Methods of manufacture of immunocompatible chorionic membrane products
Provided herein is a placental product comprising an immunocompatible chorionic 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 a chorionic membrane comprising the steps of:
a. obtaining at least one placenta comprising at least one whole or partial chorion membrane;
b. isolating the chorionic membrane from the placenta;
c. selectively depleting the chorionic membrane of a substantial portion of one or more types of functional immunogenic cells by the following steps:
i. removing all or a portion of a trophoblast layer;
ii. incubating the chorionic membrane with all or a portion of the trophoblast layer removed in a cryopreservation medium at a temperature of about 2° C. to about 8° C. for at least 30 minutes; followed directly by
iii. cryopreserving the chorionic membrane in the cryopreservation medium at about −80° C.
wherein the cryopreserved chorionic membrane comprises at least 70% viable therapeutic cells, wherein the viable therapeutic cells are native to the chorionic membrane, wherein the viable cells comprise at least two cell types consisting of MSCs, and fibroblasts; and wherein said selectively depleting step kills or inactivates functional CD14+ macrophages.
2. The method of claim 1 , wherein the selective depletion step comprises treating the chorionic membrane with at least one enzyme prior to removing the trophoblast layer.
3. The method of claim 1 , wherein the selective depletion step comprises one or more of the steps of (a) scraping the trophoblast layer from the chorionic membrane, (b) blunt dissection of the trophoblast layer from the chorionic membrane, (c) removal of the trophoblast layer from the chorionic membrane without removing the basement membrane from the chorionic membrane; or (d) combinations thereof.
4. The method of claim 1 , wherein the cryopreservation medium comprises at least one cell-permeating cryopreservative, at least one non-cell permeating cryopreservative, or a combination thereof.
5. The method of claim 4 , wherein the cell-permeating cryopreservative comprises DMSO.
6. The method of claim 5 , wherein the DMSO is in an amount of about 2% to about 20% by volume.
7. The method of claim 6 , wherein the cryopreservation medium further comprises a sufficient amount of albumin.
8. The method of claim 7 , wherein the sufficient amount of albumin is about 1% to about 15% by volume of the cryopreservation medium.
9. The method of claim 1 , wherein the step of selectively depleting the chorionic membrane of a substantial portion of one or more types of functional immunogenic cells further comprises the step of lysing red blood cells in the chorionic membrane, removing blood clots from the chorionic membrane, or a combination thereof.
10. The method of claim 1 , wherein the method provides a cryopreserved chorionic membrane that exhibits a substantial decrease in TNF-α release after lipopolysaccharide (LPS) stimulation.
11. The method of claim 10 , wherein the level of TNF-α is less than about 420pg/ml.
12. The method of claim 1 , further comprising the step of treating the chorionic membrane with one or more antibiotics.
13. The method of claim 1 , wherein about 10 ml of the cryopreservation medium is added to the chorionic membrane.
14. The method of claim 1 , further comprising the step of associating the chorionic membrane to at least one nitrocellulose substrate or support.
15. The method of claim 1 , wherein the cryopreserving the chorion membrane in the cryopreservation medium takes place between 75° C. to 85° C.
16. The method of claim 1 , wherein the cryopreserving the chorion membrane in the cryopreservation medium takes place between 70° C. to 90° C.