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.

Claims

1.-68. (canceled)

69. An unseparated amnion/chorion sheet comprising an intermediate layer in between the amnion layer and chorion layer, wherein the unseparated amnion/chorion sheet is dehydrated, and wherein the epithelial layer of the amnion layer is partially or substantially removed from the unseparated amnion/chorion sheet.

70. The composition of claim 69, wherein the composition is lyophilized, heat-dried, oven-dried, air-dried, or chemically dried.

71. The composition of claim 69, wherein about 1% to about 89% of the epithelial layer is removed from the unseparated amnion/chorion sheet.

72. The composition of claim 69, wherein about 10% to about 90% of the epithelial layer is removed from the unseparated amnion/chorion sheet.

73. The composition of claim 69, wherein about 90% to about 99% of the epithelial layer is removed from the unseparated amnion/chorion sheet.

74. The composition of claim 69, wherein about 95% to about 99% of the epithelial layer is removed from the unseparated amnion/chorion sheet.

75. The composition of claim 69, wherein the composition has a hemoglobin content of less than about 13 ng/mg of dry mass of the composition.

76. The composition of claim 69, wherein the composition has a hemoglobin content of dry mass of the composition that is reduced in an amount greater than about 50%, 60%, 70%, 80%, 90%, or 95% compared to an unprocessed dried placental tissue.

77. The composition of claim 69, wherein the composition comprises one or more additional sheets, wherein the one or more additional sheets is selected from a perforated, unseparated amnion/chorion sheet; an unperforated, unseparated amnion/chorion sheet; a perforated, separated amnion sheet; an unperforated, separated amnion sheet; a perforated, separated chorion sheet; and an unperforated, separated chorion sheet.

78. The composition of claim 69, wherein the composition comprises nucleated cells, collagens, fibronectins, fibroblasts, growth factors, cytokines, proteoglycans, glycoproteins, tissue inhibitors of metalloproteinases, and/or hyaluronic acid.

79. The composition of claim 69, wherein the composition comprises a marking selected from an embossment, a cut, a stain, a tint, and a stamp.

80. The composition of claim 69, wherein the composition is a placental graft.

81. A method of preparing a dehydrated composition comprising an unseparated amnion/chorion sheet comprising an intermediate layer in between the amnion layer and chorion layer comprising: (a) obtaining unseparated amnion/chorion tissue dissected away from the placental disc; (b) partially removing or substantially removing the epithelial layer of the amnion layer; (c) performing at least one washing step comprising washing the unseparated amnion/chorion sheet with at least one suitable washing medium to remove substantially all blood remnants; and (d) dehydrating the unseparated amnion/chorion sheet.

82. The method of claim 81, wherein the at least one suitable washing medium, wherein the washing medium 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.

83. The method of claim 81, wherein about 90% to about 99% of the epithelial layer is removed from the unseparated amnion/chorion sheet.

84. The method of claim 81, wherein about 95% to about 99% of the epithelial layer is removed from the unseparated amnion/chorion sheet.

85. The method of claim 81, wherein the epithelial layer is removed by gently scraping away the epithelial cells from the amnion layer.

86. The method of claim 81, wherein the epithelial layer is removed by rinsing the unseparated amnion/chorion sheet with an ionic detergent, a nonionic detergent, or a zwitterionic detergent.

87. The method of claim 81, wherein the dehydrating comprises lyophilizing, heat-drying, oven-drying, air-drying, or chemically dehydrating the sheet.

88. The method of claim 87, further comprising milling, mincing, grinding and/or pulverizing the composition.

89. A method of treating a wound comprising applying to the wound the composition of claim 69.

90. The method of claim 89, wherein the composition is applied so that the amnion side of the unseparated amnion/chorion sheet, the chorion side of the unseparated amnion/chorion sheet, the exposed basement membrane of the amnion, or the trophoblast layer of the chorion faces the wound.

Description

EXAMPLES

Example 1. Preparing Perforated, Unseparated Amnion/Chorion Sheets

[0123] A placenta collected from a donor mother is placed in sealed bag containing a suitable storage medium and is shipped on wet ice to the laboratory. After receipt, the placenta is stored at refrigeration temperature. The placenta is removed from its bag for processing at room temperature. The placenta is transferred to a metal tray. Using a pair of surgical scissors, the unseparated amnion/chorion is dissected from the placental disc and umbilical cord. The unseparated amnion/chorion sheet is transferred to a 1 L Nalgene plastic screw-top bottle containing about 750 mL of a sodium chloride solution and placed on a rocker platform and gently agitated for approximately 1 hour at room temperature.

[0124] The unseparated amnion/chorion is transferred to a cutting mat and cut into multiple sheets using a rotary blade. An unseparated amnion/chorion sheet is placed on a rubber or silicon mat (or other soft, nonporous surface that will allow a perforator to penetrate the tissue) with the epithelial layer of the amnion facing up. The unseparated amnion/chorion sheet is perforated, for example, by rolling a perforator device (e.g., a device having a wooden handle and steel roller with a series pins arranged in multiple rows) across the top surface of the sheet such that multiple series of holes are made that traverse the thickness of the amnion/chorion sheet.

[0125] The sheet is transferred to another 1 L bottle containing about 750 mL of a sodium chloride solution and placed on the rocker and gently agitated for about 1 hour at room temperature. The sheet is then transferred to a 1 L bottle containing about 750 mL of sterile water or a sodium chloride solution and placed on the rocker at room temperature with gentle agitation for about 1.5 hours followed by no agitation for another about 1.5 hours without changing the solution.

[0126] The sheet is then transferred to a 1L bottle containing about 750 mL of sterile water or a sodium chloride solution and stored at refrigeration temperature for a period of up to several days before lyophilization. After storage and before lyophilization, the sheet is transferred to a 1 L bottle containing about 750 mL of sterile water or a sodium chloride solution and placed on the rocker at room temperature with gentle agitation for about 1.5 hours followed by no agitation for an additional about 1.5 hours without changing the solution. The sheet is then stored in sterile water at a freezing temperature overnight and then lyophilized until dry.

Example 2. Preparing Perforated, Unseparated Amnion/Chorion Layered With a Separated Sheet

[0127] Following collection of a placenta, dissection of the unseparated amnion/chorion away from the placental disc, and washing in a sodium chloride solution, the unseparated amnion/chorion is cut into multiple sheets using a rotary blade as described in Example 1. Two sheets are placed on a mat with the epithelial layer of the amnion facing down. The chorion is gently separated from the amnion of each sheet by hand and placed on the mat. One of the separated amnion sheets and one of the separated chorion sheets is perforated, for example, by rolling a perforator device across the sheets such that several holes are made that traverse the thickness of the sheets. The other separated amnion sheet and the other separated chorion sheet are not perforated.

[0128] The perforated and unperforated separated amnion sheets and chorion sheets are transferred to a 1 L bottle containing about 750 mL of a suitable washing medium (e.g., sterile water or a sodium chloride solution), placed on a rocker, and gently agitated for about 1 hour at room temperature. The suitable washing medium is replaced with about 750 mL of the same or a different suitable washing medium and gently agitated at room temperature for about 1.5 hours followed by no agitation for another about 1.5 hours without changing the solution. The sheets are then transferred to a 1L bottle containing about 750 mL of a suitable washing medium and stored at refrigeration temperature for a period of up to several days.

[0129] Four perforated, unseparated amnion/chorion sheets are prepared according to the method of Example 1 through the step of storing at refrigeration temperature and before lyophilization. After transferring the perforated, unseparated amnion/chorion sheets; the perforated, separated amnion sheet; the perforated, separated chorion sheet; the unperforated, separated amnion sheet; and the unperforated, separated chorion sheet to about 750 mL of a suitable washing medium, the sheets are gently agitated at room temperature for about 1.5 hours followed by no agitation for an additional about 1.5 hours without changing the solution.

[0130] The four perforated, unseparated amnion/chorion sheets are each placed on a solid surface with either the epithelial layer of the amnion facing upward or downward. A separated sheet is layered on top of one of the perforated, unseparated amnion/chorion sheets such that either side of the separated sheet is facing up. After storing the layered sheets at a freezing temperature overnight, they are lyophilized until dry.

Example 3. Preparing Partial Perforations of Unseparated Amnion/Chorion

[0131] Following collection of a placenta, dissection of the unseparated amnion/chorion away from the placental disc, and washing in a suitable washing medium, the unseparated amnion/chorion is cut into two sheets using a rotary blade as described in Example 1. The sheets are then placed on a mat with the epithelial layer of one sheet facing up and the epithelial layer of the other sheet facing down.

[0132] A perforating roller device having pins of varying length is rolled across the surface of the sheets to create perforations of varied depth. For example, a roller having pins with a length ranging from 0.1 m or less to 1 mm or greater may be used to create partial perforations.

Example 4. Process A

[0133] Donor placentas were logged and quarantined in the freezer until results from serological testing were received indicating that the placentas were nonreactive. Each placenta was processed separately according to the following procedure. The bag containing the donor placenta was placed on a tray. The placenta was transferred from the bag to another tray by grasping the umbilical cord. The unseparated amnion/chorion was then carefully dissected away from the placental disc. The umbilical cord and placental disc were then discarded. Using fingertips, some visible, removable blood clots were gently lifted away from the outer surfaces of the unseparated amnion/chorion to reduce the amount of blood entering the first washing medium.

[0134] A portion of the unseparated amnion/chorion tissue was placed in a 1 L Nalgene screw-top bottle to which about 750 mL of a first suitable washing medium was added. For Process A, the first washing medium used was 26% sodium chloride maintained at room temperature. The bottle was closed and gently agitated for 1 hour at room temperature. In this example and the following examples, gentle agitation was achieved by using a rocker at about 30 RPM.

[0135] The unseparated amnion/chorion was then removed and laid flat on a soft, nonporous mat with the epithelial layer of the amnion facing up. The first washing medium was discarded. Using fingertips, additional visible, removable blood clots were gently lifted away from the tissue's outer surfaces. Blood-tinted areas of the unseparated amnion/chorion tissue were gently massaged using fingertips with care so as not to separate the tissue.

[0136] To perforate the unseparated amnion/chorion, a perforating roller was placed on top of the center of the tissue and rolled outwards over the surface of the tissue. The perforating roller used had a metal roller head with 247 rows of metal pins spaced about 6 mm apart.

[0137] Next, the perforated, unseparated amnion/chorion was placed back into the empty bottle to which about 750 mL of a second suitable washing medium was added. For Process A, the second washing medium used was purified water maintained at room temperature. The bottle was closed and gently agitated for 1.5 hours at room temperature.

[0138] The second washing medium was decanted and replaced with about 750 mL of a third suitable washing medium. For Process A, the third washing medium used was 26% sodium chloride maintained at room temperature. After closing the bottle, it was gently agitated for 1 hour at room temperature.

[0139] After removing the third washing medium, about 750 ml of a fourth suitable washing medium was added. For Process A, the fourth washing medium used was 26% sodium chloride maintained at room temperature. The sample was then refrigerated (at about 4 C.) overnight.

[0140] The next morning, the fourth washing medium was decanted and replaced with about 750 mL of a fifth suitable washing medium. For Process A, the fifth washing medium used was purified water maintained at room temperature. After closing the bottle, it was gently agitated for 1 hour at room temperature.

[0141] The tissue was removed and placed in a closed container and stored overnight at a freezing temperature (about 20 C.). The frozen tissue was then lyophilized overnight at about 46 C. The lyophilized tissue was placed into a container, labeled, and sealed.

Example 5. Process B

[0142] The steps of Process A were performed with the exception that (1) the perforating step was performed after the placental disc and umbilical cord were removed and prior to the step involving the first washing medium; (2) the first washing medium used was purified water instead of 26% sodium chloride; (3) the second washing medium used was 26% sodium chloride instead of purified water; and (4) the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order.

Example 6. Process C

[0143] The steps of Process A were performed with the exception that prior to the addition of each of the first, second, third, fourth, and fifth washing medium, the mediums were preheated in a water bath maintained at 37 C.

Example 7. Process D

[0144] The steps of Process A were performed with the exception that the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order.

Example 8. Process E

[0145] The steps of Process A were performed with the exception that (1) the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order, and (2) prior to the addition of each of the first, second, and fourth washing mediums (the washing steps performed during the first day for Process E), the mediums were preheated in a waterbath maintained at 37 C.

Example 9. Process F

[0146] The steps of Process A were performed with the exception that (1) the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order, and (2) prior to the addition of each of the first, second, third, fourth, and fifth washing mediums, the mediums were preheated in a waterbath maintained at 37 C.

Example 10. Process G

[0147] The steps of Process A were performed with the exception that the step involving a third washing medium was not performed.

Example 11. Process H

[0148] The steps of Process A were performed with the exception that (1) the step involving a third washing medium was not performed, and (2) prior to the addition of each of the first, second, fourth, and fifth washing mediums, the mediums were preheated in a waterbath maintained at 37 C.

Example 12. Process I

[0149] The steps of Process A were performed with the exception that (1) the step involving a third washing medium was not performed, and (2) for the step involving the first washing medium, the bottle containing the tissue was gently agitated for 2 hours at room temperature.

Example 13. Processes J.SUB.a.-J.SUB.i

[0150] The steps of Processes A-I were performed with the exception that the unseparated amnion/chorion tissue was not perforated. Process J.sub.a refers to the process in which the steps were as in Process A except that the unseparated amnion/chorion tissue was not perforated; Process J.sub.b refers to the process in which the steps were as in Process B except that the unseparated amnion/chorion tissue was not perforated; and so on.

Example 14. Processes K.SUB.a.-K.SUB.i .and K.SUB.Ja.-K.SUB.Ji

[0151] The steps of Processes A-I and J.sub.a-J.sub.i were performed with the exception that instead of freezing the tissue overnight followed by lyophilizing the tissue overnight, the sample was oven dried overnight at about 35 C. Process K.sub.a refers to the process in which the steps were as in Process A except that instead of freezing the tissue overnight followed by lyophilizing the tissue overnight, the sample was oven dried overnight at about 35 C.; Process Kb refers to the process in which the steps were as in Process B except that instead of freezing the tissue overnight followed by lyophilizing the tissue overnight, the sample was oven dried overnight at about 35 C.; and so on. Process K.sub.Ja refers to the process in which the steps were as in process J.sub.a except that instead of freezing the tissue overnight followed by lyophilizing the tissue overnight, the sample was oven-dried overnight at about 35 C.; Process K.sub.Jb refers to the process in which the steps were as in Process J.sub.b except that instead of freezing the tissue overnight followed by lyophilizing the tissue overnight, the sample was oven dried overnight at about 35 C.; and so on.

Example 15. Dry Mass

[0152] The dry mass per surface area (mg/cm.sup.2) of processed, unseparated amnion/chorion tissue was measured and compared to that of unprocessed, unseparated amnion/chorion tissue. The results are summarized in Table 1.

TABLE-US-00001 TABLE 1 Dry Mass per Surface Area Unseparated n includes (mg/cm.sup.2) amnion/chorion tissue n Processes Mean Range Unprocessed, lyophilized 9 n/a 11.7 5.6-20.3 Unprocessed, oven-dried 3 n/a 8.9 6.6-14.0 Processed, perforated, lyophilized 15 A, B, C, D, 9.8 4.9-16.0 E, F, H Processed, unperforated, lyophilized 9 J.sub.c, J.sub.d, J.sub.f 7.3 5.4-9.4 Processed, perforated, oven-dried 16 K.sub.c, K.sub.d, K.sub.e, 7.6 4.5-13.8 K.sub.f Processed, unperforated, oven-dried 8 K.sub.Jc, K.sub.Jd, K.sub.Jf 6.3 3.4-11.3

Example 16. Water Absorption

[0153] The water absorption potential (mg of water absorbed per cm.sup.2 of tissue) of processed, unseparated amnion/chorion tissue was compared to fresh, unprocessed, unseparated amnion/chorion tissue. A 3 cm3 cm sheet of each sample was weighed before and after rehydration. Rehydration was achieved by placing the sample in a dish and adding 100 ml distilled water over the tissue. After 80 seconds of remaining in the distilled water, the tissue was removed, laid out on a mat, and the excess surface water wicked away using a wipe. The water absorption potential was calculated by subtracting the mass of the dried sample from the mass of the rehydrated sample and dividing by the surface area of the tissue. The water absorption potential of 3 samples of fresh, unprocessed, unseparated amnion/chorion tissue was undetectable (LOD=0.1 mg), presumably because the tissue was already fully hydrated. The results of the processed samples are summarized in Table 2.

TABLE-US-00002 TABLE 2 Water absorption potential Processed, unseparated n includes (mg absorbed/cm.sup.2) amnion/chorion tissue n Processes Mean Range Perforated, lyophilized 12 C, D, F, G 23 14-34 Unperforated, lyophilized 5 J.sub.c, J.sub.d, J.sub.g 17 11-22 Perforated, oven-dried 12 K.sub.c, K.sub.d, K.sub.e, K.sub.f 10 3-15 Unperforated, oven-dried 6 K.sub.Jc, K.sub.Jd, K.sub.Je 11 9-13

Example 17. Protein content

[0154] Total protein content was determined for samples of processed, unseparated amnion/chorion tissue and for fresh, unprocessed, unseparated amnion/chorion tissue. Total protein was extracted from the tissue samples, quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, Product No. 23227), and normalized to the surface area of the sample. The results are summarized in Table 3.

TABLE-US-00003 TABLE 3 Total protein Unseparated n includes (/cm.sup.2) amnion/chorion tissue n Processes Mean Range Fresh, unprocessed 3 n/a 1057 749-1571 Processed, perforated, 18 A, C, D, 432 392-597 lyophilized F, G, H Processed, unperforated, 18 J.sub.a, J.sub.c, J.sub.d, 507 275-844 lyophilized J.sub.f, J.sub.g, J.sub.h Processed, perforated, oven- 10 K.sub.a, K.sub.c, K.sub.d, 338 131-500 dried K.sub.f, K.sub.g, K.sub.h Processed, unperforated, oven- 8 K.sub.Ja, K.sub.Jc, K.sub.Jd, 336 116-686 dried K.sub.Jf, K.sub.Jg, K.sub.Jh

Example 18. Hemoglobin

[0155] The hemoglobin content of unprocessed, unseparated amnion/chorion tissue was compared to that of processed, unseparated amnion/chorion tissue that were either perforated or unperforated. Hemoglobin content was determined using a RayBio Human Hemoglobin ELISA kit (RayBiotech, Product No. ELH-Hgb1) and normalized to the dry mass of the sample. The mean and ranges of hemoglobin content (based on the ratio of hemoglobin mass to tissue mass) are summarized in Table 4.

[0156] The limit of detection (LOD) for the hemoglobin assay as performed was considered to be 0.043 ng/mg, based on lysis of the sample in 1 mL lysis buffer per 35 mg sample and the lowest detectable hemoglobin concentration for the RayBio Human Hemoglobin ELISA kit being 1.5 ng/mL.

TABLE-US-00004 TABLE 4 Unseparated n includes ng/mg amnion/chorion tissue n Processes Mean Range Unprocessed, lyophilized 8 n/a 14.9 14.1-17.9 Processed, perforated, 7 A, C, 1.0 0.1-2.9 lyophilized F, G, H Processed, unperforated, 7 J.sub.a, J.sub.c, 2.8 <LOD-12.6.sup. lyophilized J.sub.f, J.sub.g, J.sub.h (LOD: limit of detection.)

[0157] Both (i) the difference in hemoglobin content between unprocessed and processed, perforated, unseparated amnion/chorion tissue and (ii) the difference in hemoglobin content between unprocessed and processed, unperforated, unseparated amnion/chorion tissue were statistically significant (p-values<0.01). The significant reduction in hemoglobin content observed in processed, unseparated amnion/chorion tissue compared to unprocessed tissue indicates a significant reduction in red blood cell remnants.

Example 19. Thickness

[0158] The thickness of processed, unseparated amnion/chorion tissue was compared to that of fresh, unprocessed, unseparated amnion/chorion tissue. The thickness of the intermediate layer of the tissues were also compared. A portion of each tissue was embedded, sectioned, mounted, and stained with hematoxylin and eosin (H & E). A digital image of each slide was obtained. Approximate thickness of the tissues and approximate thickness of the intermediate layer of the tissues were measured at five or six representative areas under 20 magnification. Mean thicknesses of each tissue were determined by calculating the average of the measured values. The mean and range of mean thicknesses were determined for each group of samples and are summarized in Table 5.

TABLE-US-00005 TABLE 5 Intermediate Layer Unseparated n includes Thickness (m) Thickness (m) amnion/chorion tissue n Processes Mean Range Mean Range Fresh, unprocessed 3 n/a 604 511-757 310 283-326 Processed, 7 A, F, G, H 339 210-440 104 45-196 perforated, lyophilized Processed, 5 J.sub.a, J.sub.g, J.sub.h 472 262-679 66 54-86 unperforated, lyophilized Processed, 3 K.sub.f, K.sub.i 158 122-220 42 22-65 perforated, oven-dried Processed, 3 K.sub.Jf, K.sub.Ji 243 101-432 55 26-75 unperforated, oven-dried

Example 20. Growth factors, extracellular matrix molecules, and other biomolecules

[0159] The presence of various growth factors and other biomolecules in processed, unseparated amnion/chorion tissues was determined using ELISA kits that assay for bFGF, EGF, TGF beta 1, PDGF-AA, PDGF-BB, TIMP-1, TIMP-2, and TIMP-4 (RayBiotech, RayBio ELISA kits Product Nos. ELH-bFGH, ELH-EGF, ELH-TGFb1, ELH-PDGFAA, ELH-PDGFBB, ELH-TIMP1, ELH-TIMP2, and ELH-TIMP4), and for hyaluronic acid (R&D Systems, Hyaluronan Quantikine ELISA Product No. DHYAL0). Samples of processed, unseparated amnion/chorion tissue were found to contain bFGF, EGF, TGF beta 1, PDGF-AA, PDGF-BB, TIMP-1, TIMP-2, TIMP-4, and hyaluronic acid regardless of whether they were perforated or unperforated, and of whether they were lyophilized or oven-dried.

[0160] The presence of nucleated cells and extracellular matrix molecules in processed, unseparated amnion/chorion tissue was determined by standard histological staining. Unseparated amnion/chorion tissue was prepared according to Process K.sub.i and K.sub.Ji. A portion of each tissue was embedded, sectioned, mounted, and stained. The presence of nucleated cells (H & E), collagens (Masson's Trichrome), glycosaminoglycan-containing molecules (e.g., proteoglycans, etc.) (Alcan Blue), and elastins (VanGieson) was observed and the intensity of staining for each layer of the tissues is summarized in Table 7.

TABLE-US-00006 TABLE 7 Layer of unseparated Masson Alcan amnion/chorion tissue H & E Trichrome Blue VanGieson Epithelial layer +++ + + + Amniotic basement + +++ ++ + membrane Compact layer + +++ +++ + Fibroblast layer + +++ ++ ++ Intermediate layer + ++ ++ ++ Reticular layer + + + + Chorionic basement membrane* Trophoblast layer +++ ++ + +++ including capsular decidua Scoring: +, ++, +++ indicates slight, moderate, and heavy staining, respectively. *indicates not quantified.

Example 21. Chorion Separation from Amnion and Intermediate Layer

[0161] Unseparated amnion/chorion having an intermediate layer is dissected away from the placental disc and placed in a tray with the chorion facing up. The edge of the chorion is gently pulled up using one hand while the other hand secures the amnion with the intermediate layer on the tray. With flattened fingers of one hand on the amnion/intermediate layer securing it to the base of the tray, the other hand holds the chorion close to the line of separation and pulls upwards slowly such that the chorion pulls away from the amnion/intermediate layer over a short distance. This process of peeling away of the chorion is slowly continued by repeating the same securing and pulling actions along the line of separation between the intermediate layer and the chorion until the chorion is separated away leaving the intermediate layer substantially intact on the separated amnion.

[0162] The separated amnion with intermediate layer or the separated chorion may be processed using any one of Processes A-K. In addition, the chorion may be separated from the amnion and intermediate layer before, during, or after dissecting, cutting, perforating, or washing.

Example 22. Amnion Separation from Chorion and Intermediate Layer

[0163] Unseparated amnion/chorion having an intermediate layer is dissected away from the placental disc and placed in a tray with water to swell the intermediate layer. The excess water is drained away and the unseparated amnion/chorion is oriented with the amnion facing up on the tray. Using forceps, the edge of the amnion is pulled up slightly to visualize the line of separation between the intermediate layer and the amnion. The flat end of a scalpel is then used to gently scrape from the forceps to the line of separation while gently and slowly pulling up with the forceps. This process of scraping and pulling is slowly repeated along the line of separation between the intermediate layer and the amnion until the amnion is separated away leaving the intermediate layer substantially intact on the separated chorion.

[0164] The separated chorion with intermediate layer or the separated amnion may be processed using any one of Processes A-K. In addition, the amnion may be separated from the chorion and intermediate layer before, during, or after dissecting, cutting, perforating, or washing.