Name: establishment of cancer stem cell cultures from human conventional osteosarcoma
Uploaded: 2016-10-14T13:00:00.000+00:00
Duration: 9 min 25 s
Description: Watch this Scientific Journal Video about Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma at JoVE.com

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.

的CSCs具有几个特性，允许在肿瘤散装这个特定的蜂窝子集的标识。上的这些特征，例如获得性抗性细胞毒性化疗药物为ATP结合盒多药转运蛋白28，32，33，或用于解毒酶如ALDH 32的表达的上调的表达，为表达的基础特定表面标记，如CD133，CD44，CD34，CD90，和其他30，34，35，41，几种不同的方法来分离的CSCs已经开发了42-44。这些技术之一是球形成测定法，它是基于的CSCs的非贴壁条件下生长的能力。 组织干细胞和肿瘤干细胞的形成球体的能力中，第一次研究由Reynolds等37描述对神经干细胞的鉴定。随后，吉布斯等人。我们38编这些研究，开始从实体瘤中分离的CSCs，特别是从骨肉瘤。我们已经决定使用由吉布斯等人出的球形成测定方法。从常规活检OS OSA获得的细胞系的CSC隔离。我们适于原来的方法来改善该测定法的结果，并促进其用于其它癌细胞系的再现性。参照建立球体形成实验中，我们证实了电镀40,000个细胞/孔为在测定开始时保持在隔离细胞好的做法。这招是非常重要的，以避免球状菌落从蜂窝聚集和不能从一个单一的CSC的特定和独特的能力非粘附的条件下生长，并形成一个球面菌落发起的可能性。这种能力是该测定的一个特别关键点。 我们还证明，要获得的球形成一个良好率，这是足以作为在原始方法中描述每天刷新的生长因子等份每3天，不。在这项研究中，我们还建立并广泛描述的好方法分离时非粘附的条件下培养所形成的球形菌落。这一步骤是在该测定中非常重要，因为这是非常重要的，试图尽可能多的球体尽可能在每个中形成的的隔离良好而不损坏它们。同样重要的是隔离仅球体而不是单细胞，这可能留在悬浮液用于测定的持续时间。为了克服这些关键点，我们已经开发出一种特殊的分离方法，其中，如上图所示，给了CSC隔离了良好的效果。显然，是并非所有所形成的球体可以回收的可能性，但损失百分比非常低。事实上，我们也有可能使用过滤器40微米毛孔隔离球，他们会变大（表后编约100 - 200个细胞）。 这种隔离停止球体形成，但是允许待过滤的单细胞，甲基纤维素残基的一部分，并且最小球体。这消除由彻底过滤在协议中所述进行。 此外，通过40微米网状具有最小球面菌落随之损失最大球形菌落的选择允许一个带，以形成球形菌落最高容量并以更大的干性选择的CSCs。所有这些修改进行，以提高该测定，并帮助研究人员在研究的CSCs理解和重现球体形成测定的原始方法的最关键的步骤。 在体外方法对分离肿瘤干细胞研究，这项研究的目的是显示这个球改编形成实验如何是一个很好的方法，从分离肿瘤干细胞OSA细胞系。到原来的方法和详细隔离技术改编所述提高其疗效。在很短的时间，可以得到良好的数目的CSCs的并用于多种实验。因此，能够迅速地确认干细胞样表型，尤其是，要研究双茎样表型表征操作系统的CSCs。因此，这个修改后的分析可能是孤立肿瘤干细胞和研究其生物学一个很好的技术。在未来，该方法中，与另外的修改，也可用于分离从由稀有实体肿瘤的活组织检查获得的其他有限癌细胞系的CSCs。 隔离罕见实体瘤，如OS的CSCs的可能性，不仅允许左右这个特定癌症研究改善，但也延伸到不同类型的癌症，开发其隔离更好方法的研究和对生物学的未来研究这个重要的细胞子集。因此，在我们在这项研究中所做的那样，它通过CSC生物学的研究，以改善CSC隔离的方法中，以找到分子靶和显影针对这个特定的蜂窝子集的非常具体的抗癌疗法，这可能是负责的最终目标是非常重要的维持原发肿瘤，其复发的发展，和转移的若干器官的起源。 CSC生物学的研究也是寻找治疗方法，可能是癌症，如操作系统，为此，新辅助治疗后的存活率仍然非常差的固化精辟重要。

肉瘤是罕见的恶性结缔组织肿瘤从胚胎中胚层1主要源于一组异质性。不同类型的包括骨肉瘤和软组织肉瘤。骨肉瘤，一组比较少见的原发肿瘤，由若干亚型，包括骨肉瘤（OS）的。操作系统，骨中最常见的原发性肿瘤中的一个，是间充质恶性表现出广泛的临床，组织学和分子非均质2,3。不幸的是，操作系统主要是在儿童和年轻成人4时，5和代表60％的在儿童骨肉瘤的常见的组织学亚型6,7。操作系统通常影响骨骼区域，其通过快速骨生长特征（ 例如 ，长骨的干骺端）。在OS，OS的常规，也称为延髓或中央OS的组织学不同的亚型，具有恶性肿瘤的高品位和配额SHA重的80％的8。这80％的由60％的常规的成骨细胞的OS，10％软骨细胞的OS，以及10％的成纤维细胞的OS 6，8-10。其他操作系统亚型包括间变性，毛细血管扩张，巨细胞丰富和小细胞操作系统。尽管联合手术，并在OS的管理化疗的进步，结果仍然较差，患者65-70％长期存活率无转移11,12。远处复发频繁发生作为肺转移或，较不频繁，如转移到遥远的骨骼和局部复发13。转移通常对常规治疗有抗性。这种阻力就是为什么10年无病生存率是转移性肿瘤患者约30％在诊断14，15的原因。 与正常组织，癌组织组成的细胞类型的异质集合。肿瘤内细胞似乎对应于不同的发展阶段。在任意normal组织驻留的细胞亚群与到selfrenew的能力，从而为组织稳态祖细胞和成熟细胞。同样地，癌症是在不同的发展阶段组成细胞的类似异质群体，具有不同程度的增殖和致瘤性的。这些癌细胞的子集，被称为癌症干细胞（CSCs的），构成自保持细胞到selfrenew和维持肿瘤的恶性潜能异能力，从而产生了构成肿瘤本体16上的不同的细胞谱系的一个储存器。在20世纪90年代，在急性髓细胞白血病的研究为CSC亚群17，18的存在提供了第一个令人信服的证据。社区体育会，因为从实体瘤19的大量的隔离，从而成为在癌症研究中研究最多的话题之一。肿瘤干细胞可能确实是从正常干细胞的突变的基因，使正常的出现癌干细胞20-23。多重转化突变和互动的微环境也可促进健康的祖细胞和成熟细胞获取自我更新能力和不朽的典型化CSCs的。大约有这一转型几种假说。健康祖细胞，健康成熟细胞和癌细胞，可以去分化干细胞，通过激活自我更新相关基因24-28获得干细胞样表型。尽管最近的几项研究，肿瘤干细胞的起源尚未被发现。 肿瘤干细胞的一个具体特点是，他们的能力抵御多的治疗方法，其中包括联合手术和化疗不同的药物。最近的研究表明，肿瘤干细胞也可能获得耐细胞毒性化疗药物。对于该电阻可能的解释包括ATP结合盒（ABC）多药转运的过表达（即MDR1和BCRP1），化疗代谢酶如醛脱氢酶1（ALDH1），和/或在细胞周期动力学30-33的变化的过表达。的已迄今描述的所有这些概念的直接后果是，癌症治疗将是有效的只有当CSC亚群被完全消除，而如果甚至单个CSC存活局部复发或远处转移可能发生。 的CSCs在人类肉瘤34，特别是操作系统35，或者在任何其他的骨和软组织癌症，发现具有重要的临床意义，因为它提供了一个可能的解释，为什么许多治疗似乎是有效的最初，但患者后来复发。因此，反对传统的OS未来战场的希望是基于对以下操作系统的CSCs由于定向到此子的分子特性的创新药物的发展寻找新的和具体的有针对性的治疗人口和CSC生物学研究。 在1992年，Reynolds和同事，谁正在调查的干细胞的一个子集是否存在于成年哺乳动物脑，研制分离怀疑是干细胞样细胞36,37的细胞的方法，该方法是基于对所述特定能力当非粘性的条件下生长，这些细胞以形成球形菌落。类似的技术被吉布斯和同事在2005年用于研究干细胞样细胞在骨肉瘤38亚群。分离并从不同类型的常规操作系统的原代细胞培养表征操作系统的CSCs，我们决定以适应该技术对于OS的细胞系。 这里，我们描述了球形成测定的这种适配方法，称为"sarcosphere测定法"，它可用于操作系统的CSCs从常规操作系统的人类活检衍生有限原代细胞系分离。我们还描述了所有技术üsed将验证由该测定分离的细胞系的干细胞样的CSC型:1）表征多能胚胎干细胞（ESCs的基因的表达的评价）与CD133基因，这是肿瘤干细胞的标志物的; 2）集落形成单位（CFU）测定; 3）的这些细胞分化为适当的分化条件下的成骨细胞和脂肪细胞的能力评价; 4）通过免疫染色和流式细胞仪分析间质干细胞的表面标记（MSCS）（ 即，CD44，CD105和STRO-1）的研究; 5）这些细胞的ALDH活性的评估。

<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

在与骨肉瘤（OS）的治疗目前的改进延长癌症患者的生命，但已发生转移时的五年存活率仍然不佳。癌症干细胞（CSC）理论认为有肿瘤细胞具有干细胞样的特性，包括以维持肿瘤和抗多种药物化疗的能力的肿瘤内的一个子集。因此，需要以推进靶向治疗的发展，以消除这一特定子集，以减少患者的发病率和死亡率OS生物学和发病机制的更好的理解。隔离的社区体育，社区体育建立的细胞培养，并研究其生物学对提高我们的操作系统生物学和发病机制的认识的重要步骤。源于人的操作系统的CSCs的从操作系统的活检建立已成为可能使用几种方法，其中包括以下创建nonadhere 3维的干细胞培养物的能力NT的条件。在这些条件下，肿瘤干细胞是能够创建由子干细胞形成的球形浮菌落这些殖民地被称为"细胞球"。在这里，我们描述了建立从OS活检获得常规操作系统的原代细胞培养CSC培养的方法。我们清楚地描述了分离和鉴定肿瘤干细胞所需的几个段落。

通过针穿刺或手术切除肿瘤的一小部分的获得的OS样品（ 图1A，B）准许只有一个OSA的隔离如果治疗精确地说，如（ 图2A，B），在协议部分中描述。不幸的是，从活检分离的细胞的数量是低，与来自30的输出范围 - 50％。输出取决于类型和活检（ 图5A，B）的尺寸。这些细胞必须精确处理。因此，约一个月是必要的原代培养，以100毫米的培养皿达到汇合。在此时间后，OSA从两个OS样品标记得到OSA5和OSA6（ 图6A，B）。然后，有必要传代培养的初级细胞系，以获得细胞的足够数目来执行表征分析和冷冻保存的细胞林上课。在传代培养的第 3代，当两个OSA初级细胞系达到汇合，它们被镀在用于sarcosphere测定6孔超低附着板。使用这种类型的板，因为它可以使我们保持在悬浮状态的细胞，以防止附着介导的分化的干细胞，以防止锚定依赖性细胞分裂，最后，以减少附着于基板。因此，它们的使用使我们能够创建用于癌细胞紧张状况，这是必要的肿瘤干细胞的选择。在测定开始后24小时，细胞显得彼此（ 图7）隔离。监测测定的进展的7天之后，小球状的菌落已开始形成并是可见的（ 图8）。在28天，都形成在每个孔中的几个大球形菌落可以观察到（ 图9A，B）。经过sarcospheres甲肝Ë被用于28天培养，这些大球形菌落可被分离。 图10示出了用于从6孔超低附着板分离sarcospheres和粘附条件下reculturing它们的步骤。 图11示出了分离后的浮动球形菌落。镀在正常安装板的大球形殖民地显示隔离后贴壁扩张（ 图12A，B）。从单个sarcospheres扩大细胞是可能的癌细胞与干细胞样表型。因此，分离后，很可能获得OS-肿瘤干细胞。这些细胞被命名为OSA5-的CSCs和OSA6-的CSCs（ 图13A，B）。 在这一点上，有必要与所获得的两个OS-CSC线干细胞样表型的表征来进行，如上所述。对于干细胞样表型WER的表征的分析E对于每个OS-CSC线sarcospheres分离后亚文化的第 4通道上执行。两个细胞系，OSA5-的CSCs和OSA6-的CSCs，显示出对表面的MSC标记强阳性（CD105和CD44）（ 图14A，B和图15A，B），而它们显示出对表面的MSC标记中度阳性STRO- 1（图16A，B）。我们的观察已通过与商业和分化结肠癌细胞系HCT8（ 图14C，图15C，图16C）中得到否定的结果证实。观察到完全没有在HCT8细胞系，这些表面标志物特异性和非特异性染色。 为了评估这两个操作系统的肿瘤干细胞的表型的MSC，我们还进行了流式细胞分析。无论是OS-CSC线表达高水平的CD44和CD105的。然而，在这两种细胞系的细胞中，只有1.14％表示的Stro-1。因此，该重新SULT证实的Stro-1的适度存在通过免疫荧光染色所证明。与此相反，OSA5-的CSCs的99.62％表达CD44和这些细胞的87.38％表达CD105;在OSA6-的CSCs的99.88％，在表达CD44和这些细胞的95.79％表示CD105。此外，这两种细胞系是CD45 - 。 我们评估了3 ESC标志物（Nanog的10月3/4，Sox2的）和CD133基因，另一个的CSCs标记，通过RT-PCR的表达。我们注意到，所有这些基因在两个OS-CSC线（ 图17）表达。成脂和成骨分化的实验表明这两个孤立的OSA-CSC线分化为成骨细胞（ 图18A - D和 图19A - D）的能力，并为脂肪细胞（ 图20A - D）。 此外，CFU一个<html> SSAY（ 图21）显示出的克隆效率良好的速率，与OSA5-的CSCs 13％，对于OSA6-CSC 14％。最近的几项研究表明，高水平的ALDH活性的是各种癌症的特征。此参数可作为癌症干细胞标记物，并与预后不良相关。所述ALDH活性测定表明，两个OS-CSC线具有高水平的ALDH活性（ 图22）的，而在被用作该测定的阴性对照的成纤维细胞系的低量化限观察ALDH活性。 <img alt="图1" src="/files/ftp_upload/53884/53884fig1.jpg" /> 图1. OS活检样品的例子。（A）。通过针吸活检获得的样本。 （B）。通过手术切除肿瘤的一部分的获得活检样本。884 / 53884fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53884/53884fig2.jpg" /> 图2.在OS样品的机械解体。（A）中使用佩里镊子和刺血针的样品的碎片。悬浮在CM（箭头所示），（B）片段。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53884/53884fig3.jpg" /> 图3.设备及消耗品的所需来隔离Sarcospheres。（A）。所需的所有细胞分离的设备。 1.无菌注射器用无菌膜过滤持有人; 2.两个不同的媒体:通用汽车第二SCGM; 3.无菌玻璃巴斯德吸管。装配在支撑注射器，用膜过滤器架的（B）的细节。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53884/53884fig4.jpg" /> 图4.过滤股。装配过滤单元（A）所需的几个组件（净滤波器由箭头指示）。净器（B）的40微米的网眼（1个网眼由箭头指示）的相位差观察。原始放大倍数:10X。该过滤单元组装（C - D）。过滤装置灭菌（E）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig4large.jpg" target="_blank">请点击此处查看大图已经</a>这个数字rsion。 <img alt="图5" src="/files/ftp_upload/53884/53884fig5.jpg" /> 图5.传统的操作系统的原代细胞培养，高品位的操作系统原代细胞培养的相衬观察。在（A），几个明亮的碎骨是可见的，而在（B），几个小圆角和浮动红细胞都存在。原始放大倍数:10X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53884/53884fig6.jpg" /> 图6.传统的有限骨肉瘤细胞系（OSA）。（A）OSA5和（B）OSA6。观察相衬。原始放大倍数:10X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53884/53884fig7.jpg" /> 图7. OSA5和OSA6的Sarcosphere测定从测定开始24小时后，将细胞浮，并从彼此分离的（细胞通过箭头表示）。观察在相衬。原始放大倍数:20X <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/53884/53884fig8.jpg" /> 图8. OSA5的Sarcosphere分析和OSA6 7 D. 7天进入实验，几个小spherica由单个细胞包围升菌落可以观察到。该sarcospheres（一些sarcospheres由箭头表示）出现浮在中期或小幅回落落户到井的底部。观察在相衬。原始放大倍数:20X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图9" src="/files/ftp_upload/53884/53884fig9.jpg" /> 图9. OSA5的Sarcosphere测定和OSA6 28 D. 28天之后，几个大琥珀sarcospheres在每个孔中的每个OSA细胞系，OSA5（A）和OSA6（B）的板的观察。酒吧尺寸:100微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;/ P&gt; <img alt="图10" src="/files/ftp_upload/53884/53884fig10.jpg" /> 图10.经文为Sarcospheres的分离从6孔超低附着板用于分离sarcospheres的步骤和粘合条件下的reculturing示（A）中所需要的隔离的所有设备。: 1.一个6孔超低板在每口井，2注射器网过滤器支架，3吸管，4 1000μL无菌针头，5。2无菌镊子佩里，6 2种不同的培养基形成sarcospheres 7.无菌巴斯德吸管8培养皿。 sarcospheres（ 二）集合。中所包含的每孔培养基用移液管，用无菌1000微升尖端收集。 （℃）收集在注射器中的悬浮液中。所收集的悬浮液转移到注射器使用开始自然过滤过程膜过滤器支架。 （D） 自然过滤。 （E）的拆卸从注射器的膜过滤器保持器。所有的悬浮液过滤后，净过滤器保持器被拆开并放在培养皿; （F） 拆卸膜过滤器支架。 Sarcospheres被包含在网过滤器保持器的净过滤器的孔，因此它们必须使用佩里镊子被释放。 （G，H） 从膜过滤sarcospheres的去除效果。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图11" src="/files/ftp_upload/53884/53884fig11.jpg" /> 图11. Sarcosphere隔离。隔离sarcospheres漂浮在60毫米培养皿中。观察在相衬。原始放大倍数:40X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig11large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图12" src="/files/ftp_upload/53884/53884fig12.jpg" /> 图12. Sarcosphere从OSA5（A）和OSA6（B）的下列再引入和在单层粘附的条件下，在48小时后隔离reculturing细胞系在贴壁膨胀的开始分离。Sarcospheres后 。观察在相衬。原始放大倍数:20X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig12large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图13" src="/files/ftp_upload/53884/53884fig13.jpg"/&gt; 图13. 在Sarcospheres 7天隔离后 。从OSA5（A）和OSA6（B）的细胞系Sarcospheres表明粘附扩张以下再引入和在单层粘附的条件下，在7天的隔离后reculturing。观察在相衬。原始放大倍数:20X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig13large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图14" src="/files/ftp_upload/53884/53884fig14.jpg" /> 图14.免疫荧光染色为CD105。在OSA-CSC线免疫荧光染色为CD105 OSA5-的CSCs（A）和OSA6-的CSCs（B）和在连续细胞系HCT8（℃），将其用作阴性对照。激光共聚焦显微镜常规颜色:绿色CD105和红色的骨架。原始放大倍数:10X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig14large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图15" src="/files/ftp_upload/53884/53884fig15.jpg" /> 图15.免疫荧光染色为CD44。在OSA-CSC线免疫荧光染色为CD44 OSA5-的CSCs（A）和OSA6-的CSCs（B）和在连续细胞系HCT8（℃），将其用作阴性对照。 LSCM传统颜色:绿色，CD44和红色的骨架。原始放大倍数:10X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig15large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图16. Stro1。免疫荧光染色的免疫荧光染色为STRO-1在OSA-CSC线OSA5-的CSCs（A）和OSA6-的CSCs（B）和在连续细胞系HCT8（℃），将其用作负极控制。 LSCM传统颜色:绿色STRO-1和红色骨架。原始放大倍数:10X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig16large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图17" src="/files/ftp_upload/53884/53884fig17.jpg" /> 图17.核ESC标记和的CD133基因，RT-PCR检测显示Nanog的10月3/4，Sox2的和CD133在OSA5-CSCs的（A）和OSA6-的CSCs（表达表达 <s仲&gt; B）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig17large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图18" src="/files/ftp_upload/53884/53884fig18.jpg" /> 图18.成骨分化分析- ALP在0 D（A，B）和后使用固蓝BB的ALP通过细胞化学染色所确定的诱导10天（C，D），成骨分化。在蓝，ALP +细胞;在红色，细胞核复染色用碘化丙锭。综合观察在明和荧光。原始放大倍数:20X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig18large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图19"SRC ="/文件/ ftp_upload / 53884 / 53884fig19.jpg"/&gt; 图19.成骨分化分析- HA成骨在0天分化（A，B）和后通过细胞化学染色与茜素红S.确定羟基磷灰石（HA）诱导的20天（C，D），这些细胞在对比HA的蓝/灰色，颗粒感存款被染成红色。观察在相衬。原始放大倍数:40X。酒吧尺寸:100微米<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig19large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图20" src="/files/ftp_upload/53884/53884fig20.jpg" /> 图20.成脂诱导分化含量在 0 D（A，B）和后14 D（C，D）成脂分化诱导由细胞色素决定emical染色油红O.在红，类脂囊泡（较大的囊泡是由黑/红箭头指示;小囊泡由白色/黑色箭头）;蓝色/紫色，细胞核用苏木染。观察明。原始放大倍数:40X。酒吧尺寸:100μM <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig20large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图21" src="/files/ftp_upload/53884/53884fig21.jpg" /> 图21. CFU试验。用甲苯胺蓝染色OSA-CSC线CFU检测。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig21large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图22" src="/files/ftp_upload/53884/53884fig22.jpg" /><br<html> /&gt; 图22. ALDH活性测定该 ALDH比色法检测高水平两个OS-CSC线，OSA5，肿瘤干细胞和OSA6-CSCs的ALDH活性，而法检测缺席本次活动在有限的分化细胞系成纤维细胞，纤维蛋白原。误差棒:SD。 **:P &lt;0.001对FIB; *:P &lt;0.01 VS FIB <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53884/53884fig22large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 基因 </td><td> 寡核苷酸 </td><td colspan="2"> 序列（5¹-3¹） </td><td> 扩增子的大小（bp）的 </td><td> Tₐ（℃） </td></tr><tr><td> NANOG </td><td>正向引物反向引物</td>2"&gt; CCCAGCTGTGTGTACTCAAT GGTTCAGGATGTTGGAGAGTT </td><td> 87 </td><td> 60 </td></tr><tr><td>十月3/2 </td><td>正向引物反向引物</td><td colspan="2"> GGGAGGAGCTAGGGAAAGA TCCTTCCTTAGTGAATGAAGAACT </td><td> 77 </td><td> 60 </td></tr><tr><td> SOX2 </td><td>正向引物反向引物</td><td colspan="2"> TGCAGTACAACTCCATGACC GGACTTGACCACCGAACC </td><td> 125 </td><td> 55 </td></tr><tr><td> CD133 </td><td>正向引物反向引物</td><td colspan="2"> CCAGAAGCCGGGTCATAAAT ATTCACTCAAGGCACCATCC </td><td> 127 </td><td> 56 </td></tr><tr><td colspan="6"> BP，碱基对扩增子的大小;Tₐ，退火温度</td></tr></tbody></table> 表1详细引物序列对的Nanog，十月3/4，Sox2的和CD133与扩增子的大小和退火温度的列表

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

癌症干细胞的骨肉瘤存在（的CSCs）最近被链接到他们的发病机理。在这篇文章中，我们目前使用的CSC不依条件下生长的能力从传统的骨肉瘤（OS）的人获得活检原代细胞培养肿瘤干细胞的分离。

巨蟹座的建立源于人类传统的骨肉瘤细胞培养

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

18.9K 次观看.  University of Florence. 癌症干细胞的骨肉瘤存在（的CSCs）最近被链接到他们的发病机理。在这篇文章中，我们目前使用的CSC不依条件下生长的能力从传统的骨肉瘤（OS）的人获得活检原代细胞培养肿瘤干细胞的分离。 

视频: 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.

病人衍生软组织肉瘤原代培养的建立

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

科研

癌症研究

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.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

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.

从妇科和乳腺癌肿瘤获得癌症干细胞球

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.

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

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

遗传学

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).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

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

医学

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.

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.

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.

癌症干细胞从人类前列腺癌样本隔离

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.

丰富的人力细胞系化疗耐药性卵巢癌干细胞

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.

评价的人卵巢癌细胞干细胞特性使用多和单细胞为基础的化验球

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

<meta charset="utf8"></head><body>骨肉瘤 PDX 小鼠模型的方案：研究生长和耐药性

Establishment of Patient-Derived Xenograft Mouse Model with Human Osteosarcoma Tissues

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, the prognosis for osteosarcoma patients has not significantly improved. Therefore, it is crucial to establish a robust preclinical model with high fidelity. The patient-derived xenograft (PDX) model faithfully preserves the genetic, epigenetic, and heterogeneous characteristics of human malignancies for each patient. Consequently, PDX models are considered authentic in vivo models for studying various cancers in transformation studies. This article presents a comprehensive protocol for creating and maintaining a PDX mouse model that accurately mirrors the morphological features of human osteosarcoma. This involves the immediate transplantation of freshly resected human osteosarcoma tissue into immunocompromised mice, followed by successive passaging. The described model serves as a platform for studying the growth, drug resistance, relapse, and metastasis of osteosarcoma. Additionally, it aids in screening the target therapeutics and establishing personalized treatment schemes.

<meta charset="utf8"></head><body>作者聚焦：在免疫缺陷小鼠中复制人骨肉瘤进展用于癌症研究

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma tissue.

人骨肉瘤组织患者来源的异种移植小鼠模型的建立

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite the development of new treatment plans in recent years, ...