Extraction of Growth and Differentiating Factors from Colostrum

FIELD OF THE INVENTION

The present invention relates to a novel process for extracting growth and differentiating factors from colostrum. This process is characterized by chemical steps (controlled acid hydrolysis) and physical steps (molecular filtration). The invention further includes the use of the growth and differentiating factors derived from this process in prophylactic, therapeutic, cosmetic, cosmeceutical, dermatological, pharmaceutical, medical, veterinary or surgical (burn wounds, wounds, etc.) applications.

BACKGROUND OF THE INVENTION

Colostrum is a thick, yellow fluid produced by mammary glands during the first few hours after birth. It provides life-supporting immune (gamma globulin) and growth factors that ensure the health and vitality of a newborn.

The identities and functions of many of the bioactive principles of colostrum milk remain to be elucidated. However, colostrum is known to be a source of numerous bioactive hormones and growth factors, many of which have been demonstrated to influence intestinal growth, cell differentiation, and the development of the immune and enteroendocrine systems when administered in isolation.

Growth factors may be defined as proteins of 5 to 580 kDa that possess growth modulating bioactivities. Their biological actions also include the modulation and facilitation of the expression of cellular phenotype. To exert biological effects, growth factors must interact with specific high-affinity membrane receptors that activate appropriate signal transduction/second messenger cascades. In their natural state, most growth factors are inert on human cells and have very high molecular weights (340-580 kDa). In order to become active, these growth factors need to be released from their inactive original forms either through hydrolysis or temperature change, or both.

Interestingly, even growth factors from non-human origin, such as those derived from porcine or bovine colostrum, when converted into their active forms, have been found to be active on human cells. This can be explained by the fact that the active forms of smaller molecular weight are almost completely homologous to the corresponding human growth factors. This has been found to be the case for the following families of factors, for example: IGFs (1-3), TFGs β (1-3), PDGFs (AA, AB, BB), and BMPs (1-24). These factors, when in active form, are recognized for their ability to proliferate and/or differentiate the stem cells of a newborn.

United States Patent No. 6,277,813 (Kelly) describes the extraction of a novel growth factor from porcine colostrum. The process for extracting this growth factor, identified as CDGF for "Colostrum Derived Growth Factor", includes the following steps: (1 ) separating all components of colostrum having a molecular weight below 200 kDa and discarding all components having a lower molecular weight; (2) treating the product of step 1 with dithiothreitol and boiling for 10 minutes; and (3) centrifuging the mixture of step (2) to spin down any precipitated matter and recovering the CDGF located in the supernatant.

Other methods for the extraction of growth factors are known in the art, but surprisingly, there is no expeditious or efficient method described for doing so, nor does a process appear to exist for deliberately and simultaneously extracting growth factors with highly disparate molecular weights.

There is therefore a need for a method of extracting growth and differentiating factors from colostrum that permits the separation of a great number of these factors in a manner that is efficient, reproducible and non-deleterious to their activities.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided a novel process for extracting growth and differentiating factors from colostrum. More specifically, this process is characterized by chemical steps (controlled acid hydrolysis) and physical steps (molecular filtration) which optimize recovery of measured growth factors and their ability to entice a response on human cells.

In contrast to processes that are known in the art, the process of the present invention is neither performed at boiling temperatures nor does it depend on hydrolysis for fractionation. What results from this process are novel filtrate pools containing factors that are active on human cells, even if the colostrum is of bovine origin.

In one prefered embodiment, the process includes: • Diluting the colostrum and subjecting it to partial hydrolysis by adjusting the pH to about 3.75-3.85; • vortexing the resulting colostral solution 60 minutes (30-90 minutes); • adjusting the pH of the colostral solution to about 4.52-4.55; • centrifuging the new colostral solution, and setting aside the resulting supernatant; and • running the supernatant through a filtration system comprising one or more filtration columns (ceramic membranes) in order to obtain a fraction containing pools of growth and differentiating factors, all the while ensuring that the reaction temperature never exceeds 4O0C. In another embodiment, the process further comprises lyophilizing the pools of derived growth and differentiating factors.

Generally, the process includes a filtration system which is comprised of one or more filtration columns selected from the following filtration sizes: 0.2μm, 300 kDa, 150 kDa, 50 kDa, 15 kDa and 3 or 5 kDa. More specifically, and depending on the content of pooled growth and differentiating growth factors that is sought, the filtration system is selected from one of the following: a 0.2μm column; a 300 kDa column; a 150 kDa column; a 50 kDa column; a 15 kDa column, all terminating with a 3-5 kDa column; a 0.2μm column linked with a 150 kDa column; a 0.2μm column linked with a 15 kDa column; and a 150 kDa column linked with a 15 kDa column.

The invention further includes colostral fractions extracted from the process of the present invention. Such fractions may include one or more fractions selected from the following: LP1 , LP2, LP3, LP4, LP5, LP7 (see Table 5), LP1-LP3, LP1-LP5 and LP3-LP5. Depending on the application, these fractions may be used in their native form or they may be combined with an excipient or carrier.

Advantageously, this process allows the derivation and isolation of growth and differentiating factors in a manner that has not heretofore been possible, with the result that a number of factors with highly disparate sizes (or molecular weights) can be separated in pools from one another and used in select and varied ways, such as in cosmetic, cosmeceutical, nutraceutical, dermatological, pharmaceutical, medical and veterinary applications.

Other objects, advantages and features of the present invention will become apparent upon reading of the following non restrictive description of preferred embodiments thereof, given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Schematic view of the process steps for the extraction of growth factors from colostrum, including (A) controlled acid hydrolysis and (B) molecular filtration.

Figure 2: Growth factors found in colostral fractions.

Figure 3: ELISA test results for growth factors found in colostral fractions.

Figure 4: Fibroblast growth (Hoechst) during 72 hr exposure.

Figure 5: Fibroblast growth (new pools, Hoechst) during 72 hr exposure.

Figure 6: Proliferation of Human Umbilical Vein Endothelial Cells (HUVECs) (Cyquant®).

Figure 7: Percent of proline integrated into collagen synthesis.

Figure 8: Collagen Synthesis and Deposition in monolayer cell cultures as a function of cell number.

Figure 9: Collagen synthesis and deposition by fibroblasts in fibrin gel (new LP pools).

Figure 10: Fibroblasts grown in fibrin gel for 7-9 days. In the presence of 3.3 mg/ml LP1 -3, fibroblasts formed a dense matrix as observed on phase contrast (A) whereas the cell density was limited as observed after Hoescht staining (B). Conversely, the control culture in serum-free resulted in poor matrix density (C&D), compared to A & B. At day 8, fibroblast-containing fibrin gels were released from the culture wells and observed the next day for potential contraction as observed in the presence of 3.3 mg/ml LP1-3 (E) compared to control culture (F). In the presence of LP1 -5, at 1 mg/ml, fibroblasts reorganized into a network as observed by phase contrast (G) and at 3.3 mg/ml fibrin liquefied and agregated (H). Magnification at 2OX.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions: Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The following is a list of all growth and differentiating factors detected by ELISA tests built in with factors of human origin as standards. (See also Figure 3.)

BMP-2: bone morphogenic protein 2 BMP-4: bone morphogenic protein 4 EGF: epidermal growth factor FGF-2: (basic) fibroblast growth factor basic FGF-4: fibroblast growth factor 4 HGF: hepatocyte growth factor IGF-1 : insuline-like growth factor 1 IGFBP-1 : insuline-like growth factor binding protein 1 IGFBP-3: insuline-like growth factor binding protein 3 KGF (FGF-7): keratinocyte growth factor PDGF-AA: platelet-derived growth factor-AA PDGF-AB: platelet-derived growth factor-AB PDGF-BB: platelet-derived growth factor-BB PLGF: placenta growth factor SCF: stem cell factor c-kit ligand TGF-α: transforming growth factor alpha TGF-β1 : activated transforming growth factor beta 1 TGF-β2: activated transforming growth factor beta 2 TNFα: tumor necrosis factor alpha TNFβ: tumor necrosis factor beta VEGF: vascular endothelial growth factor

Example 1 : Extraction of Growth and Differentiating Factors from Commercially Available Bovine Colostrum

The process of the invention, shown schematically in Figure 1 (A) & (B), will now be described.

1. Preliminary Preparation

When starting with a lyophilized colostral preparation (freeze dried colostrum exempt of fat, coliforms and antibiotics) it has been found that the best way to reconstitute colostrum is to dissolve 80 g per liter of water (18.2 Mega Ohms).

The best pH for extraction is between about 3.75 and 3.85. The colostrum is adjusted to this pH (with a 10 N HCI solution, for example) and then placed in an agitator (Hobart™), at the first speed for 60 minutes. The pH of the solution is readjusted, with NaOH 10 N, to a pH of about 4.52-4.55 and the solution agitated for another 15 minutes before being centrifuged for 35-45 minutes at 4 000 G. The best results were observed using a Bechman™ J6M1 centrifuging 6 liters at a time for 35-45 minutes at 3 500-4 500 G.

The combined precipitates are dissolved in water for re-extraction (2 liters of 18.2 Mega Ohms for every kg of precipitate) and centrifuged again for about 40 minutes at 4 000 G. The supematants from each bottle are added to the pooled supernatant (1 ) from the first centrifugation. The final pH of the solution sometimes needs to be readjusted as it will be 4.35-4.40 instead of the 4.52-4.55 required for optimal results. The solution is now ready for filtration and lyophilization, as described below.

2. Filtration using TAM I LAB® Filter System

Using the solution obtained in step 1 , filtration is conducted by passing the supernatant through progressively smaller filtration columns, or molecular sieves. The choice of molecular sieve will depend on the fraction that is sought. As shown in Table 1 , these fractions are identified as LP1 to LP5, depending on the filtration column selected.

Table 1 : Correspondence between Filtration Column and Fraction

In accordance with one embodiment of the present invention, in order to obtain fraction LP3 (150 kDa - 3 kDa), a first filtration is performed using a 0.20 μm column. A column of this size will eliminate unwanted factors quickly before the supernatant is passed through the 150 kDa column, which is the column that is suitable for the LP3 fraction.

Moreover, in-between fractions may also be generated. For example, fraction LP3 may be filtered on a 50 kDa molecular sieve (used to obtain LP4). The result will be a filtrate having a cutoff molecular weight of 50 kDa (i.e., 50 kDa or below). Similarly, LP4 may be filtered on a 15 kDa molecular sieve (used to obtain LP5). The result will be a filtrate having a cutoff molecular weight of 15 kDa (i.e., 15 kDa or below). The retentate should be washed down with 3 liters of pure water at the end of a filtration run in order to extract as much of the residue as possible.

3. Lyophilization

This operation must be done very carefully in order to maximize efficiency. The different fractions are divided into samples of 300-350 ml per flask (Labconco™, 1200 ml) and refrigerated at about 2-4 0C before freezing in liquid nitrogen. This method of refrigeration permits rapid freezing with a lesser quantity of liquid nitrogen.

In a Virtis™ 25EL lyophilizer the flasks must be placed one at a time but not without a vacuum of at least 50 microthors, otherwise thawing could occur. After approximately 40-48 hours, the lyophilized samples, which are in the form of a fine powder ready for encapsulation (for example, 1 g/capsule), are ready to be pooled and conserved in storage bags (such as Ziplock™ freezer bags) at a temperature of about -160C to -240C.

Using the process described above, it is possible to extract growth and differentiating factors from colostrum. Figure 2 shows the growth factors found in the following fractions: LP1 ,LP2, LP3, LP4, LP5, LP1-LP3 (also referred to as LP1- 3), LP3-LP5 (also referred to as LP3-5), and LP1-LP5 (also referred to as LP1-5).

Figure 3 reveals the quantities of certain of the growth factors identified in Figure 2. The quantities, measured through ELISA, are per kg of colostrum.

Table 2 shows the quantity of extract product per fraction for colostrum (1 kg; dry matter basis). Table 2: Quantity of Extract Product per Fraction for Colostrum

Example 2: Extraction of Growth and Differentiating Factors from Natural Colostrum (from dairy cows)

1. Preliminary Preparation

Frozen colostrum is thawed (storage temperature is -200C) then centrifuged 6 liters at a time at 200C. The layer of butter and other residues are filtered first through cheesecloth and then through a Whatman™ 541 ashless filter. A thorough removal of this layer of fat will facilitate filtration and enhance the overall extraction of the growth and differentiating factors.

This preliminary filtration is followed by acid extraction at a pH of about 3.75-3.85. It is convenient to use a 10 N HCI solution for this purpose. It is important to add 40-50% water by volume (18.2 Mega Ohms) to the supernatant in order to increase the fluidity of the supernatant for extraction. This greatly enhances filtration on the TAMILAB® system of columns (0.20 μm, 300 kDa, 150 kDa, 50 kDa, 15 kDa and 3 kDa), as will be described below.

The solution is now ready for filtration and lyophilization, as described in Example 1. Example 3: Extraction Results for Growth Factors IGF-1 and TFG-β2 (after partial hydrolysis)

Growth Factors IGF-1 and TFG-β2 were quantified in 2.5 ml fractions (hydrogenated, pH 3.9 colostrum) that were purified on HPLC. Tables 3 and 4

show the results for growth factors IGF-1 and TFG-β2, respectively.

Table 3: Quantification of IGF-1 in Retentate 21

Discussion

The results in Tables 3 and 4 are but two examples showing the specific activity of the pools of factors derived using the process of the present invention. Partial hydrolysis converts many factors from their inactive (or "pro") forms (> 450 kDa) to their active forms. Significantly, these factors, which are present in pools in the various fractions, , as verified through human ELISA testing (Figure 3), have been found to be active on human cells.

Example 4: Effect on Cell Behavior of a Variety of Purified Fractions

1. Objectives of the Study The objectives of the study were to evaluate the effect on cell behavior of a variety of fractions purified with the process of the present invention. The pools tested were termed LP1 , LP2, LP3, LP4, LP5, LP1-3, LP3-5 and LP1-5. The proliferation and growth of human fibroblasts as well as their collagen synthesis were investigated in vitro in order to select optimal pools for further study. In addition, some studies were also performed with human vascular endothelial cells.

2. Materials and Methods 2.1. CeIIs Human fibroblasts, stored in liquid nitrogen, and derived from foreskin of young adult, were used at passage 3-6. Fibroblasts were grown in Dulbecco's modified Eagles medium with 5% fetal bovine serum (FBS). Ascorbic acid and β- aminoproprionitrile were added to the cultures dedicated to the collagen synthesis assessment.

Human vascular endothelial cells, stored in liquid nitrogen and derived from umbilical veins (HUVECs), were used at passage 3-4. HUVECs were grown on gelatin-adsorbed culture dishes in Medium 199 containing 10% FBS, L-glutamine (2mM) and endothelial cells growth supplement (ECGS at 20 μg/ml). For testing the pools, serum-free Medium 199 was used with ECGS and L-glutamine to allow survival of cells. In a pilot experiment, we have tried unsuccessfully cell cultures without ECGS and serum, endothelial cells died in less than 24 hrs. 2.2. LP pool concentrations In the first set of experiments, LP pools were diluted to final concentrations of 0.1 , 1.0, 10 mg/ml. In the following sets of experiments, the final concentrations tested were 0.33, 1.0, and 3.3 mg/ml. These conditions were compared to negative control cultures free of serum. In some cases, serum-supplemented medium was used in positive control cultures.

2.3. Proliferation Test (Cyquant® Assay from Molecular Probes) Cells were seeded in wells of 24 multiwell plates at a density of 5x103 fibroblast/well and a density of 1 x104 endothelial cells/well and grown for 6-I2 hrs to allow cell adhesion in the presence of serum (5% for fibroblasts, and 10% for endothelial cells). At time zero, medium was removed and cells were rinsed twice with Hank's balanced salt solution (HBSS), then replaced by culture serum-free medium containing the LP pool to be tested at different concentrations. Control cultures were grown in parallel. After 12 or 24 hours of growth without changing medium, medium was removed, and the wells were rinsed twice in PBS. Multiwell plates were frozen at -7O0C. Two hours later, plates were thawed, the lysis buffer (solution A and B) was added with an incubation of 3-5 minutes, then fluorescence was read in a cytoplate with a BioTek FL-600 fluorometer at 480nm excitation and 520nm emission.

2.4. Cell growth (Hoechst) Cells were seeded in wells of 24 multiwell plates at a density of 1 x104 fibroblast or endothelial cells/well and grown overnight (or 24 hrs in the first set of experiment) to allow cell adhesion in the presence of serum (5% for fibroblasts, and 10% for endothelial cells). The next day (time zero), medium was removed and cells were rinsed twice with HBSS, then replaced by culture serum-free medium containing the LP pool to be tested at different concentrations. After 72 hours of growth without changing medium, medium was removed, and the wells were rinsed twice in PBS. Then, PBS was replaced by a 200μl saline-sodium citrate buffer (SSCI) solution containing 0.1 % SDS, and incubated for 1 hour at 37°C. 20μl of Hoechst 33258 solution (at 1 mg/ml) was added to the SSCI solution. After agitation (up- down), fluorescence was read in a cytoplate at 340nm excitation and 460nm emission with a sensitivity set at 100-120.

In parallel, incrementing cell density was established, incubated with Hoechst, then fluorescence was read to perform a standard curve towards number of cells versus optic density.

2.5. Statistic analyses One Way Analysis of Variance was used for the statistic analysis of quantitative data, with a p value ≤0.05. Bonferroni t-test method was used for all pairwise comparison procedures.

2.6. Collagen synthesis in monolayer fibroblast cultures Cells were seeded in wells of 24 multiwell plates at a density of 1X105 fibroblast/well and grown for overnight to allow cell adhesion in the presence of serum (5%), ascorbic acid (10μg/ml) and β-aminoproprionitrile (10μg/ml). The next day (time zero), medium was removed, rinsed with PBS, and cells were exposed to medium containing LP pools, and radioactive proline (14 C or 3 H proline). Control cultures were run in parallel. Cultures lasted for 7 days to allow collagen synthesis and deposition, for which fresh medium containing LP pools and radioactive proline was changed every other day. At medium changes, media of each condition were collected and pooled (i.e., soluble collagen). At the end of the 7 day culture period, cells and matrix were pooled (i.e., cellular, insoluble and deposited collagen), separately of the medium pools (i.e., soluble collagen). Matrix-cells and media were promptly diluted in a protease cocktail inhibitor solution. Matrix-cells pools were counted on a scintillation counter, whereas medium pools were dialyzed to remove any free radioactive proline, then counted. 2.7. Cell cultures in 3-D fibrin gel and collagen synthesis/deposition Fibrin gel was used instead of collagen gel in order to investigate the collagen/deposition by fibroblasts, since collagen itself is known to inhibit collagen synthesis. Moreover, fibrin represents the primary extracellular matrix during wound healing. A 3 mg/ml fibrinogen solution was mixed with 5x104 fibroblasts/ml and polymerized by thrombin in the wells. The fibrin gels were then covered with culture medium containing the different LP pools, and radioactive proline. The method to analyze collagen synthesis and deposition was similar to that described earlier in 2.6.

3. Results 3.1. Fibroblast proliferation Proliferation was measured at 12 and 24 hrs of cultures in the presence of the LP pools with incrementing concentrations.

At 24 hours, 1 mg/ml LP1 and LP1 -3 induced a statistically significant higher value compared to the other LPs and control culture with no serum (not shown). At 10 mg/ml, the values with LP1 were significantly higher than those with LP1-3 at the same concentration. The latter was not different statistically with 10 mg/ml LP2, but different with 10 mg/ml LP3, LP4 and LP5. The values of LP1 were similar at 1 and 10 mg/ml. The values between 1 and 10 mg/ml of LP1-3 and LP2 were also similar. The values with 1 and 10 mg/ml LP1 , LP2 and LP1-3 were significantly higher than those at 0.1 and control. The values of LP4 and LP3 were not significantly different. High dose of LP5 induced a significant inhibition compared to control and the other LP pools at 10 mg/ml.

At 12 hrs (not shown), the values of cell proliferation at 0.33, 1 and 3.3 mg/ml of LP1 -3 was statistically higher than the other conditions, except with 3.3 mg/ml LP- 2 that was similar to LP1-3. However, the values with 3.3 mg/ml LP2 were not different than LP1 , LP3, LP4 and LP5 at the same concentration. 3.2. Fibroblast growth The first assay was performed with 0.1 , 1.0 and 10 mg/ml of LP pools (not shown). There was a statistically significant increase in the presence of LP1-3 at 1 and 10 mg/ml and between 1 and 10 mg/ml LP1-3. LP1-3 did not reach the number of cells found in the control cultures in serum-supplemented medium, which was 1.5- fold increase.

A significant higher numbers of cells were found in the presence of 10 mg/ml LP1 , LP2, LP3 and LP4 compared to those pools at lower doses, control without serum, and to 10 mg/ml LP5. The presence of LP5 resulted in a significant inhibition at the highest dose (10 mg/ml).

A second set of experiment was performed with 0.33; 1.0 and 3.3 mg/ml of LP pools (Figure 4). The numbers of cells in the presence of 1.0 and 3.3 mg/ml LP1-3 were significantly higher than those of the other pools and the control cultures without serum, except 3.3 mg/ml LP2 that resulted statistically in similar number of cells than that with 3.3 mg/ml LP1 -3. The cell number with 3.3 mg/ml LP2 was not significantly different than that with 3.3 mg/ml LP3. In addition, LP1-3, LP2 and LP3 had a significant increase in cell numbers between 1 and 3.3 mg/ml.

A third set of experiment was conducted with new pools LP1-3, LP3-5 and LP5 (Figure 5). The number of cells in the presence of 3.3 mg/ml LP1-3 was significantly higher than the other conditions. The cell number with 1.0 and 3.3 mg/ml LP3-5 was significantly different than 3.3 mg/ml LP5. The cell number with 1.0 mg/ml LP1-3 and LP3-5 were not found statistically different, but different compared to LP5 and controls.

3.3. Proliferation of HUVECs The Cyquant® assay shows a significant increase in the proliferation within 12 hours in the presence of LP1-3 at 0.33, 1.0 and 3.3 mg/ml compared to the other conditions (Figure 6). However, the values for 3.3 mg/ml LP1 -3 were close to those with 3.3 mg/ml LP2, and those for 1.0 mg/ml LP1 -3 were not different than those of 1.0 mg/ml LP 1. LP2 and LP5.

3.4. Growth of HUVECs A drop in cell numbers (from 10,000 cells at seeding time to 3,700 cells after more than 72 hrs of incubation) was observed (not shown), due to the lack of serum since these cells are very dependent on it. Once again the exposure to LP1-3 enhanced significantly cell growth at the 3 doses tested, compared to the other pools and the control cultures, recovering the initial number of cells. However, the cell number with 3.3 mg/ml LP1-3 was close to that of 3.3 mg/ml LP3. LP3 also increased significantly the number of cells when used at 3.3 mg/ml. Conversely, high dose of LP1 inhibited endothelial cell growth.

3.5. Collagen synthesis and deposition in monolayer The presence of LP1-3, particularly at 1.0 and 3.3 mg/ml enhanced collagen synthesis as shown by increased radioactivity (Figure 7). Similar doses of LP3 and LP2 also increased collagen synthesis, but at a lesser degree.

Phase contrast microscopy shows the extracellular matrix deposition and cells (not shown). LP2 induced matrix between cells particularly with 3.3 mg/. LP3 also enhanced matrix deposition at all doses tested. The behavior of cells in the presence of 1.0 and 3.3 mg/ml LP1-3 appeared differently than the others with a reorganization of cells into a network, rarely seen in monolayer cell cultures.

In another set of experiment, the effec of LP1 -3, LP1 -5 and LP3-5 were compared. The CPM values were reported to the cell number at day 7. Thus, the collagen synthesis and deposition per cell was particularly enhanced in the presence of 0.33, 1.0 and 3.3 mg/ml of LP1-LP5, even above the value found in the presence of serum (Figure 8). Moreover, the values of collagen synthesis and deposition were elevated in the presence of LP1-LP3 and LP3-LP5.

3.6. 3-D cell cultures and collagen synthesis In a first set of experiments, cell and matrix pools showed an increase in collagen synthesis and deposition with LP2 and LP3, particularly at 3.3.mg/ml (not shown). LP1 -3 also enhanced collagen synthesis at 3.3 mg/ml, but less than LP2 and LP3. Phase contrast microscopic observation (not shown) shows numerous cells with extracellular matrix deposition at day 7, in the presence of 1 and 3 mg/ml LP2 and LP3 and 1 mg/ml LP1-3, all compared to control and LP1 , LP4 and LP5.

A second set of experiments was performed with new LP1-3, LP3-5 and LP5 pools. Afterwards, fibrin gels were detached from the wells to allow contraction. LP1-3 induced an increase in collagen synthesis and deposition in the cell-matrix pools , which was close to that observed in the presence of serum (Figure 9). LP3- 5 induced less collagen synthesis, higher than that in control without serumCell cultures were observed by phase contrast microscopy at day 8. One and 3.3 mg/ml LP1 -3 resulted in a dense matrix with few cells, when compared particularly with control cultures and LP-5 (Figure 10 A-D). By day 9, the contraction occurred that resulted in a floating fibrin gel. The latter was very dense in the presence of 3.3 mg/ml LP1-3 (Figure 10 E-F).

Another study was performed with LP1 -5. In the presence of LP1 -5, fibroblasts in fibrin gels were reorganized into a network, particularly at 1 mg/ml, as shown in Figure 10 G. Moreover, at higher dose (3.3 mg/ml) LP1-5, fibrin gel was likely dissolved, perhaps by fribrinolysis, and some residual fibrin particles aggregated (Figure 10 H). Measurement of collagen synthesis and deposition show less production than with LP1-3, with an increase at 3.3 mg/ml (not shown). However, considering the decrease in cell density by day 9, collagen production was more elevated in the presence of LP1 -5. 4. Discussion and Conclusion The data shows clearly that cell proliferation and growth are stimulated by the presence of LP1-3 (3.3-fold increase in cell number), even with low dose such as 0.33 mg/ml as observed in some experiments, and this is incrementing as a function of the dose. Similarly, but at a less degree, LP2, LP3, and LP3-5 stimulate cell growth and replication when 3.3 mg/ml is used. The stimulation of cell replication in the presence of LP1 appears only after 24 hours, and the consequence on cell number is perceptible when high dose of 10 mg/ml is tested. Furthermore, the proliferation and growth of vascular endothelial cells are also stimulated by the presence of LP1 -3. Assessment of endothelial cell growth shows an incrementing effect as a function of dose. LP3 and LP2 may also enhance cell replication and growth, but to a lesser degree.

The observation and quantification of collagen synthesis and deposition show different patterns in monolayer cell cultures versus 3-D cultures in fibrin gel, more specifically in the presence of LP2 and LP3. The two latter induce a significant increase in collagen synthesis and deposition by fibroblast in fibrin gel, particularly with 3.3 mg/ml. On the other hand, LP1-3 also increases, but at a less degree, collagen synthesis and deposition. LP1-3 also increases the organization of fibroblasts in monolayer and more specifically in fibrin gel (since they have matrix to attach and migrate), as observed on micrographs. This observation is confirmed by the induction of a dense contracted matrix after days in culture. This suggests that newly formed collagen deposited in fibrin is remodeled by fibroblasts. Conversely, LP3-5 is less efficient to induce newly formed collagen, compared to LP1-3. On the other hand, LP1-5 induces synthetic activity as demonstrated in monolayer cultures. Whereas in 3-D fibrin gel, a differentiation activity is exhibited that involved protease activation as observed during wound remodeling.

The effect of LP1-3 on collagen synthesis and deposition could be explained by the presence of high cell density at the start of the cell cultures, due the stimulation of cell replication as determined by the different assays. Although the LP pools are renewed at medium change during the 7-9 day period of fibroblast cultures for collagen synthesis assay, it appears that by 8 days the cell density is less than expected, and less than that observed in the control culture with serum. Thus, LP1-3 not only enhances fibroblast proliferation and growth, but also the biosynthetic activity of fibroblast towards the formation of collagen, its deposition, and its remodeling.

In conclusion, selective LP pools such as LP1-3, LP2, LP3 and LP5 have potential and specific effects on fibroblasts and endothelial cell behaviour. These pools may have a beneficial effects in wound healing and closure.

Example 5:

1. Objectives of the Study The objectives of the study were to evaluate the effect on cell behavior of the growth and differentiating factors present in three pools : LP1-LP3; LP3-LP5 and LP1-LP5. The proliferation and growth of human fibroblasts as well as their collagen synthesis were investigated in vitro for a comparative study

2. Materials and Methods 2.1. Fibroblasts Human fibroblasts were used in conditions similar to those described in Example 4. They were derived from the same batch used in the previous experiments.

2.2. LP pools concentrations LP pools were diluted to final concentrations of 0.33, 1.0, and 3.3 mg/ml. These conditions were compared to negative control cultures in serum-free medium and positive control cultures in serum-supplemented medium. 2.3. Test of proliferation (Cyquant® Assay); Cell growth (Hoechst); and Collagen synthesis in monolayer and in fibrin gel cultures (14C-proline)

Similar experimental method was used as described earlier, as well as statistic comparison.

3. Results 3.1. Fibroblast proliferation (Figure 8) Cell proliferation after 24hrs of culture was increased, more specifically with 1.0 and 3.3 mg/ml of LP1-LP3 and LP3-LP5 pools (not shown). Statistical analyses show that the values of 1.0 mg/ml LP1-LP3 and those of 3.3 mg/ml LP1 -LP3 and LP3-LP5 were significantly higher than those of the control with no serum. Due to large variations in the values with LP1-LP5 pools, the cell proliferation values were not significantly different than those of the control.

3.2. Fibroblast growth Cell growth was increasing as a function of the doses tested for the different pools (not shown). The values of 3.3 mg/ml LP1-LP3 were significantly higher than all the other conditions, except with the control cultures in the presence of serum. The values of 3.3 mg/ml LP1 -LP5 were significantly higher than all the other conditions, except LP1- LP3 and LP3-LP5 both at 3.3 mg/ml (similar), and the presence of serum (lower). The values of 3.3 mg/ml LP3-LP5 were significantly different than those of the two control cultures. Statistically, the values of 1.0 mg/ml LP 1 -LP3 were significantly different than those of the two control cultures. Moreover, the values at 0.33 mg/ml were different for LP1-LP3 and LP3-LP5, compared to control cultures with no serum.

3.3. Collagen synthesis and deposition in monolayer After 7 days in cell culture, collagen synthesis and deposition was elevated for LP1-LP3 and LP3-LP5. However, when the values were reported with respect to the cell number, collagen synthesis and deposition per cell was particularly enhanced in the presence of 0.33, 1.0 and 3.3 mg/ml of LP1 -LP5, even above the value found in the presence of serum. Observation of the cell cultures shows clearly less cells left in the presence of LP1-LP5, more specifically with the highest concentration tested compared to the other conditions. In the presence of serum, a dense population of cells was seen, for little quantities of formed collagen. Moreover, the values of collagen synthesis and deposition was higher in the presence of LP1-LP3 and LP3-LP5. Specifically, 3.3 mg/ml of LP3-LP5 enhanced collagen synthesis and deposition. The curve of LP1-LP3 resembles to that reported earlier in monolayer cell culture. The ratio of soluble collagen versus insoluble collagen was relatively constant in any conditions tested.

3.4. 3-D cell cultures and collagen synthesis and deposition First, the experiment in fibrin gel did not go properly due to a weakness in the fibrin gel formation with a limited number of cells (2 x IO5 cells/well instead of 5 x 105 cells/well). However, it is interesting to report some of the data. In fibrin gel, LP 1 - LP5 behaved differently compared to LP1-LP3 and LP3-LP5 Observation of cells shows a clearly diminished number of cells as well an organisation of the fibroblasts into a network in the presence of 1.1 mg/ml LP1 -LP5. This has not been observed with other components, and may correspond to dramatic cell differentiation. Moreover, LP1-LP5 at 3.3 mg/ml appeared to induce the dissolution of the fibrin gel and it is accompanied by cell death and loss after each medium change (radioactivity value was not determined). The latter phenomenon may be induced by excessive protease activation, in particular plasminogen activators secreted by fibroblasts that have differentiated. In one instance (not shown), LP1- LP3 slightly increased the formation of soluble and insoluble collagen. However, it did not show any stimulation when the values of cpm were reported to the number of cells. LP3-LP5 increased the collagen production per cell. On the other hand, LP 1 -LP5 appeared to enhance collagen synthesis and deposition when the values were reported to the number of cells. 4. Discussion and conclusion The three LP pools of pools stimulate cell proliferation and cell growth, particularly the LP1-LP3 at high dose of 3.3 mg/ml. Although the stimulation of cell growth by LP1-LP3 occurs, collagen synthesis and deposition was limited when compared specifically with LP1-LP5. The latter induces a significant increase of collagen formation in monolayer cell culture and in 3D fibrin gel. Moreover, the presence of LP1-LP5 results in a cell differentiation into cord-like structures, but at high doses proteases are likely to be involved.

In conclusion, the LP1-LP5 pool of factors induces cell differentiation along with synthetic activity rather than proliferative and growth activity. The synthetic activity is accurately demonstrated in monolayer cultures, while the differentiation activity is exhibited in 3D fibrin gels.

Example 6: Uses or Applications for Various Growth Factors

The growth factors that are extracted through the novel process of the present invention may be used in a number of applications, including: cosmetics, cosmeceuticals, nutraceuticals and food additives, as well as in dermatological, pharmaceutical, medical and veterinary applications. Suggested applications for the specific growth factors found in individual fractions (see Figure 1 (B) and Figure 3 for the factors found in the various fractions) are listed in Table 5.

Interestingly, fraction LP7, which is the filtrate passing through the microfilter of 3 or 5 kDa (Figure 1 (B)), may also be useful in a number of applications. LP7 has been found to contain a wealth of vitamins, trace elements, amino acids, natural peptides and salts, among other pools. It can therefore be used as a diluent in the manufacture of cosmetic products and as an effluent in the preparation of nutraceutical substances, among other applications. Table 5: Uses or Applications for Growth Factors in Specific Fractions

Although the present invention has been described hereinabove by way of

preferred embodiments thereof, it can be modified without departing from the spirit, scope and nature of the subject invention, as defined in the appended claims.