Exosome production method
A manufacturing method, exosome technology, applied in the field of exosome manufacturing system from biological samples, can solve the problems of only preparation, low yield, insufficient precision of ultrafiltration, etc., and achieve the effect of high purity
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Embodiment 1
[0148] Mice and Cell Lines
[0149] Imported H-2K d DUC18 mice with restricted and mutated (m) ERK2136-144 peptide-specific TCR (Va10.1 / Ja 48 and Vβ8.3 / Dβ2.1 / Jβ2.6) genes were bred in the experimental animal facility of Mie University, And used at 8 to 10 weeks of age. Purchased Restructuring I-A from Charles River Corporation b Restricted and OVA 323-339 peptide-specific TCR genes in OT-ll mice. This experimental protocol was approved by the Animal Experimentation Ethics Review Committee of Mie University (permission number: 23-8). HEK293, HEK293.2sus, Jurkat E6.1 and Molt-4 were purchased from American Type Culture Collection. Bone marrow MSCs (Mesenchymal Stem Cells, mesenchymal stem cells) from adults were purchased from Lonza Company.
[0150] Preparation of culture supernatant
[0151] EV-depleted fetal calf serum (dFCS) and autologous plasma were prepared by ultracentrifugation (100.000×g, 18 hours). Use 293SFM11 medium (ThermoFisher Scientific Company) in 10...
Embodiment 2
[0178] 〔Human PBMC exosomes and mouse CD8 + Isolation and biological characterization of T cell exosomes)
[0179] Isolation of exosomes
[0180]According to the method described in Example 1, EVs were prepared from the culture supernatant of human PBMC cultured for 11 days. The obtained EV was concentrated by ultrafiltration through a MWCO 750kDa filter corresponding to a filter pore size of about 50 nm in the same manner as in Example 1, and then passed through anion exchange chromatography using 100 mL of DEAE-sepharose Fast Flow (GE Healthcare) to separate. Elution of EV from the DEAE-sepharose column was performed with a linear gradient (0.15M-0.8M) of NaCl, and all fractions eluted with 0.15M-0.5M were pooled. According to the method described in Example 1, CD8 obtained from DUCl8 mice was cultivated for 7 days. + EVs were prepared from the culture supernatant (4 L) of T cells. In the same manner as in Example 1, the obtained EV was concentrated by ultrafiltration...
Embodiment 3
[0197] (Bioactivity of human T cell (PBMC) exosomes)
[0198] cytotoxic activity
[0199] The culture supernatant of human PBMCs was prepared according to the method described in Example 1, and the exosomes of human T cells (PBMC) eluted by ion exchange column were prepared according to the method of the present invention (all the exosomes eluted with 0.15M~0.5M Nacl exosomes). Likewise, exosomes eluted from an ion-exchange column from human cancer cells (MKN45) were prepared. For comparison, according to Example 1, exosomes were separated by ultracentrifugation prepared from the culture supernatant of human PBMCs. Mix 2X10 separately 4 MKN45 cells and CAFs from lung cancer (cancer-associated fibroblasts; cancer stromal fibroblasts) were added to non-adherent 24-well plates (day 0). The next day (Day 1), spheroids mimicking the tumor microenvironment were formed. On day 1, the above three exosomes were added at 2 μg / well, and on day 10, it was observed whether the spher...
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