The Utilization of Autologous Serum as a Substitute of Fetal Bovine Serum in the Expansion of Mesenchymal Stem Cells Derived from Menstrual Blood

Menstrual blood-derived mesenchymal stem cells (MB-MSCs) expressing CD44, CD90, and CD105 markers were recently introduced. These cells are a good source of stem cells for research and use in regenerative medicine due to uncomplicated collection without invasive surgical intervention or ethical issues. Cell culture is one of the most essential techniques in molecular cell biology, and the culture medium is the most critical component. Serum is one of the crucial components of the culture medium and a protective solution. One of the most common serums used in cell culture is fetal bovine serum (FBS). This serum is an unknown mixture and can contain unfavorable factors such as endotoxin, mycoplasma, viral contaminants, or prion proteins. Therefore, there is a need for a human substitute for FBS that can be utilized for clinical applications. In this study, a humanized protocol utilizing autologous serum (AS) instead of FBS is tested to investigate the expansion of MB-MSCs and exosomes released from these cells.

A menstrual blood sample was collected from the donor on the second day of menstruation. Autologous serum samples were also collected to prepare the culture medium. First, mesenchymal stem cells were extracted from menstrual blood and then cultured in an environment enriched with autologous serum at different concentrations of 10%, 15%, and 20%. The investigation was done on the cells obtained in the third passage. Cultured cells in autologous serum were analyzed regarding expansion and expression of mesenchymal surface markers (CD73 and CD105). An inverted microscope was used to study the expansion, and flow cytometry was used to investigate the expression of mesenchymal markers. Also, in the next step, the culture medium of cultured cells in autologous serum was collected for exosome isolation. Exosome isolation was done by a three-step combination method of sedimentation, size exclusion chromatography with CL-2B sepharose resin, and making it concentrated. Finally, the purified exosomes were analyzed regarding morphology, size, and expression of CD9, CD63, and CD81 markers. Transmission electron microscope (TEM), dynamic light scattering (DLS), and flow cytometry analysis were operated, respectively.

According to the observations, cell expansion was observed in all three concentrations of autologous serum, and the most favorable results were marked in the concentration of 15%. In addition, flow cytometry results indicated the expression of mesenchymal markers CD73 and CD105 in cells cultured with 15% autologous serum. Exosomes were isolated from the culture medium of mesenchymal stem cells cultured with autologous serum, and their characteristics were investigated using dynamic light scattering, transmission electron microscope, and flow cytometry techniques. The size of the exosomes ranged from 30-150 nm, and their morphology was cup-shaped. The expression of exosome markers such as CD9, CD63, and CD81 was also confirmed.

As a result, with the increase of using mesenchymal stem cells in regenerative medicine, it is imperative to ensure the safety of the process and materials used in this field. Therefore, autologous serum can be a suitable option for the culture of mesenchymal stem cells derived from menstrual blood.