Amnion derived therapeutic compositions and methods of use

A method of making an amniotic fluid derived therapeutic composition comprising a heterogeneous population of primary cells with cell viability after a freeze-thaw cycle, the method comprising:

• a) providing an amniotic fluid comprising a fluid component and amniotic cells;
• b) measuring an initial cell count in the amniotic fluid to determine an initial cell concentration of amniotic cells;
• c) centrifuging a quantity of the amniotic fluid to separate the fluid component from the amniotic cells to produce
• (i) a quantity of an acellular amniotic fluid;
• and
• (ii) removed amniotic cells, the removed amniotic cells comprise a heterogeneous population of primary cells;
• d) adding a controlled number of said removed amniotic cells to a volume of carrier fluid to produce a concentrated composition having a calculated concentration of removed amniotic cells per volume of carrier fluid;
• and
• e) providing an amniotic membrane comprising an amnion layer and chorion layer, and wherein the amnion layer comprises epithelium,
• f) separating the amnion layer from the chorion layer;
• g) micronizing the amnion layer to produce micronized amniotic layer particles comprising:
• freezing said amniotic layer without a cryoprotectant to produce frozen amniotic layer;
• micronizing said frozen amniotic layer to produce said micronized amniotic layer particles that are free of cryoprotectant;
• wherein the micronized amniotic layer particles consist essentially of epithelium having a concentration of at least 70% epithelium;
• h) adding a controlled amount of micronized amniotic layer particles to the concentrated composition having a calculated concentration of removed amniotic cells per volume of carrier fluid to produce said amniotic fluid derived therapeutic composition having a calculated concentration of micronized amniotic layer particles and a calculated concentration of amniotic cells, wherein the micronized amniotic layer particles are free of chorion;
• wherein the amniotic fluid derived therapeutic composition has a concentration of micronized amniotic layer particles of at least 0.1 mg/ml of therapeutic composition;
• wherein the amniotic fluid derived therapeutic composition has a calculated concentration of removed amniotic cells between 0.25×106 to 10.0×106 per milliliter of carrier fluid;
• wherein the amniotic layer particles comprise micronized amniotic layer particles that have an average particle size of no more than lulu;
• wherein the method results in cell viability for the amniotic fluid derived therapeutic composition that is at least 50% after a freeze-thaw cycle.