Preparation method of human adipose mesenchymal stem cell exosome for treating myocardial infarction
A technology of mesenchymal stem cells and exosomes, applied in the field of preparation of exosomes from human adipose-derived mesenchymal stem cells, to achieve stable effects, enhance heart function, and reduce compensatory cardiac load
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[0032] Example 1
[0033] The object of this embodiment is to separate the culture of human fatty mesenchymal stem cells.
[0034] Material
[0035] Preparing human fat is normal human fat after surgery (signed informed consent), PBS, BSA, IV collagenase, fetal bovine serum, CD29 + CD44 + , CD90 + CD34 - And CD45 - Monoclonal antibody.
[0036] 2. Method
[0037] 2.1 Separation of human fat mesenchymal stem cells
[0038] (1) The adipose tissue is collected under sterile conditions, and the connective tissue envelope and blood vessels of the surface of the adipose tissue are removed.
[0039] (2) Quickly cut the untraped to the paste, add 3 ml of medium containing medium, containing a medium containing a final concentration of 0.06 to 0.16% IV type collagenase and 0.015 ~ 0.02% BSA At 37 ° C constant temperature 120R / min oscillation digestion 30 ~ 35min, observe the tissue digestion at any time, during which the mix is removed every 5 to 8 minutes, and then use 100 mesh and 20...
Example Embodiment
[0057] Example 2
[0058] The object of this embodiment is to separate and purify the human fatty mixed stem cells of human fatty mesenchymal stem cells, including the following steps:
[0059] Material method
[0060] Preparing human fat (normal human fat after surgery, signed an informed consent), PBS, BSA, IV collagenase, fetal bovine serum, CD9, CD63, monoclonal antibody of CD81, α-Actinin-4 and CD40 Dimethylene deargylglycine.
[0061] 2. Collect human fat mesenchymal stem cell exosomes
[0062] (1) Collect human fat mesenchymal stem cells, inoculate, serum medium with no external body at 37 ° C, 5% CO 2 Cultivate under conditions;
[0063] (2) After 12 h, the serum medium containing 300 μmol / L dimethylactylglycine-containing immobilized stem cell exosomeric body was exchanged, and 36 h was continued to collect cell culture fluid;
[0064] (3) The cell culture liquid obtained in step (2) was discarded at 4 ° C, i.e., at 1500 g of centrifugation for 10 min, then centrifuged ...
Example Embodiment
[0079] Example 3
[0080] The object of this embodiment is to cultivate H-MSCS cell lines.
[0081] Material
[0082] Prepare human fat (normal human fat after surgery, have signed informed consent), PBS, BSA, IV collagenase, fetal bovine serum, CD29 + CD44 + , CD90 + CD34 - And CD45 - Monoclonal antibody.
[0083] 2.H-MSCS pass
[0084] (1) After the method shown in Example 1 was collected, the cells were cultured in the method to 80% to 90% in accordance with the method described in Example 1 in Example 1. At 0.25% trunca-0.02% EDTA, the cell precipitate was obtained after centrifugation;
[0085] (2) Cell precipitation with fresh non-subcontinent medium retransmitted, in accordance with 1: 2 to 4 passage, cultured to P10, each generation of cells named P1-H-MSCS, P2-H-MSCS, P3- 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) collecting P2-h-MSCs, P5-h-MSCs, P8-h-MSCs and P10-h-MSCs, were digested into single cell suspensions w...
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