Skip to main content
NCBI home page
Tissue Engineering and Regenerative Medicine logoLink to Tissue Engineering and Regenerative Medicine
. 2016 Aug 5;13(4):428–436. doi: 10.1007/s13770-016-0005-2

Indirect co-culture of stem cells from human exfoliated deciduous teeth and oral cells in a microfluidic platform

Kyung-Jung Kang 1, Seon Min Ju 1, Young-Joo Jang 1,2,✉, Jeongyun Kim 1,2,✉
PMCID: PMC6171551  PMID: 30603424

Abstract

Oral epithelial-mesenchymal interactions play a key role in tooth development and assist differentiation of dental pulp. Many epithelial and mesenchymal factors in the microenvironment influence dental pulp stem cells to differentiate and regenerate. To investigate the interaction between oral cells during differentiation, we designed a microfluidic device system for indirect co-culture. The system has several advantages, such as consumption of low reagent volume, high-throughput treatment of reagents, and faster mineralization analysis. In this study, stem cells from human exfoliated deciduous teeth were treated with media cultured with human gingival fibroblasts or periodontal ligament stem cells. When human exfoliated deciduous teeth was incubated in media cultured in human gingival fibroblasts and human periodontal ligament stem cells under the concentration gradient constructed by the microfluidic system, no remarkable change in human exfoliated deciduous teeth mineralization efficiency was detected. However, osteoblast gene expression levels in human exfoliated deciduous teeth incubated with human gingival fibroblasts media decreased compared to those in human exfoliated deciduous teeth treated with human periodontal ligament stem cells media, suggesting that indirect co-culture of human exfoliated deciduous with human gingival fibroblasts may inhibit osteogenic cytodifferentiation. This microfluidic culture device allows a co-culture system set-up for sequential treatment with co-culture media and differentiation additives and facilitated the mineralization assay in a micro-culture scale.

Key Words: Microfluidic device, Indirect co-culture, Stem cells from human exfoliated deciduous teeth, Osteogenic differentiation

Footnotes

These authors equally contributed to this work.

Contributor Information

Young-Joo Jang, Phone: 82-41-550-1936, FAX: 82-41-559-7839, Email: yjjang@dankook.ac.kr.

Jeongyun Kim, Phone: 82-41-550-1255, FAX: 82-41-561-1256, Email: jeongyunkim@dankook.ac.kr.

References

  • 1.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 2000;97:13625–13630. doi: 10.1073/pnas.240309797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003;100:5807–5812. doi: 10.1073/pnas.0937635100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet. 2004;364:149–155. doi: 10.1016/S0140-6736(04)16627-0. [DOI] [PubMed] [Google Scholar]
  • 4.Arora V, Arora P, Munshi AK. Banking stem cells from human exfoliated deciduous teeth (SHED): saving for the future. J Clin Pediatr Dent. 2009;33:289–294. doi: 10.17796/jcpd.33.4.y887672r0j703654. [DOI] [PubMed] [Google Scholar]
  • 5.Morsczeck C, Götz W, Schierholz J, Zeilhofer F, Kühn U, Möhl C, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth. Matrix Biol. 2005;24:155–165. doi: 10.1016/j.matbio.2004.12.004. [DOI] [PubMed] [Google Scholar]
  • 6.Cicconetti A, Sacchetti B, Bartoli A, Michienzi S, Corsi A, Funari A, et al. Human maxillary tuberosity and jaw periosteum as sources of osteoprogenitor cells for tissue engineering. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;104:618. doi: 10.1016/j.tripleo.2007.02.022. [DOI] [PubMed] [Google Scholar]
  • 7.Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, et al. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002;81:531–535. doi: 10.1177/154405910208100806. [DOI] [PubMed] [Google Scholar]
  • 8.Shi S, Bartold PM, Miura M, Seo BM, Robey PG, Gronthos S. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res. 2005;8:191–199. doi: 10.1111/j.1601-6343.2005.00331.x. [DOI] [PubMed] [Google Scholar]
  • 9.Chai Y, Jiang X, Ito Y, Bringas P, Han J, Rowitch DH, et al. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000;127:1671–1679. doi: 10.1242/dev.127.8.1671. [DOI] [PubMed] [Google Scholar]
  • 10.Janebodin K, Horst OV, Ieronimakis N, Balasundaram G, Reesukumal K, Pratumvinit B, et al. Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice. PLoS One. 2011;6:e27526. doi: 10.1371/journal.pone.0027526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ibarretxe G, Crende O, Aurrekoetxea M, García-Murga V, Etxaniz J, Unda F. Neural crest stem cells from dental tissues: a new hope for dental and neural regeneration. Stem Cells Int. 2012;2012:103503. doi: 10.1155/2012/103503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Arakaki M, Ishikawa M, Nakamura T, Iwamoto T, Yamada A, Fukumoto E, et al. Role of epithelial-stem cell interactions during dental cell differentiation. J Biol Chem. 2012;287:10590–10601. doi: 10.1074/jbc.M111.285874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Thesleff I, Mikkola M. The role of growth factors in tooth development. Int Rev Cytol. 2002;217:93–135. doi: 10.1016/S0074-7696(02)17013-6. [DOI] [PubMed] [Google Scholar]
  • 14.Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent. 2000;28:77–92. doi: 10.1016/S0300-5712(99)00047-0. [DOI] [PubMed] [Google Scholar]
  • 15.Deng MJ, Jin Y, Shi JN, Lu HB, Liu Y, He DW, et al. Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch. Tissue Eng. 2004;10:1597–1606. doi: 10.1089/ten.2004.10.1597. [DOI] [PubMed] [Google Scholar]
  • 16.Ohlstein B, Kai T, Decotto E, Spradling A. The stem cell niche: theme and variations. Curr Opin Cell Biol. 2004;16:693–699. doi: 10.1016/j.ceb.2004.09.003. [DOI] [PubMed] [Google Scholar]
  • 17.Bai Y, Bai Y, Matsuzaka K, Hashimoto S, Kokubu E, Wang X, et al. Formation of bone-like tissue by dental follicle cells co-cultured with dental papilla cells. Cell Tissue Res. 2010;342:221–231. doi: 10.1007/s00441-010-1046-9. [DOI] [PubMed] [Google Scholar]
  • 18.De Rosa A, Tirino V, Paino F, Tartaglione A, Mitsiadis T, Feki A, et al. Amniotic fluid-derived mesenchymal stem cells lead to bone differentiation when cocultured with dental pulp stem cells. Tissue Eng Part A. 2011;17:645–653. doi: 10.1089/ten.tea.2010.0340. [DOI] [PubMed] [Google Scholar]
  • 19.Yu J, Deng Z, Shi J, Zhai H, Nie X, Zhuang H, et al. Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium. Tissue Eng. 2006;12:3097–3105. doi: 10.1089/ten.2006.12.3097. [DOI] [PubMed] [Google Scholar]
  • 20.Aigner T, Stöve J. Collagens—major component of the physiological cartilage matrix, major target of cartilage degeneration, major tool in cartilage repair. Adv Drug Deliv Rev. 2003;55:1569–1593. doi: 10.1016/j.addr.2003.08.009. [DOI] [PubMed] [Google Scholar]
  • 21.Alexanian AR. Neural stem cells induce bone-marrow-derived mesenchymal stem cells to generate neural stem-like cells via juxtacrine and paracrine interactions. Exp Cell Res. 2005;310:383–391. doi: 10.1016/j.yexcr.2005.08.015. [DOI] [PubMed] [Google Scholar]
  • 22.Sands RW, Mooney DJ. Polymers to direct cell fate by controlling the microenvironment. Curr Opin Biotechnol. 2007;18:448–453. doi: 10.1016/j.copbio.2007.10.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Shin Y, Han S, Jeon JS, Yamamoto K, Zervantonakis IK, Sudo R, et al. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels. Nat Protoc. 2012;7:1247–1259. doi: 10.1038/nprot.2012.051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wu J, Chen Q, Liu W, Zhang Y, Lin JM. Cytotoxicity of quantum dots assay on a microfluidic 3D-culture device based on modeling diffusion process between blood vessels and tissues. Lab Chip. 2012;12:3474–3480. doi: 10.1039/c2lc40502d. [DOI] [PubMed] [Google Scholar]
  • 25.Chen Q, Wu J, Zhang Y, Lin Z, Lin JM. Targeted isolation and analysis of single tumor cells with aptamer-encoded microwell array on microfluidic device. Lab Chip. 2012;12:5180–5185. doi: 10.1039/c2lc40858a. [DOI] [PubMed] [Google Scholar]
  • 26.Schirhagl R, Fuereder I, Hall EW, Medeiros BC, Zare RN. Microfluidic purification and analysis of hematopoietic stem cells from bone marrow. Lab Chip. 2011;11:3130–3135. doi: 10.1039/c1lc20353c. [DOI] [PubMed] [Google Scholar]
  • 27.Chen Q, Wu J, Zhang Y, Lin JM. Qualitative and quantitative analysis of tumor cell metabolism via stable isotope labeling assisted microfluidic chip electrospray ionization mass spectrometry. Anal Chem. 2012;84:1695–1701. doi: 10.1021/ac300003k. [DOI] [PubMed] [Google Scholar]
  • 28.Gao D, Liu H, Lin JM, Wang Y, Jiang Y. Characterization of drug permeability in Caco-2 monolayers by mass spectrometry on a membranebased microfluidic device. Lab Chip. 2013;13:978–985. doi: 10.1039/c2lc41215b. [DOI] [PubMed] [Google Scholar]
  • 29.Kim C, Lee KS, Bang JH, Kim YE, Kim MC, Oh KW, et al. 3-dimensional cell culture for on-chip differentiation of stem cells in embryoid body. Lab Chip. 2011;11:874–882. doi: 10.1039/c0lc00516a. [DOI] [PubMed] [Google Scholar]
  • 30.Khademhosseini A, Langer R, Borenstein J, Vacanti JP. Microscale technologies for tissue engineering and biology. Proc Natl Acad Sci U S A. 2006;103:2480–2487. doi: 10.1073/pnas.0507681102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Huh D, Matthews BD, Mammoto A, Montoya-Zavala M, Hsin HY, Ingber DE. Reconstituting organ-level lung functions on a chip. Science. 2010;328:1662–1668. doi: 10.1126/science.1188302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.El-Ali J, Sorger PK, Jensen KF. Cells on chips. Nature. 2006;442:403–411. doi: 10.1038/nature05063. [DOI] [PubMed] [Google Scholar]
  • 33.Choi JK, Hwang HI, Jang YJ. The efficiency of the in vitro osteo/dentinogenic differentiation of human dental pulp cells, periodontal ligament cells and gingival fibroblasts. Int J Mol Med. 2015;35:161–168. doi: 10.3892/ijmm.2014.1986. [DOI] [PubMed] [Google Scholar]
  • 34.Min JH, Ko SY, Cho YB, Ryu CJ, Jang YJ. Dentinogenic potential of human adult dental pulp cells during the extended primary culture. Hum Cell. 2011;24:43–50. doi: 10.1007/s13577-011-0010-7. [DOI] [PubMed] [Google Scholar]
  • 35.Ju SM, Jang HJ, Kim KB, Kim J. High-throughput cytotoxicity testing system of acetaminophen using a microfluidic device (MFD) in HepG2 cells. J Toxicol Environ Health A. 2015;78:1063–1072. doi: 10.1080/15287394.2015.1068650. [DOI] [PubMed] [Google Scholar]
  • 36.An D, Kim K, Kim J. Microfluidic system based high throughput drug screening system for curcumin/TRAIL combinational chemotherapy in human prostate cancer PC3 cells. Biomol Ther (Seoul) 2014;22:355–362. doi: 10.4062/biomolther.2014.078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Kim J, Taylor D, Agrawal N, Wang H, Kim H, Han A, et al. A programmable microfluidic cell array for combinatorial drug screening. Lab Chip. 2012;12:1813–1822. doi: 10.1039/c2lc21202a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Wang H, Kim J, Jayaraman A, Han A. Microfluidic geometric meteringbased multi-reagent mixture generator for robust live cell screening array. Biomed Microdevices. 2014;16:887–896. doi: 10.1007/s10544-014-9893-x. [DOI] [PubMed] [Google Scholar]
  • 39.Tucker A, Sharpe P. The cutting-edge of mammalian development; how the embryo makes teeth. Nat Rev Genet. 2004;5:499–508. doi: 10.1038/nrg1380. [DOI] [PubMed] [Google Scholar]
  • 40.Zhang YD, Chen Z, Song YQ, Liu C, Chen YP. Making a tooth: growth factors, transcription factors, and stem cells. Cell Res. 2005;15:301–316. doi: 10.1038/sj.cr.7290299. [DOI] [PubMed] [Google Scholar]
  • 41.Lerner UH, Hänström L, Sjäström S. Stimulation of bone resorption and cell proliferation in vitro by human gingival fibroblasts from patients with periodontal disease. Bone Miner. 1990;10:225–242. doi: 10.1016/0169-6009(90)90264-G. [DOI] [PubMed] [Google Scholar]
  • 42.Sjöström S, Hänström L, Lerner UH. The bone resorbing activity released by gingival fibroblasts isolated from patients with periodontitis is independent of interleukin-1. J Periodontal Res. 2000;35:74–84. doi: 10.1034/j.1600-0765.2000.035002074.x. [DOI] [PubMed] [Google Scholar]

Articles from Tissue Engineering and Regenerative Medicine are provided here courtesy of Springer

RESOURCES