Name: isolation, culture, and characterization of dental pulp stem cells from human deciduous and permanent teeth
Uploaded: 2024-05-17T13:10:00
Description: Read this Scientific Article Protocol about Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth at JoVE.com

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.

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. Decid...

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&#160;demonstrated that Dental Pulp Stem Cells (DPSCs) could be ob...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-isobuty-l-methyl-xanthine&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>AS001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>RM471</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S staining solution</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>GRM894</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase -Staining kit</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;-MEM)&#160;</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha Minimum Essential Medium (&#945;-MEM)&#160;</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha-MEM, or Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Alpha-MEM, or Alpha Minimum Essential Medium</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Antibiotic/Antimycotic</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>012-04802</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-glycerophosphate&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>G9422-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet-Laminar flow hood</td>
			<td>Labconco Corporation,MO 64132-2696,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD90, CD105, CD73, CD34, CD45, and HLA-DR</td>
			<td>BioLegend, Inc.CA 92121,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 &#181;m )</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP025</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>REMI Elektrotechnik Limited (REMI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>1101 | 1102</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp. NJ 08701, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type I</td>
			<td>Worthington Biochem. Corp. NJ 08701, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Growth Medium</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>AT006</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Conical tubes (15 or 50 )</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td>546021P/546041P</td>
			<td>15 mL and 50 mL</td>
		</tr>
		<tr>
			<td>Cryo freezing container</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>15-350-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryolabels</td>
			<td>Label India:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial storage boxes&#160;</td>
			<td>Cryostore Storage Boxes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials (1.8 mL)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>PW1282</td>
			<td>Self standing</td>
		</tr>
		<tr>
			<td>Culture flask (25 cm&#178;)</td>
			<td>Corning Inc.NY 14831,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture flasks</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCG4/TCG6</td>
			<td>T25/T75</td>
		</tr>
		<tr>
			<td>Culture Plates</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP129/TCP008</td>
			<td>60 mm/100 mm</td>
		</tr>
		<tr>
			<td>Dental Diamond Discs</td>
			<td>Komet SC 29730, USA</td>
			<td></td>
			<td>Komet&#160;</td>
		</tr>
		<tr>
			<td>Dental Spoon Excavator</td>
			<td>Brasseler,GA 31419,USA</td>
			<td>5023591U0</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethosone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethosone</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TC185</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche Diagnostics,Mannheim,Germany.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche Diagnostics GmbH, Mannheim,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>MB106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol -70%</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td>Fisher Scientific</td>
		</tr>
		<tr>
			<td>Extraction forceps&#160;</td>
			<td>Dentsply Sirona, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>RM9954</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific Inc.,MA,USA</td>
			<td>F2442-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin-coated tissue culture plate</td>
			<td>Corning Inc.Corning, NY 14831,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences,CA 95131,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometry buffer</td>
			<td>BD Biosciences,CA 95131,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slip 22 x 22 mm</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TCP017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Lonza Group Ltd,4002 Basel, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-speed dental handpiece&#160;</td>
			<td>NSK Ltd,Tokyo 8216, Japan</td>
			<td></td>
			<td>Ti-Max Z series</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX, or 3-isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Pfizer Inc. NY 10017,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>I5523</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS)</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>I5523</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium (ITS) premix</td>
			<td>Corning Incorporated,MA 01876,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus Corp.,Tokyo 163-0914,Japan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (60% )</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>MB063</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine mixed with epinephrine</td>
			<td>DENTSPLY,NC 28277,USA</td>
			<td></td>
			<td>Citanest</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Air Liquide,75007 Paris,France</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen storage tank</td>
			<td>Cryo Scientific Systems Pvt. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Eppendorf AG,22339 Hamburg,Germany</td>
			<td>30020</td>
			<td>Accupipet-2-20 &#181;L</td>
		</tr>
		<tr>
			<td>Mini tissue grinder</td>
			<td>Bio-Rad Lab, Inc. CA 94547,USA</td>
			<td></td>
			<td>ReadyPrep mini grinders</td>
		</tr>
		<tr>
			<td>Minus 80 freezer</td>
			<td>Blue Star Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Marienfeld Superior,arktheidenfeld,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oil red O stain</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>1024190250</td>
			<td></td>
		</tr>
		<tr>
			<td>Osteogenic Differentiation Medium (ODM)&#160;</td>
			<td>STEMCELL Technologies Inc.Vancouver, BC, V5Z 1B3,Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL119</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution (PBS) without Ca++ and Mg++</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>TS1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific,MA, USA</td>
			<td></td>
			<td>Gibco</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Thermo Fisher Scientific, MA, USA</td>
			<td>P3813-1PAK</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Proline&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Blade Size 15&#160;</td>
			<td>Swann-Morton Ltd, Sheffield, S6 2BJ,UK</td>
			<td>BDF-6955C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite</td>
			<td>HiMedia Laboratories&#160; Ltd.Mumbai,India</td>
			<td>AS102</td>
			<td>4% w/v solution</td>
		</tr>
		<tr>
			<td>Sterile centrifuge tubes</td>
			<td>Tarsons Products Pvt. Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile container -20 mL</td>
			<td>3M Center, MN 55144-1000,USA</td>
			<td></td>
			<td>3 M</td>
		</tr>
		<tr>
			<td>Sterile phosphate-buffered saline (PBS)</td>
			<td>Sigma Aldrich, USA</td>
			<td>P3813-1PAK</td>
			<td>1x PBS, pH 7.4</td>
		</tr>
		<tr>
			<td>Sterile pipettes (2, 5, and 10 mL )</td>
			<td>Eppendorf AG,22339 Hamburg,Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes and tips</td>
			<td>Eppendorf India Limited</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Blade Handle</td>
			<td>Becton, Dickinson and Co.,NJ,USA</td>
			<td>371030</td>
			<td>BP Handle 3</td>
		</tr>
		<tr>
			<td>Transforming Growth Factor-beta 3 (TGF-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming Growth Factor-beta 3 (TGF-&#946;3)</td>
			<td>R&#38;D Systems, Inc.MN 55413,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue 0.4%&#160;</td>
			<td>Sigma-Aldrich Co. St. Louis, MO 63103.USA</td>
			<td>TCL046</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA&#160;</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25%</td>
			<td>Gibco-Thermo Fisher Scientific Inc.,MA 02451,USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Thermo Fisher Scientific ,MA 02451,USA</td>
			<td>BSW-01D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture, and characterization of dental pulp stem cells from human deciduous and permanent teeth

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.

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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Read this Scientific Article Protocol about Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth at JoVE.com

Isolation, Culture, and Characterization of Dental Pulp Stem Cells from Human Deciduous and Permanent Teeth

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.

In the realm of regenerative medicine and therapeutic applications, stem cell research is rapidly gaining traction. Dental pulp stem cells (DPSCs), which ...

Begynn med å feste den ekstraherte mennesketannen på plass med tanntang. Skjær deretter tannen med en diamantskive koblet til et tannhåndstykke og et vannkjølemiddel. Bruk en tanngraver til å fjerne massevevet fra tannen.Deretter bruker du et sterilt kirurgisk blad til å hakke massevevet forsiktig i små fragmenter. Plasser deretter disse fragmentene i en minivevskvern med PBS for å danne en homogen blanding. Overfør de hakkede vevsfragmentene til et 15 milliliters rør og fordøy dem ved hjelp av en blanding av kollagenase type én og dispase.Inkuber vevet i enzymblandingen ved 37 grader Celsius i to timer. Etter inkubasjon, nøytraliser enzymene. Overfør deretter det fordøyde vevet til et 15 ml rør og snurr ved 300 G i fem minutter ved romtemperatur.Kast deretter supernatanten forsiktig og suspender pelleten på nytt i fersk DMEM, supplert med 10 til 20 % FBS. For cellekultur, overfør cellesuspensjonen forsiktig til en 25 kvadratcentimeter kulturkolbe. Inkuber cellene ved 37 grader Celsius i 5 % karbondioksid.Etter to til tre dager, bruk en steril serologisk pipette, aspirer du mediet forsiktig og erstatter det med et friskt medium. Deretter, for karakterisering av DPSC-er, slå på flowcytometeret og bekreft den mesenkymale stamcellenaturen til de isolerte cellene. Den vellykkede differensieringen av DPSC-er til osteoblaster, adipocytter og kondrocytter validerer den multipotente naturen til DPSC-er.Doblingstiden for DPSC-er var i samsvar med den som ble rapportert for mesenkymale stamceller, noe som tyder på en sunn og proliferativ cellepopulasjon. Flowcytometrianalysen viste at et flertall av DPSC-ene var positive for CD-90, CD-73 og CD-105, noe som bekreftet deres identitet som mesenkymale stamceller. Dessuten var mindre enn 2 % av cellene positive for CD-34, CD-45 og HLADR, noe som bekrefter fraværet av signifikant hematopoetisk eller endotelcellekontaminering.

Research

JoVE Journal

Bioengineering

Use of Human Perivascular Stem Cells for Bone Regeneration

Bruk av menneskelige perivaskulær stamceller for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

Utvikling, utvidelse, og<em&gt; In vivo</em&gt; Overvåking of Human NK-celler fra humane embryonale stamceller (hESCs) og og Induced pluripotent stamceller (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Effektiv generasjon og redigering av mater-gratis IPSCs fra menneskelige bukspyttkjertelen celler ved hjelp av CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Isolering av Mesenchymal stamceller fra menneskelige Alveolar Periosteum og effekter av Vitamin D på Osteogenic aktivitet i Periosteum-avledet celler

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Isolering av voksen Human dermal fibroblaster fra abdominal hud og generering av indusert Pluripotent stamceller ved hjelp av en ikke-integrering metode

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

Skalerbar generasjon av modne cerebellar organoider fra humane pluripotente stamceller og karakterisering ved immunstaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Enkeltcellet optisk virkningspotensial måling i humane induserte pluripotente stamcelleavledede kardiomyocytter

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

En kulturmetode for å opprettholde quiescent humane hematopoietiske stamceller

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

Introduksjon Induksjon av humane dentalmasse stamceller mot bukspyttkjertelen avstamninger

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Formering av tann- og respiratoriske celler og organer i mikrogravitasjon

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...