Compositions Derived from Placenta and Methods of Producing the Same

Abstract

Compositions comprising unseparated amnion/chorion derived from the placenta and methods of preparing and using those compositions are provided. Washing or preservation of placental tissue according to the methods of the disclosure may allow for one or more benefits such as more efficient removal of blood remnants, retention of wound healing and tissue regeneration components, better handling characteristics, increased absorption potential, or improved healing capacity. The present invention also includes methods of healing a wound of the skin, eye, nerve, tendon, or dura comprising applying the compositions of the invention to the wound.

1 .- 68 . (canceled) 
     
     
         69 . A method of preparing an unseparated placental tissue or composition thereof comprising:
 (a) obtaining an unseparated placental tissue; and   (b) performing at least one washing step comprising washing the unseparated placental tissue with at least one suitable washing medium to remove substantially all blood remnants from the unseparated placental tissue,   wherein one of the at least one washing steps in (b) comprises washing the tissue in a sodium chloride solution overnight, wherein the sodium chloride solution contains a w/v concentration of NaCl of about 18% to about 30%.   
     
     
         70 . The method of  claim 69 , wherein the unseparated placental tissue is an unseparated amnion/chorion tissue. 
     
     
         71 . The method of  claim 70 , wherein the unseparated placental tissue comprises an intermediate layer. 
     
     
         72 . The method of  claim 69 , further comprising pretreating the unseparated placental tissue before washing and/or between any washing steps, wherein pretreating comprises picking material away from the tissue, or dabbing, rubbing, or massaging the tissue. 
     
     
         73 . The method of  claim 69 , wherein the at least one washing step is conducted under vacuum; under pressure; by agitating using a rocker, shaker, stir plate, or rotating mixer; or by soaking. 
     
     
         74 . The method of  claim 69 , wherein step (b) comprises performing at least two washing steps, at least three washing steps, at least four washing steps, at least five washing steps, at least six washing steps, at least seven washing steps, at least eight washing steps, at least nine washing steps, or at least ten washing steps. 
     
     
         75 . The method of  claim 69 , wherein the method comprises exchanging the at least one suitable washing medium at least once, at least twice, at least thrice, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times between any washing steps. 
     
     
         76 . The method of  claim 69 , wherein the at least one washing step comprises washing the tissue in the at least one suitable washing medium in an environment having a temperature of below, at, or above room temperature for about 30 minutes to about 64 hours. 
     
     
         77 . The method of  claim 69 , wherein the at least one washing step comprises heating or cooling the tissue or the at least one suitable washing medium. 
     
     
         78 . The method of  claim 77 , wherein the temperature of the environment does not change over time. 
     
     
         79 . The method of  claim 69 , wherein the at least one suitable washing medium for at least one further washing step is selected from water, a sodium chloride solution, a sodium hypochlorite solution, a hydrogen peroxide solution, Lactated Ringer's solution, Ringer's solution, phosphate-buffered saline (PBS), tris-buffered saline (TBS), Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced salt solution (GBSS), and a cell culture medium. 
     
     
         80 . The method of  claim 79 , wherein the at least one suitable washing medium for at least one further washing step is a sodium chloride solution, wherein the sodium chloride solution contains a w/v concentration of NaCl of about 0.45% to about 30%. 
     
     
         81 . The method of  claim 80 , wherein the sodium chloride solution contains a w/v concentration of NaCl of about 18% to about 30%. 
     
     
         82 . The method of  claim 69 , wherein the method comprises exchanging the suitable washing medium between a sodium chloride solution and water at least once, at least twice, at least thrice, or at least four times between any washing steps. 
     
     
         83 . The method of  claim 69 , wherein the washing the tissue in a sodium chloride solution overnight comprising washing at a refrigeration temperature. 
     
     
         84 . The method of  claim 83 , wherein after overnight refrigeration, the tissue is washed in water. 
     
     
         85 . The method of  claim 69 , further comprising dehydrating the unseparated placental tissue. 
     
     
         86 . The method of  claim 85 , wherein the dehydrating comprises lyophilizing, heat-drying, oven-drying, air-drying, and/or chemically dehydrating the tissue. 
     
     
         87 . The method of  claim 85 , further comprising cutting or powderizing the composition. 
     
     
         88 . The method of  claim 69 , further comprising layering on a surface of the tissue at least one separated amnion sheet, at least one perforated, separated amnion sheet, at least one unperforated, separated amnion sheet, at least one separated chorion sheet, at least one perforated, separated chorion sheet, at least one unperforated, separated chorion sheet, or at least one perforated, unseparated amnion/chorion sheet. 
     
     
         89 . A method of preparing an unseparated placental tissue sheet or composition thereof comprising:
 (a) obtaining an unseparated placental tissue dissected away from the placental disc; and   (b) performing at least one washing step comprising washing the unseparated placental tissue with at least one suitable washing medium to remove substantially all blood remnants from the unseparated placental tissue, wherein one of the at least one washing steps in (b) comprises washing the tissue in a sodium chloride solution overnight,   wherein the sodium chloride solution contains a w/v concentration of NaCl of about 18% to about 30%.   
     
     
         90 . A placental tissue or composition thereof produced according to the method of  claim 69 , wherein the unseparated placental tissue has a hemoglobin content of less than about 10 ng/mg, less than about 5 ng/mg, less than about 3 ng/mg, less than about 2 ng/mg, less than about 1 ng/mg, or less than about 0.5 ng/mg of dry mass of the sheet. 
     
     
         91 . A placental tissue or composition thereof produced according to the method of  claim 69 , wherein the unseparated placental tissue has a hemoglobin content that is reduced in an amount greater than about 50%, 60%, 70%, 80%, 90%, or 95%. 
     
     
         92 . A method of using the placental tissue or composition thereof of  claim 91 , wherein the sheet or composition is for treating a wound of a subject.