Related Applications

This application claims priority to U.S. Provisional Application No. 61/577,823, filed December 20, 2011 and French Patent Application Number 1261485 filed November 30, 2012. The contents of these applications are each hereby incorporated by reference in their entireties Field of the Invention

The present disclosure relates generally to assays for detecting biological molecules and to high throughput screening for compounds of therapeutic importance. More particularly, the disclosure relates to assays for detecting and quantifying collagen proteins deposited in the extracellular matrix. In another aspect, the disclosure also relates to methods for identifying compounds that regulate collagen deposition.

Background of the Invention

Collagens are one of the most widely distributed proteins in the human body. Collagens are responsible for the structural integrity of the extracellular matrix (ECM) of most connective tissues. Based on their structures and tissue distribution profiles, collagens may be classified into many different types. For example, Type I collagen is the most abundant collagen and is found in most connective tissues. Type I collagen is important for the structure of bones and the skin. Type I and Type III collagens are the major components of the liver and the lung. Type IV and VI collagens are found in the basement membranes of many tissues. The most common localization of Type V collagen is within the characteristic collagen fibrils, in association with Type I and Type III collagens.

The composition of the ECM is controlled by balancing between synthesis and degradation of the respective components of the ECM. For instance, the amount of collagens is controlled through regulation of gene expression, protein secretion, and protein degradation by metalloproteinases and cysteine proteases. Imbalance of these processes may result in various disorders in the human body.

One such disorder is fibrosis, which is characterized by excessive accumulation of collagen and other extracellular matrix components. While excessive accumulation of collagen in the lung may cause pulmonary fibrosis, uncontrolled accumulation of collagen in the skin may cause disorders such as cutis keloid formation.

A number of different conditions may cause fibrosis. These conditions include, for example, chronic infections, autoimmune reactions, allergic responses, chemical exposure, radiation, and tissue injury. The primary cellular mediator of fibrosis is fibroblast, which serves as the primary collagen-producing cells when activated. Many different factors or molecules have been shown to activate the collagen-producing cells. These molecules include, for example, transforming growth factor beta (TGF-β), insulinlike growth factor (IGF-1), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), connective tissue growth factor (CTGF) and IL-4/IL-13.

TGF-β is a growth and differentiation factor that may act both as a tumor suppressor and a contributor to tumor invasion and metastasis. See, e.g. , Akhurst, 2002, J. Clin. Invest. 109:1533-36. TGF-β has been shown to participate in wound healing by stimulating fibroblasts and other cells to synthesize and secrete extracellular matrix components, such as collagen. TGF-β may regulate transcription of collagen genes directly or indirectly. For instance, some of the genes encoding collagens contain a TGF-β response element in their non-coding regulatory region. TGF-β may also regulate transcription of collagen genes indirectly by helping other transcription factors bind to the regulatory elements of the collagen genes. See, e.g., Lindahl et al., 2002, J. Biol. Chem. 277:6153-6161.

In wound healing process, timely and accurate control of ECM components are critical for proper wound repair and regeneration. Early phases of wound healing involve the formation of a provisional ECM containing fibrin, fibrinogen, and fibronectin. Fibroblasts occupy this matrix and proliferate in response to activators generated by leukocytes that have migrated into the wound and are retained by the ECM. This event also coincides with the appearance of the myofibroblast, a specialized form of fibroblast whose differentiation is primarily driven by cytokines, such as TGF-β, and by mechanical tension. TGF-β signaling has been shown to play an important role in wound healing. Targeting components of the ECM, as well as the TGF-β signaling pathway, holds promise as a means to avoid development of fibrosis and scarring. SUMMARY

The present disclosure advances the art by providing a system for detecting and quantifying collagen deposition in the extracellular matrix. The system may be used as a platform to screen for compounds that play a role in regulating extracellular deposition of collagen.

A number of methods or kits are available for detecting and/or quantifying collagen. However, most of these methods fail to solve the inherent problems of detecting Collagen I in the matrix while being amenable to high throughput compound evaluation, as well as being able to account for cell proliferation or cell death events during the assay. The instant disclosure provides a system and a method that are compatible with multi-well plates (for example, 96 well plates) so that high throughput screening may be conducted. In another aspect, the disclosed method does not require sample manipulation using tubes, and no sample digestion is required.

In one aspect, a method of the invention comprises measuring the amount of extracellular collagen produced by a plurality of cells in one or more receptacles. For purpose of this disclosure, the cells along with the culture media in the receptacle(s) may also be referred to as a sample or samples. A first detecting molecule and a second detecting molecule may be added sequentially or simultaneously to the sample. The first detecting molecule may contain a primary antibody having binding affinity to extracellular collagen, and the second detecting molecule may have binding affinity to a cellular molecule. In one embodiment, the cellular molecule is selected so that the amount of the cellular molecule is relatively constant among each of the cells. For example, the cellular molecule may be DNA, RNA, a protein or a lipid molecule. In another aspect, the cellular molecule is an intracellular molecule. Preferably, the total amount of the cellular molecule in a given cell type is relatively stable such that the cellular molecule may be used for purpose of normalization.

For purpose of this disclosure, the term "cellular molecule" refers to a molecule that is associated with a cell, either inside a cell or on the surface of a cell. The term "constant" or "stable" means the level or amount does not fluctuate upward or downward by more than 10-15%.

A first signal may be measured directly from the first detecting molecule or indirectly from another molecule that binds to the first detecting molecule. A second signal may be measured from the second detecting molecule or from another molecule that binds to the second detecting molecule. In one embodiment, the intensity of the first signal is proportional to the amount of the extracellular collagen. In another embodiment, the intensity of the second signal is proportional to the amount of the cellular molecule. The amount of the extracellular collagen in the sample may then be determined by normalizing the first signal against the second signal. For instance, the intensity of the first signal may be divided by that of the second signal to generate a new data point, or normalized first signal.

In certain embodiments, the first and the second detecting molecules may each carry a detectable tag so that the amount of first and second detecting molecules may be detected and quantified after wash. Alternatively, a secondary antibody may be used which binds to the primary antibody. The secondary antibody may carry a detectable tag so that the amount of primary- secondary antibody complex may be detected and quantified. The use of a secondary antibody may help amplify the signal from the primary antibody. A first signal is obtained with an intensity that is proportional to the amount of the extracellular collagen in the sample(s). The second signal is also obtained whose intensity is proportional to the amount of the cellular molecule. The second detecting molecule may itself be detectable. For instance, the second detecting molecule itself may be a dye or a fluorophore. Alternatively, the second detecting molecule may be conjugated to a second detectable tag so that the amount of the second detecting molecule may be measured.

In another embodiment, the primary antibody binds specifically to extracellular collagen, but not intracellular collagen. In one aspect, the primary antibody binds specifically to Type I collagen. In another aspect, binding between the primary antibody and other types of collagen is negligible.

For purpose of this disclosure, suitable detectable tag may be any conventional fluorophores, dyes, infra-red molecule or other fluorescent molecules.

In one embodiment, when the amounts of extracellular collagen are to be measured in N samples and when N is an integer equal to or greater than 2, the first signal obtained from sample N may be normalized against the second signal obtained from the same sample N. Similarly, the first signal obtained from sample N-l may be normalized against the second signal obtained from the same sample N-l in order to control for variation in the number of cells between sample N and sample N-l. Thus, when all 96 wells in a 96-well plate contain samples, N is 96 and the same normalization principle applies to control for variation in cell numbers, among others.

By using a second detecting molecule, such as a DNA binding dye, total DNA of the cells may be measured to control for total cell number. This feature is especially beneficial in large-scale screening of compounds that affect extracellular collagen deposition. The ability to normalize within a single well or a single sample allows control for unanticipated compound toxicity, compound proliferative effects, or manipulations that may impact cell density. In one aspect, the method may include a step to determine the linear range of the second detecting molecule. In another aspect, the amount of the second detecting molecule is within this linear range such that the total number of cells is proportional or almost proportional to the level of the second signal.

The disclosed systems and methods may be used to screen for molecules (e. g., compounds) that regulate extracellular deposit of collagen. In one embodiment, a method is disclosed for identifying molecule(s) that regulates deposition of extracellular collagen in a cell. The method may include culturing a plurality of cells in two receptacles Nl and N2 (also referred to as samples Nl and N2), wherein the plurality of cells in Nl and N2 belong to the same cell type. A test agent (also termed "candidate molecule" or "test compound") may be added to sample Nl. In one aspect, no test agent is added to sample N2. In another aspect, a blank control may be added to N2 as a negative control. Both samples Nl and N2 receive a first detecting molecule and a second detecting molecule. The first detecting molecule may be a primary antibody having binding affinity to the extracellular collagen, and the second detecting molecule may have binding affinity to a cellular molecule in the plurality of cells, wherein the amount of said cellular molecule is relatively constant among each of said plurality of cells. A first signal from the first detecting molecule may then be measured in both samples Nl and N2. In one aspect, the intensity of the first signal is proportional to the amount of the extracellular collagen. A second signal from the second detecting molecule in both samples Nl and N2 may also be measured. In one aspect, the intensity of the second signal is proportional to the amount of the cellular molecule. The first signal may then be normalized against the second signal. The normalized first and second signals obtained from samples Nl and N2 may be compared to determine whether the candidate molecule regulates the deposition of extracellular collagen by the cell. As used herein, "regulate" means either increase or decrease. In one embodiment, factors that promote collagen deposit may be added to the sample to stimulate collagen deposition. In one aspect, such factors may include but are not limited to TGF , IGF-1 , PDGF or EGF. In another aspect, TGF is added at a concentration of from about 0.1 ng/ml to about 5ng/ml before the test agent(s) are added. In another aspect, no TGF or other factors are added before the test agent(s) are added. In another aspect, L-ascorbic acid is added before the test agent(s) are added. After the plurality of cells have been incubated with the test compounds for a period of time, the first and second detecting molecules may be added to measure extracellular deposition of collagen in response to the test compounds either in the presence or absence of TGF .

In one embodiment, the test compounds may inhibit extracellular deposition of the collagen. The inhibitor may exert its effect by inhibiting TGF , or it may exert its effect through other molecules. Such inhibitors may be used as an anti-fibrotic agent.

In another embodiment, some compounds may act as a promoter of extracellular deposition of collagen. Such compounds may be used as wound healing agent.

The disclosed methods may also be used to measure extracellular deposition of collagen by cultured primary cells isolated from an animal or a human. Under certain circumstances, quantification of extracellular deposition of collagen may help characterization of cell types or diagnosis of certain diseases.

BRIEF DES CRIPTION OF THE DRAWINGS

Figure 1 illustrates a schematic of an assay to measure Collagen I protein using secondary infrared antibody and DNA using DRAQ5 in human lung pulmonary fibroblasts treated with TGF .

Figure 2 shows the effects of increasing concentrations of TGF on collagen I deposition in the presence of L-ascorbic acid and dextran sulfate I in WI38 cells.

Figure 3 shows the detection of deposited Collagen I protein under varying conditions in IMR90 cells.

Figure 4 shows detection of deposited Collagen I protein under varying conditions in LL97AlMy cells.

Figure 5 shows detection of deposited Collagen I protein (green, left), DNA (red, center), and image overlay (green and red images, right) in human lung pulmonary fibroblasts treated with a dose response of TGF . Figure 6 shows quantification of Collagen I deposition induced by a dose response of TGF and quantification of DNA signal.

Figure 7 shows that a known anti-fibrotic compound inhibits Collagen I deposition induced by 500 pg/ml TGF .

Figure 8 shows that some test compounds inhibit Collagen I deposition induced in human pulmonary fibroblasts by 500 pg/ml TGF .

Figure 9 shows that some test extracts inhibit Collagen I deposition induced in human pulmonary fibroblasts by 500 pg/ml TGF .

Figure 10 shows that a known anti-fibrotic inhibits TGF induced Collagen I deposition (left) and an internal extract inhibits Collagen I deposition induced by TGF (right) in human adult dermal fibroblasts.

Figure 11 shows that an extract promotes Collagen I deposition in the absence of TGF in human adult dermal fibroblasts.

DETAILED DESCRIPTION

This disclosure provides systems and methods for detecting and quantifying extracellular collagen deposit. The disclosed system may also be used in high throughput screenings to identify compounds or other molecules that regulate collagen deposit.

Collagen I production is a major fibrotic component in fibrosis events. Most current methods for measuring cellular Collagen I primarily use SIRCOL or ELISA to measure collagen protein levels. Table I summarizes some of the methods that are commercially available.

Table I: A survey of commercially available kits to detect Collagen.

5 Enzymatic

ELISA I Insoluble rat colorimetric Yes

6 Human

bovine, Enzymatic

ELISA Procollagen I Soluble canine colorimetric No

7 Enzymatic

ELISA I Insoluble Human colorimetric Yes

8 Enzymatic

ELISA I Soluble Human colorimetric No

Many of these methods have various limitations. For instance, Methods 1 , 2 are not specific to Collagen Type I because they may detect other Collagen subtypes in addition to Collagen I. Methods 2-5 and 7 may require solubilization of the extra cellular matrix. Such solubilization can be tedious and may require overnight digestion with pepsin to release the collagen into individual fibrils for measurement of soluble collagen. Methods 6-8 may detect soluble collagen or procollagen levels instead of detecting only deposited Collagen I. Method 1 may require high speed spins or high speed centrifugation which is not amenable to 96-well plate format. In addition, ELISAs are generally not sensitive enough to be used for quantitation of cellular collagen.

A number of other methods to detect collagen levels are not mentioned in the table. Some of these methods entail radioactive incorporation assays, which require collagenase digestion and, more importantly, generate radioactive waste. Moreover, none of the known methods address the issue that addition of collagen matrix stimulants may increase cell proliferation which may, in turn, contribute to higher levels of collagen. On the other hand, when antagonists of collagen deposition are being tested, none of the methods address the issue that the antagonists may induce cell death which may result in lower levels of collagen.

The presently disclosed methods address many of these issues by having a collagen I specific assay that include a second signal to account for cell number normalization. In one embodiment, an assay is disclosed to measure deposited Collagen I (Type I Collagen) protein in the extracellular matrix produced by cells treated with TGF . Cells of interest may be grown on a cell culture platform. By way of example, the cell culture platform may be a 96-well plate or other multi-well plates. Cells may be fixed on the plate and on-cell Western blot may be performed to detect the collagen in the extracellular matrix. Collagen I may be detected using a primary antibody to Collagen I. In one aspect, a secondary antibody that binds to the primary antibody may be used to amplify the signal from the primary antibody. The secondary antibody may be linked to a detectable tag so that it may be detected when illuminated by certain light sources or when it is exposed to a specific substrate. In another aspect, the primary antibody itself may be linked to a detectable tag. By way of example, the detectable tag may be an infrared label, a fluorescent tag, or an enzyme, among others.

In another embodiment, an agent that binds to certain normalization molecule of the cells may be used as an internal control. In one aspect, the normalization molecule is present in the cells at a relatively constant level. The normalization molecule may be, for example, total DNA, total RNA, total proteins, or total lipids. In another aspect, the agent that binds to the normalization molecule is cell permeable. In another aspect, a cell-permeable DNA-binding dye may be added to the same cells that have been incubated with the anti-collagen antibody.

In another embodiment, an intercalating detectable compound is used as such an agent that binds to normalization molecules of the cells. A non-limiting example is an intercalating fluorescent dye. Intercalators commonly are hetero aromatic polycyclic molecules that insert between two base pairs in a DNA duplex. However, the intercalators of the present disclosure are not limited to heteroaromatic polycyclic molecules. Any intercalating molecule that shows a significant fluorescent enhancement or shift in emission or excitation parameter(s) in the presence of DNA fragments with little or no nonselective binding to RNA or proteins is contemplated by the present disclosure. Such intercalating dyes are known to those skilled in the art and may include, but are not limited to, the bisbenzimide dye Hoechst 33258. Another useful intercalating detectable compound may be propidium iodide. In another aspect, PicoGreen may be used as an intercalator. PicoGreen belongs to the family of unsymmetric monomethine cyanine dyes. It exhibits high binding constants with DNA and is highly fluorescent when bound to DNA, while virtually no n- fluorescent when free in solution. In another aspect, propidium iodide may be used as an intercalator.

In another embodiment, cell-permeable DNA probes such as BENA435 may be used as an intercalating detectable compound thus eliminating the need to lyse the cells in order to label the DNA. In one aspect, dyes such as YOPRO, Hoechst 33342, DAPI or DRAQ5 may be used. Note that the intercalating detectable molecule is not limited to a fluorescent dye. The amount of DNA fragment may be quantified using any methodology known to those skilled in the art. By way of example, DRAQ5 may be used as such a cell-permeable DNA-binding dye.

In another embodiment, the system disclosed herein may serve as a platform for high throughput screening to identify compounds or other molecules that may regulate extracellular deposit of collagen. Cells may be cultured in a multi-well plate and stimulants (e.g., TGF ) may be added to the one or more wells to stimulate synthesis and/or extracellular deposit of collagen. Single or combination of test molecules (e.g., test compounds) may be added along with the stimulants or at certain time after the stimulants are added. The effects of each test molecule or each combination of test molecules may be assessed using the disclosed system and method.

In one embodiment, the disclosed system may use an array of receptacles which can receive cells and other materials such as culture media. An array of receptacles may be any number of receptacles from at least one or more than one receptacle suitable for holding cells within the scope of the disclosure. Examples include but are not limited to flasks, culture dishes, tubes such as 1.5 ml tubes, 12 well plates, 96 well plates, 384 well plates or other multi-well plates, and miniaturized microtiter plates with perhaps 4000 receptacles (see U.S. Patent Application Publication 2005/0255580A1). The array of receptacles may be amendable to the addition of a protective covering thus preventing against entry of contaminants or evaporation of contents.

An embodiment of the disclosure uses a control. A control is a term of art well understood by skilled artisans. An appropriate control may be dependent on the assay parameters utilized or the experimental question under investigation. A control may be a particular set of assay conditions or the addition or elimination of a particular compound to the culture medium. A control may be considered a positive control in that the assay conditions or control compound added brings about the anticipated response. For example, known factors (or compounds) that are known to regulate collagen deposition may be used. A control may also be a negative control. A negative control may be a particular set of assay conditions or the addition or elimination of a particular compound to the culture medium that would not bring about the anticipated response. In one aspect, a negative control may be a "vehicle" control or a solvent control. For example, if the test agent is dissolved in DMSO then the vehicle control may be DMSO without test agent. A control may simply be the use of historical data. In another embodiment, the systems and methods disclosed may be used in high throughput screening (HTS) methods. HTS is the automated, simultaneous testing of thousands of distinct chemical compounds in assays designed to model biological mechanisms or aspects of disease pathologies. More than one compound, e.g. , a plurality of compounds, may be tested simultaneously, e.g., in one batch. In one embodiment, the term HTS screening method refers to assays which test the ability of one compound or a plurality of compounds to influence the readout of choice.

The term "agent", "test agent", "test compound", "drug candidate" or

"modulator" or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g. , protein, oligopeptide {e.g. , from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid {e.g. , a sphingolipid), fatty acid, polynucleotide, oligonucleotide, etc., which is employed in the assays of the invention and assayed for its ability to modulate DNA fragmentation or apoptosis. There are no particular restrictions as to the compound that can be assayed. Examples include single agents or libraries of small, medium or high molecular weight chemical molecules, purified proteins, expression products of gene libraries, synthetic peptide libraries, cell extracts and culture supernatants. An agent encompasses any combination of different agents.

An agent may include at least one or more soluble and insoluble factors, conditioned media, cell extracts, tissue extracts, explants, pH modifiers, gasses, osmotic pressure modifiers, ionic strength modifiers, viruses, DNA, RNA or gene fragments. An agent may be in the form of a library of test agents, such as a combinatorial or randomized library that provides a sufficient range of diversity or conversely are limited to similar structures or features. Agents may be optionally linked to a fusion partner, e.g. , targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test agent (called a "lead compound" or a "lead") with some desirable property or activity, e.g., inhibiting activity or modulating activity. The lead compound is then used as a scaffold to create variants of the lead compound, and further evaluate the property and activity of those variant compounds. The following examples are provided to illustrate the present disclosure, but are not intended to be limiting. The materials and methods used are presented as typical components and methods, and various substitutions or modifications may be made in view of the foregoing disclosure by one of skills in the art without departing from the principle and spirit of the present invention.

Certain experiments described in the Examples contain ingredients or materials that are in a size suitable for a small scale setting. It is important to note that these small scale tests may be scaled up and the principle of operation and the proportion of each ingredient in the system may equally apply to a larger scale system.

Example 1: Detection of deposited Collagen I protein in the extracellular matrix

This example describes an assay designed to measure deposited Collagen I protein in the extracellular matrix by cells treated with TGF . Figure 1 illustrates the assay to measure Collagen I protein using secondary infrared antibody and DNA using DRAQ5 in human lung pulmonary fibroblasts treated with TGF .

Cells were plated at 20,000 cells/ well in a 96 well plate. After treatment of TGF at 500 pg/ml, the culture media were removed and 100 ul ice cold methanol was added to fix the cells for 10 minutes. After fixing, methanol was removed and the cells were washed 3X with 100 ul D-PBS solution. After washing, 100 ul Odyssey block buffer (LiCor Biosciences, Catalog# 927-4000) was added to block the cells for 30 minutes.

Primary antibody, a mouse monoclonal anti-collagen Type I antibody (Sigma #C2456), was diluted 1 :400 in the same block buffer. 50 ul of diluted Primary antibody was added to the wells. At least one or more wells received block buffer alone without the primary antibody. These wells served as negative controls for the plate. The primary antibody was allowed to incubate with the cells for 60 minutes. After the incubation, the antibody or the control block buffer was removed and the cells in the wells were washed 3 times with 100 ul D-PBS each.

Secondary antibody was prepared in appropriate diluent under the dark so that it was protected from light. IR800 conjugated goat anti-mouse IgG (Rockland #610-132- 121) was diluted 1 :400 in the same block buffer. 50 ul of diluted secondary antibody was added to the cells and incubated with the cells for 60 min. in the dark. After the incubation, the secondary antibody was removed and the cells were washed twice with 100 ul D-PBS each.

A DNA-binding dye, DRAQ5, was diluted in DPBS at 1 : 1000. 50 ul of the diluted DRAQ was added to the cells and allowed to incubate with the cells for 5 minutes in the dark. At least one or more wells received DPBS alone without the

DRAQ5. These wells served as negative controls for the DNA-binding dye and the plate. After the incubation, the DRAQ5 was removed and the wells were washed twice with DBPS. The bottom of the plate was wiped with isopropanol and the plate was placed on an Odyssey flat bed scanner. The plate was scanned with a sensitivity of 7.5 for each signal. If one experiment is to be compared to another experiment, same sensitivity should be used for the scanning.

Two human fetal lung cell lines, IMR-90 and WI38 cells, were evaluated in the initial studies. These cell lines were selected based on their ability to produce collagen as determined by other methods (Sircol). Each of these two cell lines was cultured at both 10,000 cell per well and 20,000 cells per well in a 96- well plate. These cells produce and deposit collagen when stimulated with a dose response of TGFp. Further, Dextran Sulfate and L-ascorbic acid were included during TGFP stimulation as recommended by Chen and Raghunath (Fibrogenesis & Tissue Repair, Dec 2009, 2:7) to expedite collagen deposition. Initial experiments showed no dose response increase in collagen levels by TGFP when compared to untreated cells incubated with dextran sulfate and L-Ascorbic acid alone at either cell concentration (Figure 2).

A follow up experiment evaluated TGFP at different doses in the presence of L- ascorbic acid (LascAc) only, dextran sulfate (DxS) only, and both L-ascorbic acid and dextran sulfate together on IMR-90 cells and two other lung cell lines from idiopathic pulmonary fibrosis (IPF) patients, LL97 A1MY cells and LL29 AnHa cells, respectively. The IMR-90 cells showed no dose response effects at any of the conditions tested.

Surprisingly, the signal with the best signal to noise ratio was observed in LL97 AlMy cells with TGFP and L-ascorbic acid only, which conflicts with the recommendation of Chen and Raghunath mentioned above (Figures 3 and 4) (Fibrogenesis & Tissue Repair, Dec 2009, 2:7).

The observations described above facilitated the design of experiments where compounds may be evaluated for their ability to inhibit collagen I deposition. LL97 AlMy cells were used for lung indications and L-ascorbic acid only was included in the media to provide a good signal to noise ratio while maintaining a low background signals in untreated cells. A 4-7 fold increase in collagen deposition was typically observed from cells treated with low serum and ascorbic acid only as compared to cells treated with 500 pg/ml of TGFp.

Two additional cell lines were also used for the screening, namely, human adult dermal fibroblasts and human hepatic stellate cells. Additional optimizations were performed to obtain proper signal to noise ratio in hepatic stellate cells. These optimizations included, for example, maintaining the cell culture in Geltrex coated plates, and adapting the format to a 48 well plate to provide a more robust signal in the human hepatic stellate cell line.

The assay described here is amenable to 96-well or 48-well plate format. Plates are scanned using a LiCor Odyssey flatbed scanner with two lasers that detect the infrared signal specific for Collagen I and the DRAQ5 signal specific for DNA (Figure 5).

Both Collagen I and DNA images were scanned on the Licor Odyssey dual laser flatbed scanner and were quantified for data normalization (Collagen I signal / DNA signal) within individual wells to determine the level of Collagen I produced in each well (Figure 6). TGF does not significantly alter the DNA levels detected. (Triangles, Fig. 6) Further, Collagen I deposition induced by 500 pg/ml TGFP was inhibited by known anti-fibrotic compounds such as the Alk5 inhibitor, SB431542 (Figure 7).

The cell lines used in the examples were commercially available. (1) ATCC Item # CCL-191 LL97A (AlMy) Lung Fibroblasts, Human; lot # 4379504). (2) ATCC Item # CCL-134 LL29 (AnHa) Lung Fibroblasts, Human, lot # 58142602; (3) ATCC Item #CCL-201 CCD-8Lu Lung Fibroblasts, Human, lot #4428846; (4) ATCC #CCL- 210 CCD-19Lu Lung Fibroblasts, Human, lot # 3997377; (5) Invitrogen #C-013-5c

Primary Human Dermal Fibroblasts, adult (HDFa), lot #709590; (6) Sciencell Research laboratories #5300 Human Hepatic Stellate Cells (HHSteC). Example 2: Identification of compounds that regulate Collagen I deposit in lung cells

This example describes methods for the evaluation of compounds, extracts, or biologies for anti-fibrotic effects. Human pulmonary fibroblasts were used to determine the capability of small molecules and extracts in inhibiting TGF -induced Collagen I deposition in human pulmonary fibroblasts.

As shown in Figure 8, Compound A and Compound B both inhibit Collagen I deposition induced by 500 pg/ml TGF in human pulmonary fibroblasts. Figure 9 shows that Extract A and Extract B both inhibit Collagen I deposition induced by 500 pg/ml TGF in human pulmonary fibroblasts.

Example 3: Identification of compounds that regulate Collagen I deposit in skin or liver cells

The disclosed system and method may also be used for the evaluation of compounds, extracts, or biologies for anti-fibrotic effects in human adult dermal fibroblasts and anti-fibrotic effects in human hepatic stellate cells. This example describes some of the data obtained from studies conducted using these cells. Molecules or extracts that inhibit TGF -induced Collagen I deposition in liver cells may be used in the treatment of liver fibrosis.

Figure 10 shows the dose response curve of a known anti-fibrotic agent SB431542 inhibits TGF -induced Collagen I deposition and Extract C inhibits Collagen I deposition induced by TGF in human adult dermal fibroblasts.

Figure 11 shows that an extract promotes Collagen I deposition in the absence of TGF in human adult dermal fibroblasts. Such molecules or extracts that promote Collagen I deposition in skin cells may have therapeutic application in wound repair and healing.