Micromanipulation of culture niche permits long-term expansion of dental pulp stem cells—an economic and commercial angle Vijayendran Govindasamy & Veronica Sainik Ronald & Swapnil Totey & Salina Binti Din & Wan Mahadzir Bin Wan Mustafa & Satish Totey & Zubaidah Zakaria & Ramesh R. Bhonde Received: 31 March 2010 / Accepted: 16 June 2010 / Editor: J. Denry Sato # The Society for In Vitro Biology 2010 Abstract Stem cells isolated from dental pulp possess the capacity for self-renewal and the potential for multi-lineage differentiation. However, dental pulp stem cells have different characteristics in terms of their culture conditions. The success of stem cells culture is governed by its micro-environmental niche. Therefore, we studied the effects of culture niche on long-term expansion of dental pulp stem cells in terms of cell morphology, growth kinetics, senescence pattern, cell surface marker expression differentiation capacity, and seeding plating density of dental pulp stem cells in four different, widely used media composition Among the various basal media tested, α-minimum essential media and knock out- minimum essential media supplemented with 10% fetal bovine serum were found to be the most optimal media composition in preserving the phenotypic characteristics and differentiation potential for prolonged periods as compared with DMEM-F12 and DMEM-LG. Plating density has been shown to affect overall yield. As a conclusion, the adoption of an appropriate culture system significantly improved cell yield, thus enabling the attainment of sufficient yields for therapeutic applications economizing in terms of cost of production and minimizing seeding cell density for maximum yield. Keywords Dental pulp . Mesenchymal stromal cell . Optimum culture conditions . Differentiation Introduction Mesenchymal stromal cells (MSCs), with their ability for self-renewal and multi-lineage potential in regenerative medicine, are attracting much attention because of their therapeutic potential for the repair and replacement of tissues and organs that have lost their functions due to aging, disease, damage, and congenital defects (Langer and Vacanti 1999; Grinnemo et al. 2004; Atala 2005; Bonab et al. 2006). MSC was initially identified in bone marrow (Honczarenko et al. 2006) but later have also been isolated from several other tissues such as adipose tissue, perioste- um, tendon, synovial fluid, skin, amniotic fluid, umbilical cord, umbilical cord blood, brain, spleen, liver, kidney, lungs, muscle, thymus and pancreas, menstrual blood, and testes (Nakahara et al. 1991; Erices et al. 2000; De Bari et al. 2001; Toma et al. 2001; Salingcarnboriboon et al. 2003; Katz et al. 2005; Nakamizo et al. 2005; Kim et al. 2006; Baksh et al. 2007; De Coppi et al. 2007; Meng et al. 2007; Yamazaki et al. 2007). It is hypothesized that, in the adult, these cells are the reservoirs of reparative cells that are mobilized following injury and migrate to the wound site where, in cooperation with local cells, they participate in the repair response. Supporting their stem cell nature is the fact that single cell colonies of MSC express various markers indicating that they are capable of differentiating in V. Govindasamy : S. Totey : S. Totey : R. R. Bhonde (*) Stempeutics Research Malaysia, Sdn Bhd (773817-K) Lot 3-i-7, Enterprise 4, Technology Park Malaysia, Bukit Jalil, 57000 Kuala Lumpur, Malaysia e-mail: rrbhonde@stempeutics.com.my V. S. Ronald : Z. Zakaria (*) Cancer Research Center, Institute for Medical Research (IMR), Jalan Pahang, 50588 Kuala Lumpur, Malaysia e-mail: zubaidah@imr.gov.my S. Binti Din : W. M. B. W. Mustafa Department of Oral Surgery, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-010-9332-0