School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou, China.
Department of Stomatology, Ningbo Women and Children Hospital, Ningbo, China.
Cell Prolif. 2021 Feb;54(2):e12969. doi: 10.1111/cpr.12969. Epub 2020 Dec 17.
Various factors could interfere the biological performance of DPSCs during post-thawed process. Yet, little has been known about optimization of the recovery medium for DPSCs. Thus, our study aimed to explore the effects of adding recombinant bFGF on DPSCs after 3-month cryopreservation as well as the underlying mechanisms.
DPSCs were extracted from impacted third molars and purified by MACS. The properties of CD146 DPSCs (P3) were identified by CCK-8 and flow cytometry. After cryopreservation for 3 months, recovered DPSCs (P4) were immediately supplied with a series of bFGF and analysed cellular proliferation by CCK-8. Then, the optimal dosage of bFGF was determined to further identify apoptosis and TRPC1 channel through Western blot. The succeeding passage (P5) from bFGF pre-treated DPSCs was cultivated in bFGF-free culture medium, cellular proliferation and stemness were verified, and pluripotency was analysed by neurogenic, osteogenic and adipogenic differentiation.
It is found that adding 20 ng/mL bFGF in culture medium could significantly promote the proliferation of freshly thawed DPSCs (P4) through suppressing apoptosis, activating ERK pathway and up-regulating TRPC1. Such proliferative superiority could be inherited to the succeeding passage (P5) from bFGF pre-stimulated DPSCs, meanwhile, stemness and pluripotency have not been compromised.
This study illustrated a safe and feasible cell culture technique to rapidly amplify post-thawed DPSCs with robust regenerative potency, which brightening the future of stem cells banking and tissue engineering.
在解冻后过程中，各种因素可能会干扰 DPSCs 的生物学性能。然而，对于 DPSCs 的恢复介质的优化知之甚少。因此，我们的研究旨在探索在 3 个月冷冻保存后添加重组 bFGF 对 DPSCs 的影响及其潜在机制。
从阻生第三磨牙中提取 DPSCs 并通过 MACS 进行纯化。通过 CCK-8 和流式细胞术鉴定 CD146 DPSCs（P3）的特性。经过 3 个月的冷冻保存后，立即将回收的 DPSCs（P4）提供一系列 bFGF，并通过 CCK-8 分析细胞增殖。然后，确定 bFGF 的最佳剂量，通过 Western blot 进一步鉴定凋亡和 TRPC1 通道。用 bFGF 预处理的 DPSCs 进行传代（P5），在无 bFGF 的培养基中培养，验证细胞增殖和干性，并通过神经发生、成骨和脂肪分化分析多能性。
发现培养物中添加 20ng/ml bFGF 可通过抑制凋亡、激活 ERK 途径和上调 TRPC1 来显著促进新鲜解冻的 DPSCs（P4）的增殖。这种增殖优势可以遗传到 bFGF 预处理的 DPSCs 的后续传代（P5），同时，干性和多能性没有受到影响。
本研究说明了一种安全可行的细胞培养技术，可快速扩增具有强大再生能力的解冻后 DPSCs，为干细胞储存和组织工程的未来带来了希望。
