Compositions containing amniotic components and methods for preparation and use thereof

A method for making a composition having components of amniotic fluid, the method comprising:

• (a) transferring amniotic epithelial cells (AECs) and amniotic fluid cells (AFCs) to a cell culture system and culturing the AECs and AFCs in a defined medium essentially free of serum consisting of a base media;
• wherein the base media is 50% Iscove's Modified Dulbecco's Media (IMDM) and 50% F12 media;
• monothioglycerol (450 μM), 1 mg/ml polyvinyl alcohol, 1% penicillin/streptomycin and 1% chemically defined lipid concentrate, wherein the chemically defined lipid concentrate contains 2.0 mg/L arachidonic acid, 220 mg/L cholesterol, 70 mg/L DL-alpha-tocopherol acetate, 10 mg/L linoleic acid, 10 mg/L linolenic acid, 10 mg/L myristic acid, 10 mg/L oleic acid, 10 mg/L palmitic acid, 10 mg/L palmitoleic acid, and 10 mg/L stearic acid;
• and where a synergistic effect of the co-culture of the AECs and AFCs in the base media results in increased protein concentration and complexity when compared to a control culture of AECs alone or AFCs alone;
• (b) separating the co-cultured AECs and AFCs from the culture medium of (a) to obtain a conditioned supernatant;
• (c) removing large molecules and other cell debris from the conditioned supernatant of (b);
• (d) ensuring the sterility of the conditioned supernatant of (c);
• and
• (e) concentrating the sterile conditioned supernatant of (d) to produce a concentrated, sterile conditioned supernatant and determining total protein concentration, protein complexity or both;
• wherein the composition comprising components of amniotic fluid is the concentrated, sterile conditioned supernatant that comprises both increased protein concentration and increased protein complexity compared to the control culture of AECs alone or AFCs alone.

A method for making a composition having components of amniotic fluid derived from a co-culture of amniotic epithelial cells (AECs) and amniotic fluid cells (AFCs) and a carrier, the method comprising:

• (a) transferring amniotic fluid cells (AFCs) and amniotic epithelial cells (AECs) to a cell culture system and co-culturing the AECs and AFCs in a defined medium essentially free of serum consisting of a base media to a predetermined target total protein concentration in the culture medium, wherein the base media is 50% Iscove's Modified Dulbecco's Media (IMDM) and 50% F12 media;
• monothioglycerol (450 μM), 1 mg/ml polyvinyl alcohol, 1% penicillin/streptomycin and 1% chemically defined lipid concentrate, wherein the chemically defined lipid concentrate contains 2.0 mg/L arachidonic acid, 220 mg/L cholesterol, 70 mg/L DL-alpha-tocopherol acetate, 10 mg/L linoleic acid, 10 mg/L linolenic acid, 10 mg/L myristic acid, 10 mg/L oleic acid, 10 mg/L palmitic acid, 10 mg/L palmitoleic acid, and 10 mg/L stearic acid;
• and where a synergistic effect of the co-culture of the AECs and AFCs in the base media results in increased protein concentration and complexity when compared to a control culture of AECs alone or AFCs alone;
• (b) separating the co-cultured AECs and AFCs of (a) from the culture medium to obtain a conditioned supernatant;
• (c) removing large molecules and other cell debris from the conditioned supernatant of (b);
• (d) ensuring the sterility of the conditioned supernatant of (c);
• and
• (e) concentrating the sterile conditioned supernatant of (d) to produce a concentrated, sterile conditioned supernatant and determining total protein concentration, protein complexity or both;
• wherein the composition comprising components of amniotic fluid is the concentrated, sterile conditioned supernatant that comprises both increased protein concentration and increased protein complexity compared to the control culture of AECs alone or AFCs alone.

A method for tissue repair, the method comprising one of putting on, embedding into, filling, or injecting a tissue with a composition having components of amniotic fluid produced by a process comprising:

• (a) transferring amniotic epithelial cells (AECs) and amniotic fluid cells (AFCs) to a cell culture system and culturing the AECs and AFCs in a defined medium essentially free of serum consisting of a base media, wherein the base media is 50% Iscove's Modified Dulbecco's Media (IMDM) and 50% F12 media;
• monothioglycerol (450 μM), 1 mg/ml polyvinyl alcohol, 1% penicillin/streptomycin and 1% chemically defined lipid concentrate, wherein the chemically defined lipid concentrate contains 2.0 mg/L arachidonic acid, 220 mg/L cholesterol, 70 mg/L DL-alpha-tocopherol acetate, 10 mg/L linoleic acid, 10 mg/L linolenic acid, 10 mg/L myristic acid, 10 mg/L oleic acid, 10 mg/L palmitic acid, 10 mg/L palmitoleic acid, and 10 mg/L stearic acid;
• and where a synergistic effect of the co-culture of the AECs and AFCs in the base media results in increased protein concentration and complexity when compared to a control culture of AECs alone or AFCs alone;
• (b) separating the co-cultured AECs and AFCs in (a) from the culture medium to obtain a conditioned supernatant;
• (c) removing large molecules and other cell debris from the conditioned supernatant of (b);
• and
• (d) ensuring the sterility of the conditioned supernatant of (c);
• and
• (e) concentrating the sterile conditioned supernatant to produce a concentrated, sterile conditioned supernatant and determining total protein concentration, protein complexity or both;
• wherein the composition comprising components of amniotic fluid is the concentrated, sterile conditioned supernatant that comprises both increased protein concentration and increased protein complexity compared to the control culture of AECs alone or AFCs alone, and wherein the tissue is repaired by the putting on, embedding into, filling, or injecting of the tissue with the composition.