Method for separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells) from wharton jelly tissue of umbilical cord
A technology of Wharton's jelly tissue and umbilical cord, which is applied in the field of efficient and rapid separation and extraction of umbilical cord mesenchymal stem cells, which can solve the confusion of separation and amplification methods, complicated steps, and the difficulty of quickly extracting large quantities of high-purity hUC-MSCs, etc. question
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Embodiment 1
[0092] Example 1 Screening of composition of serum-free medium for mesenchymal stem cells
[0093] (1) Content screening of serum substitutes
[0094] Test medium: 0.1 parts by volume of β-mercaptoethanol, 10ng / ml of recombinant human basic fibroblast growth factor (b-FGF, Peprotech Company), 1 part by volume of non-essential amino acid aqueous solution (11140, Gibco Company), 1, 2, 5, 8, 10, 12, 15 or 20 parts by volume of Knockout FBS serum substitute (10828-028, Gibco Company), 89 parts by volume of a-MEM.
[0095] In the biosafety cabinet, the hUC-MSCs of the third generation isolated from the umbilical cord Huatong glue tissue of natural delivery newborns were collected and divided into 2×10 4 cells / cm 2 Inoculate at a density in a T75 cell culture flask, add 12-15ml of conventional commercially available medium to culture the cells. After culturing and observing that the cells have completely adhered to the wall, replace 15 mL of the test medium. Observe cell growt...
Embodiment 2
[0101] Example 2 Method for extraction of hUC-MSC by erythrocyte lysate assisted by collagenase
[0102] Collection and transportation of samples: Collect umbilical cord samples of spontaneously delivered newborns under aseptic conditions, put them into the umbilical cord preservation and transportation solution containing penicillin sodium, streptomycin sulfate, gentamicin and amphotericin B, (D-Hank's The concentration of penicillin sodium, streptomycin sulfate and gentamycin in the solution is 150U / ml; the concentration of amphotericin B is 300U / mL), transported to the clean cell room on ice within 6 hours;
[0103] Cleaning and disinfection of samples: In a biological safety cabinet, put fresh umbilical cord samples into a 50ml sterile centrifuge tube, wash them twice with 75% alcohol, and then wash them three times with sterile saline;
[0104] Pretreatment of umbilical cord tissue: Use ophthalmic scissors to cut the umbilical cord into small sections about 2-3cm in len...
Embodiment 3
[0111] Example 3 Method for extraction of hUC-MSC by erythrocyte lysate assisted by collagenase
[0112] The red blood cell lysate used contains 10g / L NH 4 Cl and 0.1 mM Na 2 - EDTA, pH 7.2-7.4.
[0113] Carried out with reference to the method of Example 2, the digestion solution containing collagenase type IV was digested for 12 hours, the primary cells were spread on 8 culture dishes, and the mesenchymal stem cells adhered to the wall until the second day, and the confluence rate was about 7 days up to 60%, trypsinized and subcultured; after the third passage, the cell purity is greater than 99.2%, and the viability is 99.7%.
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