A Rapid and Cost-Effective Protocol for Isolating Mesenchymal Stem Cells from the Human Amniotic Membrane

Regarding the role of mesenchymal stem cells (MSCs) in regenerative medicine, many studies have been conducted to isolate these cells from various sources. In this study, a method was developed which will use only one enzyme and in the shortest time MSCs will be isolated from the amniotic membranes and expanded.
The amniotic membrane (AM) was mechanically separated from the underlying membrane called chorionic. Then, the AM was sliced into tiny pieces and to isolate MSCs, it was digested only using collagens I instead of applying various enzymes. The isolated cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% fetal bovine serum. The expression profiles of several markers at isolated cells were determined by flow cytometry. To assess the differentiation potential, the isolated cells were cultured in osteogenic and adipogenic induction media.
The results indicated that cells isolated from the AM expressed markers of CD44, CD105 and CD166 mesenchymal cells, but did not express CD34 and CD45 hematopoietic markers. The osteoblastic differentiation of the isolated cells was proven by alizarin red and alkaline phosphatase staining methods, whereas the adipogenic differentiation of the isolated cells was proven by Oil Red-O staining.
The results of the study indicated that the isolated cells were of the MSCs family. Furthermore, it was demonstrated that MSCs can be obtained easily only by spending a short time and using one enzyme.