Name: brain organoid generation from induced pluripotent stem cells in home-made mini bioreactors
Uploaded: 2021-12-11T15:00:00
Description: Watch this Scientific Journal Video about Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors at JoVE.com

这项工作得到了俄罗斯联邦科学和高等教育部（RT-PCR分析）的075-15-2019-1669赠款以及俄罗斯科学基金会第19-15-00425号赠款（用于所有其他工作）的支持。作者还感谢Pavel Belikov在视频编辑方面的帮助。手稿中的人物是用 BioRender.com 创作的。

注意：在整个方案中使用无菌技术，不包括步骤1.2和1.3。在应用于细胞或类器官之前，将所有培养基和溶液加热至37°C。在37°C的CO2 培养箱中培养细胞，在80%湿度下在5%CO2 中培养细胞。协议方案如图 1所示。
1. 将培养皿转变为微型生物反应器
<ol><li>将无菌的15 mL离心管切割成7-8毫米高的环;高压灭菌环。</li>
	<li>将低附着力、...

<ol>
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所描述的方案有两个关键步骤，允许产生大小均匀的高质量类器官。首先，类器官从在细胞数量和细胞成熟度上几乎相同的球体生长。其次，自制的生物反应器为每个类器官提供了一个统一的环境，在这个环境中，类器官不会聚集或粘在一起。
细胞质量和细胞成熟状态对于执行实验方案至关重要。在iPSC汇合75-90%处开始神经元分化至关重要。如果细胞密度太低，iPSC可以分化成...

人类iPSC衍生的模型广泛用于神经发育和神经退行性疾病的研究1。在过去的十年中，3D脑组织模型，即所谓的脑类器官，基本上补充了传统的2D神经元培养物2。类器官在一定程度上概括了胚胎大脑的3D结构，并允许更精确的建模。许多方案已经发表，用于产生代表不同大脑区域的类器官：大脑皮层3，4，5，小脑6，中脑，前脑，下丘脑7，8，9和海马体10。已经有多个使用类器官研究人类神经系统疾病的例子11。此外，类器官在药物发现12中实施，并用于传染病研究，包括SARS-Cov-213，14。
大脑类器官的直径可达几毫米。因此，类器官的内区可能患有缺氧或营养不良，最终变得坏死。因此，许多方案包括特殊的生物反应器8、振荡器或微流体系统15。这些设备可能需要大量昂贵的细胞培养基。此外，这种设备的成本通常很高。一些生物反应器由许多机械部件组成，使其难以灭菌以重复使用。
大多数方案都受到"批处理效应"16的影响，这在从相同的iPSC获得的类器官之间产生了显着的变异性。这种变异性阻碍了需要一致性的药物测试或临床前研究。类器官的高产量足以选择大小均匀的类器官，可以部分解决这个问题。
时间因素也是一个重大问题。Matsui等人（2018）表明，脑类器官至少需要六个月才能达到成熟17。Trujillo等人（2019）还证明，只有在培养六个月后，类器官中才会发生电生理活性18。由于类器官成熟时间长，研究人员经常在完成前一个分化之前启动新的分化。多个并行的差异化过程需要额外的费用、设备和实验室空间。
我们最近开发了一种迷你生物反应器，主要解决了上述问题19。这款自制生物反应器由超低附着力或未经处理的培养皿组成，中间有一个塑料旋钮。这种塑料旋钮可防止类器官拥挤及其在培养皿中心的凝结，这是由振荡器的旋转引起的。本文描述了这种廉价而简单的自制迷你生物反应器如何能够大量产生高质量的脑类器官。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td>DMEM/F-12</td>
		</tr>
		<tr>
			<td>AggreWell400</td>
			<td>STEMCELL Technologies Inc</td>
			<td>34425</td>
			<td>24-well culture plate with microwells</td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>Neuronal supplement B</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>200 mM L-alanyl-L-glutamine</td>
		</tr>
		<tr>
			<td>Human BDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-2</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-839</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GDNF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-290</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828028</td>
			<td>Serum replacement</td>
		</tr>
		<tr>
			<td>mTESR1</td>
			<td>STEMCELL Technologies Inc</td>
			<td>85850</td>
			<td>Pliripotent stem cell medium</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Gibco</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Basal medium for neuronal cell maintenance</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15140130</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>InvivoGen</td>
			<td>ant-mpt-1</td>
			<td>Antimicrobials</td>
		</tr>
		<tr>
			<td>Purmorphamine</td>
			<td>EMD Millipore</td>
			<td>540220</td>
			<td></td>
		</tr>
		<tr>
			<td>StemMACS Y27632</td>
			<td>Miltenyi Biotec</td>
			<td>130-106-538</td>
			<td>Y27632</td>
		</tr>
		<tr>
			<td>StemMACS Dorsomorphin</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-466</td>
			<td>Dorsomorphin</td>
		</tr>
		<tr>
			<td>StemMACS LDN-193189</td>
			<td>Miltenyi Biotec</td>
			<td>130-106-540</td>
			<td>LDN-193189</td>
		</tr>
		<tr>
			<td>StemMACS SB431542</td>
			<td>Miltenyi Biotec</td>
			<td>130-106-543</td>
			<td>SB431542</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Versen solution</td>
			<td>Gibco</td>
			<td>15040066</td>
			<td>0.48 mM EDTA in PBS</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350010</td>
			<td></td>
		</tr>
	</tbody>
</table>

brain organoid generation from induced pluripotent stem cells in home-made mini bioreactors

iPSC衍生的脑类器官是一种有前途的技术，用于 体外 建模神经系统的病理学和药物筛选。这项技术最近才出现。它仍处于起步阶段，并且有一些限制尚未解决。目前的方案不允许获得足够一致的类器官用于药物发现和临床前研究。类器官的成熟可能需要长达一年的时间，这促使研究人员同时启动多个分化过程。这在空间和设备方面给实验室带来了额外的成本。此外，脑类器官通常在中心有一个坏死区，患有营养和缺氧。因此，大多数现行方案使用循环系统作为培养基以改善营养。
同时，没有用于类器官培养的廉价动态系统或生物反应器。本文描述了一种在紧凑且廉价的自制小型生物反应器中生产脑类器官的方案。该协议允许大量获得高质量的类器官。

协议方案如图1所示。该协议包括五种培养基，其中iPSCs在至少一个月内分化为脑类器官。开始分化，然后iPSCs达到75-90%的汇合度（图2A，B）。在培养基A中iPSC培养的第10-11天观察到向神经元分化的最初迹象，当时细胞开始聚集成"玫瑰花"（图2C）。在第14-15天，iPSCs分化为神经上皮祖细胞。99%的细胞在神经上皮标志物SOX1?...

Watch this Scientific Journal Video about Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors at JoVE.com

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

在这里，我们描述了一种从人类诱导多能干细胞（iPSCs）产生脑类器官的方案。为了获得大量和高质量的脑类器官，我们使用自制的迷你生物反应器。

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

迄今为止，从多能干细胞中分化的脑类器官是最接近天然人脑组织的体外3D模型。此外，脑类器官同样适用于模拟各种中枢神经系统病理学，测试药理活性物质，也可用于再生医学。同时，这项技术仍处于发展的初始阶段。该协议可以根据获得聚集体的方法和分化的进一步培养分为两组。第一种包括分化方案和方案，在培养时间和分化诱导剂方面有所不同，随后在特殊的低粘附装置（如市场管或板）中在振荡器中成熟类器官。另一组包括使用特殊的生物反应器。对各种方案的分析表明，类器官应在循环营养培养基的条件下培养。然而，由于缺乏简单而廉价的系统来获得具有标准尺寸和形态的类器官，因此需要开发这种设备来缩放该过程。在这项工作中，我们描述了我们获得和测试简单而廉价的微型生物反应器的方法，这些反应器可以获得大量高质量的类器官。将无菌的15毫升离心管切成7或8毫米高的环。高压灭菌环。将经过处理或微生物培养皿上的低聚集物分解成碎屑。将约1克塑料屑溶解在10毫升氯仿中过夜，以制备液体塑料。在无菌超低粘附力6厘米培养皿的中心制作一个塑料旋钮。有两种同样合适的方法。首先，将高压灭菌器放在中心的塑料环中，并将半毫升液体塑料涂在环的内部。第二个，没有任何塑料环，在培养皿的中心掉落半毫升的液体塑料。将盘子在层流罩中打开2或3小时，直到完全干燥。在培养基中培养诱导多能干细胞，用于多能干细胞，在35毫米培养皿中融合高达75%或90%，预先涂覆溶解在冷Dulbecco改性鹰培养基和Fisher-12培养基中的基质。准备培养基 A 加血清替代治疗。用血清替代物在培养基中培养诱导多能干细胞1天。在分化零天将多能干细胞培养基换成血清替代培养基。准备培养基A.在分化的第2天将培养基改为培养基A。在培养基A中培养细胞2周，每2天在培养皿中清爽培养基。在第14天，使用特殊的24孔培养板开始形成球体，该培养板在每个孔中含有约1， 200个微孔。准备一个带有微孔的24孔培养板。向每个孔中加入1毫升培养基A，在装有板架的摆动桶形转子中以1， 300g短暂离心5分钟。在显微镜下控制微孔中没有气泡。准备培养基B.从培养皿中取出培养基。对于细胞脱离，用PBS上制备的1.5毫升EDTA溶液处理细胞。在显微镜下控制细胞脱离。将细胞收获到15毫升管中。在管中加入5毫升混合的Dulbecco和Fisher培养基，朝向细胞。以200g离心5分钟。除去上清液并将细胞重新悬浮在2毫升培养基B中，将含有10亿个细胞的细胞悬浮液转移到具有微孔的24孔板的每个孔中。轻轻地上下移液细胞几次，并短暂离心100g1分钟以捕获微孔中的细胞。在显微镜下控制细胞均匀分布在微孔中。将板孵育过夜，使细胞聚集在球状体中。第二天早上，第15天，在显微镜下检查球体的质量，如果健康，它是透明和光滑的。小心地将每个孔中的球体收集到15毫升的管中，让球体通过重力沉淀2和3分钟，然后除去上清液。加入2毫升基质的球体，在冰上解冻和暂时解冻。通过移液轻轻混合，并在室温下孵育30分钟。为了洗涤多余的基质，将8毫升培养基B.移液器轻轻地加入管中，然后在100g下离心管1分钟。除去上清液。加入20毫升培养基B的管中，轻轻移液，然后在小型生物反应器之间拆分球状悬浮液。然后将迷你生物反应器放入15厘米的培养皿中，以防止水分蒸发和污染。将带有迷你生物反应器的培养皿放在轨道振荡器上。以70，75rpm的旋转速率培养类器官。在第16天，准备培养基C.将类器官转移到50毫升管中。5分钟，让它们落到底部。吸出上清液并加入5毫升培养基C.在迷你生物反应器中重新加入类器官。在培养基C中培养球体2周，每2天刷新培养基。在这2周内，每个迷你生物反应器选择约100个球体进行以下培养。第30天，准备培养基D.将培养基改为培养基D，这是一种成熟培养基。每2或3天刷新一次培养基，持续3周。然后使用溶解在F和GDNF中的培养基D。在培养基A和B中连续培养1个月后，获得大小和形态标准化的类器官。随着它的增长，他们需要更频繁地更换培养基，每周最多4次也会增加。经过2个月的栽培，它们的直径达到4和5毫米。又过了一个月，5毫米和6毫米，生长停止。该图显示了类器官酶的外观，用苏木精和曙红涂有清晰的部分。持续 1 个月的免疫组化分析显示存在 SOX2 阳性祖细胞簇，包括中央部分内部。在类器官的外周，神经胶质纤维酸性蛋白、微管相关蛋白2和噻嗪水解酶阳性。细胞是浓缩的，这对应于成熟形式的神经元。在某些情况下，在中央部分形成坏死区域的白色簇。这表明该协议对于获得较大的类器官具有局限性。最有可能的是，生长的限制因素是营养和氧气的扩散速率。球体的各种介质和细胞类型，并测试我们的系统是否存在某些种类的类器官。大量的脑和视网膜，来自肝癌细胞和间充质干细胞的组合类器官，来自资本爆炸间充质干细胞调用的组合类器官以及来自诱导多能干细胞和肠类器官的造血干细胞分化。它在细胞的初始组成中是可变的吗？分化因子、培养基和培养基、细胞外基质，可能还有气体混合物组成、平台旋转速率和其他参数。有可能在各种器官和组织中获得具有相同形状，大小或形态模型的类器官。微型生物反应器的使用被提议纳入这项工作。

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...