Compositions containing amniotic components and methods for preparation and use thereof

Abstract

Compositions are provided that contain biologically active components of amniotic fluid including growth factors and other proteins, carbohydrates, lipids, and metabolites. The compositions containing biologically active components of amniotic fluid can be useful for a range of therapeutic treatments including joint and soft tissue repair, regulation of skin condition, and for use in organ preservation, such as for use in organ transplant procedures. Advantages of the compositions include that they can be reproducibly produced, without the inherent variability of amniotic fluid from individual donors, and that they are free of fetal waste.

1 - 62 . (canceled) 
     
     
         63 . A method for making a composition comprising components of amniotic fluid, the method comprising:
 (a) transferring amniotic epithelial cells (AECs) and amniotic fluid cells (AFCs) to a cell culture system and culturing the AECs and AFCs in a defined medium essentially free of serum consisting of
 a base medium including Dulbecco's Modified Eagle's Medium (DMEM)/F12 Medium (DMEM/F12), or Iscove's Modified Dulbecco's Medium (IMDM), 
 one or more of monothioglycerol, lipids, or polyvinyl alcohol; 
 optionally, one or more antibiotics; 
   (b) separating the AECs and AFCs from the culture medium to obtain a conditioned supernatant;   (c) removing large molecules and other cell debris from the conditioned supernatant; and   (d) ensuring the sterility of the conditioned supernatant,   wherein the conditioned supernatant is the composition comprising components of amniotic fluid.   
     
     
         64 . The method of  claim 63 ,
 (a) wherein the AECs are attached to a surface of the culture system and the AFCs are deposited on top of the AECs; or   (b) wherein the AECs are mitotically inactivated prior to transferring to the cell culture system; or   (c) wherein the AECs and AFCs are expanded prior to transferring to the cell culture system by passaging the AECs and AFCs one, two or three times, and   (d) wherein the AECs and AFCs are derived from a mammalian tissue without having been previously frozen.   
     
     
         65 . The method of  claim 63 , wherein the lipids consist of a 1% chemically defined lipid concentrate containing arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid. 
     
     
         66 . The method of  claim 64 , wherein the mammalian tissue is a human tissue. 
     
     
         67 . A method for making a composition comprising components of amniotic fluid, the method comprising:
 (a) transferring at least two cell types selected from the group consisting of: (i) amniotic fluid cells (AFCs), (ii) amniotic epithelial cells (AECs), (iii) placenta cells, and (iv) umbilical cord cells to a cell culture system;   (b) culturing the cell types in a defined medium essentially free of serum consisting of:
 a base medium including Dulbecco's Modified Eagle's Medium (DMEM)/F12 Medium (DMEM/F12), or Iscove's Modified Dulbecco's Medium (IMDM), 
 one or more of monothioglycerol, lipids, or polyvinyl alcohol; and, 
 optionally, one or more antibiotics; 
 to a predetermined target total protein concentration in the culture medium; 
   (c) separating the cell types from the culture medium to obtain a conditioned supernatant;   (d) removing large molecules and other cell debris from the conditioned supernatant; and   (e) ensuring the sterility of the conditioned supernatant,   wherein the conditioned supernatant is the composition comprising components of amniotic fluid.   
     
     
         68 . The method of  claim 67 ,
 a. wherein the placenta cells comprise trophoblast cells, or   b. wherein the cell types (i)-(iv) are derived from a mammalian tissue without having been previously frozen; or   c. the cell types consist of at least three of the cell types (i)-(iv); or   d. the cell types consist of the four cell types (i)-(iv); or   e. wherein the lipids consist of a 1% chemically defined lipid concentrate containing arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid.   
     
     
         69 . The method of  claim 68 , wherein the mammalian tissue is a human tissue. 
     
     
         70 . A method for preservation of an organ, the method comprising surrounding the organ in a composition comprising components of amniotic fluid, wherein the composition is produced by a process comprising:
 (a) transferring at least two cell types selected from the group consisting of: (i) amniotic fluid cells (AFCs), (ii) amniotic epithelial cells (AECs), (iii) placenta cells, and (iv) umbilical cord cells to a cell culture system;   (b) culturing the cell types in a defined medium essentially free of serum consisting of:
 a base medium including Dulbecco's Modified Eagle's Medium (DMEM)/F12 Medium (DMEM/F12), or Iscove's Modified Dulbecco's Medium (IMDM), 
 one or more of monothioglycerol, lipids, or polyvinyl alcohol; and 
 optionally, one or more antibiotics; 
 to a predetermined target total protein concentration in the culture medium; 
   (c) separating the cell types from the culture medium to obtain a conditioned supernatant;   (d) removing large molecules and other cell debris from the conditioned supernatant; and   (e) ensuring the sterility of the conditioned supernatant,   wherein the conditioned supernatant is the composition comprising components of amniotic fluid; and wherein the organ is preserved in the composition.   
     
     
         71 . The method of  claim 70 ,
 a. wherein the organ is a transplantable organ; or   b. wherein the placenta cells comprise trophoblast cells, or   c. wherein the cell types (i)-(iv) are derived from a mammalian tissue without having been previously frozen; or   d. the cell types consist of at least three of the cell types (i)-(iv); or   e. the cell types consist of the four cell types (i)-(iv); or   f. wherein the lipids consist of a 1% chemically defined lipid concentrate containing arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid.   
     
     
         72 . The method of  claim 71 , wherein the mammalian tissue is a human tissue. 
     
     
         73 . A topical composition formulated to regulate a skin condition, the composition comprising:
 a. a safe and effective amount of a composition comprising components of amniotic fluid containing
 i. a concentrated conditioned supernatant, 
 ii. isolated proteins, microvesicles/exosomes, nucleic acids, or lipids; or 
 iii. both the concentrated conditioned supernatant of (i) and isolated proteins, microvesicles/exosomes, nucleic acids, or lipids of (ii); 
   b. one or more of a moisturizing agent, a vitamin B3 compound, an antioxidant, and   c. a carrier,   wherein the composition is formulated to regulate a skin condition; and   the composition is in form of a liquid, lotion, cream, gel, foam, mousse, spray, paste, powder, or solid.   
     
     
         74 . The topical composition of  claim 73 , wherein the composition comprising components of amniotic fluid in (a) is produced by a process comprising:
 (1) transferring amniotic epithelial cells (AECs) and amniotic fluid cells (AFCs) to a cell culture system and culturing the AECs and AFCs in a defined medium essentially free of serum consisting of
 a base medium including Dulbecco's Modified Eagle's Medium (DMEM)/F12 Medium (DMEM/F12), or Iscove's Modified Dulbecco's Medium (IMDM), 
 one or more of monothioglycerol, lipids, or polyvinyl alcohol; and, 
 optionally, one or more antibiotics; 
   (2) separating the AECs and AFCs from the culture medium to obtain a conditioned supernatant;   (3) removing large molecules and other cell debris from the conditioned supernatant;   (4) ensuring the sterility conditioned supernatant; and   (5) concentrating the sterile conditioned supernatant, or   (6) isolating one or more proteins, microvesicles/exosomes, nucleic acids, or lipids present in the total protein,   (7) or both concentrating the sterile conditioned supernatant and isolating one or more proteins, microvesicles/exosomes, nucleic acids, or lipids present in the total protein.   
     
     
         75 . The topical composition of  claim 73 , wherein
 a. the moisturizing agent constitutes from about 0.1 to about 20% of the composition; or   b. the moisturizing agent comprises one or more of panthenol, pantothenic acid derivatives, glycerin, glycerol, dimethicone, petrolatum, hyaluronic acid, or ceramides, and mixtures thereof;   c. the vitamin B3 compound comprises tocopherol nicotinate; or   d. the anti-oxidant comprises one or a combination of tocopherol or esters of tocopherol; or   e. the composition is applied to human skin at least once a day over at least seven days; or   f. the composition is applied to human skin at least twice a day over at least fourteen days; or   g. the composition once applied induces a cell to undergo an epithelial-to-mesenchymal transition (EMT);   h. The composition once applied induces a cell to undergo an epithelial to mesenchymal transition (EMT), wherein the cell is an epithelial cell or a progenitor cell.   
     
     
         76 . The topical composition of  claim 73 , wherein the composition as formulated:
 a. induces increased skin integrity by cell renewal;   b. enhances water content or moisture of skin;   c. reduces trans epidermal water loss, skin flaking, and scaling;   d. improves skin thickness;   e. enhances skin tensile properties;   f. reduces appearance of dermal fine lines and wrinkles;   g. improves skin texture;   h. reduces skin pores size;   i. enhances skin smoothness;   j. improves skin age spots;   k. improves skin tone; or   l. improves appearance of scars and skin abrasions; or   m. a combination thereof.   
     
     
         77 . The topical composition produced by the process of  claim 74 ,
 a. wherein the AECs are attached to a surface of the cell culture system and the AFCs are deposited on top of the AECs; or   b. wherein the AECs are mitotically inactivated prior to transferring to the cell culture system; or   c. wherein the lipids comprise a 1% chemically defined concentrate comprising arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid; or   d. wherein the AECs and AFCs are expanded prior to transferring to the cell culture system by passaging the AECs and AFCs one, two or three or more times; or   e. wherein the AECs and AFCs are derived from a mammalian tissue without having been previously frozen.   
     
     
         78 . The topical composition of  claim 77 , wherein the mammalian tissue is a human tissue. 
     
     
         79 . A method for tissue repair comprising
 a. formulating a composition comprising
 i. components of amniotic fluid, containing
 1. a concentrated conditioned supernatant, 
 2. isolated proteins, microvesicles/exosomes, nucleic acids, or lipids; or 
 3. both the concentrated conditioned supernatant of (i) and isolated proteins, microvesicles/exosomes, nucleic acids, or lipids of (ii); and 
 
 ii. a carrier, and 
   b. applying, embedding into, filling, or injecting a tissue to effect tissue repair.   
     
     
         80 . The method for tissue repair of  claim 79 , wherein the composition is a dermal, cartilage, or bone gel. 
     
     
         81 . The method for tissue repair of  claim 79 , wherein the lipids comprise a 1% chemically defined concentrate comprising arachidonic acid, cholesterol, DL-alpha-tocopherol acetate, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, and stearic acid. 
     
     
         82 . The method for tissue repair of  claim 79 , wherein the composition comprising components of amniotic fluid is produced by a process comprising:
 (a) transferring amniotic epithelial cells (AECs) and amniotic fluid cells (AFCs) to a cell culture system and culturing the AECs and AFCs in a defined medium essentially free of serum consisting of
 a base medium including Dulbecco's Modified Eagle's Medium (DMEM)/F12 Medium (DMEM/F12), or Iscove's Modified Dulbecco's Medium (IMDM), 
 one or more of monothioglycerol, lipids, or polyvinyl alcohol; and, 
 optionally, one or more antibiotics; 
   (b) separating the AECs and AFCs from the culture medium to obtain a conditioned supernatant;   (c) removing large molecules and other cell debris from the conditioned supernatant; and   (d) ensuring the sterility of the conditioned supernatant.   
     
     
         83 . The method of  claim 79 , wherein the tissue comprises dermal, scar, cartilage, tendon, ligament, muscle, bone, periodontal, cardiovascular, hematologic, pulmonary, urologic, ophthalmic, liver, or kidney tissue, or combinations thereof. 
     
     
         84 . The method of  claim 79 , wherein the tissue repair comprises on or more of:
 a. promoting cell/tissue homeostasis,   b. reducing inflammation, wounds and burns,   c. treating an infection,   d. reducing scarring,   e. effecting joint repair,   f. treating gout;   g. effecting tendon repair;   h. treating male urologic dysfunction;   i. treating Critical Limb Ischemia;   j. treating Intermittent Claudication;   k. treating Buerger's disease;   l. treating ischemic heart disease;   m. treating diastolic heart failure;   n. treating bronchopulmonary dysplasia,   o. treating chronic obstructive pulmonary disease,   p. treating an ophthalmic disorder,   q. reversing aging.   
     
     
         85 . The method of  claim 84 , wherein the infection comprises sepsis. 
     
     
         86 . The method of  claim 84 , wherein the inflammation comprises an arthritis selected from rheumatoid arthritis, psoriatic arthritis or osteoarthritis. 
     
     
         87 . The method of  claim 84 , wherein the inflammation comprises bursitis or tendinitis. 
     
     
         88 . The method of  claim 82 ,
 a. wherein the AECs are attached to a surface of the culture system and the AFCs are deposited on top of the AECs; or   b. wherein the AECs are mitotically inactivated prior to transferring to the cell culture system; or   c. wherein the AECs and AFCs are expanded prior to transferring to the cell culture system by passaging the AECs and AFCs one, two or three times; or   d. wherein the AECs and AFCs are derived from a mammalian tissue without having been previously frozen.   
     
     
         89 . The method of  claim 88 , wherein the mammalian tissue is a human tissue.