Mesenchymal stem cells cultured in serum-free medium(HuMSC Medium-serum free)

Product Description

Mesenchymal stem cells cultured in serum-free mediumIt is a serum-free and 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 components

Transportation and storage

componentABlue ice transportation,4℃ storage; componentBDry ice transportation,-20Store below ℃. Validity period12 A month.

Usage

1.Preparation of culture medium

取500mLcomponentAJoin in25mL componentBMixing evenly isMesenchymal stem cells cultured in serum-free medium.Note:If necessary, users can prepare the required amount according to the ratio.

2. Isolation and culture of primary mesenchymal stem cells (MSCs)T75Taking cultivation bottles as an example

(1) Remove the umbilical cord, disinfect and clean the outer surface of the umbilical cord to remove blood stains, and cut it into approximately2cm ~ 4cmThe little oneBlock, remove arteries and veins, make side length5mm~10mmA small piece, placed inT75Arrange the culture medium bottles neatly,Spread each bottle15~20Block.Note:It is necessary to distinguish between the outer skin side of the umbilical cord and the Huatong adhesive side to ensureHuatong glue is attached to the bottom of the culture bottle.

(2) Flip the culture bottle over and place it inCO2In the incubator,37℃standing60 min.

(3) Remove the culture bottle and slowly add it to the bottle15mLMesenchymal stem cells cultured in serum-free mediumPut it in 37℃ The5%CO2 Cultivate in an incubator.

(4) 第 7Oh my god, everyT75Add fresh to the cultivation bottleMesenchymal stem cells cultured in serum-free medium5mL.

(5) After fluid replacement, every3Half a day of liquid exchange, suction out10mL Discard the culture medium and add it again10mLfreshofMesenchymal stem cells cultured in serum-free medium.

(6) 第13Starting from the day, observe the tissue blocks, such as tissue blocksThe fusion degree of surrounding cells has reached90%, AcceptableObtain cells, otherwise continue every time3Perform a semi liquid exchange until the fusion degree of cells around the tissue block is reached90%.

3. Passage culture and cryopreservation of mesenchymal stem cellsT75cultivateTaking bottle maintenance as an example

(1) Translate into EnglishMesenchymal stem cells cultured in serum-free mediumBalance to room temperature.

(2) Cleaning: Remove the cells, aspirate the culture medium from the culture bottle and discard itT75culture flaskuse10mL~15mLPBSMoisturize the cells once.

(3) Digestion: Add 3mlDigestive fluid, after infiltrating the entire cell growth surface, is placed CO2 In the incubator,37℃digestion 2min~6min, microscopic observation, approximately 80%When the above cells shrink and become round and fall off,Add twice the volumeMesenchymal stem cells cultured in serum-free mediumTerminate digestion, blow and beat to disperse cells intoSingle cell, perform cell counting.

Note:If most of the cells shrink and become round but do not fall off, gently tap the bottle wall toIt falls off. If cells do not shed, digestion time can be prolonged.

(4) Collect:300g，5minCollect cell sediment by centrifugation and discard the supernatant.

(5) subculture/Cryopreservation: If passaging is performed, useMesenchymal stem cells cultured in serum-free mediumResuspend the cells and inoculate them at an appropriate density (recommended)8000A cell/cm₂）Inoculate the cells into a mixture that has been equilibrated to room temperature20mLMesenchymal stem cells cultured in serum-free mediumof T75Cultivate the bottle and put it in 37℃The5%CO2 Continue to cultivate in the incubator.

If cells are frozen, follow the steps（4）Collect cell sediment and add an appropriate amount of cells according to the freezing densityGently blow and mix the resuspended cells with the frozen solution, transfer them to the cell freezing tube, and place the tube in the program for further freezingWarm boxIn the middle,-80℃Overnight?24hTransfer to liquid nitrogen for long-term storage.

4. Mesenchymal stem cell revival T75Taking cultivation bottles as an example

(1) Translate into EnglishMesenchymal stem cells cultured in serum-free mediumBalance to room temperatureAdd to each culture bottle15mL ofMesenchymal stem cells cultured in serum-free medium.

(2) Retrieve the frozen cells and insert them 37℃Quickly thaw in a water bath.

(3) Immediately absorb the thawed cell suspension and transfer it 1mlto 15ml centrifuge tubeAdd gradually, drop by drop 4mL Mesenchymal stem cells cultured in serum-free mediumAfter mixing, count.

(4) According to the counting results, follow the appropriate vaccination density (recommended) 8000 A cell/cm₂）Move the cellsVaccination to already joined 15mL Mesenchymal stem cells cultured in serum-free mediumIn the cell culture container.

Note:If the cell cryopreservation solution does not contain DMSO, cells can be directly added 20mL Mesenchymal stem cells cultured in serum-free mediumIn the middle, there is no need to change the next dayLiquid.

(5) Place the cells in 37℃ ,5% CO2 Cultivate in an incubator.

(6) 12h~24hAfterwards, wait for the cells to adhere to the wall normally, discard the culture supernatant, and addFresh, equilibrated to room temperature 20mlMesenchymal stem cells cultured in serum-free mediumAccording to the growth of cellsSituation, every 3Change the solution until the cell fusion degree reaches 80%~90%.

Precautions

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, thenThe cultivation conditions should be optimized.

3. This product does not contain phenol red, serum, or animal derived ingredients. If neededAdditional additions are possible.

4. To achieve the desired cell culture effect, this product can be used directlyAdditional cell growth factors or hormones can also be added based on cell type or research needs.

5. thisproductThe effectiveness of cell culture may vary depending on the cell source, storage conditions, and sampleQuality and operator experienceThere are differences in testing.

6. The product should be stored under specified storage conditions and used within its expiration date.

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