Int J Stem Cells.  2018 Jun;11(1):96-104. 10.15283/ijsc17049.

Optimal Xeno-free Culture Condition for Clinical Grade Stem Cells from Human Exfoliated Deciduous Teeth

• Tangjit N
• Dechkunakorn S
• Anuwongnukroh N
• Khaneungthong A
• Sritanaudomchai H

• KMID: 2413505
• DOI: http://doi.org/10.15283/ijsc17049

Abstract

BACKGROUND AND OBJECTIVES
Stem cells from human exfoliated deciduous teeth (SHED) are a promising clinical resource for various tissue defects, including lumbar spondylosis, neural compression, and cleft palate. Use of media containing animal-derived serum carries potential risk of infectious diseases and unwanted immunogenicity. To increase the potential utility of SHED for clinical application, SHED was adapted to xeno-free conditions.
METHODS
Define xeno-free culture media were compared with the conventional serum containing media in the culture of SHED. Cultured SHED in different media were further characterized through proliferative capacities, cellular phenotype, and differentiation potential.
RESULTS
Selected xeno-free media were capable of supporting the growth of SHED. MSCGM-CD Bulletkit medium greatly increased the number and proliferate capacity of colony-forming unit-fibroblast than SHED cultured in other media. In addition, the characteristic surface markers expression and multipotent differentiation potential of SHED in the MSCGM-CD Bulletkit medium were comparable to those observed with serum-containing medium.
CONCLUSIONS
The xeno-free medium described herein has the potential to be further used for the safe expansion and to determine efficient way to produce clinical grade dental stem cells for therapeutic applications.

Keyword

Xeno-free media; Dental pulp stem cells; Clinical grade cells; Proliferation; Primary tooth; Osteogenesis

MeSH Terms

Cleft Palate
Communicable Diseases
Culture Media
Humans*
Osteogenesis
Phenotype
Spondylosis
Stem Cells*
Tooth, Deciduous*
Culture Media

Figure

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  Fig. 1 Isolation and morphological observation of stem cells from pulp tissue of human deciduous teeth. (A) Outgrowth of stem cells (arrow head) from dental pulp tissue of human exfoliated deciduous teeth. (B) Phasecontrast images showing a fibroblast-like morphology of the in vitro growth SHEDs. Magnification: 40×. SHED, stem cell from human exfoliated deciduous teeth.

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  Fig. 2 Morphology of SHED during the adaptation to xeno-free medium. (A) SHED cultured with FBS-containing medium. (B) The representative photomicrographs showing the morphology of SHED cultured in xeno-free media. (C) Representative graphs of cell proliferation in four different conditions. Scale bar=10 μm. SHED, stem cell from human exfoliated deciduous teeth, FBS, fetal bovine serum, DXF, MSC Growth Medium DXF, MSCGM-CD, MSCGM-CD Bulletkit, Xuri, Xuri MSC Medium Serum-free/Xeno-free.

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  Fig. 3 Colony-forming unit-fibroblast assay of xeno-free expanded SHED. (A) Representative images of the colonies stained by toluidine blue after 14 days of cultured in four different conditions. (B) The total number of colonies counted within each condition. SHED, stem cell from human exfoliated deciduous teeth.

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  Fig. 4 Multipotent differentiation ability of SHED cultured in MSCGM-CD or FBS-containing medium. (A) Phase contrast images of SHED derived osteoblast-like cells, as shown by calcified nodules formation which appeared during alizarin red staining. (B) Alcian blue staining showed proteoglycans in chondrocyte cultures differentiated from SHED grown in both media. (C) After induction with spiral ganglion neurons induction medium for 14 days. The expression of the mature neuronal marker Tuj1 (green) and spiral ganglion neurons markers TrkB (red) is shown. Cell nuclei were counterstained with DAPI (blue). Magnification: A, 40×; B, 100×; C, Scale bar=20 μm. SHED, stem cell from human exfoliated deciduous teeth, Tuj1, β-tubulin III, TrkB, tropomyosin receptor kinase B, DAPI, 4′,6-diamidino-2-phenylindole.

Reference

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