图1:猪心外膜切片制备。猪心室取样和切片示意图。心脏收集和设置:(1)采集心脏,保持心包,用冷停搏液再灌注。心室与心脏分离,避免接触心外膜,以防止损伤。(2)组织切片前必须设置包埋区,使所有组份冷却至冰冷温度。组织块制备:(3)将心脏组织放在冰冷的培养皿中,(4)用单刃钢刀解剖8mm ×8 mm的组织块。组织块嵌入:(5)切片的心外膜侧被压平在冷琼脂糖表面上,并放置在定制的3D打印环的中间。倒入5-8毫升30-37°C低熔点琼脂糖以嵌入组织块。(6)一旦凝固,将块从环上取下,并在一侧进行方正,以便与振动器的刀片(红色虚线)更好地对齐。切片切割和培养:(8)嵌入的组织块沿方形方向,并粘在振动器标本架上。 The blade is aligned to the epicardial surface and 400–500 µm thick slices are cut using a high precision vibratome. (9) Slices are allowed to recover for at least 30 min in a recovery bath. (10) Epicardial slices are cultured epicardium-up on 8-mm-high pillars cast at the bottom of a Petri dish. (11) En face immunohistochemistry of WT1 (red) and mesothelin (MSLN, green) shows the presence of a continuous epicardial layer, characterized by typical morphology and marker expression (WT1+ cells indicated with full arrowhead, WT1− MSLN+ cells indicated with empty arrowhead). This figure was created using BioRender.com. Credit:再生医学(2022)。DOI: 10.1038 / s41536 - 021 - 00202 - 7