Can Any Tooth Be Used for DPMSC Isolation? Understanding Direct Explant and Enzymatic Digestion Methods

Dental pulp mesenchymal stem cells (DPMSCs), also known as dental pulp stem cells (DPSCs), are a valuable type of mesenchymal stem cell derived from dental pulp tissue. Since their discovery in healthy adult third molars, DPMSCs have attracted increasing attention in regenerative medicine research due to their strong proliferative capacity, multilineage differentiation potential, and relatively accessible tissue source. This is also a topic often discussed on Reddit and other lab forums, where researchers ask whether all tooth samples are suitable for DPMSC isolation and how to choose between direct explant culture and enzymatic digestion.

Compared with other mesenchymal stem cell sources, DPMSCs offer several advantages, including convenient tissue collection, low ethical concerns, and promising applications in areas such as dental tissue regeneration, periodontal research, and tissue engineering.

However, obtaining high-quality DPMSCs requires careful consideration of both tooth source selection and isolation strategy. Different isolation methods may influence cell outgrowth efficiency, initial cell recovery, and subsequent experimental performance.

Previously, we introduced a commonly used DPMSC isolation workflow based on dental pulp tissue processing. Read the related DPMSC isolation and culture guide. In this article, our scientists will further discuss two commonly used approaches for DPMSC isolation — the direct explant attachment method and the enzymatic digestion method — and explain how researchers can select an appropriate isolation strategy based on their experimental requirements.

So, can DPMSCs be isolated from any type of tooth? Let’s explore the factors that influence DPMSC isolation success.

Materials Preparation

Tissue samples: Healthy third molars without dental caries or naturally exfoliated deciduous teeth.

Experimental reagents: OriCell Phosphate-Buffered Saline Solution (1X), OriCell Complete Medium For Human Dental Pulp Mesenchymal Stem Cells, OriCell Trypsin Cell Dissociation Solution (0.25%), OriCell Collagenase Type I (0.1%), and OriCell Penicillin-Streptomycin Solution (100X).

Instruments and consumables: Cell culture dishes, ophthalmic forceps, dental forceps, and dental hammer.

Pretreatment of Extracted Teeth

Extracted teeth should be transported to the laboratory on ice, and dental pulp tissue should be collected within 12 h.

Rinse the extracted teeth twice with PBS containing 2% penicillin-streptomycin solution for 1 min each time. If dental caries are present, soak the tooth in 75% ethanol for 2–3 min before rinsing.

Dental Pulp Extraction

Place the rinsed extracted tooth in a culture dish containing PBS with 2% penicillin-streptomycin solution. The PBS should fully cover the tooth.

Use forceps to scrape away other tissues and impurities from the extracted tooth.

After all impurities are removed, transfer the tooth to a new culture dish. Use a dental hammer and dental forceps to separate the tooth body and expose the pulp cavity.

Transfer the tooth fragments to a new culture dish. Use ophthalmic forceps to peel out the dental pulp and place it in sterile PBS.

Isolation of Dental Pulp Mesenchymal Stem Cells

Direct Explant Attachment Method

Use ophthalmic forceps to tear the dental pulp into filament-like tissue fragments. Spread them evenly across the culture dish and allow them to air-dry naturally for 3–5 min.

During this period, prewarm OriCell Complete Medium For Human Dental Pulp Mesenchymal Stem Cells to 37°C.

After the dental pulp tissue adheres closely to the dish surface, gently add OriCell Complete Medium For Human Dental Pulp Mesenchymal Stem Cells along the wall of the dish.

Place the dish in a 37°C incubator with 5% CO₂ and 95% relative humidity for culture.

During the first 72 h of culture, avoid moving the culture dish as much as possible. After 72 h, observe cell migration under a microscope and change the medium as needed. When cell confluence reaches 80%–90%, the cells can be passaged.

Enzymatic Digestion Method

Prewarm 0.1% Collagenase Type I to 37°C.

Cut the dental pulp tissue into fragments of approximately 1 mm². Collect the tissue fragments in a centrifuge tube and centrifuge at 265 × g for 5 min at room temperature.

Discard the supernatant. Resuspend the dental pulp tissue in 5 mL of 0.1% Collagenase Type I and digest in a 37°C water bath for 20 min. During digestion, take out the tube every 5 min and mix thoroughly. A thermostatic shaker can also be used if available.

After digestion, add an equal volume of complete medium and mix the digestion suspension thoroughly with the complete medium.

Centrifuge at 265 × g for 5 min and discard the supernatant. Resuspend the cells in prewarmed OriCell Complete Medium For Human Dental Pulp Mesenchymal Stem Cells, then seed the cells into a culture dish.

After seeding, avoid moving the culture dish as much as possible. Observe cell status after 72 h, and change the medium as needed. When cell confluence reaches 80%–90%, the cells can be passaged.

Passaging of Dental Pulp Mesenchymal Stem Cells

Human dental pulp mesenchymal stem cells should be passaged when they grow to 80%–90% confluence. Do not allow the cells to reach complete or excessive confluence, as this may cause contact inhibition and severely affect cell growth.

Passaging Procedure

1. Prewarm the complete medium and 0.25% trypsin to 37°C.
2. Aspirate the culture medium. Wash the cells 2–3 times with 1× PBS to remove residual serum, then aspirate the PBS.
3. Add an appropriate amount of 0.25% trypsin and gently rotate the culture dish to allow the trypsin to cover the cell surface. Digest for 1–2 min. Under the microscope, the cell gaps should become larger and the cells should become rounded. The digestion endpoint should be determined based on microscopic observation.
4. Gently tap the wall of the culture vessel by hand. When approximately 80% of the cells detach and float, immediately add 2–3 mL of complete medium to terminate digestion.
5. Transfer the cells into a centrifuge tube. Add 1× PBS to the culture flask and wash 1–2 times, then transfer the wash solution to the same centrifuge tube.
6. Centrifuge at 265 × g for 4 min. Aspirate the supernatant and resuspend the cells in 2–3 mL of complete medium.
7. Seed the cells according to the cell number or at a density of 2 × 10⁴ cells/cm².
8. Add complete medium and culture the cells in a 37°C incubator with 5% CO₂ and 95% relative humidity.

Complete OriCell Solution for Dental Pulp Mesenchymal Stem Cells

With 17 years of experience in stem cell culture, Cyagen OriCell provides a complete DPMSC solution to support dental pulp mesenchymal stem cell research.

In addition, Cyagen OriCell also provides complete solutions for mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, and umbilical cord blood. Please contact us for consultation or ordering information.

References

1. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(25):13625–13630.
2. Zhongyuan Union Cell & Gene Engineering Co., Ltd. Announcement on a drug candidate of an investee subsidiary entering Phase II clinical trial. Announcement No. 2023-022, May 25, 2023.