Methods of manufacture of immunocompatible amniotic membrane products
Provided herein is a placental product comprising an immunocompatible amniotic membrane. Such placental products can be cryopreserved and contain viable therapeutic cells after thawing. The placental product of the present invention is useful in treating a patient with a tissue injury (e.g. wound or burn) by applying the placental product to the injury. Similar application is useful with ligament and tendon repair and for engraftment procedures such as bone engraftment.
1. A method of cryopreserving an amniotic membrane comprising the steps of:
a. obtaining a placenta comprising an amniotic membrane;
b. isolating the amniotic membrane from the placenta;
c. selectively depleting the amniotic membrane of a substantial portion of one or more types of functional immunogenic cells by
i. incubating the amniotic membrane in a cryopreservation medium at a temperature of about 2° C. to about 8° C. for at least 30 minutes; followed directly by
ii. cryopreserving the amniotic membrane in the cryopreservation medium at about −80° C.;
wherein the amniotic membrane comprises at least 70% viable therapeutic cells, wherein the viable therapeutic cells are native to the amniotic membrane; wherein the viable cells comprise two or more cell types consisting of MSCs, fibroblasts, and epithelial cells; and wherein said selectively depleting step kills or inactivates functional CD14+macrophages.
2. The method of claim 1 , wherein the cryopreservation medium comprises a cell-permeating cryopreservative, a non-cell-permeating cryopreservative, or a combination thereof.
3. The method of claim 2 , wherein the cell-permeating cryopreservative comprises DMSO.
4. The method of claim 3 , wherein the DMSO is in amount of about 2% to about 20% by volume of the cryopreservation medium.
5. The method of claim 4 , wherein the cryopreservation medium further comprises a sufficient amount of albumin.
6. The method of claim 5 , wherein the albumin is about 1% to about 15% by volume of the cryopreservation solution.
7. The method of claim 1 , wherein the step of selectively depleting the amniotic membrane of a substantial portion of one or more types of functional immunogenic cells further comprises lysing red blood cells in the amniotic membrane, removing blood clots from the amniotic membrane, or a combination thereof.
8. The method of claim 1 , wherein the method provides a cryopreserved amniotic membrane that exhibits a substantial decrease in TNF-α release after lipopolysaccharide (LPS) stimulation.
9. The method of claim 8 , wherein the level of TNF-α is less than about 420 pg/ml.
10. The method of claim 1 , further comprising the step of treating the amniotic membrane with one or more antibiotics.
11. The method of claim 1 , wherein the amniotic membrane comprises a compact layer, a layer of fibroblasts, a spongy layer, a basement layer and an epithelial layer.
12. The method of claim 1 , wherein the viable cells further comprise stromal cells.
13. The method of claim 1 , further comprising the step of associating the amniotic membrane to at least one nitrocellulose substrate or support.
14. The method of claim 1 wherein:
a. the cryopreservation medium comprises DMSO in amount of about 2% to about 20% by volume; and
b. the step of selectively depleting the amniotic membrane of a substantial portion of one or more types of functional immunogenic cells further comprises lysing red blood cells in the amniotic membrane, removing blood clots from the amniotic membrane, or a combination thereof.
15. The method of claim 1 , wherein about 10 ml of the cryopreservation medium is added to the amniotic membrane.
16. The method of cryopreserving an amniotic membrane of claim 1 , wherein the cryopreserving the amnion membrane in the cryopreservation medium takes place between −75° C. to −85° C.
17. The method of cryopreserving an amniotic membrane of claim 1 , wherein the cryopreserving the amnion membrane in the cryopreservation medium takes place between −70° C. to −90° C.