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 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.
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.
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
2. Collection of pulp tissue
3. Digestion of pulp tissue and cell isolation
4. Cell culture
5. Characterization of DPSCs
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.
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.
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 DPSCs37,38,39,40. 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 cells40,41.
DPSCs can be procured through the enzymatic digestion (ED) technique or the outgrowth from tissue explants (OG) method, also known as the spontaneous growth technique14,15. 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.
The authors have nothing to disclose.
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.
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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