(12,) United States Patent
`(10) Patent No.:
`US 8,323,701 B2
`
`Daniel et a].
`(45) Date of Patent:
`Dec. 4, 2012
`
`USOO8323701B2
`
`distry et al.
`8/2008
`3/2002 Cubicciotti
`4/2002
`sscroff ct a].
`2/2003
`{ariri
`wmm waaa
`10/2003
`{ariri et al.
`12/2003
`{ariri et :11.
`2/2004
`{ariri et al.
`
`2/2004 Anderson et 31'
`3/2004 Ghlnelll
`3/2004 {arm et al.
`%Q004 Lunosetat
`9/2004
`{ariri
`11/2004
`{ariri
`12/2004 Crowe et a1.
`2/2005 Tseng et al.
`5/2005
`{1m et al.
`@9005 {anrietaL
`7/2005
`{ariri et a1.
`1/2006 Gandy
`7/2006
`{unter et :11.
`
`10/2006 Clarke et al.
`10/2006 W'ang
`1/2007 Abramson et al.
`1/2007
`{ariri et 211.
`
`eiu et a1,
`1/2007
`)eyumn
`2/2007
`2/2007 Sulllel‘ 6t alr
`3/2007 Tseng et a].
`6/2007
`{eidaran
`
`(Continued)
`
`
`
`7,413,734 B2
`2002/0034757 A1
`2002/0039788 A1
`2003/0032179 A1
`2mym534A1
`2003/0187515 A1
`2003/0235909 A1
`2004/0028660 A1
`
`2004/,0039453 A1
`2004/0057938 A1
`2004/]0048796 Al
`2004/0126878 A1
`2004/0171147 A 1
`2004/0219136 A1
`2004/0265293 A1
`2005/0026279 A1
`2005/0107876 A1
`2005/0118715 A1
`2005/0148034 A1
`2006/0004189 A1
`2006/0147492 A1
`
`2006/0222634 A1
`2006/0228339 A1
`2007/0020225 A1
`2007/0021704 A1
`
`2007/0021762 A1
`2007/0031471 A1
`2007/0033293 A1
`2007/0071740 A1
`2007/0134210 A1
`
`(54) PLACENTAL TISSUE GRAFTS
`
`5
`(7’)
`
`.
`,
`1m entors‘
`
`.
`-
`JOhn D291?" WOOdStOSk’ GA, (,US)’
`Rwfltmhdkmmtoflblkmm”l
`Spencer, ACWOrth. GA (US); John
`Russo, Sarasota, FL (US)
`
`,
`_
`( * ) Notice:
`
`(73) Assignce: Mimedx Group, Inc., Kennesaw, GA
`(US)
`,
`.
`.
`,
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(1)) by 394 days.
`
`(21)
`
`[\PP1~}J°* 1244289908
`.
`(22) Elledi
`
`Apr. 23, 2009
`
`(65)
`
`Prior Publication Data
`US 2010/0104539 A1
`Apr. 29, 2010
`
`Related U.S. Application Data
`(63) Continuation of application No. 12/206,508, filed on
`Sep. 8 2008.
`’
`
`(60) Provisional application No. 60/986,665, filed on Nov.
`9, 2007, prov151onal appl1cation No. 60/970,780. filed
`on Sep.
`7,
`2007, provisional
`application No.
`60/989,299, filed on Nov. 7, 2007.
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`Int. Cl.
`(2006.01)
`A61K 35/50
`U.S. Cl.
`...................... 424/583; 424/582; 623/2372
`Field of Classification Search ........................ None
`See application file for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`4,361,552 A
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`7,049,139 B2
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`7,244,444 B2
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`7,311,904 B2
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`7,311,905 B2
`12/2007 Hariri
`
`CN
`
`FOREIGN PATEI\T DOCUMENTS
`200610156533
`12/2007
`
`(Continued)
`
`OTHER PUBLICATIONS
`
`Pakkala et a1. “Function of Laminins and Laminin-Binding Integrins
`in Gingival Epithelial Cell Adhesion” Periodontol, Jul, 2002, pp.
`709-719.
`
`(Continued)
`
`Primary Examiner 7 Allison Ford
`(74) Attorney, Agent, or Firm 7 Foley & Lardner LLP
`
`ABSTRACT
`
`(57)
`Described herein are tissue grafts derived from the placenta.
`The grafts are composed of at least one layer ofanmion tissue
`where the epithelium layer has been substantially removed in
`order to expose the basement layer to ho st cells. By removing
`the epithelium layer, cells from the host can more readily
`interact with the cell-adhesion bio-active factors located onto
`top and Within of the basement membrane. Also described
`herein are methods for making and using the tissue grafts. The
`laminin structure of amnion tissue is nearly identical to that of
`native human tissue such as, for example, oral mucosa tissue.
`This includes high level of laminin-S, a cell adhesion bio-
`active factor show to bind gingival epithelia—cells, found
`throughout upper portions of the basement membrane.
`
`10 Claims, 8 Drawing Sheets
`
`MTF Ex. 1001, pg. 1
`
`

`

`US 8,323,701 132
`
`Page 2
`
`U.S. PATENT DOCUMENTS
`2007/0154515 A
`7/2007 Jolmson et al.
`2007/0190042 A
`8/2007 Edinger et a1.
`2007/0218039 A
`9/2007 Devi et al.
`2007/0231401 A
`10/2007 Tseng et a1.
`2007/02 54005 A
`11/2007 Pathak et a1.
`2007/0254013 A
`11/2007 Taguchi et a1.
`2007/0269791 A
`11/2007 'lakami et al.
`2007/0292401 A
`12/2007 Harmon et al.
`2008/0032401 A
`2/2008 Edinger‘ et a1.
`2008/0044848 A
`2/2008 Heidaran
`2008/0046095 A
`2/2008 Daniel
`2008/0069895 A
`3/2008 Liu et a1.
`2008/0181935 A
`7/2008 Bhatia et a1.
`2008/0213389 A
`9/2008 Lelkes et al.
`2008/0248005 A
`10/2008 Phan
`2010/0098743 A
`4/2010 Nikaido et a1.
`2011/0256202 A
`10/2011 'lom et al.
`
`
`
`EP
`FR
`JP
`WO
`W'O
`
` FOREIGN PATENT DOCUMENTS
`
`0773033
`5/1997
`2892311
`4/2007
`08266613
`10/1996
`2006129673
`12/2006
`2009132186
`10/2009
`
`OTHER PUBLICATIONS
`
`Rinastiti et al. “Histological evaluation of rabbit gingival wound
`healing transplanted with human amniotic membrane” Int. J. Oral
`Maxillofac. Surg. 2006, 35: pp. 247—251.
`Talmshima et a1. “Characterization of laminin isoforms in human
`amnion" Tissue and Cell 40 (2008). pp. 75-77.
`
`
`
`Talmi et a1. “Antibacterial Properties of Human Amniotic Mern—
`Dranes" Placenta (1991), 12, pp. 285-288.
`Ebihara et al. “The Functions of Exogenous and Endogenous
`naminin-S on Corneal Epithelial Cells" Exp. Eye Res. (2000) 71, pp.
`69—79.
`:ukuda et al. “Differential Distribution of Subchains ofthe Basement
`Membrane Components Type IV Collagen and Larninin Among the
`Amniotic Membrane. Cornea, and Conjunctiva” Cornea 18(1), 1999,
`3p. 73-79.
`{ao et al. “Identification of Antiangiogenic and Antiinflammatory
`)r'oteins in Human Amniotic Membrane” Cornea 19(3), 2000, pp.
`348-3 52.
`(oizumi ct a]. “Growth Factor mRNA and protein in preserved
`luman amniotic membrane” Current Eye Research, 2000, vol. 20,
`No. 3. pp. 173—177.
`Shatia et al. “The Mechansrn of Cell Interaction and Response on
`)ecellularized Human Amniotic Membrane: Implications in Wblrrrd
`Iealing” http:wawwoundsresearch.com/ar‘ticle/76 l4, pp.
`[-24,
`May 7, 2008.
`Niknejad et a1. “Properties of the Amniotic Membrane for Potential
`Jse in Tissue Engineering” European Cells and Materials, vol. 15,
`2008 (pp, 88-99).
`Toda et al. “The Potential of Amniotic Membrane/Amnion-Derived
`Cells for Regeneration of Various Tissues” J. Pharmacol Sci, 105,
`215-228 (2007).
`Park et al. “Healing of a porcine burn wound dressed with human and
`bovine amniotic membranes” Wound Rep Reg (2008) 16. 520-528.
`International Search Report dated May 30. 2012 for international
`application No. PCT/US l 2/24858.
`International Search Report andWritten Opinion dated Nov. 13, 2008
`in related PCT Application No. PCTUSOS/75608.
`
`MTF Ex. 1001, pg. 2
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 1 0f8
`
`US 8,323,701 B2
`
`START~O
`
`INITIAL TISSUE m I 10
`COLLECTION
`
`MATERIAL CHECK-IN
`AND EVALUATION
`
`120
`
`GROSS TISSUE
`PROCESSING
`
`130
`
`100
`
`J
`
`REMOVAL OF
`EI’I'I'I-IELIAL LAYER
`
`In 5
`
`FIG. 1
`
`CHE MICAL
`DECONTAMINATION
`
`1 40
`
`DEI-IYDR ATION
`
`I 50
`
`CUTTING AND
`PACKAGING
`
`__
`
`160
`
`165
`
`
`
` STERILIZATION
`1
`PRODUCT RELEASE
`
`’—_170
`
`MTF Ex. 1001, pg. 3
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 2 of8
`
`US 8,323,701 B2
`
`Tiwue shackwxn Form
`
`Fig 2
`
`
`
`Does donor number on paperwork and tissue ID
`
`Log Tissue: into Refrigerator Lag:
`
`Complete
`
`.
`
`[:1 Yes;
`
`[:1 N0
`
`
`
`MTF Ex. 1001, pg. 4
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 3 of8
`
`US 8,323,701 B2
`
`Raw Tissue: Asgnssment Form
`
`FIG 3
`
`
`
`Donor:
`
`.
`
`.
`
`lnsptect tissue:
`
`mbj'mwdegeimwwWWWMJmJMXLQLLNO~~~~~~~~~~~~~
`[:1 Yes
`1:] No
`v.i@983awfififiifléafli......................................................................................................
`
`-2925,E’Ei’iflfififflREESESEEEREEEEEEEQIE.EQ9§C,,,,,,,,E22Jig-L...........£3919............
`
`Tissue shows signs Omaa90?mwmwbmwflmflo,,,,,,,,,,,
`
`[r WWW‘DEflWiE357232273163EEF'HEaBWSQXmflEfiXF/fifiéfifi"""""""""""""""""""""""""""""""""""""""""""I
`
`I
`
`Tissue is acceptable for processing:
`
`[:3 Pass
`
`C] Fail
`
`Size offlssue:
`
`.
`
`CM X
`
`CM
`
`Comments
`
`M a mm“ WWWWWWWWWM
`
`>
`
`M7E§§§fi§§7§éeh 33363336 according f0 Séfiifééfi'ufigfie Aka}(aégfiiémjggjbéfiégffiyémdesrfiéflcy’m
`acceptable for processing.
`
`Signature:
`
`MTF Ex. 1001, pg. 5
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 4 of8
`
`US 8,323,701 B2
`
`Dehydratian Pmcew Farm
`
`
`FIG 4
`
`M WWWWWWWW W......................................~15WWW.5 n
`
`i .........................
`
`Load Technician:
`
`
`
`
`
`
`
`
`”(356mm Overi 5m T‘efifiéiaihre (69 45c ‘
`
`'
`
`
`
`Dried
`
`
`Finish Time:
`
`Finish Daie:
`
`] Totai Dehydration Time:
`
`Residual Moisture Samples Prepareij: {:1
`
`
`
`
`Unload Techniciaq
`
`
`;
`
`
`
`hours
`
`minutes
`
`MTF Ex. 1001, pg. 6
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 5 of8
`
`US 8,323,701 B2
`
`
`
`FIG. 5
`
`MTF Ex. 1001, pg. 7
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 6 0f 8
`
`US 8,323,701 B2
`
`Fibrous layer
`wf‘fiwm”””””
`Epithelial layer
`
`
`
`630
`
`Fibrous layer
`MW “620
`Epithelial layer
`
`Fibrous layer
`
`Basement mem brane layer
`
`61 0
`
`f
`
`DRYING FIXTURE
`
`lfimm600
`
`Stromal layer
`“W_WW
`Epithelial layer
`
`Stmmal layer
`W ”WWW
`Epithelial layer
`
`740
`
`(
`73 )
`
`Stromal layer
`_
`.
`Epithelial layer
`
`Stromal layer
`Epithelial layer
`
`Stromal layer
`
`
`
`‘
`
`“”720
`
`710
`
`m 700
`
`MTF EX. 1001, pg. 8
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 7 0f8
`
`US 8,323,701 B2
`
`Choriori fibroblast membrane 1
`.
`W’
`Chomon basement membrane
`J
`
`O
`
`81
`
`Amniotic fibroblast membrane
`‘““”'"‘““““m""""""
`Amniotic basement membrane
`
`
`800
`
`1
`
`DRYING FIXTURE
`
`lam-"600
`
`FIG. 8
`
`Basement membrane layer
`W 940
`lrribrous layer
`
`Fibrous layer
`,
`,
`Epithelial layer
`
`Fibrous layer
`.
`.
`Epithelial layer
`
`930
`
`W‘‘‘‘‘ 920
`
`Fibrous layer
`WMWWMMW..................................... WW 9 l 0
`Basement membrane layer
`
`FIG, 9
`
`MTF EX. 1001, pg. 9
`
`

`

`US. Patent
`
`Dec. 4, 2012
`
`Sheet 8 of8
`
`US 8,323,701 B2
`
`
`
`MTF EX. 1001, pg. 10
`
`

`

`US 8,323,701 B2
`
`1
`PLACEVTAI. TISSUE CRAFTS
`
`
`
`
`CROSS R 3FERENC 3 TO RET AT3D
`APPLICATIONS
`
`
`
`This application is a continuation application ofUS appli-
`cation Ser. No. 12/206,508, filed on Sep. 8, 2008, which
`claims priority upon US. provisional application Ser. Nos.
`60/970,780, filed Sep. 7, 2007; 60/986,665, filed Nov. 9,
`
`2007; and 60/989,299, filed Nov. 20, 2007. Each application
`is hereby incorporated by reference in its entirety for all of its
`teachings.
`
`BACKGROUND
`
`l—luman placental membrane (e.g. amniotic membrane or
`tissue) has been used for various types of reconstructive sur-
`gical procedures since the early 1900s. The membrane serves
`as a substrate material, more commonly referred to as a bio-
`logical dressing or patch graft. Such a membrane has also
`been used widely for ophthalmic procedures. Typically, such
`membrane is either frozen or dried for preservation and stor-
`age until needed for surgery.
`Suchplacental tissue is typically harvested after an elective
`Cesarean surgery. The placenta is composed of the umbilical
`cord and amniotic sac. The amniotic sac, corrnnonly referred
`to as the amniotic membrane. has two primary layers of
`tissue, amnion and chorion. Amnion tissue is innermost layer
`of the amniotic sac and in direct contact with the amniotic
`fluid. The amniotic sac contains the amniotic fluid and pro-
`tects the fetal environment. Histological evaluation indicates
`that the membrane layers ofthe amnion consist of single layer
`of epithelium cells, thin reticular fibers (basement mem—
`brane), a thick compact layer, and fibroblast layer, The fibrous
`layer of anmion (i.e.. the basement membrane) contains col-
`lagen types IV, V, and VII, and cell-adhesion bio-active fac-
`tors including fibronectin and laminins.
`Amnion tissue provides unique grafting characteristics
`when used for surgical procedures, including providing a
`matrix for cellular migration/proliferation, providing a natu-
`ral biological barrier, are non—immunogenic, and contains
`numerous bio-active molecules, which can be used as a mem-
`3rane to assist in tissue regeneration and improved healing
`outcomes in numerous applications. The membrane has the
`capability to self—adhere or, in the alternative, is susceptible of
`3(3ng fixed in place using different techniques including
`fibrin glue or suturing, Such grafts, when properly prepared,
`can be stored at room temperature for extended periods of
`ime, without need for refrigeration or freezing, until needed
`or a surgical procedure.
`Known clinical procedures or applications for such amnion
`grafts include ocular reconstruction, burns, anti-adhesion
`applications, barrier membranes, and general wound care.
`)escribed herein are membranes utilizing amnion tissue as
`juilding blocks to build membranes well suited for use in a
`variety of application such as, for example. perioplastic sur-
`gery and other surgical applications involving human mucosa
`issue. The grafts and methods described herein utilize these
`iiological molecules to enhance the performance ofthe amni-
`otic membrane.
`
`
`
`
`
`SUMMARY OF INVENTION
`
`Described herein are tissue grafts derived from the pla—
`centa. The grafts are composed ofat least one layer of amnion
`tissue where the epithelium layer has been substantially
`removed in order to expose the basement layer to ho st cells.
`
`m
`
`10
`
`15
`
`25
`
`3O
`
`35
`
`40
`
`45
`
`5O
`
`55
`
`60
`
`65
`
`2
`By removing the epithelium layer, cells from the host can
`more readily interact with the cell-adhesion bio-active factors
`located onto top and within of the basement membrane. Also
`described herein are methods for making and using the tissue
`grafts. The laminin structure of amnion tissue is nearly iden—
`tical to that of native human tissue such as, for example, oral
`mucosa tissue. This includes high level of laminin-5, a cell
`adhesion bio-active factor show to bind gingival epithelia-
`cells, found throughout upper portions ofthe basement mem—
`brane. The advantages ofthe invention will be set forth in part
`in the description which follows, and in part will be obvious
`from the description, or may be learned by practice of the
`aspects described below. The advantages described below
`will be realized and attained by means of the elements and
`combinations particularly pointed out
`in the appended
`claims. It is to be understood that both the foregoing general
`description and the following detailed description are exem-
`plary and explanatory only and are not restrictive.
`In one embodiment, there is provided a tissue graft com-
`prising a first membrane comprising an amnion and one or
`more additional membranes laminated to the first membrane,
`wherein the first membrane comprises an exposed basement
`membrane and stromal layer, and the one or more additional
`membranes are adjacent to the stromal layer,
`
`BRIEF DESCRIPTION OF TI IE DRAWINGS
`
`Tie accompanying drawings, which are incorporated in
`and constitute a part of this specification, illustrate several
`aspects described below.
`3 G. 1 is an overview flow chart of the process for making
`the tissue grafts described herein.
`3 G. 2 is an exemplary tissue check—in form used with the
`tissue grafts described herein.
`3 G. 3 is an exemplary raw tissue assessment form used
`with the tissue grafts described herein.
`3 G. 4 is an exemplary dehydration process form used with
`the tissue grafts described herein.
`3 G. 5 is a perspective view of an exemplary drying fixture
`for making the tissue grafts described herein.
`3 GS. 6—9 are representative side views of different tissue
`(Ic:
`raf’s described herein,
`3 G. 10 is a top view of a tissue graft with perforations.
`
`
`
`
`
`DETAILED D: LUSCRIPTION
`
`3efore the present articles and methods are disclosed and
`described, it is to be understood that the aspects described
`below are not limited to specific compounds, synthetic meth-
`ods, or uses as such may. of course. vary. It is also to be
`understoodthat the terminology used herein is for the purpose
`of describing particular aspects only and is not intended to be
`limiting.
`In this specification and in the claims that follow, reference
`will be made to a number ofterms that shall be defined to have
`the following meanings:
`It must be noted that, as used in the specification and the
`appended claims, the singular fonns “a,” “an” and “the”
`include plural referents unless the context clearly dictates
`otherwise. Thus, for example, reference to “a bioactive agent”
`includes mixtures of two or more such agents, and the like.
`“Optional” or “optionally” means that the subsequently
`described event or circumstance can or cannot occur, and that
`the description includes instances where the event or circum—
`stance occurs and instances where it does not. For example,
`the phrase “optionally cleaning step” means that the cleaning
`step may or may not be performed.
`
`MTF Ex. 1001, pg. 11
`
`

`

`US 8,323,701 B2
`
`3
`Titles or subtitles may be used in the specification for the
`convenience of a reader, which are not intended to influence
`the scope of the present invention. Additionally, some terms
`used in this specification are more specifically defined below.
`I. Tissue Grafts and Methods for Making Thereof
`Described herein are tissue grafts and methods of making
`and using thereof. In general, the tissue grafts are multilay-
`ered systems composed ofone or more membranes laminated
`to a base amnion, where the epithelium layer of the base
`amnion has been substantially removed. In one aspect, the
`process for preparing the amnion and removing the epithe-
`lium layer involves
`(a) obtaining a placenta from a subject, wherein the placenta
`comprises an amniotic membrane layer and a chorion tis—
`sue layer;
`(b) separating the chorion tissue layer from the amnion layer,
`wherein the amnion comprises epithelium cells adjacent to
`a basement membrane;
`((1) removing substantially all of the epithelium cells to
`expose the basement membrane of the amnion;
`(e) mounting the first membrane onto a surface of a drying
`fixture;
`(f) mounting one or more additional membranes on the first
`membrane to produce a layered tissue graft; and
`(g) dehydrating the layered tissue graft on the drying fixture.
`FIG. 1 depicts an overview (100) of the steps to harvest,
`process, and prepare placental material for later use as a tissue
`graft is disclosed. More detailed descriptions and discussion
`regarding each individual step will follow. Initially, the pla—
`centa tissue is collected from a consenting patient following
`an elective Cesarean surgery (step 110). The material is pre-
`served and transported in conventional tissue preservation
`manner to a suitable processing location or facility for check—
`in and evaluation (step 120). Gross processing, handling, and
`separation of the tissue layers then takes place (step 130).
`After the epithelium layer is substantially removed from the
`arrmion to expose the base membrane (step 135), acceptable
`tissue is then decontaminated (step 140), dehydrated (step
`150), cut and packaged (step 160), tissue is radiological ter-
`minal sterilized using gamma radiation (step 165), and
`released (step 170) to the market for use by surgeons and other
`medical professionals in appropriate surgical procedures and
`
`for wound care. Each step is described in detail below.
`Initial Tissue Collection (Step 110)
`The recovery of placenta tissue originates in a hospital,
`where it is collected during a Cesarean section birth. The
`donor, referring to the mother who is about to give birth,
`voluntarily submits to a comprehensive screening process
`designed to provide the safest tissue possible for transplanta-
`tion. The screening process preferably tests for antibodies to
`the human immunodeficiency Virus type 1 and type 2 (anti—
`IIIV—I and anti-IIIV-Z), hepatitis B surface antigens (IIB-
`sAg), antibodies to the hepatitis C virus (anti-HC V), antibod-
`ies to the human T—lyrnphotropic Virus type I and type II
`(anti—HTLV—I and anti—HTLV—II), CMV, and syphilis, using
`conventional serologieal tests. The above list oftests is exem-
`plary only, as more, fewer, or different tests may be desired or
`necessary over time or based upon the intended use of the
`grafts, as will be appreciated by those skilled in the art.
`Based upon a review of the donor’s information and
`screening test results, the donor will either be deemed accept-
`able or not. In addition, at the time of delivery, cultures are
`taken to determine the presence of bacteria, for example,
`Clostridium or Streptococcus. If the donor’s information,
`screening tests, and the delivery cultures are all satisfactory
`(i.e., do not indicate any risks or indicate acceptable level of
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`risk), the donor is approved by a medical director and the
`issue specimen is designated as initially eligible for further
`oroces sing and evaluation.
`Human placentas that meet the above selection criteria are
`oreferably bagged in a saline solution in a sterile shipment
`jag and stored in a container of wet ice for shipment to a
`orocessing location or laboratory for further processing.
`Ifthc placenta tissue is collected prior to the completion or
`obtaining of results from the screening tests and delivery
`cultures, such tissue is labeled and kept in quarantine. The
`issue is approved for further processing only after the
`required screening assessments and delivery cultures, which
`declare the tissue safe for handling and use, are satisfied and
`obtains final approval from a medical director.
`VIaterial Check-in and Evaluation (Step 120)
`Upon arrival at the processing center or laboratory, the
`shipment is opened and verified that the sterile shipment
`Jag/container is still sealed and in the coolant, that the appro-
`ariate donor paperwork is present, and that the donor number
`on the paperwork matches the ntmrber 011 the sterile shipment
`jag containing the tissue. The sterile shipment bag containing
`he tissue is then stored in a refrigerator until ready for further
`aroces sing. All appropriate forms, including a tissue check-in
`form, such as that shown in FIG. 2, are completed and chain
`of custody and handling logs (not shown) are also completed.
`Gross Tissue Processing (Step 130)
`When the tissue is ready to be processed further, the sterile
`supplies necessary for processing the placenta tissue further
`are assembled in a staging area in a controlled environment
`and are prepared for introduction into a controlled environ-
`ment. Ifthe controlled environment is a manufacturing hood,
`the sterile supplies are opened and placed into the hood using
`conventional sterile technique. If the controlled environment
`is a clean room, the sterile supplies are opened and placed on
`a cart covered by a sterile drape, All the work surfaces are
`covered by a piece of sterile drape using conventional sterile
`techniques, and the sterile supplies and the processing equip—
`ments are placed on to the sterile drape, again using conven—
`tional sterile techniques.
`Processing equipment is decontaminated according to con-
`ventional and industry-approved decontamination proce-
`dures and then introduced into the controlled environment.
`The equipment is strategically placed within the controlled
`environment to minimize the chance for the equipment to
`come in proximity to or is inadvertently contaminated by the
`issue specimen.
`Next, thcplacenta is removed from the sterile shipmentbag
`and transferred aseptically to a sterile processing basin within
`Ire controlled environment. The sterile basin contains hyper-
`isotonic saline solution (e.g., 18% NaCl) that is at room or
`near room temperature. The placenta is gently massaged to
`ielp separate blood clots and to allow the placenta tissue to
`reach room temperature, which will make the separation of
`he amnion and chorion layers from each other, as discussed
`iereinafter, easier. After having warmed up to the ambient
`emperature (after about 10-30 minutes), the placenta is then
`removed from the sterile processing basin and laid flat on a
`orocessing tray with the amniotic membrane layer facing
`down for inspection.
`The placenta tissue is examined and the results of the
`examination are documented on a “Raw Tissue Assessment
`:orm” similar to that shown in FIG. 3. The placenta tissue is
`examined for discoloration, debris or other contamination,
`odor, and signs ofdamage. The size ofthe tissue is also noted.
`A determination is made, at this point, as to whether the tissue
`is acceptable for further processing.
`
`
`
`MTF Ex. 1001, pg. 12
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`US 8,323,701 B2
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`5
`Next, ifthe placenta tissue is deemed acceptable for further
`firocessing, the amnion and chorion layers of the placenta
`issue are then carefully separated. In one aspect, the materi-
`als and equipment used in this procedure include a processing
`ray, 18% saline solution, sterile 4x4 sponges, and two sterile
`\Ialgene jars. The placenta tissue is then closely examined to
`find an area (typically a corner) in which the amnion layer can
`3e separated from the chorion layer. The amnion appears as a
`hin, opaque layer on the chorion.
`Prior to removal of the epithelium, the fibroblast layer is
`identified by gently contacting each side of the membrane
`with a piece ofsterile gauze or a cotton tipped applicator. The
`fibroblast layer will stick to the test material. The amnion is
`alaccd into processing tray fibroblast layer down. Using a
`olunt instrument, a cell scraper or sterile gauze, any residual
`alood is also removed. This step must be done with adequate
`care, again, so as not to tear the amnion or chorion tissues. The
`cleaning ofthe amnion is complete once the amnion tissue is
`smooth and opaque-white in appearance. Ifthe amnion tissue
`is cleaned too much, the jelly-like fibroblast layer can be
`removed. Any areas of the amnion cleaned too aggressively
`and appear clear will be unacceptable and will ultimately be
`discarded.
`Removal of Epithelium Layer from Amnion (Step 135:)
`The epithelium layer present on the arrmion is substantially
`removed in order to expose the basement layer ofthe amnion.
`The significance of removing the epithelium layer
`is
`described below. The term “substantially removed” with
`respect to the amount ofepithelium removed is defined herein
`as removing greater than 90%, greater than 95%, or greater
`than 99% of the epithelial cells from the amnion. The pres-
`ence or absence of epithelial cells remaining on the amnion
`layer can be evaluated using techniques known in the art. For
`example, after removal ofthe epithelial cell layer, a represen—
`tative tissue sample from the processing lot is placed onto a
`standard microscope examination slide, The tissue sample is
`then stained using Eosin Y Stain and evaluated as described
`below. The sample is then covered and allowed to stand. Once
`an adequate amount of time has passed to allow for staining,
`visual observation is done under ma gnification,
`The epithelium layer can be removed by techniques known
`in the art. For example, the epithelium layer can be scraped off
`of the amnion using a cell scraper. Other techniques include,
`but are not limited to, freezing the membrane, physical
`removal using a cell scraper, or exposing the epithelial cells to
`nonionic detergents, anionic detergents, and nucleases. The
`de—epithelialized tissue is then evaluated to determine that the
`basement membrane has not been compromised and remains
`intact. This step is performed after completion ofthe process-
`ing step and the tissue has been dehydrated as described in the
`next section. For example, a representative sample graft is
`removed for microscopic analysis. The tissue sample is place
`onto a standard slide and 100 ul of EosinY stain is applied to
`the sample and allowed to set. The tissue sample is then
`examined under magnification. Cellular material will stain
`dark indicating the presence of cells. If no stained cells are
`present, de—epithelization has been achieved.
`Chemical Decontamination (Step 140)
`The amnion is then placed into a sterile Nalgene jar for the
`next step for additional cleaning. Ifthe chorion is to be recov—
`ered and processed further, it too is placed in its own sterile
`Nalgene jar for additional cleaning. If the chorion is not to be
`kept or used further, it can be discarded in an appropriate
`biohazard container. In one aspect, the following procedure
`can be used to clean the amnion.
`Each Nalgene jar is aseptically filled with 18% saline
`hypertonic solution and sealed (or sealed with a top). The jar
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`6
`is then placed on a rocker platform and agitated for between
`30 and 90 minutes, which further cleans the tissue of con-
`aminants. If the rocker platform was not in the critical envi-
`ronment (e.g., the manufacturing hood), the Nalgene jar is
`returned to the controlled]sterile environment and opened.
`Jsing sterile forceps or by aseptically decanting the contents,
`he tissue is gently removed from the Nalgene jar containing
`he 18% hyperisotonic saline solution and placed into an
`empty Nalgene jar. This empty Nalgene jar with the tissue is
`hen aseptically filled with a pre-mixed antibiotic solution.
`)referably, the premixed antibiotic solution is comprised of a
`cocktail ofantibiotic s, such as Streptomycin Sulfate and Gen-
`amicin Sulfate. Other antibiotics, such as Polymixin B Sul—
`fate and Racitracin, or similar antibiotics now available or
`available in the future, are also suitable. Additionally, it is
`areferred that the antibiotic solution be at room temperature
`when added so that it does not change the temperature of or
`otherwise damage the tissue. This jar or container containing
`he tissue and antibiotics is then sealed or closed and placed
`on a rocker platform and agitated for, preferably, between 60
`and 90 minutes. Such rocking or agitation of the tissue within
`he antibiotic solution further cleans the tissue of contarni—
`nants and bacteria.
`Again, if the rocker platform was not in the critical envi-
`ronment (e.g., the manufacturing hood), the jar or container
`containing the tissue and antibiotics is then returned to the
`critical/sterile environment and opened. Using sterile for-
`ceps, the tissue is gently removed from the jar or container
`and placed in a sterile basin containing sterile water or normal
`saline (0.9% saline solution). The tissue is allowed to soak in
`place in the sterile water/normal saline solution for at least 10
`to 15 minutes. The tissue may be slightly agitated to facilitate
`removal ofthe antibiotic solution and any other contaminants
`from the tissue. After at least 10 to 15 minutes, the tissue is
`ready to be dehydrated and processed further.
`In the case when the chorion is to be used, the following
`exemplary procedure can be used. After separation of the
`chorion from the arrmion and removal of clotted blood from
`the fibrous layer, the chorion is rinsed in 18% saline solution
`for 30 min, During the first rinse cycle, 18% saline is heated
`in a sterile container using laboratory heating plate such that
`the solution temperature is approximately 48° C. The solution
`is decanted, the chorion tissue is placed into the sterile con—
`tainer, and decanted saline solution is poured into the con-
`tainer. The container is sealed and placed on rocker plate and
`agitated for 1 hour. After 1 hour agitation bath, remove the
`tissue and place the tissue into second heated agitation bath
`for an additional 1 hour rinse cycle. Next, the chorion tissue is
`placed into 200 m1 of 0.5% Triton-X wash solution. The
`container is sealed and agitated without heat for 2 hours. The
`tissue is next washed with deionized water (250 m1 of DI
`waterx4) with vigorous motion for each rinse. The tissue is
`removed and placed into a container of leBS w/EDTA
`solution. The container is sealed and agitated for 1 hour at
`controlled temperature for 8 hours. The tissue is removed and
`rinsed using sterile water. A visual inspection was performed
`to remove any remaining discolored fibrous blood material
`from the membrane. Membrane should have a cream white
`visual appearance with no evidence of brownish discolora-
`tion.
`Preparation of Tissue Graft and Dehydration (Step 150)
`After removal of the epithelium layer, the amnion is ready
`to be used to produce the tissue graft. In one aspect, the
`anmion layer is laid on a suitable drying fixture, where the
`exposed basement membrane is adjacent to the surface ofthe
`drying fixture. In other aspects, the amnion can be placed on
`the surface of the drying fixture such that the exposed base-
`
`MTF EX. 1001, pg. 13
`
`

`

`US 8,323,701 B2
`
`
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`7
`ment membrane is facing up. The drying fixture is preferably
`sized to be large enough to receive the amnion tissue, fully, in
`aid out, fiat fashion. In one aspect, the drying fixture is made
`of Teflon or of Delrin, which is the brand name for an acetal
`resin engineering plastic invented and sold by DuPont and
`which is also available conmiercially from Werner Machine,
`nc. in Marietta, Ga. Any other suitable material that is heat
`and cut resistant, capable of