Introduction: Development of bioartificial substitutes of the human oral mucosa and palate is strictly dependent on the availability of human keratinocyte cells cultured from small tissue biopsies1. Although cell culture methods have been improved over the last years, epithelial cells typically show low proliferation rates and cell culture protocols should be improved for an efficient use in Tissue Engineering. A promising strategy is the use of mesenchymal stem cell (MSC)-derived secretome to increase cell culture. Secretomes contain high amounts of growth factors, cytokines, proteases, transcription factors and other molecules involved in cell adhesion, migration, proliferation, and differentiation that may improve epithelial cell culture2. In this work, we evaluated several types of secretomes obtained from different MSC sources to evaluate their effect on human keratinocyte cell proliferation.
Methodology: To generate primary cell cultures of human adipose tissue, dental pulp, and human umbilical cord, biopsies were obtained and enzymatically digested. Isolated cells were collected by centrifugation and cultured under standard cell culture conditions. Afterwards, the medium containing the products released by each type of MSC was harvested and centrifuged for 10 minutes to eliminate all cellular debris and apoptotic bodies, followed by filtering. Subsequently, human keratinocytes were cultured in the presence of each type of secretome at the concentration of 0%, 25%, 50%, 75% and 100% for 24, 48, 72 and 120 hours. Cell viability and cell proliferation were evaluated in each experimental condition.
Results: Our results revealed that more than 80% cell viability in all study groups, with higher survival at the longest exposure times. Regarding proliferation, results were comparable to controls at the shortest follow-up times, but a significant increase was found at 72 and 120h of incubation in the presence of the different secretomes, with higher results in the case of secretomes obtained from dental pulp and human umbilical cord MSC.
Conclusion: Our data support the idea that MSC-derived secretome is not toxic for epithelial cell culture, and cells cultured with this product were highly viable. In addition, its use seems to increase cell proliferation, especially after long exposition times. These results open de door to the future use of these types of secretomes to obtain efficient cell cultures of human keratinocytes for use in oral mucosa and palate tissue engineering.