Preparation method of human adipose mesenchymal stem cell exosome for treating myocardial infarction

[0033] The purpose of this example is to isolate and culture human adipose-derived mesenchymal stem cells.

[0034] 1. Materials

[0035] The prepared body fat was discarded normal body fat after surgery (with signed informed consent), PBS, BSA, type IV collagenase, fetal bovine serum, CD29 + , CD44 + , CD90 + , CD34 - and CD45 - of monoclonal antibodies.

[0036] 2. Method

[0037] 2.1 Isolation of human adipose-derived mesenchymal stem cells

[0038] (1) Collect about 15 mL of adipose tissue under sterile conditions, and remove the connective tissue capsule and blood vessels on the surface of the adipose tissue;

[0039] (2) Quickly cut into paste, add 3 mL of exosome-free medium, which contains 0.06-0.16% type IV collagenase and 0.015-0.02% BSA at final concentration , 37℃, constant temperature, 120r / min, oscillating digestion for 30-35min, observe the digestion status of the tissue at any time, take it out and mix it every 5-8min during the period, after the digest...

[0058] The purpose of this example is to isolate and purify human adipose-derived mesenchymal stem cell exosomes, including the following steps:

[0059] 1. Material method

[0060] Prepare human fat (normal body fat discarded after surgery, signed informed consent), PBS, BSA, type IV collagenase, fetal bovine serum, CD9, CD63, CD81, α-actinin-4 and CD40 monoclonal antibodies and Dimethyloxalylglycine.

[0061] 2. Collection of human adipose-derived mesenchymal stem cell exosomes

[0062] (1) Collect human adipose-derived mesenchymal stem cells, inoculate them, and use exosome-free serum medium at 37°C, 5% CO 2 Start culturing under the conditions;

[0063] (2) After culturing for 12 hours, replace it with a serum medium containing 300 μmol / L dimethyloxalylglycine exosomes from human adipose-derived mesenchymal stem cells, continue culturing for 36 hours, and collect the cell culture medium;

[0064] (3) The cell culture solution obtained in step (2) was subjected to diffe...

[0080] The purpose of this example is to culture h-MSCs cell line.

[0081] 1. Materials

[0082] Prepare human fat (normal human fat discarded after surgery, signed informed consent), PBS, BSA, type IV collagenase, fetal bovine serum, CD29 + , CD44 + , CD90 + , CD34 - and CD45 - of monoclonal antibodies.

[0083] 2. Passage of h-MSCs

[0084] (1) Collect adipose tissue according to the method shown in Example 1, digest it with the digestion conditions of 0.1% type IV collagenase + 0.02% BSA group, and culture the cells according to the method described in Example 1 until 80% to 90% confluence , digested with 0.25% trypsin-0.02% EDTA, and centrifuged to obtain cell pellets;

[0085] (2) Resuspend the cell pellet in fresh exosome-free medium, passage according to 1:2~4, and culture to P10, each generation of cells is named P1-h-MSCs, P2-h-MSCs, P3-h-MSCs, respectively. h-MSCs, P4-h-MSCs, P5-h-MSCs, P6-h-MSCs, P7-h-MSCs, P8-h-MSCs, P9-h-MSCs and P10-h-MSCs;

[0086] (3) P...