冷藏条件可引起小鼠胆管解剖结构的形态学变化。(一)冷库实验设置。简单地说，用颈椎脱位(CD)扑杀小鼠，立即用UW溶液灌注肝脏，放入UW袋中。这个袋子被放在另一个装有冷生理盐水的袋子里，并被冰包围。肝脏在这些条件下维持1、5或10小时。每实验组4 ~ 5只小鼠。(B)冷藏1、5或10小时后的整个小鼠肝脏。比例尺，1厘米。(C)小鼠肝脏在冷藏0、1、5或10小时的H&E染色。比例尺，120 μm。 Far right: Digital magnification of the morphological changes in the biliary architecture. (D) Immunofluorescence of Keratin19 (K19) positive cholangiocytes (green). Scale bars, 250 μm. Isotype-negative control is shown in the top left quadrant. Far right: Digital magnification. (E) H&E staining at 10 hours shows cholangiocyte detachment in the biliary lumen. Right: Digital magnification. Far right: Immunofluorescence confirms the detached cells express the cholangiocyte marker K19 (green). Scale bars, 60 μm. (F) Number of bile ducts (BD) with detached K19-positive cholangiocytes during cold storage. **P < 0.01 (mean ± SEM). ANOVA, Sidak’s multiple comparisons test. (G) Bile ducts isolated from livers maintained 0 or 10 hours in cold storage (CS), grown as organoids in culture and photomicrographed over 7 days. Scale bar, 120 μm. (H) Quantification of the average organoid size (pxl2)从肝脏分离的胆管中生长而来，冷藏0或10小时。**P < 0.01(均值±SEM)，第7天的学生t检验(n = 4只小鼠，每只小鼠培养3个技术重复)。(I)从冷藏0或10小时的肝脏中分离的胆管生长的类器官中，每4 '，6-二氨基氨基-2-苯基吲哚(DAPI)阳性细胞中Ki67、PCNA和LGR5总数的百分比。**P < 0.01， ***P < 0.001(均值±SEM)， Student 's t检验。信贷:科学转化医学(2022)。DOI: 10.1126 / scitranslmed.abj4375
