이 연구는 건강의 국립 연구소 (CA148828), 미시간 위장 펩티드 센터의 대학과 미시간 대학 종합 암 센터의 제프리 A. 콜비 대장 암 연구와 톰 리우 기념 기금에서 YMS에 교부금에 의해 지원되었다.

1. 대장 종양의 분리 및 세포 해리 <ol><li> 장내 종양은 어떤 산발적 또는 치료에 의한 대장 암 모델에서 분리 할 수​​ 있습니다. 마우스는 CO 2 안락사되어야한다. 콜론은 다음 찬 인산 완충 식염수 (PBS)로 플러시 수집 및 세로 방향으로 열립니다. 입체 현미경을 사용하여 종양을 포함하는 영역을 식별, 가위로 해부하고, 차가운 PBS로 씻어. </li><li> EDTA 킬레이트 버퍼에 종양을 포함하는 장 조각 (2 mM의 EDTA, 5.6 mmol / L 나 2 HPO 4, 8.0 mmol / L KH 2 PO 4, 96.2 mmol / L의 NaCl, 1.6 mmol / L KCl을, 43.4 mmol / L 자당을 품다 얼음에 60 분 54.9 mmol / L D-소르비톨, 0.5 mmol / 증류수 L DL-디티 올 트레이 톨). </li><li> 종양 세포는 간엽에 연결된 상태로 유지됩니다 동안 킬레이트 화 한 후, 정상적인 창자 상피 세포의 대부분은 분리됩니다. 정상 상피 세포를 포함하는 킬레이트 버퍼를 대기음그리고 남은 종양을 씻어 5 ML 차가운 킬레이트 버퍼를 한 번 더 파편. </li><li> 킬레이트 버퍼를 대기음, 5 ML 차가운 1X PBS로 종양의 조각을 씻는다. </li><li> 1X PBS를 대기음, 소화 버퍼에 종양의 조각 (2.5 % 태아 소 혈청, 페니실린의 1 단위 / ML, 스트렙토 마이신의 1 ㎍ / ㎖, 2.5 NG / 암포 테리 신 B의 ML 200 U / ML 유형 IV 콜라​​를 품다 37 2 시간 동안 125 ㎍ / Dulbecco의 수정 된 이글 매체에 ML 타입 II 디스 파제 (dispase)) ° C. </li><li> 종양 조각은 1 분 동안 정상적인 중력 하에서 해결하고, 15 ML 팔콘 튜브에 뜨는를 수집 할 수 있습니다. 펠릿 3 분 200 XG에 원심 분리, 3 분 200 XG에 원심 분리하여 5 ML PBS로 한 번 세척하여 단일 세포 종양 서스펜션 뜨는. </li></ol> 2. 장내 종양의 문화 <ol><li> 혈구를 사용하여 고립 된 단일 종양 세포를 계산, 500 μL PBS와 종양 세포 펠렛을 resuspend을. </li><li> 센트 펠렛 종양 세포3 분 200 XG에 rifuging, 잘 당 마트 리젤 ㎕의 50 15,000 세포에서 24 - 웰 플레이트에 얼음 접시에 5 밀리그램 / ML 리겔에서 그들을 resuspend을. </li><li> 마트 리젤은 37 ° C에서 15 분 동안 중합, 500도 기초 / μL 문화 매체 (페니실린의 1 단위 / ML, 스트렙토 마이신의 1 ㎍ / ㎖, 2.5 NG / 암포 테리 신 B의 ML, 10 mmol / L HEPES 추가 할 , 2mM의 Glutamax, 1X N2 보충, 1X B27 보충제, 고급 Dulbecco의 수정 된 이글 Medium/F12 1 mmol / L N-아세틸 시스테인) 50 NG / ML 쥐 EGF를 포함. </li></ol> 3. 설립 Organoids 유지 보수 <ol><li> 일주일에 한 번 EGF 2 일 통과 organoids 1시 5분 모든을 포함하는 기저 문화 매체를 변경합니다. </li><li> 과 Passaging를 들어, 신선한 기초 문화 매체와 문화 매체를 교체하십시오. 기계적으로 잘라 팁 P1000 피펫을 사용하여 organoids와 리겔을 중단하고 15 ML 팔콘 튜브로 전송할 수 있습니다. 또한 기계적 분리는 화재 광택 파스퇴르 파이프를 사용하여 수행됩니다TTE. </li><li> 2 분 200 XG에 기초 배지와 원심 분리기 5 ㎖와 해리 organoids을 씻으십시오. </li><li> 상층 액을 버리고, 마트 리젤과 펠렛을 resuspend을하고 위에서 설명한대로 문화 매체를 추가합니다. </li></ol> 4. 보관 및 설립 Organoids의 복구 <ol><li> 장기 저장을 위해, 최소 2 년간 안정 액체 N 2 organoids을 동결. 냉동 organoids를 들어, 팁 잘라 15 ML 팔콘 튜브로 전송과 P1000 피펫을 사용하여 중단. </li><li> 2 분 200 XG에 기초 배지와 원심 분리기 5 ㎖와 해리 organoids을 씻으십시오. </li><li> 상층 액을 버리고, Dulbecco의 수정 이글 중간 (DMEM)는 20 % 태아 소 혈청 (FBS)와 10 % 디메틸 설폭 사이드 (DMSO)를 함유 펠렛을 resuspend을. </li><li> 1.5 ML의 cryotubes로 전송 세포는 다음 ° C 냉동고 냉각 속도를 달성하기 위해 -80에서 나르 겐 씨 서리가 내린 냉동 컨테이너 및 저장소에 튜브를 넣어-1 ° C / 분. 하룻밤 배양 한 후, 액체 N 2에 튜브를 전송합니다. </li><li> 복구, 냉동 organoids 급속하게 37 ° 위에서 설명한 조건 리겔과 문화에 resuspend을 C 물 목욕, 스핀 다운, 기초 배지로 세척.에서 해동 </li></ol> 5. RNA 추출, 단백질 추출 및 면역 <ol><li> RNA 추출 : 통과를 위해 위에서 설명한대로 세포가 수집됩니다. RNA는 제조 업체의 지침에 따라 PicoPure TM RNA 분리 키트에 격리됩니다. </li><li> 단백질 추출 : 위에서 설명한 (100 μL radioimmunoprecipitation 분석 버퍼에 RIPA를 용해으로 세포 펠렛을 수집, 50 mmol / L 트리스 - 염산 산도 7.5, 150 mmol / L 염화나트륨, 2 mmol / L EDTA (에틸렌 다이아 민 테트라 초산), 1 % NP-40, 0.1 1X 단백 분해 효소 억제제 % SDS). </li><li> 면역 : 세포 배양 매체를 대기음, 극저온 주조로 잘라 팁 P1000 피펫 종양 organoids를 전송 및 TIS 즉시 정지드라이 아이스로 냉동 매체 (OCT를) 고소. 냉동 섹션은 7 μm의에서 잘라 이전 26 설명 된대로 염색 하였다. </li></ol>

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우리는 공개 아무것도 없어.

이 프로토콜에 설명 된 실험 절차는 기본 생쥐 대장 종양의 분리 및 문화하실 수 있습니다. 프로토콜은 박사 Clevers 그룹 1,24,27 수행 정액 직장에서 적응하고 있습니다. 우리가 종양 organoids의 더 나은 수율을 얻을 수있는 소화 시간과 콜라 농도를 최적화. 중요한 단계는 종양의 단일 세포로 세포의 소화, 마트 리젤의 부유하고, 선택적 문화를 이용하실 수 있습니다. 종양 세포 소화 순서대로...

장 상피 세포 증식과 척추 동물의 몸 2,3의 모든 다른 조직을 능가 특별한 속도로 뒤집습니다. 장 줄기 세포 (ISC)와 대중 교통 증폭 세포를 포함하여 분할 세포는 분비 (잔,에 Paneth 및 enteroendocrine) 세포 또는 장 세포로 분화 3. ISC는 지하실의 기지에 위치해 있습니다. 에 Paneth 세포는 지하실의 맨 아래로 이동하고 다른 계통이 융모 3,4 위쪽으로 마이그레이션하는 반면, 긴 수명입니다. 여기에 세포는 미생물을 포함한 창자 내용에 노출되어 anoikis에 의한 세포 사멸 메커니즘을 통해 융모 끝에서 창고입니다. 콜론 융모와에 Paneth 세포를 부족하지만, 항상성을 유지하기위한 메커니즘은 4와 비슷합니다. Wnt 신호 전달 경로는 장내 증식과 ISC 유지 보수 4에 중요한 역할을에 연루되어있다. 일 삭제전자 전사 인자 TCF4, Wnt 신호의 다운 스트림 이펙터, 장 줄기 세포 및 조직 5의 후속 고장의 손실로 연결됩니다. 마찬가지로, Wnt의 억제 DKK1의 유전자 발현은 상피 증식을 감소시키고 분비 세포 계보는 6 고갈. 반대로, Wnt의 작용제 R-spondin-1의 과발현은 장 토굴 세포 7의 강력하고 신속한 확산을 유도합니다. 장 항상성에 대한 Wnt 신호의 중요성을 감안할 때, Wnt의 통로 돌연변이는 자주 결장암 8에서 관찰된다. 대장 암은 미국에서 9 암 사망의 세 번째 주요 원인이다. 붉은 고기와 알콜을 가진 과잉 섭취는 신체 활동을 감소시키고, 상속 및 체세포 돌연변이가 대장 암 10,11의 위험 인자로 간주됩니다. 대장 용종증 대장균 (APC) 유전자, 키 Wnt 신호 계수, 펜실베이니아의 대다수 변이가족, 산발적 및 대장염과 연관된 대장 암 12,13와 tients. Axin2과 β-catenin의 등 Wnt 신호 전달 경로에 관여 다른 요인의 변이는 대장 암 14,15에서 관찰된다. 그러나 대장 암에 대한 정확한 메커니즘과 효과적인 치료는 여전히 부족하다. 대장 암에 대한 분자 메커니즘의 조사를 용이하게하기 위해, 공격적인 세포 유형으로 양성에서 암 진행의 여러 단계를 나타내는 인간의 대장 암 세포 라인은 16-18 설립되었습니다. 다른 전이성 특성을 가진 마우스 대장 암 세포주도 19,20 사용할 수 있습니다. 그들은 밀접하게 생체 상태를 모방하고 더 생리학 관련 데이터에게 21을 생성하기 때문에 그럼에도 불구하고, 일차 전지 또는 organoid 문화가 변형 세포 라인을 선호합니다. 대부분의 대장 암 유래 세포주는 lackin, 플레이트에 부착 된 단층이나 세포 현탁액으로 성장정점 - 기저 방향과 세포 간의 긴밀한 접합 g. 차별화 된 세포는 융모 끝을 도달하고 22을 흘리다대로 또한, 생체의 정상 및 종양의 장 상피 세포가 사멸 되나 anoikis의 자연 형태를 겪는다. 이러한 기능은 세포 라인에 요점을 되풀이하기 어려운 있지만, 대장 암 (23)의 발달 과정에서 중요한 역할을합니다. 이러한 기능은 기본 organoids에 유지됩니다. 또한, 종양 organoid 문화는 생체 내 연구에 비해 유전자의 종양의 자율적 인 기능을 평가하는 효율적인 시스템을 제공합니다. 소장의 생체 내에서 유전자 조작은 주로 소장 특정 드라이버를 사용하여 형질 전환 및 / 또는 녹아웃 마우스를 만들 통해 시간이 소요되는 과정입니다. 그러나, 종양 organoids은 쉽게 바이러스 매개 유전자 조작에 순종함으로써 정확한 분자 메커니즘을 평가하기위한 훌륭한 도구입니다. 주 장내 종양 organoid 문화 HAVE가 가능하고 강력한 기술로 입증되었습니다. 기본 장 세포 배양은 단일 성인 Lgr5 + 줄기 세포 24에서 체외 토굴 - 융모 구조로 기능성 장 organoids을 설정할 수 있습니다. 이 organoids은 이식과 재생 25 손상된 결장 조직에 접목 할 수 있습니다. 배양 조건의 추가 적응 마우스 콜론과 인간의 소장 1 가능한 결장에서 유사한 상피 organoids을 만들었다. 기초 배지와 EGF는 기본 마우스 대장 종양 organoids 1 성장을위한 충분한 반면 차 정상 대장 상피 문화, 기초 배지뿐만 아니라에 대한 EGF, Noggin의, R-spondin과 Wnt3a 등의 성장 인자가 필수적이다. 여기에서 우리는 자세한 프로토콜, 격리 문화, 대장 종양 organoids을 생성하는 방법에 대해 설명합니다.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

여러 인간과 생쥐 대장 암 세포주가 설립되어 같은 여러 세포 레이어, 기저 정점 극성을 구별 할 수있는 능력, 그리고 anoikis으로 결장 종양의 생리적 무결성은 대장 암 유래 세포주에서 유지되지 않습니다. 본 연구 사토 T 등 1., 대장 종양의 중요한 생리적 기능을 유지에서 적응 배양 마우스 기본 대장 종양 organoids하는 방법을 보여줍니다. 이 방법은 종양 세포가 정상에 비해 영양 상태 제한에 성장을 유지한다는 원칙에 따라 마우스 결장 종양 조직 수집, 인접한 정상 대장 상피 세포 분리, 단일 세포로 결장 종양 세포의 소화, 마트 리젤로 결장 종양 세포를 포함하고, 선택적 문화로 구성 상피 세포. 유전자 변형 생쥐에서 분리 된 경우, 기본 종양 organoids 종양 자치 funct를을 평가하는 매우 유용한 시스템을 제공특정 유전자의 이온. 또한, 종양 organoids는 유전자 전달을 묵상 바이러스 유전자 조작 의무이다, 대장 종양에 관여하므로 신호 전달 경로는 광범위하게 발현 또는 최저로 조사 할 수 있습니다. 기본 종양 organoids 문화 결장 종양에 대한 메커니즘과 치료 양식을 연구하는 생리 적절하고 실현 가능한 방법을 제공합니다.

/ + 마우스가 그림 1에 나타나있다 3 개월 송출 분에서 대장 종양 organoid 형성의 시간 경과. 0 일에서 단일 세포 도금 (그림 1A)에 따라 몇 시간을 관찰 할 수 있습니다. 1 일에, 내화물 핵을 가진 대장 종양의 상피 세포가 관찰 될 수있다 살아 남았다. 3 일에서 세포의 크기가 두 배로. 매일 6시, organoid의 크기가 10 배 이상 확대하고 중간에 세포 사멸의 흔...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

기본 생쥐 대장 종양 organoid을 설정하는 간단한 방법이 설명되어 있습니다. 이 방법은 대장 종양 세포가 생존하고 상피 정상적인 결장하지 않는 반면, 제한된 성장 인자를 포함하는 미디어 organoids로 성장하는 기능을 사용합니다.

기본 마우스 대장 종양의 체외 Organoid의 문화

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

in-vitro-organoid-culture-of-primary-mouse-colon-tumors

Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K 조회수.  University of Michigan. 기본 생쥐 대장 종양 organoid을 설정하는 간단한 방법이 설명되어 있습니다. 이 방법은 대장 종양 세포가 생존하고 상피 정상적인 결장하지 않는 반면, 제한된 성장 인자를 포함하는 미디어 organoids로 성장하는 기능을 사용합니다.

비디오: In vitro Organoid Culture of Primary Mouse Colon Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

면역 결함 마우스의 확립과 인간 Retinoblastoma 종양의 전파

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

연구

의학

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

인간의 기본 섬유 아세포의 유도를위한 피부 펀치 생검 절편 문화

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

인간의 전정 신경초종의 차 문화

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

기본 종양 및 / 또는 전임상에 대한 관련 전이 및 치료제의 임상 개발의 전 생체 치료 응답

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

마우스 대장 수입 성의 엔딩의 생체 기능 특성에

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

miRNA의를 사용하여 유방암 조직에서의 miRNA 발현의 임상 병리학 적 분석<em&gt; 현장에서</em&gt; 하이브리드

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

개 동맥과 정맥에서 차 내피 세포의 분리 및 문화

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

근육의 불균형: 테스트 및 기능 편심 햄 스트링 운동 인구 강도 훈련

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

메타볼로믹스 및 리피도믹스 분석을 위한 뇌종양의 현장 샘플링 및 추출

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...