Skip to main content

A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth

Part of the Methods in Molecular Biology book series (MIMB,volume 1553)


This chapter describes a simplified method that allows the systematic isolation of multiple types of dental stem cells such as dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), and stem cells of the apical papilla (SCAP) from a single tooth. Of specific interest is the modified laboratory approach to harvest/retrieve the dental pulp tissue by minimizing trauma to DPSC by continuous irrigation, reduction of frictional heat from the bur rotation, and reduction of the bur contact time with the dentin. Also, the use of a chisel and a mallet will maximize the number of live DPSC for culture. Steps demonstrating the potential for multiple cell differentiation lineages of each type of dental stem cell into either osteocytes, adipocytes, or chondrocytes are described. Flow cytometry, with a detailed strategy for cell gating and analysis, is described to verify characteristic markers of human mesenchymal multipotent stromal cells (MSC) from DPSC, PDLSC, or SCAP for subsequent experiments in cell therapy and in tissue engineering. Overall, this method can be adapted to any laboratory with a general setup for cell culture experiments.

Key words

  • Dental Pulp Stem Cells (DPSC)
  • Stem Cells from Apical Papilla (SCAP)
  • Periodontal Ligament Stem Cells (PDLSC)
  • Mesenchymal Stromal Cells (MSC)
  • Lineage differentiation
  • Flow cytometry
  • Cell differentiation
  • Dental pulp
  • Periodontal ligament
  • Periapical tissue

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Barry FP, Murphy JM (2004) Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 36(4):568–584

    CrossRef  CAS  PubMed  Google Scholar 

  2. Chamberlain G, Fox J, Ashton B, Middleton J (2007) Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 25(11):2739–2749

    CrossRef  CAS  PubMed  Google Scholar 

  3. Doorn J, Moll G, Le Blanc K, van Blitterswijk C, de Boer J (2012) Therapeutic applications of mesenchymal stromal cells: paracrine effects and potential improvements. Tissue Eng Part B Rev 18(2):101–115

    CrossRef  CAS  PubMed  Google Scholar 

  4. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317

    CrossRef  CAS  PubMed  Google Scholar 

  5. Ramamoorthi M, Bakkar M, Jordan J, Tran SD (2015) Osteogenic potential of dental mesenchymal stem cells in preclinical studies: a systematic review using modified ARRIVE and CONSORT guidelines. Stem Cells Int 2015:378368

    CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Ito K, Yamada Y, Nakamura S, Ueda M (2011) Osteogenic potential of effective bone engineering using dental pulp stem cells, bone marrow stem cells, and periosteal cells for osseointegration of dental implants. Int J Oral Maxillofac Implants 26(5):947–954

    PubMed  Google Scholar 

  7. Sakai K, Yamamoto A, Matsubara K, Nakamura S, Naruse M, Yamagata M, Sakamoto K, Tauchi R, Wakao N, Imagama S, Hibi H, Kadomatsu K, Ishiguro N, Ueda M (2012) Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms. J Clin Invest 122(1):80–90

    CAS  PubMed  Google Scholar 

  8. Nishino Y, Ebisawa K, Yamada Y, Okabe K, Kamei Y, Ueda M (2011) Human deciduous teeth dental pulp cells with basic fibroblast growth factor enhance wound healing of skin defect. J Craniofac Surg 22(2):438–442

    CrossRef  PubMed  Google Scholar 

  9. Iohara K, Zheng L, Wake H, Ito M, Nabekura J, Wakita H, Nakamura H, Into T, Matsushita K, Nakashima M (2008) A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp. Stem Cells 26(9):2408–2418

    CrossRef  PubMed  Google Scholar 

  10. Gandia C, Arminan A, Garcia-Verdugo JM, Lledo E, Ruiz A, Minana MD, Sanchez-Torrijos J, Paya R, Mirabet V, Carbonell-Uberos F, Llop M, Montero JA, Sepulveda P (2008) Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce infarct size in rats with acute myocardial infarction. Stem Cells 26(3):638–645

    CrossRef  PubMed  Google Scholar 

  11. Gronthos S, Mankani M, Brahim J, Robey PG, Shi S (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 97(25):13625–13630

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  12. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364(9429):149–155

    CrossRef  CAS  PubMed  Google Scholar 

  13. Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Wang S, Shi S (2006) Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One 1:e79

    CrossRef  PubMed  PubMed Central  Google Scholar 

  14. Krasner P, Rankow HJ (2004) Anatomy of the pulp-chamber floor. J Endod 30(1):5–16

    CrossRef  PubMed  Google Scholar 

Download references


The authors are grateful to Xin Ran Liu for the schematic illustrations. The authors thank the Flow Cytometry and Cell Sorting facility, Department of Microbiology and Immunology, McGill University. The authors would like to thank the following funding agency: Natural Sciences and Engineering Research Council of Canada (NSERC).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Simon D. Tran D.M.D., Ph.D. .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Bakkar, M. et al. (2017). A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth. In: Di Nardo, P., Dhingra, S., Singla, D. (eds) Adult Stem Cells. Methods in Molecular Biology, vol 1553. Humana Press, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6754-4

  • Online ISBN: 978-1-4939-6756-8

  • eBook Packages: Springer Protocols