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 manufacturing a composition comprising a chorionic membrane, the method comprising the steps of:
a. obtaining a chorionic membrane;
b. refrigerating the chorionic membrane in a cryopreservation medium at a temperature of about 2° C. to about 8° C. for about 30 min to about 60 min prior to cryopreserving the chorionic membrane in a cryopreservation medium to selectively deplete the chorionic membrane of a substantial portion of functional CD14+ macrophages, wherein the refrigerating does not comprise killing or removing substantially all native therapeutic cells; and
c. cryopreserving the chorionic membrane in the cryopreservation medium at a temperature of −75° C. to −85° C.,
wherein the cryopreserved chorionic membrane comprises at least 70% viable native therapeutic cells, wherein the viable native therapeutic cells comprise mesenchymal stem cells and fibroblasts.
2. The method of claim 1 , wherein the refrigerating step further comprises removing substantially all vascularized tissue or vascularized tissue-derived immunogenic cells from the chorionic membrane.
3. The method of claim 1 , wherein the refrigerating step further comprises depleting the chorionic membrane of a substantial portion of trophoblasts by treating the chorionic membrane with a protease and removing a layer comprising the trophoblasts.
4. The method of claim 1 , wherein the refrigerating step further comprises depleting the chorionic membrane of a substantial portion of trophoblasts by any of scraping the trophoblasts from the chorionic membrane, blunt dissection of a layer comprising the trophoblasts from the chorionic membrane, and removal of the trophoblast layer without removing the basement membrane from the chorionic membrane.
5. The method of claim 1 , wherein the cryopreservation medium comprises a cell-permeating cryopreservative, a non-cell-permeating cryopreservative, or a combination thereof.
6. The method of claim 5 , wherein the cryopreservation medium comprises a cell-permeating cryopreservative comprising DMSO in amount of about 2% to about 20%.
7. The method of claim 1 , wherein the step of cryopreserving comprises reducing the temperature at a rate of less than about 5° C./min.
8. The method claim 1 , wherein the refrigerating step further comprises treating the chorionic membrane with at least one of an anti-TNF-α antibody or IL-10.
9. The method of claim 1 , further comprising the step of removing substantially all vascularized tissue or vascularized tissue-derived immunogenic cells from the chorionic membrane by lysing red blood cells.
10. The method of claim 9 , wherein said removing substantially all vascularized tissue or vascularized tissue-derived immunogenic cells further comprises treating the chorionic membrane with an anticoagulant.
11. The method of claim 1 , wherein the chorionic membrane further comprises an amniotic membrane, and wherein the method further comprises the step of removing at least a portion of the amniotic membrane from the chorionic membrane.
12. The method of claim 11 , wherein at least about 15.9% of the amniotic epithelial cells are retained in the chorionic membrane.
13. The method of claim 1 , wherein the refrigerating step further comprises:
a. removing substantially all vascularized tissue or vascularized tissue-derived immunogenic cells from the chorionic membrane;
b. treating the chorionic membrane with a protease; and
c. removing a layer comprising trophoblasts by any of scraping the trophoblasts from the chorionic membrane, blunt dissection of the trophoblast layer from the chorionic membrane, and removal of the trophoblast layer without removing the basement membrane from the chorionic membrane.
14. A method of treating a tissue injury comprising administering a chorionic membrane produced by the method of claim 1 to the tissue injury of a subject.