The human placenta is essential for successful reproduction. There is great variation in the anatomy and development of the placenta in different species, meaning that animal models provide limited information about human placental development and function. Until recently, it has been impossible to isolate trophoblast cells from the human placenta that proliferate in vitro. This has limited our ability to understand pregnancy disorders. Generating an in vitro model that recapitulates the unique features of the human placenta has been challenging. The first in vitro model system of human trophoblast that could be cultured long term and differentiated to syncytiotrophoblast (SCT) and extravillous trophoblast (EVT) was a two-dimensional (2D) culture system of human trophoblast stem cells. Here, we describe a protocol to isolate trophoblast from first-trimester human placentas that can be grown long term in a three-dimensional (3D) organoid culture system. Trophoblast organoids can be established within 2−3 weeks, passaged every 7–10 d, and cultured for over a year. The structural organization of these human trophoblast organoids closely resembles the villous placenta with a layer of cytotrophoblast (VCT) that differentiates into superimposed SCT. Altering the composition of the medium leads to differentiation of the trophoblast organoids into HLA-G+ EVT cells which rapidly migrate and invade through the Matrigel droplet in which they are cultured. Our previous research confirmed that there is similarity between the trophoblast organoids and in vivo placentas in their transcriptomes and ability to produce placental hormones. This organoid culture system provides an experimental model to investigate human placental development and function as well as interactions of trophoblast cells with the local and systemic maternal environment.

人类胎盘对于成功繁殖至关重要。不同物种的胎盘解剖结构和发育存在很大差异，这意味着动物模型提供的有关人类胎盘发育和功能的信息有限。直到最近，还不可能从体外增殖的人胎盘中分离出滋养层细胞。这限制了我们了解妊娠疾病的能力。生成概括人类胎盘独特特征的体外模型一直具有挑战性。第一个可以长期培养并分化为合体滋养层（SCT）和绒毛外滋养层（EVT）的人滋养层体外模型系统是人滋养层干细胞的二维（2D）培养系统。这里，我们描述了一种从妊娠早期人类胎盘中分离滋养细胞的方案，该胎盘可以在三维 (3D) 类器官培养系统中长期生长。滋养细胞类器官可在 2-3 周内建立，每 7-10 天传代一次，并培养一年以上。这些人类滋养层类器官的结构组织与绒毛胎盘非常相似，具有一层细胞滋养层 (VCT)，可分化为叠加的 SCT。改变培养基的组成会导致滋养细胞类器官分化为 HLA-G+ EVT 细胞，这些细胞会快速迁移并侵入培养它们的 Matrigel 液滴。我们之前的研究证实，滋养层类器官和体内胎盘在转录组和产生胎盘激素的能力方面存在相似性。