Name: production and administration of therapeutic mesenchymal stem/stromal cell (msc) spheroids primed in 3-d cultures under xeno-free conditions
Uploaded: 2017-03-18T13:00:00.000+00:00
Duration: 11 min 5 s
Description: Watch this Scientific Journal Video about Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions at JoVE.com

This work was funded in part by grant P40RR17447 from the National Institute of Health and award RP150637 from the Cancer Prevention and Research Institute of Texas. We would like to thank Dr. Darwin J. Prockop for his support on the project.

1. MSC分离和扩增<ol><li>获得早期中心代MSCs编制和成体干细胞（分布<a href="http://medicine.tamhsc.edu/irm/msc-distribution.html" target="_blank">http://medicine.tamhsc.edu/irm/msc-distribution.html</a> ）15作为冷冻瓶。另外，隔离骨髓穿刺以下例行协议14 MSC和存储为冷冻小瓶。 </li><li>制备完全培养基（CCM），它是最小必需培养基α（αMEM）补充有15-20％的溢价选择FBS，2mM L-谷氨酰胺和1x青霉素 - 链霉素。消毒通过过滤介质。 </li><li>放置30毫升CCM成150mm的细胞培养皿，在含有5％CO 2的潮湿培养箱中孵育盘30分钟，在37℃。 </li><li>孵育含有2分钟约10 6个干细胞在37℃水浴中的冷冻小瓶中。 </li><li>使用5毫升血清吸管转移小瓶的内容到培养皿，然后从培养皿1-2毫升的CCM洗小瓶几次以捕获剩余的细胞。放置隔夜菜在适当的孵化器。 </li><li>小心地用室温磷酸盐缓冲盐水（PBS）洗涤培养物两次，并用3毫升预热0.25％胰蛋白酶/乙二胺四乙酸（EDTA）溶液分离的MSC。支队通常需要在孵化器，大约3-4分钟。 </li><li>中和胰蛋白酶在用6毫升的CCM预热至37℃的菜，将细胞悬液转移到50毫升锥形管中。用PBS洗涤的结合起来，最大限度地提高细胞产量。 </li><li>离心10分钟将细胞（450 XG）在室温和吸出上清液。 </li><li>重悬在CCM中的一个小体积的细胞，并计数使用血细胞计数器和台盼蓝的活细胞。 </li><li>种子150毫米的菜肴适当数量为100个/厘米2 </sup&gt; 30毫升温CCM和孵化培养，直到大约70-80％汇合，通常是7天中每2-3天的变化和收获24-48小时之前。 </li><li>收获细胞如上述用胰蛋白酶/ EDTA和所有的细胞结合成一个单一的锥形管与适当的介质。离心再次并重新悬浮细胞成用于细胞计数的相同培养基的小体积。使用标准的CCM（含有FBS）或各种市售XF培养基配方，这里称为XF介质-1（XFM-1）和XF培养基-2（XFM-2），两者都具有和不具有人血清白蛋白（HSA，13毫克/毫升）的补充。 </li></ol>在3-D激活MSC球体2.准备下XF条件悬滴文化<ol><li>以产生约25,000个细胞的球形体，淡化收获的MSC到CCM，XFM-1，XFM-1 + HSA（13毫克/毫升），XFM-2，或XFM-2 + HSA（13毫克/毫升）在714细胞/微升。 </li><li>细胞吸取35微升/滴于150mm的倒置培养皿盖的下侧。多个液滴可通过使用多道移液器容易地吸取。 注意:如果大或小球体的需要，改变细胞浓度或滴体积（25-45微升）适当。 </li><li>转移20毫升室温的PBS入菜的基极和在一个连续和稳定运动翻转盖，含有该液滴，使液滴顶点朝下。小心地将盖培养皿的底部上。 </li><li>转移盘进入孵化器3天，而不会干扰它允许适当的球形组装。 </li><li> 72小时后，通过仔细地除去在盖和反相它使得滴，再次，朝上开始球体收获。 </li><li>角盖到大约10-20°及推液滴，含有该球状体，以使用细胞升降板边缘。 </li><li>传送球体和介质入15ml锥形管与1000微升管TTE。使用室温下PBS中并用相同的尖端洗涤板并保证球体的最大恢复的吸移管。 </li><li>对于实时PCR测定（协议3），离心球悬浮液在450×g离心在室温下5分钟，吸出上清液。用PBS洗涤球体和重复离心和愿望步骤中都。用于输送到动物（协议8），使球在管（3-4〜分钟）的底部不离心沉淀。 </li></ol>抗炎和抗癌标记3.实时PCR <ol><li>使用具有均化列和无RNA的DNA酶设置RNA提取试剂盒来分离从单层的MSC不含DNA的总RNA（协议1）和用PBS洗涤并离心该MSC球状体（协议2）。 </li><li>裂解至少100,000单层MSC和从每个条件在单独15毫升20球状体锥形管与含有β巯基乙醇（1:100）RLT缓冲液中。 </li><li>横贯FER的充分混合裂解物进1.5毫升无RNase管并放入一个冷冻器（-80℃）下至少3小时在球体裂解以帮助。 </li><li>在冰上解冻，涡旋的细胞裂解物短暂，并按照制造商的说明用市售的哺乳动物总RNA分离试剂盒提取RNA。 </li><li>量化和评估使用分光光度计分离的RNA的质量。 </li><li>使用高容量cDNA的反转录试剂盒或根据等同于制造商的说明来产生的cDNA的实时PCR。 </li><li>放置cDNA样品，市售设计实时PCR引物/探针（基因表达分析），和PCR主混合物在冰上。使用的引物/探针GAPDH（对照），PTGS2（COX-2，抗发炎），TNFAIP6（TSG6，消炎），TRAIL（抗癌），和IL-24（抗肿瘤）。 </li><li>根据制造商的基因表达测定法和快速李海说明准备实时PCR反应RSAL预混。 </li><li>遵循了实时PCR仪的准则用于产生实验文件和执行运行。 </li><li>通过计算使用GAPDH作为内对照和单层干细胞作为基准8，14基因表达的相对变化（相对量，RQ）分析使用ΔΔCT法的数据。 </li></ol> 4.收集CM为免疫调节的测定和抗癌潜力<ol><li>收获球体和CM然而，如上所述成一个15毫升的锥形管，用细胞升降机和吸管，不与PBS洗涤培养皿的盖子，因为这会稀释CM。 </li><li>离心在450 xg离心在室温下5分钟，小心收集用移液器将无细胞上清液，并且将上清液转移到1.5毫升管中。 </li><li>离心10,000 XG在室温下5-10分钟，小心收集CLarified CM用吸管，并传送CM进入新的1.5毫升管储存在冷冻（-80℃）。使用它的多种测定时冷冻之前以制备CM的等分试样。 注意:在此之前执行的下游测定，PGE 2浓度，CM的抗炎潜力的良好指标，可以使用根据制造商的说明商业ELISA试剂盒确定。可以使用发布的协议14来决定TSG6浓度。 </li></ol> 5.巨噬细胞测定来评估MSC-CM的抗炎潜力<ol><li>制备巨噬细胞中，它由含有L-丙氨酰-L-谷氨酰胺和补充有10％的溢价选择FBS和1x青霉素 - 链霉素的Dulbecco改良的Eagle培养基（DMEM）的。过滤消毒网上平台。 </li><li>获得的约10 6的J774巨噬细胞冷冻小瓶中，并孵育小瓶在37℃水浴中2分钟。 </li><lI&gt;转移小瓶的内容入15ml锥形管中，在200-300 XG并在室温下添加9毫升巨噬细胞培养基中，离心5分钟。 </li><li>吸出上清液，暂停的巨噬细胞在1ml巨噬细胞培养基中，巨噬细胞转移到装有30ml巨噬细胞培养基的150mm的陪替氏培养皿。 </li><li>改变介质每2天孵化在孵化器中的细胞，直到大约70-80％汇合。 注:巨噬细胞不紧紧粘附在陪替氏培养皿，因此应小心不要失去细胞与培养基的变化。 </li><li>由巨噬细胞介质喷涂到用吸管将细胞收获巨噬细胞。转移细胞到50ml锥形管中并离心5分钟，在200-300 XG和室温。 </li><li>吸出上清液并悬浮细胞在小体积的巨噬细胞中的用于与血球细胞计数。调至浓度为与巨噬箱200,000个细胞/ ml的dium。 </li><li>以启动设置为巨噬细胞的测定法中，添加480微升的巨噬细胞中的成12孔板的孔中。 </li><li>加入20μl的巨噬细胞中的成非刺激对照的孔和20μl的非空调或MSC条件培养基中，上面制备，在实验孔。通过轻敲板混合孔中的介质。 </li><li>转移500μl的巨噬细胞悬浮液的非刺激的巨噬细胞的对照孔。 </li><li>刺激并加入1剩余的巨噬细胞:500-1:0.1的1000毫克/毫升脂多糖（LPS）和室温下培养5-10分钟。 </li><li>转移500微升的刺激巨噬细胞的入井与非空调或MSC条件培养基。通过几次稳步摇动板混合的孔中。 </li><li>板块转移到孵化器为16-18小时。 </li><li>收获在500 XG的巨噬细胞条件培养基，离心，在室温下5分钟。 </li><li>转让上清液到新的管中，测定用于肿瘤坏死因子-α（TNF-α）和白细胞介素-10（IL-10）的含量按照制造商的说明书的商业ELISA试剂盒。 </li></ol> 6.脾细胞检测，以评估球体-CM的免疫调节潜力<ol><li>准备完整的脾细胞中，它由罗斯维尔公园纪念研究所（RPMI）-1640培养基补充有10％热灭活FBS，1×青霉素 - 链霉素，2-巯基乙醇。过滤消毒网上平台。 </li><li>从安乐死-雄性BALB / c小鼠收获脾脏，通过在腹部的左侧在脾脏制备的切口，前之间大约中途和后腿14。转移脾70微米的细胞过滤分离脾14。 </li><li>通过推动通过70微米的过滤器脾脏到无菌培养皿全光照隔离脾细胞/淋巴细胞克软橡胶/塑料柱塞从2-3结束毫升注射器14。使用磨转圈分离脾脏。 </li><li>用冷PBS洗细胞过滤几次，然后从培养皿转移细胞到50ml锥形管中。在4℃下填充用冷PBS离心（500 XG）的管10分钟。 </li><li>吸出上清液，并从红细胞温育用5-10毫升红细胞溶解溶液（每脾）5-10分钟清除细胞。 </li><li>通过加入冷PBS到管和离心机在500 xg离心5-10分钟，并在4℃停止裂解。 </li><li>暂停在完整脾细胞培养基中分离细胞，通过40微米的过滤器进行过滤，并使用血球计数细胞。脾细胞培养基添加到细胞悬浮液达到10 6个细胞/ ml的最终细胞浓度 注:此方法通常会产生每只小鼠脾约3×10 7脾细胞。 </li><li&gt;刺激脾细胞的合适数量为5分钟2微克/毫升抗CD3e抗体。 注:脾细胞的量需要取决于如由研究者测定的实验条件的数目（见步骤6.9）。对于每一个实验样品/孔，需要10 6脾细胞。 </li><li>转移10 6脾细胞（刺激或非刺激的）转换成一个12孔板的孔中，并添加非条件（对照）或MSC-CM成以1最终稀释孔:10-1:20。 </li><li>收获后24小时，离心分离脾细胞的CM，在500×g离心，在室温下5分钟。 </li><li>按照制造商的说明商业ELISA试剂盒将上清转移到新的管中，测定为干扰素γ（IFN-γ）的内容。 </li></ol> 7.前列腺癌检测，以评估球体-CM的抗癌潜力<ol><li>制备完全RPMI生长培养基是补充有10 RPMI-1640培养基％FBS，1×青霉素 - 链霉素。 </li><li>获得的LNCaP前列腺癌细胞的冷冻小瓶中并孵育小瓶2分钟在37℃水浴中。 </li><li>转移小瓶的内容到装有30ml使用机动移液器完全RPMI生长培养基和5ml的血清吸管培养皿。与从盘介质洗小瓶几次并将培养皿放入孵化器。 </li><li>细胞达到汇合之前，用室温的PBS洗培养两次，并用3毫升预热0.25％胰蛋白酶/ EDTA溶液的分离LNCaP细胞。 </li><li>中和与完全RPMI生长培养基皿的胰蛋白酶，将细胞用洗涤转移一起到50毫升锥形管中。 </li><li>离心细胞，在450×g离心在室温下10分钟，吸出上清液。 </li><li>重悬浮细胞进入完全RPMI生长培养基的体积小，并使用血细胞计数器计数的癌细胞。 </li><lI&gt;转移LNCaP细胞到96孔盘以5,000个细胞/孔在120μl的含有非空调或球体-CM的25％的完全RPMI生长培养基。孵育在孵化72小时。 </li><li>以执行使用细胞增殖测定试剂盒，细胞生长测定中，吸从井介质和板转移到冷冻（-80℃）下至少3小时。 <ol><li>解冻板并井加入100微升含有180 mM氯化钠，1mM的EDTA和0.2mg / ml的核糖核酸酶A.细胞裂解试剂溶解在每个单元</li><li>对于标准曲线，如上所述裂解已知量LNCaP细胞的。 </li><li> 1小时后，添加100微升含1 1×细胞裂解缓冲液:100稀释细胞增殖测定试剂，并测量荧光每个孔中，以确定细胞数量。 </li></ol></li></ol> 8.完整的球体腹腔交付<ol><li>上面（协议2），重悬T作为描述的准备球体干细胞他球体无菌汉克的补充有0.2-0.5％的HSA保持XF条件平衡盐溶液（HBSS）中。 HSA的溶液有助于防止注射期间球粘附于塑料。 </li><li>转移40-120球状体分为15毫升锥形管中，并通过加入6毫升HBSS / HAS到管洗涤细胞。允许3-4分钟的球下降。准备球状体每只动物的一个独立的锥形管中。此外，对于试验确定球体保留（见下文8.18）和转移后生产的治疗因素（见下文8.19），如果需要，准备球体的额外管。 </li><li>吸出上清液，用100-200微升无菌的HBSS补充有0.2-0.5％的HSA叠加球体，并将管在冰上。 </li><li>简要地麻醉用在100％的氧气2％异氟醚将动物直至捏趾反射的消失在感应室约2分钟。 </li><li>将动物的BSL-2柜，背里面ASPECT下（腹朝上），1.5％异氟醚/氧气混合物通过鼻锥的连续流。 </li><li>请用消毒剂如70％酒精下鼠标腹部。 </li><li>除去20G的静脉内（IV）导管的帽并与导管打孔组件执行腹膜内注射。用一只手按住鼠标皮肤，另一个进行注射。定位大约在连接与针尖朝向中线指向一个弯曲膝关节和生殖器区域人造线的中间注入点。 注:导管细总成具有比普通针较高的耐药性，因此保健已采取不注射针过深到腹部，这可能导致伤害内脏。皮肤的渗透，然后肌肉/腹膜可以导管放置过程中显现出来。 </li><li>轻柔地从导管细组装金属打孔/针。 CHECK代表血液，尿液和粪便的细高跟，并丢弃它。在针血液表示内部器官（多个）的穿刺。 </li><li>持注射期间在直立位置的塑料导管，以允许流体流动。 </li><li>通过使用移液管有100-200微升末端通过导管注入大约100微升无菌HBSS / HAS的确认塑料导管的适当的位置。放置导管注射器适配器形成紧密的密封内部枪头的末尾。缓缓注入腹腔内的流体。 注:在正确定位导管流体将腹腔内自由流动，只有导管的微小调整。导管的放置不当（皮下或肠内，或者如果针受伤腹膜器官如肾脏的尖端）会增加电阻，降低流动。皮下放置会导致形成皮肤下面有明显的"泡沫"。 </li><li>收集ŧ他MSC球状体，在100-200微升HBSS / HAS（步骤8.3）的制备，用200μl移液管尖端，并如上所述通过导管转移球的总体积（40-120球）进入腹膜腔。 </li><li>洗用与上述相同的尖端来收集任何残留的球状体，并将它们注入到腹膜腔中含有用100μlHBSS / HAS的球管。 </li><li> （可选）注入另外100微升HBSS / HAS的进入腹膜腔洗导管。 </li><li>放置在防腐剂在导管浸泡的纱布垫，慢慢从腹腔取出导管，并丢弃它。 </li><li>轻轻按摩用手指腹膜腔，以确保即使在球状体的分布。 </li><li>让动物密切监督下恢复，并注意从注射部位出血。 </li><li>检查任何剩余球体导管和15ml管中。这是正常现象，观察在几个领域导管/管。 注:MSC球体递送描述的技术可以用于在正常动物或在各种损伤模型的使用被采用。该技术已成功地用于注射在小鼠角膜损伤和内毒素血症模型的球体，以及在酵母聚糖诱导的腹膜炎模型8。 </li><li>为了量化保留球体（可选）的数量，使用基于DNA的细胞数量定量试剂盒/试剂。持在15ml试管中的用于导管和穿过导管大力分配的HBSS / HAS迫使任何剩余的球回15ml试管。 <ol><li>离心15ml试管在500 xg离心在室温下5分钟，吸出上清液。传送含有球体粒料管成冷冻（-80℃）下至少3小时。 </li><li>解冻管并通过加入500微升含有180 mM氯化钠，1mM的EDTA和0.2mg / ml的核糖核酸酶A.细胞裂解试剂裂解球体</li><li>对于控制，FRE结和裂解球状体的已知量（优选相当于用于单个注射制备球状体的数量），如上所述。 </li><li> 1小时后，转移100μl的细胞裂解物中的，一式两份，在96孔微量培养板，并加入到每孔100μl含有来自试剂盒1:50稀释的细胞增殖测定试剂的1×细胞裂解缓冲液中。 10分钟后，测量荧光每个孔中，以确定未由实验样品（多个）与对照样品进行比较注射球体的数目。 </li></ol></li><li>评估通过测量转移球产生的PGE2的浓度和TSG6注射（可选）准备球状体的激活水平。转印40-120球通过导管中，如上所述，为含有1或2毫升αMEM补充有2％FBS和1x青霉素/链霉素12孔或6孔平板。放置在孵化器板6小时。 <ol><li>传送自介质孔后6小时到1.5或15ml试管并在450 xg离心离心管在室温下5分钟。 </li><li>传送用吸管和离心机的无细胞上清液到新的1.5毫升管以10,000rpm XG在室温下5-10分钟。 </li><li>转移澄清CM（上清液）到新的1.5毫升管和测定使用根据制造商的说明商业ELISA试剂盒的PGE 2浓度（CM的抗炎潜力的良好的指标）。可以使用发布的协议14来决定TSG6浓度。 </li></ol></li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+Mesenchymal+Stromal+Cells'+Paracrine+Immunomodulatory+Effects.">Unraveling the Mesenchymal Stromal Cells' Paracrine Immunomodulatory Effects.</a> Transfus.Med.Rev. 30 (1), 37-43 (2016).</li><li>Nemeth, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+attenuate+sepsis+via+prostaglandin+E(2)-dependent+reprogramming+of+host+macrophages+to+increase+their+interleukin-10+production.">Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production.</a> Nat.Med. 15 (1), 42-49 (2009).</li><li>Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+two+negative+feedback+loops+place+mesenchymal+stem/stromal+cells+at+the+center+of+early+regulators+of+inflammation.">Concise review: two negative feedback loops place mesenchymal stem/stromal cells at the center of early regulators of inflammation.</a> Stem Cells. 31 (10), 2042-2046 (2013).</li><li>Krampera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cell+'licensing':+a+multistep+process.">Mesenchymal stromal cell 'licensing': a multistep process.</a> Leukemia. 25 (9), 1408-1414 (2011).</li><li>Saleh, F. A., Genever, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+round:+multipotent+stromal+cells,+a+three-dimensional+revolution?.">Turning round: multipotent stromal cells, a three-dimensional revolution?.</a> Cytotherapy. 13 (8), 903-912 (2011).</li><li>Tsai, A. C., Liu, Y., Yuan, X., Ma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compaction,+fusion,+and+functional+activation+of+three-dimensional+human+mesenchymal+stem+cell+aggregate.">Compaction, fusion, and functional activation of three-dimensional human mesenchymal stem cell aggregate.</a> Tissue Eng.Part A. 21 (9-10), 1705-1719 (2015).</li><li>Yeh, H. Y., Liu, B. H., Hsu, S. 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The authors declare they have no competing financial interests.

在一些研究和临床应用中使用的最佳的MSC应高度活化以最大化他们的利益，和化学成分确定的XF的条件下优先制备，以尽量减少潜在抗原从异种介质组分如胎牛血清的交付。在这里所描述的协议中，我们已经表明方法1）通过形成球状体的激活在三维培养的MSC，2）实现的MSCs XF条件下的3D激活，3）评价在关于他们抗球体MSCs的激活水平抗炎，免疫调节和抗肿瘤潜力，和4）提供激活的MSC作为完整的球体...

间充质干细胞/基质细胞（MSCs）已经显示关于各种再生医学的方法的巨大潜力。的MSC最初分离的骨髓的基质组分，但至今已从许多其它成体组织，包括脂肪组织1，2，3获得。有趣的是，主要分离方法涵盖MSCs的显着属性，以紧密附着到组织培养塑料中的胎牛血清（FBS）的存在。虽然这种传统的隔离技术允许在二维（2D）培养容易和迅速扩展的MSC，它也很人工和无视导致重要的细胞特征4潜在损失的天然三维（3D）环境的意义，5 6。因此，干细胞在三维文化的研究，比传统的二维培养更符合生理，已经出现在搜索"丢失/减弱"MSC特性。此外，极大的兴趣已经上升到识别（XF）无异物化学定义的条件MSC文化和活化，从而使细胞临床应用更适合。 许多研究已经发表表明无论在生物材料和球形聚集体或球状体MSC的3D培养。在生物材料的MSC最初设计用于组织工程的方法与细胞种子支架替换损坏的组织，而MSC的球体培养物被视为一种方式来理解用于在临床前或临床试验中的治疗的细胞给药后在体内的MSC行为4，5，7。有趣的是，干细胞形式球状体自发时不允许粘附到组织培养塑料8，9，10。传统上，细胞聚集是由旋转烧瓶方法或液体覆盖技术，最初用于癌症生物学中的努力，试图模仿肿瘤微环境的方法促进。最近，其他方法已经浮现演示在培养皿的细胞聚集预涂具有特定的化学物质，以防止细胞-塑料粘附4，5，6。之一，以产生MSC球状体的最简单和最经济的方法是将培养他们在悬滴，这是经常用于产生从胚胎干细胞的胚状体的技术。与悬滴培养技术，细胞粘附于组织培养塑料是通过在介质的上的组织培养皿盖的下侧下降的细胞悬浮并允许重力促进细胞aggreg防止通货膨胀在下拉的顶点。球体大小可以通过改变细胞浓度或者滴体积，使得悬滴培养尤其容易控制可以容易地操纵。 关于MSC的3D培养早期的研究表明在细胞中的3D相比，他们的2D对应6，8，9的特性基团的差异。同时，报告表明，干细胞在体内的有益效果依靠其成为通过微环境线索激活，并且作为响应，以产生抗炎和免疫调节因子11的能力。有趣的是，许多这些因素，如前列腺素E2（PGE 2），肿瘤坏死因子刺激的基因6（TSG6）和肝细胞生长因子（HGF）是在更大的数量由MSC球体比传统的二维的MSCs铺平了道路产生的理念使用3D培养激活细胞8，12，13。此外，基因活化在3D培养出现注射入小鼠12后概括机制，至少部分，细胞活化。通过激活之前将其在实验中使用的MSC，该细胞的作用可以被延长，更突出在体内传统的MSC效果往往延迟和短暂的，并且可以被描述为"打并运行"。在过去的几年中，使用MSC球状体重要的功能性研究已经表明，它们可以抑制炎症反应和通过影响效应细胞如巨噬细胞，树突细胞，嗜中性粒细胞和T细胞使得球状体致敏的MSC 2的一个有吸引力的形式调节体内免疫，3。此外，生产抗癌分子，如int的erleukin-24（IL-24），肿瘤坏死因子相关凋亡诱导配体（TRAIL），在干细胞的相对的三维培养物提高到单层的MSC，这可能对靶向癌症疗法8，10，14被利用的现象。 由于传统的MSC文化不仅需要利用组织培养塑料也FBS，另一个障碍，使MSC球体更适合临床使用已被克服。为了解决这一难题，我们最近发现形成的MSC球状体的特定化学成分确定的XF的条件下，建立所得到的MSC球体被激活以产生相同的抗炎和抗肿瘤分子作为在条件与FBS 14产生的球状体。这里，这些研究结果在演示中使用XF 3D培养预活化的MSCs的产生几个详细方案提出媒体。此外，协议呈现描述评估MSC的活化水平的问候其抗炎，免疫调节和抗肿瘤作用，与递送完整球体到小鼠的实用方法一起有效的方法。

<table><tbody><tr><td>MEM-&amp;alpha; (minimal essential medium alpha)</td><td>ThermoFisher/Gibco</td><td>12561049; 12561056; 12561072</td><td>minimal essential medium for preparation of MSC growth medium (CCM)</td></tr><tr><td>FBS (fetal bovine serum), premium select</td><td>Atlanta Biologicals</td><td>S11595; S11510; S11550; S11595-24</td><td>component of complete culture media for all types of cells</td></tr><tr><td>L-glutamine</td><td>ThermoFisher/Gibco</td><td>25030081; 25030149; 25030164</td><td>component of complete culture media for all types of cells</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisher/Gibco</td><td>15070063</td><td>component of complete culture media for all types of cells</td></tr><tr><td>Sterilization Filter Units, 0.22 &amp;micro;m PES membrane</td><td>MilliporeSigma</td><td>SCGPU01RE; SCGPU02RE; SCGPU05RE; SCGPU10RE; SCGPU11RE</td><td>media sterilization</td></tr><tr><td>150 mm cell culture dish</td><td>Nunc</td><td>D8554&amp;nbsp;SIGMA</td><td>cell culture</td></tr><tr><td>Thermo Forma water-jacketed CO&lt;sub&gt;2&lt;/sub&gt; humidified incubator</td><td>Thermo Fisher</td><td>Model 3110</td><td>incubation of cultured cells</td></tr><tr><td>Early passage MCSs</td><td>Center for the preparation and Distribution of Adult Stem Cells at The Texas A&amp;amp;M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott &amp;amp; White</td><td>NA</td><td>preparation of 2D and 3D cultures of MSCs</td></tr><tr><td>water bath</td><td>VWR</td><td>89501-468</td><td>warming media to 37 &amp;deg;C</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>492000904</td><td>manual liquid handling</td></tr><tr><td>Pipete-Aid</td><td>Drummond Scientific Company</td><td>4-000-300</td><td>handling sereological pipetes</td></tr><tr><td>Costar sterile serological pipet (5, 10, 25 and 50 ml)</td><td>Corning</td><td>4487; 4101; 4251; 4490</td><td>liquid handling</td></tr><tr><td>PBS (phosphate buffered saline), pH 7.4</td><td>ThermoFisher/Gibco</td><td>10010023; 10010072; 10010031; 10010049</td><td>cell culture processing</td></tr><tr><td>0.25% trypsin/EDTA solution</td><td>ThermoFisher/Gibco</td><td>25200056; 25200072; 25200114</td><td>lifting adherent cells and dispersing cell aggregates</td></tr><tr><td>15 ml conical tube</td><td>Corning/BD Falcon</td><td>352097</td><td>cell centrifugation</td></tr><tr><td>50 ml conical tube</td><td>Corning/BD Falcon</td><td>352098</td><td>cell centrifugation</td></tr><tr><td>Eppendor refrigerated centrifuge</td><td>Eppendorf/Fisher Scientific</td><td>Model 5810R</td><td>cell centrifugation</td></tr><tr><td>hemocytometer</td><td>Fisher Scientific</td><td>26716</td><td>cell counting</td></tr><tr><td>trypan blue</td><td>Sigma-Aldrich</td><td>T8154 SIGMA</td><td>dead cell exclusion during cell counting in hemacytometer</td></tr><tr><td>Defined xenofree MSC medium-1 (XFM-1)</td><td>ThermoFisher/Gibco</td><td>A1067501</td><td>Xeno-free media specifically formulated for the growth and expansion of human mesenchymal stem cells</td></tr><tr><td>Defined xenofree MSC medium-2 (XFM-2)</td><td>Stem Cell Technologies</td><td>5420</td><td>Defined, xeno-free medium for human mesenchymal stem cells</td></tr><tr><td>HSA (Human serum albumin)</td><td>Gemini</td><td>800-120</td><td>Component of xeno-free MSC media</td></tr><tr><td>rHSA (recombinant Human serum albumin)</td><td>Sigma-Aldrich</td><td>A9731&amp;nbsp;SIGMA</td><td>Component of xeno-free MSC media</td></tr><tr><td>Total RNA isolation Mini Kit</td><td>Qiagen</td><td>74104</td><td>Total RNA extraction</td></tr><tr><td>Qiashredder</td><td>Qiagen</td><td>79654</td><td>Sample homogenization prior to total RNA extraction</td></tr><tr><td>RNAse-free DNase Set&amp;nbsp;</td><td>Qiagen</td><td>79254</td><td>On-column DNA elimination during total RNA extraction</td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M6250&amp;nbsp;ALDRICH</td><td>inhibition of RNAses in RLT buffer</td></tr><tr><td>Vortex</td><td>VWR</td><td>97043-562</td><td>mixing sample</td></tr><tr><td>Spectrophotometer</td><td>Biorad</td><td>NA</td><td>RNA concentration and quality</td></tr><tr><td>High capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher/Applied Biosystems</td><td>4368814</td><td>transcription of total RNA into cDNA</td></tr><tr><td>Gene Expression Assays</td><td>ThermoFisher/Applied Biosystems</td><td>varies</td><td>primer/probe combination for real-time PCR</td></tr><tr><td>Fast Universal PCR Master Mix</td><td>ThermoFisher/Applied Biosystems</td><td>4352042; 4364103; 4366073; 4367846</td><td>master mix for real-time PCR reaction</td></tr><tr><td>Real-time PCR system (ABI Prism 7900 HT Sequence Detection System)</td><td>ABI Prizm</td><td>NA</td><td>real-time PCR</td></tr><tr><td>1.5 ml centrifuge tube</td><td>Eppendorf</td><td>22364111</td><td>cell centrifugation, sample collection and storage</td></tr><tr><td>(-80 &amp;deg;C) freezer</td><td>Thermo Fisher</td><td>Model Thermo Forma 8695</td><td>sample storage</td></tr><tr><td>PGE2 (Prostaglandin E2) ELISA Kit</td><td>R&amp;amp;D Systems</td><td>KGE004B</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s modified Eagle medium)</td><td>ThermoFisher/Gibco</td><td>10566-016; 10566-024;10566-032</td><td>macrophage culture media</td></tr><tr><td>J774 mouse macrophages</td><td>ATCC</td><td>TIB-67</td><td>mouse macrophage cell line</td></tr><tr><td>12-well plate</td><td>Corning</td><td>3513</td><td>&lt;em&gt;in vitro&lt;/em&gt; macrophage stimulation</td></tr><tr><td>LPS (lipopolysaccharide)</td><td>Sigma-aldrich</td><td>L4130</td><td>&lt;em&gt;in vitro&lt;/em&gt; macrophage stimulation</td></tr><tr><td>Mouse TNF-a ELISA kit</td><td>R&amp;amp;D Systems</td><td>MTA00B</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>Mouse IL-10 (interleukin 10) ELISA kit</td><td>R&amp;amp;D Systems</td><td>M1000B</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>RPMI-1640 medium</td><td>ThermoFisher/Gibco</td><td>11875-085</td><td>splenocyte culture media</td></tr><tr><td>BALB/c mice</td><td>The Jackson Laboratory</td><td>651</td><td>&lt;em&gt;in vivo&lt;/em&gt; spheroid delivery; splenocyte preparation</td></tr><tr><td>Anti-Mouse CD3e Functional Grade Purified</td><td>eBioscience</td><td>145-2C11</td><td>&lt;em&gt;in vitro&lt;/em&gt; splenocyte stimulation</td></tr><tr><td>70 &amp;mu;m strainer&amp;nbsp;</td><td>Corning</td><td>352350</td><td>Splenocyte preparation</td></tr><tr><td>Red blood cell lysis solution (1x)</td><td>Affymetrix eBioscience</td><td>00-4333</td><td>removal of red blood cells during splenocyte isolation</td></tr><tr><td>Mouse IFN-&amp;#947; (interferon gamma) ELISA kit</td><td>R&amp;amp;D Systems</td><td>MIF 00</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>LNCaP prostate cancer cells&amp;nbsp;</td><td>ATCC</td><td>CRL-1740</td><td>study the effect of 3D MSCs on cancer cell lines &lt;em&gt;in vitro&lt;/em&gt;</td></tr><tr><td>DNA-based cell proliferation assay kit</td><td>ThermoFisher</td><td>C7026</td><td>cell number measurement based on DNA content</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5150</td><td>component of lysis reagent</td></tr><tr><td>EDTA (ethylenediaminetetraacetic acid)</td><td>ThermoFisher</td><td>FERR1021</td><td>calcium chelator, component of lysis reagent</td></tr><tr><td>Rnase A</td><td>Qiagen</td><td>19101</td><td>RNA degradation for measurement of DNA</td></tr><tr><td>Filter-based multi-mode microplate reader</td><td>BMG Technology</td><td>NA</td><td>Microplate assays (ELISA, cell quantification, &lt;em&gt;etc.&lt;/em&gt;)</td></tr><tr><td>HBSS (Hanks balanced salt solution), no calcium, no magnesium, no phenol red</td><td>ThermoFisher/Gibco</td><td>14175079</td><td>resupsension of MSC spheroids prior to &lt;em&gt;in vivo&lt;/em&gt; injections</td></tr><tr><td>Isoflurane</td><td>MWI Vet Supply</td><td>502017</td><td>Anesthesia for &lt;em&gt;in vivo&lt;/em&gt; injections</td></tr><tr><td>Oxygen, compressed gas</td><td>Praxair</td><td>NA</td><td>For use with isoflurane</td></tr><tr><td>Thermo Forma BSL-2 cabinet</td><td>Thermo Fisher</td><td>Model 1385</td><td>Sterile cell culture</td></tr><tr><td>Safety I.V. catheter/needle stiletto, 20 G , 1 inch</td><td>Terumo</td><td>SR*FNP2025</td><td>Delivery of shperoids into peritoneal cavity</td></tr><tr><td>Sterile micropipette tips</td><td>Eppendorf</td><td>varies</td><td>liquid/cells handling</td></tr></tbody></table>

production and administration of therapeutic mesenchymal stem/stromal cell (msc) spheroids primed in 3-d cultures under xeno-free conditions

间充质干/基质细胞（MSCs）持有生物工程和再生医学很大的希望。的MSC可以从多个成人组织通过其强粘附到组织培养塑料中分离，然后在体外进一步扩大，最常使用的胎牛血清（FBS）。因为FBS可引起干细胞成为免疫原性的，其中的MSC培养物的存在限制了细胞的临床和实验的应用程序。因此，研究用人化学定义无异物（XF）媒体MSC文化是非常有价值的。 MSC的许多有益效果已被归因于它们对调节炎症和免疫能力，主要通过免疫调节因子的分泌，如肿瘤坏死因子刺激的基因6（TSG6）和前列腺素E2（PGE 2）。然而，干细胞需要活化以产生这些因素，并且自MSC的效果往往短暂的，极大的兴趣已经出现以发现的激活预细胞PRIO方式R以它们的使用，从而消除了滞后时间用于体内活化。这里，我们提出的协议能够有效地激活或首要的MSC在三维（3D）培养物 化学上确定的XF的条件下，并施用在体内这些预活化的MSC。具体地讲，我们首先描述的方法生成使用XF介质球形MSC微组织或在悬滴"球体"和演示如何球体和条件培养基（CM）可收获用于各种应用。第二，我们描述的基因表达的屏幕和在体外功能测定迅速评估MSC活化在球状体的水平，强调细胞的抗炎和抗肿瘤潜力。第三，我们描述了一种新的方法来注入完好的MSC球体成用于体内效力测试小鼠腹腔。总体来说，此方案得到克服根据化学定义XF CON预激活干细胞的主要挑战条件和提供了一个灵活的系统管理MSC球体的疗法。

在目前的工作中，采用悬滴文化生成球形紧凑微型组织或XF条件下激活干细胞的"球体"。 图1中的试验用路线图描绘的MSCs被鼓励自组装悬浮于悬滴72小时，在此之后，球状体，或在CM装入球体源性治疗因子时变成球体，可以收集和潜在地在两个利用研究和临床应用。大量需要的球生产细胞可以在一个星期内通过以低密度接种的干细胞，通常100-200细胞每厘米2?...

Watch this Scientific Journal Video about Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions at JoVE.com

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

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Developmental Biology

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

11.8K 次观看.  University of Mary Hardin-Baylor. The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

视频: Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

生产尺寸控制肿瘤细胞球体采用微孔板大数

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

科研

生物工程

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

奇托桑膜上人类牙周韧带细胞球体的形成

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

发育生物学

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>通过 3D 打印的邮票状设备生成组织球体

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

通过 3D 打印的邮票状设备生成组织球体

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

生物化学

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

从 iPSC 高效生成人肠道类器官的基质比较

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

作者聚焦：使用 iPSC 推进用于药物开发的类器官生成

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

生产和治疗间充质干行政/基质细胞（MSC）球体底漆在三维培养下无异物条件

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生产尺寸控制肿瘤细胞球体采用微孔板大数

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

奇托桑膜上人类牙周韧带细胞球体的形成

间质干细胞培养和交付自体条件:用于骨科应用程序的智能方法

通过 3D 打印的邮票状设备生成组织球体

通过 3D 打印的邮票状设备生成组织球体

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

大规模、自动化生产脂肪来源的干细胞球体，用于 3D 生物打印

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究

基底膜基质用于人类干细胞维持和肠道类器官生成的比较研究