Alberta Diabetes Institute, University of Alberta, Edmonton, AB, T6G 2T9, Canada.
Department of Surgery, University of Alberta, Edmonton, AB, T6G 2B7, Canada.
Stem Cell Res Ther. 2023 Jun 6;14(1):154. doi: 10.1186/s13287-023-03382-9.
Induced pluripotent stem cells (iPSCs) offer potential to revolutionize regenerative medicine as a renewable source for islets, dopaminergic neurons, retinal cells, and cardiomyocytes. However, translation of these regenerative cell therapies requires cost-efficient mass manufacturing of high-quality human iPSCs. This study presents an improved three-dimensional Vertical-Wheel® bioreactor (3D suspension) cell expansion protocol with comparison to a two-dimensional (2D planar) protocol.
Sendai virus transfection of human peripheral blood mononuclear cells was used to establish mycoplasma and virus free iPSC lines without common genetic duplications or deletions. iPSCs were then expanded under 2D planar and 3D suspension culture conditions. We comparatively evaluated cell expansion capacity, genetic integrity, pluripotency phenotype, and in vitro and in vivo pluripotency potential of iPSCs.
Expansion of iPSCs using Vertical-Wheel® bioreactors achieved 93.8-fold (IQR 30.2) growth compared to 19.1 (IQR 4.0) in 2D (p < 0.0022), the largest expansion potential reported to date over 5 days. 0.5 L Vertical-Wheel® bioreactors achieved similar expansion and further reduced iPSC production cost. 3D suspension expanded cells had increased proliferation, measured as Ki67 expression using flow cytometry (3D: 69.4% [IQR 5.5%] vs. 2D: 57.4% [IQR 10.9%], p = 0.0022), and had a higher frequency of pluripotency marker (Oct4NanogSox2) expression (3D: 94.3 [IQR 1.4] vs. 2D: 52.5% [IQR 5.6], p = 0.0079). q-PCR genetic analysis demonstrated a lack of duplications or deletions at the 8 most commonly mutated regions within iPSC lines after long-term passaging (> 25). 2D-cultured cells displayed a primed pluripotency phenotype, which transitioned to naïve after 3D-culture. Both 2D and 3D cells were capable of trilineage differentiation and following teratoma, 2D-expanded cells generated predominantly solid teratomas, while 3D-expanded cells produced more mature and predominantly cystic teratomas with lower Ki67 expression within teratomas (3D: 16.7% [IQR 3.2%] vs.. 2D: 45.3% [IQR 3.0%], p = 0.002) in keeping with a naïve phenotype.
This study demonstrates nearly 100-fold iPSC expansion over 5-days using our 3D suspension culture protocol in Vertical-Wheel® bioreactors, the largest cell growth reported to date. 3D expanded cells showed enhanced in vitro and in vivo pluripotency phenotype that may support more efficient scale-up strategies and safer clinical implementation.
诱导多能干细胞（iPSC）有望成为再生医学的一场革命，因为它可以作为胰岛、多巴胺能神经元、视网膜细胞和心肌细胞的可再生来源。然而，这些再生细胞疗法的转化需要高效、经济地大规模生产高质量的人类 iPSC。本研究提出了一种改进的三维垂直轮®（3D 悬浮）细胞扩增方案，并与二维（2D 平面）方案进行了比较。
使用仙台病毒转染人外周血单核细胞，建立无支原体和病毒污染的 iPSC 系，且不存在常见的遗传重复或缺失。然后在 2D 平面和 3D 悬浮培养条件下扩增 iPSC。我们比较评估了 iPSC 的细胞扩增能力、遗传完整性、多能性表型，以及体外和体内的多能性潜力。
使用垂直轮®生物反应器扩增 iPSC 可实现 93.8 倍（IQR 30.2）的扩增（与 2D 相比为 19.1 [IQR 4.0]，p<0.0022），这是迄今为止报道的 5 天内最大的扩增潜力。0.5 L 垂直轮®生物反应器可实现类似的扩增，并进一步降低 iPSC 的生产成本。3D 悬浮扩增的细胞增殖增加，使用流式细胞术检测 Ki67 表达（3D：69.4%[IQR 5.5%]比 2D：57.4%[IQR 10.9%]，p=0.0022），且多能性标志物（Oct4NanogSox2）的表达频率更高（3D：94.3%[IQR 1.4%]比 2D：52.5%[IQR 5.6%]，p=0.0079）。q-PCR 遗传分析表明，在长期传代（>25 代）后，iPSC 系中 8 个最常见突变区域不存在重复或缺失。2D 培养的细胞显示出初始多能性表型，在 3D 培养后转变为幼稚多能性表型。2D 和 3D 细胞均能进行三系分化，并在形成畸胎瘤后，2D 扩增的细胞主要产生实体性畸胎瘤，而 3D 扩增的细胞形成更成熟和主要为囊性的畸胎瘤，畸胎瘤内 Ki67 表达较低（3D：16.7%[IQR 3.2%]比 2D：45.3%[IQR 3.0%]，p=0.002），这与幼稚表型一致。
本研究在垂直轮®生物反应器中使用 3D 悬浮培养方案，在 5 天内实现了近 100 倍的 iPSC 扩增，这是迄今为止报道的最大细胞生长。3D 扩增的细胞表现出增强的体外和体内多能性表型，这可能支持更有效的规模化策略和更安全的临床实施。
