in accordance with the present invention there may be employed conventional molecular biology , microbiology , and recombinant dna techniques within the skill of the art . such techniques are explained fully in the literature . see , e . g ., sambrook et al , 2001 , “ molecular cloning : a laboratory manual ”; ausubel , ed ., 1994 , “ current protocols in molecular biology ” volumes i - iii ; celis , ed ., 1994 , “ cell biology : a laboratory handbook ” volumes i - iii ; coligan , ed ., 1994 , “ current protocols in immunology ” volumes i - iii ; gait ed ., 1984 , “ oligonucleotide synthesis ”; hames & amp ; higgins eds ., 1985 , “ nucleic acid hybridization ”; hames & amp ; higgins , eds ., 1984 ,“ transcription and translation ”; freshney , ed ., 1986 , “ animal cell culture ”; irl press , 1986 , “ immobilized cells and enzymes ”; perbal , 1984 , “ a practical guide to molecular cloning .” where a range of values is provided , it is understood that each intervening value , to the tenth of the unit of the lower limit unless the context clearly dictates otherwise , between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention . the upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the invention , subject to any specifically excluded limit in the stated range . where the stated range includes one or both of the limits , ranges excluding either both of those included limits are also included in the invention . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention , the preferred methods and materials are now described . it must be noted that as used herein and in the appended claims , the singular forms “ a ,” “ and ” and “ the ” include plural references unless the context clearly dictates otherwise . the anti - inflammatory properties of amnion - derived cytokine solution ( accs ) are currently being used to assist in wound healing in human clinical trials . accs contains more than 200 proteins , cytokines , and growth factors in solution and has been shown to reduce inflammation in several animal models ( see examples below ). in addition , accs is presently being tested in several human clinical trials involving inflammation resulting from radiation burns and skin grafts in diabetic patients being treated for burns . amp cell compositions are prepared using the steps of a ) recovery of the amnion from the placenta , b ) dissociation of the epithelial cells from the amniotic membrane using a protease , c ) culturing of the cells in a basal medium with the addition of a naturally derived or recombinantly produced human protein ( i . e . human serum albumin ) and no non - human animal protein ; d ) selecting amp cells from the epithelial cell culture , and , optionally e ) further proliferation of the cells , optionally using additional additives and / or growth factors ( i . e . recombinant human egf ). details are contained in u . s . pat . no . 8 , 278 , 095 , issued oct . 2 , 2012 , u . s . pat . no . 8 , 058 , 066 , issued nov . 15 , 2011 and u . s . pat . no . 8 , 088 , 732 , issued jan . 3 , 2012 , all of which are incorporated herein by reference . culturing of the amp cells — the amp cells are cultured in a basal medium . such medium includes , but is not limited to , epilife ® culture medium for epithelial cells ( cascade biologicals ), opti - pro ™ serum - free culture medium , vp - sfm serum - free medium , imdm highly enriched basal medium , knockout ™ dmem low osmolality medium , 293 sfm ii defined serum - free medium ( all made by gibco ; invitrogen ), hpgm hematopoietic progenitor growth medium , pro 293s - cdm serum - free medium , pro 293a - cdm serum - free medium , u1tramdck ™ serum - free medium ( all made by cambrex ), stemline ® t - cell expansion medium and stemline ® ii hematopoietic stem cell expansion medium ( both made by sigma - aldrich ), dmem culture medium , dmem / f - 12 nutrient mixture growth medium ( both made by gibco ), ham &# 39 ; s f - 12 nutrient mixture growth medium , m199 basal culture medium ( both made by sigma - aldrich ), and other comparable basal media . such media should either contain human protein or be supplemented with human protein . as used herein a “ human protein ” is one that is produced naturally or one that is produced using recombinant technology . “ human protein ” also is meant to include a human derivative or preparation thereof , such as human serum , which contains human protein . in specific embodiments , the basal media is imdm highly enriched basal medium , stemline ® t - cell expansion medium or stemline ® ii hematopoietic stem cell expansion medium , or opti - pro ™ serum - free culture medium , or combinations thereof and the human protein is human serum albumin is at least 0 . 5 % and up to 10 %. in particular embodiments , the human serum albumin is from about 0 . 5 to about 2 %. in a specific embodiment the human albumin is at 0 . 5 %. the human albumin may come from a liquid or a dried ( powder ) form and includes , but is not limited to , recombinant human serum albumin , plasbumin ® normal human serum albumin and plasmanate ® human blood fraction ( both made by talecris biotherapeutics ). in a most preferred embodiment , the cells are cultured using a system that is free of non - human animal products to avoid xeno - contamination . in this embodiment , the culture medium is imdm highly enriched basal medium , stemline ® t - cell expansion medium or stemline ® ii hematopoietic stem cell expansion medium , opti - pro ™ serum - free culture medium , or dmem culture medium , with human serum albumin ( i . e . plasbumin ® normal human serum albumin ) added up to amounts of 10 %. the invention further contemplates the use of any of the above basal media wherein animal - derived proteins are replaced with recombinant human proteins and animal - derived serum albumin , such as bsa , is replaced with human serum albumin . in preferred embodiments , the media is serum - free in addition to being animal - free . optionally , other factors are used . in one embodiment , recombinant human epidermal growth factor ( hegf ) at a concentration of between 0 . 01 - 1 μg / ml is used . in a particular embodiment , the hegf concentration is around 10 - 20 ng / ml . all supplements are human clinical grade . generation of accs — the amp cells of the invention can be used to generate accs . in one embodiment , the amp cells are isolated as described herein and 1x10 6 cells / ml are seeded into t75 flasks containing between 5 - 30 ml culture medium , preferably between 10 - 25 ml culture medium , and most preferably about 10 ml culture medium . the cells are cultured until confluent , the medium is changed and in one embodiment the accs is collected 1 day post - confluence . in another embodiment the medium is changed and accs is collected 2 days post - confluence . in another embodiment the medium is changed and accs is collected 4 days post - confluence . in another embodiment the medium is changed and accs is collected 5 days post - confluence . in a preferred embodiment the medium is changed and accs is collected 3 days post - confluence . in another preferred embodiment the medium is changed and accs is collected 3 , 4 , 5 , 6 or more days post - confluence . skilled artisans will recognize that other embodiments for collecting accs from amp cell cultures , such as using other tissue culture vessels , including but not limited to cell factories , flasks , hollow fibers , or suspension culture apparatus , or collecting accs from sub - confluent and / or actively proliferating cultures , are also contemplated by the methods of the invention . it is also contemplated by the instant invention that the accs be cryopreserved following collection . it is also contemplated by the invention that accs be lyophilized following collection . it is also contemplated by the invention that accs be formulated for sustained - release , timed - release , targeted - release , extended - release , etc ., following collection . skilled artisans are familiar with cryopreservation lyophilization , and sustained - release / timed - release / targeted - release / extended - release formulation methodologies . the accs of the invention is characterized by assaying for physiologically relevant cytokines secreted in the physiologically relevant range of about 5 - 16 ng / ml for vegf , about 3 . 5 - 4 . 5 ng / ml for angiogenin , about 100 - 165 pg / ml for pdgf , about 2 . 5 - 2 . 7 ng / ml for tgfβ2 , about 0 . 68 μg / ml for timp - 1 and about 1 . 04 μg / ml for timp - 2 . it is also contemplated by the invention that accs , including pooled accs , be concentrated prior to use . the appropriate level of concentration required will be dependent upon the intended use and therefore will need to be empirically determined . the compositions of the invention can be prepared in a variety of ways depending on the intended use of the compositions . for example , a composition useful in practicing the invention may be a liquid comprising an agent of the invention , i . e . accs in solution , in suspension , or both ( solution / suspension ). the term “ solution / suspension ” refers to a liquid composition where a first portion of the active agent is present in solution and a second portion of the active agent is present in particulate form , in suspension in a liquid matrix . a liquid composition also includes a gel . the liquid composition may be aqueous or in the form of an ointment , salve , cream , or the like . an aqueous suspension or solution / suspension useful for practicing the methods of the invention may contain one or more polymers as suspending agents . useful polymers include water - soluble polymers such as cellulosic polymers and water - insoluble polymers such as cross - linked carboxyl - containing polymers . an aqueous suspension or solution / suspension of the present invention is preferably viscous or muco - adhesive , or even more preferably , both viscous and muco - adhesive . the accs may be formulated as sustained - release / controlled - release / timed - release / targeted - release , etc ., compositions . skilled artisans are familiar with methodologies to create such compositions of therapeutic agents , including protein - based therapeutic agents such as accs . in addition , accs may be formulated as a spray , gel , slave , etc . pharmaceutical compositions — the present invention provides pharmaceutical compositions of accs and a pharmaceutically acceptable carrier . the term “ pharmaceutically acceptable ” means approved by a regulatory agency of the federal or a state government or listed in the u . s . pharmacopeia or other generally recognized pharmacopeia for use in animals , and more particularly , in humans . the term “ carrier ” refers to a diluent , adjuvant , excipient , or vehicle with which the composition is administered . such pharmaceutical carriers can be sterile liquids , such as water and oils , including those of petroleum , animal , vegetable or synthetic origin , such as peanut oil , soybean oil , mineral oil , sesame oil and the like . suitable pharmaceutical excipients include starch , glucose , lactose , sucrose , gelatin , malt , rice , flour , chalk , silica gel , sodium stearate , glycerol monostearate , talc , sodium chloride , dried skim milk , glycerol , propylene , glycol , water , ethanol and the like . the composition , if desired , can also contain minor amounts of wetting or emulsifying agents , or ph buffering agents . these compositions can take the form of solutions , suspensions , emulsion , tablets , pills , capsules , powders , sustained - release formulations and the like . examples of suitable pharmaceutical carriers are described in “ remington &# 39 ; s pharmaceutical sciences ” by e . w . martin , and still others are familiar to skilled artisans . the pharmaceutical compositions of the invention can be formulated as neutral or salt forms . pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric , phosphoric , acetic , oxalic , tartaric acids , etc ., and those formed with free carboxyl groups such as those derived from sodium , potassium , ammonium , calcium , ferric hydroxides , isopropylamine , triethylamine , 2 - ethylamino ethanol , histidine , procaine , etc . one of skill in the art may readily determine the appropriate concentration , or dose , of accs for a particular purpose . the skilled artisan will recognize that a preferred dose is one which produces a therapeutic effect , such as preventing and / or down - regulating the inflammatory response associated with ischemic injury , in a donor tissue or organ . of course , proper doses of accs will require empirical determination at time of use based on several variables including but not limited to the type of donor tissue or organ and the like . the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention , and are not intended to limit the scope of what the inventors regard as their invention . efforts have been made to ensure accuracy with respect to numbers used ( e . g ., amounts , temperature , etc .) but some experimental errors and deviations should be accounted for . unless indicated otherwise , parts are parts by weight , molecular weight is average molecular weight , temperature is in degrees centigrade , and pressure is at or near atmospheric . amnion epithelial cells were dissociated from starting amniotic membrane using the dissociation agents pxxiii the average weight range of an amnion was 18 - 27 g . the number of cells recovered per g of amnion was about 10 - 15 × 10 6 for dissociation with pxxiii method of obtaining selected amp cells — amnion epithelial cells were plated immediately upon isolation from the amnion . after ˜ 2 - 3 days in culture non - adherent cells were removed and the adherent cells were kept . this attachment to a plastic tissue culture vessel is the selection method used to obtain the desired population of amp cells . adherent and non - adherent amp cells appear to have a similar cell surface marker expression profile but the adherent cells have greater viability and are the desired population of cells . adherent amp cells were cultured in basal medium supplemented with human serum albumin until they reached 120 , 000 - 150 , 000 cells / cm 2 . at this point , the cultures were confluent . suitable cell cultures will reach this number of cells between ˜ 5 - 14 days . attaining this criterion is an indicator of the proliferative potential of the amp cells and cells that do not achieve this criterion are not selected for further analysis and use . once the amp cells reached 120 , 000 - 150 , 000 cells / cm 2 , they were collected and cryopreserved . this collection time point is called p0 . the amp cells of the invention can be used to generate accs , including pooled accs . the amp cells were isolated as described above and about 1 × 10 6 cells / ml were seeded into t75 flasks containing ˜ 10 ml culture medium as described above . the cells were cultured until confluent , the medium was changed and accs was collected ˜ 3 days post - confluence . optionally , the accs is collected again after 3 days , and optionally again after 3 days . skilled artisans will recognize that other embodiments for collecting accs from confluent cultures , such as using other tissue culture vessels , including but not limited to cell factories , flasks , hollow fibers , or suspension culture apparatus , etc . are also contemplated by the methods of the invention ( see detailed description above ). it is also contemplated by the instant invention that the accs be cryopreserved , lyophilized , irradiated or formulated for sustained - release , etc ., following collection . it is also contemplated that accs be collected at different time points ( see detailed description for details ). accs was obtained essentially as described above . in certain embodiments , accs was collected multiple times from an amp cell culture derived from one placenta and these multiple accs collections were pooled together . such pools are referred to as “ sp pools ” ( more than one accs collection / one placenta ). in another embodiment , amp cell cultures were derived from several placentas , i . e . from 5 or 10 placentas . the amp cells from each placenta were cultured and one accs collection from each culture was collected and then they were all pooled . these pools are termed “ mp1 pools ” ( one accs collection / placenta , multiple placentas ). in yet another embodiment , amp cell cultures were derived from several placentas , i . e . from 5 or 10 placentas . the amp cells from each placenta were cultured and more than one accs collection was performed from each amp cell culture and then pooled . these pools are termed “ mp2 pools ” ( more than one accs collection / placenta , multiple placentas ). inflammatory model — use of accs to prevent onset of periodontal disease in an animal model objective : the aim of this study was to evaluate the preventive role of accs in porphyromonas gingivalis ( p . gingivalis )- induced experimental periodontitis in rabbits methods : eight new - zealand white rabbits were distributed into 3 groups : 1 . untreated ( n = 2 ), 2 . control ( unconditioned accs culture media ) ( n = 3 ), and 3 . accs ( n = 3 ). at baseline , all rabbits received silk ligatures bilaterally tied around mandibular second premolars under general anesthesia . the assigned test materials , accs or control , in volumes of 10 μl were topically applied to the ligated sites with a blunt needled - hamilton syringe from the time of ligature ; control animals received ligature , but no treatment . topical p . gingivalis - containing slurry ( 1 ml ) was subsequently applied to induce the periodontal inflammation . the application of test materials and p . gingivalis continued for 6 weeks on an every - other - day schedule . at 6 weeks , following euthanasia , the mandibles were surgically harvested . morphometric , radiographic and histologic evaluations were performed . results : macroscopic evaluations including soft tissue assessments , crestal bone and infrabony measurements showed significant periodontal breakdown induced by p . gingivalis in control and no treatment groups at 6 weeks compared to historical ligature - alone groups ( p = 0 . 05 , p = 0 . 03 , respectively ). accs application significantly inhibited soft tissue inflammation and prevented both crestal bone loss and infrabony defect formation compared to untreated and control groups ( p = 0 . 01 , p = 0 . 05 , respectively ). histologic assessments and histomorphometric measurements supported the clinical findings ; accs treated animals demonstrated significantly less inflammation in soft tissue and less bone loss compared to the untreated and control groups ( p = 0 . 05 ). conclusions : topical accs application prevents periodontal inflammatory changes and bone loss induced by p . gingivalis as shown both at clinical and histopathological level . accs has potential as a therapeutic approach for the prevention of periodontal diseases inflammatory model — use of accs to stop progression of or reverse periodontal disease in an animal model objective : the aim of this study was to evaluate the therapeutic actions of accs in the treatment of periodontitis induced by p . gingivalis . methods : the study was conducted using a two - phase rabbit periodontitis protocol : 1 — disease induction ( 6 weeks ) and 2 — treatment ( 6 weeks ). periodontal disease was induced in 16 new - zealand white rabbits by every - other - day application of topical p . gingivalis to ligatured mandibular premolars . at the end of phase 1 , 4 randomly selected rabbits were sacrificed to serve as the baseline disease group . for phase 2 , the remaining 12 rabbits were distributed into 3 groups ( n = 4 ), 1 — untreated , 2 — control ( unconditioned accs culture media ) and 3 — accs treatment . at the end of phase 2 , morphometric , radiographic and histologic evaluations were performed on harvested mandibles . results : the baseline disease group exhibited experimental periodontitis evidenced by tissue inflammation and bone loss . at the end of phase 2 , the untreated group showed significant disease progression characterized by increased soft and hard tissue destruction ( p = 0 . 05 ). the tissue inflammation and bone loss was significantly reduced by topical accs compared to baseline disease and untreated groups ( p = 0 . 05 ; p = 0 . 002 , respectively ). the control treatment also arrested disease progression compared to untreated group ( p = 0 . 01 ), but there was no improvement in periodontal health compared to baseline disease ( p = 0 . 4 ). histopathological assessments revealed similar findings ; accs stopped the progression of inflammatory process ( p = 0 . 003 ) and reversed bone destruction induced by p . gingivalis ( p = 0 . 008 ). the accs - treated group had minimal osteoclastic activity limited to crestal area compared to untreated and control groups , which showed a profound osteoclastogenic activity at the bone crest as well as at interproximal sites . conclusions : topical application of accs stopped the progression of periodontal inflammation and resulted in tissue regeneration in rabbit periodontitis indicating its potential therapeutic efficacy . inflammatory model — evaluate the efficacy of topically applied accs to inhibit irritant 12 - o - tetradecanoylphorbol - 3 - acetate ( tpa ) skin inflammation in mice method : topical treatment was given twice daily to the following groups : 1 . tpa + topical control ; 2 . tpa + accs ; 3 . tpa + clobetasol 0 . 05 topical solution ( the strongest available topical corticosteroid ); 4 . accs alone ; 5 . no treatment ( the other untreated ear was measured ). the endpoints for the study were ear thickness and ear weight at the end of the experiment . the thicker the ear and the more it weighs correlates with the degree of inflammation . results : topically applied accs was effective at reducing the inflammation induced by tpa . the anti - inflammatory activity of topical accs reached the same level as clobetasol ( a class 1 potent topical corticosteroid ) by 3 days after beginning application . conclusion : accs has a strong anti - inflammatory effect when applied to skin . inflammatory model — evaluate the efficacy of intralesional injection of accs to inhibit irritant ( tpa ) skin inflammation in mice method : intralesional injection into the ear was given once daily to the following groups : 1 . tpa + intralesional control ; 2 . tpa + intralesional accs ; 3 . tpa + intralesional kenalog ( 10 mg / ml ) ( a potent intralesional corticosteroid ); 4 . accs intralesional injection alone ; 5 . saline sham injections to the normal untreated ear . the endpoints for the study were ear thickness and ear weight at the end of the experiment . the thicker the ear and the more it weighs correlates with the degree of inflammation . results : intralesional injection of accs was effective at reducing the inflammation induced by tpa at all time points beginning on day 2 of daily injections . intralesional kenalog ( 10 mg / ml ) injections induced a hematoma at the site of injection , which led to some inflammation and that is why there is not a substantial difference in ear thickness when comparing tpa + kenalog with tpa + control . conclusions : intralesional accs did reduce skin inflammation but the topically applied accs in example 1 above had a more potent effect . there was no difference in ear weight using either accs or intralesional kenalog compared with tpa + control . an art - accepted animal model for chronic granulating wound was used to study the effects of accs on chronic wound healing ( hayward pg , robson mc : animal models of wound contraction . in barbul a , et al : clinical and experimental approaches to dermal and epidermal repair : normal and chronic wounds . john wiley & amp ; sons , new york , 1990 .). results : accs was effective in not allowing proliferation of tissue bacterial bioburden . accs allowed accelerated healing of the granulating wound significantly faster than the non - treated infected control groups . accs compositions are tested in animal models of organ transplant to evaluate their ability to prevent , modulate , reduce , treated or ameliorate ischemic injury in the harvested organ . standard animal models of organ transplantation are found in the scientific literature as well as in “ handbook of animal models in transplantation research ”, edited by donald v . cramer , luis podesta and leonard makowka , published in 1993 by crc press ( incorporated herein by reference ). accs compositions are tested for their ability to prevent , modulate , reduce , treated or ameliorate ischemic tissue injuries . the experimental approach includes testing accs compositions in a hind limb ischemia model . this model utilizes a controlled tension tourniquet circumferentially around the proximal thigh of a mouse for 3 hours . reperfusion is initiated by release of tension on the tourniquet . immediately following reperfusion , accs compositions are injected into the left ventricle . perfusion - restoration of blood flow is monitored by laser doppler flow imaging . immunohistochemistry and quantitative pcr are used to assess accelerated tissue neovascularization and angiogenesis . preservation / storage — use of accs as a storage preservative for donated tissues and organs the successful outcome of the majority of corneal transplants depends on the presence of a viable corneal endothelium . since human corneal endothelial cells do not readily proliferate , preservation of the endothelium is a primary aim of methods of corneal storage . although some cryopreserved corneas have been transplanted successfully , the complexity of the standard cryopreservation technique and its potential for causing endothelial damage have limited its application . because of its anti - inflammatory properties , accs is tested for its ability to preserve corneas for transplant . the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof . any equivalent embodiments are intended to be within the scope of this invention . indeed , various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description . such modifications are also intended to fall within the scope of the appended claims . throughout the specification various publications have been referred to . it is intended that each publication be incorporated by reference in its entirety into this specification .
the invention is directed to methods of modulating ischemic injury in tissues and organs , including donor tissue and organs and intact tissue and organs . the invention is further directed to methods of increasing time to ischemic injury in such tissues and organs . the invention is further directed to storing and preserving donor tissues and organs . such methods utilize compositions comprising amnion - derived cellular cytokine solution . the accs compositions may be formulated for sustained - release , targeted - release , timed - release , extended - release , etc . and may be used alone or in combination with various suitable active agents .