Culture and Growth Characterization of Human Mesenchymal Stem Cells from Dental Pulp

Revathi, S (2011) Culture and Growth Characterization of Human Mesenchymal Stem Cells from Dental Pulp. Masters thesis, Ragas Dental College & Hospital, Chennai.


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INTRODUCTION: Stem cells are defined as ‘cells that have the ability to perpetuate themselves through self-renewal and to generate mature cells of a particular tissue through differentiation’. Stem cell research has been gaining momentum for the past two decades and in the past few years it has generated significant interest due to the success achieved in culturing human embryonic stem cells, and in manipulating their differentiation in vitro. Methods developed to assess and identify stem cells within a heterogeneous population using microarrays and comparison of data sets in silico have further facilitated their clinical application. The human body contains several loci or compartments called ‘stem cell niches’, inhabited by a significant number of stem cells. The dental pulp, contained within the ‘sealed niche’ of the pulp chamber, is an extremely rich site for stem cells. Stem cells in dental pulp reside in a perivascular micro-environment, where they are quiescent but have the potential to express basic stem cell characteristics. These stem cells are called DPSC (dental pulp stem cells), in permanent teeth and SHED (stem cells from human exfoliated deciduous), in deciduous teeth. DPSC and SHED are mesenchymal stem cells similar to the first mesenchymal stem cells isolated from bone marrow, (bone marrow mesenchymal stem cells; BM-MSCs) that have the capacity to form single-cellderived colony clusters called Colony Forming Unit-fibroblast (CFU-F) in vitro. Accumulated knowledge of the phenotypic characteristics of BM-MSCs has contributed significantly to the isolation of putative stem cell populations from dental pulp of human permanent teeth and deciduous teeth. As stem cells are self-renewing and multi-potent, they can potentially generate any tissue in their lifetime. The ability to expand stem cells and induce their differentiation in culture is a key property for their use in therapy, and is of great clinical interest in many diseases including Parkinson’s, cardiovascular disease and correction of neural degeneration following brain injury. In dentistry, stem cells are being used for the regeneration of dentin, creation of biologically viable scaffolds for the replacement of orofacial bone and cartilage, craniofacial regeneration-including the temporomandibular joint, and regeneration of periodontal ligament and cementum. As dental pulp derived stem cells produce neurotropic factors, they have the potential to be used in neural regeneration. Clinically, DPSC have an advantage over other types of adult stem cells in that they are easy to access and are extracted during life. Also, exfoliated deciduous teeth can be secured at a young age and the cells obtained, stored for future use. A stem cell bank can be created from the DPSC / SHED without using procedures that are invasive. The first step in the use of stem cells for therapy is their characterization. This is achieved by studying the phenotypic features, growth pattern and markers of cellular differentiation. The present study was done to isolate and expand the mesenchymal cell population from the dental pulp of both deciduous and permanent teeth, to ascertain the feasibility, standardize the procedure and characterize their growth properties and morphology. AIM OF THE STUDY: To isolate, culture and study the morphology and growth characteristics of mesenchymal stem cells from the dental pulp of permanent teeth (DPSC) and exfoliated deciduous teeth (SHED). OBJECTIVES OF THE STUDY: 1. To isolate and culture mesenchymal stem cells from permanent teeth (DPSC) and exfoliated deciduous teeth (SHED) using enzyme disaggregation technique. 2. To compare the phenotypic characteristics of cells obtained from the dental pulp of permanent teeth (DPSC) and exfoliated deciduous teeth (SHED). 3. To ascertain the population doubling time of cells obtained from the dental pulp of permanent teeth (DPSC) and exfoliated deciduous teeth (SHED). 4. To ascertain the capacity to form Colony Forming Units (CFUs). of cells obtained from the dental pulp of permanent teeth (DPSC). Materials for tissue Culture: Reagents: Growth medium- 1. Mesenchymal Stem Cell Medium (MSC Medium)- α- modified minimal essential medium (α-MEM) Fetal Bovine Serum (Invitrogen TM). Antibiotics- - Penicillin-100 IU/ml. - Streptomycin-100μg/ml. 2. D-PBS (Potassium chloride-0.2g/l, Potassium phosphate monobasic- 0.2g/l,Sodium chloride-8g/l, Sodium phosphate dibasic-1.15g/l). 3. Distilled water. 4. De-ionized water. 5. Collagenase (type I, filtered) (CLS-1- Worthington Biochemical Corporation TM). 6. Dispase (neutral protease, grade II) (Roche TM). 7. Trypsin 1:125. (Tissue culture grade, Hi media TM). 8. Ethylene-di-amine-tetra-acetic acid. (Hi Media TM). Equipment: 1. Culture dishes. (TarsonsTM), 2. 24-well plates. (Cell star TM), 3. Disposable pipettes and pipette tips, 4. Glass pipettes, 5. BP blade no. 15, 6. Centrifuge tubes, 7. Leak-proof screw-cap vials, 8. Scott Duran bottles, 9. Laminar flow cabinets, 10. Carbon dioxide incubator. (Thermo electron Corporation. Forma series II water jacketed-HEPA class 100), 11. Phase contrast microscope. ( Olympus CKX41 TM ), 12. Digital camera. (Kodak AF3X, 8.2 mega pixels, 3x optical zoom), 13. Improved Neubauer counting chamber, 14. Laboratory centrifuge. (R-86 Remi TM), 15. Cyclomixer. (C101 Remi TM), 16. Electronic balance. (Dhona 200D TM), 17. Prabivac vaccum pump, 18. Cellulose acetate filter (pore size 0.2μm), 19. Autoclave, 20. Hot air oven, 21. Micromotor (Marathon TM), 22. Contra-angled Hand piece (NSK TM), 23. Carborundum discs, 24. Chisel, 25. Mallet, Methodology: Tissue collection: Permanent teeth - Impacted third molars/premolars extracted for orthodontic reasons from patients attending Ragas Dental College and Hospital, Chennai. Deciduous teeth: Non-infected, exfoliating deciduous teeth extracted in the Department of Pedodontia, Ragas Dental College and Hospital, Chennai. Consent was obtained from patients above the age of eighteen years and from the parents of children for the collection of teeth [Annexure I].The study was approved by the Institutional Review Board, Ragas Dental College and Hospital, Chennai. Transportation of tissue to laboratory for culturing: Teeth extracted under sterile condition were transferred to serum-free α-Minimal Essential Medium(α-MEM), with added antibiotics (Penicillin-100 IU, Streptomycin-100μg/ml), at a pH of 7.2 to 7.4 and maintained at 4°C with the help of ice-packs. They were transported in leak-proof, sterilized culture vials. SUMMARY AND CONCLUSION: 1. Protocol for the culture of mesenchymal stem cells from the dental pulp of permanent and deciduous teeth using α-MEM (Mesenchymal Stem Cell media) was standardized. 2. The population of cells derived from permanent teeth (DPSC) and dedciduous teeth (SHED) showed distinctly different ratios of fibroblastoid:epithelioid cells. The cells from the permanent tooth pulp showed a higher proportion of spindle shaped fibroblastoid cells whereas a higher proportion of epithelioid cells were seen in the deciduous pulp culture. The difference was statistically significant at 5% level, p<0.05. 3. The estimation of growth curve and population doubling time was done for two DPSC cell lines (26 hours, 27 hours) and one SHED cell line (22 hours). 4. The seeding efficiency of DPSC was 88.9% (14th permanent tooth pulp) and 91.7% (15th permanent tooth pulp) and was higher as compared to SHED which was 84.25% (10thdeciduous tooth pulp). 5. Colony formation is a feature of stem cells. The colony forming efficiency of DPSC was 17%. Permanent and deciduous teeth are viable sources of stem cells. The permanent teeth are easier to culture because of a lower chance of contamination with oral microflora. The growth characteristics of the cells obtained from both these sources are similar. However, there was a difference in the proportion of fibroblastoid: epithelioid cells between the cultures obtained from the permanent and deciduous teeth. This needs to be studied using specific markers to ascertain if they are indicative of two distinct populations or just morphologic variations representative of different stages of growth.

Item Type: Thesis (Masters)
Uncontrolled Keywords: Culture and Growth Characterization ; Human Mesenchymal Stem Cells ; Dental Pulp.
Subjects: DENTAL > Oral Pathology and Microbiology
Depositing User: Kambaraman B
Date Deposited: 12 May 2018 05:55
Last Modified: 12 May 2018 05:55

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