PO.TB04.01 · 肿瘤生物学
在ProstaCult™类器官培养基中扩增源自人体组织的多谱系前列腺类器官
Expansion of multilineage prostate organoids derived from human tissue in ProstaCult™ organoid medium
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
类器官已成为研究正常与肿瘤细胞生物学的强大模型；然而，由于难以在长时间内维持含雄激素受体（AR）表达细胞的多谱系类器官，将类器官技术应用于培养人前列腺上皮细胞一直颇具挑战。为解决这一问题，我们开发了无血清的ProstaCult™类器官培养基（人源），以稳健地长期扩增人前列腺上皮细胞形成类器官。将良性人前列腺组织解离，直接接种于Matrigel®穹顶培养中，或在接种前先于EpiCult™ Plus培养基中贴壁培养至多4代。所得类器官每10至12天以小片段传代。通过流式细胞术和免疫细胞化学分析培养物中泛上皮标志物EpCAM、腔面标志物CD26、角蛋白8（K8）和雄激素受体（AR）蛋白，以及基底标志物CD271和K14的表达。我们的结果表明，前列腺上皮细胞可在10至12天内生成由极化上皮构成的多谱系类器官，同时包含基底细胞（EpCAM+、K14+、CD271high/CD26low）和腔面细胞（EpCAM+、AR+、K8+、CD271low/CD26high）。这些人前列腺类器官可扩增至少7代并维持适当的标志物表达（n=5），且可冻存而不丧失长期扩增潜力。类器官对从培养基中去除二氢睾酮（DHT）有反应，在3代中生成的腔面细胞总数下降43±7%（均值±SEM；n=4；p≤0.01，配对t检验），且AR表达下调（n=4）。同样，enzalutamide处理导致3代中生成的腔面细胞总数下降24±9%（均值±SEM；n=4）。这些结果表明，ProstaCult™类器官培养基（人源）能够在体外稳健地长期维持含AR+腔面细胞的多谱系类器官，为前列腺癌研究提供了一个优化且标准化的模型系统。
查看英文原文 English abstract
Organoids have emerged as a powerful model for studying normal and tumour cell biology; however, the application of organoid technology for culturing human prostate epithelial cells has been challenging due to the difficulty in maintaining multilineage organoids with androgen receptor (AR)-expressing cells in culture for extended periods of time. To address this, we developed serum-free ProstaCult™ Organoid Medium (Human) to robustly expand human prostate epithelial cells long-term as organoids. Benign human prostate tissue was dissociated and seeded either directly into Matrigel® dome cultures or pre-cultured up to 4 passages in adherent culture in EpiCult™ Plus medium prior to seeding. The resulting organoids were passaged as small fragments every 10 - 12 days. Cultures were analyzed by flow cytometry and immunocytochemistry for the expression of pan epithelial marker EpCAM, luminal markers CD26, keratin 8 (K8), and androgen receptor (AR) protein, and basal markers CD271 and K14. Our results demonstrate that prostate epithelial cells can generate multilineage organoids consisting of a polarized epithelium with both basal (EpCAM + , K14 + , CD271 high /CD26 low ) and luminal cells (EpCAM + , AR + , K8 + , CD271 low /CD26 high ) in 10 - 12 days. These human prostate organoids can be expanded for at least 7 passages while maintaining appropriate marker expression (n = 5) and can be cryopreserved without losing long-term expansion potential. The organoids were responsive to dihydrotestosterone (DHT) removal from the culture medium, with a 43 ± 7% decrease in the total number of luminal cells generated over 3 passages (mean ± SEM; n = 4; p ≤ 0.01, paired t-test) and a downregulation of AR expression (n = 4). Similarly, enzalutamide treatment resulted in a 24 ± 9% decrease in the total number of luminal cells generated over 3 passages (mean ± SEM; n = 4). These results demonstrate that ProstaCult™ Organoid Medium (Human) enables robust long-term maintenance of multilineage organoids containing AR+ luminal cells in vitro , providing an optimized and standardized model system for prostate cancer research.
利益披露 Disclosure
A. Tsai, None..
A. C. Eaves, None..
S. A. Louis, None..
J. Stingl, None.