이 프로토콜의 개발은 재생 의료에 대한 캘리포니아 연구소 (CIRM, TR1-01246)와 파킨슨 연합에 의해 투자되었다.

펀치 생검이 표준 절차 3 (예를 들어, 4mm 라운드 Visipunch 계기로 얻은)를 사용하여 얻은 피부는 얼음에 완전한 DMEM 20 % FBS 매체에 보관해야합니다. 일단 샘플을 실험실에 도착, 가능한 한 빨리 조직 검사를 처리합니다. 1. 피부 펀치 생검의 준비 단계 1.1-1.3는 생명 공학 안전성 캐비닛 내부 수행해야 할 <ol><li> 사전 준비 : 6 잘 플레이트의 각 웰에 젤라틴 0.1 %의 1 ML을 추가합니다. 30-60 분 동안 옆에 접시를 설정합니다. 젤라틴 용액을 흡입하고 각 well에 완료 DMEM/20 % FBS 매체 800 μl를 추가합니다. 잘의 전체 표면은 매체와 적용되어 있는지 확인합니다. </li><li> 멸균 10cm 조직 문화 접시의 뚜껑을 반전하고 뚜껑의 중앙에 1.5 DMEM의 ML / 20 % FBS 미디어를 추가하고 혈청 피펫 팁 용지 드롭을 확산. </li><li> 멸균 집게를 사용하여, 장소 t그는 피부 접시에 미디어 조각을 생검. </li></ol> 2. 피부 펀치 생검의 해부 9 2.1-2.2 단계는 수평 층류 후드 안쪽에 수행해야 할 <ol><li> 거꾸로 뚜껑에 10cm 접시의 바닥 부분을 놓고, 피부가 층류 후드 해부 현미경 조직 검사 전송할 수 있습니다. </li><li> 장소에 생검을 보유하는 하나의 메스를 사용하여 한 방향으로 회전 운동으로 잘라 두 번째 메스 같은 반에서 조각을 절단하여 날카로운 모서리를 가진 12-15 균등하게 크기의 조각으로 4 mm 라운드 피부 생검을 해부하다. 비정형 가장자리와 조각이 좋지 첨부 / 세포 부산물에 기여한다. </li></ol> 3. 해부 피부의 전송 조직 배양 플레이트에 조각 생검 단계 3.1-3.6 생명 공학 안전성 캐비닛 내부 수행해야 할 <ol><li> 10cm 조직 문화 접시의 바닥 부분을 배치거꾸로 뚜껑 및 전송 접시 위에 다시 생물 안전 캐비닛에에. </li><li> 마른 우물에 800 μL 및하지를 포함하는 준비된 6 잘 플레이트의 각 웰에 뾰족한 집게, 장소 2-3 생검 조각을 사용. 조각이 잘 하단에 부착 얻기 위해 운동을 도청하거나 슬라이딩을 사용합니다. 메스 집게에서 모든 생검 조각을 제거하는 데 유용합니다. </li><li> 37 ° C 배양기에서 6 잘 플레이트를 놓습니다. 어떤 증발 매체를 대체하는 매 2 일 1 ~ 200 μl를 추가, 미디어 코팅 첫 주에 대한 우물의 바닥의 영화가 있는지 확인하기 위해 매일 모니터링합니다. </li><li> 일주일이 지난 뒤, 전체 DMEM/20 % FBS 및 변경 매체 2-3 일마다 2 ㎖에 미디어의 양을 증가시킵니다. </li><li> 일단 섬유 아세포는 섬유 아 세포 2X T75 플라스크 (통로 1)에 잘 trypsinize 및 통로 6 잘 플레이트의 가장자리에 도달하는 시점에 각 우물에 합류한다. 조직의 조각뿐만 아니라 전송할 수 있습니다. 그들은 (을)를 부착하지 않으며 세척 할 수다음 미디어의 변화 동안 유타. </li><li> 일단 섬유 아 세포가 합류하다, 배 T175 플라스크 (통과 2) 완전한 DMEM 매체 동결 플러스 1X10 6 세포 / 유리 병 ml 당 10 %의 DMSO로 전송할 수 있습니다. </li></ol> 4. 면역 염색을 통해 특성화 섬유 아세포를 <ol><li> 8 잘 챔버 슬라이드 문화 섬유 아 세포는 젤라틴으로 코팅. </li><li> 세포가 자랄 때 80 %에있을 때, 각 우물에서 매체를 대기음. </li><li> 상온에서 십분 4 % 파라 포름 알데히드로 세포를 고정합니다. </li><li> 1X PBS로 우물 3 번 씻으십시오. </li><li> 0.3 %의 150 μL와 세포를 Permeabilize 트리톤 실온에서 5 분간 X-100 (PBS)입니다. </li><li> 1X PBS로 우물 3 번 씻으십시오. </li><li> 실온에서 1 시간 동안 200 μL 차단 솔루션 (PBS + 5 % 정상 염소 혈청 (벡터, S-1000))를 추가합니다. </li><li> 솔루션을 차단하는 안티 SERPHIN-1 (안티 - 토끼, 단클론 항체, 시그마, S5950-200 μL)를 1:250 희석을 준비합니다. </li><li>차단 솔루션을 spirate 및 AntiSERPINH-1의 1:250 희석 150 μl를 추가합니다. 실온에서 1-2 시간을 품다, 4 ° C에서 하룻밤. </li><li> 1X PBS로 우물 3 번 씻으십시오. </li><li> 솔루션을 차단에 1:200 염소 안티 - 토끼 단클론 항체 (알렉사 플 루어 염소 안티 - 토끼 IgG의 (H + L), Invitrogen의, A11008)를 준비합니다. </li><li> 각 well에 1:200 염소 안티 - 토끼 단클론 항체의 150 μl를 추가합니다. 상온에서, 어둠 속에서 1 시간 동안 배양한다. </li><li> 1XPBS 한 번 우물을 씻으십시오. </li><li> PBS를 대기음,주의 깊게 그냥 슬라이드를 나타 내기 위해 챔버를 들어 올립니다. </li><li> DAPI (벡터, H-1200)를 포함하는 설치 매체의 2 ~ 3 방울 슬라이드를 장착합니다. </li><li> 형광 현미경 슬라이드를 분석합니다. </li></ol>

<ol>
	<li>Nguyen, H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress.">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li><li>Mak, S. K., Tewari, D., Tetrud, J. W., Langston, J. W., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+skin+fibroblasts+from+a+Parkinson's+disease+patient+with+an+alpha-synuclein+triplication.">Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.</a> Journal of Parkinson's Disease. 1, 175-183 (2011).</li><li>Punch Zuber, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=biopsy+of+the+skin.">biopsy of the skin.</a> Am. Fam. Physician. 65, 1155-1164 (2002).</li><li>Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+fibroblast+cultures.">Establishment of fibroblast cultures.</a> Curr. Protoc. Cell Biol. Chapter 2, Unit 2, 1 (2001).</li><li>Kuroda, K., Tajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP47+is+a+useful+marker+for+skin+fibroblasts+in+formalin-fixed,+paraffin+embedded+tissue+specimens.">HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin embedded tissue specimens.</a> J. Cutan. Pathol. , 241-246 (2004).</li></ol>

저자는 공개 아무것도 없어.

이 프로토콜을 통해 피부 섬유 아세포 상대적으로 순수 배양을 얻을 수 있습니다. 섬유 아세포는 타원형 세포 핵에 둥근 길쭉한, 스핀들 같은 세포 기관의 특성을 형태 학적 특징을 가지고 때 합류 섬유 아세포는 정렬 및 번들에서 성장. 매체는 섬유 아세포의 성장 예를 들어, 각질 추가 보충 및 성장 인자를 필요로하거나 세포 분열 덜 활성화되어 다른 세포 집단 반면이 상대적으로 순수한 ...

<table border="1">
<tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica</td>
 <td>S6E</td>
 </tr>
 <tr>
 <td>10 cm Tissue Culture Petri dish</td>
 <td>VWR</td>
 <td>25382-166</td>
 </tr>
 <tr>
 <td>6-well Tissue Culture plate </td>
 <td>VWR</td>
 <td>73520-906</td>
 </tr>
 <tr>
 <td>Stainless steel disposable sterile scalpels (2), blade No#15, Miltex</td>
 <td>VWR</td>
 <td>21909-660</td>
 </tr>
 <tr>
 <td>Sterile pointed-tip forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Conical tubes</td>
 <td>VWR</td>
 <td>21008-940</td>
 </tr>
 <tr>
 <td>2 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-894</td>
 </tr>
 <tr>
 <td>5 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-896</td>
 </tr>
 <tr>
 <td>10 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-898</td>
 </tr>
 <tr>
 <td>Pasteur Pipettes</td>
 <td>VWR</td>
 <td>14672-380</td>
 </tr>
 </tbody>
</table>
Table A. Table of specific reagents and equipments.
 <table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company </td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1X DMEM: High Glucose</td>
 <td>Invitrogen/Gibco</td>
 <td>11960-069</td>
 </tr>
 <tr>
 <td>20% Fetal Bovine Serum</td>
 <td>Invitrogen/Gibco</td>
 <td>26140-079</td>
 </tr>
 <tr>
 <td>1X L-Glutamine</td>
 <td>Invitrogen/Gibco</td>
 <td>25030-164</td>
 </tr>
 <tr>
 <td>1X MEM Non-essential Amino Acids </td>
 <td>Invitrogen/Gibco</td>
 <td>11140-076</td>
 </tr>
 <tr>
 <td>1X Penicillin/Streptomycin</td>
 <td>Invitrogen/Gibco</td>
 <td>15140-163</td>
 </tr>
 </tbody>
 </table>
Table B. Reagents.

skin punch biopsy explant culture for derivation of primary human fibroblasts

질병을 가진 개인에서 파생 된 조직 및 세포 라인 질병 관련 세포의 표현형을 연구하는 이상적인 소스입니다. 이 프로토콜에서 환자 유래 섬유 아세포가 성공적으로 모델 병 1 유도 만능 줄기 세포의 유도에 사용되었습니다. 이 섬유 아세포의 초기 구절은 특정 질병 경로, 기계 2 및 후속 약물 검사 방식을 연구하기 위해 셀 기반 기능 분석에 사용할 수 있습니다. 효소 절차를 통해 제시된 프로토콜의 장점은 1) 효소 처리를하여 노인 환자, 파킨슨 병, 2에 영향을 예를 들면 환자) 더 도전적인 방법론을 통해 기술적으로 간단한 방법에서 파생 된 조직의 소량의 기술의 재현성 및 3아르 ) 시간 고려 사항 :이 프로토콜은 15 ~ 20 분을 소요하고 생검 도착 후 바로 수행 할 수 있습니다. 효소 처리는 4 시간까지 걸릴와의 문제가있을 수 있습니다세포 생존 제대로 처리하지 세포의 후속 첨부 overdigestion, 감소. 이 프로토콜은 4 mm 인간의 피부 섬유 아세포 문화 유도를위한 생검의 해부 및 준비를 설명하고 환자 파생 된 조직 샘플을 다룰 때 중요한 매우 높은 성공률을 가지고 있습니다. 이 문화에서는, 각질이 준비 후 첫 일주일 이내에 생검 조직에서 마이그레이션 할 수 있습니다. 섬유 아 세포는 각질의 첫 번째 파생물 후에 7-10 일이 나타납니다. 20 % FBS와 보충 DMEM 높은 포도당 미디어 각질 섬유 아세포에 섬유 아세포의 성장 각질을 자랄 것입니다 하시 더군요. 이 구절 후 각질 섬유 아세포 마커 SERPINH1 (HSP-47)를 표현 비교적 균질 한 섬유 아세포 배양 결과 중 희석되었다. 이 방법을 사용하여 15-20000000 섬유 아세포는 세포 은행을위한 4-8 주 얻을 수 있습니다. 피부 절개는 15 ~ 20 분 소요, 세포는 다음 하루에 한 번 모니터링하는현미경, 미디어 2-3 일마다 부착과 세포의 파생물 후 변경됩니다.

각질 박리 후 48 시간 자마자 생검 조각의 성장. 처음으로 섬유 아세포의 파생물은 주일 정도 처리 한 후 관찰 할 수있다. 우물 confluency에 도달하면, 섬유 아세포는 세 150cm 플라스크 (T150)에 도달하기 위해 두 개 더 구절에 대한 계대하고 세포를 냉동 보존됩니다. 우리는이 방법 15-20000000 세포를 생성합니다. 섬유 아세포는 소포체로 번역 콜라겐 특정 분자 보호자입니다 안티 SERPINH1 (또한 HSP-47라고도...

Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

인간의 피부 펀치 생검에서 섬유 아세포의 절편 배양 프로토콜은 낮은 통로 번호로 만 15-20 세포 은행을위한 4-8 주 이내에 피부 세포를 유도하기 위해 기술적으로 강력하고 간단한 방법입니다.

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 ...

skin-punch-biopsy-explant-culture-for-derivation-primary-human

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40.8K 조회수.  The Parkinson's Institute. 인간의 피부 펀치 생검에서 섬유 아세포의 절편 배양 프로토콜은 낮은 통로 번호로 만 15-20 세포 은행을위한 4-8 주 이내에 피부 세포를 유도하기 위해 기술적으로 강력하고 간단한 방법입니다.

비디오: Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

인간 Mammary 섬유아 세포에서 암종 - 관련 섬유아 세포의 실험 세대 (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

연구

의학

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

파킨슨 병 분야에서 Mitochondrial Phenotypes을 모니터링하기위한 기본 인간 섬유 아세포의 사용

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

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 ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

차 사람 골수 및 악성 종양을 연구하는 세 가지 차원 조직 배양 모델

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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 ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

수정 된 버그 스트롬 기술을 이용하여 인간의 골격 근육 생검 절차

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

전체 조직과 조직 검사에서 인간 상피 Enteroids과 Colonoids 설립

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

사람의<em&gt; 광근의 Lateralis</em&gt; 웨일 - Blakesley Conchotome를 사용하여 골격 근육 생검

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

피부 마이크로 이식 세대를위한 새로운 세분화 방법 후 조직 특성

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light Sheet Microscopy에 의한 심근 성 심근염의 Murine 모델에서의 백혈구 침윤의 정량적 시각화

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...