Method for separating and culturing human umbilical cord mesenchymal stem cells

Abstract

The invention provides a method for separating and culturing human umbilical cord mesenchymal stem cells, which comprises the following steps: taking umbilical cord of fetus born after the normal period of gestation by caesarean section, flushing by PBS under aseptic condition, removing the bloodstain; using an aseptic scissor in an animal cell medium to cut the washed umbilical cord to tissue blocks, removing blood vessel and crushing, then fully mixing with the animal cell medium and oscillating, centrifuging, mixing the centrifuged deposition and a MesenCult-XF complete medium and oscillating, and uniformly planting in a cell culture dish, placing in an incubator with the temperature of 37 DEG C and 5% of CO2 for culturing for 5 days, sucking the tissue blocks out and discharging, refilling the MesenCult-XF complete medium, and replacing the medium every three days for continuously culturing to obtain the umbilical cord mesenchymal stem cells. According to the invention, the animal serum is used, the operation is simple, the repeatability is good, the security is high, and the obtained cell enables stabilization through multitime passage.

Description

technical field

[0001] The invention relates to a method for separating and culturing human umbilical cord mesenchymal stem cells, and belongs to the technical field of cell separation and culturing of biological products. Background technique

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from the mesoderm and ectoderm in the early stages of development, with the ability of self-renewal and multidirectional differentiation. MSCs currently studied are mainly derived from adult bone marrow and umbilical cord blood. For adult bone marrow-derived mesenchymal stem cells, because the number and proliferation and differentiation potential of adult bone marrow decrease with age, and the virus infection rate is high, and bone marrow collection from donors requires bone marrow aspiration, so its acquisition is subject to certain restrictions. In recent years, a large number of studies have shown that there are a large number of mesenchymal stem c...

Examples

Embodiment 1

[0027] Take about 15ml of the umbilical cord of the full-term cesarean section fetus, and wash it repeatedly with PBS under sterile conditions to remove the residual blood. In RPMI MEDIUM 1640 medium, use sterile scissors to cut it into a tissue piece about 1.0 cm in length, remove the blood vessel and cut it as much as possible, put the tissue piece in a 50ml centrifuge tube, mix thoroughly with RPMI MEDIUM 1640 medium, and then centrifuge. The speed of the centrifuge is 2000 rpm, centrifuge for 5 minutes, discard the supernatant, and repeat 3 times. After centrifugation, about 20ml of umbilical cord tissue fragments are obtained, and the centrifuged sediment is mixed with MesenCult-XFMediums complete medium and shaken to evenly plant on the cells Petri dish and place at 37 °C, 5% CO 2 in the incubator. On the 5th day of culture, all the tissue pieces were sucked out and discarded, and the culture medium was added, and adherent spindle-shaped or polygonal cells were observed...

Embodiment 2

[0029] Take about 30ml of the umbilical cord of the full-term cesarean section fetus, and wash it repeatedly with PBS under sterile conditions to remove the residual blood. Use sterile scissors in DMEM medium to cut tissue pieces about 1.5 cm in length, remove the blood vessels and cut them into pieces as much as possible. Put the tissue pieces in a 50ml centrifuge tube, mix and oscillate fully with DMEM medium, and then centrifuge. The centrifuge speed is Centrifuge at 1600 rpm for 10 minutes, discard the supernatant, and repeat 4 times to obtain about 30ml of umbilical cord tissue fragments. Mix and shake the centrifuged part with MesenCult-XF Mediums complete medium and plant it evenly in a cell culture dish, and place 37°C, 5% CO 2 in the incubator. On the 5th day of culture, all the tissue pieces were sucked out and discarded, and the culture medium was added, and adherent spindle-shaped or polygonal cells were observed under the microscope, and the culture medium was re...

Embodiment 3

[0031]Take about 15ml of the umbilical cord of the full-term cesarean section fetus, and wash it repeatedly with PBS under sterile conditions to remove the residual blood. In RPMI MEDIUM 1640 medium, use sterile scissors to cut it into a tissue piece about 1.5cm long, remove its blood vessels and use a tissue homogenizer to fully and thoroughly mince it, and place the tissue homogenate in a 50ml centrifuge tube after mincing Mix well with RPMI MEDIUM 1640 medium and then centrifuge at a speed of 2200 rpm, centrifuge for 10 minutes, discard the supernatant, and repeat twice to obtain about 40ml of umbilical cord tissue fragments. Mediums complete medium mixed and shaken evenly planted in T-75cm 2 culture flask and place at 37 °C, 5% CO 2 in the incubator. On the 5th day of culture, all the tissue pieces were sucked out and discarded, and the culture medium was added, and adherent spindle-shaped or polygonal cells were observed under the microscope, and the culture medium was ...

technical field

[0001] The invention relates to a method for separating and culturing human umbilical cord mesenchymal stem cells, and belongs to the technical field of cell separation and culturing of biological products. Background technique

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from the mesoderm and ectoderm in the early stages of development, with the ability of self-renewal and multidirectional differentiation. MSCs currently studied are mainly derived from adult bone marrow and umbilical cord blood. For adult bone marrow-derived mesenchymal stem cells, because the number and proliferation and differentiation potential of adult bone marrow decrease with age, and the virus infection rate is high, and bone marrow collection from donors requires bone marrow aspiration, so its acquisition is subject to certain restrictions. In recent years, a large number of studies have shown that there are a large number of mesenchymal stem c...

Examples

Embodiment 1

[0027] Take about 15ml of the umbilical cord of the full-term cesarean section fetus, and wash it repeatedly with PBS under sterile conditions to remove the residual blood. In RPMI MEDIUM 1640 medium, use sterile scissors to cut it into a tissue piece about 1.0 cm in length, remove the blood vessel and cut it as much as possible, put the tissue piece in a 50ml centrifuge tube, mix thoroughly with RPMI MEDIUM 1640 medium, and then centrifuge. The speed of the centrifuge is 2000 rpm, centrifuge for 5 minutes, discard the supernatant, and repeat 3 times. After centrifugation, about 20ml of umbilical cord tissue fragments are obtained, and the centrifuged sediment is mixed with MesenCult-XFMediums complete medium and shaken to evenly plant on the cells Petri dish and place at 37 °C, 5% CO 2 in the incubator. On the 5th day of culture, all the tissue pieces were sucked out and discarded, and the culture medium was added, and adherent spindle-shaped or polygonal cells were observed...

Embodiment 2

[0029] Take about 30ml of the umbilical cord of the full-term cesarean section fetus, and wash it repeatedly with PBS under sterile conditions to remove the residual blood. Use sterile scissors in DMEM medium to cut tissue pieces about 1.5 cm in length, remove the blood vessels and cut them into pieces as much as possible. Put the tissue pieces in a 50ml centrifuge tube, mix and oscillate fully with DMEM medium, and then centrifuge. The centrifuge speed is Centrifuge at 1600 rpm for 10 minutes, discard the supernatant, and repeat 4 times to obtain about 30ml of umbilical cord tissue fragments. Mix and shake the centrifuged part with MesenCult-XF Mediums complete medium and plant it evenly in a cell culture dish, and place 37°C, 5% CO 2 in the incubator. On the 5th day of culture, all the tissue pieces were sucked out and discarded, and the culture medium was added, and adherent spindle-shaped or polygonal cells were observed under the microscope, and the culture medium was re...

Embodiment 3

[0031]Take about 15ml of the umbilical cord of the full-term cesarean section fetus, and wash it repeatedly with PBS under sterile conditions to remove the residual blood. In RPMI MEDIUM 1640 medium, use sterile scissors to cut it into a tissue piece about 1.5cm long, remove its blood vessels and use a tissue homogenizer to fully and thoroughly mince it, and place the tissue homogenate in a 50ml centrifuge tube after mincing Mix well with RPMI MEDIUM 1640 medium and then centrifuge at a speed of 2200 rpm, centrifuge for 10 minutes, discard the supernatant, and repeat twice to obtain about 40ml of umbilical cord tissue fragments. Mediums complete medium mixed and shaken evenly planted in T-75cm 2 culture flask and place at 37 °C, 5% CO 2 in the incubator. On the 5th day of culture, all the tissue pieces were sucked out and discarded, and the culture medium was added, and adherent spindle-shaped or polygonal cells were observed under the microscope, and the culture medium was ...

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