Patent RU-2732238-C2 (RU)
Title: Method of culturing stem cells
Inventor: GLADKOV Alexei
IPC: C12N5/0775
Filed: 2016-12-06
Granted: 2020-09-14
Abstract: FIELD: biotechnology.SUBSTANCE: invention relates to cultivation of mesenchymal stem cells using a bone marrow aspirate obtained from a donor. Method comprises a step of collecting 2–3 cmof first aspirate of bone marrow, introduction of 2–3 cmof first bone marrow aspirate into separation vessel, which is in vertical position, which is statically maintained at virtually the same temperature as the body temperature, for approximately 2 hours, to separate the first bone marrow aspirate in the separation vessel into 3 layers in a vertical position. That is followed by a bone marrow aspirate recovery stage for recovering a second bone marrow aspirate located in an intermediate layer between the first bone marrow aspirate, divided into 3 layers in separation vessel at bone marrow aspiration separation stage. That is followed by a stage of the first stem cell recovery for introducing a second bone marrow aspirate recovered at a bone marrow aspirate recovery stage and a culture medium, containing a solution of mineral salts, to which penicillin (approximately 100 units/ml), amphotericin (approximately 100 ng/ml), streptomycin (approximately 100 mcg/ml), L-glutamine (approximately 2–4 ml) and 20 % embryonal bovine serum and amino acids into a first culture vessel having a capacity of about 20–30 cmand a surface area of about 25–36 mm, statically holding the first culture vessel for 12–24 hours to attach the first mesenchymal stem cells to the lower surface of the first culture vessel and proliferation of the first mesenchymal stem cells, when the total surface area of the first mesenchymal stem cells in relation to the lower surface of the first culture vessel reached a first ratio by increasing the flattened shape of the first mesenchymal stem cells on the lower surface of the first culture vessel, extraction of the first mesenchymal stem cells from the first culture vessel. That is followed by a stem cell centrifugation step for administering the first mesenchymal stem cells recovered at the first stem cell recovery step into a separation vessel, placing the separation vessel into a centrifuge and centrifuging the first mesenchymal stem cells into the layers in a vertical position. That is followed by a second stem cell recovery step for isolating the second mesenchymal stem cells located in the lower layer, after dividing the first mesenchymal stem cells into layers in a separation vessel at the stem cell centrifugation stage, and a third stem cell recovery step for introducing the second mesenchymal stem cells, recovered at the second stem cell recovery step and the culture medium into a second culture vessel having a high capacity of about 40–60 cm, and larger area of lower surface, which is approximately 50–72 mm, than those of first culture vessel, statically holding the second culture vessel for 36–48 hours to attach the second mesenchymal stem cells to the lower surface of the second culture vessel and proliferation of the second mesenchymal stem cells, and, when total surface area of the second mesenchymal stem cells relative to the lower surface of the second culture vessel reached a second set ratio by enlarging the flat shape of the second mesenchymal stem cells on the area of the lower surface of the second culture vessel, extraction of the second mesenchymal stem cells from the second culture vessel.EFFECT: invention widens the range of equipment.7 cl, 17 dwg