이 프로젝트에 대한 자금은 연구 및 망막 연구 재단 클레이튼 재단에 의해 제공됩니다.

1. retinoblastoma의 tumorspheres의 준비 <ol><li> IRB 승인 프로토콜에 따라 retinoblastoma 종양 샘플을 구합니다. </li><li> 신선한 정의 줄기 세포 미디어를 준비 (Neurobasal - A 미디어, 1X B - 27 보충 마이너스 비타민 A, 1X 아닌 필수 아미노산 솔루션 1X L - 글루타민, 20 NG / ML EGF, 10 NG / ML bFGF). </li><li> 기계식 멸균, 조직의 크로스 커팅 기법을 사용하여 메스 문화 처리 6 잘 플레이트와 종양 조직을 disaggregate. 즉시 조직에 정의된 줄기 세포 미디어 100 μL를 추가하고 부드럽게 메스를 사용하여 다진 조직을 확산. 잘 정의된 줄기 세포 미디어의 5 ML을 추가합니다. </li><li> 37 품어 ° C, 5 humidified 인큐베이터에서 % CO 2 및 일일 점검. 일부 tumorspheres는 2~4일 과정 이상의 증가와 함께, 거의 즉시 중단 종양 조직에서 발생한다. </li><li> 주간, 5 분 300xg에서 원심 분리기의 tumorspheres은 신선한 정의 줄기 세포 미디어에 resuspend하고, pipet 위아래로 10 배 이상 큰, 중앙 괴사 분야를 방해하고 새로운, 건강 분야는 양식 수 있습니다. </li></ol> 2. 주사에 대한 종양 세포의 준비 <ol><li> 경우 15 ML 원뿔 관에 부드럽게 교양 tumorspheres, pipet의 분야를 넣고. serological pipet 분야를 혼란 10-15 시간으로 부드럽게 위아래로 pipet. </li><li> 새로운 튜브에 hemacytometer하고, 나누어지는 주사 적어도 초당 50,000 전지를 사용하여 주입하는 세포를 계산합니다. 가능한 세포의 개수에 따라 가능한 한 많은 주사 준비. </li><li> 5 분 300xg에서 원심 분리기. 조심스럽게 5 ML PBS에서 뜨는 및 resuspend을 대기음. 원심 분리와 열망을 반복하고, 사출 당 10 μL PBS에 resuspend. </li></ol> 3. 동물의 준비 <ol><li> 인슐린 주사기를 사용하여, 쥐 마취 칵테일의 intraperitoneal 주사 (케타민을 37.6 MG / ML, xylazine 1.92 MG / ML, acepromazine 0.38 MG / ML) g 체중 당 1 μL (gbw)에서.를 관리 진정이 될 수있는 동물 5 분 동안 기다리십시오. 하트 비트에 대한 확인 및 온수 패드에 동물을 놓으십시오. 복구까지 항상 온수 패드에 동물을 유지합니다. </li><li> 진정 작용 후, 오른쪽 눈에 proparacaine HCL 1 방울을 관리할 수 있습니다. 온수 패드에 동물을 교체하고 1 분 기다립니다. </li><li> 오른쪽 눈에 HCL을 phenylephrine 1 방울을 관리할 수 있습니다. 온수 패드에있는 동물을 교체하고 5 분간 기다립니다. 학생의 지연이 발생하지 않은 경우 1, 드롭을 다시 관리하고 다른 5 분 기다립니다. </li><li> &gt; 삼십분 후 진정 작용에서 발생할 수있는 운동이나 꿈틀의 흔적에 대한 동물을 모니터링합니다. 운동의 초기 징조에, 1 μL / 케타민을 HCL의 gbw (10 MG / PBS에 ML에 희석) 관리하고 5 분 기다립니다. </li></ol> 4. 종양 세포의 주입 <ol><li> 최대 직면한 오른쪽 눈과 머리는 거즈에 휴식과 현미경을 통해 관찰하면 오른쪽 눈이 fundus (학생이 동시에 확대되어야합니다)의 붉은 반사를 얻을 중심으로 자사의 측면에있는 현미경 anesthetized 동물을 플레이스 . </li><li> 부드럽게 눈꺼풀에 두 손가락으로 동시에 아래로 밀어와 절차의 나머지를 위해 꾸준히이 위치를 고수하여 바로 세계를 프로프. </li><li> 피어스 갈 거예요 플라나의 영역에서 각막 limbus에 인접 결막 및 공막엔 (그림 1)를 통해 단 유리 구멍에 맥락막 통해 옆으로, Luer - 잠금 주사기에 부착된 멸균 30 게이지 바늘을 세계 사용합니다. 오프닝에서 바늘을 제거합니다. </li><li> 알코올 면봉으로 해밀턴 바늘을 소독. 해밀턴 주사기로 세포 현탁액을 내리고 바늘이 망막 근처의 유리체에서 렌즈 뒤에 현미경을 통해 시각 때까지 개방에 바늘을 삽입합니다. 이 위치에 꾸준히 바늘을 누른 상태에서 두 번째 사람의 도움으로 플런저를 천천히 우울. 바늘을 제거합니다. 필요한 경우, 개막으로부터 액체를 흡수하기 위해 면봉을 사용합니다. </li><li> 부드럽게 마우스 눈꺼풀을 닫을 수있는 눈꺼풀의 압력을 풀어주고있다. </li><li> 각각의 눈에 hypromellose 1 방울을 관리하고 복구를 위해 온수 패드로 동물을 반환합니다. </li></ol> 5. 주입 마우스 및 종양의 수확의 모니터링 <ol><li> 매일, 보통 2~4주 포스트 분사 명백한된다 주입 leukocoria 눈을 (화이트 젖꼭지 반사) 및 / 또는 periocular 팽창을 확인합니다. </li><li> 일단 종양 성장 동물 제도적 지침에 따라, 안락사 감지합니다. </li><li> 눈 및 종양은 눈꺼풀이 적용하는 경우, palpebral 균열 (fissure)에 직교이 incisions을 만들고 피부 날개를 리프트에 메스를 사용합니다. </li><li> 실체 현미경을 사용하여 시각화에서 각막과 렌즈를 제거 limbus에서 원주 절개를하고, 신중하게 트위를 사용하여 다른 조직에서 종양 질량의 대부분을 해부하다zers. 무균 RPMI - 1640 미디어 종양을 놓으십시오. </li><li> 천천히 소켓에서 열린 세계를 당길 족집게를 사용합니다. 종양 침공은 시각 수 있도록 연결된 시신경을 보장하기 위해, 신경이 노출까지 세계를 당겨하고 가능한 한 오랫동안 신경을 절단하는 가위를 사용합니다. histological 시험에 대한 일반적인 처리를 위해 포르말린 10 % 놓습니다. </li></ol> 6. 대표 결과 : Retinoblastoma의 tumorspheres 거의 즉시 그들은 종양 덩어리에서 해방되면서 disaggregated 조직에서 나타나기 시작합니다. 2-4일 내에 더 많은 tumorspheres는 형태와 크기로 증가하기 시작합니다. 분야는 정기적으로하고 집계합니다 (그림 2) 주변의 잘 정의된, 보조 멤브레인을 전시하는 경향이 있습니다. 동물은 일반적으로 세계와 종양이 성장함에 따라 5~8주 주입 후 주변 조직의 팽창의 확대에 의해 다음, 사주 후 사출 (그림 3B) 이내 leukocoria과 함께 선물 (그림 3C). <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="그림 1"/> 그림 1. 프로토콜에 언급된 기능을 강조 마우스 눈의 크로스 - 단면. <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="그림 2"/> 그림 2. 체외에서 인간 retinoblastoma 세포의 문화) 4X에서 몇 군데, 그리고 B) 10X 목적의 확대. Retinoblastoma 기본 종양 세포는 일반적인 회전 타원체 모양과 선명한 외부 막과 tumorspheres을 생산합니다. 스케일 바) 500 μm의를 대표하고, B) 200 μm의. <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="그림 3"/> 그림 3 Rag2의 눈 -. / -, B를 A) 정상적인 기능을 보여주는 마우스) 유리 곳에 종양이 대량 나타내는 leukocoria, 및 C)은 큰 종양의 질량 관련 periocular의 팽만, intraocular 출혈과 함께 지구를 작성.

<ol>
	<li>Ch&#232;vez-Barrios, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+and+Nonmetastatic+Models+of+Retinoblastoma.">Metastatic and Nonmetastatic Models of Retinoblastoma.</a> Am. J. Pathol. 157, 1405-1412 (2000).</li><li>Suber, M. L., Hurwitz, M. Y., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+consequences+of+intraocular+administration+of+modified+adenoviral+vectors.">Immune consequences of intraocular administration of modified adenoviral vectors.</a> Hum. Gene Ther. 12, 833-838 (2001).</li><li>Mallam, J. N., Hurwitz, M. Y., Mahoney, T., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+Retinal+Cells+Using+Adenoviral+Vectors:+Dependence+on+Receptor+Expression.">Efficient Gene Transfer into Retinal Cells Using Adenoviral Vectors: Dependence on Receptor Expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1680-1687 (2004).</li></ol>

동물 실험은 의학 IACUC위원회의 베일러 대학에서 정한 지침과 규정에 따라 수행되었다.

여기 설명한 기술들은 intraocular, intravitreal 환경에서 전파 retinoblastoma의 종양을 용이하게합니다. intraocular 사출 기술은 retinoblastoma 유도 세포 라인 한뿐 아니라 intraocular 유전자 치료 2,3에 대한 바이러스 벡터를 제공하는에서 종양을 만들기 위해 과거에 사용되고 있습니다. 이 기술은 현재 성공적으로 기본 종양 및 사출 tumorspheres의뿐만 아니라 이종 이식 종양의 일련 전파의 세포를 직접 주입하여 인간 retinoblastoma의 종양을 생산하기 위해 이용되었다. 종양 형성 (보통 leukocoria)의 표시 증거는 대개 먼저 궤도를 둘러싼 세계 및 / 또는 조직의 intraocular 출혈과 팽창이 5-8주 이내에 개발하는 후 4 주 이내에 기록됩니다. 주입된 생쥐의 소수 민족은 눈꺼풀의 영구 폐쇄에 이르는, 주입 눈을 다치게. 이러한 경우에는 팽창은 종양 형성의 유일한 기호이다. 설립 이종 이식 종양의 전파는 매우 성공하지만 murine 이종 이식 종양의 확립, 모든 인간의 retinoblastoma의 종양과 함께 성공되지 않았습니다. 이 관찰은 invasiveness과 차별화 또는 다른 알려지지 않은 요소의 수준으로 기본 종양의 특정 특성, murine 안구 환경에서 계속 이러한 종양의 능력에 영향을 미치는있을 것입니다. 인간 retinoblastoma 종양에서 얻은 수있는 조직의 양은 매우 작은, 그리고 제한된 수명, 고체 종양의 조직학의 손실과 세포 표현형 변화로 체외에서 문화 인간 retinoblastoma 세포 수있는 능력에 상당한 제한이있다. 이 프로토콜은 인간의 종양을 전파하고 인간의 질병의 murine 모델을 수립하는 비교적 간단한 방법을 제공합니다. 이것은 체외에서 retinoblastoma와 환자의 종양 이외의 더 이상 유지 보수의 생물학의 생체내 실험에서 추가하실 수 있습니다.

<table border="1"><tbody><tr><th> 이름 </th><th> 회사 </th><th> 카탈로그 # </th><th> 댓글 </th></tr><tr><td> HCL 2.5 % Phenylephrine </td><td> 바우 &amp; 롬 </td><td> 053-11 </td><td> 안과 솔루션 </td></tr><tr><td> 30가 니들 </td><td> BD </td><td> 305128 </td><td> 정기 베벨 </td></tr><tr><td> 10 - ML Luer - 잠금 주사기 </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 3/10-cc의 인슐린 주사기 </td><td> BD </td><td> 328431 </td><td></td></tr><tr><td> 알코올 면봉 </td><td> BD </td><td> 326895 </td><td></td></tr><tr><td> 6 자 플레이트, 조직 문화 치료 </td><td> BD </td><td> 353046 </td><td></td></tr><tr><td> Proparacaine HCL 0.5 % </td><td> 버틀러 AHS </td><td> 017239 </td><td> 안과 솔루션 </td></tr><tr><td> 케타민을 HCL 100mg/mL </td><td> 포르 다지 </td><td> 4402A </td><td></td></tr><tr><td> 10 - μL 해밀턴 주사기 </td><td> 해밀턴 회사 </td><td> 7648-01 </td><td></td></tr><tr><td> 32 - GA 해밀턴 니들 </td><td> 해밀턴 회사 </td><td> 7803-04 </td><td> 지정 길이 - 0.5 &quot; </td></tr><tr><td> Neurobasal - 미디어 </td><td> Invitrogen </td><td> 10888-022 </td><td></td></tr><tr><td> B - 27 보충 마이너스 비타민 A, 50X </td><td> Invitrogen </td><td> 12587-010 </td><td></td></tr><tr><td> RPMI - 1640 미디어 </td><td> Mediatech </td><td> 10-040 - CV </td><td></td></tr><tr><td> 비 필수 아미노산 솔루션, 100X </td><td> Mediatech </td><td> 25-025 - CI </td><td></td></tr><tr><td> L - 글루타민, 100X </td><td> Mediatech </td><td> 25-005 - CI </td><td></td></tr><tr><td> 일회용 # 11 메스 </td><td> Miltex </td><td> 4-411 </td><td></td></tr><tr><td> 쥐 아나스타샤 콤보 </td><td> N / A </td><td> N / A </td><td> 자체 약국 제제 (케타민을 37.6 MG / ML, xylazine 1.92 MG / ML, acepromazine 0.38 MG / ML) </td></tr><tr><td> 재조합 인간 표피 성장 인자 (EGF) </td><td> STEMCELL 기술 </td><td> 02,633 </td><td> 10 μg / ML 주식 솔루션에 Reconstitute </td></tr><tr><td> 재조합 인간 기본 Fibroblast의 성장 인자 (bFGF) </td><td> STEMCELL 기술 </td><td> 02,634 </td><td> 10 μg / ML 주식 솔루션에 Reconstitute </td></tr><tr><td> OMS - 75 운영 현미경 </td><td> Topcon 의료 장비 </td><td> OMS - 75 </td><td> 이 모델은 단종되었습니다 </td></tr><tr><td> 포르말린 10% </td><td> VWR </td><td> 95042-908 </td><td></td></tr></tbody></table>

establishment and propagation of human retinoblastoma tumors in immune deficient mice

정의 줄기 세포 미디어에 Culturing의 retinoblastoma 종양 세포는 제한된 시간 동안 성장 유지 수있는 기본 tumorspheres에 상승을 제공합니다. 원래 종양에 비해 이러한 교양 tumorspheres은 현저하게 다른 세포 phenotypes 발생할 수 있습니다. 교양 세포가 새로운 종양을 형성의 기능을 가지고 데모는 교양 세포 모델에게 원래 종양의 생물학을 보장하는 것이 중요합니다. / - - 면역 결함 쥐들이 여기 Rag2를 사용하여 생체내 인간의 retinoblastoma의 종양 증식을위한 프로토콜을 제시한다. 낮은 통로 또는 murine 눈 유리체 캐비티에 직접 주입 갓 수확 인간 retinoblastoma의 종양에서 얻은 세포의 교양 인간 retinoblastoma의 tumorspheres는 2~4주 이내에 종양을 형성. 이러한 종양은 수확과 중 추가로 생체내에 murine 눈을 passaged이나 체외에서 tumorspheres로 성장하실 수 있습니다. 전파가 성공적으로 추가되므로 실험에 대한 인간의 retinoblastoma 조직의 지속적인 소스를 수립 최소한 세 구절에 대해 실시되었습니다. 웨슬리 S. 본드와 Lalita Wadhwa는 공동 첫번째 작가입니다.

Watch this Scientific Journal Video about Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice at JoVE.com

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

방법은 생쥐에서 인간의 retinoblastoma 종양을 전파하기 위해 설명되어 있습니다. 종양 세포에 직접 면역 결함 생쥐의 눈을에 주입하고 있습니다. 이차 종양이 성공적으로 직접적으로 인간의 종양과 교양 tumorspheres에서 수확을 모두 세포를 사용하여 설립되었습니다.

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 ...

establishment-propagation-human-retinoblastoma-tumors-immune

Research

JoVE Journal

Medicine

11.7K 조회수.  Baylor College of Medicine. 방법은 생쥐에서 인간의 retinoblastoma 종양을 전파하기 위해 설명되어 있습니다. 종양 세포에 직접 면역 결함 생쥐의 눈을에 주입하고 있습니다. 이차 종양이 성공적으로 직접적으로 인간의 종양과 교양 tumorspheres에서 수확을 모두 세포를 사용하여 설립되었습니다.

비디오: Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

검증 된 내시경 채점 시스템을 사용하여 대장염과 대장 종양의 위험을 평가하기 위해 마우스의 유연한 대장 내시경 검사

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

연구

의학

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

면역 본래대로 사람 MUC1 유전자 변형 마우스의 침략 과도 셀 방광 암의 유도 : 면역 요법 개발을위한 모델

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

형광 SAPC-DOPS Nanovesicles을 사용하여 뇌 종양 및 관절염의 생체 광학 이미징

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

라이브 쥐의 대퇴부 골수 흡인

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

만들기 해부학 적으로 정확하고 인간의 두뇌 종양의 재현 두개 내 이종 이식

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

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 ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

닭에서 인간의 다발성 골수종 이종 이식 모델의 설립은 종양의 성장, 침략과 혈관 신생을 공부하기

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

마우스에서 가속 죽상 경화증의 유도: "와이어 부상" 모델

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Cytometry 사용 하 여 인간 지방 조직에서 면역 세포의 특성

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.