Patent ID: 8377692

Claim:
A process for making activated human mononuclear phagocytic leukocytes suitable for human administration, the method comprising: (i) suspending monocytes isolated from a blood sample of a human individual in a medium suitable for cell culture; (ii) processing human skin segments obtained from the same individual by: (A) freezing said skin fragments, thawing and soaking them in a medium suitable for cell culture and containing antibiotics, (B) removing the epidermis from the skin fragments and cutting them into segments, yielding human dermis segments, C) sonicating the human dermis segments in a medium suitable for cell culture and containing antibiotics, yielding sonicated dermis segments, (D) soaking the sonicated dermis segments in fresh medium suitable for cell culture and containing antibiotics, and (E) rinsing the dermis segments with fresh medium suitable for cell culture; (iii) activating the isolated monocytes suspended in step (i) by coincubating them with the rinsed human dermis segments resulting from step (ii), thus obtaining an incubation mixture containing activated human mononuclear phagocytic leukocytes; (iv) removing the human dermis segments from the incubation mixture resulting from step (iii) and sedimenting the obtained activated human mononuclear phagocytic leukocytes by centrifugation; and (v) washing and resuspending in fresh medium suitable for cell culture, the activated human mononuclear phagocytic leukocytes resulting from step (iv), thus obtaining a culture of activated human mononuclear phagocytic leukocytes; wherein at least 80% of the activated human mononuclear phagocytic leukocytes are viable according to a Trypan Blue exclusion assay; wherein the suspension of activated human mononuclear phagocytic leukocytes comprises less than 200 Eu bacterial endotoxin per mL of suspension; wherein the activated human mononuclear phagocytic leukocytes in the top quartile of granularity contain at least 4 granules per cell; wherein at least 40% activated human mononuclear phagocytic leukocytes express CD14; and wherein the activated human mononuclear phagocytic leukocytes produce at least 17 pg interleukin-1β per 10 6 activated human mononuclear phagocytic leukocytes expressing CD14.