Abstract：
Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.
摘要：
小鼠神经元CAD5细胞系有效地繁殖各种病毒株。以前,我们已经证明，它也可以分化成形态上类似神经元的细胞。这里,我们证明，在相同的条件下，慢性感染朊病毒的CAD5细胞会发生分化。为了让我们的模型更真实，我们触发了通过缓慢摇动CAD5细胞悬液产生的3D培养中的分化。球体在1周内形成，并在不到3周的培养中完全发育。成熟的球状体的中值尺寸为〜300μm，可以培养长达12周。分化标记GAP43，酪氨酸羟化酶，β-III-微管蛋白和SNAP25支持球状体细胞的分化状态。它们中的大多数被发现在细胞周期的G0/G1期，这是典型的分化细胞。此外，细胞膜上一半的PrPC被N端截短，与分化的CAD5贴壁细胞相似。最后,我们证明，球体可以从朊病毒感染的CAD5细胞中产生。通过免疫组织化学证实了朊病毒的存在，蛋白质印迹和种子扩增试验。我们还证实，球状体可以从头感染朊病毒。我们的分化CAD5细胞的3D培养模型成本低，易于生产和栽培数周。我们预计它可能用于抗朊病毒化合物的测试和未来的朊病毒形成动力学研究。
