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Methodological Stumbles in the Culture of Stem Cells from the Dental Pulp: An In vitro Study
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Elena Padín Iruegas3 , 4
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Evelyn Quiroz Pavón5
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Xavier Andrés Villao-León6
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Byron Velásquez-Ron7
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Mario Pérez-Sayáns2 , 3 , 8
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Authors Info & Affiliations
Abstract
Background
Human dental pulp is a valuable source of multipotent stem cells with considerable regenerative cell potential. The protocol for isolating dental pulp stem cells involves extracting healthy teeth, dissecting the pulp, enzymatically digesting it with collagenase, and culturing the cells in a specialized medium. Cell growth is monitored using microscopy and staining to assess viability and contamination.
Objective
This study aimed to describe methodological complications in culturing dental pulp stem cells.
Material and Methods
A sample of eight healthy third molars were extracted: group 1 (n=4) included molars, group 2 (n=3) comprised partially erupted molars, and group 3 (n=1) included molars with pericoronitis. Extracted molars were dissected, and the pulp was enzymatically digested with collagenase placed in Dulbecco's modified Eagle's medium-low glucose culture medium, fetal bovine serum, porcine skin gelatin, reduced L-glutathione, penicillin-streptomycin, and amphotericin-B. Observation under inverted microscopy using a 40X lens and gram and trypan blue staining was performed.
Results
Undissolved particles were observed in the medium, possibly related to the addition of gelatin or L-glutathione at the start of the culture, negatively affecting cell growth and observation. In the initial days of the experiment, there were floating cells in groups 1 and 2, but no cells were found adhering to the container surfaces. In group 3, there was an absence of cells, and particles and undigested tissue remnants were observed. Gram staining revealed the presence of Gram-positive bacteria in groups 1 and 2, and trypan blue staining did not allow the observation of cells in the Neubauer chamber.
Conclusion
Common difficulties include issues related to medium manipulation, pH regulation, presence of undissolved particles, lack of cell adherence, bacterial contamination, and difficulty in cell reproduction. Therefore, standardization of protocols and careful selection of reagents used are necessary.
