Methods of preparing lyophilized human tissues

Abstract

Described herein are methods of preparing human amniotic membrane tissue grafts derived from the placenta. The grafts are composed of three layers as seen in the amniotic membrane in utero. These grafts are processed using physiologic solutions, lyophilized and terminal sterilized (via gamma irradiation in a frozen state) that thereby preserves the graft in such a manner as to retain the naturally occurring biological properties of the amniotic membrane and offer a sterile graft for transplantation. By dehydration via lyophilization and terminal sterilization in a frozen state, the graft has the advantage of storage at ambient temperatures for prolonged periods of time prior to transplantation.

Claims

1. A method of preparing a tissue to be a biological dressing or a tissue graft, said method comprising, in listed order: processing and cleaning, consisting of using only a physiologic saline solution and physical manipulation, an amniotic membrane maintaining the following layers in the following order: a) an epithelium layer; b) a basement membrane layer; and c) a stroma layer comprising an upper compact layer, a middle fibroblast layer, and a lower spongy layer; freezing and dehydrating while lyophilizing, without heat or chemicals, the amniotic membrane after said cleaning to create a frozen, dehydrated amniotic membrane; and in a frozen state, sterilizing the frozen, dehydrated amniotic membrane using gamma irradiation.

2. The method of claim 1, further comprising: separating the amniotic membrane from a placental tissue before said cleaning.

3. The method of claim 2, further comprising: obtaining human placental tissue from which to extract the amniotic membrane.

4. The method of claim 1, further comprising: before said sterilizing, packaging the amniotic membrane.

5. The method of claim 4, further comprising: storing the tissue at room temperature after said sterilizing.

6. The method of claim 5, further comprising: wrapping or replacing a damaged tissue with the tissue.

7. The method of claim 6, wherein said wrapping or replacing the damaged tissue with the tissue comprises wrapping or replacing a damaged tendon, damaged oral tissue, or damaged gastrointestinal mucosa, damaged epidermal tissue with the tissue.

8. The method of claim 4, further comprising: sectioning the amniotic membrane before packaging.

9. The method of claim 1, further comprising: wrapping or replacing a damaged tissue with the amniotic membrane after said sterilizing.

10. The method of claim 9, wherein said wrapping or replacing comprises wrapping or replacing a damaged tendon, damaged oral tissue, damaged gastrointestinal mucosa, or damaged epidermal tissue.

11. A method of preparing a tissue to be a biological dressing or a tissue graft, said method comprising: step for processing an amniotic membrane consisting of using only physiologic saline solution and physical manipulation; step for cleaning, consisting of using only physiologic saline solution and physical manipulation, an amniotic membrane maintaining the following layers in the following order: a) an epithelium layer; b) a basement membrane layer; and c) a stroma layer comprising an upper compact layer, a middle fibroblast layer, and a lower spongy layer; step for freezing and dehydrating while lyophilizing, without heat or chemicals, the amniotic membrane to create a frozen, dehydrated amniotic membrane, after said step for cleaning; and step for sterilizing the frozen, dehydrated amniotic membrane in a frozen state using gamma irradiation, after said step for freezing and dehydrating while lyophilizing.

12. The method of claim 11, further comprising: step for obtaining human placental tissue from which to extract the amniotic membrane.

13. The method of claim 11, further comprising: before said step for sterilizing, step for packaging the amniotic membrane.

14. The method of claim 13, further comprising: step for storing the tissue at room temperature after said step for sterilizing.

15. The method of claim 13, further comprising: step for sectioning the amniotic membrane before said step for packaging.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate the following aspects.

(2) FIG. 1 is a process overview flowchart for the recovery and manufacture of the tissue grafts described herein.

(3) FIG. 2 is an exemplary histological representation sketch of the layers of the tissue grafts.

DETAILED DESCRIPTION OF THE INVENTION

(4) First it is to be understood that the aspects of the methods to be described are not limited to specific compounds, exact methods described or uses as such may vary. FIG. 1 depicts a process overview flowchart for the recovery and manufacture of the tissue grafts described herein.

(5) Step 1: Informed Consent Obtained, Donor Pre-Screened for Eligibility and Suitability

(6) Initially, the potential female donor is approached and written informed consent is obtained following standard industry practices and the guidelines set forth by AATB. The potential donor must fully understand the donation process and give their informed consent to the process and to the testing of their blood for diseases that may affect the suitability of their placenta tissues for use.

(7) After written consent is obtained, the donor is pre-screened for eligibility and suitability to donate her placenta at delivery. Screening involves assessment for risk factors to communicable diseases as specified in the Food and Drug Administration's (FDA) Donor Eligibility Guidance documents and regulations for Good Tissue Practices (GTP) for human tissue products intended for human transplantation. Additionally, standards put forth by the American Association of Tissue Banks (AATB) are also used as guidelines for donor eligibility criteria. After initial pre-screening information is reviewed, a determination is made as to whether the donor is eligible to donate and plans are made for the recovery of the placenta upon delivery.

(8) Step 2: Recover Placenta for Donor Meeting Pre-Screening Criteria

(9) The recovery of the placenta takes place in a hospital or birthing center where it is collected during a live Cesarean section or vaginal delivery birth. The placenta is recovered by the operating room or delivery personnel and placed in sterile designated containers, labeled with distinct donor identification information. The packaged placenta is then either shipped on wet or dry ice to the processing facility or it is temporarily stored in a freezer until it can be shipped to the processing facility.

(10) Step 3: Test Donor Blood for Infectious Diseases Per Criteria

(11) Blood samples are drawn from the donor within 7 days before or after delivery and sent to a CLIA accredited contract laboratory to be tested for communicable diseases such as hepatitis, AIDS, Syphilis, and West Nile Virus using FDA licensed test kits, where appropriate. The specific list of tests for these communicable diseases that are performed are per the current FDA Donor Eligibility and GTP requirements and per AATB's standards.

(12) Step 4: Inspect & Release Placenta if/when all Criteria Met

(13) The placenta container is inspected at the processing facility upon receipt. All donor medical history charts are compiled and reviewed to include completed behavioral, sexual, medical, clinical, and health questionnaires, results of serology testing for communicable diseases, pre-natal history and medical records and hospital/birthing center delivery records. Once all donor criteria are met, the placenta is released for processing.

(14) Step 5: Process Tissue to Separate Amnion

(15) The placenta is thawed (if previously frozen) in a controlled environment and all processing methods are conducted in a controlled environment following FDA's GTP and AATB standards. Equipment and the facilities used are cleaned and decontaminated and all major processing steps are documented following industry practice, FDA regulations and AATB standards. Critical processes are validated per FDA requirements. Work areas are draped prior to usage and supplies and instruments are either sterile or clean with known manufacturing methods to control incoming bioburden.

(16) The placenta is inspected for abnormalities and pre-processing samples are taken to determine the baseline Bioburden. Placentas passing inspection are further processed by separating the amnion layers from both sides of the placenta from the chorion layers and the rest of the placenta. The isolated amnion layers in the form of continuous sheets of tissue are then further processed.

(17) Step 6: Soak, Rinse & Clean Amnion with Physiologic Saline Solutions

(18) The isolated amnion sheets are further processed by briefly soaking for up to 1 hour at room temperature in a physiologic saline solution such as Earle's Balanced Salt Solution (1×) or Hank's Balanced Salt Solution (1×) with periodic gentle mixing to disassociate blood from the amnion. During this time, multiple subsequent rinses with Earle's or Hank's, as needed, may be performed to further remove the blood during the soak time. During or upon completion of the soak and rinse processes, any remaining blood clots or blood on the surface of the amnion may be manually removed. The resultant amnion sheets are now ready for lyophilization.

(19) Step 7: Lyophilize (Freeze Dry)

(20) The amnion sheets are spread out on parchment paper with the stromal side face down, placed on lyophilization trays, placed in sealed lyophilization bags and lyophilized at approximately −40 to −45° C. for 20-48 hours using standard lyophilization methods until moisture is removed and desired drying is achieved. Freeze dry time may vary depending on the thickness of the tissue and the size of the amnion sheets.

(21) Step 8: Cut, Section & Package

(22) The freeze dried (lyophilized) amnion sheets are sectioned into desired sizes (e.g. 4 cm×4 cm, 4 cm×6 cm, etc.) using any sharp cutting device (e.g. a scalpel, a device similar to a pizza cutter, etc.), packaged and sealed (e.g. heat sealed, vacuum sealed, etc.).

(23) Step 9: Inspect & Test

(24) Routine quality inspections of each unit are performed to ensure that manufacturing criteria are met in the areas of size, appearance, package integrity, etc. Standard residual moisture tests may be performed on random units to ensure consistency of the freeze drying process.

(25) Step 10: Sterilize—Gamma Irradiate Amniotic Membrane while Frozen

(26) The lyophilized amnion products are shipped to a contract sterilizer to be gamma irradiated by a validated method to achieve a Sterility Assurance Level (SAL) of 10.sup.−6. The gamma irradiation is performed on dry ice with product in the frozen state to circumvent damage to the tissue. Sterilized product is returned to the possession of the processor for a final inspection.

(27) Step 11: Final Inspection

(28) Sterilized product returned from the contract sterilizer is inspected at by the processing facility for final release. Inspection will include package integrity inspection and final labeling inspection, to name a few. Units passing inspection are released for storage, distribution and subsequent transplantation.