Methods of manufacture of immunocompatible amniotic membrane products

Abstract

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 manufacturing a placental product comprising the steps of:
 a. obtaining a placenta;   b. selectively depleting the amniotic membrane of a substantial portion of one or more types of immunogenic cells, wherein
 i. the one or more types of immunogenic cells comprise functional CD14+ macrophages; and 
 ii. the step of selective depletion preserves the viability of a substantial portion of native therapeutic cells selected from MSCs, fibroblasts, epithelial cells, and a combination thereof; and 
   c. cryopreserving the amniotic membrane in a cryopreservation medium; and   d. optionally, removing substantially all vascularized tissue or vascularized tissue-derived immunogenic cells from the placenta, or amniotic membrane thereof.   
     
     
         2 . The method of  claim 1 , wherein said depleting step comprises removing trophoblasts from the chorionic membrane and retaining the chorionic membrane. 
     
     
         3 . The method of  claim 1 , wherein the chorionic membrane is not retained. 
     
     
         4 . The method of  claim 1 , wherein said depleting step comprises selectively killing or inactivating the functional CD14+ macrophages. 
     
     
         5 . The method of  claim 4 , wherein said depleting step comprises refrigerating the placenta for a period of time before the step of cryopreservation. 
     
     
         6 . The method of  claim 5 , wherein the step of refrigerating comprises incubating the placenta in the cryopreservation medium for the period of time. 
     
     
         7 . The method of  claim 6 , wherein the period of time is a period of time sufficient to allow the placenta or amniotic membrane thereof to equilibrate. 
     
     
         8 . The method of  claim 5 , wherein the period of time is at least: about 10 min, about 20 min, about 30 min, about 40 min, or about 50 min. 
     
     
         9 . The method of  claim 5 , wherein the period of time is: about 10 min to about 120 min, about 20 min to about 90 min, or 30 min to about 60 min. 
     
     
         10 . The method of any of  claims 5 - 9 , wherein the refrigerating comprises incubating the placenta for the period of time at a temperature of about −10° C. to about 15° C. or about 2°-8° C. 
     
     
         11 . The method  claim 4 , wherein said the depleting step comprises treating the placenta with an anti-TNF-α antibody. 
     
     
         12 . The method  claim 4 , wherein said depleting step comprises treating the placenta with IL-10. 
     
     
         13 . The method of any of the preceding claims, wherein the step of cryopreserving comprises reducing the temperature at a rate of less than any of: about 10° C./min, about 5° C./min, about 3° C./min, about 2° C./min, or about 1° C./min. 
     
     
         14 . The method of  claim 13 , wherein the cryopreservation medium comprises a volume of at least any of: about 20 ml, about 30 ml, about 40 ml, about 45 ml, or about 50 ml. 
     
     
         15 . The method of any of  claims 1 - 12 , wherein the cryopreservative comprises a cell-permeating cryopreservative, a non-cell-permeating cryopreservative, or a combination thereof, optionally wherein the permeating cryopreservative comprises:
 a. DMSO; or   b. DMSO in a majority amount.   
     
     
         16 . The method of  claim 15 , wherein the cell-permeating cryopreservative:
 a. does not comprise glycerol in a majority amount; or   b. does not comprises a substantial amount of glycerol.   
     
     
         17 . The method of  claim 15 , wherein the cell-permeating cryopreservative comprises a DMSO in amount of: about 2% to about 20%, about 5% to about 20%, about 5% to about 15%, or about 7% to about 13%. 
     
     
         18 . The method of  claim 15 , wherein the cryopreservation medium further comprises albumin, optionally wherein the albumin is HSA. 
     
     
         19 . The method of  claim 17 , wherein the cryopreservation medium further comprises albumin, optionally wherein the albumin is HSA. 
     
     
         20 . The method of  claims 15 - 19 , wherein the step of cryopreserving comprises reducing the temperature at a rate of less than any of: about 10° C./min, about 5° C./min, about 3° C./min, about 2° C./min, or about 1° C./min. 
     
     
         21 . The method of any of  claims 1 - 12 , wherein the step of removing substantially all vascularized tissue or vascularized tissue-derived immunogenic cells from the placenta, or amniotic membrane thereof comprises lysing red blood cells, removing blood clots or a combination thereof from the placenta. 
     
     
         22 . The method of  claim 21 , wherein said removing substantially all vascularized tissue or vascularized tissue-derived immunogenic cells comprises treating the placenta with an anticoagulant. 
     
     
         23 . The method of  claim 22 , wherein the anticoagulant is a citrate. 
     
     
         24 . The method of any of  claims 1 - 12  further comprising the step of treating the placenta with one or more antibiotics. 
     
     
         25 . The method of any of  claims 1 - 12 , wherein the method comprises retaining the basement layer, the compact layer, the layer of fibroblast, and the spongy layer of the amniotic membrane. 
     
     
         26 . The method of  claim 25  wherein at least about 15.9% to about 100%, or about 25% to about 100%, or about 35% to about 100%, or about 50% to about 100%, or about 75% to about 100%, or about 90% to about 100%, or about 95% to about 100%, or substantially all amniotic epithelial cells are not removed from the placenta. 
     
     
         27 . A placental product produced by the method of any of  claims 1 - 26 . 
     
     
         28 . The placental product of  claim 27 , wherein the membrane is dermatologically acceptable after thawing. 
     
     
         29 . A method of treating a patient comprising administering to an injury a placental product of  claim 28  to the injury. 
     
     
         30 . The method of  claim 29 , wherein the injury is a surgical procedure. 
     
     
         31 . The method of  claim 30  wherein the surgical procedure is a tendon repair or an engraftment. 
     
     
         32 . The method of  claim 30 , wherein the injury is an ulcer, optionally a foot ulcer, optionally a diabetic foot ulcer. 
     
     
         33 . The method of any of  claims 29 - 32  wherein the patient has diabetes. 
     
     
         34 . The method of any of  claims 29 - 32  wherein the injury is associated with ischemia.