Amnion derived therapeutic compositions and methods of use
Therapeutic compositions are described for the treatment of a variety of conditions including heart, eye, lungs, organs, joints, dermal, nerve, and the like. A therapeutic composition may be a fluid comprising amniotic fluid or micronized amniotic particles. A therapeutic composite may be a dispersion of micronized amniotic membrane combined with a fluid, such as plasma, saline, amniotic fluid, combinations thereof and the like. In another embodiment, the therapeutic composite is a mixture of micronized amniotic membrane particles combined with an amniotic rich stem cell fluid. An amniotic rich or concentrated stem cell fluid comprises at least 0.5×10 6 amniotic stem cells per milliliter of fluid or composition. A therapeutic composite may be used to treat any number of conditions through topical application, surgical introduction, and/or injection.
1. 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 0.25×10 6 to 10.0×10 6 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.
2. The method of claim 1 , wherein the amniotic fluid derived therapeutic composition has a calculated concentration of removed amniotic cells between 0.25×10 6 to 3.0×10 6 per milliliter of carrier fluid.