BACKGROUND Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. METHODS Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis. RESULTS This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%). CONCLUSION This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

中文翻译：

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标准化的符合GMP的可缩放生产的人体胰腺类器官。

背景技术类器官是在体外生长的三维细胞簇，概括了天然器官的关键特征。在再生医学中，类器官技术代表了一种有前途的方法，可用于替代严重受损的器官，例如1型糖尿病患者的胰腺。在化学定义的无血清培养基中分离人胰腺类器官（hPOs）将是该方法的一个重要里程碑。方法从废弃的胰腺组织开始，我们开发了一种大规模方法来获得临床上相关量的未分化类器官，避免了酶消化和操作者依赖的胰管挑选步骤。hPO身份通过分子和流式细胞仪分析进行表征。结果这项工作表明，有可能大规模生产类器官。我们在隔离，扩展和冻结来自五个捐赠者的hPO方面引入了一些创新。首先，选择能够在低通量下获得大量hPO的原料（胰岛贫化的胰腺）。另一方面，我们引入了机械离解，并取消了拣选步骤，以排除依赖于操作员的步骤，而不会影响培养的成功率（100％成功率）。另一个重要的改进是用Rspo1重组分子替代了R-spondin-1（Rspo1）条件培养基，从而获得了扩展培养基的明确组成。最后，我们实现了符合GMP的冻结协议。hPOs在7天内随直径和面积呈指数增长，分别增长了三倍和八倍。免疫表型分析和基因表达分析显示，hPOs由导管细胞（82.33±8.37％），腺泡细胞（2.80±1.25％）和胰腺祖细胞（5.81±2.65％）组成。结论这项工作代表了符合GMP的hPO生产的里程碑，并最终将其作为1型糖尿病疗法用于临床。