WO1995015763A1 - Procede de preparation d'un extrait de serum et de facteur de croissance plaquettaire - Google Patents

Procede de preparation d'un extrait de serum et de facteur de croissance plaquettaire Download PDF

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Publication number
WO1995015763A1
WO1995015763A1 PCT/CA1994/000601 CA9400601W WO9515763A1 WO 1995015763 A1 WO1995015763 A1 WO 1995015763A1 CA 9400601 W CA9400601 W CA 9400601W WO 9515763 A1 WO9515763 A1 WO 9515763A1
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platelet
serum
extract
plasma
concentration
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PCT/CA1994/000601
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English (en)
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Paul Brazeau
Thierry Abribat
Michel Ibea
Michèle BOUSHIRA
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Universite De Montreal
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Priority to AU79885/94A priority Critical patent/AU7988594A/en
Publication of WO1995015763A1 publication Critical patent/WO1995015763A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum

Definitions

  • the invention relates to a process for the pro ⁇ duction of a whole blood derived serum and/or platelet extracts and their clinical uses in wound dressing,
  • the very first event that normally occurs in a wound is blood extravagation, that results in platelet aggregation and impregnation of the wound with platelet and serum constituents.
  • these constituents are polypeptide growth factors, which are known to play a major role in tissue regeneration.
  • Platelet ⁇ gran- ules which are released by aggregated platelets, are one of the richest physiological source of platelet- derived growth factor (PDGF) and transforming growth factor ⁇ (TGF ⁇ ), while serum contains high amounts of insulin-like growth factor I (IGF-I), IGF-II and their binding proteins (IGF-BPs) (Strovbant P & Waterfield MD, Embo.J., 1984, 2 :2963-2967; Assoian RK et al., J. Biol. Chem., 1983, 258:7155-7160: Sara VR et al., Physiol. Rev., 1990, 70:591-614) .
  • IGF-I insulin-like growth factor I
  • IGF-BPs binding proteins
  • PDGFs include PDGF, platelet derived angiogene- sis factor (PDAF), TGF ⁇ and platelet-factor-4 (PF-4), which is a chemoattractant for neutrophils (Knighton et al. , In Growth Factors and Other Aspects ofWound Healing: Biological and Clinical Implications, Alan R. Liss, Inc., pp. 319-329 (1988)).
  • PDGF is a mitogen and chemoattractant for fibroblasts and smooth muscle cells and is a stimulator of protein synthesis in cells of mesenchymal origin, including fibroblasts and smooth muscle cells.
  • PDGF is also a nonmitogenic chemoattractant for endothelial cells.
  • TGF ⁇ is a chemoattractant for macrophages and monocytes. Depending upon the presence or absence of other growth factors, TGF ⁇ increases the tensile strength of healing dermal wounds. TGF ⁇ also inhibits endothelial cell mitosis, and stimulates collagen and glycosaminoglycan synthesis by fibroblasts. Other growth factors, such as EGF, TGF ⁇ , the
  • HBGFs and osteogenin are also important in wound heal ⁇ ing.
  • EGF which is found in gastric secretions and saliva
  • TGF ⁇ which is made by both normal and transformed cells, are structurally related and may recognize the same receptors, which mediate cell pro ⁇ liferation on epithelial cells. Both factors acceler ⁇ ate re-epithelialization of skin wounds.
  • Surgical adhesives and tissue sealants which contain plasma proteins are known and are used for sealing internal and external wounds in order to reduce blood loss and maintain hemostasis.
  • Such sealants typically contain blood clotting factors and other blood proteins.
  • Stroetmann, U.S. Patent Nos. 4,427,650 and 4,427,651 describes the preparation of an enriched plasma derivative in the form of a powder that contains fibrinogen, thrombin and/or prothrombin, and a fibrinolysis inhibitor, and may also contain other ingredients, such as a platelet extract.
  • Fibrin glue which is also called fibrin sea ⁇ lant, is primarily formulated for clinical topical application and is used to control bleeding and promote wound healing.
  • the clinical uses of fibrin glue have recently been reviewed (Gibble et al., Transfusion, 1990, 3J):741-747; Lerner et al., J. Surg. Res., 1990, 48:165- 181).
  • Fibrin glues are commercially available.
  • IMMUNO AG Vienna, Austria
  • BEHRINGWERKE AG Germany
  • Fibrin glues are prepared from plasma. The precise components of each fibrin glue are a function of the particular plasma fraction used as a starting material. Typically fibrin glue contains a mixture of proteins that, upon mixing with thrombin, form a clot.
  • fibrin glue can be prepared from plasma by cryoprecipitation followed by fractionation, to yield a composition that forms a sealant or clot upon mixture with thrombin or an activator of thrombin.
  • Fractiona ⁇ tion of plasma components can be effected by standard protein purification methods, such as ethanol, polyeth ⁇ ylene glycol, and ammonium sulfate precipitation, and ion exchange, and gel filtration chromatography.
  • Fibrin glues generally include a fibrinogen concentrate, which contains fibronectin, Factor XIII, von Willebrand factor, and dried human or bovine throm ⁇ bin. It is prepared in lyophilized form and is mixed with a solution of calcium chloride immediately prior to use. Upon mixing, the components coagulate on the tissue surface and form a clot that includes cross- linked fibrin.
  • Factor XIII which is present in the fibrinogen concentrate, catalyzes the cross-linking, fibrin glue, by sealing tissues face to face, prevents air or fluid leaks, and thereby induces hemostasis.
  • fibrin glue promotes wound healing. It does no, however possess true wound healing properties. Because fibrin glue is suitable for both internal and external injuries and is useful to maintain hemostasis, it would be desirable to enhance its wound healing properties.
  • One aim of the present invention is to provide for an easy process for the isolation of whole blood derived serum/platelet extract containing non-denatured growth factors at a high concentration of about 54 to about 99.8% , which extract is suitable for use in a wound dressing composition.
  • Another aim of the present invention is to pro ⁇ vide for a process for the production of growth factors extracts nearly 100% identical to their human counter ⁇ parts.
  • Another aim of the present invention is to pro- vide a blood derived serum/platelet extract for use in a wound dressing composition being characterized by a total absence of potential viral transmission of human origin (Hepatitis, HIV, etc.).
  • a further aim of the present invention is to provide for pharmaceutical compositions for promoting wound healing.
  • the novel process in accordance with the present invention comprises the steps of: a) concentrating a platelet-rich-plasma prepared from whole blood by passing through a neutral support for adsorbing the platelets or by cen- trifuging whole blood at about lOOOg to about 3000g, preferably at 1500g; b) releasing the concentrated platelets of step a) by ultra-sound shock; c) precipitating the concentrated plasma of step a), a platelet-poor-plasma or a serum by adding an amount of acetone solution to obtain an ace ⁇ tone concentration of about 68 to about 95% at a temperature of about -15 to 5°C; thereby obtaining a solid plasma or serum precipitate; d) the plasma or serum precipitate of step c) is reconstituted to a concentration of about 0.5 to 6% of protein weight/volume and mixed to the released platelets of step b) to obtain a platelet-serum-extract of a concentration which substantially has a physiological concentration to give a maximum syner
  • the preferred wound dressing includes a combination of the main growth factors present at the wound site, i.e. PDGF, TGF ⁇ s and IGFs.
  • a process for the production of a whole blood derived serum/platelet extract is described along with its biological potencies as a wound healing enhan- cer when associated with a fibrin matrix and/or biode ⁇ gradable gelifying matrix.
  • the whole blood derived serum/platelet extract characterized by a total absence of potential viral transmission of human origin may be prepared from decontaminated human whole blood or whole blood of porcine or bovine origin or of other closely related species.
  • Fig. 1 is a reaction scheme of a process in accordance with one embodiment of the present inven ⁇ tion
  • Fig. 2 is a Western blot for the identification of IGFBPs in serum, platelet and serum/platelet extracts
  • Fig. 3 is a SDS-PAGE analysis of serum, plate ⁇ let and serum/platelet extracts
  • Fig. 4 illustrates the effect of a serum/plate- let extract (PSE) on a MTT test of human fibroblast survival
  • Fig. 5 illustrates the wound breaking strength of (A) control vs fibrin glue-treated rats (experiment 1) and of (B) control vs PSE-treated rats in presence or absence of fibrin glue (experiment 2);
  • Fig. 6 illustrates the dose-effect relationship of platelet/serum extracts (PSE) on wound breaking strength in rats
  • Fig. 7 illustrates the morphometric analysis of 14 day-old wounds treated with PSE alone, PSE in fibrin vs control wounds.
  • Fig. 1 illustrates the reaction scheme of a process in accordance with one embodiment of the pre ⁇ sent invention.
  • PRP is a platelet-rich-plasma, preferably of porcine origin prepared by low speed centrifugation in order to pellet and remove erythrocytes and leukocytes.
  • PE is a platelet extract.
  • NS is a mono- or poly-atomic neutral support, having an amorphous structure or a crystalline lattice (with or without defect) selected from the following polygons, but not limited to this list: hexagon, cubic, mono- or tri-clinic, romboedric and orthorhombic.
  • the support may be of carbon or heteroatomic matter. It may have a single or several anomeric phases and may include also in its network one or more of the following heteroa- tomes: cobalt, Nickel, sulfur, oxygen, tungsten, alu- minum, silicon, nitrogen, fluorine among others.
  • the preferred support is graphite powder or activated car ⁇ bon.
  • FM is a neutral filtering membrane especially designed using a non-soluble and hydrophobic organic polymer. This membrane offers a wide filtering surface which confers its almost non-saturable filtering capac ⁇ ity.
  • US is representing ultra-sound shock
  • ii n or " sp” is a platelet-poor-plasma resulting from a unique passage of support-adsorbed- platelet containing plasma through the filtering mem ⁇ brane.
  • PA-1 and PA-2 are each respectively pre ⁇ cipitating agent 1 and 2 of the following chemical for- mulas:
  • Q is a radical homo- or hetero-cyclic, alkyl, silyl, cycloalkyl, alkenyl, aryl, aralkyl including at least one functional group selected from the following, but not limited to this list, acid, alcohol, amine, amide, alde ⁇ hyde, ketone, enol, oxime, halide, imine, enamine, ester, ether, phosphite, phosphate, nitro, nitroso, sulfamide, sulfate, sulfite, sulfoxyde, sulfone, thiosulfone, thioether, and nitrile;
  • M is an ammonium or phosphonium ion or any positive ion resulting from the chemical ioni- sation of elements of groups 1 to 3 of the
  • Y is a carboxylate, a sulfate, a nitrate, a halide, a hexafluorophosphate, or a tetrafluoroborate ion, or any stable anion resulting from the Lewis acid-base interaction; and x and n are identical or different and are any number between 1 and 6.
  • the precipitating agents PA-1 and PA-2 may be used as such or combined together in a mixture with one or more agents, preferably in aqueous suspension or solution of a pH range from 1 to 12.
  • the preferred precipitating agent PA-1 is an amount of acetone solution where the resulting acetone concentration is about 68 to about 72%.
  • the preferred precipitating agent PA-2 is an amount of acetone solution where the resulting acetone concentration is about 84 to about 95%.
  • the process of the present invention excludes from the platelet-rich-plasma (PRP) step any manipula ⁇ tion or rinsing of the free platelets by successive centrifugation.
  • PRP platelet-rich-plasma
  • the first step of the process of the present invention consists mainly in the preparation of the platelet extract (PE) based on the use of ultrasound as a shock treatment for the release of the material of filtered platelet. In addition to its low cost, this step is simple and can be carried out in less than two hours, even using large quantities of material.
  • the second step of the process of the present invention consists mainly in the preparation of the serum extract (SE) while overcoming the drawbacks of the prior art procedures.
  • This step allows for the chemical transformation of a platelet-poor-plasma or of a serum in a hyperactive growth factor concentrate of molecular weight between 0 and 50 kDa.
  • SE may be prepared in advance or in parallel and may be stored and reconstituted in water or in any appro ⁇ priate physiological buffer such as saline, phospho- saline, Krebs or Hepes.
  • physiological buffer such as saline, phospho- saline, Krebs or Hepes.
  • porcine whole blood was used in accordance with the present invention due to its availability and its contamination-free charac- teristic.
  • young Landrace, Yorkshire, male cas ⁇ trated piglets (2 month-old, 15kg B.W. were purchased from a local breeder). They were housed for 2-3 days in our facilities with free access to water and food.
  • ketamine hydrochloride (10 mg/kg BW)
  • fluothane by intratracheal administration.
  • a cannula was aseptically inserted into one carotid artery and animals were exsanguinated.
  • ACD acid-citrate-dextrose
  • ACD acid- citrate-dextrose
  • the collected platelet-rich-plasma (PRP) was immediately mixed with synthetic activated carbon
  • Q is the amount of activated carbon to add (g)
  • k is an empirically determined constant (between 0.01 and 0.80, preferably 0.082)
  • [PT] is the platelet concen ⁇ tration per liter of PRP and V the PRP volume (L).
  • Activated carbon was used as a neutral support to selectively adsorb platelets.
  • Other supports such as silicon nitride, silicon carbide, or activated carbon G-60TM (Aldrich) or Carbon Decolorizing AlkalineTM (Norit A, Fischer Scientific, Nepean, Ont.) were also successfully used.
  • the mixture activated carbon-PRP was gently mixed for 2 min., then filtered through a Whatman folded filter paper 2VTM (Whatman International Ltd, England).
  • the filter containing platelets and acti ⁇ vated carbon, was washed twice with phosphosaline buffer (PBS, pH 7.0) in order to completely eliminate proteins from serum. The content of the filter was then transferred into a glass vial containing picopure distilled water.
  • PBS phosphosaline buffer
  • the vial was subjected to a shock treatment consisting of either heating at 100°C for one min. and then cooled at 25°C or of two ultra-sound treatment with a sonicator (KontesTM 40-watt; output control:60; monitor scale:20) for about 5 sec. to 10 min. with a pause of 30 sec. in between.
  • the mixture was finally centrifuged at 2,000 to
  • the activated carbon pellet was reconstituted in picopure water, centrifuged, and both supernatants were pooled and lyophilized.
  • the result ⁇ ing powder was the platelet extract (PE) .
  • the precipitate, corre ⁇ sponding to the platelets was resuspended in phosphate buffer saline (PBS) to obtain a final concentration of 10 ⁇ platelet equivalent per ml (pq/ml), which corre ⁇ sponds to the first washing step.
  • PBS phosphate buffer saline
  • the resuspended platelets were centrifuged for a second time at the conditions mentioned above.
  • the precipitated platelets were resuspended in picopure, distilled water to a final concentration of 10 ⁇ plate ⁇ let equivalent per ml (pq/ml).
  • the solution was cooled to about -5 to about 4°C in a ice bath and was subjected to a shock treatment consisting of two ultra-sound treatment with a sonica- tor (KontesTM 40-watt; output control:60; monitor scale:20) for 45 sec. with a pause of 30 sec. in between.
  • the treated solution was frozen using liquid nitrogen and lyophilized overnight.
  • the lyophilized platelets were reconstituted in picopure water, centri ⁇ fuged, and both supernatants were pooled and lyophilized.
  • the resulting powder was the platelet extract (PE).
  • a serum extract was prepared by double acetone protein precipitation in acidic conditions.
  • Whole serum was first acidified with formic acid (88% formic acid, Fisher Scientific, Neapan, Ontario) to pH 1.9- 2.0, then mixed with acetone (Anachemia, Montreal, Canada) to a final concentration of about 68% to about 72% acetone, the preferred acetone concentration being 70%.
  • the mixture was filtered through* a Whatman filter paper 2VTM.
  • the filtrate was collected and acetone was added to a final concentration of about 84% to about 95%, the preferred final concentration being 90%.
  • the mixture was stirred up and again filtered through a Whatman filter paper 2VTM.
  • the filter content was washed with pure acetone, then with ether in order to completely remove acetone.
  • Ether was air-evaporated, the final powder was reconstituted in picopure water (>18.2 megaohms) (1% w/v), filter-sterilized through 0.2 ⁇ m filters (Nalgene, Rochester, NY) and kept frozen at -75 to about -85°C.
  • the serum precipitate may be reconstituted to a concentration of about 0.5 to 6% of protein weight/volume, preferably to a concentration of about 1 to 2% of protein weight/volume (for example,
  • the PE was reconsti ⁇ tuted with the 1% SE to a final concentration of 10 10 platelet equivalent per ml.
  • the mixture was centri ⁇ fuged at 5000 g for 15 min. at +4°C, then filter ster ⁇ ilized through 0.2. ⁇ m filters and either stored at -75°C or lyophilized.
  • the final product was termed platelet-serum-extract (PSE).
  • PSE platelet-serum-extract
  • the PE was reconstituted in picopure water (>18.2 megaohms) (10 ⁇ platelet equivalent/ml).
  • PDGF antibody used in the kit. Results were therefore converted in porcine PDGF equivalent by dividing them by 0.38.
  • Transforming growth factor ⁇ l and ⁇ 2 were meas ⁇ ured in PSE using the Quantikine testTM commercially available (Amersham International, V.K.). Before meas ⁇ urement, extracts were mixed 1:2 either in distilled water or in trifluoroacetic acid (TFA) 1%, incubated for 1 hour at room temperature, lyophilized and recon ⁇ stituted with water to their initial volume. This pro ⁇ cedure was conducted in order to determine if TGF ⁇ was present in extracts as an active or latent, high molecular weight form.
  • TGF ⁇ was present in extracts as an active or latent, high molecular weight form.
  • Insulin-like growth factor-I and II were measured in SE by radioimmunoassay after for ⁇ mic acid-acetone extraction (Bowsher R.R. et al., Endocrinology, 1991, -128:805-814). Briefly, SE (100 ⁇ l) was acidified with formic acid 8.0 M, containing 0.5% Tween 20TM (Biorad Lab., Richmond, CA), then precipi ⁇ tated with acetone (350 ⁇ l).
  • the supernatant was then diluted in RIA buffer for IGFs measurements (1/60 for IGF-I and 1/300 for IGF-II, in a 30 mM phosphate buffer, pH 7.4, containing 0.02% protamine sulfate, grade II, 10 mM EDTA, 0.02% sodium azide and 0.25% bovine serum albumin RIA grade, all buffer components from Sigma Chemicals, St Louis, MO) .
  • IGF-I was then measured in neutralized extracts by double antibody RIA using recombinant human IGF-I (Bachem California) for iodination and standards, and polyclonal anti-hIGF-I antiserum kindly provided by Dr. Underwood (University of North Carolina) through the National and Pituitary Program of the NIDDK, as previ- ously described by Brazeau P. and Abribat T. (75" 1 Ann. Meet, of the Endocrine Soc., Las Vegas, NA, June 9-12, 1993, Abst. 1327) according to the following procedure. hIGF-I RIA PROCEDURE
  • IGF-II was also measured in neutralized extracts in the same RIA buffer by double antibody RIA (Brazeau P. and Abribat T., 75" 1 Ann.Meet, oftheEndocrineSoc., Las Vegas, NA, June 9-12, 1993, Abst 1327) using recom ⁇ binant human IGF-II (Bachem California) for iodination and standards, and monoclonal anti hIGF-II antibodies diluted 1/50 K (Amano International Enzymes Co) accord ⁇ ing to the following procedure.
  • IGF-II RIA 0.5% and cross reactivity of IGF-I in the IGF-II RIA was 3.5%.
  • ED50 from standard curves were typically 70 pg/tube. and 90 pg/tube for IGF-I and IGF-II RIAS, respectively.
  • IGFBP IGF-binding proteins
  • TBS Tris-buffered saline
  • NonidetTM P-40 Calbiochem Co, La Jolla, CA
  • Sheets were trans ⁇ ferred into sealed plastic bags containing 1.5. 10 ⁇ cpm 12 ⁇ I-IGF-I in 20 ml. After an overnight incubation with continual gentle agitation, sheets were washed twice in TBS-0.1% Tween 20TM, then 3 times in TBS, and finally autoradiographed at -70°C on X-ray films for 48 h. For quantitative analysis, bands of the nitrocellulose sheets corresponding to those on autoradiograms were cut and counted in a gamma counter.
  • PDGF levels averaged 3.5 ⁇ 0.5 ng/ml (mean ⁇ SEM of 7 measurements).
  • PDGF levels unexpectedly varied from 3.16 to 14.2 ⁇ g/1.
  • non extracted (water extracted) TGF ⁇ 2 content was 4145 ⁇ 542 pg/ml
  • acid extracted TGF ⁇ 2 content was identical (4169 ⁇ 489 pg/ml) sug- gesting that TGF ⁇ 2 was entirely present in PE in its active form of about 25 KD.
  • TGF ⁇ 1 levels unexpectedly varied from 0.873 to 3.9 ⁇ g/1.
  • TGF ⁇ 2 levels unexpectedly varied from 5.8 to 46.0 ng/1.
  • IGF-I and IGF-II concentrations averaged 200 ⁇ 16 ng/ml and 452 ⁇ 82 ng/ml respectively (mean ⁇ SEM of 7 measurements) .
  • IGFBPs could be detected both in SE and PSE but not in PE.
  • the major IGFBP in serum, IGFBP3 doublet migrating at 37 and 41 KD
  • IGFBP2 doublet migrating at 37 and 41 KD
  • IGFBP2 three other IGFBPs in serum (IGFBP2, 1 and 4, migrating at 31, 27, and 22 KD respectively) were found in SE and PSE, at a concentration of 15-25% that in serum.
  • IGF-I concentration unexpectedly varied from 16.9 to 88 ⁇ g/1 and IGF-II concentration varied from 47.9 to 356 ⁇ g/1.
  • TGF ⁇ 1 level unexpectedly varied from 1.98 to 21.5 ⁇ g/1 and TGF ⁇ 2 level varied from 0.027 to 0.290 ⁇ g/1.
  • Total protein content was 5.3 ⁇ 0.5 mg/ml in PE (mean ⁇ SEM of five determinations), 6.3 ⁇ 0.8 mg/ml in SE (mean ⁇ SEM of five determinations) and 12.2 ⁇ 0.8 mg/ml in PSE (mean ⁇ SEM of six determinations).
  • PE, SE and PSE were sub ⁇ jected to a SDS-PAGE electrophoresis on 12% polyacryla- mide gels, and silver-stained according to the method described by Morissey JH (Anal. Biochem., 1981, 117:307- 310).
  • a protein staining of typical PE, SE and PSE is shown in Fig. 3.
  • the major contaminant in PSE was identified as a 50-55 KD weighing group of proteins of seric origin.
  • PE porcine primary cultured fibroblasts
  • both PE and SE stimulated cell proliferation in a dose-dependent manner between 1 and 100 ⁇ l of extracts.
  • Combination of PE and SE was synergic on cell proliferation, that is the effect of PSE was more than the addition of the effects of PE and SE.
  • This synergy was observed at the doses of 1 and 10 ⁇ l/ml, but not 100 ⁇ l/ml. In fact, the 100 ⁇ l/ml dose was less potent than the 10 ⁇ l/ml dose in all PSE tested.
  • a dose response study from 0 to 200 ⁇ l/ml revealed that PSE induced a dose-related linear stimu ⁇ lation of cell proliferation from 0 to 10 ⁇ l/ml, the effect being progressively blunted between 50 and 200 ⁇ l/ml.
  • the maximal effect on cell proliferation was estimated to be at 10-50 ⁇ l/ml of PSE.
  • RPMI 1640 RPMI 1640 containing 10% FCS and 1% penicillin-streptomycin at a density of 10 ⁇ cells in one ml per well. Twenty four hour later, medium was replaced by a test medium (RPMI 1640TM, 0.5% FCS and 1% penicillin-streptomycin) containing test samples (0 to 200 ⁇ l/ml SE, PE or PSE; 0 to 100 ng/ml porcine PDGF-R & D system, MN, Cat.#125PD). Maximal stimulation of proliferation was evaluated by adding 10% FCS in control wells. Each dose of each test sample was assayed in triplicate.
  • both PE and SE stimulated cell proliferation in a dose-dependent manner between 1 and 100 ⁇ l of extracts.
  • Combination of PE and SE was synergic on cell proliferation, that is the effect of PSE was more than the addition of the effects of PE and SE.
  • This synergy was observed at the doses of 1 and 10 ⁇ l/ml, but not 100 ⁇ l/ml. In fact, the 100 ⁇ l/ml dose was less potent than the 10 ⁇ l/ml dose in all PSE tested.
  • a dose response study from 0 to 200 ⁇ l/ml revealed that PSE induced a dose-related linear stimu ⁇ lation of cell proliferation from 0 to 10 ⁇ l/ml, the effect being progressively blunted between 50 and 200 ⁇ l/ml.
  • the maximal effect on cell proliferation was estimated to be at 10-50 ⁇ l/ml of PSE. 3 .3 M. T. T . Test
  • a quantitative colorimetric assay for porcine primary cultured fibroblasts (PPCF) and human foreskin fibroblasts (HFF) survival was developed in order to assess potential cytotoxicity of the extracts.
  • the assay is dependent on the reduction of the tretrazolium bromide, from Sigma Chemical, St Louis, MO) by the mitrochondrial deshydrogenase of viable cells to form a blue formazan product.
  • the assay was run in 96 well plates in DMEM/Ham F12 medium (Gibco). Cells were plated at confluency (4.0 x 10 4 cells/well) and the procedure was conducted exactly as described by Hansen M.B. et al. (J. Immunol. Methods, 1989, 111:203-210). All test samples (0 to 100 ⁇ l/ml) were tested in tripli- cate.
  • MTT tests were performed on a dose range (0 to 100 ⁇ l/ml) of PSE. As shown in Fig. 4, even high concentrations of PSE (50, 75, 100 ⁇ l) had no negative effect on cell survival, indicating an absence of cytotoxicity. In fact, an increase in optical density was noted at concentrations higher than 50 ⁇ l/ml, that reflected a good cellular health.
  • Sprague-Dawley male rats (275-300g) were pur- chased from Charles River Canada Inc. (St Constant, Quebec) . They were housed in individual cages and fed ad libitum. Each experimental group contained 7 to 8 rats. Wounds were performed as previously described by Garrel DR et al. (J. Surg. Res., 1991, 5_l:297-302) . Briefly, one 6 cm-full-thickness cutaneous incision was made longitudinally on the dorsal skin of each rat under pentobarbital anesthesia. The incisions were immediately closed with Dermalon 4.0TM sutures placed 1 cm apart.
  • Serum and platelet extract were prepared as described above. Except otherwise specified, they were applied onto the wounds in combination with a fibrin matrix purchased from Haemacure Biotech Inc. (Pointe- Claire, Quebec). Platelet-Serum extracts were used to reconstitute a lyophilized thrombin powder (1 ml extract for 100 IU thrombin) in presence of calcium chloride (40 mM) . This mixture was applied directly onto the wound in combination with a bovine fibrinogen solution (40-50 mg/ml) in picopure water. For a 6 cm- long wound, 250 ⁇ l of each preparation was used (total volume: 500 ⁇ l) .
  • the dose-effect relationship of growth factor extract (27,83 and 250 ⁇ l of PSE, 10 10 platelet equiva ⁇ lent/ml in 1% SE) in 500 ⁇ l fibrin matrix, vs control (BSA, 60 mg/ml) was determined.
  • SE lyophilized and reconstituted in 250 ⁇ l sterile water) in 500 ⁇ l fibrin matrix, vs control (BSA, 60 mg/ml) were determined.
  • PSE as a wound healing enhancer was tested in a model of rat surgical wound. Measured parameters included wound breaking strength 14 days post wounding, as well as histological examination of the scars.
  • Morphological evaluation includes aspect of the epidermis and of the cells within the scar, whereas morphometric evaluation includes thickness of the epi ⁇ dermis surrounding the scar, width of the scar at the level of both superficial and reticular dermis and cel ⁇ lularity, by counting fibroblasts per microscopic field, magnification x 750.
  • PSE increased wound ' breaking strength in vivo when administered in a fibrin matrix. This effect was associated with increased scar width and cellular- ity. In all observed histological sections, no sign of inflammation, of chelo ⁇ d or of tumoral foci could be detected.
  • a seric and a platelet extract yielded a preparation containing at least sig ⁇ nificant amounts of PDGF, TGF ⁇ 2, IGF-I, II and IGFBPs.
  • growth factors were only characterized by immunoreactivity in accordance with one embodiment of the present invention, numerous reports confirm that platelets and serum are two major sources of growth factors. Platelets ⁇ granules are the richest in vivo source of TGF ⁇ and PDGF. Porcine platelets contain two isotypes, TGF ⁇ 1 and ⁇ 2. Human and porcine TGF ⁇ 1 have total sequence identity and TGF ⁇ 2 has approxi ⁇ mately 70% homology with TGF ⁇ 1.
  • TGF ⁇ s are natu- rally found as a latent, high molecular weight, inac ⁇ tive complexes in platelets.
  • the measurement of non-extracted and acid-extracted platelet extracts yielded the same amount of TGF ⁇ 1 and ⁇ 2, indicating that TGF ⁇ 1 and ⁇ 2 are present in our extracts in its active form of about 25 KD. Since activation of TGF ⁇ in the latent complex has been shown to occur by treatment with acid, urea or heat in vitro, it was concluded that the heating step of the PE preparation was responsible for this activa- tion.
  • PDGF is a disulfide-linked dimer with a molecu ⁇ lar weight 30-32 KD.
  • the subunits of the dimer are two related polypeptides designated the A and B chains.
  • human platelet PDGF has been shown to consist of PDGF-AB and PDGF-BB
  • porcine platelet PDGF consists primarily of PDGF-BB homodimers. Because of its higher affinity for the type B PDGF receptor, PDGF-BB is more potent than PDGF-AB in stimulating cell proliferation in vitro.
  • PDGF AB and BB have equal potency as wound healing enhancers.
  • Both PDGF and TGF ⁇ s are known as stimulators of wound healing. They both induce extracellular matrix synthesis, granulation tissue formation and increase wound breaking strength in a variety of animal models. Both recombinant TGF ⁇ 2 and PDGF-BB are currently tested in clinical trials as therapeutic agents for the healing of chronic ulcers with promising initial results.
  • platelet ⁇ gran- ules have been shown to contain a number of agents that might play a role in the process of wound healing.
  • these substances are Platelet Factor 4 (PF4) , a platelet-derived Endothelial Cell Growth Factor (pdECGF), an Epidermal Growth Factor-like protein (EGF), and traces of IGF-I, IGF-II and IGFBP3.
  • PF4 Platelet Factor 4
  • pdECGF platelet-derived Endothelial Cell Growth Factor
  • EGF Epidermal Growth Factor-like protein
  • Insulin-like growth factors I and II are two related peptides of approximately 7500 KD and of iden ⁇ tical sequence in human and pig. In serum, they circu ⁇ late in large amount, tightly bound to specific, high affinity binding proteins. IGF-I is the most important mediator of the biological actions of growth hormone and possesses strong systemic and local anabolic actions. Both IGFs and IGFBPs are present in wound fluid in the course of wound healing. When adminis- tered alone or in combination with IGFBPs, IGF-I has been shown to stimulate wound strength and granulation tissue formation in models of normal or impaired wound healing.
  • the analytical results showed that the process of extraction in accordance with the present invention successfully recovered IGFs and IGFBPs from serum, and PDGF and TGF ⁇ from platelets.
  • other bioactive substances such like PF4, pdECGF or EGF might also be present and might participate in the biological activity of PSE.
  • the process of the present invention for the manufacture of PSE is new, simple and inexpensive. It involves a few number of steps that can be performed in less than 24 hours to produce a mixture made of at least 5 important growth factors for wound healing (PDGF, TGF ⁇ , IGF-I, IGF-II, IGFBPs).
  • porcine origin of the blood in accordance with one embodiment of the present inven- tion, guaranties: 1) a total absence of potential transmission of human viral agents (hepatitis, HIV, etc.); 2) the production of growth factors nearly 100% identical to their human counterparts and, 3) a reli ⁇ able, largely available and highly reproducible source of raw material.
  • both the platelet and serum extracts of the present invention stimulated fibroblast proliferation in a dose dependent manner, and coadmin- istration of both extracts resulted in a synergic effect.
  • SE and PE contained IGFs and PDGF, respectively, it is suggested that the synergy was due to interactions between these two growth factors. This synergy was first reported in cultured 3T3 fibroblasts, then confirmed in vivo in animal models of wound heal- ing. It has been explained by the complementary roles of PDGF and IGFs in the cell cycle, PDGF being a compe ⁇ tence factor and IGFs being a progression factor.
  • PSE induced a dose-dependent increase of por ⁇ cine primary cultured fibroblasts (PPCF) or 3T3 up to 10 ⁇ l/ml, while the stimulatory effect was inhibited at doses equal or above 50 ⁇ l/ml.
  • PSE was not cytotoxic at doses up to 100 ⁇ l/ml and stimulated protein (collagen) synthesis in a dose dependent fash ⁇ ion between 0 and 100 ⁇ l/ml.
  • platelet/serum extract of the present invention is a powerful stimulator of granulation tissue formation. Its potential therapeutical applications include the treatment of chronic ulcers and surgical wounds, and the regeneration of injured soft tissues.
  • SE and PE of the present invention might be used alone or advantageously in combination (PSE) because they work synergically.
  • PSE in vivo, they have to be adminis ⁇ tered topically in a matrix slowly releasing them, such as fibrin glue, collagen, polymers among others.
  • the bioactive formulation is 50 to 1000 ⁇ l of a preparation made of 10 to 1000 IU/ml thrombin, 20 to 200 mg/ml fibrinogen, and 50 to 500 ⁇ l/ml SE (0.1 to 5%), PE (10 8 to 10 12 platelet equiva ⁇ lent/ml buffer) or PSE (10 8 to 10 12 platelet equiva- lent/ml SE 0.1 to 5%) in presence of CaCl2 (1 to 100 mM final) .
  • Q K.D X [PRP] . V. 10-9
  • K is an empirically determined constant of 0.082
  • D x is a ratio of the density of the neutral support with respect to the density of carbon (1 for graphite)
  • [PRP] is the platelet concentration per liter of PRP (464 X 10 9 )
  • V is the PRP volume in L (3)
  • the adsorbed platelet are washed twice with three liters of phos- phosaline buffer pH 7.4, then the washed filtrate is transferred in a beaker.
  • the membrane is rinsed with 1 L of distilled water and the temperature of the washed filtrate in the beaker is raised to 100°C for 1 min.
  • the washed filtrate in the beaker is filtered and/or centrifuged; the supernatant is lyophilized.
  • the lyophilisate constitutes the solid platelet extract (PE) which can be reconstituted.
  • PPCF control 75858 763 100 0.21 0.017 100
  • EXAMPLE II Preparation of Platelet Extract The procedure as described in Example I was carried out, except that carbure of silicium was used instead of graphite. This material has a density con ⁇ stant of 3.23, which resulted in a Q value of 335g of carbure of silicium used.
  • the precipitated carbon is kept and carefully mixed with the three liters of PRP. After a 2 min. mixing, the platelet-adsorbed-carbon mixture is fil- tered through a Whatman filter paper 2VTM. The fil ⁇ tered PPP is discarded.
  • the adsorbed platelet are washed twice with 450 ml of phosphosaline buffer pH 7.4, then the washed filtrate is transferred in a beaker.
  • the adsorbed platelet are resuspended in picopure water (>18.2 megaohms) to obtain a final con ⁇ centration of 10 pq/ml and the temperature of the beaker is cooled to about 0-4°C using an ice bath.
  • the adsorbed platelet are subjected to a first ultra-sound treatment with a sonicator (KontesTM 40- watt; output control:60; monitor scale:20) for about 45 sec, a pause of 30 sec. and a second ultra-sound treatment for about 45 sec.
  • a sonicator KontesTM 40- watt; output control:60; monitor scale:20
  • the washed filtrate in the beaker is centri- fuged for 5 min. at 5000g; the precipitate is resus- pended in a minimum quantity of picopure water and cen ⁇ trifuged again under the same conditions. Both super- natants are combined, frozen in liquid nitrogen and then lyophilized.
  • the lyophilisate constitutes the solid platelet extract (PE) which is reconstituted in picopure water (>18.2 megaohms) to obtain a ' final con ⁇ centration of 10 10 pq/ml, centrifuged for 30 min. at 10,000g.
  • the supernatant is filtered first through 0.8 ⁇ m and 0.2 ⁇ m filters (Nalgene, Rochester, NY). The preparation is aliquoted and kept at -80°C.
  • TGF ⁇ 2 46.0 ng/1
  • PPCF control 76982 1706 100 0.21 0.017 100
  • Example IV The procedure as described in Example IV is carried out, except that activated carbon DarcoTM G-60 (Aldrich) was used instead of carbon.
  • the resuspended platelets are centrifuged for a second time at the conditions mentioned above.
  • the precipitated platelets are resuspended in picopure water (>18.2 megaohms) to a final concentration of 10*" platelet equivalent per ml (pq/ml).
  • the solution is cooled to about 0 to about 4°C in a ice bath and is subjected to a shock treatment consisting of two ultra-sound treatment with a sonica- tor (KontesTM 40-watt; output control: 60; monitor scale: 20) for 45 sec. with a pause of 30 sec. in between.
  • a shock treatment consisting of two ultra-sound treatment with a sonica- tor (KontesTM 40-watt; output control: 60; monitor scale: 20) for 45 sec. with a pause of 30 sec. in between.
  • the treated solution is frozen using liquid nitrogen and lyophilized overnight.
  • the lyophilized platelets are reconstituted in picopure water to obtain a final concentration of 10 ⁇ pq/ml.
  • the membrane fragments are eliminated by ultracentrifugation or by filtration through 0.8 ⁇ m, 0.45 ⁇ m and 0.2 ⁇ m filters (Nalgene, Rochester, NY). The preparation is aliquoted and kept at -80°C.
  • a 300 ml of whole serum was 'first acidified with 18.2 ml of formic acid (88% formic acid, Fisher Scientific, Neapan, Ontario) at room temperature. After 5 min. of agitation at room temperature, 700 ml of pure acetone was added. The agitation was continued for another 5 min., then the suspension was filtered through a Whatman filter paper 2VTM.
  • the filtrate was collected and slowly trans ⁇ ferred in a beaker containing 1400 ml of pure acetone constantly agitated. The order in the addition is of extreme importance.
  • the precipitate is filtered through a Whatman filter paper 2VTM, washed three times with 500 ml of pure acetone, then three times with 500 ml of ether, then dried.
  • the solid mass is 1.7 g and is reconsti- tuted in 170 ml of picopure water (18.2 Ohm) (1% w/v), filter-sterilized through 0.22 ⁇ m filters (Nalgene, Rochester, NY) and aliquoted under laminar flux (Canadian Cabinets Co, Ltd., V6 MW99.C30TM). This solution of pH 6.5 and 1% of final concentration, con- stitutes the serum extract (SE).
  • the acidified serum is cooled at 4°C in a cold room. A quantity of 75% acetone is added and agitated magnetically for an hour to precipitate the proteins. The suspension is filtered through a Whatman filter paper 2VTM.
  • the filtrate was collected arid slowly trans ⁇ ferred in a beaker containing an amount of pure acetone to obtain a final concentration of 90% acetone. After an incubation of one hour, the suspension is filtered through a Whatman filter paper 2VTM.
  • the precipitate is washed twice with pure ace ⁇ tone, and then a third time with anhydride ether, then dried using a lyophilizer.
  • the solid mass is reconstituted in picopure water (>18.2 megaohms) to obtain a final concentration of 1%, filter-sterilized through 0.22 ⁇ m filters (Nalgene, Rochester, NY) and aliquoted under laminar flux (Canadian Cabinets Co, Ltd., V6 MW99.C30TM) and kept at -80°C.
  • PPCF control 74087 1761 100 0.21 0.017 100 1 ul/ml 0.20 0.006 93 10 ul/ml 0.17 0.007 84
  • Example VIII The procedure as described in Example VIII is carried out, except that the acidified serum is cooled at 0°C instead of 4°C. Also, the first precipitation using acetone is conducted such that the acetone is added dropwise until a final acetone concentration of 72% is achieved, the second acetone addition is effected under the same conditions until a final ace ⁇ tone concentration of 75% is achieved, and a third ace ⁇ tone addition is effected under the same conditions until a final acetone concentration of 90% is achieved.
  • Example I, II, III, IV, V or VI is reconstituted with SE of Example VII, VIII, IX, or X to obtain a final concentration of 10 ⁇ platelet per ml without neutralization.
  • the mixture is centrifuged at 10,000g for 15 min. at +4°C, then filter sterilized through 0.22 ⁇ m filters and aliquoted under laminar flux.
  • EXAMPLE XII
  • Example I, II, III, IV, V or VI is reconstituted with SE of Example VII, VIII, IX, or X to obtain a final concentration of 10 ⁇ platelet per ml.
  • the mixture is neutralized using a Amberlite IRA- 400(OH)TM resin, centrifuged at 10,000g for 15 min. at +4°C, then filter sterilized through 0.22 ⁇ m filters and aliquoted under laminar flux.

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Abstract

L'invention concerne un procédé, permettant de préparer un extrait de plaquettes et de sérum, qui consiste à : a) concentrer un plasma riche en plaquettes préparé à partir de sang total passant au travers d'un support neutre permettant d'absorber les plaquettes ou par centrifugation du sang total entre 1000 et 3000g environ; b) libérer les plaquettes concentrées de l'étape a) par onde de choc ultrasonore; c) faire précipiter le plasma concentré de l'étape a), un plasma pauvre en plaquettes ou un sérum par adjonction d'une certaine quantité de solution d'acétone pour obtenir une concentration en acétone allant de 68 à 95 % environ à une température de -15 à 5 °C environ, ce qui donne un précipité solide de plasma ou de sérum; d) reconstituer ce précipité de plasma ou de sérum à une concentration d'environ 0,5 à 6 % de poids de protéines/volume et le mélanger aux plaquettes libérées de l'étape b) pour donner un extrait de plaquettes et de sérum à concentration physiologique, en pratique, pour offrir une synergie maximum permettant de promouvoir la guérison d'une blessure. L'invention concerne aussi une composition pharmaceutique, promouvant la guérison des blessures, qui contient en concentration efficace un extrait de plaquettes, un extrait de sérum ou un extrait de plaquettes et de sérum, préparé selon le procédé propre à l'invention et associé à un vecteur pharmacologiquement acceptable.
PCT/CA1994/000601 1993-12-08 1994-11-01 Procede de preparation d'un extrait de serum et de facteur de croissance plaquettaire WO1995015763A1 (fr)

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Cited By (18)

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WO1997034614A1 (fr) * 1996-03-20 1997-09-25 Theratechnologies Inc. Procede de preparation d'extrait de facteurs de croissance plaquettaire pour preparations cicatrisantes
US6110484A (en) * 1998-11-24 2000-08-29 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
WO2005105121A1 (fr) * 2004-05-05 2005-11-10 Synthes Gmbh Utilisation de plaquettes ou de plasma riche en plaquettes (prp)
EP1848447A2 (fr) * 2004-08-20 2007-10-31 Alberto Gorrochategui Barrueta Agent de regeneration d'un tissu biologique et methode de preparation et d'utilisation
US20110008458A1 (en) * 2009-07-09 2011-01-13 Orogen Biosciences, Inc. Process for removing growth factors from platelets
EP2389942A1 (fr) * 2010-05-25 2011-11-30 GWO REI Biomedical Technology Corp. Facteurs de croissance inactivés de manière virale contenant du lysat plaquettaire à teneur réduite en PDGF et VEGF et leur procédé de préparation
US20120114760A1 (en) * 2004-07-02 2012-05-10 James Gandy Compositions for treating wounds and processes for their preparation
US8293530B2 (en) 2006-10-17 2012-10-23 Carnegie Mellon University Method and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8529958B2 (en) 2006-10-17 2013-09-10 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8529960B2 (en) 2002-03-18 2013-09-10 Carnell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8821858B2 (en) * 2005-02-09 2014-09-02 Gw Ip, Llc Lyophilized platelet rich plasma for the use in wound healing (chronic or acute) and bone or tissue grafts or repair
US10166258B2 (en) 2008-09-16 2019-01-01 Mayo Foundation For Medical Education And Research Compositions containing platelet contents
CN110464740A (zh) * 2019-09-02 2019-11-19 广州准优生物科技有限公司 Prp产品及制备方法、prp与调节性t细胞复合制剂及应用
CN115463249A (zh) * 2022-08-11 2022-12-13 中南大学湘雅医院 一种负载富血小板血浆水凝胶及其制备方法
US11604026B2 (en) 2019-03-14 2023-03-14 Terumo Bct Biotechnologies, Llc Lyophilization loading tray assembly and system
US11634257B2 (en) 2017-10-09 2023-04-25 Terumo Bct Biotechnologies, Llc Lyophilization container and method of using same
US11891620B2 (en) 2014-05-16 2024-02-06 Mayo Foundation For Medical Education And Research Cell culture media compositions for primary cells
US11994343B2 (en) 2023-07-11 2024-05-28 Terumo Bct Biotechnologies, Llc Multi-part lyophilization container and method of use

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Cited By (36)

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Publication number Priority date Publication date Assignee Title
WO1997034614A1 (fr) * 1996-03-20 1997-09-25 Theratechnologies Inc. Procede de preparation d'extrait de facteurs de croissance plaquettaire pour preparations cicatrisantes
AU725091B2 (en) * 1996-03-20 2000-10-05 Theratechnologies Inc. Process for the preparation of platelet growth factors extract for wound healing compositions
US6110484A (en) * 1998-11-24 2000-08-29 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
US6277394B1 (en) 1998-11-24 2001-08-21 Cohesion Technologies, Inc. Collagen-polymer matrices with differential biodegradability
US8529960B2 (en) 2002-03-18 2013-09-10 Carnell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
WO2005105121A1 (fr) * 2004-05-05 2005-11-10 Synthes Gmbh Utilisation de plaquettes ou de plasma riche en plaquettes (prp)
US20120114760A1 (en) * 2004-07-02 2012-05-10 James Gandy Compositions for treating wounds and processes for their preparation
EP1848447A2 (fr) * 2004-08-20 2007-10-31 Alberto Gorrochategui Barrueta Agent de regeneration d'un tissu biologique et methode de preparation et d'utilisation
EP1848447A4 (fr) * 2004-08-20 2009-07-29 Barrueta Alberto Gorrochategui Agent de regeneration d'un tissu biologique et methode de preparation et d'utilisation
US8821858B2 (en) * 2005-02-09 2014-09-02 Gw Ip, Llc Lyophilized platelet rich plasma for the use in wound healing (chronic or acute) and bone or tissue grafts or repair
US8529958B2 (en) 2006-10-17 2013-09-10 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8293530B2 (en) 2006-10-17 2012-10-23 Carnegie Mellon University Method and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8529961B2 (en) 2006-10-17 2013-09-10 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US9364503B2 (en) 2006-10-17 2016-06-14 Carmell Therapeutics Corporation Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US8911789B2 (en) 2006-10-17 2014-12-16 Carnegie Mellon University Methods and apparatus for manufacturing plasma based plastics and bioplastics produced therefrom
US10166258B2 (en) 2008-09-16 2019-01-01 Mayo Foundation For Medical Education And Research Compositions containing platelet contents
US10925901B2 (en) 2008-09-16 2021-02-23 Mayo Foundation For Medical Education And Research Compositions containing platelet contents
US20110008458A1 (en) * 2009-07-09 2011-01-13 Orogen Biosciences, Inc. Process for removing growth factors from platelets
US8734854B2 (en) * 2009-07-09 2014-05-27 Orogen Biosciences Inc. Process for removing growth factors from platelets
US9511118B1 (en) 2009-07-09 2016-12-06 Pgfx Patent Holdings, Llc Process for removing growth factors from platelets
WO2011148326A1 (fr) * 2010-05-25 2011-12-01 Gwo Rei Biomedical Technology Corp. Lysat plaquettaire appauvri en pdgf et vegf et contenant des facteurs de croissance viralement inactivés, et procédé de préparation de celui-ci
US9034646B2 (en) 2010-05-25 2015-05-19 Zheng Yang Biomedical Technology Co., Ltd. Virally-inactivated growth factors-containing platelet lysate depleted of PDGF and VEGF and preparation method thereof
CN102985101B (zh) * 2010-05-25 2014-07-30 国维联合科技股份有限公司 含有病毒去活化的生长因子且pdgf与vdgf耗尽的血小板裂解物及其制备方法
CN102985101A (zh) * 2010-05-25 2013-03-20 国维联合科技股份有限公司 含有病毒去活化的生长因子且pdgf与vdgf耗尽的血小板裂解物及其制备方法
EP2389942A1 (fr) * 2010-05-25 2011-11-30 GWO REI Biomedical Technology Corp. Facteurs de croissance inactivés de manière virale contenant du lysat plaquettaire à teneur réduite en PDGF et VEGF et leur procédé de préparation
US11891620B2 (en) 2014-05-16 2024-02-06 Mayo Foundation For Medical Education And Research Cell culture media compositions for primary cells
US11634257B2 (en) 2017-10-09 2023-04-25 Terumo Bct Biotechnologies, Llc Lyophilization container and method of using same
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US11815311B2 (en) 2019-03-14 2023-11-14 Terumo Bct Biotechnologies, Llc Lyophilization container fill fixture, system and method of use
CN110464740A (zh) * 2019-09-02 2019-11-19 广州准优生物科技有限公司 Prp产品及制备方法、prp与调节性t细胞复合制剂及应用
CN115463249A (zh) * 2022-08-11 2022-12-13 中南大学湘雅医院 一种负载富血小板血浆水凝胶及其制备方法
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