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

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 capacity^1,2. 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 exploration^4,5. 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 type^7,11. 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 tissue^12,13,14.

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 suspensions^15,16. 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

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 involved in this study.

1. Extraction and transport of teeth

1. Collection of deciduous and permanent teeth
   1. Explain to the participants the procedure's purpose and the potential use of the extracted teeth for research purposes.
   2. Perform teeth extraction under local anesthesia by administering an anesthetic agent in the vicinity of the tooth to be extracted. Specifically, 1.75 mL of lidocaine mixed with epinephrine (see Table of Materials) at a 1:200,000 ratio was injected for this study. Selecting teeth that do not exhibit severe dental caries or periodontitis is preferable^20,21.
2. Transportation of the extracted teeth
   NOTE: Working under aseptic conditions is recommended to avoid contamination. This includes wearing personal protective equipment, such as gloves and a lab coat, and working in a sterile environment, such as a biohazard laminar flow hood.
   1. After the tooth is extracted, rinse it with sterile phosphate-buffered saline (PBS) solution to remove the blood and other debris. Using sterile forceps and scalpel, carefully remove any attached soft tissue remnants from the tooth's surface (Figure 2A).
   2. Immerse the tooth in a disinfectant solution, such as 70% ethanol, for 10 s. After the disinfection, rinse the tooth with sterile PBS to wash away any residual disinfectant.
   3. Place the tooth in a sterile container containing the transport medium.
      NOTE: The transport medium consisted of Alpha-MEM, a commercial culture medium supplemented with antibiotics: 100 U/mL penicillin/streptomycin (see Table of Materials). This medium supplied essential nutrients to the cells within the extracted tooth, ensuring their survival until laboratory processing. The antibiotics served to inhibit bacterial growth.
   4. Maintain a temperature of 4 °C during transport to slow cellular metabolic activities and delay cell death. Transport the tooth to the laboratory for the next steps, including processing and isolating the dental pulp stem cells.

2. Collection of pulp tissue

1. Secure the tooth using dental forceps to ensure it stays in place during the procedure. Use a diamond disc to cut the tooth in a dental handpiece (see Table of Materials) connected to a water coolant (Figure 2B).
   NOTE: The goal is to expose the pulp chamber without causing unnecessary damage to the pulp tissue within.
2. Employ a dental excavator (see Table of Materials) to remove the pulp tissue. This instrument enables the extraction of pulp without inflicting damage, thereby preserving the viability of the dental pulp stem cells (Figure 2C).
3. Place the obtained tissue on a glass Petri dish. Wash the pulp tissue with phosphate-buffered saline.

3. Digestion of pulp tissue and cell isolation

1. Tissue mincing and digestion
   1. With a sterile surgical blade, carefully mince the pulp tissue into small fragments (Figure 2D) and place it in a mini tissue grinder with PBS to obtain a finely homogenized mixture. This process increases the surface area of the tissue, facilitating more efficient enzyme action during digestion.
   2. Transfer the minced homogenous tissue fragments into a 15 mL tube and enzymatically digest utilizing a mixture of 3 mg/mL collagenase type I and 4 mg/mL dispase (see Table of Materials). These enzymes were dissolved in Hank's Balanced Salt Solution (HBSS) to achieve the desired concentrations.
   3. Incubate the pulp tissue in the enzyme mixture (step 3.1.2) for about 2 h at 37 °C. After incubation, neutralize the enzymes, and collect the cells by centrifugation (step 3.2.1) for further culture.
2. Centrifugation and resuspension
   1. After incubation, transfer the digested tissue to a 15 mL centrifuge tube and spin at 300 x g for 5 min at room temperature to pellet the cells. This step separates the cells from the remaining undigested tissue fragments and enzymes. Carefully discard the supernatant medium, ensuring not to disturb the cell pellet at the bottom of the tube.
   2. Resuspend the cell pellet in a fresh culture medium. This medium provides the necessary nutrients and environment for the isolated cells to survive and proliferate.

4. Cell culture

1. Plating and incubation of cells
   1. Carefully transfer the resuspended cells to a 25 cm² culture flask.
      NOTE: All material from a single pulp is transferred into a single 25 cm² cell culture flask. A media volume of 5-7 mL ensures sufficient coverage and nutrient availability for the cells. Ensure that the culture medium for DPSCs is Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10%-20% fetal bovine serum (FBS) (see Table of Materials) to provide necessary growth factors. Further, supplement the medium with 1% penicillin/streptomycin to prevent bacterial contamination. Ensure the cells are evenly distributed throughout the flask to promote homogeneous growth.
   2. Incubate the flask at 37 °C in an atmosphere containing 5% CO₂.
2. Medium change and monitoring confluency
   1. Change the culture medium every 2-3 days to provide fresh nutrients and remove metabolic waste. To do this, carefully aspirate the old medium using a sterile serological pipette and replace it with a fresh medium.
   2. Regularly monitor the cell culture under a microscope to track the cells' growth and check for any signs of contamination.
   3. Continue this process until the cells reach 80%-90% confluency, the point at which the cells cover most of the flask surface area but are not overly crowded.
3. Cell trypsinization and passaging or freezing
   1. Once the cells reach 80%-90% confluency, remove the culture medium and wash the cells with phosphate-buffered saline (PBS) to remove the residual medium and non-adherent cells.
   2. Add a solution of 0.25% trypsin-EDTA to the flask to detach the cells from the flask's surface. Incubate for 2-5 min at 37 °C until the cells lift off.
   3. Neutralize the trypsin by adding an equal volume of culture medium, then gently pipette the cell suspension up and down to ensure complete cell detachment.
   4. Centrifuge the cell suspension at 300 x g for 5 min at room temperature to pellet the cells.
   5. Resuspend the cells in a fresh culture medium and either replate them (passage) for further growth or freeze them for future use. When freezing, use a freezing medium that contains a cryoprotectant, like DMSO, to protect the cells from damage during freezing.

5. Characterization of DPSCs

1. Flow cytometry analysis
   1. To confirm the mesenchymal stem cell nature of the isolated cells, perform flow cytometry analysis^22,23.Initially, trypsinize and collect cells as described in step 4.3.
   2. Determine the cell viability by the trypan dye exclusion technique. Perform the analysis using a cell viability analyzer (see Table of Materials) during the 2^nd and 8^th passages. Perform phenotypic analysis via a flow cytometer during the 3^rd and 7^th passages.
   3. After incubation, wash the cells in a flow cytometry buffer to remove excess antibodies and analyze the cells using a flow cytometer.
      NOTE: The flow cytometry buffer is composed of phosphate-buffered saline (1x PBS), 1%-2% fetal bovine serum (FBS), and 0.1% sodium azide (NaN₃). The FBS or BSA blocks the non-specific binding of antibodies, while the sodium azide acts as a preservative. A high percentage of cells should express the mesenchymal stem cell markers and lack the hematopoietic markers, confirming their identity as DPSCs²⁴.
   4. Detach the DPSCs and stain them with immunofluorescence antibodies for flow cytometry analysis.
      NOTE: The selection of markers for flow cytometry analysis of mesenchymal stem cells (MSCs) is based on the consensus established by the International Society for Cellular Therapy (ISCT), which states that MSCs should express CD105, CD73, and CD90, and lack the expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA class II²⁵. These markers are commonly used in studies investigating dental pulp stem cells²⁶.The concentrations of antibodies for staining can vary depending on the specific antibody used, and it is recommended to follow the manufacturer's instructions (see Table of Materials) for antibody dilutions and incubation conditions²⁵.
   5. Establish the classification criteria^27,28 for the expression of CD markers as mentioned: less than 10%, no expression; between 11% and 40%, low expression; a range of 41% to 70%, moderate expression; above 71%, high expression.
   6. Additionally, confirm the absence of hematopoietic markers by incubating a separate cell aliquot with antibodies against CD34 and CD45.

6. Multilineage differentiation

NOTE: The following steps outline protocols for osteogenic, adipogenic, and chondrogenic differentiation of dental stem cells. Begin by seeding cultures at a density of 1 x 10⁵ cells per well in a fibronectin-coated tissue culture plate with complete medium (CM). Monitor cell growth until 80%-90% confluency is achieved before initiating the desired differentiation protocol. To evaluate the DPSCs' multilineage differentiation potential, initiate the differentiation process towards osteoblasts, adipocytes, and chondrocytes by seeding cells into 24-well plates and culturing them in appropriate differentiation media.

1. Osteogenic differentiation
   1. Initiate by culturing DPSCs in a regular growth medium under standard conditions until they reach 70%-80% confluence.
   2. Remove the growth medium from the cells, then wash the cells once with 1x PBS. Add the Osteogenic Differentiation Medium (ODM) to the cells.
      NOTE: The ODM consists of Alpha Minimum Essential Medium (α-MEM) supplemented with 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, 50 µg/mL ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone (see Table of Materials).
   3. Incubate the cells in the osteogenic medium at 37 °C with 5% CO₂. Change the osteogenic medium every 2-3 days.
      NOTE: After 14 to 21 days, the DPSCs should have differentiated into osteoblast-like cells. This can be confirmed by checking for an increase in the expression of osteoblast-specific markers, such as alkaline phosphatase, or by using staining techniques to detect mineralized matrix, such as Alizarin Red S staining.
   4. Fix the cells by incubating them with 70% ice-cold ethanol for 1 h at -20 °C.
   5. Stain the fixed cells with 40 nM Alizarin Red S (pH 4.2) staining solution at room temperature in the dark for 10-15 min. Visualize the staining under a microscope to confirm osteogenic differentiation (Figure 3). Confirm the differentiation by staining the cells with Alizarin Red S, which identifies calcium deposits indicative of mineralization^29,30.
2. Adipogenic differentiation
   1. As detailed in the earlier section, culture DPSCs under standard conditions in a humidified atmosphere with 5% CO₂ at 37 °C. Passage the cells when they reach around 70%-80% confluence.
   2. Aspirate the medium and wash the cells once with 1x PBS. Replace the washed-out medium with adipogenic differentiation medium. This medium generally consists of DMEM, 10% FBS, 10 µg/mL insulin, 1 µM dexamethasone, 200 µM indomethacin, and 0.5 mM IBMX (see Table of Materials).
   3. Change the differentiation medium every 3-4 days. After about 3 weeks, wash the adipogenic-differentiated DPSCs with PBS and fix them with 4% paraformaldehyde (PFA) for 20 min at room temperature.
   4. Stain the fixed cells with Oil Red O (see Table of Materials) to visualize lipid droplet accumulation (Figure 4). To further confirm adipogenic differentiation, perform gene expression analysis of adipogenic markers such as peroxisome proliferator-activated receptor gamma (PPARγ) and adiponectin^31,32.
3. Chondrogenic differentiation
   1. DPSCs cultures maintained in a humidified atmosphere with 5% CO₂ at 37 °C are used for Chondrogenic differentiation. Passage the cells when they reach around 70%-80% confluence.
   2. Aspirate the medium and wash the cells once with 1x PBS. Replace the washed-out medium with chondrogenic differentiation medium, which typically contains high glucose Dulbecco's Modified Eagle Medium (DMEM), 1% ITS, 100 nM dexamethasone, 50 µg/mL ascorbate-2-phosphate, 40 µg/mL proline, and 10 ng/mL Transforming Growth Factor-beta 3 (TGF-β3) (see Table of Materials).
   3. Maintain the cells in the chondrogenic differentiation medium, changing the medium every 2-3 days. After about three weeks, the DPSCs should have differentiated into chondrocyte-like cells.
   4. Fix the cells by incubating them with 4% paraformaldehyde (PFA) for 20 min at room temperature. After fixation, stain the cells with Alcian Blue (see Table of Materials) and visualize them under a microscope to confirm chondrogenic differentiation (Figure 5).
   5. Confirm the differentiation of DPSCs into chondrocytes using techniques such as Alcian Blue staining for proteoglycans or gene expression analysis of chondrogenic markers like aggrecan, SOX9, and type II collagen^33,34.

Representative 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.

Colony-Forming Unit (CFU) assays
CFU assays were conducted to evaluate the self-renewal capacity of the DPSCs. A single-cell suspension was plated at a low density and cultured for 14 days. The colonies formed were then stained and counted. The results show many colonies, indicating a high proportion of self-renewing cells within the DPSC population.

Cell doubling time
The cell doubling time was calculated based on the growth curve of the DPSCs. Cells were seeded at a specific density and counted regularly to construct a growth curve. The doubling time was then calculated using the exponential growth phase of the curve. The results show a doubling time consistent with that reported for MSCs, suggesting a healthy and proliferative cell population (Figure 6).

Flow cytometry analysis
The flow cytometry analysis showed that a large majority of the DPSCs (e.g., >95%) were positive for CD90, CD73, and CD105, confirming their identity as MSCs (Figure 7). Moreover, less than 2% of the cells were positive for CD45, CD34, and HLA-DR, confirming the absence of significant hematopoietic or endothelial cell contamination.

Osteogenic differentiation
Upon completion of osteogenic differentiation, the cells exhibited a mineralized matrix, which is visualized by Alizarin Red S staining (Figure 3). The presence of red coloration in the stained cells indicates the successful deposition of a mineralized matrix, an essential characteristic of osteogenic differentiation. The inability to successfully achieve differentiation staining could potentially be linked to challenges faced during the sample collection procedure.

Adipogenic differentiation
During the adipogenic differentiation, the DPSCs accumulated lipid droplets, indicative of adipogenesis. The lipid droplets can be observed with Oil Red O staining (Figure 4). A successful differentiation resulted in orange-red stained lipid droplets within the cells.

Chondrogenic differentiation
Lastly, after chondrogenic differentiation, DPSCs produced glycosaminoglycans, a critical cartilage component confirmed by Alcian Blue staining, which binds to these glycosaminoglycans. Cells successfully undergoing chondrogenesis exhibited blue coloration upon staining (Figure 5).

The cells that failed to exhibit respective color changes in the staining procedures might be due to the sub-optimal differentiation, possibly due to issues with the differentiation media or the initial quality of the DPSCs. Ensuring the quality of media and supplements and the health and passage number of DPSCs used is important. The successful differentiation of DPSCs into osteoblasts, adipocytes, and chondrocytes validates these cells' multipotent nature and underscores their potential in regenerative medicine and therapeutic applications.

Figure 1: Overview of dental pulp stem cell isolation, characterization, and differentiation. This figure provides a schematic representation of the workflow for isolating, characterizing, and differentiating dental pulp stem cells (DPSCs). The process begins with the extraction of the tooth (or teeth), followed by the isolation of the pulp tissue, from which the DPSCs are derived. After isolation, the DPSCs undergo various characterization tests to confirm their stem cell identity and assess their viability, proliferation rate, and other key attributes. The final stage represented in this diagram involves the induction of differentiation, where the DPSCs are treated with specific factors to guide their transformation into specialized cell types, such as osteoblasts, adipocytes, or chondrocytes. Please click here to view a larger version of this figure.

Figure 2: Pulp retrieval from extracted teeth. (A) An extracted tooth prepared for pulp tissue extraction. (B) Sectioning of the extracted tooth using a diamond bur. (C) Removal of the pulp tissue from the pulp chamber. (D) Fragmentation of the pulp tissue using a surgical blade. Please click here to view a larger version of this figure.

Figure 3: Osteogenic differentiation of dental pulp stem cells (DPSCs) evidenced by Alizarin Red S staining (40x). The bright red staining reveals the deposition of the mineralized matrix, a characteristic feature of osteoblasts, confirming successful osteogenic differentiation. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 4: Adipogenic differentiation of dental pulp stem cells (DPSCs) demonstrated by Oil Red O staining. The presence of bright red droplets represents lipid accumulation, a hallmark of adipocytes, confirming successful adipogenic differentiation. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 5: Chondrogenic differentiation of dental pulp stem cells (DPSCs) demonstrated by Alcian Blue Staining (40x). The intense blue color indicates the presence of a proteoglycan-rich extracellular matrix, a characteristic of chondrocytes, confirming successful chondrogenic differentiation. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 6: Cell doubling time for dental pulp stem cells (DPSCs). This figure illustrates the growth curve of DPSCs over time. Cells were initially seeded at a specific density, and cell counts were taken at regular intervals (represented on the x-axis) to monitor the proliferation rate. Please click here to view a larger version of this figure.

Figure 7: Stem cell surface marker expression in DPSC culture. This figure demonstrates the expression of specific surface markers on DPSCs. The mesenchymal stem cell markers CD44, CD90, and CD105 show high levels of expression, indicating the presence of mesenchymal traits in these cells. Conversely, the expression of hematopoietic markers (CD34 and CD45) and the major histocompatibility complex class II molecule (HLA-DR) is almost negligible, confirming the absence of hematopoietic lineage cells in the culture. Please click here to view a larger version of this figure.

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 obtained from various sources, including infected vital human teeth afflicted by conditions like periodontitis, root resorption, pericoronitis, and tooth fractures, as well as supernumerary and misaligned teeth. This finding was further corroborated by subsequent studies identifying infected vital teeth as potential sources of DPSCs^37,38,39,40. However, Bernardi et al.¹⁰ pointed out that increased root resorption could detrimentally affect the viability of these stem cells. A significant hurdle in the process is preserving the cells and preventing their degradation, which starts immediately after tooth extraction or exfoliation. Furthermore, it is important to note that teeth affected by severe conditions such as periapical abscesses, tumors, or cysts should not be utilized to extract stem cells^40,41.

DPSCs can be procured through the enzymatic digestion (ED) technique or the outgrowth from tissue explants (OG) method, also known as the spontaneous growth technique^14,15. Researchers use these strategies to investigate the efficiency of cell proliferation or the maintenance of morphological and phenotypic attributes of isolated stem cells¹⁷. Studies have found that stem cells isolated via the OG method show a slower proliferation rate and weaker expression of stem cell markers¹⁸. Conversely, DPSCs isolated through the ED method demonstrated faster proliferation, differentiation, and increased expression of additional surface markers than those isolated by the OG method. Interestingly, dental pulp immature stem cells (IDPSCs) extracted from deciduous teeth using the OG technique showed that cell culture encouraged the selective proliferation of IDPSCs, thereby preventing early differentiation⁴².

The purification of DPSCs is crucial for obtaining highly regenerative cells and requires specific cell markers. Cell selection is performed using either fluorescence or magnetic-activated cell sorting methods. Besides the aforementioned markers, numerous other surface antigens are also used due to the heterogeneous nature of DSCs⁴³. However, no single marker has been identified to segregate the subpopulations.

Compared to mesenchymal stem cells derived from bone marrow, DPSCs have been recognized as a promising source of multipotent cells due to their multilineage potential⁴⁴. This stemness is likely a result of the relative immaturity of the source tissues, as wisdom teeth, often the source of DPSCs extraction, are the last permanent teeth to develop and are thus less mature than bone marrow⁴⁵. This protocol successfully explores the multipotent nature of dental pulp stem cells by inducing these cells to undergo adipogenesis, osteogenesis, and chondrogenesis. Notably, DPSCs are the only cell type that upregulates DSPP expression when cultured under osteogenic conditions, indicative of their future differentiation into odontoblasts⁴⁶.

Dental stem cells, isolated from various sources, showcase many proliferation and differentiation potentials⁴⁷. The significant strides made in in vitro systems have profoundly impacted stem cell biology and therapeutics. Despite these advances, challenges remain when translating stem cell therapies from bench to bedside. Particular attention must be paid to the survival and stability of stem cells to ensure that preservation techniques do not compromise the viability of these cells. While dental stem cell research has yielded promising results in animal models, there is a pressing need to extend these findings to human trials. By continuing to drive rigorous research and development, dental stem cells are poised to play a substantial role in advancing the field of stem cell banking.

While widely used, the enzymatic digestion approach for isolating dental pulp stem cells (DPSCs) presents several limitations. Foremost, the process can compromise cell viability, as enzymes like collagenase and dispase can inadvertently damage cell membranes, yielding fewer live stem cells¹⁷. Additionally, this method often results in the degradation of the vital extracellular matrix (ECM), which is crucial in preserving the inherent properties of stem cells⁴⁸. The procedure may also alter cell surface markers, hampering the accurate identification and characterization of DPSCs⁴³. Beyond these biological constraints, the approach is time-consuming, elevating the risks of microbial contamination due to prolonged incubations at 37 °C. Moreover, the inconsistency between enzyme batches can lead to variable results, challenging the reproducibility of the isolation procedure. The financial burden cannot be overlooked, as high-grade enzymes suitable for tissue digestion are expensive⁴⁹. Furthermore, the aftermath of the digestion might see residual enzyme activity that could detrimentally affect cell cultures, influencing their health, proliferation, and differentiation. Lastly, the potential for enzymatic digestion to preferentially isolate certain DPSC subpopulations over others might inadvertently skew the resultant cell pool, influencing their therapeutic potential⁵⁰.

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