Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Human Cell, Feb 2017

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.

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Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Human Cell Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine Dai Murabayashi 0 1 Mai Mochizuki 0 1 Yuichi Tamaki 0 1 Taka Nakahara 0 1 0 Department of Developmental and Regenerative Dentistry, School of Life Dentistry at Tokyo, The Nippon Dental University , 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159 , Japan 1 & Taka Nakahara Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the Periodontal ligament cell; Serum-free culture; Hypoxia susceptibility; Mesenchymal stem; Cytotoxic - capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine. Introduction Mesenchymal stem cells (MSCs) comprise a multipotent cell population capable of extensive proliferation and differentiation into a variety of cell types including osteoblasts, adipocytes, chondrocytes, myoblasts, and neurons [ 1, 2 ]. MSCs can be efficiently isolated from several tissues including bone marrow, adipose tissue, and umbilical cord (Wharton’s jelly) [ 3–5 ]. In addition, teeth, which are usually discarded during common extraction procedures, represent a source of multiple types of dental tissue-derived MSCs [6]. We previously reported the successful isolation and characterization of four types of human dental MSCs derived from dental pulp, periodontal ligament (PDL), apical papilla, and dental follicle, which were all collected from extracted mature or immature wisdom teeth [ 7 ]. Notably, these dental MSCs exhibited greater proliferative ability than iliac bone marrow-derived MSCs along with similar multidifferentiation potential and gene/protein expression profiles. Furthermore, we and other researchers have reported that PDL-derived MSCs (PDLSCs) in particular are beneficial for stem cell therapy in preclinical trials with large animals [ 8–10 ]. In conventional in vitro MSC culture, supplementation of culture media with fetal bovine serum (FBS) is essential for facilitating various cell behaviors such as cell attachment to culture dishes, cell growth, and cytodifferentiation. However, the use of FBS involves potential risks such as xenogeneic immune response and pathogenic infection [ 11, 12 ]; accordingly, several FBS-free culture media for expanding MSCs have been developed for commercial use [ 13–15 ]. In addition, low oxygen tension in the cell culture environment has been shown to exert a positive effect on cell growth and proliferation, enabling enhanced numbers of MSCs to be obtained in vitro culture [ 16, 17 ]. Hypoxic culture conditions, therefore, represent a potential alternative method for substantially accelerating MSC expansion. The cellular phenotypes and behaviors of dental MSCs including PDLSCs, such as cell growth, gene/protein expression, cytodifferentiation potential, genomic stability, cytotoxic reactions, and in vivo tissue generation under serum-free and hypoxic culture conditions, remain poorly understood. Here, we aimed to characterize the stem cell properties of PDLSCs under serum-free and hypoxic culture conditions, and to establish practical cell culture methods for obtaining cells for clinical applications and cell-based therapies. Materials and methods (...truncated)


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Dai Murabayashi, Mai Mochizuki, Yuichi Tamaki, Taka Nakahara. Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine, Human Cell, 2017, pp. 169-180, Volume 30, Issue 3, DOI: 10.1007/s13577-017-0161-2