SECRETOME ACTIVITY ANALYSIS Russian patent published in 2023 - IPC G01N33/50 

FIELD: cell biology; medicine.

SUBSTANCE: present invention relates to cell biology and medicine, in particular to methods for determination of activity of supernatant of a mammalian cell culture for use in the treatment of an inflammatory condition. For implementation of the specified methods, first, SH-SY5Y cells containing an expression cassette containing an activating protein 1 (hereinafter – AP-1) promotor functionally connected to a nucleic acid molecule encoding reporter protein are incubated in a cultural medium containing or consisting of the specified supernatant, and promotor activity of AP-1 promotor is measured. Then, A549 cells containing an expression cassette containing NF-kB promotor functionally connected to a nucleic acid molecule encoding reporter protein are incubated in a cultural medium containing or consisting of the specified supernatant, and promotor activity of NF-kB promotor is measured. Next, A549, HEK293, Ha-Cat, and/or SH-SY5Y cells are incubated in a cultural medium containing or consisting of the specified supernatant, and amount of phosphorylated heat shock protein 27 (hereinafter – HSP27) released with eucaryotic cells into the cultural medium is measured. In this case, a decision is made that supernatant of the mammalian cell culture has a potential for use in the treatment of an inflammatory condition, if promotor activity of at least one of AP-1 and NF-kB promotors is at least 50% higher than promotor activity of AP-1 and NF-kB promotors, measured when eucaryotic cells are cultivated in a cultural medium not containing the specified supernatant, and if amount of HSP27 released into the cultural medium is at least 20% higher than amount of phosphorylated HSP27 released into the cultural medium, when eucaryotic cells are cultivated in a cultural medium not containing the specified supernatant.

EFFECT: present invention allows for expansion of the arsenal of methods for activity analysis for measuring biological activity and therapeutic efficiency of conditioned cultural media by means of monitoring of expression of specific proteins and/or regulation of specific promotors.

10 cl, 13 dwg, 1 tbl, 8 ex