Extraction and Characterization of Dental Pulp Stem Cells from Rat Mandibles to Develop a Multi‐potential Cell Bank for Tissue Graft Technologies

Abstract

Establishment of well-characterized cell banks for use in both experimental and clinical settings has long been a challenge with primary multi-potent cells. Vital to this process is the initial selection of tissue for harvesting, as tissue types display varying differentiation capacity profiles. Additionally, the quantity of a tissue available and the required method for extraction will dictate the quality of the downstream cell populations. To examine the practicality of developing a primary cell bank that could be utilized for regenerative therapies, both as standalone and in combination with biomaterials, in multiple tissues, we determined dental pulp-derived stem cells (DPSCs) to be a promising candidate. As neural crest lineage cells, DPSCs should be capable of differentiating to mesoderm and ectodermal cell types, thereby offering a robust therapeutic reservoir. Mandibles were collected from euthanized rats at the completion of an IACUC approved study, soft tissue was removed from the bone structure, and samples were placed in 70% ethanol to prevent contamination during sample transfer to cell culture cabinet. The dental pulp canal was exposed using a scalpel blade on both ends, allowing for the pulp tissue to be extruded by placing pressure on one end of the canal. Whole tissue was then dissected and placed directly into T25 culture flasks. Cells were expanded in both α-MEM and DMEM/F12-based mediums to assess the impact of media on differentiation potential. Additionally, media was supplemented with 20% FBS to encourage population growth initially, which was reduced to 10% for subsequent passages. Characterization of α-MEM cultured cells using flow cytometry demonstrated expression of the key stem cell markers CD90 and CD73. CD29, an integrin associated with angiogenesis, was also positively expressed indicating that these cell populations may have undergone some degree of differentiation. This was supported by cell morphology that revealed rounded cell shapes distributed through-out that appeared to resemble endothelial-like cells as opposed to the spindled shape of stem cells. Comparative morphological observations of cultures grown in DMEM/F12 revealed that these population maintained spindled morphology exclusively and exhibited enhanced growth rates based on passaging intervals. Cryopreservation and subsequent thawing further showed that banked cells are viable and can be expanded. Given these data, DPSCs offer a means for developing stem cell aliquot banks that are multi-potent and readily expanded. Furthermore, the expansion of these cells should be carried out using DMEM/F12-based media in order to preserve stem characteristics. The methods employed avoid enzymatic degradation of cells during collection and allow for the generation of large culture population from relatively small quantities of tissue, thereby presenting a promising biological therapeutic agent for application in a wide variety of tissue injuries.