Tan Justin J Y, Thuya Win Lwin, Zhu Hongyu, Kristo Jessie G, Common John E, Wu Chunyong, Ho Paul C L, Kang Lifeng
Department of Pharmacy, Faculty of Science, National University of Singapore, Lower Kent Ridge Road, 18 Science Drive 4, Singapore 117543, Singapore.
Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore.
Biomater Adv. 2025 Dec;177:214423. doi: 10.1016/j.bioadv.2025.214423. Epub 2025 Jul 21.
Three-dimensional (3D) cell-culturing methods have usually been considered superior to two-dimensional (2D) culturing for in-vitro tissue formation intended for tissue engineering and drug research applications, including hair follicle (HF) development. However, cellular interactions within 3D cultures are generally more complex and therefore, may require further investigation. Apart from grafting in-vitro cultured (2D and 3D) dermal papilla cells directly onto the skin of animals to study the impact of 3D culturing on hair inductivity, molecular studies remain lacking in the understanding of how 2D and 3D culturing methods influence the morphogenesis of early stage HF models. The proposition that 3D cultures is always superior to 2D cultures for mimicking HF at its early developmental stage remains unknown. Therefore, this study aimed to investigate the influence of 3D and 2D culturing methods on the morphogenesis of HFs. 3D-cultured spheroids were assumed to exhibit greater expressions of HF-associated proteins and more expected drug-induced expression responses than 2D cultures. Dermal papilla cells and keratinocytes were cultured together in 2D and 3D cultures, where polyethylene glycol diacrylate microwell arrays were designed to provide the 3D culturing environment. Both 2D and 3D cultures were treated with either minoxidil or dihydrotestosterone (DHT) and the expressions of four hair proteins were analyzed. The results showed that 3D cultures responded in more expected ways than 2D cultures when exposed to minoxidil, demonstrating a significant increase in trichohyalin (AE15, one of the 4 proteins) as expected, while 2D cultures exhibited a significant down-regulation. On the other hand, surprisingly, DHT treatment significantly reduced all protein expressions in 2D culture as expected, but did not significantly alter protein expression in 3D culture, suggesting that 2D cultures could respond better than 3D cultures in DHT treatment.
对于旨在用于组织工程和药物研究应用（包括毛囊发育）的体外组织形成而言，三维（3D）细胞培养方法通常被认为优于二维（2D）培养。然而，3D培养中的细胞相互作用通常更为复杂，因此可能需要进一步研究。除了将体外培养的（2D和3D）真皮乳头细胞直接移植到动物皮肤上以研究3D培养对毛发诱导性的影响外，在理解2D和3D培养方法如何影响早期毛囊模型的形态发生方面，分子研究仍然缺乏。关于在毛囊早期发育阶段模拟毛囊时3D培养总是优于2D培养这一观点仍不明确。因此，本研究旨在探讨3D和2D培养方法对毛囊形态发生的影响。与2D培养相比，3D培养的球体被认为会表现出更高的毛囊相关蛋白表达以及更预期的药物诱导表达反应。将真皮乳头细胞和角质形成细胞在2D和3D培养中共同培养，其中聚乙二醇二丙烯酸酯微孔阵列被设计用于提供3D培养环境。2D和3D培养均用米诺地尔或二氢睾酮（DHT）处理，并分析四种毛发蛋白的表达。结果表明，在暴露于米诺地尔时，3D培养比2D培养的反应更符合预期，如预期的那样，毛透明蛋白（AE15，4种蛋白之一）显著增加，而2D培养则表现出显著下调。另一方面，令人惊讶的是，DHT处理如预期显著降低了2D培养中的所有蛋白表达，但在3D培养中并未显著改变蛋白表达，这表明在DHT处理中2D培养比3D培养反应更好。
