WO2024150259A1

WO2024150259A1 - Biometric model of fibrosis

Info

Publication number: WO2024150259A1
Application number: PCT/IT2024/050004
Authority: WO
Inventors: Elena Veronesi; Tiziana PETRACHI; Massimo Dominici
Original assignee: Fondazione Democenter Sipe
Priority date: 2023-01-12
Filing date: 2024-01-12
Publication date: 2024-07-18

Abstract

Method for producing a biomimetic model of fibrosis which comprises the steps of: associating at least one cell type with at least one bio-ink containing collagen, obtaining a first compound; depositing said first compound in a culture medium obtaining a first deposited compound; keeping said first deposited compound in said culture medium for a period of time longer than 1 day, preferably comprised between 2 and 5 days; adding a profibrotic factor TGF-beta (TGFP) to said first deposited compound, obtaining a second added compound; keeping said second added compound in said culture medium for an additional period of time longer than 1 day, preferably comprised between 2 and 7 days; a biomimetic model of fibrosis which comprises at least one cell type associated with at least one bio-ink containing type I collagen and at least one profibrotic factor TGFβ is added to said bio-ink.

Description

BIOMETRIC MODEL OF FIBROSIS

Field of the invention

The invention concerns a method for producing a three-dimensional biomimetic model of fibrosis which can be generally used to test both the efficacy of anti- fibrotic drugs and/or therapies, and also the pathological mechanisms underlying the condition and the three-dimensional model of fibrosis.

Background of the invention

Fibrosis is a tissue repair process that sees the involvement of the immune system, the activation of which is followed by the abnormal deposition of the extracellular matrix (ECM), with severe alteration of the architecture of the affected tissue and loss of organ function (Sacchi M., et al, 2019 & Rockey D.C., et al, 2015).

Fibrosis can affect all biological organs and tissues, such as the lung, pancreas, kidney, liver, heart, skin, eye and stomach. Inflammatory system cells (macrophages and T cells), epithelial cells, fibroblasts and endothelial cells are the main cell types involved in fibrosis.

Fibrosis is characterized by the activation of several molecular pathways, among which the most important is the TGF beta (TGF0) pathway which plays an important role in both physiological and also pathological conditions (MengX.M., et al, 2014 .

Under physiological conditions, there is a balance between the ECM synthesis proteins of and the ECM degradation proteins. In pathological conditions, on the other hand, we see an alteration of this balance, with excessive secretion and deposition of ECM. The pathogenesis of fibrosis originates from an inflammatory process resulting in an increased release of inflammatory mediators (cytokines, chemokines and others), which in turn recruit inflammatory cells such as lymphocytes, polymorphonuclear leukocytes, eosinophils, basophils, mast cells and macrophages (Wynn T.A., et al, 2004). In addition, fibroblasts acquire a particular contractile activity following TGF0 stimulation, becoming myofibroblasts (Rockey

D.C., et al, 1993). The fibroblasts and myofibroblasts thus formed, therefore, are stimulated to synthesize ECM proteins, in particular interstitial collagen type I and III, fibronectin and basement membrane proteins, such as laminin (Hinz B., et al, 2007).

This transformation of fibroblasts into myofibroblasts is characterized by the activation of smooth muscle a-actin (aSMA) and is often used to identify pathological fibroblasts. Some authors have equated aSMA+ cells with contractile myofibroblasts, hypothesizing that these cells are the main source of ECM {Sun KH., et al, 2016).

In this context, the fibrotic process in the kidney is poorly studied due to the lack of adequate animal models Duffield J.S., et al, 2014). It is known, however, that renal fibrosis can be localized in the glomerulus or in the interstitium, and constitutes approximately 15-30% of cases of chronic renal failure. Often glomerular fibrosis can result in interstitial fibrosis. In this case, the normal function of the renal tubule is impaired, until the ECM also starts to involve the nephrons and their ability to support the capillaries. This condition leads to a progressive reduction in kidney volume and impaired perfusion. TGFp plays a decisive role also in renal fibrosis. In fact, it can be produced both by resident renal cells and also by infiltrating leukocytes. TGFP stimulates myofibroblast activation and the transition of the mesangial cells, the interstitial fibroblasts, and the tubular epithelial cells into fibrogenic cells capable of secreting ECM {Cha M.H., et al, 2010). In the case of the skin, fibrosis manifests itself with an abnormal accumulation of ECM components in the dermis, leading to an alteration of the architecture of the skin and the impairment of its functionality. Skin fibrosis can be a physiological process that occurs during wound repair (scar formation) or be linked to a pathological process. In the latter case, the normal balance between ECM synthesis and degradation is disrupted, leading to the formation of, for example, keloids or scleroderma. The triggering cause of this fibrotic mechanism is currently poorly defined; however, as with the other types of fibrosis, the immune system and the activation of the TGFp pathway are involved {Ludwicka A., et al, 1995). Once secreted, TGFp leads to the activation of neutrophils, macrophages and fibroblasts which in turn continue to release TGF {Wahl S.M., et al, 1987). TGFp is abundantly present in wound exudate throughout the entire tissue repair process, in the fibroblasts of hypertrophic scars resulting from bums, and in the keloids {Schimid P., et al, 1998). In addition, high levels of TGFP in perilesional areas can be detected in patients affected by scleroderma (Querferl C., et al, 1999}. All these data attest to the importance of the role of TGF[3 in the initial stages of a general fibrotic process.

Although two-dimensional models are the ones most commonly used for the study of the pathological mechanisms underlying fibrosis, this state of the art has some disadvantages.

One disadvantage is that two-dimensional models give extremely partial information, lacking important and necessary details, such as their complexity and three-dimensional organization for example, leading to incomplete information about the fibrotic development and process.

Another disadvantage is that the cell-cell and cell-ECM interaction is missing. These interactions represent one of the key mechanisms underlying the development of the fibrotic process and their absence means that two-dimensional models are poorly predictive and representative of the disease. Another disadvantage is that two-dimensional models fail to faithfully reproduce the behavior of the cell during the fibrotic process. This aspect also means that two-dimensional models are not very representative of the disease, since they cannot reproduce every aspect of cellular behavior during the pathogenic process. In view of the importance of the ECM in the fibrotic process, it is possible to use three-dimensional models capable of reproducing cell-cell and cell-matrix interactions and other cellular processes, such as migration and chemotaxis, in all three dimensions.

One of these is an in vitro model of skin fibrosis in which healthy dermal fibroblasts are cultured and stimulated to become fibrotic, hence “diseased”, through the addition of the growth factor TGF[3 with a specific concentration of lOng/ml in the culture medium. At the same time, a concentrated type I collagen hydrogel 3 mg/ml is created manually in vitro. The diseased fibroblasts are then detached from the culture medium and seeded onto the surface of the hydrogel. Confirmation of the success of the fibrosis model is verified with Western blot, with which the presence of the aSMA protein is detected (Alsharabasy A.M., et al, 2021}.

A second three-dimensional model is obtained through 3D printing technology. In this model, also called “scaffold”, the cells are added to a bio-ink consisting of type I collagen. The concentration of collagen chosen for the extrusion printing technology is fundamental for a successful creation of the three-dimensional model. An ink containing type I collagen with a concentration of less than 20 mg/ml, hence defined as “low concentration”, means that the result of the printing, that is, the model, cannot have a height greater than l-2mm. This limited height would not make the three-dimensional model usable. Even a too high concentration, above 50 mg/ml, is not considered the best condition for three- dimensional printing, since the viscosity and hardness of the construct, caused by high concentrations of collagen, can alter its mechanical properties and affect the ability of the cells to deposit the ECM, as well as limit the ability of the cells to migrate within the scaffold (Stepanovska J., et al, 2021).

Kang D., et al, 2022 describes a method similar to the above, in which type I collagen is deposited by means of inkjet printing, at low concentration. However, this state of the art also has some disadvantages.

One disadvantage is that the insertion into a hydrogel of fibroblasts that are already fibrotic, and therefore already diseased, reproduces only the last stage of the disease, preventing the study of the initial stages of fibrosis. In addition, such insertion may introduce an influence variable into the results obtained by the model. In fact, the ability of cells to deposit matrix, migrate and differentiate could be conditioned by the early presence of the TGF0 factor in the culture.

Another disadvantage of the state of the art is that using a low concentration of type I collagen, as defined above, does not allow to correctly and truthfully recreate the pathology of fibrosis in vitro, which is characterized by a high deposition of type I collagen, so high as to make the affected organ fibrotic and, therefore, unusable.

Another disadvantage is that the confirmation of the successful creation of the fibrosis model is only assessed through quantification of the aSMA protein. This protein is not only an indication of the presence of the fibrotic process, but it can also be produced by the fibroblasts that undergo physiological senescence, or be associated with other physiological or pathological cellular processes. Furthermore, the aSMA protein, studied individually, cannot be considered to be a marker of choice for the presence of the fibrotic process. The model does not allow to study any component of the ECM, the deposition of which is the main characteristic of the pathology, as well as the cause of the reduced or lack of functionality of the affected organ.

Another disadvantage is that the manual reproduction of hydrogel does not allow to create standardized models; these manually obtained models have characteristics that are, therefore, influenced by the skill of the operator who produces them. This variability can compromise the correct interpretation of the results.

Another disadvantage is that using an incorrect concentration of type I collagen in a three-dimensional model of fibrosis causes the lack of the adequate stimulation for the fibroblasts to deposit ECM. The use in the model of only the initial factor of the fibrosis (TGFP) causes a repeatable or so-called “loop” process to fail thanks to the use of the initial effector (TGFP) and final effector (highly concentrated collagen I) of the pathogenic process of fibrosis, in which the perennial co- stimulation of TGFp and highly concentrated type I collagen means that the three- dimensional model does not face attempts at spontaneous resolution, which would make it useless in the function of testing the effectiveness of anti-fibrotic drugs.

Summary of the invention

The purpose of the invention is to overcome the disadvantages disclosed above, by providing a method for the in vitro production of a three-dimensional biomimetic model of fibrosis obtained with 3D extrusion printing technology, which is able to reproduce fibrosis in the kidney and dermis as realistically as possible, so that it can be used to test the effectiveness of anti-fibrotic drugs and/or therapies. Another purpose is to develop a three-dimensional biomimetic model of fibrosis obtained with the method according to the invention.

According to one aspect of the invention, there is provided a method for producing a three-dimensional model of fibrosis in accordance with the characteristics of claim 1. According to another aspect of the invention, there is provided a three- dimensional model of fibrosis, in accordance with the characteristics of claim 12.

The invention allows to achieve the following advantages:

- Create a three-dimensional biomimetic model of fibrosis, offering the possibility of recreating in vitro the correct development of the disease as it occurs physiologically in humans and reproducing the effect of the presence of a high deposition of ECM;

- Obtain a repeatable or so-called loop process thanks to the use of the initial effector (TGF[3) and final effector (highly concentrated collagen I) of the pathogenetic process of fibrosis;

- Control the shape and sizes, mechanical properties and three-dimensional architecture of the model created, in such a way as to make it predictive of the pathological process and highly reproducible; - Perform tests in high-throughput, studying the effect of drugs on a three- dimensional model predictive of the disease;

- Achieve reduced times in the preparation of the model;

- Perform even a long-term culture, for a period of time that can be defined according to the type of fibrosis to be reproduced. - Reproduce different models of fibrosis, such as fibrosis of the skin, kidney, lung, pancreas, liver, heart, skin, eye and stomach.

Other characteristics and advantages of the invention will become more apparent from the detailed description of some preferred, but not exclusive, embodiments of a method for producing a biomimetic model of fibrosis and a three-dimensional biomimetic model, shown as a non-restrictive example in the attached drawings wherein:

- Fig. 1 and fig. 2 are scanning electron microscopy views of the fibrosis model obtained with fibroblasts isolated from the dermis following the administration of TGF[3 for 72 hours;

- Fig. 3 and fig. 4 are scanning electron microscopy views of the fibrosis model obtained with fibroblasts isolated from kidney following the administration of TGF[3 for 72 hours;

- Fig. 5 and fig. 6 are views of a qualitative analysis of the fibronectin protein in the three-dimensional biomimetic model of fibrosis isolated from skin;

- Fig. 7 is a graphic representation of a quantitative analysis of the fibronectin protein in the three-dimensional biomimetic model of fibrosis isolated from skin;

- Fig. 8 and fig. 9 are views of a qualitative analysis of the fibronectin protein in the three-dimensional biomimetic model of fibrosis obtained with fibroblasts of renal origin;

- Fig. 10 is a graphic representation of a quantitative analysis of the fibronectin protein in the three-dimensional biomimetic model of fibrosis obtained with fibroblasts of renal origin;

- Figs. 11 and 12 are views of a qualitative analysis of collagen deposition observed in bright field;

- Figs. 13 and 14 are views of a qualitative analysis of collagen deposition observed in polarized light; - Fig.15 is a graphic representation of a quantitative analysis of collagen deposition in the three-dimensional biomimetic model of fibrosis obtained with fibroblasts of dermal origin;

In detail, in fig. 1, arrows Fl and F2 indicate fibroblasts that have a physiological architecture and that interconnect with each other. Fig. 2 shows the ECM which appears homogeneous, with collagen fibrils aggregated together and compact. Following stimulation with TGFp, the collagen fibrils are no longer so clearly distinguishable, due to the deposition by the skin fibroblasts of a dense and compact matrix that covers the model, as indicated by the arrows F3, F4, F5 and F6. In fig. 3, the three-dimensional biomimetic model not stimulated with TGFp and defined as control (CNTRL) appears compact and regular.

In fig. 4, the three-dimensional biomimetic model following the administration of TGFp for 72 hours has an irregular architecture partly covered by ECM; the irregular surface shows the presence of depressions and protrusions, which indicate a remodeling of the collagen.

Fig. 6 shows that the stimulation of the model with TGFp for 72 hours significantly increases the expression of fibronectin in fibroblasts of dermal origin, compared to the negative control, that is, without TGFp, shown in fig. 5.

In fig. 7, the result is confirmed with the quantification of fibronectin in fibroblasts of dermal origin through image analysis, carried out with Zen Pro Software by Zeiss. From the results obtained, it can be seen that stimulation with TGFp induces an increase in positivity more than 4 times greater than the negative control (CNTRL) in a statistically significant manner (p-value < 0.01). Fig. 9 shows, through immunofluorescence staining, a significant increase in the expression of fibronectin compared to the control in fig. 8 (CNTRL) following stimulation of fibroblasts of renal origin with TGFp for 72 hours.

In fig. 10 the graph confirms the result. In fact, the positivity of the area affected by fibronectin expression is approximately 1500 times greater than the control, in a statistically significant manner (p-value <0.001).

In Fig. 12, following staining with Picrius Sirius Red, it is possible to observe denser areas affected by darker coloration compared to the negative control, not stimulated with TGFP, shown in fig. 11. In fig. 14 it is possible to observe that stimulation with TGFp for 72 hours induces greater collagen deposition compared to the model not stimulated with TGFp and shown in fig. 13.

This datum is confirmed by the graph in fig.15, which reports the quantification of the newly deposited collagen fibers, with a deposition of collagen by dermal fibroblasts stimulated with TGFp for 72 hours twenty times greater than that of non-stimulated fibroblasts.

Detailed description of some examples of preferred embodiments

The method for producing a biomimetic model of fibrosis according to the invention comprises the steps of associating at least one cell type with at least one bio-ink containing collagen, thus obtaining a first compound, depositing the first compound in a culture medium so as to obtain a first deposited compound, and keeping the first deposited compound in the culture medium for a period of time longer than 1 day, preferably comprised between 2 and 5 days.

In the preferred embodiment of the method, it was found that the deposited compound gave the best results when it was kept in the culture medium for three days.

Once the three-day period was completed, a profibrotic factor TGFp was added to it, obtaining a second added compound.

The second added compound was also kept in the culture medium for an additional period of time longer than 1 day, preferably comprised between 2 and 7 days, even more preferably for a period of time of three days.

According to the method, the collagen of the ink is used in a concentration comprised between 20 and 45 mg/ml, preferably 33.3 mg/ml, in an initial instant of deposition of the first compound.

With such a high concentration of collagen, a model with higher rigidity is obtained compared to what is achieved with the methods of the state of the art. Higher rigidity means that the model obtained is closer to real fibrotic tissue, which is much more rigid than a healthy tissue.

The model obtained therefore has the right mechanical properties to simulate the real fibrotic tissue. In particular, the bio-ink has an elastic modulus of approximately 100-400 kPa, which is much lower than the elastic modulus of a healthy tissue (approximately 900 kPa). The elastic modulus was measured by means of a contactless-type rheometer and further confirmed through the procedure described in Gutierrez E, Groisman A (2011 ) Measurements of Elastic Moduli of Silicone Gel Substrates with a Microfluidic Device. PLOS ONE 6(9): e25534. (https://doi.org/10.1371/journal.pone.0025534). It should also be said that the bio-ink has a high viscosity, which is suitable to make the model moldable, that is, obtainable by means of printing. The bio-ink can come in the form of slurry.

Another advantage of such a high concentration is that it does not require crosslinking of the collagen during preparation. Another advantage of the high concentration is that the model lasts longer than the 7 days usually required to carry out the controls. Still according to the method, the model can comprise, as cell type, fibroblasts or fibroblasts in association with stromal progenitors and/or endothelial cells and/or cells of the immune system and/or cancer cells and/or epithelial cells; the person of skill will understand that the model can also provide combinations of the cell types indicated above. The cell type used to obtain the model can comprise fibroblasts chosen from fibroblasts of dermal origin, fibroblasts of renal origin.

As an alternative to the above, the person of skill will understand that it is also possible to use fibroblasts from sources other than those indicated, provided that they are all of human origin. Stromal progenitors, endothelial cells, immune system cells and cancer cells can also be chosen originating from the dermis or kidney, or from a different source, as long as they are of human origin.

The fibroblasts used can be in a quantity greater than 1,000,000 cells, for example a quantity comprised between 1,000,000 and 25,000,000 cells for each ml of bio-ink, in particular in an initial instant in the step of depositing the first compound. In particular, the quantity of cells for each ml of bio-ink can be greater than 5,000,000, greater than 10,000,000, greater than 15,000,000, for example up to 20,000,000 or 22,000,000. For exam pie, it is possible to provide quantities of between 1,000,000 and 5,000,000 cells, between 5,000,000 and 10,000,000 cells, between 10,000,000 and 15,000,000 cells, between 15,000,000 and 20,000,000 cells or between 20,000,000 and 25,000,000 cells for each ml of bio-ink. One possible variant provides that there are between 1,000,000 and 3,000,000 cells for each ml of bio-ink. Other possible variants provide quantities of between 3,000,000 and 6,000,000 cells, between 6,000,000 and 9,000,000 cells, between 9,000,000 and 12,000,000 cells, between 12,000,000 and 15,000,000 cells, between 15,000,000 and 18,000,000 cells, between 18,000,000 and 21,000,000 cells, or between 21,000,000 and 25,000,000 cells. By the term “associate” we mean introducing the cell type inside the bio-ink, the latter being usable in a bio-plotter for the creation of three-dimensional models. According to the invention, the bio-ink, after the cells have been associated, can be used, as stated, in a bio-plotter, that is, to print with a technology chosen from extrusion printing, ink-jet printing, laser-assisted printing. Extrusion printing is the technology best suited to the high concentrations of collagen, as well as the viscosity of the bio-ink that is used. Good results are also achieved with ink-jet printing and laser-assisted printing.

Advantageously, the extrusion printing step occurs with pneumatic extrusion, in particular by means of pneumatic extruder means. Alternatively, it is possible to provide that the bio-ink is dispensed through manual dispensing, for example by means of a suitable dispensing tool that can be activated with a controlled dispensing force.

Usefully, extrusion printing technology is implemented using selected parameters, in particular a printing pressure comprised between 0.5 and 1 bar, a range in which 0.7 bar is generally the optimal parameter; a printing speed comprised between 5 and 10 mm/s, optimal speed: 7 mm/s; setting the temperature of the printing head within a range comprised between 2°C and 6°C, preferably 4°C. The temperature of the printing surface is preferably set to 39°C. Printing is advantageously carried out using a truncated cone needle with an internal diameter comprised between 150 gm and 400 gm, preferably of 250 pm.

Advantageously, at the end of the step of adding, in which the second added compound is obtained, the pro fibrotic factor TGFP is in a concentration comprised between 5 and 30 ng/ml, preferably lOng/ml.

The method according to the invention allows to obtain a three-dimensional biomimetic model of fibrosis, hereafter in brief “model”, which comprises a cell type that is associated with at least one bio-ink containing type I collagen and that is added with the profibrotic factor TGFp. Type I collagen is, usefully, in a concentration comprised between 20 and 45 mg/ml.

The model is made using a bio-plotter, depositing multiple overlapping layers of cells and bio-ink. The model therefore advantageously comprises a plurality of layers. Furthermore, it is preferable for the model to outline a three-dimensional shape. The overlapping layers are preferably four and overall outline a three- dimensional figure, advantageously full cylindrical, without however excluding other possible full three-dimensional shapes.

From the above description it is inferred that the method according to the invention becomes repeatable (so-called “loop”) thanks to the use of the initial effector TGFP and final effector, collagen I, of the pathogenic process of fibrosis.

The combination of perennial co-stimulation of the cells with TGF and the particularly high concentration of type I collagen prevents the model from reaching spontaneous resolution, that is, the fibrotic pathology from being cured.

A preferred embodiment of the method according to the invention is indicated in the following example 1.

Example 1

Three-dimensional biomimetic model of dermal fibrosis.

The three-dimensional biomimetic model, in short “model”, of fibrosis was obtained through bio-printing technology with printing by means of extrusion technique, which provides the layer-by-layer deposition of cells immersed in a bioink.

To create the model, it was provided to use healthy primary fibroblasts isolated from the dermis, and a bio-ink consisting of 33.3 mg/ml concentrated type I collagen (Lifeink, Advanced matrix).

After the fibroblasts were inserted into the bio-ink with a concentration of 2.5 million per ml in an initial instant in the step of depositing the first compound, the latter, that is, the compound added with the fibroblasts, was transferred into a 30 ml printing syringe subsequently housed in the printing head of the bio-plotter instrument (Envisiontec) and kept at the controlled temperature of 4°C.

For the printing of the model, a CAD project was developed to create a figure of a cylinder with a diameter of 8 mm.

The CAD thus created was suitably processed by means of slicing, applying a slicing height of 240 pm. For each printing process, 4 layers of material were deposited, each 240 pm thick, so the model had a total height of approximately 0.96 mm.

The printing was performed in liquid, that is, by depositing the material in a Petri dish containing a few ml of culture medium, preheated on the printing plate at 39°C for at least 30 minutes.

According to the example, the culture medium was composed of Dmem (Gibco, Cat. N°41965-039), 10% Fetal Bovine Serum (Coming, Cat. N°35-079CV), 1% Penicillin/Streptomycin (Gibco, Cat. N° 15140-122), 2% Glutamine (Gibco, Cat. N°.25030-024). After printing, the model was kept in liquid and on the hot printing plate for at least 30 minutes to allow the completion of the process of self-organization into fibrils and the temperature stabilization of the collagen.

After 30 minutes, the plate with the model was removed from the printing plate and transferred into an incubator to provide the appropriate cell culture conditions (37°C, 5% CO2).

The first added compound was subjected to the extrusion printing process using a printing pressure comprised between 0.65 and 0.8 bar, identifying 0.7 bar as a generally optimal parameter; printing speed comprised between 6 and 8 mm/s, optimal speed: 7 mm/s; using a conical needle with an internal diameter of 250 pm (25 GA Nordson, PN 7018391); setting the printing head temperature to 4°C and the printing surface temperature to 39°C.

The deposition creates the three-dimensional biomimetic model, consisting of four layers of dermal fibroblasts encapsulated in the type I collagen matrix, which has a cylindrical outline, typically full, with a diameter of 8 mm and height of 0.96 mm.

The model thus obtained was kept in culture for three days in the culture medium. In this step, the fibroblasts proliferate and have the possibility to interact with each other and with the collagen matrix.

After three days of culture, the model was stimulated by adding it with the profibrotic factor TGF0 (P eprotech, Cat.N° 100-21) at the concentration of 10 ng/ml at the end of the adding step, obtaining a second added compound. In order to be able to evaluate the efficacy of the method according to the invention through direct comparison, it is possible to create a comparison model by depriving it of the addition of TGFP (see fig.1 ).

Both the model of the invention as well as the comparison model were kept in the same culture medium for an additional three days, during which the model according to the invention modifies its micro-architecture in a peculiar way compared to the comparison model.

On the sixth day, both models were processed according to different procedures, as a function of a Scanning Microscope (SEM) analysis, or for fixation in formalin for evaluation in immunofluorescence, or for inclusion in paraffin for evaluation with Picrius Sirius Red, or for extraction of the RNA and analysis with real-time

PCR.

For the analysis of the sample with the Scanning Microscope (SEM), a fixation in glutaraldehyde (Sigma, 49629-250ml) at 4%, a subsequent ascending scale inclusion of alcohols and a dehydration were provided. The SEM exploits the interaction between an electron beam and the atoms that make up the sample under examination, and allows to generate images with very high magnifications, exceeding the resolution limit of optical microscopy.

In the present invention, the SEM allows, in particular, to evaluate how TGFP affects the micro-architecture of the model. The samples were mounted on a support with a conductive tape and are metallized with a gold/palladium coating in order to increase their conductivity.

The micrographs were obtained from the SEM with an acceleration of the electron beam of lOkV, under medium vacuum conditions, and using a secondary electron (SE) detector at 1 OOOx magnification.

For the immunofluorescence evaluation of the fibronectin expression, the following were provided: fixing the sample in formalin (Sigma, F1635-500ml) at 4% buffered for 20 minutes at room temperature, and the specific use of the fibronectin rabbit antibodies pAb ab2413 1 :200 (Abeam) and secondary Donkey anti-rabbit IgG-h +1 DyLight 594 conjugated (Bethyl).

By using specific antibodies, immunofluorescence is a technique capable of highlighting the localization and expression levels of the protein of interest.

According to the invention, the protein of interest is fibronectin, one of the proteins most expressed in the ECM during the evolution of the fibrotic process.

Immunofluorescence allows to evaluate how TGFp can modify the expression of the protein.

The samples stained with anti-fibronectin antibody were observed with Axiozoom V 16 (Zeiss) at 180X magnification and the red signal corresponds to positivity, that is, to the index of the binding of the antibody and therefore of the presence of fibronectin.

Signal quantification was achieved through the Image Analysis plugin of the software ZEN Pro (Zeiss).

For inclusion in paraffin, there was provided the fixation in gluteraldehyde (Sigma, 49629-250ML) at 2.5% of the samples and the subsequent inclusion of the latter in paraffin for the evaluation of the collagen fibers through staining with Picrius Sirius Red on 4pm micro-sections obtained with the microtome (Leica).

Staining with Picrius Sirius Red is one of the most widely used histology techniques to study the collagen network following observation with polarized light, which exploits the normal birefringence of collagen fibers that will appear red or green on a black field when observed in polarized light.

This staining allows to evaluate how stimulation with TGFp can impact the neodeposition of collagen fibers.

The samples were observed with Axiozoom V 16 in transmitted light with the addition of the polarizing filter and the green fibers were quantified with the software Zen Pro (Zeiss).

The quantification of the signal corresponding to the deposition of collagen fibers was carried out using the Image Analysis ZEN PRO plugin. The analysis protocol was designed in such a way as to isolate the green and red signal from the black background, and to evaluate how much section of the analyzed image was affected by fluorescence.

The result of the analysis expressed in pm² was reported as a percentage, considering the negative control as 100% of the expression.

For the extraction from the RNA models, the models were degraded in the solvent TRIzol® (Ambion, Life Technologies) and then extracted using a special kit that allows to extract the largest amount of RNA available from both models (Direct-zolTM RNA MicroPrep, Zymo Research). The kit consists of a series of micro-extracting columns equipped with a filter capable of separating and retaining only the RNA.

In more detail, each sample was immersed in 800 pl of TRIzol at room temperature for 5 minutes.

TRIzol degrades the protein structure of the models, dissolving the membrane and cell structures, extracting and preserving the nucleic acids.

The degradation process was speeded up by disintegrating the solution with the pipette several times during the 5 minutes of action of the TRIzol at room temperature, then the samples were stored for at least one night at -80°C.

The extraction and purification of the RNA was achieved using the Direct-zol kit and following the manufacturer’s instructions.

In particular, the extract in TRIzol derived from 2 biomimetic models of fibrosis was loaded into a micro-extraction column. A second column was used for the extraction of the RNA from two comparison models.

The RNA obtained was quantified with Nanodrop spectrophotometric analysis (Thermo Fisher) and was subsequently reverse- transcribed into cDNA using a reverse transcription kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher).

For each sample, 600 ng of RNA were reverse-transcribed, using a mix of reagents as provided by the protocol supplied by the manufacturer. This mix consists of a solution of oligo(dT) primers that appear with the messenger RNA poly(A) sequence, a reaction buffer, an RNase inhibitor (Ribolock), a mix of dNTPs and reverse transcriptase (Revertaid M-MuLV RT, 200 U/mL). The cDNA obtained from the different types of samples was subjected to Real Time PCR analysis, loading it in triplicate into a 48-well plate at the final concentration of 1 ng/pL, together with the specific primers (sense and antisense) for the gene to be studied and a suitable reaction mix, called Fast SYBR™ Green Master Mix (Thermo Fisher).

The mix Fast SYBR™ Green Master Mix (Thermo Fisher) contains the DNA polymerase enzyme, dNTPs, a fluorescent green double-stranded DNA intercalant (Sybr green), and other components that ensure cDNA amplification and the most reproducible and reliable analysis possible. As provided by state of the art, the expression of an endogenous gene whose levels are known to be stable was also evaluated for each gene analyzed, so as to have a method of normalization of the result, useful for comparing the expression of the gene of interest between different samples.

For the fibronectin gene, the beta- actin gene was used as endogenous. The Real Time PCR reaction was carried out using a StepOne™ Real-Time PCR System thermal cycler and the amplification results were analyzed using the software StepOne™.

A threshold cycle value of 1.085 was set for the fibronectin gene and for its endogenous. The expression comparison between the different samples was obtained by subtracting, for each sample, the value of the fluorescence signal (ARn) at the threshold cycle (Ct) of the gene of interest from that of the endogenous gene, and obtaining the value ACt; subtracting the value ACt of the sample of interest from that of a reference sample and obtaining the AACt; determining the fold change, that is, the relative variation in expression of the gene of interest between the two samples, calculated as relative quantification (Rq) = 2-AACt.

Example 2

Three-dimensional biomimetic model of kidney fibrosis.

To create the model, it was provided to use healthy primary fibroblasts isolated from the kidney, and a bio-ink consisting of 33.3 mg/ml concentrated type I collagen (Lifeink, Advanced matrix).

For the printing of the model, a CAD project was developed to create a figure of a cylinder with a diameter of 8 mm. The CAD thus created was suitably processed by means of slicing, applying a slicing height of 240 pm. For each printing process, 4 layers of material were deposited, each 240 pm thick, so the model had a total height of approximately 0.96 mm.

According to the example, the culture medium was the “Complete Fibroblast Medium” (CliniSciences).

After printing, the model was kept in liquid and on the hot printing plate for at least 30 minutes to allow the completion of the process of self-organization into fibrils and the temperature stabilization of the collagen.

After 30 minutes, the plate with the model was removed from the printing plate and transferred into an incubator to provide the appropriate cell culture conditions (37°C, 5% CO2). The first added compound was subjected to the extrusion printing process using a printing pressure comprised between 0.65 and 0.8 bar, identifying 0.7 bar as a generally optimal parameter; printing speed comprised between 6 and 8 mm/s, optimal speed: 7 mm/s; using a conical needle with an internal diameter of 250 pm (25 GA Nordson, PN 7018391); setting the printing head temperature to 4°C and the printing surface temperature to 39°C.

The deposition creates the three-dimensional biomimetic model consisting of four layers of kidney fibroblasts encapsulated in the type I collagen matrix, which has a cylindrical outline, typically full, with a diameter of 8 mm and height of 0.96 mm.

The model thus obtained was kept in culture for three days in the culture medium.

In this step, the fibroblasts proliferate and have the possibility to interact with each other and with the collagen matrix.

After three days of culture, the model was stimulated by adding it with the profibrotic factor TGFp (Peprotech, Cat.N⁰100-21) at the concentration of 10 ng/ml at the end of the adding step, obtaining a second added compound.

In order to be able to evaluate the efficacy of the method according to the invention through direct comparison, it is possible to create a comparison model by depriving it of the addition of TGFp (see fig.3).

On the sixth day, both models were processed according to different procedures, as a function of a Scanning Microscope (SEM) analysis, or for fixation in formalin for evaluation in immunofluorescence, or for extraction of the RNA and analysis with real-time PCR.

For the analysis of the sample with the Scanning Microscope (SEM), a fixation in glutaraldehyde (Sigma, 49629-250ml) at 4%, a subsequent ascending scale inclusion of alcohols and a dehydration were provided.

The SEM exploits the interaction between an electron beam and the atoms that make up the sample under examination and allows to generate images with very high magnifications, exceeding the resolution limit of optical microscopy.

In the present invention, the SEM allows, in particular, to evaluate how TGFp affects the micro-architecture of the model.

The samples are mounted on a support with a conductive tape and are metallized with a gold/palladium coating in order to increase their conductivity.

The micrographs were obtained from the SEM with an acceleration of the electron beam of 1 OkV, under medium vacuum conditions, and using a secondary electron (SE) detector at lOOOx magnification.

For the immunofluorescence evaluation of the fibronectin expression, the following were provided: fixing the sample in formalin (Sigma, F1635-500ml) at 4% buffered for 20 minutes at room temperature, and the specific use of the fibronectin rabbit antibodies pAb ab2413 1 :200 (Abeam) and secondary Donkey anti-rabbit IgG-h +1 DyLight 594 conjugated (Bethyl). By using specific antibodies, immunofluorescence is a technique capable of highlighting the localization and expression levels of the protein of interest.

The samples stained with anti-fibronectin antibody were observed with Axiozoom V 16 (Zeiss) at 180X magnification and the red signal corresponds to positivity, that is, to the index of the binding of the antibody and therefore of the presence of fibronectin. Signal quantification was achieved through the Image Analysis plugin of the software ZEN Pro (Zeiss).

In more detail, each sample was immersed in 800 pl of TRIzol at room temperature for 5 minutes. TRIzol degrades the protein structure of the models, dissolving the membrane and cell structures, extracting and preserving the nucleic acids.

The degradation process was speeded up by disintegrating the solution with the pipette several times during the 5 minutes of action of TRIzol at room temperature, then the samples were stored for at least one night at -80°C. The extraction and purification of the RNA was achieved using the Direct-zol kit and following the manufacturer’s instructions.

The RNA obtained was quantified with Nanodrop spectrophotometric analysis (Thermo Fisher) and was subsequently reverse-transcribed into cDNA using a reverse transcription kit (RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher).

For each sample, 600 ng of RNA were reverse-transcribed, using a mix of reagents as provided by the protocol supplied by the manufacturer.

This mix consists of a solution of oligo(dT) primers that appear with the messenger RNA poly(A) sequence, a reaction buffer, an RNase inhibitor (Ribolock), a mix of dNTPs and reverse transcriptase (Revertaid M-MuLV RT, 200 U/mL).

The cDNA obtained from the different types of samples was subjected to Real Time PCR analysis, loading it in triplicate into a 48-well plate at the final concentration of 1 ng/pL, together with the specific primers (sense and antisense) for the gene to be studied and a suitable reaction mix, called Fast SYBR™ Green Master Mix (Thermo Fisher).

The mix Fast SYBR™ Green Master Mix (Thermo Fisher) contains the DNA polymerase enzyme, dNTPs, a fluorescent green double-stranded DNA intercalant (Sybr green), and other components that ensure cDNA amplification and the most reproducible and reliable analysis possible.

As provided by state of the art, the expression of an endogenous gene whose levels are known to be stable was also evaluated for each gene analyzed, so as to have a method of normalization of the result, useful for comparing the expression of the gene of interest between different samples. For the fibronectin gene, the beta-actin gene was used as endogenous.

The Real Time PCR reaction was carried out using a StepOne™ Real-Time PCR System thermal cycler and the amplification results were analyzed using the software StepOne™.

A threshold cycle value of 1.085 was set for the fibronectin gene and for its endogenous.

The expression comparison between the different samples was obtained by subtracting, for each sample, the value of the fluorescence signal (ARn) at the threshold cycle (Ct) of the gene of interest from that of the endogenous gene, and obtaining the value ACt; subtracting the value ACt of the sample of interest from that of a reference sample and obtaining the AACt; determining the fold change, that is, the relative variation in expression of the gene of interest between the two samples, calculated as relative quantification (Rq) = 2-AACt. In practice, it has been verified that the invention achieves the intended purposes.

The invention as conceived is susceptible to modifications and variants, all of which are within the scope of the inventive concept.

For example, it can be provided that instead of printing with a bio-plotter, the bio-ink can be delivered manually by means of a suitable delivery tool, for example a syringe, which can be activated through a controlled delivery force.

Furthermore, all the details can be replaced with other technically equivalent elements.

In practical embodiments, any other materials, as well as shapes and sizes, can be used depending on requirements, without departing from the scope of protection of the following claims.

Claims

1. Method for producing a biomimetic model of fibrosis, characterized in that it comprises the steps of:

- associating at least one cell type with at least one bio-ink containing collagen at a concentration comprised between 20 and 45 mg/ml, obtaining a first compound;

- depositing, by means of extrusion printing, said first compound in a culture medium obtaining a first deposited compound;

- keeping said first deposited compound in said culture medium for a period of time longer than 1 day, preferably comprised between 2 and 5 days; - adding to said first deposited compound a profibrotic factor TGFP stimulating said cell type, obtaining a second added compound;

- keeping said second added compound in said culture medium for an additional period of time longer than 1 day, preferably comprised between 2 and 7 days.

2. Method as in claim 1, wherein said at least one cell type is fibroblasts or fibroblasts in association with stromal progenitors and/or endothelial cells and/or immune system cells and/or tumor cells and/or epithelial cells and/or combinations thereof.

3. Method as in claim 1, wherein said at least one bio-ink containing collagen is in a concentration equal to approximately 33.3 mg/ml in an initial instant in the step of depositing said first compound.

4. Method as in claim 2, wherein said fibroblasts, stromal progenitors, endothelial cells, immune system cells, tumor cells and/or combinations thereof are of human origin.

5. Method as in claim 4, wherein said at least one cell type comprises fibroblasts selected from fibroblasts of dermal origin, fibroblasts of renal origin.

6. Method as in claim 2, wherein said fibroblasts are in a quantity greater than 1,000,000 cells for each ml of said bio-ink in an initial instant in the step of depositing said first compound.

7. Method as in claim 6, wherein said fibroblasts are in a quantity comprised between 1,000,000 and 25,000,000 cells for each ml of said bio-ink in an initial instant in the step of depositing said first compound.

8. Method as in claim 7, wherein said fibroblasts are in a quantity comprised between 1,000,000 and 3,000,000 cells for each ml of said bio-ink in an initial instant in the step of depositing said first compound.

9. Method as in claim 1, wherein said extrusion printing provides pneumatic extrusion printing by means of pneumatic extruder means.

10. Method as in claim 9, wherein said extrusion printing comprises a printing pressure comprised between 0.5 and 1 bar; a printing speed comprised between 5 and 10 mm/s; a temperature of the printing head between 2°C and 6°C.

11. Method as in claim 10, wherein said extrusion printing comprises a printing pressure equal to 7 bar; a printing speed equal to 7 mm/s; a temperature of the printing head equal to 4°C.

12. Method as in any claim 9, 10 or 11, wherein said extrusion printing also comprises a truncated cone needle with an internal diameter comprised between 150 pm and 400 pm.

13. Method as in claim 12, wherein said extrusion printing comprises a truncated cone needle with an internal diameter equal to approximately 250 pm.

14. Method as in any claim hereinbefore, wherein said profibrotic factor TGFp is in a concentration comprised between 5 and 30 ng/ml at the end of the step of adding, in which said second added compound is obtained.

15. Method as in claim 14, wherein said profibrotic factor TGFp is in a concentration equal to lOng/ml at the end of the step of adding, in which said second added compound is obtained.

16. Biomimetic model of fibrosis obtainable with a method as in any claim hereinbefore, characterized in that it comprises at least one cell type associated with at least one bio-ink containing type I collagen and in that said bio-ink comprises at least one profibrotic factor TGFp.

17. Model as in claim 16, also comprising a plurality of layers.

18. Model as in claim 16 or 17, wherein it outlines a three-dimensional shape.

19. Model as in claim 16, 17 or 18, wherein said at least one bio-ink containing collagen is in a concentration comprised between 20 and 45 mg/ml, preferably equal to approximately 33.3 mg/ml.

20. Model as in any claim from 16 to 19, wherein said at least one bio-ink has an elastic modulus of approximately 100-400 kPa.