Tissue Engineering and Regenerative Medicine

, Volume 16, Issue 5, pp 513–523 | Cite as

Three-Dimensional Spheroid Formation of Cryopreserved Human Dental Follicle-Derived Stem Cells Enhances Pluripotency and Osteogenic Induction Properties

  • Hyo-Jung Kim
  • Iel-Yong Sung
  • Yeong-Cheol Cho
  • Min-Su Kang
  • Gyu-Jin Rho
  • June-Ho Byun
  • Won-Uk Park
  • Myeong-Gyun Son
  • Bong-Wook Park
  • Hyeon-Jeong LeeEmail author
  • Young-Hoon KangEmail author
Original Article



Enhancement and maintenance of the stemness of mesenchymal stem cells (MSCs) is one of the most important factors contributing to the successful in vivo therapeutic application of these cells. In this regard, three-dimensional (3D) spheroid formation has been developed as reliable method for increasing the pluripotency of MSCs. Moreover, using a new protocol, we have previously shown that dental tissues of extracted wisdom teeth can be effectively cryopreserved for subsequent use as a source of autologous stem cells. The main purpose of this study is to analyze the stemness and in vitro osteogenic differentiation potential of 3D spheroid dental MSCs compared with conventional mono-layer cultured MSCs.


In this study, MSC-characterized stem cells were isolated and cultured from long-term cryopreserved dental follicles (hDFSCs), and then 2D hDFSCs were cultured under 3D spheroid-forming conditions using a newly designed microchip dish. The spheroids (3D hDFSCs) thus produced were investigated and characterized with respect to stemness, MSC marker expression, apoptosis, cell cycle analysis, extracellular matrix (ECM) production, and osteogenic and adipogenic differentiation properties.


In terms of MSC and senescence markers, spheroid cells showed no difference when compared with 2D hDFSCs; however, 3D hDFSCs were observed to have a higher proportion of cell cycle arrest and a larger number of apoptotic cells. Moreover, spheroids showed substantially increased levels of pluripotency marker (early transcription factors) and ECM protein expression. Compared with 2D hDFSCs, there was also a notable enhancement in the osteogenic induction potential of spheroids, although no differences were observed with respect to in vitro adipogenesis.


To the best of our knowledge, this is the first study to demonstrate the application of a spheroid culture system for dental follicle-derived stem cells using a microchip dish. Although further studies are needed, including in vivo transplantation, the results obtained in this study indicate that spheroid hDFSCs derived from cryopreserved dental follicle tissues could be used as a valuable source of autologous stem cells for bone tissue regeneration.


Mesenchymal stem cells Dental follicle stem cells Spheroid Stemness Osteogenesis 



This work was supported by the grant from National Research Foundation (2017R1D1A1B03035677), Republic of Korea.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interest.

Ethical statement

This study protocol was approved by the institutional review board of Changwon Gyeongsang National University Hospital (IRB No. GNUH IRB-2018-11-002-001) and informed consent was confirmed by IRB.


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Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society 2019

Authors and Affiliations

  1. 1.Department of Oral and Maxillofacial Surgery, College of MedicineUniversity of UlsanDong-gu, UlsanRepublic of Korea
  2. 2.Department of Oral and Maxillofacial SurgeryChangwon Gyeongsang National University HospitalSeongsan-gu, ChangwonRepublic of Korea
  3. 3.Department of Theriogenology and Biotechnology, College of Veterinary Medicine and Research Institute of Life ScienceGyeongsang National UniversityJinju-siRepublic of Korea
  4. 4.Department of Dentistry, Institute of Health ScienceGyeongsang National University School of MedicineJinjuRepublic of Korea
  5. 5.Department of Dental TechnologyJinju Health CollegeJinjuRepublic of Korea
  6. 6.Department of DentistryHanil HospitalJinjuRepublic of Korea

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