Contributions
Abstract: PB1549
Type: Publication Only
Session title: Hematopoiesis, stem cells and microenvironment
Background
Mesenchymal stromal cells (MSC) are one of the cell lines for potential use in the treatment of a number of hematological and non-hematological diseases. The basis of successful cultivation is the study of factors influencing the activity of culture's growth. It is known that apoptosis processes can affect the growth parameters of MSC. Apoptosis is programmed cell death resulting from the implementation of its genetic program in response to external signals. One of the universal methods for detecting apoptotic cells is the analysis of the lipid composition of their membrane. Phosphatidylserine molecules are found in large quantities on the surface of the cell membrane at the stage of apoptosis. It can be detected by flow cytometry. Currently, the method of studying programmed cell death by staining cells with annexin 5 conjugated with fluorescein isothiocyanate (AnxV-FITC) and vital DNA dyes (7-aminoactinomycin D (7-AAD) is widely used.
Aims
To assess the relationship between the growth activity of MSC cultures and the number of cells at the stage of apoptosis.
Methods
MSC were obtained from the donors bone marrow (n = 15), whose age ranged from 24 to 46 (median, 26.5) years. Cultivation of MSC was carried out on the surface of plastic flasks under conditions of 5% carbon dioxide at a temperature of 37 ° C. When confluent growth of cultures was achieved, the cells were detached from the plastic using a solution of trypsin. The growth activity of cell cultures was determined by the time the cells of primary seeding (PO) reached the confluent monolayer and the period of cell doubling at passage 1 (P1). Apoptotic cells were distinguished from necrotic cells by spontaneous binding to AnxV-FITC and lack of 7-AAD staining (AnxV-FITC+ 7-AAD-) by laser flow cytometry using a BD FACS Canto II analyzer.
Results
The relative number of MSC with the AnxV-FITC+ 7-AAD- characteristic in the primary culture ranged from 1.3 to 7.9 (median 2.3)% of the total number of MSC, P1 - from 1.7 to 6.0 (median 3.8)%. The period of reaching of confluent monolayer for the primary culture varied from 336 to 528 (median - 456) h, the time of cell doubling on P1 - from 53.4 to 63.3 (median - 58.9) h. A direct relationship between the number of AnxV-FITC+ 7 -AAD- MSC and the time the cells reached the confluent monolayer was found (Pearson's coefficient, r = 0.73). Also a direct correlation between the number of MSC with the characteristic AnxV-FITC+ 7-AAD- and the cell doubling time at P1 was determined (r = 0.83). With increasing of number cells with markers of apoptosis, the longer it takes to achieve confluent growth.
Conclusion
The number of apoptotic MSC spontaneously binding to AnxV-FITC and unstained with 7-AAD has a direct effect on the activity of culture growth.
Keyword(s): Annexin A5, Apoptosis, Mesenchymal cells
Abstract: PB1549
Type: Publication Only
Session title: Hematopoiesis, stem cells and microenvironment
Background
Mesenchymal stromal cells (MSC) are one of the cell lines for potential use in the treatment of a number of hematological and non-hematological diseases. The basis of successful cultivation is the study of factors influencing the activity of culture's growth. It is known that apoptosis processes can affect the growth parameters of MSC. Apoptosis is programmed cell death resulting from the implementation of its genetic program in response to external signals. One of the universal methods for detecting apoptotic cells is the analysis of the lipid composition of their membrane. Phosphatidylserine molecules are found in large quantities on the surface of the cell membrane at the stage of apoptosis. It can be detected by flow cytometry. Currently, the method of studying programmed cell death by staining cells with annexin 5 conjugated with fluorescein isothiocyanate (AnxV-FITC) and vital DNA dyes (7-aminoactinomycin D (7-AAD) is widely used.
Aims
To assess the relationship between the growth activity of MSC cultures and the number of cells at the stage of apoptosis.
Methods
MSC were obtained from the donors bone marrow (n = 15), whose age ranged from 24 to 46 (median, 26.5) years. Cultivation of MSC was carried out on the surface of plastic flasks under conditions of 5% carbon dioxide at a temperature of 37 ° C. When confluent growth of cultures was achieved, the cells were detached from the plastic using a solution of trypsin. The growth activity of cell cultures was determined by the time the cells of primary seeding (PO) reached the confluent monolayer and the period of cell doubling at passage 1 (P1). Apoptotic cells were distinguished from necrotic cells by spontaneous binding to AnxV-FITC and lack of 7-AAD staining (AnxV-FITC+ 7-AAD-) by laser flow cytometry using a BD FACS Canto II analyzer.
Results
The relative number of MSC with the AnxV-FITC+ 7-AAD- characteristic in the primary culture ranged from 1.3 to 7.9 (median 2.3)% of the total number of MSC, P1 - from 1.7 to 6.0 (median 3.8)%. The period of reaching of confluent monolayer for the primary culture varied from 336 to 528 (median - 456) h, the time of cell doubling on P1 - from 53.4 to 63.3 (median - 58.9) h. A direct relationship between the number of AnxV-FITC+ 7 -AAD- MSC and the time the cells reached the confluent monolayer was found (Pearson's coefficient, r = 0.73). Also a direct correlation between the number of MSC with the characteristic AnxV-FITC+ 7-AAD- and the cell doubling time at P1 was determined (r = 0.83). With increasing of number cells with markers of apoptosis, the longer it takes to achieve confluent growth.
Conclusion
The number of apoptotic MSC spontaneously binding to AnxV-FITC and unstained with 7-AAD has a direct effect on the activity of culture growth.
Keyword(s): Annexin A5, Apoptosis, Mesenchymal cells