WO2014016829A1 - Insoluble fibrinogen particles (ifp) and coatings : composition, fabrication methods, and applications for harvesting and culturing mammalian cells - Google Patents

Insoluble fibrinogen particles (ifp) and coatings : composition, fabrication methods, and applications for harvesting and culturing mammalian cells Download PDF

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WO2014016829A1
WO2014016829A1 PCT/IL2013/050620 IL2013050620W WO2014016829A1 WO 2014016829 A1 WO2014016829 A1 WO 2014016829A1 IL 2013050620 W IL2013050620 W IL 2013050620W WO 2014016829 A1 WO2014016829 A1 WO 2014016829A1
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ifp
fibrinogen
cells
composition
blood
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Gerard Marx
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Mx Biotech Ltd.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745Blood coagulation or fibrinolysis factors
    • C07K14/75Fibrinogen
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

A new matrix is presented which is composed of insoluble fibrinogen particles (i FP). The i FP are composed of >75% fibrinogen, are insoluble in physiological buffers, exhibit a density of 1.20 ± 0.03 gm/ml and are selected for those with diameters between 30-300 microns. The present invention also provides a novel 3- stage procedure for preparing i FP from a fibrinogen-rich precipitate. The i FP are fabricated under conditions designed to optimize the attractiveness of i FP for a variety of normal stem and cancer cells. A number of biomedical applications are evident for harvesting cells of interest using devices with i FP.

Description

Title: Insoluble Fibrinogen Particles (iFP) and Coatings: Composition,

Fabrication Methods, and Applications for Harvesting and Culturing

Mammalian Cells

FIELD OF THE INVENTION

The present invention relates to insoluble, fibrinogen particles (iFP), and fibers, particles or filters coated with insoluble fibrinogen, and to methods for preparing such iFP, and fibers, particles or filters coated with insoluble fibrinogen, without employing thrombin or other enzymes.

BACKGROUND OF THE INVENTION

There is a widespread demand for materials that could permit the harvesting and growth of either normal stem cells from bone marrow or cancer cells from tissue samples. It has been estimated that the market for stem cell and cancer cell products could reach over $10 billion over the next 10 years.

Based on the availability of autologous stem cells from tissue sources such as bone marrow or blood, techniques are being developed for tissue regenerative therapies. Animal data and clinical studies indicate that implantation of stem cells could be the basis for treatment of acute myocardial infarction, advanced coronary artery disease, and chronic heart failure (1-4). The augmentation of other tissues with stem cells is currently under development in many institutions.

Similarly, there is a demand for a matrix that could be used to isolate and grow various types of cancer cells for diagnostic and metabolic studies (5-8).

Thus, it would be desirable to have available a matrix that could be used to effectively and conveniently harvest stem cells from normal tissue sources, such as blood, bone marrow or adipose tissue (liposuction), or to capture cancer cells from biopsy tissue or blood.

The "plastic plate" method is most often used to isolate and grow cells. In that the plastic is relatively inert to such cells, various proteins, such as fibronectin or collagen, have been employed as coatings to render the plastic more "cell friendly" but yields are low, selectivity is poor, costs are high and the attached cells must be eventually desorbed from the plastic by trypsinization.

Various 3-D matrices (collagen, tricalcium phosphate, derivatized PEG, chitosan, alginates, PLA/PGA copolymers ) have been proposed as matrices for the culturing of attachment-dependent cells (9-11). However, these are not very potent in terms of being attractive to select cells or have other disadvantages, such as toxic degradation products or not being biocompatible or requiring complex equipment and reagents. Many techniques are labor intensive, or require electromagnetic hardware as well as libraries of monoclonal antibodies and are expensive. Thus, there are ongoing efforts to develop more cost effective, selective and cell-friendly matrices.

Fibrinogen is possible candidate protein from which a matrix or coating for cell culturing applications could be fabricated (12-15). However, native fibrinogen is soluble in aqueous buffer. Thus, it is usually mixed with thrombin to generate insoluble fibrin, from which beads, foams, tubes and sheets have been prepared.

For commercial scale preparations, Cohn fractionation with cold ethanol is the main process by which fibrinogen is precipitated from pooled blood plasma . The resulting precipitate (fraction I paste) can be dissolved by appropriate buffers at 37°C, often with the addition of formulants and antifibrinolytic compounds (such as aprotinin). However, instability, extensive labor costs and rigorous virus

inactivation/removal processes make fraction I paste a difficult pathway for preparing soluble fibrinogen, as required for fibrin sealant.

Moreover, fraction I paste does not store well. Even at -20°C, I paste undergoes extensive thrombin activation and cross-linking by factor XIII, both which are co-precipitated during the ethanol precipitation step. Consequently, the fraction I paste is often discarded.

Where soluble, native fibrinogen is desired, such as used in fibrin sealant kits, great efforts have gone into retaining the solubility and functionality of virus- inactivated, dried fibrinogen (23,24). Because of its sensitivity to heat, procedures for preparing soluble fibrinogen concentrates are usually derived from cryoprecipitate or through a special isolation procedures performed at low temperatures (25-31). Different groups developed proprietary formulating agents to permit mild heat pasteurization and resolubilization of dried fibrinogen by adding salts, chaotropic agents, detergents, sugars and other formulants .

Previously, insoluble, but biodegradable adryomycin-enriched microbeads were prepared from fibrinogen by heating to 90-160°C in an oil emulsion system

(19). As such high temperatures were employed, these fibrinogen-based materials lost much cell attachment activity and would not be suitable for cell culture purposes.

The solubility of lyophilized fibrinogen in aqueous buffers and its stickiness when it becomes wetted, make it inconvenient to use as a cell culture matrix.

Recently, electrospun fibrinogen scaffolding was prepared and used as a tissue engineering scaffold (20). The electrospinning process requires complex equipment for fabrication, and the resultant scaffold is rather delicate. As it has not been heat treated or cross-linked, it tends to dissolve. Thus for practical cell culture applications, electrospun fibrinogen scaffolding requires treatment with a cross-linking agent (such as gluteraldehyde or transglutaminase). Also, a gel comprised of soluble fibrinogen mixed with hyaluronic acid has been described for encapsulating and implanting bone marrow-derived stem cells (21).

A dried fibrin composition, of fibrinogen, thrombin and factor XIII as well as medically active ingredients, was prepared by freeze-drying without heating (22).

Other variants of fibrin were described for drug delivery, organ culture and tissue engineering (23-33). For example, fibrin microbeads (FMB) were prepared by mixing solutions of concentrated fibrinogen with thrombin, in a hot (below 80°C) oil emulsion system. The thrombin induced fibrin coagulation within the aqueous droplets that were dispersed throughout the oil phase, wherein coagulation and factor XIII induced cross-linking occurred. The rate of stirring and temperature, as well as added detergents or formulants, all impacted on the yield and size distribution of the dry, as well as the cell attachment potency, of spheroidal fibrin microbeads (FMB). The FMB presented cell many attachment epitopes and were quite attractive to a variety of mesenchymal cells (33-37). From an industrial perspective , the FMB fabrication process was laborious and required many washing steps to remove residual oil and surfactants. One could expect that the FMB oil emulsion process would be difficult and expensive to scale up for industrial production.

SUMMARY OF THE INVENTION

The present invention overcomes the above drawbacks of the background art by providing, in at least some embodiments thereof, a new matrix comprising, insoluble fibrinogen particles (iFP), and in at least some embodiments, fibers, particles or filters coated with insoluble fibrinogen. The iFP and devices coated with insoluble fibrinogen are preferably fabricated under specific conditions without the use of enzymes, oils or detergents, to render the fibrinogen insoluble by forming aggregates, while retaining its attractiveness to cells.

It should be noted that although the discussion centers around iFP for clarity, optionally fibers, particles or filters coated with insoluble fibrinogen, fabricated under specific conditions to retain the cell attachment properties of native fibrinogen, could also be so prepared and/or employed as described herein.

According to at least some embodiments of the present invention, iFP could optionally be employed to harvest attachment-prone cells from mixtures of normal cells, even from such mixtures containing a small fraction of mesenchymal or stem cells (<1 %).

According to other embodiments of the present invention, a composite of adherent normal stem cells and iFP may optionally be provided, for example for tissue reconstruction applications. The iFP could be employed to harvest normal stem cells from blood, bone marrow, adipose aspirates (liposuction), then employed for tissue regeneration purposes.

Also, iFP could optionally be incorporated into a device used to capture the few cancer stem cells (i.e. 100-1000 per liter blood) suspended among the many other

12

non-cancer cells in whole blood ( red blood cells 4-6 x 10 cells per liter; white blood cells (3-10 x 109 cells per liter; platelets 150-400 x 109 cells per liter). According to at least some embodiments of the present invention, iFP preferably comprise more than 75% fibrinogen, are insoluble in physiological buffer, such as phosphate buffered saline, pH 7.0 (PBS), or any other buffer which maintains a physiological pH, for example in the range of pH 6.5-7.5. Optionally and more preferably, the iFP exhibit a density of 1.20 ± 0.03 gm/ml (range 1.17-1.23 ) are more preferably selected for those with diameters between 30-300 microns.

According to at least some embodiments of the present invention, a novel procedure for preparing iFP from a fibrinogen-rich precipitate, such as Cohn fraction I paste obtained during blood plasma fractionation. The 3-stage iFP fabrication process optionally and preferably involves removal of water and contaminating proteins, controlled heating under inert gas atmosphere or in vacuuo, and milling followed by sieving. Water removal preferably comprises suspending the fibrinogen-rich I paste in a series of buffers containing increasing levels of water-miscible solvents, more preferably with homogenization and removal of supernatant liquids at each step, to generate a washed precipitate. Non-limiting examples of such a series of solvents would be: 12-15% ethanol in PBS buffer, pH 7.0, followed by 70% ethanol in PBS buffer, followed by 95% ethanol, followed by 100% acetone. The PBS buffer may optionally contain 1 mM EDTA as a chelating agent.

For denaturing and rendering it insoluble, the washed precipitate is preferably subjected to heating 60-70°C for 5-12 hours, under an inert gas (N2, He) atmosphere, or in vacuuo, to produce a dry matrix.

For milling, the heat-dried matrix is preferably milled or ground so as to break into particles, then sieved, to obtain discretely sized particles (ranging from 30-300 μιη diameter).

The washed protein mass from stage 1 (water removal) preferably comprises a significant majority of fibrinogen (for example and without limitation, preferably greater than 75% fibrinogen, preferably >85% fibrinogen, as determined by electrophoresis (SDS-PAGE) or % clottable protein. The heating stage renders the precipitate insoluble and prevents oxidation, but retains the cell attraction properties of native fibrinogen.

The iFP fabrication process preferably does not employ clotting enzymes (such as thrombin and factor XIII) or additives (such as oils or surfactants). Rather, stage 1 washings remove most water, as well as most contaminating albumin, IgG and trace elements Ca(II), Zn(II), Cu(II). Stage 2 heating to 60-70°C under an inert gas atmosphere renders the fibrinogen insoluble but prevents oxidation and retains cell attachment potency of native fibrinogen. Stage 3 is used to prepare and collect particles of desired size, which differ for various applications.

Thus, according to at least some embodiments of the present invention, there is provided a relatively simple process for preparing an insoluble fibrinogen-based matrix without using thrombin, or oils with surfactants. The process is preferably mild to retain the cell attraction properties of native fibrinogen, and is also scalable to produce industrial quantities of final product.

For example, preferably a series of solvent washing steps was employed to increase the fibrinogen levels in the paste from an initial -60% to -85%, concomitantly removing contaminating proteins (albumin, IgG) and divalent cations. The washed precipitate was then heated for 5-12 hr at 60-70°C under an inert gas atmosphere to partially denature the fibrinogen but minimize protein oxidation to prevent loss of cell attraction potential (see Fig 1 for schematic diagram).

Tests demonstrated that the resultant non-porous particles were insoluble in physiologic buffers, such as PBS, had a density - 1.20+0.03 and did not clot or clump together when mixed with thrombin. Thus, they could be suspended in aqueous cell culture buffers, but due to their density, tended to settle to the bottom when not agitated.

As no lytic enzymes were employed in the process, the iFP of the present invention retained the fibrinopeptides A & B normally clipped off by thrombin. Also as no thrombin was employed and Ca+2 was removed, little factor Xlll-induced cross- linking occurred. Rather, after water removal with solvents and controlled heating steps, the D-domains of native (soluble) fibrinogen monomers became denatured resulting in an insoluble matrix composed of misfolded monomers aggregated into an insoluble matrix. The dried, non-porous matrix could be milled and sieved to generate a high yield of granular particles of desired size for cell culture applications (30-300 um diameter). As a result of specific treatments during fabrication, the iFP present the 4 cell attachment (Haptide) epitopes per fibrinogen monomer, for the adherance of cells (53, 54). The present invention in at least some embodiments provides a 3-stage procedure for preparing iFP from a fibrinogen-rich precipitate, such as Cohn I paste, comprising:

(i) Suspending and homogenizing the Cohn I paste in a buffer containing 12- 15% ethanol in PBS buffer, pH 7.2, 1 mM EDTA (as a non-limiting example of a chelator), maintained at ~ 4°C. This permits albumin and IgG co-precipitated in the Cohn I paste, to dissolve from the precipitate. Filtration or centrifugation permits isolation of the washed precipitate, discarding the supernatant. This step also removes contaminating metabolites, molecules and divalent cations.

This washing/ homogenizing process is optionally and preferably repeated, with 60- 70% ethanol, then 95% ethanol, then with 100% acetone at ambient temperature. After homogenizing, the solvent with water are decanted and discarded, to generate a washed, dehydrated precipitate.

(ii) Heating the washed precipitate for 5-12 hr at 60-70°C under inert gas

atmosphere or under vacuuo, to generate a dry matrix.

(iii) Milling/grinding the dry matrix and sieving to obtain fibrinogen particles having a mean diameter of 30-300 micron.

For large-scale production, the most economic procedure would to obtain the fraction I paste from a commercial blood fractionator. For small batches, fibrinogen can be precipitated from plasma by mixing 8-12% ethanol at -2 to 4°C, as is well known to those skilled in the arts of blood fractionation.

Tests demonstrated that the iFP exhibited a density of 1.20 ±0.03 and were insoluble in PBS buffer for over 1 week. However, they were degraded quickly in 1 N NaOH (Fig 2) or collagenase. Moreover, the iFP dissolved in 4 N urea, confirming their monomeric make-up. By contrast, cross-linked fibrin did not dissolve in 4 N urea. If so desired, according to at least some embodiments of the present invention, the iFP can be prepared by adding a contrast agent or drug to stage 1 paste to become incorporated within the dried iFP matrix.

As summarized in Table 1, but without wishing to be limited in any way, the iFP according to at least some embodiments of the present invention differ significantly from the fibrin-type FMB (fibrin microbeads) of the background art in a number of respects (29-33,36,37).

Table 1. Parameters comparing the iFP of this invention, with the previously described FMB (29-33).

Figure imgf000009_0001

The process described here for fabricating iFP (versus that for FMB) offers distinct advantages, in that it requires only a single source material (fibrinogen stock such as fraction I paste) and a simpler, 3-stage fabrication process to generate an insoluble, non-porous, dense ( p = 1.2 ± 0.03), 3-D matrix useful for cell culture applications. Moreover, the iFP process described here can be more economic in terms of reduced costs for materials and labor.

Fibrinogen is susceptible to air oxidation during heating, which could diminish its attractiveness to mesenchymal cells but render it more inflammatory in terms of attraction of leukocytes, macrophages and the like. In recognition of these effects, oxidative divalent cations such as Cu(II) are removed by including a chelating agent (such as EDTA) in the ethanol washings. After air drying, the washed precipitate is subjected to heating 60-70°C under an inert gas atmosphere to prevent oxidation of amino acid side chains. This results in a matrix composed of misfolded fibrinogen monomers which retain the cell attachment activity of native fibrinogen.

The present invention, in at least some embodiments, also provides a novel composition comprising iFP enriched with an agent, such as a barium(II) salt or iodine-based contrast material, which were added to washed I paste during fabrication, to generate radio-opaque iFP that could be monitored by X-ray or other imaging technique. Similarly, drugs could be added during the process ( Row chart of Fig 1) to generate drug-enriched iFP .

The insoluble fibrinogen particles (iFP) constitute a novel insoluble matrix fabricated from fibrinogen-rich blood plasma fraction, without requiring thrombin or other enzymes or cross-linking agents (see Figure 1 for a flow diagram). Essentially, the iFP of this invention are a form of fibrinogen which has become transformed from its native, fully hydrated, monomeric state, into an aggregate of dehydrated, partially heat-denatured or misfolded monomers. The resultant non-porous matrix has not been cross-linked by transglutaminase enzymes (such as factor XIII) or chemical reagents (such as gluteraldehyde).

Rather, the transformation of hydrated, soluble, native fibrinogen into insoluble, non-porous fibrinogen is due to a specific series of treatments, namely: water extraction with solvents (such as a series of ethanol washes, for example optionally 12-15% ethanol wash followed by 60-70% ethanol wash, then in a 100% acetone wash , then heating (60- 70°C) under inert gas atmosphere, or under vacuuo (for example optionally 0.1-10 Torr). The process generates a matrix composed of entangled, misfolded, monomers of non-oxidized fibrinogen which is not soluble, but whose surface presents many cell attachment epitopes (4 cell binding sites per monomer).

Previous work demonstrated that mild heating (~52°C) resulted in the denaturation of the D-domains of fibrinogen, which exposed cell attachment (Hap tide) epitopes (33). As a consequence of the procedures employed here during fabrication, the iFP can be expected to elicit high cell attachment activity from normal mesenchymal type cells and for a variety of cancer cells.

Cells attached to iFP could be identified by various standard techniques, such as propidium iodide staining to detect cell nuclei by fluorescent microscopy, as well as histologic or immuno- staining procedures familiar to those skilled in the art. Thus, the present iFP invention is expected to provide a novel, cost-effective tool to harvest attachment-prone cells from bone marrow, blood or other normal or cancer biological sample.

According to at least some embodiments of the present invention, special devices which incorporate iFP could be built and employed to harvest attachment- prone cells from circulating blood. The iFP could be employed to harvest normal stem cells from blood, bone marrow, adipose aspirates (liposuction) for tissue regeneration purposes. Also, a Trap containing iFP could be used to capture cancer stem cells or other fibrinogen attachment-prone cells from tissue aspirates, biopsy samples or blood.

According to additional embodiments of the present invention, coatings of insoluble fibrinogen may optionally be prepared and coated onto pharmaceutically acceptable synthetic fibers, particles, meshes or filters, optionally by adsorption of fibrinogen, and then preparing an insoluble fibrinogen coating, functionally equivalent to the 3-dimensional iFP particles described herein, to render the devices potent for cell attachment. Optionally and preferably, the devices can be treated so as to functionalize their surface with aliphatic diamines or polyamines, to maximize fibrinogen adsorption capacity. After the fibrinogen adsorption, the water extraction stage starts by washing the fibrinogen-coated devices in 12-15% ethanol, followed by a 60- 70% ethanol wash, followed by a 100% acetone wash. The fibrinogen- coated fibers, particles, meshes or filters are then heat treated (60-70°C) under an inert gas atmosphere as described herein for preparing iFP. The resultant fibers, particles, meshes and filters which are coated with insoluble fibrinogen equivalent to iFP, can be packed into a device resembling a Trap (Fig 5) and used to capture attachment- prone cells from a suspension of mixed cells , as in bone marrow maintained in sterile bags (Fig 6) or harvested from a pateint's blood system (Fig 7), by pumping the suspension or blood through the "iFP Trap". Expectedly, such devices coated with insoluble fibrinogen would not be thrombogenic, as indicated by tests which showed that the iFP did not induce the coagulation of citrated whole blood (Example 5).

According to at least some embodiments of the present invention, there is provided a composition comprising fibers, particles or filters coated with insoluble fibrinogen prepared as described herein, with attached normal cells or cancerous cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Flow diagram outlining the preparation of insoluble fibrinogen particles (iFP) from commercial fraction I paste, which contains around 60% fibrinogen. The ethanol washings (12-15% followed by 60-70% ethanol in PBS with 1 mM EDTA), thereby removing water, divalent cations as well as co-precipitated albumin and IgG, leaving the washed precipitate composed of ~ 85% fibrinogen. The acetone wash removes most of the remaining water. The washed precipitate is air dried, then heated at 60- 70°C for 5-12 hr, in an inert gas (nitrogen or helium) atmosphere. Alternatively, the material could be heated under vacuuo. The resultant solid is milled and sieved to select particles of a size useful for cell culture applications (50-300 um diameter).

FIG. 2. Testing iFP in buffers. The iFP could be suspended in PBS buffer and settled in the absence of mixing (left tube). The iFP remained stable in PBS for more than 1 week. By contrast, the iFP in 1 N NaOH was totally degraded within 1 hr (right tube).

FIG. 3. Exposure of either native fibrinogen solution or iFP to thrombin. After mixing with an aliquot of thrombin (10 U/ml), the fibrinogen solution formed a fibrin clot within 1 min , as seen by the clot interface (tube 2). By contrast, the iFP particles did not aggregate or dissolve in PBS (Tube 3) and did not aggregate or form large clumps after exposure to thrombin (tube 4). Tests with iFP added to whole blood confirmed that iFP did not induce clotting of whole blood. FIG. 4. Measuring the density of iFP in a series of sucrose solutions. In PBS buffer, the iFP settled and clearly had a density greater than water (left tube). In sucrose solution with density >1.18, the iFP remained suspended and did not settle (right tube).

FIG. 5. Prototype "iFP Trap" connected to tubing, showing the housing for entrapping the iFP within mesh/wool with pores/channels >20 um, through which the blood is to be pumped.

FIG. 6. Schematic diagram of an example of an iFP-packed Trap connected to harvest select cells from whole blood units or cell suspensions in sterile plastic bags. The peristaltic pump non-invasively drives the suspended cells from the donor bag, through the "iFP Trap" with pores/channels >20 um, into the recipient bag, sterility being maintained by the sealed system. The plasma as well as the non-adherent cells pass through the Trap. Fig. 7. Schematic drawing showing an exemplary, illustrative "iFP Trap" hooked up via a pheresis pump apparatus, to harvest circulating cancer stem cells from a patient.

Figure 8 shows a schematic block diagram of circulating cancer cells binding to either soluble fibrinogen within the blood plasma or pumped through the iFP immobilized in a Trap.

DETAILED DESCRIPTION OF SOME EMBODIMENTS

According to at least some embodiments, the present invention is directed to the relatively simple preparation of a dry matrix composed of fibrinogen which is insoluble in physiological buffer and not active in terms of coagulation, but which could be used for cost-effective cell culture applications. The iFP of the present invention are prepared by removing water from fraction I paste, by suspending in a series of solvents (for example ethanol and/or acetone, preferably in a series of washes), followed by mild heating. The process removes contaminating proteins (such as albumin and IgG) and divalent cations (such as Ca+2 and Cu+2) from the commercial I paste. In order to minimize oxidation of the fibrinogen during heating, the process is performed in an atmosphere of nitrogen or helium or under vacuuo, thereby minimizing the oxidative loss of the methionine, tyrosine and tryptophan moieties which form the cell attachment epitopes.

Commercial fraction I paste, which contains -60% fibrinogen of total protein, is often discarded by commercial blood fractionators. As presented here, the iFP process could convert fraction I paste into a stable matrix useful for cell culturing and biomedical applications. The water is optionally and preferably removed by mixing the gummy I paste with an increasing proportion of water miscible organic solvents i.e. 12%-15% ethanol at 4°C, then 60-70% ethanol, finally 100% acetone (all whose supernatants contain extracted water that is discarded). The 1st washing step also removes much contaminating albumin and IgG, as well as divalent cations. This washing step also minimizes the generation of thrombin and prevents the activation of factor XIII, thereby inhibiting cross-linking. The resultant washed I paste, composed of >80% fibrinogen, is further dehydrated by suspending in 70% ethanol, then 100% acetone. The washed precipitate is then heated to a maximum of 70° C under inert gas atmosphere to prevent oxidation. After milling and sieving, one obtains non-porous, particles having a mean diameter of 30-300 microns. The above-described process generates matrix (as free floating particles or particles adhered to surfaces) composed of denatured fibrinogen, which is not soluble in physiological buffers, but which presents the cell attachment, Haptide epitopes (53-55). Thus, fraction 1 paste, which is often discarded, could be converted into useful biomedical devices designed to capture attachment-prone normal and cancer cells.

An alternative water-removal step could be employed, such as freeze- vacuum drying the I paste after the 12%-15% ethanol washing steps. The material would be then heated 60-70° C under inert gas atmosphere or under vacuuo. Expectedly, other standard water removal procedures known to those skilled in the art, followed by heating to a maximum of 70° C could be employed to achieve a "cell-friendly", iFP matrix or insoluble fibrinogen coating.

The iFP are different in a number of respects to previously described fibrinogen-based materials and their methods of production (32-46). For example, the oil emulsion procedure used to prepare FMB (49-52), required 2 starting materials

(fibrinogen and thrombin), which were formulated with detergents or surfactants, then added into a rapidly stirred, heated oil bath to generate an emulsion of aqueous droplets dispersed in the oil phase. Workup required separation of the FMB from the oil, then multiple washing steps with various solvents and buffers to remove residual oil and detergents.

By contrast, the iFP have not been exposed to any oils, detergents or formulants during the fabrication process. The solvents that were employed to remove the water (i.e. ethanol, acetone) are themselves evaporated by heating under inert gas atmosphere. Overall, the iFP process is much simpler, easier to execute (less labor and simpler equipment), and requires only one starting material. From a practical perspective, the process for preparing iFP is simpler and cheaper than that for preparing an equivalent amount of FMB. A comparison of parameters showing up differences between iFP and FMB is provided in Table 1.

The present invention, according to at least some embodiments, also provides a composition of insoluble fibrinogen particles containing a reagent (such as drug or contrast agent), wherein the reagent is admixed prior to the dehydration process. Suitable agents that can be added during processing include but are not limited to drugs, antibiotics, polysaccharides, synthetic molecules, enzyme inhibitors, radiolabels, radiopaque compounds, fluorescent compounds and the like.

The iFP, according to at least some embodiments of the present invention, could be used to harvest attachment-prone cells from a biological sample, such as suspended in blood, bone marrow or fatty tissue aspirate (liposuction) by suspension culture techniques. After an appropriate incubation period, the cells attached to the iFP can be harvested by sedimentation and the non-adherent cell removed by decantation. Thus, the iFP with cells bound thereto may be used for further study or manipulation.

US Patent No 6,777,231 (7) describes adipose-isolated stem cells derived from adipose tissue (liposuction). From the perspective of using iFP to obtain adipose- derived stem cells, there are significant differences between the iFP method described here and that of the '231 patent. For example, the '231 patent describes a method of isolating and growing cells by placing the adipose aspirate on plastic plates. Each "passage", wherein the adherent cells are subjected to a trypsinization treatment, digests adherent cells off the plastic to become suspended in the buffer medium, then to be transferred to a fresh plastic plate. Each "passage" requires such trypsinization to remove the cells which were tightly adherent to the plastic surface. Such a procedure necessarily subjects the cells to stress and is not optimal for cell survival.

By contrast, the IFP cell culture method described here does not require trypsinization, as the cells are not harvested alone. Rather, a measured amount of iFP (i.e. 10 mg/30 ml) are mixed into bone marrow, blood or adipose liposuction and incubated in a suspension culture under mild shaking conditions. The fraction of cells that attached to the iFP are harvested as a "composite" containing "naked" iFP as well as iFP with attached cells. The composite is separated by sedimentation from the supernatant with the unattached cells. After decantation of the supernatant with unattached cells, the cells attached to the iFP can be harvested under appropriate conditions and subsequently employed for biomedical procedures, without undergoing the trauma of trypsinization. Where required to obtain a greater cell number, more fresh iFP can be added to the suspension and culturing continued. Thus, the composite of stem cells attached to iFP is harvested but not "isolated" as defined and demonstrated by the '231 patent and are distinct from such.

A device (Trap) containing iFP has been designed for the removal of attachment prone cells from tissue aspirates or blood. In the prototype model, the iFP are immobilized by a plastic mesh which is packed within the Trap, through which the cell suspension can be pumped. The internal channels of the plastic mesh are small enough to trap the iFP but large enough to permit the perfusion of red blood cells and the like (i.e. >20 micron pores). The immobilized iFP present a large surface to which attachment-prone cancer stem cells (11) are exposed to maximize the attachment of cells, a technique that is analogous to affinity chromatography for adsorption of specific molecules, such as immunochromatography for the purification of particular antibodies.

Similarly, fibers, particles or filters coated with insoluble fibrinogen aggregates could be employed in a similar manner within a Trap. Thus, a Trap packed with iFP or materials coated with insoluble fibrinogen aggregates as described herein, could be employed to harvest even a small number of cancer stem cells (100-1000 per liter) dispersed in a mixture of suspended cells or whole blood (>109 cells per liter). From the point of view of attaching cells, the iFP and devices coated with insoluble fibrinogen both elicit equivalent attachment responses from various normal and cancer cell types.

Some non-limiting embodiments of the present invention is described in the following examples which are set forth to aid understanding the invention, and should not be construed to limit in any way the invention as defined in the claims.

EXAMPLE 1

Preparing insoluble fibrinogen particles (iFP) from Fraction I paste

The Cohn fractionation method is a standard procedure for precipitating fibrinogen from citrated or heparinized blood plasma or from even a purified fibrinogen solution. Typically, the plasma or fibrinogen solution is cooled to 0-4°C and 8-10% ethanol is added. The resulting precipitated fibrinogen (fraction I paste) is collected by centrifugation (21,22).

Using commercial fraction I paste (-60% clottable protein) as a starting material, the new process involves the following steps as a non-limiting example:

The fraction I paste is suspended in a cold (4°C) 4X volume PBS buffer, pH 7.2, ~1 mM EDTA (i.e. 400 ml to 100 gm paste) containing 10%-15% ethanol, and homogenized with a blender, to generate a slurried precipitate, which is incubated ~ 1 hr at ~4°C. Centrifugation or filtration permits the collection of the precipitate, discarding the supernatant. This washing treatment removes water, as well as contaminating albumin, IgG and divalent cations, rendering the washed fraction I paste >80% fibrinogen. For further removal of such contaminants, these washing and seperation steps can be repeated.

The twice washed washed I paste is suspended in 4x volume of 60-70% ethanol, then incubated 30-60 min and the liquid decanted. Then, the precipitate is suspended in 100 % acetone, mixed for -30 min and the solvent decanted. These steps extracted most water from the protein.

The resultant precipitate is air dried, then heated within a range 60-70°C under an inert gas atmosphere (nitrogen or helium) or under vacuuo for 5-12 hrs to remove any residual water and solvents and to denature the fibrinogen monomers, without oxidation. No formulants or protectants are added.

The resulting dry matrix is subjected to a grinding/milling step followed by sieving to obtain particles of desired size. Tests indicated that these particles are insoluble in physiological buffers, and are termed "insoluble fibrinogen particles (iFP)" (See Fig 1 for flow diagram).

An alternate process for preparing iFP is by way of freeze-drying (lyophilization) of the ethanol-washed precipitate (30). The lyophilized solid would then be washed with 12-15% ethanol, then heated to 60-70°C under an inert gas atmosphere (nitrogen or helium), or under vacuum (~ 0.1 to 10 Torr) 5-12 hrs, to render the fibrinogen insoluble without oxidation. The above procedures incorporate pathogen and virus inactivation steps (exposure to organic solvents and heating 60-70°C), resulting cumulatively in >10 12 virus kill suitable for preparing safe blood products for clinical applications. Variant procedures to those knowledgeable in the blood fractionation arts, such as precipitation of blood plasma with glycine, or ammonium sulfate to obtain fraction I paste, could be used to collect fibrinogen-rich fraction I paste intended to fabricate iFP.

EXAMPLE 2

Testing the solubility of iFP

The iFP were tested for insolubility by placing 5 -10 mg iFP into 1 ml PBS buffer. The supernatant can be monitored visually or spectrophotometrically at 280 nm.

Whereas normal, lyophilized fibrinogen dissolved within 1 hour with a characteristic extinction coefficient of E1% 28o = 15, the iFP did not significantly dissolve in PBS buffer for up to 1 week at room temperature.

By contrast, the iFP dissolved in 1 N NaOH within 1 hr (Fig 2).

EXAMPLE 3

Measuring the density of iFP A sample of iFP was hydrated for 30 min in PBS. A series of 1 ml sucrose solutions of pre-determined density (density range 1.05 to 1.25) were placed in test tubes, and 50 ul aliquots of the hydrated, iFP were layered on the surface. The tubes were shaken, then allowed to stand for 1 hr. Particles that settled to the bottom of the tube were denser than the solution; those that floated to the top were less dense than the solution ; the iFP that remained suspended had a density equivalent to the sucrose solution. Thus, it was determined that the iFP has a density 1.20, ranging between 1.17 to 1.23. EXAMPLE 4

Measuring the effect of thrombin on native fibrinogen versus iFP

A set of tubes containing 10 mg/ml native fibrinogen in PBS buffer (Fig 3, tubes 1 & 2,) was compared to samples of 10 mg iFP suspended in PBS (Fig 3, tubes 3 & 4). An aliquot of 20 ul thrombin (20 U/ml) was added to tubes #2 and #4 and mixed. The sample in tube #2 clotted within 1 min. By contrast, the iFP in tube #4 did not aggregate or coagulate or form large clumps, but remained disperse (Fig 3). These results demonstrated that unlike native fibrinogen, the iFP were no longer active in terms of coagulation reactions induced by thrombin. This demonstrated that the iFP differed significantly from soluble, native fibrinogen.

EXAMPLE 5

Measuring the effect of iFP on whole blood

Two tubes, each containing 200 ul citrated whole blood, were prepared. The 1st tube was the control; the second tube also contained 5 mg iFP. The tubes were shaken and periodically examined for clot formation.

For up to 24 hr, none of the samples exhibited blood clots. The iFP remained as particles suspended within the free flowing blood. As a positive control, 10 U/ml thrombin was added to the 1st tube, which clotted within 10 minutes.

Conclusion: iFP can be exposed to whole blood without inducing blood clotting or aggregating. EXAMPLE 6

Harvesting attachment-prone cells from bone marrow to iFP

An optional example of a generic process for using iFP to harvest cells under sterile cell culture conditions is as follows: The iFP are sterilized by standard g-irradiation technique or by suspending in 70% ethanol for 8 hrs, then rinsed in sterile PBS buffer. A sample of -100 million cells from 1 ml bone marrow or adipose aspirate

(liposuction) is mixed with 0.1 gm sterile iFP in a rotating 50 ml sterile tube with 10- 20 ml medium and slowly rotated (-10 rpm, 30° tilt) in a sterile cell culture incubator with 5-7% CO2 air mixture, 37°C, for 24 hrs. The tube is removed and kept static to permit sedimentation of the iFP with attached cells. The supernatant, along with unattached cells is removed by aspiration; fresh medium is added and suspension culturing of cells-on-iFP is continued or samples are removed for staining.

The cells attached to iFP could be characterized by immuno-staining for markers (such as CD14, CD 31 , CD34, CD 29, CD 49e, vimentin and fibronectin and others). Variations of culture medium, cell sources, culture conditions and immunostaining techniques can be performed by workers skilled in the cell culture arts.

EXAMPLE 7

Fabricating iFP admixed with anticancer antibodies

In order to increase the efficacy of the matrix for selectively harvesting cancer cells suspended in whole blood, the iFP is fabricated by admixing with IgG or IgM with affinity toward cancer cell surface markers (such as for breast, prostate, bone, brain cancers), into the matrix obtained after the 1st rinse of the fraction I paste with 10-15% ethanol buffer. The IgG or IgM becomes dehydrated and intermixed with the fibrinogen molecules. After heat-drying (as in example 1), the resultant matrix is more potent in terms of harvesting cancer cells suspended in blood.

EXAMPLE 8

"iFP Trap" for harvesting attachment-prone cells

A molded plastic canister (Trap) of volume -15 ml (like a chromatography column), is packed with 0.5 gm iFP immobilized within a plastic mesh of nylon or other pharmaceutically acceptable material. The plastic mesh would be porous to cells such as erythrocytes or other circulating cells (i.e. pores or channels >20 microns) . The sterile Trap housing terminates in screw- caps on both ends with nipples or dockings permitting hook-up to sterile tubing (Fig 5). The tubing could be connected to a peristaltic pump which would drive the cells suspension through the iFP trap, as from one sterile bag into the other (Fig 6). Thus, the iFP could be employed to capture the attachment-prone cells from suspensions of many types of cells, as suspended in whole blood or bone marrow. EXAMPLE 9

Harvesting cancer stem cells from circulating blood with an "iFP Trap".

In an optimized set-up, the patient would be hooked up to the blood pheresis machine and the whole blood (citrated or heparinized) would be pumped through the sterile iFP Trap maintained at 37°C (Fig 7). After a suitable time (i.e. 4 hrs) to permit ~4x passage of patient's blood through the system, the iFP Trap would be removed. The Trap can be opened by twisting off the screw caps; the iFP with attached cells can be removed and stained by histologic or immunochemical procedures to identify various markers or count attached cells.

Essentially, the "iFP Trap" could be used to harvest attachment prone cells in blood, such as circulating cancer stem cells or transformed endothelial-type cells. The principle underlying of this device is that cells (such as cancer stem cells) that normally circulate in the blood with fibrinogen loosely bound thereto (in equilibrium), could be presented with attractive, alternate surface of insoluble iFP. Attachment responses would effectively immobilize the cells, as indicated in Figure 8.

Figure 8 shows a schematic block diagram of circulating cancer cells binding to either soluble fibrinogen within the blood plasma or pumped through the iFP immobilized in a Trap.

When the cell-containing suspension, aspirate or blood is pumped through the iFP Trap, most cells and plasma would pass through the pores or channels that are >20 uM , but the attachment-prone cells which bind circulating fibrinogen loosely (reversibly) would bind preferentially to the immobilized iFP, permitting the capture of attachment-prone cells. Therapeutically, such a device could help reduce the level of circulating cancer cells and also help in the diagnosis and treatment of cancer.

Example 10

Alternately, a pharmaceutically acceptable fiber, mesh or filter can be coated with fibrinogen, either by adsorption , then treated in a manner equivalent to that used to produce iFP (Example 1) by washing sequentially with 12-15% ethanol, then 70% ethanol, then acetone, then dried and heated to 60-70°C under inert gas atmosphere for 5-12 hrs. The resultant fibers, mesh or filters coated with insoluble fibrinogen can then be packed into a Trap and hooked up to donor and recipient bags, as illustrated (Fig 6). Passage of suspended cells through such an packed Trap, with pores or channels >20 um, would permit flow-through of most blood cells but capture the cells prone to attach to the immobilized fibrinogen coating. In order to minimize blood coagulation, optionally, the internal surfaces of the housing of the iFP Trap could be treated with a solution of heparin (10 U/ml) or other anticoagulant.

The choice of shape (fiber, particle, mesh or filter) would be dictated by the ultimate intention for the cell-material composite. Thus, linear fibers coated with insoluble fibrinogen, equivalent to iFP, would be chosen to regenerate nerve fibers. For jaw-bone reconstruction, a pharmaceutically acceptable mesh coated with insoluble fibrinogen (equivalent to iFP) with attached mesenchymal cells would be more suitable. For ex vivo applications, filters coated with insoluble fibrinogen equivalent to iFP, could be useful where the object is to capture attachment-prone cells by binding to insoluble fibrinogen, thereby separating them from the other types of cells, which would pass through the device.

Variations in the design of the heparinized housing of the Trap packed with materials, such as fibers, meshes or filters coated with insoluble fibrinogen

(equivalent to iFP), can be useful for a variety of cell culture and medical applications.

Overall, the choice of free-form 3-dimensional iFP or fibers, mesh or filters coated with denatured, insoluble fibrinogen (equivalent to iFP) , would be dictated by technical considerations, notably the cell-attachment kinetics, the selectivity of the various packings, the geometry of the intended biomedical application as well as the overall cost.

References:

All publications and patents mentioned hereinabove are hereby incorporated by reference in their entirety. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated from a reading of the disclosure by one skilled in the blood protein and cell culture arts, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

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Claims

What is claimed:
1. A composition comprising insoluble fibrinogen particles (iFP) comprising more than 75% fibrinogen in particle form which are insoluble in aqueous, physiological buffers.
2. The composition of claim 1, prepared without using enzymes or cross-linking reagents.
3. The composition of claims 1 or 2, wherein said fibrinogen comprises
aggregates of denatured fibrinogen monomers which have not been cross- linked by factor XIII.
4. The composition of any of claims 1-3, wherein said particles exhibit a density of up to 2 gm/ml.
5. The composition of claim 4, wherein said density is from 1 to 2 gm/ml.
6. The composition of any of claims 1-5, wherein said particles have diameters between 30-300 microns.
7. The composition of any of claims 1-6, wherein said particles further comprise a drug, a growth factor or a contrast agent.
8. The composition of claim 7, wherein said drug is selected from the group consisting of adryamicin, tetracycline, Doxorubicin, and carboplatin.
9. The composition of claim 7, wherein said contrast agent is selected from the group consisting of barium salt and iodinated compound.
10. The composition of claim 7, wherein said growth factor is selected from the group consisting of BMP, VEGF, insulin, heparin.
11. A method for preparing iFP from blood plasma paste comprising the steps:
(i) washing the paste to prepare washed precipitate (ii) Heating the precipitate at 60-70°C to generate a dry matrix; (iii) Milling the dry matrix, followed by sieving, to select particles having a mean diameter of 30 to 300 microns.
12. The method of claim 11, wherein said heating comprises heating under inert gas atmosphere or under vacuuo for 5-12 hr.
13. The method of claim 12, wherein said inert gas atmosphere comprises nitrogen or helium.
14. The method of any of claims 11-13, wherein the step of washing the paste further comprises washing the paste with one or more water miscible organic solvents in a buffer and removing water from the precipitate.
15. The method of claim 14, wherein said organic solvent is selected from the group consisting of selected from the group consisting of ethanol, methanol, acetone and propanol.
16. The method of claim 15, wherein said step of removing water from the
precipitate comprises a plurality of washing steps performed sequentially, first with a first aqueous ethanol buffer comprising up to 20% ethanol, a second aqueous ethanol buffer comprising up to 70% ethanol, then with 100% acetone.
17. The method of claim 15, wherein said removing said water comprises
suspending the plasma fraction paste in a buffer containing up to 20% ethanol, with homogenization to form a slurry, followed by filtration or centrifugation of the slurry.
18. The method of claim 15, wherein said removing said water comprises freeze- drying.
19. The method of any of claims 15-18, wherein said buffer further comprises a chelating agent.
20. The method of any of claims 11-19, further comprising providing the blood plasma paste comprising one or more of fibrinogen-rich Cohn blood fraction pastes prepared from plasma by precipitation with ethanol, ammonium sulfate, glycine or arginine .
21. A method for capturing attachment-prone cells from tissue such as bone
marrow, blood or biopsy tissue, by mixing iFP of claim 1 with a cell suspension of or from said tissue, under conditions for attachment to iFP of select cell types.
22. The method of claim 21, further comprising sedimentation of the iFP with attached cells to remove the non-attached cells remaining in the supernatant.
23. The method of claim 21, further comprising culturing and expanding the attached cells on iFP.
24. The method of claim 23, further comprising identifying the attached cells on iFP histologically or immunochemically.
25. The method of claim 24, further comprising monitoring the cells on iFP with microscopy or cell sorting (FACS) techniques.
26. A composition comprising iFP of claim 1, with attached normal cells or cancerous cells.
27. A sterile Trap of plastic, non-thrombogenic housing with terminal couplings on the housing, which is packed with iFP of claim 1, designed for sterile hook-up to the tubing of a blood pumping (pheresis) device.
28. A method for harvesting fibrinogen attachment-prone cells from tissue aspirate or a suspension of mixed cells, by peristaltic pumping the suspension through an iFP-containing, sterile Trap of claim 27 at a rate to permit attachment of some types of cells to the immobilized iFP, but with channels large enough to permit exit of the liquid (plasma or culture medium) along with unattached cells.
29. A method for harvesting fibrinogen attachment-prone cancer cells from a patient's blood, by pumping patient's blood through an iFP-containing Trap of claim 27, comprising sterile connection of said trap to a "blood pheresis machine" via tubing , to withdraw patient blood from an appropriate vein/artery, pump it through the iFP Trap , then return the blood to the patient.
30. A composition of pharmaceutically acceptable, non-thrombogenic fibers, meshes, particles or filters which are coated with insoluble fibrinogen prepared according to the method of any of claims 11-20, meshes or filters.
31. A composition of pharmaceutically acceptable, non-thrombogenic synthetic fibers, meshes, particles or filters whose surfaces have been functionalized with aliphatic diamines or polyamines, to increase their ability to adsorb fibrinogen, which is then rendered as an insoluble coating.
32. The composition of claim 31, wherein said insoluble fibrinogen-coated fibers, particles, meshes or filters, elicit attachment responses from cancer cells.
33. The composition of claim 32, wherein said insoluble fibrinogen-coated fibers, are enriched with insoluble IgG or IgM with specificity to cancer cell surface antigens.
34. The composition of claim 32, wherein said insoluble fibrinogen-coated fibers, particles, meshes or filters can be used to capture attachment-prone cells from blood or tissue aspirates, such as bone marrow or liposuctions.
35. A trap containing either dispersed 3 -dimensional iFP of any of claims 1-0 or insoluble fibrinogen-coated devices of claims 30-34, with pores or channels >20 um to permit blood flow, with terminal couplings on the housing designed to be hooked up in a sterile manner, to the tubing of a blood pumping (pheresis) device.
36. A composition comprising insoluble fibrinogen-coated fibers, particles, meshes or filters of any of claims 30-34, with attached normal cells or cancerous cells.
37. A method of using insoluble fibrinogen-coated fibers, particles, meshes or filters of any of claims 30-34, with attached normal cells or cancerous cells, for clinical diagnostic purposes.
PCT/IL2013/050620 2012-07-22 2013-07-22 Insoluble fibrinogen particles (ifp) and coatings : composition, fabrication methods, and applications for harvesting and culturing mammalian cells WO2014016829A1 (en)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
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WO2001048016A1 (en) * 1999-12-23 2001-07-05 Csl Limited Separation of fibrinogen from plasma proteases
US6552172B2 (en) * 2001-08-30 2003-04-22 Habto Biotech, Inc. Fibrin nanoparticles and uses thereof
WO2005036180A1 (en) * 2003-10-08 2005-04-21 The Government Of The United States Of America As Represented By The Secretary Of Department Of Health And Human Services Analysis methods using biomarkers concentrated with biomarkers attractant molecules

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US6150505A (en) * 1997-09-19 2000-11-21 Hadasit Medical Research Services & Development Ltd. Fibrin microbeads prepared from fibrinogen, thrombin and factor XIII
WO2001048016A1 (en) * 1999-12-23 2001-07-05 Csl Limited Separation of fibrinogen from plasma proteases
US6552172B2 (en) * 2001-08-30 2003-04-22 Habto Biotech, Inc. Fibrin nanoparticles and uses thereof
WO2005036180A1 (en) * 2003-10-08 2005-04-21 The Government Of The United States Of America As Represented By The Secretary Of Department Of Health And Human Services Analysis methods using biomarkers concentrated with biomarkers attractant molecules

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