Product Description
Mesenchymal stem cell serum-free culture medium is a serum-free, animal derived human umbilical cord mesenchymal stem cell culture product, mainly composed of amino acids, vitamins, inorganic salts, albumin, transferrin, insulin, trace elements, cytokines, etc.
This product can be used for primary culture and multiple passages of mesenchymal stem cells, while maintaining their potential for multi-directional differentiation, such as the ability to differentiate into bone cells, chondrocytes, and adipocytes, without the need for serum or serum substitutes.
Order Information
product name | Item number | specifications |
Mesenchymal stem cells cultured in serum-free medium | AC01L212 | 500mL + 25mL |
Product components
components | specifications | Storage temperature |
A. Mesenchymal stem cells cultured in serum-free basal medium | 500 mL | 4℃ |
B. Mesenchymal stem cells without serum additives | 25 mL | -20℃ |
Transportation and storage
Component A is transported on blue ice and stored at 4 ℃; Component B is transported by dry ice and stored below -20 ℃. Valid for 12 months.
usage method
1. Preparation of culture medium
Take 500mL of component A and add 25mL of component B. Mix well to obtain serum-free culture medium for mesenchymal stem cells. Note: If necessary, users can prepare the required amount according to the ratio.
2. Isolation and culture of primary mesenchymal stem cells (using T75 culture bottle as an example)
(1) Take out the umbilical cord, disinfect and clean the outer surface of the umbilical cord to remove blood stains, cut it into small pieces of about 2cm~4cm, remove the arteries and veins, and make small pieces with a side length of 5mm~10mm. Place them neatly in T75 culture medium bottles, and lay 15~20 pieces in each bottle. Note: It is necessary to distinguish between the outer skin side of the umbilical cord and the Huatong glue side, ensuring that the Huatong glue side is attached to the bottom of the culture bottle.
(2) Flip the culture bottle over and place it in a CO2 incubator. Let it stand at 37 ℃ for 60 minutes.
(3) Remove the culture bottle and slowly add 15mL of serum-free culture medium for mesenchymal stem cells to the bottle. Incubate in a 37 ℃, 5% CO2 incubator.
(4) On the 7th day, add 5mL of fresh serum-free culture medium for mesenchymal stem cells to each T75 culture bottle.
(5) After fluid replacement, perform a half change every 3 days, aspirate 10mL of culture medium and discard it, then add 10mL of fresh serum-free culture medium for mesenchymal stem cells.
(6) Starting from the 13th day, observe the tissue block. If the fusion degree of cells around the tissue block reaches 90%, the cells can be harvested. Otherwise, continue to exchange the medium every 3 days until the fusion degree of cells around the tissue block reaches 90%.
3. Passage culture and cryopreservation of mesenchymal stem cells (using T75 culture bottle as an example)
(1) Equilibrate the serum-free culture medium of mesenchymal stem cells to room temperature.
(2) Cleaning: Remove the cells, aspirate the culture medium from the culture bottle and discard it. Rinse the cells once with 10mL~15mL PBS in each T75 culture bottle.
(3) Digestion: Add 3ml of digestion solution to infiltrate the entire cell growth surface, then place it in a CO2 incubator and digest at 37 ℃ for 2-6 minutes. Observe under a microscope. When more than 80% of the cells shrink and become round and fall off, add twice the volume of serum-free culture medium for mesenchymal stem cells to terminate digestion, blow and disperse the cells into single cells, and perform cell counting.
Note: If most of the cells shrink and become round but do not fall off, gently tap the bottle wall to make them fall off. If cells do not shed, digestion time can be prolonged.
(4) Collection: 300g, centrifuge for 5 minutes to collect cell sediment, discard supernatant.
(5) Passage/Cryopreservation: If passage operation is performed, resuspend the cells in serum-free mesenchymal stem cell culture medium, and inoculate the cells into T75 culture bottles containing 20mL of serum-free mesenchymal stem cell culture medium that has been equilibrated to room temperature at an appropriate seeding density (recommended 8000 cells/cm2). Place the cells in a 37 ℃, 5% CO2 incubator for further cultivation.
If the cells are frozen, add an appropriate amount of cell freezing solution to the cell pellet collected in step (4) according to the freezing density, gently blow and resuspend the cells, mix well, and transfer them to a cell freezing tube. Place the tube in a programmed cooling box at -80 ℃ overnight, and transfer it to liquid nitrogen for long-term storage after 24 hours.
4. Mesenchymal stem cell recovery (using T75 culture bottle as an example)
(1) Equilibrate the serum-free culture medium of mesenchymal stem cells to room temperature, and add 15mL of serum-free culture medium to each culture bottle.
(2) Remove the frozen cells and quickly thaw them in a 37 ℃ water bath.
(3) Immediately transfer 1ml of thawed cell suspension to a 15ml centrifuge tube, add 4mL of serum-free mesenchymal stem cell culture medium dropwise, mix well, and count.
(4) According to the counting results, inoculate the cells into a cell culture container containing 15mL of serum-free mesenchymal stem cell culture medium at an appropriate seeding density (recommended 8000 cells/cm2).
Note: If the cell cryopreservation solution does not contain DMSO, the cells can be directly added to 20mL of serum-free mesenchymal stem cell culture medium without changing the solution the next day.
(5) Cultivate the cells in a 37 ℃, 5% CO2 incubator.
(6) After 12-24 hours, wait for the cells to adhere normally, discard the culture supernatant, and add 20ml of fresh, equilibrated room temperature serum-free mesenchymal stem cell culture medium. According to the growth of cells, change the medium every 3 days until the cell fusion degree reaches 80% to 90%.
matters needing attention
1. This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
2. This product does not contain antibiotics and it is not recommended to add antibiotics. If antibiotics must be added, the cultivation conditions should be optimized.
3. This product does not contain phenol red, serum, or animal derived ingredients. Additional ingredients can be added if necessary.
4. In order to achieve the desired cell culture effect, this product can be used directly or additional cell growth factors or hormones can be added according to the cell type or research needs.
5. The effect of cultivating cells with this product may vary due to factors such as cell source, storage conditions, sample quality, and operator experience.
6. The product should be stored under specified storage conditions and used within its validity period.
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This product is only for scientific experimental research and should not be used in clinical diagnosis, treatment, or other fields.
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