Université de technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu, CS 60319, 60203, Compiègne Cedex, France; CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan.
Université de technologie de Compiègne, CNRS, Biomechanics and Bioengineering, Centre de recherche Royallieu, CS 60319, 60203, Compiègne Cedex, France.
J Biotechnol. 2021 Mar 20;330:45-56. doi: 10.1016/j.jbiotec.2021.02.009. Epub 2021 Feb 19.
The limited availability of primary human β-cells/islets and their quality (due to donor diversity) restrict the development of in vitro models for diabetes research. Human induced pluripotent stem cells (hiPSCs) may be a promising cell-source for diabetes studies, anti-diabetic drug screening and personalized therapies. However, achieving levels of maturity/functionality that are comparable to the in vivo situation and islets rebuilt from iPSCs is still challenging. Here, we compare and discuss two strategies for culturing human pancreatic β-cells derived from hiPSCs in microfluidic biochips. First, we confirmed that the protocol in conventional Petri 2D monolayer led to insulin, PDX1 and MAFA positive staining, to C-Peptide productive cells, and to tissue responsive to high/low glucose and GLP1 stimulation. This protocol and its subsequent modifications (including extracellular matrix coating, cell adhesion time, cell inoculation density, flow rate) was not successful in the 2D biochip culture. We proposed a second strategy using 3D spheroids created from honeycomb static cultures. Spheroids in static experiments carried out over 14 days demonstrated that they expressed high levels of β-cell markers (INS mRNA) and higher α-cell markers (GCG mRNA and glucagon positive staining), when compared to Petri 2D cultures. Furthermore, the 3D spheroids were specifically able to secrete insulin in response to both high/low glucose stimulation and GLP1 exposure. The spheroids were successfully inoculated into biochips and maintained for 10 days in perfusion. The 3D biochip cultures increased mRNA levels of GCG and maintained high levels of β-cell markers and responsiveness to both high/low glucose and GLP1 stimulation. Finally, C-peptide and insulin secretion were higher in biochips when compared to static spheroids. These results illustrate the promising potential for hiPSCs derived β-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.
人原代β细胞/胰岛数量有限，且其质量（因供体多样性而异）限制了糖尿病研究体外模型的发展。人诱导多能干细胞（hiPSC）可能成为糖尿病研究、抗糖尿病药物筛选和个性化治疗的有前途的细胞来源。然而，要达到与体内情况和由 iPSC 重建的胰岛相当的成熟/功能水平仍然具有挑战性。在这里，我们比较并讨论了两种在微流控生物芯片中培养人诱导多能干细胞衍生的胰腺β细胞的策略。首先，我们证实，传统的 Petri 2D 单层培养方案可诱导 hiPSC 分化为胰岛素、PDX1 和 MAFA 阳性细胞，产生 C-肽阳性细胞，并使组织对高/低糖和 GLP1 刺激产生反应。该方案及其后续改进（包括细胞外基质包被、细胞黏附时间、细胞接种密度、流速）在 2D 生物芯片培养中并不成功。我们提出了第二种策略，使用来自蜂窝状静态培养的 3D 球体。静态实验中培养 14 天的球体表明，与 Petri 2D 培养相比，它们表达高水平的β细胞标志物（INS mRNA）和更高水平的α细胞标志物（GCG mRNA 和胰高血糖素阳性染色）。此外，3D 球体能够特异性地对高/低糖刺激和 GLP1 暴露作出胰岛素分泌反应。球体成功接种到生物芯片中，并在灌注条件下维持 10 天。3D 生物芯片培养增加了 GCG 的 mRNA 水平，并维持了高水平的β细胞标志物和对高/低糖及 GLP1 刺激的反应性。最后，与静态球体相比，生物芯片中的 C 肽和胰岛素分泌更高。这些结果表明，hiPSC 衍生的β细胞及其基于球体的胰腺类器官芯片模型在胰腺疾病/糖尿病建模和抗糖尿病药物筛选方面具有很大的应用潜力。
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