td>Formaldehyde-solution 4%</td>
			<td>SAV-Liquid Produktion GmbH, Flintsbach, Germany</td>
			<td>Formaldehyd 37/40 %, Cat.: 1000411525005</td>
			<td>Dilute to 4% from concentrated solution. Buffer to neutral pH. Wear appropriate eye-, hand- and respiratory protection. Process tissue samples fixed in formaldehyde solution under an exhaust hood and wear protective goggles and laboratory gloves.</td>
		</tr>
		<tr>
			<td>Graph paper (for calibration)</td>
			<td>B&#252;romarkt B&#246;ttcher AG, Jena, Germany. www.bueromarkt-ag.de</td>
			<td>Penig Millimeterpapier A4, Cat.: 2514</td>
			<td>Any type of graph paper (scaled in millimeter) will do.</td>
		</tr>
		<tr>
			<td>Laboratory beakers (5ml, 10 ml, 50 ml, 100 ml)</td>
			<td>NeoLab Migge GmbH, Heidelberg, Germany</td>
			<td>Becherglas SIMAX&#174; , niedrige Form, Borosilikatglas 3.3 Cat.: E-1031, E-1032, E-1035, E-1036</td>
			<td>Any kind of glass- or plastic beakers of 5 &#8211; 100 ml volume will do.</td>
		</tr>
		<tr>
			<td>Laboratory scale(s)</td>
			<td>Mettler Toledo GmbH, Gie&#223;en, Germany</td>
			<td>PM6000</td>
			<td rowspan="2">Any standard laboratory scales with measuring ranges between 0.1 mg to approximately 20 g, respectively between 100 mg to approximately 500 g will do</td>
		</tr>
		<tr>
			<td></td>
			<td>Sartorius AG, G&#246;ttingen, Germany</td>
			<td>BP61S</td>
		</tr>
		<tr>
			<td>Microtome blades</td>
			<td>Engelbrecht Medizin &#38; Labortechnik, Ederm&#252;nde, Germany</td>
			<td>FEATHER Microtome blasdes S35, Cat.:14700</td>
			<td>Any kind of single-use microtome blades will do.</td>
		</tr>
		<tr>
			<td rowspan="2">Morphometry/planimetry software/system</td>
			<td>National Institute of Health (NIH)</td>
			<td>ImageJ</td>
			<td>Download from https://www. imagej.nih.gov/ij/ (1997).</td>
		</tr>
		<tr>
			<td>Zeiss-Kontron, Eching, Germany</td>
			<td>VideoplanTM image analysis system</td>
			<td>Out of stock</td>
		</tr>
		<tr>
			<td>Photo camera</td>
			<td>Nikon</td>
			<td>D40</td>
			<td>Any kind of digital photocamera that can be mounted to a tripod&#160; will do.</td>
		</tr>
		<tr>
			<td>Plastic transparencies</td>
			<td>Avery Zweckform GmbH, Oberlaindern, Germany</td>
			<td>Laser Overhead-Folie DINA4 Cat.:&#160; 3562</td>
			<td>Any (laser)-printable plastic transparency will do.</td>
		</tr>
		<tr>
			<td>Random number tables</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Random number tables can conveniently be generated (with defined numbers of random numbers and within defined intervals), using random number generators, such as: https://www.random.org/</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Plano GmbH, Wetzlar, Germany</td>
			<td>T5016</td>
			<td>Any kind of razor blades will do.</td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>B&#252;romarkt B&#246;ttcher AG, Jena, Germany. www.bueromarkt-ag.de</td>
			<td>Office-Point Lineal 30 cm, Kunststoff, transparent, Cat.: ln30</td>
			<td>Any kind of cm-mm-scaled ruler will do as well.</td>
		</tr>
		<tr>
			<td>Saline (0.9%)</td>
			<td>Carl Roth GmbH, Germany</td>
			<td>Natriumchlorid, &#62;99% Cat.: 0601.1</td>
			<td>To prepare 0.9% saline, dissolve 9 g NaCl in 1000 ml of distilled water at 20&#176;C.</td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Aesculap AG &#38; Co KG, Tuttlingen, Germany</td>
			<td>BRAUN Surgical blades N&#176;22</td>
			<td>Any kind of scalpel blades will do.</td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>Hewlett-Packard</td>
			<td>hp scanjet 7400c</td>
			<td>Any type of standard office scanner capable of scanning with resolutions from 150-600 dpi will do.</td>
		</tr>
		<tr>
			<td>Slicing devices</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>Examples forself constructed slicing devices can be found in Knust, et al. Anatomical record. 292, 113-122, doi: 10.1002/ar.20747 (2009) and in the supplemental data file of Albl et al.&#160; Toxicol Pathol. 44, 414-420, doi: 10.1177/0192623316631023 (2016).</td>
		</tr>
		<tr>
			<td>Spherical casting molds (e.g., in 25.5 mm diameter)</td>
			<td>Pralinen-Zutaten.de, Windach, Germany</td>
			<td>Pralinen-Hohlkugeln Vollmilch, 25.5 mm</td>
			<td>Spherical casting molds can as well be be self-constructed, or obtained from other confectioner suppliers (for for pralines). The casting molds indicated here are actually the package/wrapping of hollow pralines bodies (first eat the pralines and then use the package for generation of i-sector sections)</td>
		</tr>
		<tr>
			<td>Thin wire</td>
			<td>Basteln &#38; Hobby Schobes, Stra&#223;furth, Germany. www,bastel-welt.de</td>
			<td>Messingdraht (0.3 mm) Cat.: 216464742</td>
			<td>Any other kind of thin wire will also do.</td>
		</tr>
		<tr>
			<td>Tissue paper</td>
			<td>NeoLab Migge GmbH, Heidelberg, Germany</td>
			<td>Declcate Task Wipes-White, Cat.: 1-5305</td>
			<td>Any other kind of laboratory tissue paper will do as well.</td>
		</tr>
		<tr>
			<td>Waterproof pen</td>
			<td>Staedler Mars GmbH &#38; Co KG, N&#252;rnberg, Grmany</td>
			<td>Lumocolor permanent 313, 0.4 mm, S, black, Cat.: 313-2</td>
			<td>Any other kind of waterproof pen will do as well.</td>
		</tr>
	</tbody>
</table>

sampling strategies and processing of biobank tissue samples from porcine biomedical models

변환 의료 연구에서 돼지 모델 꾸준히 더 인기가 되고있다. 특히 유전자 변형된 돼지의 개별 동물의 높은 가치를 고려 하 고 모델과이 모델의 사용 가능한 동물의 자주 제한 수, 적절 하 게 처리 조직 샘플의 (biobank) 컬렉션의 설립에 적합 한 샘플링, 시간 시점에서 지정 되지 않은 분석을 포함 하 여 후속 분석 방법의 광범위 한 의미 있는 접근을 활용 모델의 변환 값을 나타냅니다. 돼지 해부학의 특성에 관하여 포괄적인 지침 최근 대표, 다른 돼지 장기와 조직에서 높은-품질 샘플의 표준화 된 세대에 대 한 설립 되었습니다. 이러한 지침의 결과 다른 연구와 조사 사이 그들의 comparability 재현성에 대 한 필수적인 전제 조건은 없습니다. 기본 데이터, 장기 무게 볼륨, 결정의 샘플링 위치와 조직 샘플의 숫자의 생성으로 그들의 방향, 크기, 처리 및 트리밍 방향, 녹음 관련 요인이 가해질 및 분자, 질적, 양적 형태소 분석에 대 한 견본의 유용성을 결정. 여기, 한 설명, 실용적인, 단계별 데모의 가장 중요 한 기술 담당자의 세대에 대 한, 돼지 조직에서 다목적 biobank 견본 제공 됩니다. 여기에 설명 된 방법이 포함 기관/조직 볼륨 및 밀도, 조직의 수축의 넓이의 포인트-세기, 결정에 의해 parenchymal 장기에 대 한 볼륨-가중치 체계적인 무작위 샘플링 절차의 응용 프로그램 결정 샘플, 및 "Orientator" 및 "Isector" 방법, 및 수직 제복에 의해 생성 된 균일 한 임의의 등방성 (IUR) 섹션 등 양적 stereological 분석에 대 한 무작위로 지향 샘플 세대의 조직학 포함 관련 무작위 (VUR) 섹션입니다.

조직/기관 밀도의 결정에 대 한 잠수 기술
그림 12A -B 는 밀도의 대표 결정 돼지 신장 단계 1.1 (그림 1, 그림 2)에서 설명 된 침수 기술을 사용 하 여 볼륨을 보여줍니다. 더 대표 추가 돼지 장기 및 조직의 밀도 측정 결과 표 2에 표시 됩니?...

Watch this Scientific Journal Video about Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models at JoVE.com

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

샘플링 전략 및 돼지 생물 의학 모델에서 Biobank 조직 샘플의 처리

실용적인 응용 프로그램 및 다운스트림 분석 biobank 프로젝트에서의 광범위 한 스펙트럼에 대 한 돼지 동물 모델의 대표적인 조직 샘플의 세대를 위한 방법의 성능을 시연, volumetry를 포함 하 여 체계적인 무작위 샘플링 그리고 차동 질적 및 양적 형태 론 적과 분자 분석 유형에 대 한 조직 샘플의 처리.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

sampling-strategies-processing-biobank-tissue-samples-from-porcine

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Medicine

15.5K 조회수.  LMU Munich. 실용적인 응용 프로그램 및 다운스트림 분석 biobank 프로젝트에서의 광범위 한 스펙트럼에 대 한 돼지 동물 모델의 대표적인 조직 샘플의 세대를 위한 방법의 성능을 시연, volumetry를 포함 하 여 체계적인 무작위 샘플링 그리고 차동 질적 및 양적 형태 론 적과 분자 분석 유형에 대 한 조직 샘플의 처리.

비디오: Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

바이오 메디컬 응용 프로그램을위한 알긴산 Microspheres의 생성

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

연구

의학

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

지역 Gyrification 인덱스를 계산하기위한 단계별 자습서 : MR 이미지에서 두피 접기를 측정하는 방법

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

정렬 줄기 세포 Assays 및 흐름에 대한 기본 뇌 종양 조직의 처리

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

신선한 수술 샘플에서 분리 및 인간의 심실 심근 기능 특성

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.

The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques

This article describes bronchoscopic techniques in the bovine lung under experimental conditions, i.e. bronchoscopically guided inoculation, bronchoalveolar lavage, bronchial brushing, and transbronchial lung biopsy.

생물 의학 연구에 소 폐 : 시각적으로 유도 기관지, Intrabronchial 접종 및<em&gt; 생체</em&gt; 샘플링 기법

There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

임상 샘플에서 RNA 바이러스의 편견 깊은 시퀀싱

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

비 강 및 기관지 체액의 흡수: 인간의 호흡기 점 막 및 실험실 처리 샘플의 정밀 샘플링

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

조직 및 혈액 샘플링 절차의 표준화에 의해 닫힌된 가슴 돼지 심근 경색 모델의 개선

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Human Subcutaneous Adipose Tissue Sampling Using a Mini-Liposuction Technique

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate training and strict adherence to aseptic technique, subcutaneous adipose samples can be safely and efficiently obtained in a non-clinical setting by researchers. Following the administration of local anesthetic lateral to the umbilicus, a 14 G needle attached to a 5 or 10 mL syringe is inserted through the skin into the subcutaneous tissue. Under suction, the syringe is moved in a reciprocating, slicing motion to isolate fragments of adipose tissue. Withdrawing the plunger is enough to ensure that adipose tissue fragments are aspirated through the needle into the syringe. A single biopsy can collect about 200 mg of tissue. This biopsy technique is very safe for both participants and research staff. Following the biopsy, participants can resume most everyday activities, although they should avoid swimming and overly strenuous activities for 48 h to avoid excessive bleeding. Participants can safely undergo 2 biopsies within a single day, meaning that the technique can be applied in before-after acute intervention studies.

The manuscript and associated video demonstrate a percutaneous biopsy technique to obtain samples of subcutaneous adipose tissue from areas surrounding the umbilicus. This method is a low-risk and efficient way to investigate a range of parameters (e.g., gene or protein expression, enzyme activity, lipid content) within adipose tissue.

미니 지방 흡입 기술을 사용한 인간 피하 지방 조직 샘플링

Studies on adipose tissue are useful in understanding metabolic and other conditions. Human subcutaneous adipose tissue is accessible. With appropriate ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

의식 쥐의 쇄골 하 정맥에서 반복적 인 혈액 샘플링

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...