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    7 views37 pages

    ECM Cancer

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    Sherry
    ecm

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    © All Rights Reserved
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    of 37
    «2 United States Patent Naughton et al. 'US009506038B2 US 9,506,038 B2 *Nov. 29, 2016 (10) Patent No.: (4s) Date of Patent: oa ow @ 3) wo ey @y 6s) @) (oo) on) (2) (58) 66) sps0.946 A 20070077232 AL DoDwon20466 AL EXTRACELLULAR MATRIX COMPOSITIONS FOR THE TREATMENT OF CANCER, Applicant: Histogen, tne., San Diego, CA (US) K. Naughton, San Diego, CA (US); Emmett Pinney, Poway, CA Inventors c (ws) Assignee: Mistogen, Ine., San Diego, CA (US) Notice: Subject to any disclaimer, the tem ofthis patent is extended or adjusted under 38 USC. 1540) by 0 days. This patent is subject to a terminal dise climes, Appl. No. 14/711,646 iled: May 13, 2015 Prior Publication Data US 2015/0246076 Al Sep. 3, 2015 Related U.S. Application Data Continuation of application No. 12/822,126, fled on Jun. 23, 2010, now Pat. No. 9,034,312, which is a ‘continuation-in-part of application No. 12°363,479, filed on Jan. 30, 2008, now Pat. No. 8,852,637 Provisional application No. 61/114,966, filed on Nov. 14, 2008, Int. cl. CRN 00 CRN 509 AGIK 35/13 AGIK 35/12 AGIK 3324 AGIK 3533 (2006.01) Q01001), (201501), (201501) (2006.01) (201501) CTAN 50693 (2013.01), ABIK 33/24 (2013.01); A6IK 35/12 201301): ABIK 3913 (2013.01): ABIK 393 (2013.01) Field of Classification Search CPC. CI2N 510693; -ABIK 35/12; AGIK 35/33: AGIK 35/13 ‘See application file for complete search history References Cited USS. PATENT DOCUMENTS. 1/1999 ones eta 142007 Navghton etal 9008 Fuga FOREIGN PATENT DOCUMENTS A 2002-s30069 $2000 3 2007-528282 $2006, w 2006240031 A 93006 Ru 33112 C2 122007 Wo wosunrms al ‘Fim. wo woossoe2s “tL ig9s WO WOdSHE A Los WO WOoeIDIOL AL Ie WO wo 20626652 42 "32006 WO — WO 2DOSSDRL AZ — $2006 WO Wo 3mene76ss a2 62006 wo “Wonsors A? 92006 (OTHER PUBLICATIONS Keita ot al. Act Ci Bras 11-16, 2008. Pavelic eal. Cancer Res 46:3653-362, 1986* Gould sad Auchincloss. Immunology Today 20(2)77-82, 1999 ‘Azinzaleh et al. Hemology and Cell Therapy 38(4) 331-83, toon Albets etal, Molecular Bislogy of the Cel. 4h elton. New YorksGarland Scienve, 2002 Barker CR, eas The topoisomerase I-Hp90 complex: « new chemotherapeutic target int} Canes, Su. 1; 2006, SID 2685-2698 (Abstr. ‘Moiscenko, MD VME: Modem tactics treatment of patients with ‘maligna aoplsms of bone metastases; (Mantal fr physician Oncology Instat, Pov RU, Dee 21, 2012 8 pages (rans: ‘ion alana tal: “Low Oxygen Stimulates Proliferation of Fbroblasts Seeded os Single Cells", J. Cell. Physiol. 1098, vol 184, p. sts. alana tal: “Low Gren Tension Scimulates Collagen Syohess dnd Colt I Transcription Through the Action of TOF-131"s 1 Cll. Physi, 2002, vo. 101, p. 42-80, (Chemicdl Encylopedia: vol 2, Fitorship Soviet Eacyelopadia, M, 1090, pp 432-438 English Transation. Intentional Search Report (ISR) issued on Mar. 20, 2009 Re eTUS20090032677 * cited by examiner Primary Esaminer — Marcia $ Noble (74) Auornes. Agent, oF Firm — DLA Piper LLP (US) on ‘The present invention is directed to methods of inhibiting cancer cell growth or proliferation by contacting the cancer cel with an extracellular matrix (ECM) eompesition. Also provided are methods of delivering a chemotherapeut ‘agent to a cancer cell by contacting a cancer ell with an ‘extracellular matrix composition containing a chemother peutic agent. Also provided are compositions containing ECM and a chemotherapeutic agent ABSTRACT 14 Claims, 18 Drawing Sheets U.S. Patent Nov. 29, 2016 FIG. 1 Sheet 1 of 18 US 9,506,038 B2 tumor weight (gm) 0.45 2 0.35 0.25 ° © e fo 5085 5 Notreatment treated with ECM U.S. Patent Nov. 29, 2016 Sheet 2 of 18 US 9,506,038 B2 FIG. 2 0.60 0.50 2 & tumor weights (gm) ° 8 ° 8 0.10 0.00 untreated tweated U.S. Patent Nov. 29,2016 Sheet 3 of 18 US 9,506,038 B2 FIG. 3 B16/CAM a —€ 05 © 0.4 S03 * = 0. . Bo 1, Ss no treatment mixed with on ECM ECM U.S. Patent Nov. 29,2016 Sheet 4 of 18 US 9,506,038 B2 FIG. 4 ee C6TUMCAM.1 tumor weighstam) 3 8 Rotreatment = — ECM. ECM+cis Cisplatin U.S. Patent Nov. 29,2016 Sheet 5 of 18 US 9,506,038 B2 FIG. 5 C6 TUMCAM.2 250 > 200 - = & 150 - 2 100 50 + o- oe Nott cM EcMieeis cisplatin U.S. Patent Nov. 29,2016 Sheet 6 of 18 US 9,506,038 B2 FIG. 6A U.S. Patent Nov. 29, 2016 Sheet 7 of 18 US 9,506,038 B2 FIG. 6B U.S. Patent tumor weight (mg) Nov. 29, 2016 0.300 0.250 0.150 0.100 0.050 | Sheet 8 of 18 FIG. 7 US 9,506,038 B2 Gs US 9,506,038 B2 Sheet 9 of 18 Nov. 29, 2016 U.S. Patent FIG. 8 FIG. 9 US 9,506,038 B2 Sheet 10 of 18 Nov. 29, 2016 U.S. Patent FIG. 10 FIG. 11 U.S. Patent tumor mass mm3 (LXWXW) 800 600 400 200 Nov. 29, 2016 —— day9 Sheet 11 of 18 FIG. 12 C6 Glioma day 15 ly day18 US 9,506,038 B2 —eNotxt ECM. ae ECM ACIS seas US 9,506,038 B2 Sheet 12 of 18 Nov. 29, 2016 U.S. Patent 134, FIG. ] ei: ; FIG. 13B US 9,506,038 B2 Sheet 13 of 18 Nov. 29, 2016 U.S. Patent FIG. 13C FIG. 13D U.S. Patent tumor mass (LXWXW/2) Nov. 29,2016 Sheet 14 of 18 300 250 200 150 100 50 FIG, 14 MDA435 Cells dayl1 day18 day21 day25 US 9,506,038 B2 no Txt ECM ke ECM Cis seas U.S. Patent Nov. 29,2016 Sheet 15 of 18 US 9,506,038 B2 FIG, 15 U.S. Patent Nov. 29,2016 Sheet 16 of 18 US 9,506,038 B2 FIG. 16 BioNeusis treated B16 tumcam 09 | | 10mg/mLBNeu os 8162-3e6/e8g 100uL/dose 07 06 as 04 ‘tumor weight (gm) 03 02 on NoTxT BNot Single dose U.S. Patent Nov. 29,2016 Sheet 17 of 18 US 9,506,038 B2 1.200 ° ° re oe. 8 8 8 Tumor Weight in grams ° S 8 8 No Treatment FIG. 17 Dose Curve (B16 tumor CAM) | i | = Amg/mL —-10mg/mL =—-100me/mL. U.S. Patent Nov. 29,2016 Sheet 18 of 18 US 9,506,038 B2 % reduction in tumor mass vs unfractionated FIG. 18 Tumor Inhibition Activity by Molecular Weight 40 30 : of . >100k <100k 30k <10k Molecular Weight Fractions of concentrated BN US 9,506,038 B2 1 EXTRACELLULAR MATRIX COMPOSITIONS FOR THE TREATMENT OF CANCER CROSS-REFERENCE TO RELATED 'APPLICATIONS| ‘This application is a continuation application of US. application Set. No. 121822,126 filed Jun. 23, 2010, now issued as US. Pat. No. 9,034,312; which is continuation- ‘ncpart application of U.S. application Ser. No. 12363,479 filed Jan. 30, 2009, now issued as U.S. Pat. No. 8.852.637, which clsims the benefit under 35 USC. §119(@) to US. Application Ser. No, 61/114,966 filed Nov. 14, 2008. The ‘disclosure ofeach ofthe prior applications is considered par, ‘of and is incomporated by reference inthe disclosure of this, application, BACKGROUND OF THE INVENTION Field ofthe Invention ‘The present invention relates penerally to use of extra cellule matrix compositions and more specifically, the use ‘of extrcelular matrix compositions to inhibit cell proif= ‘eration. Background Information ‘he extracellular mattix (BCM) is @ complex structural ‘entity surrounding and supporting cells that are found in vivo within mammalian tissues. The FCM is often refered to as the connective tissue, The ECM is primaily composed ‘of thive major classes of biomolecules inchuding stractoral proteins such 2s collagens and elastin, specialized proteins such as fibillns, ibroncetins, and laminins, and proteogly- ‘Growth of ECM compositions in vitro and their use in 3 varity of therapeutic and medical applications have beea described in the art. One therapeutic application of such ECM compositions inchides treatment and repaie of soft tissue and skin defects such as wrinkles and scars, The repair or augmentation of soft tissue defects caused by defiets, stich as, acne, surgical scarring or aging has proven to be very difficult. A number of materials have been tsed to correct soft tissue defects with varying degrees of success, however, no material as been completely sae and celfective. For example, silicon causes a variety of physi ‘logical and clinical problems including long. tem side ‘effets, sch as nodules, recurring cellulitis and skin ules Callagea compositions have also been use 2s an inject= ‘able material for soft tissue augmentation. Collagen i the ‘main protein of connective tissue and the most abundant protein in mammals, making up about 25% of the total protein content. There are curently 28 types of collagen ‘described in literature (Gee, eg, Tables 1 and 2 infra, fora tailed listing). However, over 90% ofthe eollagen in the body are Collagens I, I, II, and IV. Different collagen materials have been used for treatment ‘of soft tissue defects, such as reconstituted inoctabe bovine collagen, crosslinked collagen, or other xenogeneic colle ens. However, several problems exist with such collagens. ‘Aconimon prablem includes the complexity and high cost ‘of producing the implant materials 10 remove potentially jmmunogenic substances to avoid allergic reactions in the subject, Adlitionaly, treatments using such collagens have not proven long lasting ‘Other materials have also been described that may be wed for soft tissue repair or augmentation, such as, biocompal- ible ceramic particles in aqueous pels (US. Pat No. 5,204, 0 o 2 382), thermoplastic and/or thermosetting materials (US. at No. $278,202), and lactic acid based polymer blends US. Pat, No. 4,235,312), Additionally, use of naturally secreted extracellular matrix compositions have also been described (U'S. Pat, No, 6,284,284), However, such mate- ‘als have all proven to have limitations. Accordingly, new materials are needed for soft tissue repair and augmentation that overcome the deficiencies of prior materials. The need exists o provide a safe, injectable, Jong lasting, bioabsorbable, soft issue repair and augsien- sation material Ta vitro cultured ECM compositions can additionally be used to treat damaged tissue, sueh as, damaged cardiac ‘muscle and related tissue. The compositions are useful as implants or biological coatings on implantable devices, such as, stents or vascular prosthesis to promote vascularization jn organs, such as the heart and related tissue ‘Coronary heart disease (CHD) also ealled coronary artery disease (CAD), isehiaemic heart disease, and atherosclerotic heart disease, is characterized by a narrowing of the small blood vessels that supply blood and oxygen to the hear. Conmnary heart disease #8 usally caused by a condition called atherosclerosis, which occurs whee fatty material and plaque builds up on the walls of arteries causing the arteries fo narrow. As the coronary arteries narrow, blood flow to the heart can slow down of stop, causing chest pain (stable angina), shortness of breath, heart atack, and other symp- toms. Coronary heart disease (CHD) is the leading cause of oath in the Unite States for men and women, According 0 the American Heart Association, more than 15 million people have some form of the condition, While the symip- foms and signs of coronary heat disease ae evident in the advanced state ofthe disease, most individuals with eoro- ‘ary heart disease show no evidence of disease for decades as the disease progresses before a sudden heart attack ‘curs, The disease is the most common eause of sudden ‘eat, and i also the most common reason for death of men fand swomen over 20 years of age. According #9 present trends in the United States, hal of healthy 40-year-old males will develop CHD in the future, as well at one in three healthy 40-year-old women, ‘Curent methods for improving blood flow in a diseases or ollerise damaged heat involve invasive surgical tec niques. such as, coronary by-pass surgery. angioplasty, and endarterectomy. Such procedures naturally involve high- degrees of inherent risk during and after sirgery, and often only provide a temporary remedy 10 cardiac ischemia Aecoringly. new treatment options are required to increase the success of eurtently available techniques for testing (CHD and related symptoms In vitro cultured ECM compositions ean additionally be sed to repair andlor regenerate damaged cells oF issue, such as ehondral or osteochondral cells. Osteochondral tissue is any tissue that relates to or contains bone or cartilage. The compositions of the present invention are ‘set For treatment of osteochondral defects, such as degen: eative connective tissue diseases, such as rheumatoid and for osteoarthritis as well as defeots in patients who have cartilage defects due to trauma. Cureat attempts at repuiring osteochondral defects ‘include implantation of human ehonerocytes in bioeompat- idle and biodegradable hydrogel grafts in attempts. 10 improve the possibilities to restore articular cartilage lesions. Additionally, the technique of chondrocyte cultire in alginate beads or » mateix including polysuiphated alg- inate has been described to penerate a yaline-ike cartlag- US 9,506,038 B2 3 ‘neous tissue, However, attempts at repairing enchondral Fesions of anicular cardlage by implantation of human ‘autologous chondrocytes have had limited success. Accont- ingly, new treatment options are required 10 increase the suecess of currently avilable techniques for tating ‘ostechondml defects. In viteo cultured ECM compositions are also useful in tissue culture aystems for generation of engineered tissue ‘implants, The field of tissue engineering involves the use of ‘ell culture technology to generate new biological issues oF repair damaged tissues, Fueled in part, by the stem cell revolution, tissue engineering technology offers the promise ‘of ise regeneration and replacement following trsuma oF treatment of degenerative diseases. 1 ean also be used in the ‘context of cosmetic procedures. Tissue engineering techniques can be used 10 generate both autologous and heterologous tissue or cells using & varity of cll types and culture techniques. In creating an ‘autologous implant, donor tissue may’ be harvested and ‘dissociated into individual eells, and subsequently atached ‘and cultured on. substrate to be implanted at the desired site ff the functioning tissue, Many isolated cell types can be ‘expanded in vito using cell culture techniques, however, anchorage dependent cells require specifie environments, ‘often inclading the presence ofa thrce-dimensional seaold (o act as a template for growth. Current tissue engineering technology provide generally, ‘ariical implants, Successful cell tansplantation therapy ‘depends on the development of suitable substrates for both Jn vito and in vivo tisse culture, Thus the development of an extracellular matrix that contains only natural materials ‘and that suitable for implantation would have more ofthe ‘chanicterstcs ofthe endogenons tissue. Accordingly, gen= ‘eration of natural extracellular matrix astral isan ongoing challenge in the field of tissue engineering SUMMARY OF THE INVENTION ‘The present invention is based in parton the discovery that extricefular matrix (ECM) compositions ean inhibit the growth of eancer cells in vivo. Accordingly, in one embodi- ‘ment ofthe present invention, there are provided methods of Inhibiting cancer cell rowih or proliferation, by contacting the cancer cell with an extracellular mateix composition as described herein, In another embodiment of the present invention, there are provided methods of delivering a chemotherapeutic agent 10 coll or tissue, including contacting the cell or tissue with ‘an extricellular matrix composition containing a chemo- therapeutic agent Instill another embodiment ofthe present invention there are provided compositions containing ECM and a chemo- therapeutic agent, In some embodiments, the composition Jurker contains an immunomodulatory agent In yet another embodiment ofthe present invention there are provided methods of inhi ‘ell by contacting the cancer cell with an extracelluae ‘matrix composition as described herein la Various embodi- ‘ments, the cancer cel is metastatic, for example, the cancee cell may be from a metastatic tumor, such as @ metastatic Primary tumor. BRIBE DESCRIPTION OF THE DRAWINGS FIG. 1 shows a plot of tumor weight of B16 cells in the presence and absence of ECM in the CAM assay. 0 o 4 FIG. 2 shows a plot of tumor weight of B16 cells presence and absence of ECM in the CAM assay. FIG. 3 shows a plot of tumor weight of BI cells grown in the absence of ECM, mixed with ECM, or on ECM in the CAM assay, FIG. 4 shows a plot of tumor weight of Cé eels in the presence and absence of ECM, with or without cisplatin ia the CAM assay. PIG, 5 shows a plot of tumor weight of C6 cells in the presence and absence of FCM, with or without cisplatin in the CAM assy. PIGS, 68 and 62 show photographs of histologic analysis of C6 glioma tumors grown in the presence or abseace of ECM. FIG, 7 shows a plot of tumor weight of MDA 435 cells ‘grown in the presonee and absence of ECM in the CAM assay. TFIG. & shows photograph of tamor growth of C6 eels with no treatment in nde mice, FIG. 9 shows photograph of tumor growth af C6 cells \with ECM treatment in-nude mice. FIG. 10 shows a photograph of tumor growth of C6 cells with ECM plus cisplatin twstment in nude mice FIG. 1 shows a photograph of tumor growth of C6 cells with cisplatin treatment in nude mice FIG. 12 shows a plot of the growth of C6 glioma tumors ‘in mude mice inthe presence or absence of FCM, with or ‘without cisplatin, FIGS, 13A-D show photographs of tumor growth of MDA 435 cells in aude mice with no treatment (FIG. 134), ‘ECM treatment (FIG. 13B), hECM plos cisplatin treatment (1G. 18C) and BECM with cisplatin treatment (FIG. 13D). FIG. 14 shows a plot of the growth of MDA 435 tumors in aude mice in the presence or absence of ECM, with or without cisplatin, FIG. 18 shows a photograph of tumor growth of B16 cells ‘with (Specimen on right) and without ECM (specimen on Jeft) in rodents FIG. 16 shows a plot of tumor weight of BIG cells inthe presence and absence of liquid ECM in the CAM assay. FIG. 17 shows a dose response curve of tumor weight of B16 cells grown in the presence of liquid ECM ia the CAM FIG, 18 shows a plot of tumor weight of BIS cells grown, in liquid ECM of various molecular weight fractions in the CAM assay, the DETAILED DESCRIPTION OF THE. INVENTION ‘The present invention relates to methods of using extra ccllular mate compositions fo te inhibition of growth or proliferation of cuacer cells alone or as biological vehicle for the delivery of a chemotherapeutic agent Before the present compositions and methods are eseribod, itis to be understood that this invention is not Timited t particular eompositions, methods, and experimen- tal conditions described, as such compositions, methods, and conditions may vary. [tis albo to be understood that the ‘erminology used herein is for purposes of describing parr ‘icular embodiment only and is not intended to be lititing, since the scope ofthe present invention willbe limited ony in the appended claims. As use in this specification andthe appended claims, the singular forms“s", "an", and “the” inelode plural references unless the context clearly dictates otherwise. Thus, for ‘example, references to “the method” includes one or more US 9,506,038 B2 5 methods, andlor steps of the type described herein whieh ‘will become apparent to those persons skilled in the art upon reading this diselosure and so forth Tn one embodiment of the present invention, there are provided methods of inhibiting cancer cell growih or pro- Fiferation, in which the method includes contacting, the ‘cancer cell with an extracellular mix composition. In some embodiments, the cancer cell is metastatic. In some ‘embodiments, the ECM may be a soluble fraction, and in ‘ther embodiments the ECM may be a non-solube faction, Insill other embodiments, the ECM may be a combination ‘of soluble and non-soluble fractions. In cetain embodiments, the cancer eel is from a tumor, such as @ primary or secondary tumor. In certain embodi- rents, the tumor is metastatic. In some aspects, the cancer Js selevied from the group consisting of melanoma, glioms, and adenocarcinoma, In other aspects the cancer i cancer fof the adrenal gland, bladder. bone, bone marrow, bra, spine, breast, cervi, gall bladder, ganglia, gastrointestinal teoct, stomach, colon, hear, kidney, liver, lung, muscle ‘ovary, pancreas, parathyroid, penis, prostate, salivary lands, skin, spleen, esis, thymus, thyroid, or uterus. In one spect the cancer is breast cancer; in another aspect the ‘cancer is melanoma; and in another aspect the cancer is ‘colon cancer In another embodiment of the present invention, there are provided methods of delivering # chemotherapeutic agent to ‘cancer cell, wherein the method includes contscting 8 ‘cancer cell with an extracellular matrix composition con- taining a chemotherapentic agent, Tn one aspect, the contacting is in the surgical margin of the area from which a tumor has been excised, In sich ‘embodiments, 2 tumor is removed from a subject using Standard surgical techniques knowa to the clinician, ECM ‘compositions ofthe inveation may be placed inthe space left ‘upon excision of the tumor (resection), so that the ECM is jn contact with the surgical margin, In certain embodiments, the FCM may inhibit the growth of cancer cells residing in the tumor margin, In some embodiments, the ECM compo- sition may’ further contain a chemotherapeutic agent with oF ‘without an immunomodulatory agent. The use of the inven= tion ECM compositions in treatment of cancer following surgical revection has the advantage of delivering localized ‘and sustsined-release delivery ofa chemotherapeutic anor ‘immunomodulatory agent tothe tumor ste. Additional ben- ‘fits can include improved healing of surgical wounds and Improved cosmetic outcome. Instill another embodiment of the present invention there ‘are provided compositions containing ECM and a chemo= therapeutic agent, In some embodiments, the composition Jurker contains an immunomodulatory agent ‘In some embodiments the extracellular matrix compos! tions are generated hy’ culturing cells under hypoxic eondi- tions on a two- or three-dimensional surface in a suitable growth medium. The culturing method prodvces both Soluble and non-soluble factions which may be used sepa- rately orin combination t obtain physiologically acceptable ‘compositions having variety of applications. In other ‘embodiments, the cells may be cultured under standard oF normal oxygen conditions “The compositions of the present invention have a variety ‘of applications including, but not limited to, inhibiting growth or proliferation of cancer cells, delivery of there peutic agents, promoting repair andlor regeneration of dam- ‘aged cells oF tissues, use in patches to promote tissue regeneration, use in tissue culture systems for culturing cell, sch a stem cells, use in surface costings used i 0 o 6 association with implantable devices (ext, pacemakers, Stents, stent grafls, vascular prostheses, heart valves, shunts, drug delivery ports of catheters), promoting soft tissue repair, augmentation, andor improvement ofa skin surface, seh as wrinkles, use asa biological anticadhesion agent or fas biological vehicle for cell delivery or maintenance at a site of delivery. ‘The invention is based in part, om the discovery that cells tured on two~ oF throe-dimessional surfaces under con- ditions that stimulate the early embryonic environment (hypoxia and reduced gravitational forees) prior 10 angio- genesis produces extracellular matrix compositions with {etal properties, inluding generation of embryonie proteins Growth of cells under hypoxie conditions demonstrate a ique ICM with fetal properties and growth factor expres- sion. Unlike the culturing of ECM under traditional culture conditions, over 500 genes are differentially expressed in ECM cultured under hypoxic conditions. This results in a cultured ECM that has different properties and a different 1 composition. For example, aa ECM produced hhyponie conditions is similar to fetal mesenchymal tissue in that is eltively rich in collagens type Il IV, and ' and glycoproteins suchas fibronectin, SPARC, thromabo- spondin, and hyaluronic acid. Hypoxia also enhances expression of fsetors which regu late wound healing and organogenesis, such as VEGF, FGE-7, and TGE-, as well as multiple Wat factors includ. ing wais 2b, 4 7a, 10a, and 11, Cultured embryonie human FCM also stimulates an increase of metabolic ctivity in ‘human fibroblasts in vitro, as measured by increase eazy matic activity. Additionally, there is an increase in cell ‘number in response to the cultured embryonic ECM, Tn various embodiments, the present invention involves methods for making extracellular mateix compositions that include one or more embryonie proteins and applications thereol: In particular the compositions are generated by culturing cells under hypoxic conditions on a. two- oF three-dimensional surface ina suitable growth medium. The compositions are derived by growing cells on a framework resulting ina multi-layer ell culture system, Cells grown on framework suppon, in accordance with the present inven- ‘ion, grow in motiple layers, forming a cellar matrix. Growih of the cultured cells under hypoxic conditions results in differential gene expression as the result of byponie culturing conditions versus traditional culture Extracellular matrix (ECM) is a composition of proteins ‘and biopolymers that substantially comprise tissue that is produced by cultivation of cells. Stromal cells, such as {Mhroblass, are an anchorage dependant cell type requiring srowth while attached to materials and surfaces suitable for cell culture. The ECM materials produced by the cultured cells are deposited in a three-dimensional arrangement pro- viding spaces for the omnation of tssue-likesirictures "The cultivation materials providing thrse-dimensionsl architoctures are refered to af scaffolds. Spaces for depo- sition of ECM. are in the form of openings within, for example woven mesh or iatestitial spaces created in a ‘compacted configuration of spherical beads, called miero- ‘As used herein, “extracellular matrix composition” includes both soluble and non-soluble Iracions or any portion or combination thersof. The non-soluble fraction ‘includes those scereted extracelhlar matrix proteins and biological components that are deposited on the support or scaffold, The soluble fraction refers to culture media ia ‘which cells have been cultured and into wich the cells have secreted active agent(s) and includes those proteins and US 9,506,038 B2 7 biological components not deposited on the seaffold. Both Jfactions may be colleted, and optionally furlher processed. ‘and used individually of in combination in a variety of applications as described herein, FCM may also be obtained from eadaver tissues (US. Pat, Nos. 6,284,284 and 6,372, 494), The soluble fraction may be concentrated or diluted t0 achieve an optimal concentration. In certain embodiments, the concentration may be about 1 mg/m, about $ mg/m, about 10 mg/mL, about $0 mg/mL, about 100 mg/mL. of about 200 mg/ml. The concentration may be ina range of ‘bout 0.1 mg/l. to 1000 agin, or about 1 to 200 mga, ‘or about 10 mg/l. to 100 mg ‘The soluble fraction may be further fraetionated accont- to molecular weight (eg. by dialysis, filtration, oF matography) so that the majority ofthe solute moleciles in fetion meet 2 particular molecular weight cuof. 1 will be recognized that the eutll values are not absate but that a inajoniy (eg. 70%, 80%, 80%, oF 98%) of the solute molecules conlsined ina given fraction meet the molecular ‘weight cutoff. Accordingly in certain embodiments of the methods provided herein, the ECM composition may include a soluble fraction in which 70%, or 80%, or 90% of the solute molecules contained therein have a molecular ‘weight of less than 5,000 Daltons, or les than 10,000 Daltons, or less than 30,000 Daltons, of less than 100.000 Daltons. In other embodiments, the ECM may include 2 soluble fraction in which the solute molecules contained therein have a molecular weight of greater than $.000 Daltons, or greater than 10,000 Daltons, or greater than 30,000 Daltons. or greater than 100,000 Daltons. In other ‘embodiments, the ECM may include a soluble fraction in, ‘hich the solute moleetles contained therein havea molec Jar weight between 10,000 and 100,000 Daltons, oF 30,000 {© 100,000 Daltons, or 10,000 0 30,000 Daltons ‘The two- or threedimensional support or scalfold used (0 culture stromal cells may be of eny material andor shape that: (a) allows cells to attach to it (oF can bo modified to allow cells to attach to it}: and (b) allows cells to grr ia more than one layer (ie, form a three dimensional tisste). nother embodiments, a substantially two-dimensional shect ‘of membrane may be used to culture cells that are sufi

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