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Tooth Storage, Dental Pulp Stem Cell Isolation, and Clinical Scale Expansion without Animal Serum Emily J. Eubanks,* Susan A. Tarle,* and Darnell Kaigler, DDS, MS, PhD*† Abstract Introduction: Dental pulp stem cells (DPSCs) have therapeutic potential for dentin and dental pulp regeneration. For regenerative approaches to gain clinical acceptance, protocols are needed to determine feasible ways to store teeth, isolate DPSCs, and expand them to clinical scale numbers. Methods: In this study, 32 third molars were obtained from patients and immediately placed in saline or tissue culture medium followed by overnight storage at 4 C or immediate isolation of DPSCs. Upon isolation, cells were expanded in medium containing either fetal bovine serum (FBS) or human serum (HS). Cell proliferation (population doubling time [PDT]), cell surface marker expression, and multipotency were compared between DPSCs in FBS and DPSCs in HS. Results: The time frame of storage and storage medium did not affect the ability to isolate DPSCs. However, using HS instead of FBS in the initial isolation of DPSCs significantly decreased (P < .01) the isolation success rate from 89% (FBS) to 23% (HS). Yet, incorporating fibronectin in the DPSC initial isolation (using HS) significantly (P < .01) increased the isolation success rate to 83%. Interestingly, it was found that the proliferation rate was significantly (P < .05) higher for DPSCs in HS (PDT = 1.59 0.46) than that for DPSCs in FBS (PDT = 2.84 2.5). Finally, there was no difference in the expression of CD73, CD90, CD105, or multipotency (as measured by osteogenic, adipogenic, and chondrogenic differentiation) between DPSCs in FBS and DPSCs in HS. Conclusions: These findings show a clinically feasible method of storing third molars for the isolation of DPSCs. Additionally, DPSCs can be isolated and expanded to clinical scale numbers in media devoid of FBS and still maintain their phenotypic properties. (J Endod 2014;40:652–657)
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n the context of regenerative therapies, dental stem cells hold great promise for cell therapy approaches in dentistry; studies have already shown encouraging results in their ability to regenerate bone and periodontal tissues (1–5). Specifically, the identification of dental pulp stem cells (DPSCs) provides promise for regenerating lost dental pulp and tooth tissues because of their ability to differentiate into dentin and pulplike tissue (6–12). Current methodologies for tooth storage, cell isolation, and expansion of DPSCs have limitations regarding their use therapeutically (13). First, if DPSCs are to be used clinically, a feasible way to store teeth after extraction needs to be established. Second, most current protocols for the expansion of these cells incorporate animal serum (14, 15). Again, from a therapeutic standpoint, the use of animal serum should be reduced or eliminated from clinical cell therapy protocols in order for these approaches to meet safety and regulatory guidelines. There have been a number of cryopreservation protocols evaluated for DPSC storage after their isolation (8, 16–19). Yet, one of the practical challenges for cell therapies using DPSCs is the potential degradation of the pulp tissue between the time of tooth extraction and DPSC isolation and/or cryopreservation. It has been shown that these cells can remain viable for up to 5 days after extraction (20). Although these are important findings, the storage of teeth and subsequent isolation and the storage of dental stem cells are not very practical in a standard dental practice setting because of the lack of available reagents and solutions (ie, tissue culture medium) needed for storage. The feasibility of storage and expansion of these cells would be enhanced if there was a readily available storage solution into which clinicians could place the teeth until DPSC isolation could occur. There are currently companies attempting to address this issue with proprietary tooth storage solutions for DPSC ‘‘banking’’ (www.store-a-tooth.com). Nonetheless, further studies are clearly needed to examine the feasibility and safety of dental stem cell banking for therapeutic indications. Efficient, generalizable, user-friendly, predictable, and safe processes need to be developed in order to store teeth extracted in the dental office. Thus, identifying an optimum storage medium, determining the length of time cells remain viable in this medium, and establishing standardized cell isolation and expansion protocols are of great importance. The hypothesis underlying the proposed study is that DPSCs can be isolated from extracted teeth that have been stored in a medium devoid of animal serum. Additionally, after DPSC isolation, these cells can be expanded to clinical scale numbers without ever being exposed to animal serum; media containing human serum (HS) is used instead.
Key Words Cell therapy, dental pulp, dental pulp stem cells, human serum, regeneration, saline, tooth storage From the *Department of Periodontics and Oral Medicine, University of Michigan, Ann Arbor, Michigan; and †Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan. Address requests for reprints to Dr Darnell Kaigler, Department of Periodontics and Oral Medicine, University of Michigan, 1011 North University Avenue, Ann Arbor, MI 48109. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.005
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Methods Tooth Storage and DPSC Isolation Thirty-two third molars were obtained from tooth extractions from patients ranging from ages 15–22 years. Teeth were placed in either a sterile saline solution (n = 25) or alpha minimum essential medium (a-MEM) containing 15% fetal bovine serum (FBS) (n = 7). From these teeth, DPSCs were isolated immediately (n = 16) or stored for 24 hours at 4 C before being isolated (n = 9). The isolation of DPSCs was performed as previously described (7). Briefly, the crown of the tooth was cut just above the cementoenamel junction to open up the contents of the pulp. The pulp cells in the chamber and canals were cleaned out using various instruments, avoiding nerve tissue, and placed in Iscove modified Dulbecco medium (IMDM) without serum. After isolation, the cell suspension was placed in a conical tube and centrifuged at 1600 rpm for 5 minutes at room temperature. The supernatant was
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Basic Research—Biology aspirated, and the pellet was resuspended in a Dispase II–Collagenase solution. The solution was placed at 37 C for 60 minutes, inverting the tube at 15-minute intervals. IMDM was added to the cells before the suspension was centrifuged at room temperature for 5 minutes at 1600 rpm. The cell pellet was resuspended in IMDM without serum. This cell suspension was placed in a T25 tissue culture flask in 1 of the following 3 conditions: containing 15% FBS + a-MEM (n = 30), containing 15% human serum (HS) + a-MEM (n = 20), or containing 15% HS + a-MEM, which had a fibronectin (FN) coating on the bottom of the flask (n = 17).
Cell Proliferation Population Doubling Time Cells counts were performed at each passage, and the population doubling time (PDT) was calculated and compared between conditions. In order to determine PDT, the following calculation was from P0 to P1 Þðlog 2Þ , where P0 is the number of cells at the used: ð# days ðlog P1 logP0 Þ initial passage and P1 is the number of cells at the next passage. Cell Surface Marker Expression Flow cytometry to determine the expression levels of the cell surface markers CD90, CD73, and CD105 was performed. DPSCs were harvested from T150 flasks, washed, and aliquoted equally into tubes. Cells were first incubated with a blocking solution containing CD16/CD32 at 4 C for 10 minutes followed by washing. Cells were then incubated with the specific antibodies conjugated with fluorochromes (Biolegends, San Diego, CA) at 4 C for 30 minutes.
After washing, these cells were analyzed on a Beckman Coulter MoFlo flow cytometer.
Differentiation Staining For multipotent potential, osteogenic, adipogenic, and chondrogenic pathways were evaluated using von Kossa, alcian blue, and oil red O stains. Cells were plated in 12-well plates at 30,000 cells/well and cultured for 4 weeks with the appropriate media being replaced every 2–3 days. Cells were fixed to the plates using 10% formalin for osteogenic and adipogenic induction and cold methanol for chondrogenic induction. For von Kossa staining, 5% silver nitrate was added to each well and incubated in the dark for 30 minutes at room temperature. After this incubation period, wells were washed and then exposed to ultraviolet light for 30 minutes. Sodium thiosulfate (1%) was added to each well and allowed to sit for 3–4 minutes at room temperature. Oil red O stain and alcian blue stain were added to the appropriate wells and allowed to incubate at room temperature for 30 minutes. To the wells with alcian blue stain, after the incubation period, the stain was removed, and 0.1 N hydrochloric acid was used to wash the wells. Staining of the cells was recorded by photography. Statistical Analysis Statistical analysis was performed with the use of Instat software (GraphPad Software, San Diego, CA). All data were reported as mean standard deviation unless otherwise noted. Statistically significant differences were determined by 2-tailed Student t tests, and statistical significance was defined as P < .05.
Figure 1. The effect of tooth storage conditions on the ability to isolate DPSCs. (A) The percentage of teeth that yielded DPSCs was compared between teeth stored in media containing FBS (n = 7) versus those stored in sterile saline (n = 25) before DPSC isolation. (B) The number of days it took to yield 1 million cells was compared between teeth stored in media containing FBS versus those stored in sterile saline. (C) The percentage of teeth that yielded DPSCs was compared between teeth stored in saline overnight at 4 C (n = 9) versus those in which DPSCs were isolated immediately after tooth extraction (n = 16). (D) The number of days it took to yield 1 million cells was compared between teeth stored overnight in saline at 4 C versus those in which DPSCs were isolated immediately after tooth extraction.
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Basic Research—Biology Results Tooth Storage for DPSC Isolation Thirty-two third molar teeth were collected from 14 patients for the isolation of DPSCs. Before isolation, teeth were stored in a-MEM media containing FBS or in sterile saline to determine if saline was a sufficient storage solution for extracted teeth before the isolation of DPSCs. After placement of the teeth in the respective storage solution, DPSCs were immediately isolated. Regardless of the storage medium used, there was no difference in the ability to successfully isolate DPSCs (Fig. 1A). Additionally, the number of days from the initial isolation of DPSCs to the first passage of cells (approximately 1 million cells) was evaluated between teeth stored in the different solutions, and there was no difference between the 2 (Fig. 1B). As such, for the remainder of the DPSC isolations, sterile saline was used as the storage medium before isolation. We next wanted to determine if DPSCs could be isolated from extracted teeth after storage for 24 hours at 4 C. Thus, the ability to isolate DPSCs immediately (within 1 hour) after extraction was compared with the ability to isolate DPSCs from teeth stored in saline for 24 hours at 4 C. It was determined that DPSC isolation was not affected by the length of time (1 vs 24 hours) of tooth storage before DPSC isolation (Fig. 1C). In evaluating the numbers of days to the first passage of cells between the 2 different storage times, it was also determined that regardless of the storage time, it took between 14 and 16 days to reach the first passage of cells (approximately 1 million cells) (Fig. 1D). After finding that neither the storage medium (saline vs FBS) nor the time of storage (1 vs 24 hours) affected the ability to isolate and expand DPSCs, for all additional experiments, all teeth were stored at 4 C for 24 hours in sterile saline before DPSC isolation. DPSC Isolation in Human Serum versus FBS After determining that sterile saline was sufficient tooth storage medium for DPSC isolation, we next wanted to determine if DPSCs could be isolated without using FBS in the cell culture media. DPSCs from the
same tooth were isolated in a-MEM supplemented with either 15% FBS or 15% HS. Cells isolated in HS did not appear to gain good initial adherence to tissue culture flasks relative to those in FBS. To promote initial cell attachment, FN was coated on the tissue culture flasks before isolation. With this coating, there was a significant increase in the ability to successfully isolate DPSCs in HS (Fig. 2A). The morphology of the cells in HS appeared no different than those cultured in FBS (Fig. 2B), presenting with a characteristic fibroblastlike, spindle appearance. It should also be noted that if DPSCs were successfully isolated, they did not require FN-coated plates for additional expansion, and, thus, this substrate was only used for initial isolation and not beyond the initial passage. Additionally, in this media, it took the same length of time from the initial isolation to reach 1 million cells (Fig. 2C). After subculture and continual cell expansion, the PDTs for these were calculated and showed a significantly lower PDT for the DPSCs isolated and expanded in HS (1.59 0.46 days) relative to those in FBS (2.84 2.5 days) (Fig. 2D). Additionally, after cell expansion, DPSCs were frozen at 80 C for >6 months in HS and upon thawing were not different in their proliferation rate relative to cells expanded and thawed in FBS (data not shown).
Multipotency of DPSCs in HS Cell surface marker expression of CD73, CD90, CD105, and CD45 were evaluated for DPSCs because these markers are all considered important determinants in defining ‘‘stemness’’ associated with mesenchymal stem cells (21). After isolation and cell expansion (up to 5 passages or 95%), CD90 (>98%), and CD105 (>85%) (Fig. 3A and B). Additionally, between the conditions, there were no differences noted in the expression of these cell surface markers regardless of the serum source or isolation condition (with or without FN) (Fig. 3C).
Figure 2. DPSC isolation and expansion without animal serum. (A) The ability to isolate DPSCs in media with either animal serum (FBS, n = 30), HS (n = 20), or HS plus FN (n = 17) as a surface coating was evaluated. (B) Photomicrographs of DPSCs cultured in media containing FBS or HS. (C) The number of days it took to yield 1 million cells was compared between DPSCs expanded in FBS versus those expanded in HS. (D) Cell proliferation, as measured by the PDT, between DPSCs expanded in FBS versus those expanded in HS. *P < .05 relative to FBS condition.
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Figure 3. Stem cell surface marker expression of passage 5 DPSCs. DPSC populations expanded in media containing (A) FBS or (B) HS consistently yielded high proportions of cells that were positive for the mesenchymal stem cell markers CD73, CD90, and CD105. (C) There was no difference in the cell surface marker expression of CD73, CD90, or CD105 between DPSCs from the same tooth expanded in media containing FBS (n = 3) or HS (n = 3).
The ‘‘stemness’’ of DPSCs was also assessed through their capacity to differentiate toward different cellular lineages after culture under adipogenic, chondrogenic, and osteogenic conditions. After 3 weeks of culture, commitment toward different tissue lineages was evaluated through lineage-specific staining of induced cells. DPSCs were stained with oil red O, alcian blue, and von Kossa. In the uninduced control culture conditions, oil red O, alcian blue, and von Kossa staining were all negative. In adipogenic conditions, oil red O staining was used to detect intracellular lipid-rich vacuoles and morphologic changes in cell shape. The results confirmed that cells were differentiated toward an adipogenic lineage (Fig. 4A). Similarly, in cells grown under chondrogenic conditions, the presence of chondrogenic proteoglycans was indicated by positive alcian blue staining (Fig. 4A), confirming chondrogenic differentiation. Under osteogenic culture conditions, deposition of mineralized matrix indicative of osteoblasts was evident through positive von Kossa staining (Fig. 4A). There was no qualitative difference between cells isolated and expanded in the presence of FBS versus those that were expanded in HS (Fig. 4B).
Discussion Cell therapy is a promising approach for the regeneration of dental pulp–derived tooth tissues. Yet, in order for this approach to become JOE — Volume 40, Number 5, May 2014
clinically feasible, practical methodologies for tooth storage and cell isolation are in need of development. In this study, we determined that the overnight storage of extracted third molars in saline at 4 C was a sufficient condition to maintain teeth for the isolation and expansion of DPSCs. Additionally, DPSCs could be isolated and expanded using HS, instead of animal serum, without changes in proliferation, cell morphology, expression of stem cell markers, or multipotency. Finally, it was also determined that in the absence of animal serum, the use of FN as a surface coating was important for the initial establishment of cell cultures at the time of DPSC isolation. To our knowledge, this is the first study showing both the clinical feasibility of tooth storage for DPSC isolation and the expansion of DPSCs under conditions completely devoid of animal serum. There have been recent reports describing different storage and cryopreservation protocols for teeth after their extraction to be used for the isolation of DPSCs (8, 20, 22). Perry et al (8) conducted a time course study evaluating extracted third molars in 3 different storage solutions for up to 5 days to evaluate if viable DPSCs could be isolated and expanded after storage. There was a time-dependent reduction in the number of DPSCs that could be isolated from extracted teeth as the length of time of storage increased; yet, phosphate-buffered saline was shown to be a sufficient media for storage. Because most
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Figure 4. Multipotency of DPSC populations is shown through phenotypic expression of lineage specific markers. Photographs and corresponding photomicrographs of DPSCs from the same tooth expanded in (A) FBS or (B) HS show multipotent differentiation as measured by phenotypic expression of osteogenic mineralized matrix (von Kossa), chondrogenic proteoglycans (alcian blue), and adipogenic lipid vacuoles (oil red O). Low-magnification images were taken at 40; high-magnification images are shown at 200 (3 independent experiments were performed).
clinicians performing third molar extractions would not readily have phosphate-buffered saline available, we evaluated sterile saline as an overnight storage media at 4 C. Our study produced similar findings to the findings of Perry et al in showing that these conditions were sufficient for the maintenance of viable DPSCs for isolation. Another important finding of our study was that DPSCs were isolated in media devoid of animal serum; human serum was used instead. Animal serum is a rich source of cell adhesion molecules, namely FN, and these molecules play important roles in promoting cell attachment and proliferation (23–29). In our DPSC isolations, the additional step of providing an FN coating upon which the cells could attach made a significant difference in the isolation success rate of DPSCs. After the establishment of an initial cell culture of DPSCs and through subsequent subculturing and cell expansion, FN was not needed for the promotion of cell attachment and proliferation. In fact, DPSCs cultured in human serum proliferated faster than those cultured in FBS from the same tooth. Recently, 2 other reports have evaluated the expansion of stem cells from exfoliated deciduous teeth (30) and DPSCs (31) using human serum as a replacement for animal serum. In addition to using stem cells from exfoliated deciduous teeth instead of DPSCs, a major difference in our study and that of Ferro et al (30, 32) was that they supplemented the growth media with not only human serum but also exogenous growth factors including platelet-derived growth factor, fibroblast growth factor, insulinlike growth factor, and 656
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epidermal growth factor. Pisciotta et al (31) showed that HS promoted the expansion of DPSCs and osteogenic differentiation of these cells; yet, these cell populations were sorted before cell expansion for c-Kit, CD34, and STRO-1. Neither of these studies evaluated the storage conditions of the teeth before DPSC isolation. In contrast, our study evaluated the feasibility of tooth storage as well as the isolation and expansion of DPSCs without any exposure to animal serum. Additionally, clinical scale numbers of DPSCs could be predictably achieved in that up to 50 million cells could be generated within 3 weeks of the initial tooth isolation. In the context of therapeutic feasibility, another important consideration is the preservation of expanded cells. Cryopreservation protocols have been examined for ‘‘banking’’ of various mesencyhmal stem cells from different tissues normally discarded after removal, including the placenta (33), amniotic fluid (34), adipose tissue (35), and teeth (2, 8). However, these freezing protocols typically incorporate animal serum, and, thus, we evaluated HS as not only a cell expansion substitute for animal serum but also a component of the freezing media. After >6 months of cryopreservation of expanded DPSCs in 20% human serum, 10% dimethyl sulfoxide, and basal media, DPSCs maintained viability and could be continually expanded up to 12 passages without undergoing significant cell senescence (data not shown). Evaluations of longer freezing periods and the use of alternatives to potentially cytotoxic cryopreservants such as dimethyl sulfoxide will have important therapeutic implications and are currently under investigation. In this study, we have determined that at 4 C overnight storage of teeth in saline is a viable option for maintaining teeth for DPSC isolation. Additionally, DPSCs can be isolated and significantly expanded without the incorporation of animal serum in the media but, instead, through using human serum. Additional studies are underway to further evaluate these cells and their dentin and pulp-tissue regenerative potential in vivo.
Acknowledgments Supported by a Career Award for Medical Scientists from the Burroughs Wellcome Fund (DK) and the University of Michigan School of Dentistry Pathways Program. The authors thank the University of Michigan Wisdom Tooth clinic for the provision of teeth and David Adams and Ann Marie Deslauriers at the University of Michigan BRCF Flow Cytometry Core. The authors deny any conflicts of interest related to this study.
References 1. Mao JJ, Giannobile WV, Helms JA, et al. Craniofacial tissue engineering by stem cells. J Dent Res 2006;85:966–79. 2. Papaccio G, Graziano A, d’Aquino R, et al. Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J Cell Physiol 2006;208:319–25. 3. Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364:149–55. 4. Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A 2003;100:5807–12. 5. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res 2009;88:792–806. 6. Hara K, Yamada Y, Nakamura S, et al. Potential characteristics of stem cells from human exfoliated deciduous teeth compared with bone marrow-derived mesenchymal stem cells for mineralized tissue-forming cell biology. J Endod 2011;37: 1647–52. 7. Gronthos S, Mankani M, Brahim J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–30. 8. Perry BC, Zhou D, Wu X, et al. Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use. Tissue Eng Part C Methods 2008;14:149–56.
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Basic Research—Biology 9. Sakai VT, Zhang Z, Dong Z, et al. SHED differentiate into functional odontoblasts and endothelium. J Dent Res 2010;89:791–6. 10. Osathanon T, Sawangmake C, Nowwarote N, et al. Neurogenic differentiation of human dental pulp stem cells using different induction protocols. Oral Dis 2013 Apr 17 [Epub ahead of print]. 11. Wang Y, Zhao Y, Jia W, et al. Preliminary study on dental pulp stem cell-mediated pulp regeneration in canine immature permanent teeth. J Endod 2013;39:195–201. 12. Shi S, Robey PG, Gronthos S. Comparison of human dental pulp and bone marrow stromal stem cells by cDNA microarray analysis. Bone 2001;29:532–9. 13. Tirino V, Paino F, d’Aquino R, et al. Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives. Stem Cell Rev 2011;7:608–15. 14. Hilkens P, Gervois P, Fanton Y, et al. Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells. Cell Tissue Res 2013;353:65–78. 15. Khanna-Jain R, Mannerstrom B, Vuorinen A, et al. Osteogenic differentiation of human dental pulp stem cells on beta-tricalcium phosphate/poly (l-lactic acid/caprolactone) three-dimensional scaffolds. J Tissue Eng 2012;3(1):2041731412467998 http://dx.doi.org/10.1177/2041731412467998. Epub 2012 Dec 2. 16. Ma L, Makino Y, Yamaza H, et al. Cryopreserved dental pulp tissues of exfoliated deciduous teeth is a feasible stem cell resource for regenerative medicine. PLoS One 2012;7:e51777. 17. Lizier NF, Kerkis A, Gomes CM, et al. Scaling-up of dental pulp stem cells isolated from multiple niches. PLoS One 2012;7:e39885. 18. Kodonas K, Gogos C, Papadimitriou S, et al. Experimental formation of dentin-like structure in the root canal implant model using cryopreserved swine dental pulp progenitor cells. J Endod 2012;38:913–9. 19. Umemura E, Yamada Y, Nakamura S, et al. Viable cryopreserving tissue-engineered cell-biomaterial for cell banking therapy in an effective cryoprotectant. Tissue Eng Part C Methods 2011;17:799–807. 20. Woods EJ, Perry BC, Hockema JJ, et al. Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology 2009;59:150–7. 21. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–7.
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22. Lee SY, Huang GW, Shiung JN, et al. Magnetic cryopreservation for dental pulp stem cells. Cells Tissues Organs 2012;196:23–33. 23. Mauney JR, Kirker-Head C, Abrahamson L, et al. Matrix-mediated retention of in vitro osteogenic differentiation potential and in vivo bone-forming capacity by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion. J Biomed Mater Res A 2006;79:464–75. 24. Steele JG, McFarland C, Dalton BA, et al. Attachment of human bone cells to tissue culture polystyrene and to unmodified polystyrene: the effect of surface chemistry upon initial cell attachment. J Biomater Sci Polym Ed 1993;5:245–7. 25. Freshney RI. Culture of Animal Cells. New York: Wiley-Liss Inc; 2000. 26. Price PJ, Gregory EA. Relationship between in vitro growth promotion and biophysical and biochemical properties of the serum supplement. In Vitro 1982;18: 576–84. 27. Shahdadfar A, Fronsdal K, Haug T, et al. In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem Cells 2005;23:1357–66. 28. Barnes D, Sato G. Methods for growth of cultured cells in serum-free medium. Anal Biochem 1980;102:255–70. 29. Barnes D, Sato G. Serum-free cell culture: a unifying approach. Cell 1980;22: 649–55. 30. Ferro F, Spelat R, Beltrami AP, et al. Isolation and characterization of human dental pulp derived stem cells by using media containing low human serum percentage as clinical grade substitutes for bovine serum. PLoS One 2012;7:e48945. 31. Pisciotta A, Riccio M, Carnevale G, et al. Human serum promotes osteogenic differentiation of human dental pulp stem cells in vitro and in vivo. PLoS One 2012;7: e50542. 32. Ferro F, Spelat R, Falini G, et al. Adipose tissue-derived stem cell in vitro differentiation in a three-dimensional dental bud structure. Am J Pathol 2011;178: 2299–310. 33. Chien CC, Yen BL, Lee FK, et al. In vitro differentiation of human placenta-derived multipotent cells into hepatocyte-like cells. Stem Cells 2006;24:1759–68. 34. Soncini M, Vertua E, Gibelli L, et al. Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med 2007;1:296–305. 35. Goh BC, Thirumala S, Kilroy G, et al. Cryopreservation characteristics of adiposederived stem cells: maintenance of differentiation potential and viability. J Tissue Eng Regen Med 2007;1:322–4.
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Tooth Storage, Dental Pulp Stem Cell Isolation, and Clinical Scale Expansion without Animal Serum Emily J. Eubanks,* Susan A. Tarle,* and Darnell Kaigler, DDS, MS, PhD*† Abstract Introduction: Dental pulp stem cells (DPSCs) have therapeutic potential for dentin and dental pulp regeneration. For regenerative approaches to gain clinical acceptance, protocols are needed to determine feasible ways to store teeth, isolate DPSCs, and expand them to clinical scale numbers. Methods: In this study, 32 third molars were obtained from patients and immediately placed in saline or tissue culture medium followed by overnight storage at 4 C or immediate isolation of DPSCs. Upon isolation, cells were expanded in medium containing either fetal bovine serum (FBS) or human serum (HS). Cell proliferation (population doubling time [PDT]), cell surface marker expression, and multipotency were compared between DPSCs in FBS and DPSCs in HS. Results: The time frame of storage and storage medium did not affect the ability to isolate DPSCs. However, using HS instead of FBS in the initial isolation of DPSCs significantly decreased (P < .01) the isolation success rate from 89% (FBS) to 23% (HS). Yet, incorporating fibronectin in the DPSC initial isolation (using HS) significantly (P < .01) increased the isolation success rate to 83%. Interestingly, it was found that the proliferation rate was significantly (P < .05) higher for DPSCs in HS (PDT = 1.59 0.46) than that for DPSCs in FBS (PDT = 2.84 2.5). Finally, there was no difference in the expression of CD73, CD90, CD105, or multipotency (as measured by osteogenic, adipogenic, and chondrogenic differentiation) between DPSCs in FBS and DPSCs in HS. Conclusions: These findings show a clinically feasible method of storing third molars for the isolation of DPSCs. Additionally, DPSCs can be isolated and expanded to clinical scale numbers in media devoid of FBS and still maintain their phenotypic properties. (J Endod 2014;40:652–657)
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n the context of regenerative therapies, dental stem cells hold great promise for cell therapy approaches in dentistry; studies have already shown encouraging results in their ability to regenerate bone and periodontal tissues (1–5). Specifically, the identification of dental pulp stem cells (DPSCs) provides promise for regenerating lost dental pulp and tooth tissues because of their ability to differentiate into dentin and pulplike tissue (6–12). Current methodologies for tooth storage, cell isolation, and expansion of DPSCs have limitations regarding their use therapeutically (13). First, if DPSCs are to be used clinically, a feasible way to store teeth after extraction needs to be established. Second, most current protocols for the expansion of these cells incorporate animal serum (14, 15). Again, from a therapeutic standpoint, the use of animal serum should be reduced or eliminated from clinical cell therapy protocols in order for these approaches to meet safety and regulatory guidelines. There have been a number of cryopreservation protocols evaluated for DPSC storage after their isolation (8, 16–19). Yet, one of the practical challenges for cell therapies using DPSCs is the potential degradation of the pulp tissue between the time of tooth extraction and DPSC isolation and/or cryopreservation. It has been shown that these cells can remain viable for up to 5 days after extraction (20). Although these are important findings, the storage of teeth and subsequent isolation and the storage of dental stem cells are not very practical in a standard dental practice setting because of the lack of available reagents and solutions (ie, tissue culture medium) needed for storage. The feasibility of storage and expansion of these cells would be enhanced if there was a readily available storage solution into which clinicians could place the teeth until DPSC isolation could occur. There are currently companies attempting to address this issue with proprietary tooth storage solutions for DPSC ‘‘banking’’ (www.store-a-tooth.com). Nonetheless, further studies are clearly needed to examine the feasibility and safety of dental stem cell banking for therapeutic indications. Efficient, generalizable, user-friendly, predictable, and safe processes need to be developed in order to store teeth extracted in the dental office. Thus, identifying an optimum storage medium, determining the length of time cells remain viable in this medium, and establishing standardized cell isolation and expansion protocols are of great importance. The hypothesis underlying the proposed study is that DPSCs can be isolated from extracted teeth that have been stored in a medium devoid of animal serum. Additionally, after DPSC isolation, these cells can be expanded to clinical scale numbers without ever being exposed to animal serum; media containing human serum (HS) is used instead.
Key Words Cell therapy, dental pulp, dental pulp stem cells, human serum, regeneration, saline, tooth storage From the *Department of Periodontics and Oral Medicine, University of Michigan, Ann Arbor, Michigan; and †Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan. Address requests for reprints to Dr Darnell Kaigler, Department of Periodontics and Oral Medicine, University of Michigan, 1011 North University Avenue, Ann Arbor, MI 48109. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2014 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.01.005
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Methods Tooth Storage and DPSC Isolation Thirty-two third molars were obtained from tooth extractions from patients ranging from ages 15–22 years. Teeth were placed in either a sterile saline solution (n = 25) or alpha minimum essential medium (a-MEM) containing 15% fetal bovine serum (FBS) (n = 7). From these teeth, DPSCs were isolated immediately (n = 16) or stored for 24 hours at 4 C before being isolated (n = 9). The isolation of DPSCs was performed as previously described (7). Briefly, the crown of the tooth was cut just above the cementoenamel junction to open up the contents of the pulp. The pulp cells in the chamber and canals were cleaned out using various instruments, avoiding nerve tissue, and placed in Iscove modified Dulbecco medium (IMDM) without serum. After isolation, the cell suspension was placed in a conical tube and centrifuged at 1600 rpm for 5 minutes at room temperature. The supernatant was
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Basic Research—Biology aspirated, and the pellet was resuspended in a Dispase II–Collagenase solution. The solution was placed at 37 C for 60 minutes, inverting the tube at 15-minute intervals. IMDM was added to the cells before the suspension was centrifuged at room temperature for 5 minutes at 1600 rpm. The cell pellet was resuspended in IMDM without serum. This cell suspension was placed in a T25 tissue culture flask in 1 of the following 3 conditions: containing 15% FBS + a-MEM (n = 30), containing 15% human serum (HS) + a-MEM (n = 20), or containing 15% HS + a-MEM, which had a fibronectin (FN) coating on the bottom of the flask (n = 17).
Cell Proliferation Population Doubling Time Cells counts were performed at each passage, and the population doubling time (PDT) was calculated and compared between conditions. In order to determine PDT, the following calculation was from P0 to P1 Þðlog 2Þ , where P0 is the number of cells at the used: ð# days ðlog P1 logP0 Þ initial passage and P1 is the number of cells at the next passage. Cell Surface Marker Expression Flow cytometry to determine the expression levels of the cell surface markers CD90, CD73, and CD105 was performed. DPSCs were harvested from T150 flasks, washed, and aliquoted equally into tubes. Cells were first incubated with a blocking solution containing CD16/CD32 at 4 C for 10 minutes followed by washing. Cells were then incubated with the specific antibodies conjugated with fluorochromes (Biolegends, San Diego, CA) at 4 C for 30 minutes.
After washing, these cells were analyzed on a Beckman Coulter MoFlo flow cytometer.
Differentiation Staining For multipotent potential, osteogenic, adipogenic, and chondrogenic pathways were evaluated using von Kossa, alcian blue, and oil red O stains. Cells were plated in 12-well plates at 30,000 cells/well and cultured for 4 weeks with the appropriate media being replaced every 2–3 days. Cells were fixed to the plates using 10% formalin for osteogenic and adipogenic induction and cold methanol for chondrogenic induction. For von Kossa staining, 5% silver nitrate was added to each well and incubated in the dark for 30 minutes at room temperature. After this incubation period, wells were washed and then exposed to ultraviolet light for 30 minutes. Sodium thiosulfate (1%) was added to each well and allowed to sit for 3–4 minutes at room temperature. Oil red O stain and alcian blue stain were added to the appropriate wells and allowed to incubate at room temperature for 30 minutes. To the wells with alcian blue stain, after the incubation period, the stain was removed, and 0.1 N hydrochloric acid was used to wash the wells. Staining of the cells was recorded by photography. Statistical Analysis Statistical analysis was performed with the use of Instat software (GraphPad Software, San Diego, CA). All data were reported as mean standard deviation unless otherwise noted. Statistically significant differences were determined by 2-tailed Student t tests, and statistical significance was defined as P < .05.
Figure 1. The effect of tooth storage conditions on the ability to isolate DPSCs. (A) The percentage of teeth that yielded DPSCs was compared between teeth stored in media containing FBS (n = 7) versus those stored in sterile saline (n = 25) before DPSC isolation. (B) The number of days it took to yield 1 million cells was compared between teeth stored in media containing FBS versus those stored in sterile saline. (C) The percentage of teeth that yielded DPSCs was compared between teeth stored in saline overnight at 4 C (n = 9) versus those in which DPSCs were isolated immediately after tooth extraction (n = 16). (D) The number of days it took to yield 1 million cells was compared between teeth stored overnight in saline at 4 C versus those in which DPSCs were isolated immediately after tooth extraction.
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Basic Research—Biology Results Tooth Storage for DPSC Isolation Thirty-two third molar teeth were collected from 14 patients for the isolation of DPSCs. Before isolation, teeth were stored in a-MEM media containing FBS or in sterile saline to determine if saline was a sufficient storage solution for extracted teeth before the isolation of DPSCs. After placement of the teeth in the respective storage solution, DPSCs were immediately isolated. Regardless of the storage medium used, there was no difference in the ability to successfully isolate DPSCs (Fig. 1A). Additionally, the number of days from the initial isolation of DPSCs to the first passage of cells (approximately 1 million cells) was evaluated between teeth stored in the different solutions, and there was no difference between the 2 (Fig. 1B). As such, for the remainder of the DPSC isolations, sterile saline was used as the storage medium before isolation. We next wanted to determine if DPSCs could be isolated from extracted teeth after storage for 24 hours at 4 C. Thus, the ability to isolate DPSCs immediately (within 1 hour) after extraction was compared with the ability to isolate DPSCs from teeth stored in saline for 24 hours at 4 C. It was determined that DPSC isolation was not affected by the length of time (1 vs 24 hours) of tooth storage before DPSC isolation (Fig. 1C). In evaluating the numbers of days to the first passage of cells between the 2 different storage times, it was also determined that regardless of the storage time, it took between 14 and 16 days to reach the first passage of cells (approximately 1 million cells) (Fig. 1D). After finding that neither the storage medium (saline vs FBS) nor the time of storage (1 vs 24 hours) affected the ability to isolate and expand DPSCs, for all additional experiments, all teeth were stored at 4 C for 24 hours in sterile saline before DPSC isolation. DPSC Isolation in Human Serum versus FBS After determining that sterile saline was sufficient tooth storage medium for DPSC isolation, we next wanted to determine if DPSCs could be isolated without using FBS in the cell culture media. DPSCs from the
same tooth were isolated in a-MEM supplemented with either 15% FBS or 15% HS. Cells isolated in HS did not appear to gain good initial adherence to tissue culture flasks relative to those in FBS. To promote initial cell attachment, FN was coated on the tissue culture flasks before isolation. With this coating, there was a significant increase in the ability to successfully isolate DPSCs in HS (Fig. 2A). The morphology of the cells in HS appeared no different than those cultured in FBS (Fig. 2B), presenting with a characteristic fibroblastlike, spindle appearance. It should also be noted that if DPSCs were successfully isolated, they did not require FN-coated plates for additional expansion, and, thus, this substrate was only used for initial isolation and not beyond the initial passage. Additionally, in this media, it took the same length of time from the initial isolation to reach 1 million cells (Fig. 2C). After subculture and continual cell expansion, the PDTs for these were calculated and showed a significantly lower PDT for the DPSCs isolated and expanded in HS (1.59 0.46 days) relative to those in FBS (2.84 2.5 days) (Fig. 2D). Additionally, after cell expansion, DPSCs were frozen at 80 C for >6 months in HS and upon thawing were not different in their proliferation rate relative to cells expanded and thawed in FBS (data not shown).
Multipotency of DPSCs in HS Cell surface marker expression of CD73, CD90, CD105, and CD45 were evaluated for DPSCs because these markers are all considered important determinants in defining ‘‘stemness’’ associated with mesenchymal stem cells (21). After isolation and cell expansion (up to 5 passages or 95%), CD90 (>98%), and CD105 (>85%) (Fig. 3A and B). Additionally, between the conditions, there were no differences noted in the expression of these cell surface markers regardless of the serum source or isolation condition (with or without FN) (Fig. 3C).
Figure 2. DPSC isolation and expansion without animal serum. (A) The ability to isolate DPSCs in media with either animal serum (FBS, n = 30), HS (n = 20), or HS plus FN (n = 17) as a surface coating was evaluated. (B) Photomicrographs of DPSCs cultured in media containing FBS or HS. (C) The number of days it took to yield 1 million cells was compared between DPSCs expanded in FBS versus those expanded in HS. (D) Cell proliferation, as measured by the PDT, between DPSCs expanded in FBS versus those expanded in HS. *P < .05 relative to FBS condition.
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Figure 3. Stem cell surface marker expression of passage 5 DPSCs. DPSC populations expanded in media containing (A) FBS or (B) HS consistently yielded high proportions of cells that were positive for the mesenchymal stem cell markers CD73, CD90, and CD105. (C) There was no difference in the cell surface marker expression of CD73, CD90, or CD105 between DPSCs from the same tooth expanded in media containing FBS (n = 3) or HS (n = 3).
The ‘‘stemness’’ of DPSCs was also assessed through their capacity to differentiate toward different cellular lineages after culture under adipogenic, chondrogenic, and osteogenic conditions. After 3 weeks of culture, commitment toward different tissue lineages was evaluated through lineage-specific staining of induced cells. DPSCs were stained with oil red O, alcian blue, and von Kossa. In the uninduced control culture conditions, oil red O, alcian blue, and von Kossa staining were all negative. In adipogenic conditions, oil red O staining was used to detect intracellular lipid-rich vacuoles and morphologic changes in cell shape. The results confirmed that cells were differentiated toward an adipogenic lineage (Fig. 4A). Similarly, in cells grown under chondrogenic conditions, the presence of chondrogenic proteoglycans was indicated by positive alcian blue staining (Fig. 4A), confirming chondrogenic differentiation. Under osteogenic culture conditions, deposition of mineralized matrix indicative of osteoblasts was evident through positive von Kossa staining (Fig. 4A). There was no qualitative difference between cells isolated and expanded in the presence of FBS versus those that were expanded in HS (Fig. 4B).
Discussion Cell therapy is a promising approach for the regeneration of dental pulp–derived tooth tissues. Yet, in order for this approach to become JOE — Volume 40, Number 5, May 2014
clinically feasible, practical methodologies for tooth storage and cell isolation are in need of development. In this study, we determined that the overnight storage of extracted third molars in saline at 4 C was a sufficient condition to maintain teeth for the isolation and expansion of DPSCs. Additionally, DPSCs could be isolated and expanded using HS, instead of animal serum, without changes in proliferation, cell morphology, expression of stem cell markers, or multipotency. Finally, it was also determined that in the absence of animal serum, the use of FN as a surface coating was important for the initial establishment of cell cultures at the time of DPSC isolation. To our knowledge, this is the first study showing both the clinical feasibility of tooth storage for DPSC isolation and the expansion of DPSCs under conditions completely devoid of animal serum. There have been recent reports describing different storage and cryopreservation protocols for teeth after their extraction to be used for the isolation of DPSCs (8, 20, 22). Perry et al (8) conducted a time course study evaluating extracted third molars in 3 different storage solutions for up to 5 days to evaluate if viable DPSCs could be isolated and expanded after storage. There was a time-dependent reduction in the number of DPSCs that could be isolated from extracted teeth as the length of time of storage increased; yet, phosphate-buffered saline was shown to be a sufficient media for storage. Because most
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Figure 4. Multipotency of DPSC populations is shown through phenotypic expression of lineage specific markers. Photographs and corresponding photomicrographs of DPSCs from the same tooth expanded in (A) FBS or (B) HS show multipotent differentiation as measured by phenotypic expression of osteogenic mineralized matrix (von Kossa), chondrogenic proteoglycans (alcian blue), and adipogenic lipid vacuoles (oil red O). Low-magnification images were taken at 40; high-magnification images are shown at 200 (3 independent experiments were performed).
clinicians performing third molar extractions would not readily have phosphate-buffered saline available, we evaluated sterile saline as an overnight storage media at 4 C. Our study produced similar findings to the findings of Perry et al in showing that these conditions were sufficient for the maintenance of viable DPSCs for isolation. Another important finding of our study was that DPSCs were isolated in media devoid of animal serum; human serum was used instead. Animal serum is a rich source of cell adhesion molecules, namely FN, and these molecules play important roles in promoting cell attachment and proliferation (23–29). In our DPSC isolations, the additional step of providing an FN coating upon which the cells could attach made a significant difference in the isolation success rate of DPSCs. After the establishment of an initial cell culture of DPSCs and through subsequent subculturing and cell expansion, FN was not needed for the promotion of cell attachment and proliferation. In fact, DPSCs cultured in human serum proliferated faster than those cultured in FBS from the same tooth. Recently, 2 other reports have evaluated the expansion of stem cells from exfoliated deciduous teeth (30) and DPSCs (31) using human serum as a replacement for animal serum. In addition to using stem cells from exfoliated deciduous teeth instead of DPSCs, a major difference in our study and that of Ferro et al (30, 32) was that they supplemented the growth media with not only human serum but also exogenous growth factors including platelet-derived growth factor, fibroblast growth factor, insulinlike growth factor, and 656
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epidermal growth factor. Pisciotta et al (31) showed that HS promoted the expansion of DPSCs and osteogenic differentiation of these cells; yet, these cell populations were sorted before cell expansion for c-Kit, CD34, and STRO-1. Neither of these studies evaluated the storage conditions of the teeth before DPSC isolation. In contrast, our study evaluated the feasibility of tooth storage as well as the isolation and expansion of DPSCs without any exposure to animal serum. Additionally, clinical scale numbers of DPSCs could be predictably achieved in that up to 50 million cells could be generated within 3 weeks of the initial tooth isolation. In the context of therapeutic feasibility, another important consideration is the preservation of expanded cells. Cryopreservation protocols have been examined for ‘‘banking’’ of various mesencyhmal stem cells from different tissues normally discarded after removal, including the placenta (33), amniotic fluid (34), adipose tissue (35), and teeth (2, 8). However, these freezing protocols typically incorporate animal serum, and, thus, we evaluated HS as not only a cell expansion substitute for animal serum but also a component of the freezing media. After >6 months of cryopreservation of expanded DPSCs in 20% human serum, 10% dimethyl sulfoxide, and basal media, DPSCs maintained viability and could be continually expanded up to 12 passages without undergoing significant cell senescence (data not shown). Evaluations of longer freezing periods and the use of alternatives to potentially cytotoxic cryopreservants such as dimethyl sulfoxide will have important therapeutic implications and are currently under investigation. In this study, we have determined that at 4 C overnight storage of teeth in saline is a viable option for maintaining teeth for DPSC isolation. Additionally, DPSCs can be isolated and significantly expanded without the incorporation of animal serum in the media but, instead, through using human serum. Additional studies are underway to further evaluate these cells and their dentin and pulp-tissue regenerative potential in vivo.
Acknowledgments Supported by a Career Award for Medical Scientists from the Burroughs Wellcome Fund (DK) and the University of Michigan School of Dentistry Pathways Program. The authors thank the University of Michigan Wisdom Tooth clinic for the provision of teeth and David Adams and Ann Marie Deslauriers at the University of Michigan BRCF Flow Cytometry Core. The authors deny any conflicts of interest related to this study.
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