METHODS OF ISOLATING AND CULTIVATING HUMAN RETINAL PROGENITOR CELLS Russian patent published in 2023 - IPC A61K35/30 C12N5/73 A61P27/02 

FIELD: pharmaceutical industry; medical industry.

SUBSTANCE: group of inventions relates to a method of isolating primary retinal cells from a human sample and to a method of culturing isolated primary human retinal cells to obtain a population of non-immortalized human retinal progenitor cells. A method of isolating primary retinal cells from a human sample, being one or a pair of human eyeballs obtained from a human donor, wherein the said one or pair of human eyeballs have normal morphology including intact eyeball(s), clear cornea, normal shape, or any of these combination comprising the following: (a) isolating a retinal sample comprising a plurality of primary retinal cells from a human sample within 8 hours of collecting one or a pair of eyeballs from a human donor, wherein the human donor is a fetus with a gestational age of between 12 weeks and 28 weeks, (b) mechanically dissociating the plurality of primary retinal cells in the retinal sample isolated at step (a), without treating the plurality of primary retinal cells with protease, resulting in a separated cell suspension and cell aggregates, and (c) determining the viability, number and morphology of primary retinal cells obtained from a retinal sample, where there are at least 30×10⁶ viable primary retinal cells obtained. A method of culturing isolated primary human retinal cells to obtain a population of non-immortalized human retinal progenitor cells includes: (a) plating into one or more culture flasks or coated plates containing culture medium, in a first passage, a plurality of primary retinal cells obtained by the method described above, with obtaining a plurality of cultured retinal cells; (b) seeding one or more culture flasks or coated plates containing culture media in a second passage of a plurality of cultured retinal cells obtained in the first passage; (c) seeding one or more culture flasks or coated plates containing culture medium in the third passage of a plurality of cultured retinal cells obtained in the second passage; and (d) cryopreservation of multiple cultured retinal cells; wherein one or more coated culture flasks are inoculated at a density of 0.5×10⁴ cells per square centimeter (cm²) to 5×10⁶ cells per cm², the seeding density in the first passage is greater than the seeding density in the second passage, and the seeding density in the second passage is greater than the seeding density in the third passage, resulting in a population of non-immortalized human retinal progenitor cells, where the culture the medium includes enhanced DMEM/F12, KnockOut DEMEM/F12, UltraCULTURE, ReNcell or neurobasal medium, and wherein the culture medium is further supplemented with at least one of the following: N-2, B27, Stempro, EGF, bFGF, GlutaMAX I, L-glutamine or gentamicin.

EFFECT: above methods, containing a combination of an 8-hour restriction after collecting eyeballs and a purely mechanical dissociation step, can effectively increase the number of cells, reduce the risk of cell lysis, increase cell viability, and increase the final yield.

19 cl, 2 dwg, 6 ex