Patent Application
20220196635
The various embodiments of the present disclosure relate generally to methods and systems for forming assays of microscale cell-laden matrices, and more particularly to fibrin assays formed using an aqueous two-phase system.
Studying the process of remodeling events such as tissue degradation or formation at the cellular level can provide valuable information to the overall process of wound healing. Even more so, the differences between normal and pathogenic wound healing of various tissues has broad applications in studying many different cell types and diseases. In particular, following tissue damage, fibrin will form a temporary scaffold at the injury that enables fibroblasts to migrate to the site for matrix remodeling. The fibrotic remodeling events that occur after can result in a variety of complications, for example, excessive collagen accumulation promoting fibrotic scarring.
Current approaches available to evaluate such fibrotic remodeling events require large volumes that hinder high-throughput adaption or fail to consider key contributing factors such as specific environmental factors, epigenetics, or senescence. An approach that can control the crosslinking process within fibrotic systems by separating the enzyme, thrombin, and the protein, fibrinogen, in separate and distinct phases can form microscale matrices for high-throughput screening for fibrosis and other dysregulated wound healing diseases.
The present disclosure relates to methods for forming microscale cell-laden matrices and systems for making an assay having microscale cell-laden matrices. An exemplary embodiment of the present disclosure provides a method for forming a microscale cell-laden matrix using an aqueous two-phase system (“ATPS”) comprising a mixture of a first material and a second material having a phase boundary between the first and second materials. The method can comprise mixing an enzyme with the first material, mixing a protein with the second material, and mixing a suspension comprising cells with the first material. The enzyme, protein, and suspension can comprise cells to generate the cell-laden matrix. The first material can be a first polymer selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, and ficoll. The second material can be a second polymer comprising dextran, polyvinyl pyrrolidone, polyvinyl alcohol, and ficoll. The second material can be selected to be different from the first material.
In any of the embodiments disclosed herein, at least one of the enzyme or the cells in the first material can be configured to diffuse into the second material.
In any of the embodiments disclosed herein, the mixture can comprise up to about 300 microliters (μL) of volume.
In any of the embodiments disclosed herein, the enzyme can comprise a plasma enzyme from the group consisting of prothrombin, thrombin, amylase, pepsin, lipoprotein lipase, and pseudo-choline esterase.
In any of the embodiments disclosed herein, the protein can comprise a plasma protein from the group consisting of fibrinogen, fibronectin, collagen, albumin, globulin, and plasminogen activator inhibitor type 1.
In any of the embodiments disclosed herein, the suspension comprising cells can comprise fibroblasts, fibrocytes, osteoblasts, myofibroblasts, epithelial cells, endothelial cells, immune cells, mesenchymal cells, cancer cells, and stem cells.
In any of the embodiments disclosed herein, the method can further comprise mixing the mixture with a third material comprising one or more additives.
In any of the embodiments disclosed herein, the method can further comprise imaging the cell-laden matrix and the one or more additives.
In any of the embodiments disclosed herein, the one or more additives can comprise transforming growth factor beta 1 (TGF-β1).
In any of the embodiments disclosed herein, the method can further comprise adding, to the cell-laden matrix, a digestive agent.
In any of the embodiments disclosed herein, the method can further comprise imaging the cell-laden matrix and the digestive agent.
In any of the embodiments disclosed herein, the method can further comprise detecting one or more remodeling events of the cell-laden matrix selected from the group consisting of matrix degradation, matrix growth, matrix proliferation, matrix cell invasion, matrix cell contraction, matrix cell type, and matrix cell density.
An exemplary embodiment of the present disclosure provides a cell-laden matrix assay system comprising a solid support comprising at least one defined area and an aqueous two-phase system mixture for forming a cell-laden matrix. The mixture can comprise a first material having an enzyme and one or more cells, a second material having a protein, and a phase boundary between the first and second materials. The first material can be a first polymer selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, and ficoll. The second material can be a second polymer comprising dextran, polyvinyl pyrrolidone, polyvinyl alcohol, and ficoll. The second material can be selected to be different from the first material.
In any of the embodiments disclosed herein, the at least one defined area can comprise up to about 300 microliters (μL) of volume.
In any of the embodiments disclosed herein, forming the cell-laden matrix can comprise the enzyme, the protein, and at least one cell.
In any of the embodiments disclosed herein, said solid support can be selected from the group consisting of a plate, a multiwell plate, a microfluidic device, and a slide.
In any of the embodiments disclosed herein, the system can further comprise a third material comprising one or more additives.
In any of the embodiments disclosed herein, the system can further comprise a digestive agent.
In any of the embodiments disclosed herein, the system can further comprise a detection system selected from the group consisting of label-free image processing, colorimetric, fluorescent, fluorescence polarization or lifetime readings, refractive index change, and electrochemical detection systems.
In any of the embodiments disclosed herein, the enzyme can comprise a plasma enzyme from the group consisting of prothrombin, thrombin, amylase, pepsin, lipoprotein lipase, and pseudo-choline esterase. The protein can comprise a plasma protein from the group consisting of fibrinogen, fibronectin, collagen, albumin, globulin, and plasminogen activator inhibitor type 1. The one or more cells can comprise a fibroblast, a fibrocyte, an osteoblast, a myofibroblast, an epithelial cell, a mesenchymal cell, a cancer cell, and a stem cell.
These and other aspects of the present disclosure are described in the Detailed Description below and the accompanying drawings. Other aspects and features of embodiments will become apparent to those of ordinary skill in the art upon reviewing the following description of specific, exemplary embodiments in concert with the drawings. While features of the present disclosure may be discussed relative to certain embodiments and figures, all embodiments of the present disclosure can include one or more of the features discussed herein. Further, while one or more embodiments may be discussed as having certain advantageous features, one or more of such features may also be used with the various embodiments discussed herein. In similar fashion, while exemplary embodiments may be discussed below as device, system, or method embodiments, it is to be understood that such exemplary embodiments can be implemented in various devices, systems, and methods of the present disclosure.
The following detailed description of specific embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, specific embodiments are shown in the drawings. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
To facilitate an understanding of the principles and features of the present disclosure, various illustrative embodiments are explained below. The components, steps, and materials described hereinafter as making up various elements of the embodiments disclosed herein are intended to be illustrative and not restrictive. Many suitable components, steps, and materials that would perform the same or similar functions as the components, steps, and materials described herein are intended to be embraced within the scope of the disclosure. Such other components, steps, and materials not described herein can include, but are not limited to, similar components or steps that are developed after development of the embodiments disclosed herein.
As shown in
As used herein, the first material 202 and the second material 204, which constitute the aqueous two-phase system (“ATPS”) 200, can include any suitable polymer/polymer ATPS system. Polymer/polymer ATPS systems can include two nonionic polymers, such as, polyethylene glycol (PEG) mixed with any one of polypropylene glycol (PPG), polyvinyl pyrrolidone (PVP), poly(vinyl methyl ether) (PVME), poly(vinyl methyl ethyl ether) (PVMEE) polyether sulfones (PES), polyvinyl alcohol (PVA), polypropylene glycol dimethyl ether (PPGDME), UCON, Ficoll, Dextran, Pullulan, Maltodextrin, and hydroxypropyl starch. As an alternative, a two nonionic polymer ATPS system may also include dextran mixed with any one of PVP, PVA, PPG, UCON, Ficoll, hydroxypropyl starch, and Natrosol. In some embodiments, a two nonionic polymer ATPS system may include PPG mixed with any one of polyethylene glycol methyl ether (PEGME), polyethylene glycol dimethyl ether (PEGDME), PVA, and Ficoll. In some embodiments, the polymer/polymer ATPS systems can include one nonionic polymer and one ionic polymer such as PEG/dextran sulphate, PEG/polyacrylic acid (PAA), PEG/polyacrylamide (PAM), PEG/carboxymethyl dextran, PVP/PAM, and PVA/acrylic polymers. In any of the embodiments herein, the polymer/polymer ATPS system can include two ionic polymers such as dextran sulphate/polystyrene sulfonate (PSS) or dextran sulphate/diethylaminoethanol (DEAE)-dextran. In addition, suitable ATPS systems may include but are not particularly limited to, water/ethylene oxide propylene oxide (EOPO), PEG/high-concentration salt, PEG/levan, PEG/ammonium sulfate, PEG/sodium sulfate, PEG/magnesium sulfate, PEG/potassium phosphate, and PEG/sodium carbonate.
The two materials used to form the aqueous two-phase system 200 are preferably polyethylene glycol/dextran. In some embodiments, the enzyme 102 can be mixed in the polyethylene glycol phase and the protein 104 may be characterized by being concentrated in the dextran phase, and the proteins 104 concentrated in the dextran phase may be isolated using a pipette, or other suitable methods. As an alternative, the enzyme 102 can be concentrated in the dextran phase while the protein 104 can be mixed in the polyethylene glycol phase. In certain embodiments, additives can also be included in either the polyethylene glycol phase or the dextran phase.
As used herein, the enzyme 102 can include any substance composed wholly or largely of protein or polypeptides that catalyzes or promotes, more or less specifically, one or more chemical or biochemical reactions. In certain embodiments, the ATPS 200 can include one or more enzymes from blood plasma or other bodily fluids. Suitable plasma enzymes can include, but are not limited to, prothrombin, thrombin, amylase, pepsin, lipoprotein lipase, and pseudo-choline esterase. In general, thrombin is an activated enzyme, also known as α-thrombin, which results from the proteolytic cleavage of prothrombin (factor II). As an alternative, or in addition thereto, the enzyme may include cell types that produce endogenous prothrombin.
In some embodiments, the ATPS 200 can include one or more proteins 104, including soluble proteins found in the plasma of normal humans or animals. These include but are not limited to coagulation proteins, albumin, lipoproteins and complement proteins. In particular, plasma proteins can include fibrinogen, albumin, globulin, and plasminogen activator inhibitor type 1.
Referring back to
In some embodiments, the cell suspension 106 can include cells that can form matrixes when mixed with the enzyme 102 and the protein 104. Cell suspension 106 can be made up of a variety of cells found in normal humans or animals. Suitable cells for forming matrixes can include, but are not limited to fibroblasts, fibrocytes, osteoblasts, myofibroblasts, epithelial cells, endothelial cells, immune cells, mesenchymal cells, cancer cells, and stem cells. In certain embodiments, cell suspension 106 can include cells from a particular subject. For instance, studying fibrosis on a subject that has been exposed to smoking can include mixing human lung fibroblasts from a subject with a history of smoking. Such ATPS assay can provide insight into idiopathic pulmonary fibrosis.
Mixing the enzyme 102, the protein 104, and cell suspension 106 can generate the cell-laden matrix 100. In certain embodiments, mixing thrombin with fibrinogen and fibroblasts can result in a fibrin cell-laden matrix. As would be appreciated, changing the enzyme 102, protein 104, or cell suspension 106 can generate a variety of cell-laden matrices using an ATPS mixture.
In some embodiments, using method 10 to create an ATPS assay of a subject may include collecting body fluid from a subject to study a disease. Here, the body fluid may include, but is not particularly limited to, at least one selected from the group consisting of whole blood, serum, peritoneal fluid, breast milk, and urine. The disease may include, but is not particularly limited to, at least one selected from the group consisting of fibrosis, pulmonary fibrosis, cancer, sepsis, arteriosclerosis, rheumatoid arthritis, dermatomyositis, polymyositis, mixed connective tissue disease, systemic lupus erythematosus, sarcoidosis, scleroderma, and pneumonia.
Referring back to
In some embodiments, a mixture of the first and second materials 202, 204 comprising the enzyme 102, the protein 104, and cell suspension 106 can comprise up to about 300 microliters of volume. Preferably, the mixture can range from about 0.5 μL to about 300 μL, and more preferably from 100 to 200 μL. In any of the embodiments herein, a volume of the first material comprising the enzyme 102 and optionally comprising the one or more additives 108 can be between about 50 and 200 μL mixed with a volume of the second material 204 comprising the protein 104 and optionally comprising the cell suspension 106 can be between about 0.5 μL and 50 μL, and preferably from 10 μL to about 50 μL.
In some embodiments, following formation of the cell-laden matrix 100, method 10 can further comprise imaging the cell-laden matrix 100 and the progression of cellular remodeling. Imaging techniques can include phase-contrast microscopy, fluorescent imaging, brightfield microscopy, confocal microscopy, 4D live-cell imaging (e.g., confocal with time-lapse microscopy), and other suitable cellular imaging techniques. In some embodiments, the imaging of matrix degradation can be achieved with label-free methods such as with absorbance or brightfield microscopy. As would be appreciated, such imaging methods can be automated by implementing a cellular imaging library in combination with artificial intelligence or machine learning methods.
In some embodiments, cellular remodeling events can also be detected by a detection system selected from the group consisting of label-free image processing, colorimetric, fluorescent, fluorescence polarization or lifetime readings, refractive index change, and electrochemical detection systems.
As would be appreciated, forming the cell-laden matrix can be done using a range of volumes while keeping ratios of the enzyme 102, protein 104, and cell suspension 106 the same or similar. In some embodiments, the cell-laden matrix can be formed from at least one cell within the cell suspension 106 such that the cell-laden matrix can be analyzed on a single-cellular level.
In
Output contraction times and final assay areas from image processing analysis were used to plot kernel density estimates showing interplay between contraction time and final assay area.
In any of the embodiments disclosed herein, the system and method can be used for diagnosing diseases associated with alterations in fibrinolysis and/or fibrin remodeling including cellular deposition of novel ECM material and subsequent contraction of the material. For example, a body fluid sample from a patient with such a disease can be used in combination with a system and/or method disclosed herein.
In any of the embodiments disclosed herein, the system and method can be used to identify potential therapeutic compounds that alter fibrinolysis and/or fibrin remodeling, including high-throughput analyses of such therapeutic compounds. High throughput multi-well analyses can be adapted for use with a method or system disclosed herein for diagnostic and/or drug discovery applications.
In any of the embodiments disclosed herein, the system and method can be used in combination with one or more imaging methods for high throughout multi-well analyses.
The following examples further illustrate aspects of the present disclosure. However, they are in no way a limitation of the teachings or disclosure of the present disclosure as set forth herein.
A stock solution of DEX (20% w/w dextran T500; Sigma) was prepared in phosphate buffered saline (PBS) on a rocker overnight. A stock solution of PEG (6% w/w, 35 k MW; Sigma) was prepared in fully supplemented culture media with 10% deionized water to balance osmolality. Both stock solutions were passed through a 0.22 um sterilizing syringe filter before storage. PEG working solutions were stored for up to 2 weeks at 4° C. Thrombin (Human Alpha Thrombin; Enzyme Research Labs) was also added to the PEG solution at a concentration of 0.1 U/mL immediately preceding experiments. Fibrinogen-DEX solutions were prepared by diluting fibrinogen stock solution (human fibrinogen 3; Enzyme Research Labs) to a final concentration of 4 mg/mL in a sterile solution of 4% 10×DMEM, 15% DEX stock solution (to a final concentration of 3% dextran), and 50% cell suspension in growth media. For all experiments excluding cell concentration evaluation, the cell suspension was diluted for 1000 cells per microliter in the final fibrinogen-DEX solution.
Normal human lung fibroblasts (NHLF lot #0000580583; Lonza) from a 79-year-old female with a history of smoking, and idiopathic pulmonary fibrosis fibroblasts (IPF lot #0000627840; Lonza) from a 52-year-old male were cultured in fibroblast growth media (FGM; Lonza). Cells were passaged at 80-90% confluence and were sub-cultured in 1:3 ratios by trypsinization. When at the desired confluence, cells were washed with PBS and 0.05% trypsin solution was added to the flask. Cells were incubated for 2 min, diluted with fibroblast growth media, and then harvested and centrifuged (200× g, 5 min) in a conical tube. The supernatant was aspirated and the cell pellet was re-suspended in serum-free culture media. When used in fibrin degradation experiments, cells were re-suspended at 2× the final desired concentration (1000 cells/ul unless otherwise indicated). All experiments were conducted with cells at or below passage 12. In all experiments, media was changed every 48 hours and any media additives (plasminogen, TGF-β1, drugs, etc.) were included.
Working solutions of PEG with 0.1 U/mL of thrombin were warmed to 37° C. and pipetted into a 96-well plate. For production of droplets, fibrinogen-DEX solutions with cell suspension were maintained at 37° C. and pipetted directly into the PEG-thrombin media using either a manual pipette or a semi-automated 96-channel pipette (Viaflo-96; Integra). All assays utilized a volume of 1 μl unless otherwise noted. Following dispensing of the DEX phase, the plates were placed in an incubator at 37° C. for 30 min to allow the thrombin to enzymatically crosslink the fibrinogen into a fibrin matrix (
In order to print fibrin into letters and arbitrary shapes, a 6% PEG solution containing 0.1 U/ml of thrombin was pipetted into a 6-well plate and warmed to 37° C. A 6% DEX solution containing 8 mg/mL fibrinogen was pipetted directly into the PEG phase to manually draw the desired shapes. After 30 min, darkfield images were taken on a stereoscope (Leica S6 E) to visualize the printed fibrin scaffold.
For cell viability measurements, 1 μl fibrin scaffolds were printed each containing 5000 cells total. The fibroblast-laden scaffolds were maintained in serum free media for 24 hours before live/dead staining (ReadyProbes™ Cell Viability Imaging Kit; Invitrogen). NucBlue and NucGreen (staining for total cells and dead cells respectively) were applied according to manufacturer directions. Dead control scaffolds were treated with 70% ethanol for 15 min prior to staining. Scaffolds were fluorescently imaged to assess viability. For calculation of percent viability, the density counting workflow in ilastik was used to count the number of total cells and dead cells.
After each experiment, brightfield images for every time point were downloaded in jpeg format from the automated cell culture monitoring system. Python's OpenCV library was implemented for the masking approach illustrated in
For each experimental replicate, a sigmoid curve was fit using the curve fit function from the SciPy library in Python. The logistic function given by the equation in
In order to evaluate fibrin degradation rate with a known anti-fibrinolytic stimulus, various concentrations of transforming growth factor type 131 (Human Recombinant TGF-β1; Peprotech) were added to the assay media after ATPS polymers were rinsed out of the microplates.
To evaluate the capability of this assay to test the fibrinolytic effects of therapeutic stimuli, a variety of drug compounds were introduced to the fibrinolysis assays after the wash step. This included 400 μM pirfenidone (Selleck Chem), 0.4 μM nintedanib (Selleck Chem), 100 μM hydrogen peroxide (Sigma), and 20 μM diethyl-pythiDC (AOBIOUS). These concentrations were established in preliminary experiments that evaluated a range of concentrations used in prior literature. Stimuli were freshly mixed for each media change during experiments, and a minimum of four replicates were tested per experimental condition.
All experimental values are reported as means±standard deviation. ANOVA tests were performed using the statsmodels library in Python 3 with the Tukey test for post-hoc pairwise comparisons.
The development and characterization of the cell-mediated fibrinolysis assay was focused on establishing a microplate-compatible fibroblast-laden fibrin scaffold and verifying the ability to distinguish between subtly different fibrinolytic environments. First, an ATPS approach was implemented to enable accurate printing of unprecedentedly small cell-laden fibrin scaffolds. Then, an automated image processing approach quantified fibrin degradation data from label-free brightfield images. Next, the established fibrinolytic effects of cell density and TGF-β1 were used to validate the assay's capability to distinguish between conditions. Finally, the microscale cell mediated fibrinolysis assay was implemented to evaluate the effects of anti-fibrotic therapeutics on fibroblasts from normal and diseased donors.
Biological environments establish fibrin matrices through coagulation, where a cascade of clotting factors activates thrombin, which enzymatically crosslinks fibrinogen into fibrin. Similarly, in vitro fibrin scaffolds are formed by exposing monomeric fibrinogen to thrombin. Fibrin has been used extensively in a wide variety of tissue engineering applications, but it is generally implemented as a bulk cast hydrogel. The conventional bulk casting procedure mixes thrombin and fibrinogen solutions by micropipette; however, this method cannot consistently handle small volumes (under 100 μl) due to adhesion of the partially coagulated mixture to pipette tips.
There have been a few applications of fibrin bio-printing that control crosslinking by alternating between layers of fibrinogen and thrombin, but this poses limitations to accuracy and reproducibility due to lack of control over fibrinogen's exposure to thrombin. There have also been a variety of applications for fibrin microbeads where oil immersions were used to disperse microbeads during crosslinking in oil-suspended droplets, but this results in inconsistent size and cells must be added separately after the microbeads have been washed. Reliable microscale volume and microplate compatibility were necessary to enable high-throughput adaptation in this assay. Precise control over cell seeding density was also vital for this approach due to its effect on remodeling rate.
A new approach to maintain fibrinogen in a distinct droplet and control diffusion of thrombin into fibrinogen during the polymerization process was established by implementing an ATPS with PEG and DEX. Above their critical concentrations, these soluble polymers thermo-dynamically drive aqueous systems to form two distinct phases. A previous ATPS microscale adaptation from related research for collagen contraction demonstrated consistency in response between the conventional 100 μL assay and ATPS microscale volumes. This work specifically took advantage of the short length scales for time-dependent and burst stimulation profiles, which would not be possible with conventional approaches due to diffusion constraints. A similar ATPS adaptation suited the approach described herein, and enabled fabrication of microscale fibrin scaffolds with standard liquid handling equipment to facilitate microplate compatibility.
During the initial optimization of PEG and DEX concentrations, lower concentrations were found to be unstable and resulted in fissure of the ATPS droplet. In order to maintain stable separation of phases during polymerization, minimum assay concentrations of 6% 35 kDa PEG and 3% T500 dextran were determined for stability during crosslinking (
The necessity for this ATPS environment in the microscale fibrin degradation assay described herein comes from the capability of aqueous two-phase partitioning to control the timing of thrombin diffusion into the fibrinogen droplet. This control over timing restricts enzymatic crosslinking of cell-laden fibrin matrices until after the droplets have been dispensed (
After plasminogen was added to the wells, various activators and inhibitors produced by cells regulate the conversion of plasminogen into plasmin. Control conditions for each experiment verified rapid matrix degradation with the addition of exogenous plasmin and no matrix degradation when plasminogen is omitted. As the assay proceeds, the fibrin matrix gradually degrades with activated plasmin cleaving fibrin into soluble fibrin degradation products (
Due to the relative opacity of the fibrin scaffolds, pixels within the assay area are significantly darker than those in the background of microscope images. This enabled an analysis approach based on pixel intensities within the assay area. Many established hemostasis assays take advantage of fibrin's attenuation of light for quantification. These assays generally implement plate readers to measure absorbance during coagulation and fibrinolysis. Evaluation of this assay in a microplate reader may therefore serve as an alternative to brightfield analysis. However, the approach favored evaluation of pixel intensity from brightfield images so that the micrographs could serve as validation of assay progression. Unfortunately, the commercial image analysis package embedded in the live cell imaging system was not able to reliably discern the microprinted fibrin scaffold. An alternative image analysis pipeline was developed using Python's OpenCV library.
In order to isolate the assay area from background, a thresholding approach was sufficient because of the significant difference in pixel brightness. Here, any pixels brighter than the specified threshold were classified as background. A closing morphological filter was applied to the thresholded images to remove noise left by the thresholding process.
The effects of assay volume were also evaluated. Assay volumes between 0.5 μl and 8 μl could be consistently printed and viewed within the field-of-view of a 4× microscope objective (
In prior microscale adaptation of collagen contraction, different assay volumes were found to maintain consistent contraction rates as long as cell density was maintained. Fibrinolysis trends in the experiments described herein also depend on cell density rather than assay volume. While the pixel intensity of higher volume assays had lower starting values, this reflected the presence of more fibrin which resulted in decreased transmission of light through those assays (
The consistency in degradation rates between volume conditions indicates uniformity in fibrin organization. Fibrin network morphology has a significant impact on fibrinolysis rate, where tight fibrin conformations degrade at a slower rate than scaffolds with looser fibrin conformations and thicker fibers. This suggests that at the concentration of thrombin used in the present assays, ATPS-mediated control over the diffusion of thrombin into the fibrinogen-containing phase results in consistent fibrin organization across the range of assay volumes tested.
Cell seeding density was also evaluated. Conditions with higher seeding densities demonstrated significantly faster fibrinolysis (
TGF-β1 is an established pro-fibrotic stimulus with well-characterized anti-fibrinolytic effects. Various concentrations of TGF-β1 were used to stimulate NHLF cells in the fibrin assays (
In NHLF cells, a significant effect of cell passage number on fibrinolysis was also noticed. Higher passage numbers exhibited progressively longer 50% degradation times with slower fibrin degradation rates. These incidental observations are consistent with prior studies which demonstrate inhibition of fibrinolysis in senescent fibroblasts in vivo and in vitro due in part to the upregulation of PAI-1.
Having established baseline cell response measurements for fibrinolysis of the bioprinted fibrin micro-scaffolds, fibrinolytic profiles were compared between normal and diseased lung fibroblasts with a number of stimulants and inhibitors. Hydrogen peroxide is a reactive oxygen species (ROS) known to be produced by cells in response to TGF-β1 stimulation, while nintedanib and pirfenidone are the only two FDA-approved therapies for IPF. Diethyl-pythiDC, an experimental anti-fibrotic drug, is an inhibitor of certain prolyl 4-hydroxylases that play a role in post-translational modification of collagen and other proteins. The plasmin control condition was included in graphs for reference but was excluded from statistical analysis in the interest of focusing on therapeutic conditions of interest.
A general comparison between normal and diseased fibroblasts (
Stimulation with hydrogen peroxide (H2O2) alone demonstrated highly significant decreases in the rates of fibrinolysis (P<0.1) suggesting a critical role of ROS in the process of cell-mediated fibrinolysis. In contrast, conditions that included TGF-β1 showed no significant difference upon further stimulation with exogenous hydrogen peroxide. This non-additive effect is consistent with a notion that the effects of adding exogenous H2O2 and exogenous TGF-β1 converge. That is, TGF-β1-triggered increase in endogenous H2O2 production, may mask effects of any exogenous H2O2 addition. Such effects may also work in concert with ROS-induced reduction in TGF-β1 receptors.
The two FDA-approved IPF drugs, nintedanib and pirfenidone, did not show a significant impact on fibrinolysis. These therapeutics have established anti-fibrotic effects, so these results indicate that the mechanism of action for nintedanib and pirfenidone has little relation to fibrinolytic activity of lung fibroblasts. Given the limitation that nintedanib and pirfenidone can slow but not stop or reverse IPF, the ability to test IPF-relevant pathways such as fibroblast-mediated fibrinolysis-associated processes that these drugs do not target may provide opportunities for developing co-therapeutics or alternatives with enhanced efficacy. The experimental drug diethyl-pythiDC significantly (P<0.05) delayed fibrinolysis. Diethyl-pythiDC is a selective inhibitor of prolyl 4-hydroxylase, an enzyme best known for structure-stabilizing modifications of collagen that also acts on a variety of proteins including hypoxia inducible factor 1. The ability of diethyl-PythiDC to reduce fibroblast-mediated fibrinolysis is a novel finding and demonstrates the utility of the assay described herein. Given the many physiological factors present in blood or expressed by many types of cells positively (e.g. proteases such as uPA, tPA, cathepsins, FXIa, FXIIa, kallikreins) and negatively (e.g. serpins such as PAI-1 and α2-antiplasmin, α2-macroglobulin) impact fibrinolysis, it is possible that this fibrin printing and fibrinolysis assay will be of broad utility.
This work describes an approach for ATPS-based printing of microscale cell-laden fibrin scaffolds. A droplet comprised of the heavier phase partitions cells and fibrinogen while the bulk phase provides thrombin to promote localized enzymatic crosslinking, leading to controlled production of microliter-scale fibrin constructs. Automated label-free image processing quantified rates of cell-mediated fibrin degradation from time-course brightfield images. Primary human lung fibroblasts were found to degrade the fibrin scaffold at a rate dependent on source of cells, cell density, and the presence of soluble factors. Given the variety of contributors to dysregulation of fibrinolysis seen in cancer, fibrosis, and metabolic disease; this phenotypic assay for cell-mediated fibrin degradation provides a potentially valuable research tool for further studies in these and other fields. Additionally, the technique developed here for aqueous two-phase printing of cell-laden fibrin by in situ enzymatic cross-linking can be broadly applied in bio-printing and tissue engineering applications.
Human primary lung fibroblasts were used in all experiments presented in this paper. Unless otherwise noted, experiments utilized normal human lung fibroblasts (NHLF B lot #0000580583; Lonza) from a 79-year-old female with a history of smoking. For experiments evaluating donor variability, the following cells were utilized: NHLF A (NHLF lot #0000608197; Lonza) from a 67-year-old male, IPF A (IPF lot #0000627840; Lonza) from a 52-year-old male, and IPF B (IPF lot #6F5002; Lonza) from an 83-year-old male. All cells were cultured in fibroblast growth media (FGM; Lonza). Cells were passaged at 80-90% confluence and were sub-cultured in 1:3 ratios by trypsinization. When at the desired confluence, cells were washed with PBS and 0.05% trypsin solution was added to the flask. Cells were incubated for 2 min, and then harvested and centrifuged (200 μg, 5 min) in a conical tube. The supernatant was aspirated and the cell pellet was re-suspended in FBS-free culture media. When used in fibrin degradation experiments, cells were re-suspended at 2× the final desired concentration (2500 cells/ul unless otherwise indicated). To promote collagen production and contraction in addition to fibrin degradation, serum (bovine or human) were added at 2% or higher up to about 10%. Higher cell densities (about 2500 cells/ul and higher up to about 10,000 cells/ul) also promote collagen production. All experiments were conducted with cells at or below passage 8 except for high passage experiments conducted at passage 12. In all experiments, media was changed every 48 hours and any media additives (plasminogen, TGF-β1, drugs, etc.,) were included.
ATPS printing of fibrin micro-scaffolds has previously described above. Briefly, working solutions of PEG with 0.1 U/mL of thrombin were warmed to 37° C. and pipetted into a 96-well plate. For production of droplets, fibrinogen-DEX solutions with cell suspension were maintained at 37° C. and 4 μl per assay (unless otherwise noted) was pipetted directly into the PEG-thrombin media using a semi-automated repeater pipette (Repeater E3X; Eppendorf). Following dispensing of the DEX phase, the plates were placed in an ambient air incubator at 37° C. for 30 min to allow the thrombin to enzymatically crosslink the fibrinogen into a fibrin matrix (
Contracted cell-ECM spheroids were harvested after 9-12 days of culture. These structures were prepared for histology, stained, and imaged as previously described for cultured spheroids. Briefly, the spheroids were washed with PBS and fixed in 4% paraformaldehyde (Alfa Aesar) for 1 hour at room temperature. The structures were stained with 0.5% methylene blue solution in PBS for 10 minutes at room temperature to aid in visualization during histology. Samples were placed in a cryomold containing optimal cutting temperature (OCT) compound, and flash frozen in cooled isopentane. 10 um sections were obtained using a CryoStar NX70 cryostat (Thermo Fisher Scientific).
Upon warming to room temperature, the sections were washed with PBS, permeabilized with 0.2% Triton-X 100, and blocked for 1 hour at room temperature with 4% bovine serum albumin (BSA) (Millipore Sigma). Sections were stained for 30 min at room temperature with Sirius red (0.1% of Sirius red in saturated aqueous picric acid), as previously described for collagen bundle staining. The samples were then washed with PBS, stained with DAPI for 15 minutes at room temperature, and coverslipped. Samples were imaged using a DMi8 microscope (Leica) equipped with 10× and 20× air objectives. Fluorescence was detected using Texas Red channel settings as previously described. Mean fluorescence intensity was quantified in ImageJ as the average pixel intensity within the sections.
RNA extraction and qPCR: Cells from 12 wells at the indicated time points were pooled together per condition and lysed with 350 μl of RLT lysis buffer. RNA was extracted using a RNeasy Mini Kit (Qiagen, #74104) and was performed according to the manufacturer's instructions. RNA sample concentration was measured using a NanoDrop OneC Spectrophotometer (Thermo Fisher Scientific). A High-Capacity RNA-to-cDNA Kit (Applied Biosystems, #4387406) was used for reverse transcription; 400 ng of RNA for each sample was mixed with 10 μl primer, 1 μl reverse transcriptase enzyme and nuclease-free water to bring the final reaction volume to 20 μl. The reaction was performed for 60 minutes at 37° C., followed by 5 minutes at 95° C. using a Veriti Thermal Cycler (Applied Biosystems). qPCR was performed using a QuantStudio 3 Real-Time PCR System (Applied Biosystems). Each reaction consisted of 1 μl cDNA, 10 μl TaqMan Fast advanced master mix (Applied Biosystems, #4444556), 1 μl primer, and 6 μl nuclease-free water. TaqMan primers (Applied Biosystems) for smooth muscle actin (ACTA2, Hs00426835_g1), plasminogen activator (PLAT, Hs00263492_m1), Plasminogen activator inhibitor-1 (SERPINE1, Hs00167155_m1), collagen type I (COL1A1, Hs00164004_m1), plasminogen activator (PLAU, Hs01547054_m1), and Ki67 (MKI67, Hs01032443_m1) were utilized. The QuantStudio 3 was programmed with a 2-minute hold at 95° C., followed by 40 cycles of 95° C. for 1 second and 60° C. for 20 seconds. Each sample was run with biological triplicates. The relative gene expression was calculated using the 2-AACT method, with glyceraldehyde-3-phosphate dehydrogenase as the housekeeping gene (GAPDH, Hs02786624_g1). Fold changes were normalized with respect to the time zero timepoint with no TGF-β1 stimulation and are reported as the mean with the error bars representing the minimum and maximum values.
After each experiment, the final projected areas of the cell-ECM spheroids were determined from brightfield images taken at the final time point using a benchtop imaging system (2× objective; EVOS M7000; ThermoFisher). These images were then segmented through a process of pixel classification, thresholding, and morphological filtering in order to isolate the cell-ECM construct area from the background.
Pixel classification implemented Ilastic, a freely available image classification tool developed by the European Molecular Biology Laboratory. Ilastik's pixel classification utility implements a random forest classifier for quick and robust segmentation. In order to train the classifier, 10 characteristic images were selected to include different stages of ECM remodeling. In this step, each individual pixel is assigned a probability for belonging to layers for the background or the cell-ECM construct. Ilastik enables interactive training of the random forest classifier via user annotations of the training images. All default features (G=0.3 through 10 for intensity, edge, and texture) were utilized for this interactive training by methodically annotating mislabeled areas of each training image. Care was taken to equally annotate background and cell-ECM areas in order to prevent the algorithm from weighting features inappropriately. Through this iterative training method, the user can evaluate interactive predictions by the algorithm and then draw additional annotations to correct mistakes. When additional training annotations no longer improved background noise and edge feature fit of the predicted mask over the cell-ECM area, the trained classifier was saved for future use. With each experiment, this trained classifier was reloaded and classification performance was evaluated on representative images (not from the training set) before use.
This pixel classification workflow performs semantic segmentation, and therefore returns a probability map for the background and cell-ECM area for each image. The probability map was transformed into background and cell-ECM area objects through thresholding. Thresholding of these probability masks then enabled generation of a single mask to isolate the cell-ECM area. A closing morphological filter with a 25×25 kernel was then applied to each mask in order to remove noise. The area from segmented masks was then used to quantify cell-ECM contraction.
Various concentrations of transforming growth factor type β1 (Human Recombinant TGF-β1; Peprotech) were used for validation due to its established anti-fibrinolytic and pro-fibrotic qualities. TGF-β1 was added at indicated concentrations in the assay media which was used to rinse and remove ATPS polymers after incubation.
As described above in example 12, the customized high-throughput image analysis approach can also be applied for phenotypic evaluation of all experiments. In order to evaluate ECM remodeling behavior with established stimuli; experiments implemented different conditions of TGF-β1, FBS, and cell seeding density. TGF-β1 was introduced at concentrations of 0, 0.5, 2, and 10 ng/mL; however the highest concentration did not contract within the duration of the experiment and was therefore omitted from analysis. In order to evaluate the remodeling effects of serum, fetal bovine serum (FBS, Lonza) at concentrations of 0, 1, 2, 4, and 8% by volume of the cell culture media was added during the washing step after fibrin crosslinking. For cell seeding density experiments, fibroblasts were suspended at appropriately modified concentrations in the dextran phase of the ATPS fibrin printing formulation so that assays were printed with concentrations of 1, 2, 4, and 8 thousand cells per microliter within a 4 μl assay.
In order to evaluate the capability of this assay to test the fibrinolytic and anti-fibrotic effects of therapeutic stimuli, a variety of drug compounds were introduced to the assays after the wash step. This included 10 μM TM5275 (MedChemExpress), 1 μM nintedanib (Selleck Chem), and 500 μM pirfenidone (Selleck Chem); all diluted and stored according to supplier data sheet recommendations. These concentrations were established in preliminary experiments that evaluated a range of concentrations used in prior literature. These stimuli were freshly mixed for each media change during experiments, and a minimum of four replicates were tested per experimental condition.
All experimental values are reported as means±standard deviation. ANOVA tests were performed using the statsmodels library in Python 3 with the Tukey test for post-hoc pairwise comparisons. Experiments involving two independent variables (such as therapeutic stimulus and TGF-β1) implemented two-way ANOVA to evaluate the significance of combined effects.
In tissue repair, fibrin formation is followed by fibroblast migration, fibrinolysis and matrix remodeling. In lung fibrosis, fibrinolytic activity is decreased and undegraded fibrin is commonly reported in human IPF patient lungs. The method to print microscale cell-laden fibrin gels to quantify cell-mediated fibrinolysis is described above in examples 1 through 11. The study conditions described above, however, do not provide readouts of fibroplasia such as collagen deposition. Furthermore, the fibrinolysis assay did not show significant change in response to treatment with fibrosis drugs such as nintedanib and pirfenidone.
Published larger-scale fibrin-based fibroplasia assays were used for skin keloids. Specifically, the plasminogen addition step was replaced with addition of FBS and TGF-β1 instead. Under these revised conditions, fibrin was gradually replaced by a collagen-rich ECM. Surprisingly, after 3-7 days, contraction and detachment of the remodeled cell-laden matrix into a compact cell-ECM spheroid can be observed. While early contraction of the fibrin gel itself has been reported and inhibition of collagen gel contraction by incorporation of fibrin has been reported, there are no reports of detachment and contraction of the ECM produced by cells embedded in fibrin gels. Here, the method tests whether this unexpected contraction, detachment, and spheroid formation process could be utilized as a convenient, image-based, direct readout of fibroplasia in fibrin droplet assays.
Biological environments establish fibrin matrices through coagulation, where a cascade of clotting factors activates thrombin to enzymatically convert fibrinogen into fibrin. Similarly, synthetic fibrin scaffolds are formed by exposing monomeric fibrinogen to activated thrombin. In the technique described above for generating fibrin micro-scaffolds utilized an ATPS with PEG and dextran to improve control over fibrin formation, which enables printing of unprecedentedly small cell-laden fibrin matrices with standard liquid handling equipment. As described above, fibroblast-mediated fibrinolysis can be initiated by addition of exogenous plasminogen comparable to levels found in serum. Here, decreasing availability of plasminogen to levels that may better reflect tissue levels by supplementing the media with FBS. These adjustments significantly altered the trajectory of the microscale fibrin remodeling process compared to the fibrin forming approach and to other published fibrin fibroplasia assays. Rather than strictly degrading the scaffold into fibrin degradation products and dissociated cells, these conditions induced deposition of significant amounts of collagen followed by contraction of the cell-ECM construct into a fibrotic spheroid.
In this approach, the microscale format enabled microwell plate implementations with convenient automated live imaging. In order to fit the printed microscale fibrin drops within the field of view of a 4× objective, ATPS printing was implemented as described above. The ATPS-based bioinks allowed partitioning of fibrinogen into the denser, DEX-rich, droplet phase while thrombin was allowed to diffuse in gradually from the less dense, PEG-rich, bulk phase solution. This controlled mixing of enzyme with fibrinogen delayed crosslinking of cell-laden fibrin matrices until after the fibrinogen droplets were dispensed (
During assay progression, remodeling is visually apparent in brightfield images as opaque fibrin transitioning into a translucent fibrous matrix and eventually contracting into a dense spheroid (
Downstream signaling effects of TGF-β1 include inhibition of fibrinolysis, increased fibroblast activation, increased synthesis and deposition of ECM, inhibition of ECM breakdown, and increased contractility. This section describes how TGF-β1 treatment impacts histologic staining of collagen, expression of key genes associated with fibroplasia, and size of the final contracted cell-ECM spheroids.
Final organization of deposited collagen. The most commonly used commercially-available method for quantification of deposited collagen is the Sircol™ insoluble collagen assay kit, which implements the dye Sirius Red F3B due to its high specificity for collagen. These kits, however, are optimized for use on fixed quantities of excised tissue. Due to the low assay volume and variability in assay final size (
Picrosirius red (PSR) utilizes the same anionic dye as Sircol™ assay kits to visualize collagen in paraffin embedded tissue sections. Under light microscopy, PSR stained collagen appears red and can be used for qualitative evaluation of collagen organization. A variety of quantitative approaches for morphometric assessment of collagen networks implement polarized light to visualize fiber alignment; however, signal strength and hue under linear polarized light are heavily dependent on sample orientation. Fluorescent imaging of PSR stained tissues with standard red filter sets yields a strong red fluorescence signal that is sensitive, collagen-specific, and is unaffected by sample orientation.
In order to evaluate deposited collagen, contracted cell-ECM spheroids were collected after 12 days of culture. Intermediate time points could not be sectioned due to adhesion of flat fibrin scaffolds to the microplate. Fluorescent micrographs demonstrate relatively homogenous collagen distribution for the interior of the contracted assay with higher deposition around the edge (
Well-established mechanisms have linked TGF-β1 signaling to exaggerated extracellular deposition of type I collagen in fibrosis. Here, histologic evaluation indicates consistency in ECM deposition between conditions, suggesting that the conveniently visualized contracted spheroid size is correlated with the total amount of collagen accumulated during assay progression.
In order to further evaluate the factors contributing to altered ECM remodeling with TGF-β1 stimulation, qPCR was used to determine mRNA expression for proteins involved in fibrinolysis and collagen deposition.
Quantification of mRNA for SERPENE1, which encodes for the protein plasminogen activator inhibitor type 1 (PAI-1), demonstrated significant time-dependent and dose-dependent increases in expression in response to TGF-β1 (
Time course measurements also show significant increases in expression of the genes for tPA and uPA relative to the initial time point, but the effect of TGF-β1 stimulation is inverted between these two plasminogen activators. uPA demonstrated relative upregulation compared to the control time series, while tPA demonstrated a relative downregulation (
In addition to its effects on the fibrinolytic system, TGF-β1 also has established roles in myofibroblast activation and collagen synthesis. Myofibroblasts are collagen-producing cells that express the contractile protein alpha smooth muscle actin (aSMA). Increases in myofibroblast activation and myofibroblast resistance to apoptosis have been identified as major contributors to IPF pathogenesis. Evaluation of ACTA2 mRNA demonstrated significant time-course increases in aSMA expression as well as increased expression for the highest concentration of TGF-β1 (
COL1A1 encodes the pro-alpha1(I) chain, which is a primary component of type I collagen. Quantification of mRNA for COL1A1 demonstrated dose-dependent increase in COL1A1 in response to TGF-β1 (
Expression of MKI67 mRNA was evaluated as a marker for proliferation. MKI67 expression was significantly upregulated with higher concentrations of TGF-β1, indicating increased cellular proliferation relative to the control condition (
TGF-β1 is a key regulator of ECM remodeling and dysregulation of TGF-β function is closely associated with fibrosis. The assay reveals multiple effects of TGF-β1 on fibroblasts including its ability to impact ECM remodeling through regulation of the fibrinolytic system and upregulated collagen synthesis.
In order to evaluate fibrosis in vitro, conventional approaches generally quantify specific contributors, such as activation of myofibroblasts or concentration of soluble collagen, using multi-step post-culture procedures. Here, the extent to which the unexpected, cell-driven ECM contraction and spheroid formation process could be used as a label-free approach was tested to assess fibroplasia.
An image processing pipeline was established in order to automate quantification and to allow consistent human bias-free analysis. Due to transitions in cell-ECM construct appearance over the course of the experiment, the built-in image segmentation software in the live-cell imager could not provide accurate segmentation. To overcome this image analysis challenge, Ilastic, a freely available image classification tool developed by the European Molecular Biology Laboratory was utilized.
Ilastik's pixel classification tool utilizes a random forest algorithm that can be interactively trained through iterations of user annotations on a small set of training images. To ensure consistent performance over the duration of the experiments, training images with a variety of features taken at different time points throughout the course of the assay were selected. When additional training annotations no longer improved background noise and edge feature fit, the trained pixel classification algorithm was saved for future use. Ilastik performs semantic segmentation, which returns probability maps that can be converted into masks by thresholding. In order to remove remaining background noise, opening and closing morphological filters were applied to the masks.
The projected area of the final contracted cell-ECM spheroid was the primary readout evaluated in the analysis, which demonstrated a significant dose-dependent increase with TGF-β1 stimulation (
The effects of serum concentration on matrix remodeling were evaluated by varying volumetric percentage of FBS in the cell culture media. FBS contains a complex mix of growth factors, hormones, cytokines, proteases, zymogens, co-factors, latent TGF-β1, and inhibitors that influence cellular activity. In the context of fibrin remodeling, an important component of FBS is plasminogen which can be activated by fibroblasts into plasmin for cell-mediated fibrinolysis. Assay media conditions ranging from serum-free to 8% FBS were evaluated. FBS-free conditions did not induce contraction within the duration of the experiments. In the absence of FBS, cell-ECM constructs also maintained their opaque appearance, indicating minimal fibrin degradation.
The projected-area of the final, contracted cell-ECM spheroids exhibited dose-dependent increases in response to FBS (
The initial fibroblast seeding density used in assays is also relevant to IPF. Fibroblasts from fibrotic lungs have particularly proliferative phenotypes, resulting in higher numbers of fibroblasts and myofibroblasts. Over a fibroblast seeding density range of between 1000 and 8000 cells/μl, a cell number-dependent increase was observed in the final projected cell-ECM spheroid area as expected (
Prior studies have observed altered fibrogenic response in aged and diseased pulmonary fibroblasts compared to fibroblasts from younger and normal donors. PAI-1 production and TGF-β1 signaling have both been implicated in this pathogenic alteration in behavior. Primary human pulmonary fibroblasts from 2 normal donors and 2 IPF diseased donors were tested. For one of the normal donors, the PAI-1 production and TGF-β1 signaling were also compared at a higher passage (p11).
Sections of the final contracted cell-ECM spheroids showed consistency in organization of collagen (
Here, the response of lung fibroblasts from one normal and one IPF donor was tested using three different drugs that target different pathways (
Nintedanib and pirfenidone are the two current FDA-approved therapeutics for IPF. In clinical use, however, these drugs do not halt or reverse fibrosis, and merely slow the progression of fibrotic scarring in the lungs. To address the need for alternative treatment strategies, several recent reviews have proposed components of the fibrinolytic system as potential targets for therapeutic intervention. Inhibition of PAI-1 is of particular interest, as its increased expression in IPF has been associated with worse clinical outcome. TM5275 is a small molecule inhibitor of PAI-1, which has been shown to minimize the extent of fibrotic remodeling in an animal model of pulmonary fibrosis and trigger apoptosis in TGF-β1 treated (but not untreated) fibroblasts and myofibroblasts. Consistent with these prior observations, TM5275 decreased the spheroid area for all the TGF-β1 treated conditions (P<0.05) but not in conditions that omitted TGF-β1. This difference in response may be related to PAI-1 upregulation with TGF-β1, where elevated levels could enable TM5275 to inhibit PAI-1 more effectively.
Fibrosis is the aggregate outcome of multiple dysregulated pathways. The ability of the contracting scar-in-a-drop assay to robustly detect effects of three different anti-fibrotic agents that work through disparate pathways, including the only 2 FDA-approved drugs, is encouraging for broader drug testing applications in the future.
Despite its importance in wound healing and fibrosis, fibrin gels have seen limited applications within fibroplasia assays. Instead, current prevailing assays focus on specific aspects of collagen production or cellular activation.
Macromolecular crowding agent-based approaches, so-called scar-in-a-jar assays, have recently been the focus of several publications. The scar-in-a-jar assays are possible to perform in high throughput format but have difficulty detecting effects of some drugs when the readout is based on ECM production. The readouts are always based on methods that requires additional procedures such as staining or immunoassays. The ATPS bioprinting method uses macromolecules, particularly dextran, for ATPS-based fibrin printing; this may appear to mimic the scar-in-a-jar assay. The polymers in this assay, however, are quickly washed out after the 30 min cross-linking reaction, although the presence of some residual dextran is not completely ruled out. The assay is also different in allowing contraction of the cell-produced ECM.
Wound closure involves contraction at the macroscopic scale and fibrosis involves mechanical activation of cytokines and mechanotransduction making assays that provide readouts of mechanical function of cells important. The collagen contraction assay is the classic assay of this type and has been used extensively over the years. These assays, however, are also not sensitive to effects of anti-fibrotic drugs such as pirfenidone and the well-to-well variability can be quite large. Furthermore, collagen is known to inhibit fibroplasia by limiting collagen production by cells. This contracting scar-in-a-drop assay is beneficial in starting from a collagen-free gel to allow uninhibited cellular collagen deposition followed by contraction once fibrin has sufficiently degraded. This integrated approach enables this assay to replicate, in vitro, more of the biological wound healing process compared to prior models, showing the cumulative impact of multiple steps of an abnormal scarring process.
In summary, this paper reports a unique microscale fibrosis assay that induces fibroplasia in fibrin gels, uninhibited by presence of pre-existing collagen, that in later stages undergo a dramatic ECM contraction. The convenience of direct visual readouts of fibroplasia coupled with high sensitivity to multiple anti-fibrotic drugs makes this assay promising for drug testing applications. Given the variety of diseases that involve fibroplasia and mechano-transduction such as fibrotic diseases, cancer, and cardiovascular diseases; this phenotypic assay provides broad utility beyond IPF. This paper focuses on evaluating the projected area of cell-ECM spheroids after contraction. Given the central role of cell and tissue mechanics in fibrosis, there may also be opportunities to analyze contraction dynamics to gain additional information and readouts from this assay in the future.
It is to be understood that the embodiments and claims disclosed herein are not limited in their application to the details of construction and arrangement of the components set forth in the description and illustrated in the drawings. Rather, the description and the drawings provide examples of the embodiments envisioned. The embodiments and claims disclosed herein are further capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purposes of description and should not be regarded as limiting the claims.
Accordingly, those skilled in the art will appreciate that the conception upon which the application and claims are based may be readily utilized as a basis for the design of other structures, methods, and systems for carrying out the several purposes of the embodiments and claims presented in this application. It is important, therefore, that the claims be regarded as including such equivalent constructions.
Furthermore, the purpose of the foregoing Abstract is to enable the United States Patent and Trademark Office and the public generally, and especially including the practitioners in the art who are not familiar with patent and legal terms or phraseology, to determine quickly from a cursory inspection the nature and essence of the technical disclosure of the application. The Abstract is neither intended to define the claims of the application, nor is it intended to be limiting to the scope of the claims in any way.
This application is a continuation application of U.S. patent application Ser. No. 17/349,297, filed on 16 Jun. 2021, which claims the benefit of U.S. Provisional Application Ser. No. 63/039,736, filed on 16 Jun. 2020, which is incorporated herein by reference in its entirety as if fully set forth below.
This invention was made with government support under grant/award number R21AG061687 awarded by the National Institutes of Health, grant/award number R01HL136141, awarded by the National Institutes of Health, and N66001-13-C-2027, awarded by the Department of the Navy. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63039736 | Jun 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17349297 | Jun 2021 | US |
| Child | 17514264 | US |
