This protocol emphasizes the extraction, culture, and preservation of multipotent stem cells from dental pulp through enzymatic digestion. Additionally, it demonstrates their potential to differentiate into osteoblasts, adipocytes, and chondrocytes, highlighting the importance of precision and consistency in the process.
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Method Article
Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth
In This Article
Summary
Abstract
In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which are present in both deciduous and permanent teeth, have emerged as a vital stem cell source due to their accessibility, adaptability, and innate differentiation capabilities. DPSCs offer a readily available and abundant reservoir of mesenchymal stem cells, showcasing impressive versatility and potential, particularly for regenerative purposes. Despite their promise, the main hurdle lies in effectively isolating and characterizing DPSCs, given their representation as a minute fraction within dental pulp cells. Equally crucial is the proper preservation of this invaluable cellular resource. The two predominant methods for DPSC isolation are enzymatic digestion (ED) and outgrowth from tissue explants (OG), often referred to as spontaneous growth. This protocol concentrates primarily on the enzymatic digestion approach for DPSC isolation, intricately detailing the steps encompassing extraction, in-lab processing, and cell preservation. Beyond extraction and preservation, the protocol delves into the differentiation prowess of DPSCs. Specifically, it outlines the procedures employed to induce these stem cells to differentiate into adipocytes, osteoblasts, and chondrocytes, showcasing their multipotent attributes. Subsequent utilization of colorimetric staining techniques facilitates accurate visualization and confirmation of successful differentiation, thereby validating the caliber and functionality of the isolated DPSCs. This comprehensive protocol functions as a blueprint encompassing the entire spectrum of dental pulp stem cell extraction, cultivation, preservation, and characterization. It underscores the substantial potential harbored by DPSCs, propelling forward stem cell exploration and holding promise for future regenerative and therapeutic breakthroughs.
Introduction
Stem cell research has flourished in biomedical science due to its promising applications in regenerative medicine and tissue engineering. Dental pulp stem cells (DPSCs), derived from the pulp tissue of both human deciduous and permanent teeth, have attracted significant interest as a source of stem cells due to their ready availability and multipotent capacity1,2. These cells have the potential to differentiate into various cell types, including adipocytes, osteoblasts, and chondrocytes, as confirmed by numerous studies3.
Over the past few decades, research ....
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Protocol
The protocol outlined herein conforms to the guidelines of the institutional human research ethics committee (IRB, Pushpagiri Research Center, Kerala). The use of extracted teeth was conducted following ethical standards to ensure the integrity, dignity, and rights of the participants. The participants selected for this study were healthy individuals under 30 years of age who required tooth extraction for orthodontic treatment. Those with extensive dental caries or severe periodontitis were excluded from the study. Deciduous teeth were collected from children who required the extraction of retained teeth. Informed written consent was also obtained from the subjects in....
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Results
The successful execution of the outlined protocol yielded dental pulp stem cells (DPSCs) capable of multilineage differentiation, demonstrating their multipotency.
Viability assays
The viability of the DPSCs was assessed using a Trypan Blue exclusion assay at various time points. The results show consistently high viability (greater than 95%) throughout the culture period, demonstrating the robustness of our isolation and culture protocol.
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Discussion
The protocol outlines the isolation, culture, and characterization of dental pulp stem cells (DPSCs) from human deciduous and permanent teeth. It includes a description of the storage and proliferation of these cells, as well as the assessment of their in vitro differentiation potential into osteoblasts, adipocytes, and chondrocytes35.
Chen et al.36 demonstrated that Dental Pulp Stem Cells (DPSCs) could be ob.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors are grateful to Dr. Mathew Mazhavancheril, Director and Head of the Pushpagiri Research Centre in Thiruvalla, for his support in documenting the procedures at the Research Centre.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 3-isobuty-l-methyl-xanthine | Sigma-Aldrich Co. St. Louis, MO 63103.USA | I5879 | |
| Acetic acid | Sigma-Aldrich Co. St. Louis, MO 63103.USA | AS001 | |
| Alcian Blue | Sigma-Aldrich Co. St. Louis, MO 63103.USA | RM471 | |
| Alizarin Red S staining solution | Sigma-Aldrich Co. St. Louis, MO 63103.USA | GRM894 | |
| Alkaline phosphatase -Staining kit | Thermo Fisher Scientific ,MA 02451,USA | ||
| Alpha Minimum Essential Medium (α-MEM) | Thermo Fisher Scientific ,MA 02451,USA | Gibco | |
| Alpha Minimum Essential Medium (α-MEM) | Thermo Fisher Scientific ,MA 02451,USA | Gibco | |
| Alpha-MEM, or Alpha Minimum Essential Medium | Thermo Fisher Scientific ,MA 02451,USA | Gibco | |
| Alpha-MEM, or Alpha Minimum Essential Medium | Thermo Fisher Scientific ,MA 02451,USA | Gibco | |
| Antibiotic/Antimycotic | Sigma-Aldrich Co. St. Louis, MO 63103.USA | P4333 | |
| Ascorbate-2-phosphate | Sigma-Aldrich Co. St. Louis, MO 63103.USA | 012-04802 | |
| Beta-glycerophosphate | Sigma-Aldrich Co. St. Louis, MO 63103.USA | G9422-10G | |
| Biosafety cabinet-Laminar flow hood | Labconco Corporation,MO 64132-2696,USA | ||
| CD90, CD105, CD73, CD34, CD45, and HLA-DR | BioLegend, Inc.CA 92121,USA | ||
| Cell strainer (70 µm ) | HiMedia Laboratories Ltd.Mumbai,India | TCP025 | Cell strainer |
| Centrifuge | REMI Elektrotechnik Limited (REMI) | ||
| Centrifuge | HiMedia Laboratories Ltd.Mumbai,India | 1101 | 1102 | |
| CO2 Incubator | Thermo Fisher Scientific ,MA 02451,USA | ||
| Collagenase type I | Worthington Biochem. Corp. NJ 08701, USA | ||
| Collagenase type I | Worthington Biochem. Corp. NJ 08701, USA | ||
| Complete Growth Medium | HiMedia Laboratories Ltd.Mumbai,India | AT006 | DMEM |
| Conical tubes (15 or 50 ) | Thermo Fisher Scientific, MA, USA | 546021P/546041P | 15 mL and 50 mL |
| Cryo freezing container | Thermo Fisher Scientific ,MA 02451,USA | 15-350-50 | |
| Cryolabels | Label India: | ||
| Cryovial storage boxes | Cryostore Storage Boxes | ||
| Cryovials | Thermo Fisher Scientific ,MA 02451,USA | ||
| Cryovials (1.8 mL) | Thermo Fisher Scientific ,MA 02451,USA | PW1282 | Self standing |
| Culture flask (25 cm²) | Corning Inc.NY 14831,USA | ||
| Culture flasks | HiMedia Laboratories Ltd.Mumbai,India | TCG4/TCG6 | T25/T75 |
| Culture Plates | HiMedia Laboratories Ltd.Mumbai,India | TCP129/TCP008 | 60 mm/100 mm |
| Dental Diamond Discs | Komet SC 29730, USA | Komet | |
| Dental Spoon Excavator | Brasseler,GA 31419,USA | 5023591U0 | |
| Dexamethasone | Sigma-Aldrich Co. St. Louis, MO 63103.USA | D4902-25MG | |
| Dexamethosone | Sigma-Aldrich Co. St. Louis, MO 63103.USA | D4902-25MG | |
| Dexamethosone | Sigma-Aldrich Co. St. Louis, MO 63103.USA | D4902-25MG | |
| Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich Co. St. Louis, MO 63103.USA | TC185 | |
| Dispase | Roche Diagnostics,Mannheim,Germany. | ||
| Dispase | Roche Diagnostics GmbH, Mannheim,Germany | ||
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific, MA, USA | ||
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific ,MA 02451,USA | ||
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific ,MA 02451,USA | ||
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific ,MA 02451,USA | ||
| Ethanol (70%) | HiMedia Laboratories Ltd.Mumbai,India | MB106 | |
| Ethanol -70% | Thermo Fisher Scientific ,MA 02451,USA | Fisher Scientific | |
| Extraction forceps | Dentsply Sirona, USA | ||
| Fetal bovine serum (FBS) | Thermo Fisher Scientific Inc.,MA,USA | F2442-500ML | |
| Fetal bovine serum (FBS) | Thermo Fisher Scientific Inc.,MA,USA | F2442-500ML | |
| Fetal bovine serum (FBS) | HiMedia Laboratories Ltd.Mumbai,India | RM9954 | |
| Fetal bovine serum (FBS) | Thermo Fisher Scientific Inc.,MA,USA | F2442-500ML | |
| Fetal bovine serum (FBS) | Thermo Fisher Scientific Inc.,MA,USA | F2442-500ML | |
| Fibronectin-coated tissue culture plate | Corning Inc.Corning, NY 14831,USA | ||
| Flow cytometer | BD Biosciences,CA 95131,USA | ||
| Flow cytometry buffer | BD Biosciences,CA 95131,USA | ||
| Glass cover slip 22 x 22 mm | HiMedia Laboratories Ltd.Mumbai,India | TCP017 | |
| Hank's Balanced Salt Solution (HBSS) | Lonza Group Ltd,4002 Basel, Switzerland | ||
| High-speed dental handpiece | NSK Ltd,Tokyo 8216, Japan | Ti-Max Z series | |
| Horse Serum | Thermo Fisher Scientific ,MA 02451,USA | ||
| IBMX, or 3-isobutyl-1-methylxanthine | Sigma-Aldrich Co. St. Louis, MO 63103.USA | ||
| Indomethacin | Pfizer Inc. NY 10017,USA | ||
| Insulin-Transferrin-Selenium (ITS) | Thermo Fisher Scientific ,MA 02451,USA | I5523 | |
| Insulin-Transferrin-Selenium (ITS) | Thermo Fisher Scientific ,MA 02451,USA | I5523 | |
| Insulin-Transferrin-Selenium (ITS) premix | Corning Incorporated,MA 01876,USA | ||
| Inverted microscope | Olympus Corp.,Tokyo 163-0914,Japan | ||
| Isopropanol (60% ) | Sigma-Aldrich Co. St. Louis, MO 63103.USA | I9516 | |
| Isopropyl alcohol | Sigma-Aldrich Co. St. Louis, MO 63103.USA | MB063 | |
| Laminar flow hood | Thermo Fisher Scientific ,MA 02451,USA | ||
| Lidocaine mixed with epinephrine | DENTSPLY,NC 28277,USA | Citanest | |
| Liquid Nitrogen | Air Liquide,75007 Paris,France | ||
| Liquid nitrogen storage tank | Cryo Scientific Systems Pvt. Ltd. | ||
| Micropipettes | Eppendorf AG,22339 Hamburg,Germany | 30020 | Accupipet-2-20 µL |
| Mini tissue grinder | Bio-Rad Lab, Inc. CA 94547,USA | ReadyPrep mini grinders | |
| Minus 80 freezer | Blue Star Limited | ||
| Neubauer counting chamber | Marienfeld Superior,arktheidenfeld,Germany | ||
| Oil red O stain | Sigma-Aldrich Co. St. Louis, MO 63103.USA | 1024190250 | |
| Osteogenic Differentiation Medium (ODM) | STEMCELL Technologies Inc.Vancouver, BC, V5Z 1B3,Canada | ||
| Paraformaldehyde (PFA) | Sigma-Aldrich Co. St. Louis, MO 63103.USA | TCL119 | |
| Penicillin-Streptomycin | Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA | ||
| Phosphate Buffered Solution (PBS) without Ca++ and Mg++ | HiMedia Laboratories Ltd.Mumbai,India | TS1101 | |
| Phosphate-buffered saline (PBS) | Thermo Fisher Scientific | Gibco | |
| Phosphate-buffered saline (PBS) | Thermo Fisher Scientific,MA, USA | Gibco | |
| Phosphate-buffered saline (PBS) | Thermo Fisher Scientific, MA, USA | ||
| Phosphate-buffered saline (PBS) | Thermo Fisher Scientific, MA, USA | P3813-1PAK | 1x PBS, pH 7.4 |
| Proline | Sigma-Aldrich Co. St. Louis, MO 63103.USA | ||
| Scalpel Blade Size 15 | Swann-Morton Ltd, Sheffield, S6 2BJ,UK | BDF-6955C | |
| Sodium Hypochlorite | HiMedia Laboratories Ltd.Mumbai,India | AS102 | 4% w/v solution |
| Sterile centrifuge tubes | Tarsons Products Pvt. Ltd. | ||
| Sterile container -20 mL | 3M Center, MN 55144-1000,USA | 3 M | |
| Sterile phosphate-buffered saline (PBS) | Sigma Aldrich, USA | P3813-1PAK | 1x PBS, pH 7.4 |
| Sterile pipettes (2, 5, and 10 mL ) | Eppendorf AG,22339 Hamburg,Germany | ||
| Sterile pipettes and tips | Eppendorf India Limited | ||
| Surgical Blade Handle | Becton, Dickinson and Co.,NJ,USA | 371030 | BP Handle 3 |
| Transforming Growth Factor-beta 3 (TGF-β3) | R&D Systems, Inc.MN 55413,USA | ||
| Transforming Growth Factor-beta 3 (TGF-β3) | R&D Systems, Inc.MN 55413,USA | ||
| Trypan Blue 0.4% | Sigma-Aldrich Co. St. Louis, MO 63103.USA | ||
| Trypan Blue 0.4% | Sigma-Aldrich Co. St. Louis, MO 63103.USA | TCL046 | |
| Trypan Blue 0.4% | Sigma-Aldrich Co. St. Louis, MO 63103.USA | TCL046 | |
| Trypsin-EDTA | Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA | ||
| Trypsin-EDTA 0.25% | Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA | ||
| Water bath | Thermo Fisher Scientific ,MA 02451,USA | BSW-01D |
References
- Bakopoulou, A., About, I. Stem cells of dental origin: current research trends and key milestones towards clinical application. Stem Cells International. 2016, 4209891(2016).
- Yamada, Y., Nakamura-Yamada, S., Konoki, R., Baba, S.
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