Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth
Summary
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.
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 and therapeutic applications of stem cells have surged. The expansive potential of stem cells calls for diversifying the sources from which they are obtained. Several factors influence the efficiency, viability, and stemness of chosen cells. Despite the existence of various known stem cell reservoirs, such as bone marrow and adipose tissues, the invasive procedures, site morbidity, and ethical concerns linked to these sources often limit their exploration4,5. Among the various stem cell sources, dental stem cells have gained attention due to their easy accessibility, high plasticity, and diverse potential applications. Human dental pulp stem cells, in particular, have been extensively researched for their therapeutic prospects6. Teeth, commonly discarded as medical waste, hold a wealth of mesenchymal stem cells7. Safeguarding this valuable stem cell pool requires collective efforts from patients, dentists, and doctors to ensure that these resources are not wasted, making each dental pulp stem cell available for future regenerative requirements.
Dental pulp-derived stem cells, such as human adult dental pulp stem cells (DPSCs) and stem cells from exfoliated human deciduous teeth (SHED), are located in the perivascular niche of the dental pulp. These cells are believed to originate from cranial neural crest cells and exhibit early markers for both mesenchymal stem cells (MSCs) and neuroectodermal stem cells. DPSCs and SHEDs have demonstrated multipotency and the ability to regenerate diverse tissue types8.
Potential sources of dental stem cells encompass healthy deciduous and permanent teeth. Stem cells constitute only about 1% of the total cell population in the pulp, highlighting the importance of effective isolation and expansion techniques9. Consequently, the extraction and expansion of these stem cells are pivotal steps in DPSC isolation10. Extracted or exfoliated teeth need to be stored in a nutrient-rich transport medium, such as phosphate-buffered saline (PBS) or Hanks-buffered saline solution (HBSS).
Obtaining dental pulp can be achieved through various methods, contingent on the tooth type7,11. For deciduous teeth with resorbed roots, extraction can be performed via the root apex. Similarly, sterile barbed broaches can be used to obtain pulp from permanent teeth with an immature open apex. In cases of permanent teeth with fully developed roots, accessing the pulp chamber involves separating the dental crown from the root. This is accomplished by cutting the tooth using a diamond disc at the cementoenamel junction. This incision exposes the pulp chamber, enabling retrieval of the pulp tissue12,13,14.
Dental pulp stem cells (DPSCs) can be isolated through enzymatic digestion (ED) or outgrowth from tissue explants (OG), also known as spontaneous growth. The ED method employs enzymes, primarily collagenase I and dispase, to break down the tissue into single-cell suspensions15,16. The OG method, simpler and quicker, entails chopping the pulp fragments and directly placing them into a culture plate, allowing cells to grow from the tissue explants17. Researchers have utilized and compared both techniques to assess cell proliferation rates, preservation of isolated stem cell properties, differentiation, and surface marker expression18. Establishing and standardizing protocols for acquiring DPSCs with high efficiency and stemness can pave the way for effective and safe therapies19. This protocol encompasses extracting DPSCs using enzymatic digestion, lab processing, preservation, and cell differentiation with colorimetric staining for adipogenesis, osteogenesis, and chondrogenesis.
The protocol outlined in this article presents a step-by-step procedure, beginning with the initial isolation of dental pulp from the tooth, followed by culture and maintenance of DPSCs in the laboratory, and concluding with their characterization using specific stem cell markers (Figure 1). The techniques for inducing these stem cells into different cell lineages, highlighting their multipotency, are also described.
Protocol
Representative Results
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…
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.
Materials
| 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 |
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