Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

Jeanette Wihan; Isabell Karnatz; Isabelle S&#233;bastien; Ralf Kettenhofen; Benjamin Schmid; Christian Clausen; Benjamin Fischer; Rachel Steeg; Heiko Zimmermann; Julia C. Neubauer

doi:10.3791/65085

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Production of Human Neurogenin 2-Inducible Neurons in a Three-Dimensional Suspension Bioreactor

在三维悬浮生物反应器中产生人神经原蛋白2诱导神经元

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07:21 min

March 17, 2023

DOI: 10.3791/65085-v

Jeanette Wihan¹, Isabell Karnatz¹, Isabelle Sébastien¹, Ralf Kettenhofen¹, Benjamin Schmid², Christian Clausen², Benjamin Fischer¹, Rachel Steeg³, Heiko Zimmermann^1,4,5,6, Julia C. Neubauer^1,4

¹Fraunhofer Project Center for Stem Cell Process Engineering,Fraunhofer Institute for Biomedical Engineering IBMT, ²Bioneer A/S, ³Fraunhofer UK Research Ltd, Technology and Innovation Centre, ⁴Fraunhofer Institute for Biomedical Engineering IBMT, ⁵Department of Molecular and Cellular Biotechnology,Saarland University, ⁶Facultad de Ciencias del Mar,Universidad Católica del Norte

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Overview

This article describes a protocol for generating human induced pluripotent stem cell-derived neurons using a benchtop 3D suspension bioreactor. The method facilitates high cell yield and rapid neuronal differentiation in a physiological environment, offering potential for large-scale applications.

Key Study Components

Area of Science

Neuroscience
Stem Cell Biology
Cell Culture Techniques

Background

Human induced pluripotent stem cells can differentiate into various cell types.
3D culture systems improve cell interactions and viability.
Large-scale applications are necessary for efficient cell production.
Previous methods primarily utilized 2D culture systems.

Purpose of Study

To develop a cost-effective protocol for rapid neuronal differentiation.
To enhance cell yield and maintain low batch-to-batch variability.
To lay the groundwork for automated large-scale bioreactor systems.

Methods Used

The study utilizes a benchtop 3D suspension bioreactor.
Human induced pluripotent stem cells are the biological model for differentiation.
The protocol includes critical steps for cell detachment, resuspension, and differentiation.
Neuronal differentiation is induced after a pre-cultivation phase, followed by media changes.
Cells are cryopreserved after two days of differentiation for subsequent maturation.

Main Results

The protocol resulted in neuronal cells that maintained viability and functionality.
Cell aggregates grew substantially during the initial days of differentiation.
Distinct neuronal markers confirmed the identity of cryopreserved cells.
The study found that prolonged culture beyond a certain point did not increase cell yield.

Conclusions

This study demonstrates a scalable approach for producing neurons from iPSCs.
The methods introduced may advance our understanding of neuronal development.
Future applications could significantly benefit from automation in bioreactor environments.

Frequently Asked Questions

What are the advantages of using a 3D bioreactor for neuronal differentiation?

3D bioreactors improve cell-cell and cell-matrix interactions, resulting in higher yields and more physiologically relevant cellular environments compared to 2D cultures.

How is the induction of neuronal differentiation achieved?

Neuronal differentiation is initiated by replacing the culture medium with a neural induction medium after pre-cultivation of aggregates.

What are the key steps in the protocol?

Key steps include detaching human iPSCs, resuspending aggregates, inducing differentiation, and performing media changes regularly.

What types of data are obtained from this method?

The method yields neuronal cells that can be characterized by molecular markers and viability assessments, providing insights into their maturity.

How can this protocol be scaled for larger studies?

The approach is suited for automation in bioreactors, allowing for increased throughput and efficiency in cell production for research and therapeutic applications.

What are the limitations of this study?

Prolonged cultivation past optimal time frames may not enhance yields, as aggregates can become resistant to enzymatic detachment.

本文描述了在台式 3D 悬浮生物反应器中生成人类诱导多能干细胞衍生神经元的协议。

使用生物反应器将诱导多能干细胞或IPSC培养方案从2D转换为3D，可以产生大量细胞，用于大规模应用，例如高通量筛选。这种在生理3D环境中的快速神经元分化方案改善了起始细胞相互作用，并在较短的持续时间内提供高细胞产量，批次间=批次间差异低，从而降低成本和时间。欧洲诱导多能干细胞库正在应用类似的方法，在多个谱系（包括其他脑细胞和心肌细胞）中快速且经济高效地生成分化细胞。

当人诱导的多能干细胞或人 iPSC 培养物汇合 60% 至 80% 时开始预培养。从人iPSC中完全吸出培养基，并用1X DPBS轻轻冲洗细胞两次。将两毫升预热的胰蛋白酶EDTA溶液加入6厘米培养皿中，并在培养箱中以37摄氏度孵育细胞三分钟。

然后，轻轻敲击培养皿以促进细胞分离，或者如果细胞没有脱离，则再孵育一到两分钟。然后，通过加入五毫升含有 ROCK 抑制剂的无饲养层 IPSC 维持培养基，将细胞重悬在每个培养皿中。将细胞悬液转移到15毫升或50毫升的管中，并通过移液轻轻混合以确保细胞奇异化。

使用自动细胞计数器确定100微升细胞悬液中的细胞数，并将所需体积的细胞悬液转移到50毫升管中。将细胞以300 G离心三分钟。接下来，吸出上清液并将细胞重悬于两毫升含有 ROCK 抑制剂的无饲养层 IPSC 维持培养基中。

在每个 50 毫升的试管中填充 18 毫升的培养基。然后，将20毫升细胞悬液分配到每个生物反应器管中。将试管放入生物反应器系统并设置无限期的培养参数。

通过生物反应器显示屏启动预培养程序。为了第二天更换培养基，让聚集体在生物反应器管中沉淀约五分钟，然后小心地吸出上清液。每管加入15毫升不含ROCK抑制剂的新鲜无饲养层IPSC维持培养基，并在台式生物反应器中继续培养24小时。

要开始神经元分化，让聚集体沉淀在生物反应器管中并小心地从细胞中吸出上清液，在管中留下约五毫升上清液。然后，加入35毫升由神经基础培养基和每毫升2微克多西环素组成的神经诱导培养基。将试管放回台式生物反应器中并继续培养。

如前所述，连续两天每天执行介质更换。如前所示吸出上清液后，将聚集体转移到无菌的15毫升或50毫升管中，并用DPBS轻轻冲洗聚集体两次。尽可能小心地吸出上清液，不要干扰聚集体。

然后根据沉淀大小向沉淀中加入两到五毫升预热的细胞解离酶，并将细胞在37摄氏度的水浴中孵育约10分钟。每两分钟轻轻重悬沉淀的聚集体，直到聚集体解离。加入预热的神经基础培养基，体积是先前添加的细胞解离酶的三倍，并小心重悬细胞以确保细胞奇异化。

确定细胞数量并将相应体积的细胞悬液转移到15毫升或50毫升管中以进行冷冻保存。以300 G离心至细胞三分钟。吸出上清液并将细胞沉淀轻轻重悬于含有10%二甲基亚砜的相应体积的冷冻培养基中。

将细胞悬液分装在合适的小瓶中进行冷冻保存。立即将小瓶转移到装有2-丙醇的预冷慢速冷冻容器中，并将容器置于零下80摄氏度过夜。第二天将小瓶置于零下150摄氏度下长期储存。

人iPSCs的贴壁培养物分离、单化并转移到悬浮液后，聚集体在24小时内形成并继续生长。经过两天的转基因诱导，早期的神经元可以被冷冻保存以供随后成熟。在悬浮的最初几天观察到人iPSCs的持续增殖，并且在诱导两天后细胞培养数量达到峰值。

然而，长时间悬浮培养超过四天并没有提高细胞产量，因为聚集体对酶奇异化的抵抗力越来越强。此外，与第0天相比，总直径在第二天增加了50%，在第五天几乎翻了一番。尽管直径的增加限制了聚集体的营养供应，但在分化的第二天或第五天，细胞的活力没有受到影响。

神经元标志物β三微管蛋白和微管相关蛋白（MAP2）的时间基因表达谱和免疫化学染色证实了两天冷冻保存细胞的神经元细胞身份。此外，神经元培养物富含微管相关蛋白tau转录物（MAPT），并显示出多能性调节转录因子POU5F1的表达伴随下降解冻后的第一周内形成了致密的神经炎网络，这也表明神经元培养物的成熟度增加。该协议为转化为大规模和全自动生物反应器奠定了基础，这表明未来大规模应用的细胞输出能力进一步显着增加。

一旦用神经原蛋白2证实，使用线性决定转录因子来加速分化已被应用于许多不同的细胞类型和疾病，以促进iPSC建模。