Effective procedures for the purification of Leydig cells (LCs) can facilitate functional studies and transplantation therapies. However, current methods to purify LCs from testes are still far from satisfactory. Here, we found that testicular autofluorescence existed in the interstitium along with the gradual maturation of LCs from birth to adulthood. These autofluorescent cells were further isolated by fluorescence-activated cell sorting (FACS) and determined to be composed of LCs and macrophages. To further purify LCs, we combined two fluorescence channels of FACS and successfully separated LCs and macrophages. Of note, we confirmed that the obtained LCs not only possessed high purity, viability and quantity but also had intact steroidogenic activity and excellent responsiveness to luteinizing hormone. Moreover, subcutaneous transplantation of isolated LCs could alleviate the symptoms of testosterone deficiency in castrated mice. In summary, we established an effective autofluorescence-based method for isolating LCs. This method will aid in the future success of using LCs for basic and translational applications.

用于纯化 Leydig 细胞 (LC) 的有效程序可以促进功能研究和移植治疗。然而，目前从睾丸中纯化 LC 的方法仍远不能令人满意。在这里，我们发现睾丸自发荧光存在于间质中，同时 LC 从出生到成年逐渐成熟。这些自发荧光细胞通过荧光激活细胞分选 (FACS) 进一步分离，并确定由 LC 和巨噬细胞组成。为了进一步纯化 LC，我们结合了 FACS 的两个荧光通道，成功分离了 LC 和巨噬细胞。值得注意的是，我们证实所获得的 LC 不仅具有高纯度、活力和数量，而且具有完整的类固醇生成活性和对促黄体激素的出色反应。而且，分离的 LC 皮下移植可以缓解去势小鼠睾酮缺乏的症状。总之，我们建立了一种有效的基于自发荧光的分离 LC 的方法。这种方法将有助于未来成功地将 LC 用于基础和转化应用。