This study was supported by ITT (Istituto Toscano Tumori) Grant Proposal 2010.

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The authors declare that they have no competing financial interest.

암줄 기세포는 종양의 대부분이 특정 세포 부분 집합의 식별을 허용하는 여러 속성이 있습니다. 이러한 표현식, ATP 결합 카세트 다제 유출 수송차 28, 32, 33, 또는 ALDH 32 해독 효소의 발현의 상향 조절을위한의 과발현을위한 화학 요법 제를 세포 독성 취득 된 저항 이러한 특성에 기초 된 CSC를 분리 예컨대 CD133, CD44, CD34, CD90, 및 기타 30, 34, 35, 41, 여러 가지의 방법으로서, 특정 표면 마커의 42-44를 개발되었다. 이러한 기술 중 하나는 비 - 부착 조건 하에서 성장 된 CSC의 용량에 기초한 구 형성 분석이다. 구체를 형성하는 조직 줄기 세포 및 CSC 추가의 능력은 제 레이놀즈 등. (37)에 의해 신경줄 기세포의 식별에 관한 연구에서 설명 하였다. 그 후, 깁스 등. (38) 우리를이러한 연구 에디션은 골 육종에서, 특히, 고형 종양에서 암줄 기세포를 분리 시작합니다. 우리는 깁스 등의 알에 도시 된 구 형성 분석 방법을 사용 할 수 있습니다. 기존의 OS 생검에서 얻은 OSA 세포주에서 암줄 기세포를 분리하기로 결정했습니다. 우리는 원래의 방법이 분석의 결과를 개선하기위한 다른 암세포에 대한 재현성을 용이하게하도록 적응. 구 형성 검정의 설립을 참조하여, 우리는 40,000 세포를 도금하는 것은 / 웰의 분석의 시작 부분에서 분리 세포를 유지하는 것이 좋다 것을 확인 하였다. 이 트릭은 구 식민지가 세포 응집하지 않은 부착 조건에서 성장하고 구형 콜로니를 형성하는 하나의 CSC의 특정 독점 용량에서 발생 가능성을 방지하는 것이 매우 중요합니다. 이 기능은이 분석의 특히 중요한 포인트입니다. 또한 구 형성의 양호한 비율을 구하는 것을 증명원래의 방법에 기재된 바와 같이 매일 성장 인자의 분취 량을 매 3 일 아닌 새로 고침하기에 충분하다. 본 연구에서는, 우리는 또한 설립 광범위하게 비 접착 조건에서 배양 할 때 형성되는 구형 콜로니를 분리하기위한 좋은 방법을 설명했다. 이 웰을 손상시키지 않고 각각 형성 가능한 분야의 많은을 분리하려고하는 것이 매우 중요하기 때문에이 단계는이 분석에서 중요하다. 단지 구체 아니라 분석 기간 동안 현탁액에 남아 있었던 단일 세포를 분리하는 것이 중요하다. 이 중요한 점을 극복하기 위해, 우리는 위의 그림과 같이, CSC 격리를위한 좋은 결과를 준, 특정 분리 방법을 개발했습니다. 분명히이 형성하는 모든 분야가 복구 될 수없는 가능성이 있지만, 손실 비율이 매우 낮다. 실제로, 우리는 그들이 결정 (커질 후 구를 분리하여 40 ㎛의 기공 필터를 사용할 수있을에드 약 100-200 세포). 이 격리 영역 형성을 정지하지만, 단 전지의 메틸 잔기의 일부가 가장 작은 구체를 여과 할 수있다. 프로토콜에 기재 한 바와 같이 제거는 철저한 여과에 의해 수행된다. 또한, 가장 작은 구형 콜로니 그에 따른 손실이 40 ㎛의 메쉬를 통해 큰 구형 콜로니의 선택은 하나의 구형 콜로니를 형성 할 수있는 높은 용량과 더 stemness로 된 CSC를 선택할 수 있습니다. 이러한 모든 수정은 분석 향상과 이해 ​​구 형성 검정의 원래있어서 가장 중요한 단계를 재현 된 CSC 공부 연구자 있도록 하였다. 분리 암줄 기세포에 대한 시험 관내 방법에 관한 연구 중 본 연구에서 암줄 기세포를 분리이 적응 구 형성 분석은 좋은 방법이 될 수있는 방법을 보여 목표OSA 세포주. 일본어 방법 및 상세한 분리 기술에 대한 적응은 효능 향상을 설명했다. 단시간에, CSC 추가의 상당수가 얻어 질 수 있고 여러 가지 실험에 사용 하였다. 따라서, 급속 OS-CSC 추가 특징 이중 스템 형 표현형을 연구하기 위해, 특히, 줄기 등의 표현형을 확인하는 것이 가능하다. 따라서,이 수정 된 분석은 암줄 기세포를 분리 및 생물학 연구를위한 좋은 방법이 될 수 있습니다. 미래에,이 방법은 추가로 적응도 희귀 고형 종양의 생검에 의해 얻어지는 다른 유한 암 세포주에서 암줄 기세포를 분리하는데 사용될 수있다. 이러한 OS 희귀 고형 종양에서 CSC 추가 분리 가능성뿐만 아니라, 특정 암에 대한 연구의 개선을 허용하지만, 또한 분리를위한 더 좋은 방법을 개발하는 암의 다양한 유형의 연구 및 생물학의 앞으로의 연구를 확장 이 중요한 세포의 부분 집합. 따라서, 우리와 같은본 연구에서 수행 한, 아마에 대한 책임이 특정 세포의 부분 집합에 관한 매우 구체적인 항암 치료, 분자 표적을 발견하고 개발의 최종 목표로, CSC 생물학의 연구를 통해 CSC 분리의 방법을 개선하는 것이 중요하다 주 종양의 유지 재발의 개발 및 여러 기관에서 전이의 기원. CSC 생물학의 연구는 항암 치료 후 생존율이 매우 가난한 유지하는 등의 OS와 같은 암의 치료에 예리한 될 수 치료법을 찾는 것이 중요합니다.

육종은 배아 중배엽 (1)에서 주로 발생하는 드문 악성 결합 조직 종양의 이종 그룹입니다. 다른 유형은 골 육종 및 연부 조직 육종을 포함한다. 뼈 육종은 비교적 드문 차 종양의 그룹, 골육종 (OS)를 포함한 여러 아형,로 구성되어 있습니다. OS, 뼈의 일반적인 기본 종양 중 하나는, 광범위한 임상 적, 조직 학적 및 분자 이질성 (2, 3)을 나타내는 중간 엽 악성 종양이다. 불행히도, OS 5, 어린이 및 청년 4에서 주로 발생하며 60 %를 차지 어린 시절 뼈 육종의 일반적인 조직 학적 아형은 6, 7. OS는 일반적으로 영향을 빠른 뼈의 성장을 특징으로 골격 영역 (예를 들면, 긴 뼈의 골 간단). 또한 수질 또는 중앙 OS라는 OS, 기존 OS의 조직 학적으로 서로 다른 아형 중 악성 종양의 높은 학년과 할당량 샤가80 % 8 재. 이 80 %는 60 % 종래 골아 OS 10 % chondroblastic OS, 및 10 % 섬유 아세포 OS 6, 8-10로 구성되어있다. 다른 OS 서브 타입은 역 형성, telangiectatic, 거대 세포가 풍부한 작은 세포 OS를 포함한다. 결합 수술 OS 관리 화학 요법의 진보에도 불구하고, 결과는 전이 11,12없이 환자 65-70%의 장기 생존율 불량 남아있다. 먼 재발 자주 전이로, 또는 덜 빈번 폐 전이 발생할 먼 뼈와 지방 재발 13. 전이는 종종 기존 치료에 저항. 이 저항은 10 년 무병 생존율 진단 14, 15, 전이성 질환을 가진 환자의 약 30 %가되는 이유이다. 정상 조직과 같이, 암 조직은 세포 유형의 이종의 집합으로 구성된다. 종양 내 세포 발달의 각 단계에 대응하는 것으로 보인다. 상관 없음 내ormal 조직 따라서 조직 항상성을위한 전구 세포 및 성숙한 세포를 제공 selfrenew 할 수있는 능력 세포의 서브 모집단을 상주. 마찬가지로, 암의 증식과 종양 전위 상이한 정도와 개발의 다른 단계에서 세포의 유사한 이종 집단으로 구성된다. 이러한 암 세포의 서브 세트는 암 줄기 세포 (CSC 추가)를, 종양 부피 (16)를 구성하는 다른 세포 계통을 따라서, 종양의 악성 가능성 selfrenew 및 유지 배타적 능력 세포 selfsustaining 생성 리저버를 구성 되나. 1990 년대에, 급성 골수성 백혈병에 대한 연구는 CSC의 소집단 (17), (18)의 존재에 대한 최초의 강력한 증거를 제공했다. 암줄 기세포는 이후함으로써 암 연구에서 가장 연구 주제 중 하나가되고, 고형 종양 (19)의 큰 숫자에서 고립되었다. 암줄 기세포는 실제로 정상 만드는 유전자의 돌연변이에 의해 정상 줄기 세포에서 발생할 수20-23 암 줄기 세포. 미세 환경과 여러 변형 돌연변이 및 상호 작용은 암줄 기세포를 예표 selfrenewal 용량과 불멸을 획득 ​​건강 전구 세포와 성숙 세포에 기여할 수있다. 이러한 변화에 대한 여러 가지 가설이있다. 건강 선조 건강한 성숙 세포 및 암세포 selfrenewal 관련 유전자 24-28을 활성화하여 줄기 같은 표현형을 획득하는 세포를 줄기 탈분화있다. 몇 가지 최근의 연구에도 불구하고, 암줄 기세포의 기원은 아직 발견해야합니다. 암줄 기세포의 특별한 특징은 능력이 다른 약물과 결합 된 수술과 화학 요법으로 구성 다중 치료 접근 방식을, 저항하는 것입니다. 최근의 연구는 암줄 기세포는 화학 요법 제를 세포 독성에 내성을 획득 할 수 있다는 것을 보여 주었다. 이 저항에 대한 가능한 설명은 ATP 바인딩 카세트 (ABC) 다중 약물 수송의 과발현을 포함한다 (즉,MDR1 및 BCRP1) 알데히드 데 하이드로게나 제 1 (ALDH1) 및 / 또는 세포주기 동력학 30-33 변화로서 항암제 대사 효소의 과발현. 지금까지 설명 된 모든 이러한 개념의 직접적인 결과는 CSC의 하위 집단이 완전히 제거 된 경우에만 단 한 번의 CSC가 살아 경우 국소 재발이나 원격 전이가 발생할 수 있습니다 동안 암 치료가 효과적 일 것입니다. 이 환자 나중에 재발 많은 치료 초기에 효과적인 것으로 보인다 이유에 대한 가능한 설명을 제공하지만, 때문에 인간의 육종 (34), 특히 OS (35), 또는 다른 뼈와 연부 조직 암에서의 암줄 기세포의 발견은 큰 임상 적 중요성을 가지고있다. 따라서, 기존의 OS에 대한 미래의 전투에 대한 희망이 하위의 분자 특성에 OS-암줄 기세포 감사에 관한 혁신적인 약물의 개발에 따라 새로운 특정 표적으로 한 치료를 찾을 수 있습니다인구와 CSC 생물학의 연구에. 1992 년 줄기 세포의 일부는 성숙한 포유 동물의 뇌에 존재 여부를 조사 하였다 레이놀즈와 동료, 줄기 형 셀 (36), (37)로 의심되는 세포를 분리하는 방법을 개발 하였다.이 방법의 특정 기능에 기초 비 접착 조건 하에서 성장 될 때이 세포 구형 콜로니를 형성한다. 비슷한 기술이 골 육종 (38)의 줄기와 같은 세포의 모집단을 연구하기 위해 2005 년에 깁스와 동료에 의해 사용되었다. 분리 종래 OS의 종류의 일차 세포 배양 물로부터 OS-CSC 추가 특성화하기 위해 OS 세포주에이 기술을 적용하기로 결정했다. 여기서는 종래의 OS의 생검 인간 유래의 유한 차 세포주로부터 OS-CSC 추가를 분리하는데 사용될 수있다 &quot;sarcosphere 분석&quot;이라 구 형성 분석이 적응 방법을 서술. 우리는 또한 모든 기술 유를 설명이 분석에 의해 단리 세포주의 줄기 등 CSC 표현형 확인 나오지 : 그리고 암줄 기세포의 마커 인 CD133 유전자의) 다 능성 배아 줄기 세포 (는 ESC를 특성화하는 유전자의 발현의 1) 평가하는 단계; 2) 집락 형성 단위 (CFU) 분석; 3) 적당한 분화 조건에서 조골 세포 및 지방 세포로 분화하도록 이들 세포의 기능 평가; 면역 형광 염색 및 유세포 분석에 의한 중간 엽 줄기 세포 표면 마커 (MSC들) (즉, CD44, CD105 및 STRO-1) ~ 4) 연구; 이들 세포의 활성 ALDH 5) 평가.

<table><tbody><tr><td>Dulbecco&#039;s Phosphate Buffered Saline with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>LONZA</td><td>BE17-513F</td><td>_</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline&amp;nbsp; without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; (DPBS)</td><td>LONZA</td><td>BE17-512F</td><td>_</td></tr><tr><td>Porcine Trypsin 1:250</td><td>BD Difco</td><td>215310</td><td>Solvent: DPBS. Stock concentration: Powder</td></tr><tr><td>Ethylenediamine tetraacetic acid disodium salt dihydrate(EDTA)</td><td>Sigma-Aldrich</td><td>E4884</td><td>Solvent: DPBS. Stock concentration: Powder</td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C0130</td><td>Solvent: Buffer Solution, pH 7.4. Stock concentration: Powder</td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>BDH Chemicals-VWR</td><td>10323</td><td>_</td></tr><tr><td>Nutrient Mixture F-12 Ham</td><td>Sigma-Aldrich</td><td>F6636</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>2-Phospho-L-ascorbic acid trisodium salt</td><td>Sigma-Aldrich</td><td>49752</td><td>Solvent: DPBS. Stock concentration: 5 mg/mL</td></tr><tr><td>&amp;beta;-Glycerol phosphate disodium salt pentahydrate</td><td>Sigma-Aldrich</td><td>50020</td><td>Solvent: DPBS. Stock concentration: 1 M</td></tr><tr><td>Insulin. Human Recombinant</td><td>Sigma-Aldrich</td><td>91077</td><td>Solvent: NaOH 0.1 M. Stock concentration: 10 mM</td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I5879</td><td>Solvent: DMSO. Stock concentration: 500 mM</td></tr><tr><td>Indomethacin</td><td>Sigma-Aldrich</td><td>I7378</td><td>Solvent: DMSO. Stock concentration: 200 mM</td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td>Solvent: DMSO. Stock concentration:&amp;nbsp; 1 mM / 100 &amp;micro;M. Store in liquid nitrogen to maintain the biological activity</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td>_</td></tr><tr><td>Fetal Bovine Serum South America&amp;nbsp;</td><td>EUROCLONE</td><td>ECS0180L</td><td>_</td></tr><tr><td>Penicillin-Streptomycin (PEN-STREP) 10,000 U/mL</td><td>LONZA</td><td>DE17-602E</td><td>_</td></tr><tr><td>Methyl cellulose</td><td>Sigma-Aldrich</td><td>274429</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: 2%</td></tr><tr><td>Putresceine dihydrochloride</td><td>Sigma-Aldrich</td><td>P5780</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>apo-Transferrin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T-1147</td><td>Solvent: DPBS. Stock concentration: 25 mg/mL</td></tr><tr><td>Human Epidermal Growth Factor (EFGF)</td><td>Sigma-Aldrich</td><td>E5036</td><td>Solvent: DPBS pH 7.4. Stock concentration: 10 &amp;micro;g/mL</td></tr><tr><td>Fibroblast Growth Factor-Basic Human</td><td>Sigma-Aldrich</td><td>F0291</td><td>Solvent: DPBS + 0.2% BSA. Stock concentration: 25 &amp;micro;g/mL</td></tr><tr><td>Selenous Acid</td><td>Sigma-Aldrich</td><td>211176</td><td>Solvent: DPBS. Stock concentration: 30 mM</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P8783</td><td>Solvent: ETOH. Stock concentration: 10 mM</td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>198161</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>Oil Red O</td><td>ICN Biochemicals</td><td>155984</td><td>Solvent: 2-Propanol. Stock concentration: Powder</td></tr><tr><td>Naphtol AS-MX Phosphate Disodium Salt</td><td>Sigma-Aldrich</td><td>N5000</td><td>Solvent: DMSO. Stock concentration: Powder</td></tr><tr><td>Fast Blue BB Salt</td><td>Sigma-Aldrich</td><td>F3378</td><td>Solvent: Tris HCL, pH 9.0. Stock concentration: Powder</td></tr><tr><td>Fast Red Violet LB Salt</td><td>Sigma-Aldrich</td><td>F3381</td><td>Solvent: Tris HCL, pH 9.1. Stock concentration: Powder</td></tr><tr><td>Bovine Serum Albumin, Fraction V (BSA)</td><td>Sigma-Aldrich</td><td>A-4503</td><td>Solvent: DPBS. Stock concentration: 2%</td></tr><tr><td>Alizarin Red S</td><td>ICN Biochemicals</td><td>100375</td><td>Solvent: Ultrapure dH&lt;sub&gt;2&lt;/sub&gt;O. Stock concentration: Powder</td></tr><tr><td>Formaldehyde solution</td><td>Sigma-Aldrich</td><td>533998</td><td>4%</td></tr><tr><td>Triton-100X</td><td>MERCK</td><td>11869</td><td>Solvent: DPBS. Stock concentration: 0.2%. Danger - Use only under chemical hood</td></tr><tr><td>Calcein</td><td>MERCK</td><td>2315</td><td>Solvent: DPBS . Stock concentration: 200 &amp;micro;g/mL</td></tr><tr><td>2-Propanol</td><td>MERCK</td><td>109634</td><td>Danger - Use only under chemical hood</td></tr><tr><td>Ab-CD105&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal [SN6] to CD105 (FITC)</td><td>Abcam</td><td>ab11415</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD44&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; [F10-44-2] to CD44 (PE/Cy7&amp;reg;) )</td><td>Abcam</td><td>ab46793</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD45&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse monoclonal&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; [MEM-28] to CD45 (PerCP))&amp;nbsp;</td><td>Abcam</td><td>ab65952</td><td>Liquid. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)</td></tr><tr><td>Ab-CD105&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Human CD105 Purified Antibody)&amp;nbsp;</td><td>Invitrogen</td><td>MHCD10500</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>Ab-CD44&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Anti-CD44 Antibody)&amp;nbsp;</td><td>Abcam</td><td>EPR1013Y(ab51037)</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>Ab-Stro-1&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Mouse anti-STRO-1)&amp;nbsp;</td><td>Invitrogen</td><td>398401</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Immunofluorescence staining (IF)</td></tr><tr><td>Alexa Fluor 488&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (Anti-Rabbit IgG (Alexa Fluor 488 Donkey Anti-Rabbit IgG (H+L))</td><td>Invitrogen</td><td>A-21206</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>FITC Anti-Mouse IG (FITC-Rabbit Anti Mouse IgG (H+L))</td><td>Invitrogen</td><td>61-6511</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application:&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Flow Cytometry (Flow Cyt)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Immunofluorescence staining (IF)</td></tr><tr><td>Alexa Fluor 635 Phalloidin&amp;nbsp;</td><td>Invitrogen</td><td>A34054</td><td>Solvent: DPBS. Stock concentration: Lyophilized. Application: Immunofluorescence staining (IF)</td></tr><tr><td>AutoMACS&amp;trade;Running Buffer MACS Separation Buffer</td><td>Miltenyi</td><td>130,091,221</td><td>Liquid. Store at 4 &amp;deg;C</td></tr><tr><td>QIAzol&amp;reg;Lysis Reagent</td><td>QIAGEN</td><td>79306</td><td>Danger - Use only under chemical hood</td></tr><tr><td>QUANTITECT&amp;reg;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Reverse Transcription Kit</td><td>QIAGEN</td><td>205314</td><td>_&amp;nbsp;</td></tr><tr><td>Chlorophorm</td><td>Sigma-Aldrich</td><td>C2432</td><td>Liquid. Danger - Use only under chemical hood</td></tr><tr><td>Laminar flow hood</td><td>GELAIRE</td><td>BSB6A</td><td>_&amp;nbsp;</td></tr><tr><td>Chemical hood</td><td>ARREDI TECNICI&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Villa</td><td>Modello&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; DYNAMICA</td><td>_&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>EPPENDORF</td><td>5415R</td><td>_&amp;nbsp;</td></tr><tr><td>Laser Scanning Confocal Microscopy LSM 5109 Meta&amp;nbsp;</td><td>ZEISS</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>iCycler PCR Thermalcycler&amp;nbsp;</td><td>BIORAD</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>CyFlow&amp;reg;SPACE&amp;nbsp;</td><td>(PARTEC)</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Inverted Micrposcope Axiovert 200M&amp;nbsp;</td><td>ZEISS</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Freezing container ,</td><td>Nalgene</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Original Pipet-Aid</td><td>pbiBrand</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Micropipettes</td><td>EPPENDORF</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Glass Pasteur Pipette</td><td>SIGMA</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>VICTOR3&amp;trade;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>PERKIN ELMER</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Conical tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (15 and 50 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>352096 (for 15 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 352070 (for 50 mL)</td><td>_&amp;nbsp;</td></tr><tr><td>24-Well Clear Flat Bottom TC-Treated Multiwell-Cell-Culture-Plate</td><td>BD FALCON</td><td>353047</td><td>_&amp;nbsp;</td></tr><tr><td>6-Well Clear Flat Bottom Ultra Low Attachment Multiple-Well-Plates</td><td>CORNING</td><td>3471</td><td>_&amp;nbsp;</td></tr><tr><td>Serological pipettes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (5 and 10 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>357543 (for 5 mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 357551 (for 10mL)</td><td>_&amp;nbsp;</td></tr><tr><td>Syringe (5mL)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>B|BRAUN</td><td>4617053V</td><td>_&amp;nbsp;</td></tr><tr><td>Petri dish 100X20 mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353003</td><td>_&amp;nbsp;</td></tr><tr><td>R&amp;ouml;hren Tubes&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; (3.5 mL, 55x12mm, PS)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>SARSTEDT</td><td>55,484</td><td>_&amp;nbsp;</td></tr><tr><td>Petri dish 60X15 mm&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353004</td><td>_&amp;nbsp;</td></tr><tr><td>Cryovials 1.5 mL&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>NALGENE</td><td>5000-1020</td><td>_&amp;nbsp;</td></tr><tr><td>Cell-Culture Flasks 25 cm&amp;sup2;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>BD FALCON</td><td>353014</td><td>_&amp;nbsp;</td></tr><tr><td>Nylon Net Filter, Hydrophilic</td><td>MERCK</td><td>NY4104700</td><td>_&amp;nbsp;</td></tr><tr><td>Swinnex Filter Holder</td><td>MERCK</td><td>SX0002500</td><td>_&amp;nbsp;</td></tr><tr><td>Perry tweezer&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Lancet</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td><td>_&amp;nbsp;</td></tr><tr><td>Dounce</td><td>_</td><td>_</td><td>_</td></tr></tbody></table>

establishment of cancer stem cell cultures from human conventional osteosarcoma

골육종 (OS)에 대한 치료에 현재 향상 암 환자의 생명을 연장했지만 전이가 발생했을 때, 5 년 생존율은 여전히 ​​좋지. 암 줄기 세포 (CSC) 이론은 종양을 유지하고 약제 요법에 저항 할 수있는 능력을 포함한 줄기 같은 특성을 갖는 종양 내 종양 세포의 서브 세트가 있음을 보유하고있다. 따라서, OS의 생물학 및 병인에 대한 이해는 특정 서브 세트를 박멸 및 환자군 질병률 및 사망률을 감소시키는 치료 표적의 개발을 진행시키기 위해 필요하다. , 암줄 기세포를 분리 암줄 기세포의 세포 배양을 수립, 그들의 생물학을 공부하는 것은 OS 생물학 및 병인에 대한 우리의 이해를 향상시키는 중요한 단계입니다. OS의 생검에서 인간 유래 된 CSC-OS의 설립 nonadhere 하에서 3 차원 줄기 세포 배양 물을 만들 수있는 능력을 포함하여, 여러 가지 방법을 사용 가능하게 된NT 조건. 이러한 조건에서, CSC 추가 딸 줄기 세포에 의해 형성된 콜로니 구형 플로팅을 만들 수있다; 이 식민지는 &quot;세포 분야&quot;라고되어있다. 여기서는 OS 생검에서 얻어진 종래의 OS의 일차 세포 배양 물로부터 CSC 배양을 설정하는 방법을 설명한다. 우리는 명확하게 암줄 기세포를 분리하고 특성화하는 데 필요한 여러 구절을 설명합니다.

Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

뼈 육종 암 줄기 세포의 존재 (CSC 추가)는 최근에 발병에 연결되어있다. 이 글에서, 우리는 비 접착 조건에서 성장하는 암줄 기세포의 능력을 사용하여 기존의 골육종 (OS)의 인간 생체 검사에서 얻은 기본 세포 배양에서 암줄 기세포의 분리를 제시한다.

암의 설립은 인간 기존의 골육종에서 세포 배양 줄기

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

establishment-cancer-stem-cell-cultures-from-human-conventional

Research

JoVE Journal

Cancer Research

18.9K 조회수.  University of Florence.  뼈 육종 암 줄기 세포의 존재 (CSC 추가)는 최근에 발병에 연결되어있다. 이 글에서, 우리는 비 접착 조건에서 성장하는 암줄 기세포의 능력을 사용하여 기존의 골육종 (OS)의 인간 생체 검사에서 얻은 기본 세포 배양에서 암줄 기세포의 분리를 제시한다. 

비디오: Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

환자에서 파생 된 연 조직 육 종의 기본 문화 설립

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

연구

암 연구학

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

육종 환자 유래 이종이식에서 종양 을 발하는 세포의 격리 및 특성화

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

부인과 및 유방암 종양에서 암 줄기 세포 구체 획득

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

마우스 횡문근에서 분리 된 원발성 종양 세포에서 종양 구체 유도 및 치료

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

유전학

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

의학

An Orthotopic Mouse Model of Ovarian Cancer using Human Stroma to Promote Metastasis

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant transgenic mouse models of ovarian cancer exist, these mice are expensive and take a long time to develop tumors. Intraperitoneal injection xenograft models lack human stroma and do not accurately model ovarian cancer metastasis. Even patient derived xenografts (PDX) do not fully recapitulate the human stromal microenvironment as serial PDX passages demonstrate significant loss of human stroma. The ability to easily model human ovarian cancer within a physiologically relevant stromal microenvironment is an unmet need. Here, the protocol presents an orthotopic ovarian cancer mouse model using human ovarian cancer cells combined with patient-derived carcinoma-associated mesenchymal stem cells (CA-MSCs). CA-MSCs are stromal progenitor cells, which drive the formation of the stromal microenvironment and support ovarian cancer growth and metastasis. This model develops early and diffuses metastasis mimicking clinical presentation. In this model, luciferase expressing ovarian cancer cells are mixed in a 1:1 ratio with CA-MSCs and injected into the ovarian bursa of NSG mice. Tumor growth and metastasis are followed serially over time using bioluminescence imaging. The resulting tumors grow aggressively and form abdominal metastases by 14 days post injection. Mice experienced significant decreases in body weight as a marker of systemic illness and increased disease burden. By day 30 post injection, mice met endpoint criteria of &gt;10% body weight loss and necropsy confirmed intra-abdominal metastasis in 100% of mice and 60%-80% lung and parenchymal liver metastasis. Collectively, orthotopic engraftment of ovarian cancer cells and stroma cells generates tumors that closely mimic the early and diffuse metastatic behavior of human ovarian cancer. Furthermore, this model provides a tool to study the role of ovarian cancer cell: stroma cell interactions in metastatic progression.

<meta charset="utf8"></head><body>전이를 촉진하기 위해 인간 기질을 사용하는 난소암의 정소성 마우스 모델

Orthotopic engraftment of ovarian cancer cells mixed with human stromal cells provides a mouse model that exhibits rapid, diffuse metastatic behavior that is characteristic of human ovarian cancer. This model also allows for the study of tumor cell and stromal cell interactions, as well as their role in tumor progression and metastasis.

전이를 촉진하기 위해 인간 기질을 사용하는 난소암의 정소성 마우스 모델

Ovarian cancer is characterized by early, diffuse metastasis with 70% of women having metastatic disease at the time of diagnosis. While elegant ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

인간 세포주에서 Chemoresistant 난소 암 줄기 세포에 대한 심화 학습

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

암의 설립은 인간 기존의 골육종에서 세포 배양 줄기

요약

더 많은 비디오 탐색

이 비디오의 챕터

환자에서 파생 된 연 조직 육 종의 기본 문화 설립

육종 환자 유래 이종이식에서 종양 을 발하는 세포의 격리 및 특성화

부인과 및 유방암 종양에서 암 줄기 세포 구체 획득

마우스 횡문근에서 분리 된 원발성 종양 세포에서 종양 구체 유도 및 치료

환자 유래 세포 배양 및 CD133의 절연

전이를 촉진하기 위해 인간 기질을 사용하는 난소암의 정소성 마우스 모델

전이를 촉진하기 위해 인간 기질을 사용하는 난소암의 정소성 마우스 모델

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

인간 세포주에서 Chemoresistant 난소 암 줄기 세포에 대한 심화 학습

다중 및 단일 세포 기반 분야 분석 실험을 사용하여 인간의 난소 암 세포의 줄기 세포 특성의 평가