在这里,我们描述了一种从人类多能干细胞(hPSCs)生成肾类器官的方案。该方案在两周内产生肾脏类器官。所得的类肾类器官可以在大型旋转瓶或多孔磁力搅拌板中培养,以进行平行的药物测试方法。
由hPSCs产生的肾类器官提供了无限的肾组织来源。人肾类器官是研究肾脏疾病和损伤,开发基于细胞的疗法以及测试新疗法的宝贵工具。对于此类应用,需要大量均匀的类器官和高度可重复的测定。我们以之前发布的类肾类器官方案为基础,以改善类器官的整体健康状况。这种简单,强大的3D方案涉及在含有脂质,胰岛素 – 转铁蛋白 – 硒 – 乙醇胺补充剂和聚乙烯醇的最小组分培养基中形成均匀的胚体,使用GSK3抑制剂(CHIR99021)3天,然后在含有敲除血清置换(KOSR)的培养基中培养。此外,搅拌测定可以减少胚体的结块并保持均匀的大小,这对于减少类器官之间的变异性非常重要。总体而言,该协议为产生大量肾类器官提供了一种快速,高效且具有成本效益的方法。
近年来,已经开发了许多将人类多能干细胞分化为肾类器官的方案1,2,3,4,5。类肾类化合物为研究新的再生医学方法,模拟肾脏相关疾病,进行毒性研究和治疗药物开发提供了重要工具。尽管它们具有广泛的适用性,但肾脏类器官具有局限性,例如缺乏成熟,体外长期培养能力有限,以及缺乏在人肾中发现的几种细胞类型6,7,8。最近的工作重点是通过修改现有方案9,10,11,12来提高类器官成熟水平,延长培养期并扩大肾细胞群的复杂性。在我们已建立的方案5,13的当前迭代中,我们将方案第一阶段的培养基组分修改为补充胰岛素 – 转铁蛋白 – 硒 – 乙醇胺(ITSE),脂质,聚乙烯醇(E5-ILP)和CHIR99021的无血清基础培养基(图1)。这些变化提供了一种完全定义的、无血清的低蛋白培养基,其成分比我们以前的培养基组成5,13少,并且没有额外的生长因子。因此,与我们之前发布的版本相比,第一阶段培养基的制备劳动强度较低,并且可以减少批次之间的差异5。先前的研究表明,胰岛素和转铁蛋白在无血清培养14,15中都很重要,然而,高水平的胰岛素可以抑制中胚层分化16。我们保持了原始方案中规定的低胰岛素水平,并在测定的第二阶段进一步降低了KOSR(含胰岛素)的水平。根据肾类器官形成的其他方案,较低水平的KOSR有利于维持肾组织增殖和分化之间的平衡17。此外,我们还降低了II期培养基13中的葡萄糖浓度。
我们的方法描述了一种用于肾类器官悬浮测定的设置,从原始出版物5,13中所述的初始〜60%融合hPSC 100mm培养板中产生多达约1,000个类器官。该协议可以很容易地扩展到从多个100 mm或150 mm板开始,以进一步增加类器官的数量。
先前的研究表明,初始方案步骤对于中间中胚层分化5,19,20 至关重要,因此,在此阶段实施严格的培养基组成至关重要。从方案的第一阶段去除未定义的组分,例如血清,白蛋白,无蛋白杂交瘤培养基II,可能有助于提高测定21之间的一致分化效率。
肾细胞的代谢状态对其功能至关重要…
The authors have nothing to disclose.
这项研究由美国国立卫生研究院R01 DK069403,UC2 DK126122和P30-DK079307以及ASN肾脏研究基金会Ben J. Lipps研究奖学金计划资助。
2-Mercaptoethanol | Thermo Fisher | 21-985-023 | |
Anti-adherence rinsing solution | STEMCELL Technologies | 7010 | |
CHIR99021 | STEMCELL Technologies | 72054 | 10 mM stock in DMSO |
Corning disposable spinner flasks | Fisher Scientific | 07-201-152 | |
Corning Ultra-Low Attachment 6-well plates | Fisher Scientific | 07-200-601 | |
Corning Slow-Speed Stirrers | Fisher Scientific | 11-495-03 | Multi plate magnetic stirrer for spinner flask culture |
Dispase | STEMCELL Technologies | 7923 | Aliquot and freeze |
DMEM, low glucose, pyruvate, no glutamine, no phenol red | Thermo Fisher | 11054020 | |
DPBS 1x, no calcium, no magnesium | Thermo Fisher | 14-190-250 | |
Egg / Oval Stirring Bars | 2mag | PI20106 | |
Excelta General-Purpose Tweezers | Fisher Scientific | 17-456-103 | Keep sterile in the cell culture hood |
EZBio Single Use Media Bottle, 250mL | Foxx Life Sciences | 138-3211-FLS | Used to make PVA 10% |
Falcon Standard Tissue Culture Dishes (100 mm) | Thermo Fisher | 08-772E | |
Fisherbrand Sterile Aspirating Pipet 2mL | Fisher Scientific | 14-955-135 | |
Fisherbrand Cell Lifters – Cell lifter | Fisher Scientific | 08-100-240 | |
Fisherbrand Multi Function 3D Rotators | Fisher Scientific | 88-861-047 | Orbital shaker |
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix | Thermo Fisher | A1413302 | BME. Aliquot on ice and freeze. Another suitable matrix alternative is Matrigel or Cultrex. |
Gentle Cell Dissociation Reagent | STEMCELL Technologies | 7174 | GCDR |
GlutaMAX Supplement | Thermo Fisher | 35-050-061 | L-glutamine supplement. |
HEPES (1M) | Thermo Fisher | 15-630-080 | |
Insulin-Transferrin-Selenium-Ethanolamine | Thermo Fisher | 51-500-056 | ITSE |
KnockOut Serum Replacement – Multi-Species | Thermo Fisher | A3181502 | KOSR. Aliquot and freeze |
Lipid Mixture 1, Chemically Defined | Millipore-Sigma | L0288-100ML | |
MEM Non-Essential Amino Acids Solution | Thermo Fisher | 11140-050 | |
MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 500mL | Fisher Scientific | S2GPU05RE | |
MilliporeSigma Stericup Quick Release-GP Sterile Vacuum Filtration System 250mL | Fisher Scientific | S2GPU02RE | |
MIXcontrol MTP / Variomag TELEcontrol MTP Control Unit | 2mag | VMF 90250 U | |
MIXdrive 6 MTP / Variomag TELEdrive 6 MTP Microplate Stirring Drive | 2mag | VMF 40600 | 6MSP |
MP Biomedicals 7X Cleaning Solution | Fisher Scientific | MP0976670A4 | Tissue culture suitable detergent. Make a 5% solution in water |
mTeSR1 | STEMCELL Technologies | 85850 | hPSC medium.TeSR-E8, NutriStem XF, and mTeSR Plus medium have also been tested and are suitable alternatives. |
Nunc 50 mL Conical, Sterile Centrifuge Tubes | Fisher Scientific | 12-565-270 | |
Nunc 15mL Conical Sterile Centrifuge Tubes | Fisher Scientific | 12-565-268 | |
Penicillin-Streptomycin | Thermo Fisher | 15-140-122 | Aliquot and freeze |
Plasmocin | Invivogen | ant-mpt | Anti-mycoplasma reagent. Aliquot and freeze |
pluriStrainer® 200 µm | Fisher Scientific | NC0776417 | Cell strainer |
pluriStrainer® 500 µm | Fisher Scientific | NC0822591 | Cell strainer |
Poly(vinyl alcohol) 87-90% hydrolyzed (PVA) | Millipore-Sigma | P8136-250G | 10% in DPBS stirring at 98 degrees C until disolves, make in 138-3211-FLS |
ROCK inhibitor Y-27632 (ROCKi) | STEMCELL Technologies | 72304 | 10 mM stock in DPBS |
Sterile Disposable Serological Pipets – 10mL | Fisher Scientific | 13-678-11E | |
Sterile Disposable Serological Pipets – 25mL | Fisher Scientific | 13-678-11 | |
Sterile Disposable Serological pipette – 5 mL | Fisher Scientific | 13-678-12D | |
TeSR-E5 | STEMCELL Technologies | 5916 | Serum-free, low protein base medium for E5-ILP |
Variomag distriBOX 2 Distributor | 2mag | VMF 90512 | If you use more than one MIXdrive |