Method of regulating a fibrotic state and drug screening method

Abstract

The present invention relates to a method of regulating a fibrotic state in cells in cell culture, the cells preferably being fibroblasts, fibrocytes, fibroblast-like cells and / or progenitors of fibroblasts, the method including inducing a fibrotic state in the cells by performing the cell culture under the following conditions: culturing the cells in a scaffold comprising the cells at a density of from 3000 to 6000 cells / mm3, preferably 3400 cells / mm3 cells, for 4 hours to 14 days, wherein the scaffold comprising the fibroblasts is cultured in cell culture medium; and / or culturing the fibroblasts at hypoxic conditions, preferably at an oxygen concentration of less than 21%, more preferably at an oxygen concentration of 20% to 1%, more preferably at an oxygen concentration of 19 % to 1%; most preferably at an oxygen concentration of 18 % to 1%; and / or culturing the fibroblasts at starving conditions. The present invention also relates to a drug-screening method.

Description

[0001] Method of Regulating a Fibrotic State and Drug Screening Method

[0002] The present invention relates to a method of regulating a fibrotic state in fibroblasts in cell culture, including inducing a fibrotic state in the fibroblasts. The invention also relates to a method including regenerating the induced fibrotic state. The invention also relates to a drugscreening method. The present invention also relates to bioreactors for organ-on-a-chip applications. The method(s) may relate to the bioreactors.

[0003] Fibroproliferative disorders constitute a group of medical conditions characterized by excessive tissue fibrosis, which is the overproduction and accumulation of fibrous connective tissue. These disorders often result from an abnormal wound-healing process where fibroblast proliferation and overactivation, as well as extracellular matrix deposition, exceed normal levels found in physiological tissue regeneration and wound healing. Key cellular players in these disorders include fibroblasts and myofibroblasts, which are normally responsible for the remodeling of the wound matrix and the synthesis of collagen, as well as other extracellular matrix components in the remodeling phase of wound healing. The pathophysiology of fibroproliferative disorders involves a complex interplay of inflammatory cytokines, growth factors, and mechanical stress signals. Transforming Growth Factor-beta (TGF-|3) is a pivotal cytokine that drives fibroblast activation and differentiation into myofibroblasts, which is a key mechanism in both wound healing as well as fibrotic tissue remodeling. This myofibrotic differentiation is marked by the expression of alpha-smooth muscle actin (a-SMA), enhancing the contractile ability of these cells, which contributes to pathological tissue stiffness.

[0004] Fibroproliferative disorders can be categorized as either local or systemic and include pathologies such as idiopathic pulmonary fibrosis (IPF), systemic sclerosis (SSc), liver cirrhosis, or fibrosis of musculoskeletal tissues such as synovial, tendinous, or fat pad tissues. In IPF, chronic and progressive fibrosis leads to the destruction of lung architecture and impaired gas exchange. Systemic sclerosis is characterized by skin thickening and can involve internal organs, leading to functional impairment. In local fibrosis of the patellar fat pad, adipose tissue is replaced by fibrous tissue, which is similar to systemic fibroproliferative disorders, where normal tissue is replaced by excessive fibrotic tissue. Tendon fibrosis can occur as a result of chronic inflammation, injury, or after surgical interventions. Synovial fibrosis involves the abnormal accumulation of fibrous tissue in the synovium. Fibroblast-like synoviocytes in the synovial membrane become overactive, producing excessive collagen and other extracellular matrix components. Even though these pathologies are localized and tissue-specific, similar underlying pathomechanisms also govern more systemic proliferative disorders such as idiopathic pulmonary fibrosis or systemic sclerosis.

[0005] The treatment approach is often multidisciplinary, involving the use of anti-fibrotic agents, immunosuppressants, and supportive care. Antifibrotic agents, like pirfenidone and nintedanib, are used in IPF to slow disease progression. In SSc, immunosuppressive therapy is commonly employed to reduce inflammation and fibrosis. Physical therapy and pulmonary rehabilitation are essential supportive care measures, especially in lung fibrosis. Treatment of local fibrosis (i.e., Hoffa fat pad) can involve physical therapy, anti-inflammatory medications, and in some cases, surgical intervention to remove the fibrotic tissue or release the joint pressure.

[0006] Research in fibroproliferative disorders is focused on understanding the molecular mechanisms driving fibrosis and identifying novel therapeutic targets. The role of epigenetics, microRNAs, and the extracellular matrix are areas of active investigation. Despite advances, these disorders remain challenging to treat, necessitating ongoing research and development of new therapeutic strategies.

[0007] In view of the above, the disclosed invention provides a method for generating a pro-fibrotic environment in the absence of pro-fibrotic cytokines (i.e., TGF- or PDGF).

[0008] The invention further discloses a, e.g. musculoskeletal, organ-on-a-chip device configured to provide biomechanical stimuli (e.g., fluid flow and / or shear and / or compressive loading) that modulate pro-fibrotic mechanisms (e.g., myofibrotic differentiation of stromal fibroblasts).

[0009] Furthermore, the invention discloses a drug screening method, such as a method involving the use of an organ-on-a-chip device for determining an attenuating effect of anti-fibrotic agents (e.g., human platelet lysate) on modelling of musculoskeletal adipose tissue fibrosis.

[0010] The disclosed invention may combine tissue-relevant fluid-flow, mechanical stimulation, and heterotypic 3D co-culture in a biomechanical system to model cellular mechanisms of tissue fibrosis. A system featuring a cell culture compartment (also termed cell chamber) and a pressure (in embodiments a pneumatic) actuation mechanism was developed to generate a distinct physio-chemical niche that promotes mechanisms of tissue fibrosis. The system is configured for the investigation and manipulation and / or stimulation of cells, e.g. pathological stromal cells (e.g., pre-fibroblasts, fibroblasts or fibrocytes) and eventually myofibroblast differentiation mechanisms found in tissue fibrosis.

[0011] Particularly, the present invention relates in one aspect to a method of regulating a fibrotic state in cells in cell culture. The cells may be fibroblasts, fibroblast-like cells and / or fibroblast progenitors. The method may include inducing a fibrotic state in the cells by performing the cell culture under the following conditions: o culturing the cells in a scaffold comprising the cells at a density of 3,000 to 6,000 cells / mm3, preferably 3400 cells / mm3cells, for 4 hours to 14 days, wherein the scaffold comprising the fibroblasts is cultured in cell culture medium; and / or o culturing the fibroblasts at hypoxic conditions, preferably at an oxygen concentration of less than 21%, more preferably at an oxygen concentration of 20% to 1%, more preferably at an oxygen concentration of 19% to 1%, most preferably at an oxygen concentration of 18 % to 1%; and / or o culturing the fibroblasts at starving conditions from 0.1 to 3 g / l D-glucose, most preferably at a concentration of 2 g / L.

[0012] The total volume of the scaffold may be 40 pL to 80 pL, preferably 50 pL to 70 pL, more preferably 55 pLto 65 pL, for example 60 pL. This is, however, not limiting and the total volume of the scaffold may be adapted as appropriate. "Starving conditions", as used herein, refer to a state of the cell's microenvironment, where after initial addition of a complete culture medium at day 1 and medium replacement at day 7 at a concentration of 4.5 g / L glucose, the cell metabolism is adjusting the nutrient (i.e., glucose) to natural concentrations without the dilution or extrinsic depletion of glucose.

[0013] The fibroblasts may be cultured at a pH-value of physiological or slightly acidic range from 6.0 to 7.4.

[0014] The "fibrotic state in cells" as used in the present disclosure is understood as follows: Fibrosis in the IFP is marked by an overaccumulation of ECM, mainly collagen types I and III, causing the IFP to thicken and stiffen. This not only diminishes its cushioning ability but also may lead, e.g., to knee discomfort and impairment. Myofibroblasts, identified by a-smooth muscle actin (a-SMA, also known as ACTA2), are vital in this mechanism, contributing to the stiffness through contractile forces and ECM protein secretion. Therefore, ACTA2 is used as a reporter of the fibrotic state.

[0015] The cell culture medium may be animal-product or animal-product-free cell culture medium. The cell culture medium may comprise calf serum (fetal or non-fetal), serum substitutes or defined culture medium supplements.

[0016] Performing the cell culture may be carried out under the following conditions: o in the absence of externally applied mechanical and / or hydraulic deformation and / or compression of the scaffold comprising the cells; and / or o in the absence of externally applied medium flow through the scaffold comprising the cells.

[0017] Externally applied, as used herein, may mean that the referenced factor, e.g., deformation or flow, does not stem from the cells themselves. For example, a naturally occurring compaction of the scaffold by the cells themselves would not be considered an external application of deformation.

[0018] Additionally or alternatively, the scaffold comprising the cells may be subject to liquid flow that allows fibrotic state formation either as a periodical stimulus or as a stimulus pattern comprising several intervals with intermittent resting periods per day, for example up to 50 pl / s through an area of 3.6 mm2, up to 0.42 dyn / cm2, at a frequency from 0.1 to 2 Hz, preferably at an actuation frequency of 0.5 Hz to 2 Hz, more preferably 1 Hz.

[0019] A deformation and / or compression of the scaffold may be applied to regenerate and / or reverse the fibrotic state of the fibroblasts: o such that it results in reducing a height of the scaffold up to 50%, preferably 10% to 30%, more preferably 15%; and / or o at a frequency between 0.1 and 2 Hz, preferably at a frequency of 0.5 Hz or more, more preferably 1 Hz; and / or o for a duration of 15 minutes up to 4 hours per pressurization interval, and up to 3 pressurization intervals per day. Additionally or alternatively, the scaffold comprising the fibroblasts may be subject to liquid flow to regenerate and / or reverse the fibrotic state either as a periodical stimulus or as a stimulus pattern comprising several intervals with intermittent resting periods per day, for example up to 500 pl / s, e.g. more than 50 pl / s and up to 500 pL / s, through an area of 3.6 mm2, up to 4.2 dyn / cm2(0.42 Pa), at a frequency between 0.1 and 2 Hz, preferably at an actuation frequency of 0.5 Hz to 2 Hz, more preferably 1 Hz. For example, 0.05 Pa to 0.42 Pa, 0.1 Pa to 0.42 Pa, 0.2 Pa to 0.42 Pa, or 0.3 Pa to 4.2Pa may be used.

[0020] The scaffold comprising the cells may be subject to liquid exchange (preferably only, or essentially only, for cell culture medium exchange) and / or supplementation with fresh cultivation medium and / or nutrients with time intervals of 1 hour to 14 days, preferably 6 hours to 10 days, more preferably 48 hours to 8 days, most preferably 7 days to allow fibrotic state formation. This may result in the fibrotic state.

[0021] Method steps and / or conditions that are mutually exclusive may be performed after one another. For example, after a fibrotic state inducing phase of cell culture with a fluid flow that allows fibrotic state formation (e.g. up to 50 pL / s through 3.6 mm2as specified above) a regeneration phase may follow with cell culture at a fluid flow that regenerates the fibrotic state (e.g. at 500pL / s through 3.6 mm2as described above).

[0022] The cell culture medium may comprise less than 100 pg / ml of pro-inflammatory and / or pro- fibrotic additives, most preferably no pro-inflammatory additives that are known as contributing to inducing a fibrotic state. Optionally, the cell culture medium comprises less than 100 pg / ml TGF- 1, PDGF, IL-6, IL1|3 or TNF-a.

[0023] The cells may comprise one or a combination of the following cell types: o Fibroblasts o Fibrocytes o Fibroblast progenitors o Pericytes o Myogenic progenitor cells o Myofibroblasts o Adipose-tissue-derived fibroblast-like cells o Adipose-tissue-derived stem cells o Adipose-tissue-derived myeloid or lymphoid cells o Bone-marrow-derived stem cells o Synovium-tissue-derived fibroblast like cells o Synovium-tissue-derived myeloid or lymphoid cells o Synovium-tissue-derived endothelial cells o Synovium-tissue-derived stem cells o Dermal-tissue-derived myeloid or lymphoid cells o Dermal-tissue-derived endothelial cells o Dermal-tissue-derived stem cells o Fibroblast-like and / or fibroblast cells derived from meniscus, discus, tendon or ligament tissues.

[0024] The method(s) may include providing the scaffold comprising the cells in a cell chamber of a chip, the chip being configured to apply optional deformation and / or compression and / or optional fluid shear to the scaffold comprising the cells in the cell chamber to regenerate and / or reverse the fibrotic state of the cells.

[0025] The chip may comprise a pressure chamber adjacent to the cell chamber and separated from the cell chamber by a flexible membrane, the pressure chamber being configured to be pressurized by adding fluid, i.e. liquid or gas, to the pressure chamber, e.g. silicone oil, water, or pressurized filtered air. Pressurizing the pressure chamber may result in application of deformation and / or compression to the scaffold comprising the cells via the membrane. The chip and the pressure chamber may be configured to be coupled to any suitable supply structure, e.g. tubing, for pressurization.

[0026] The chip may be a chip as described with reference to the figures.

[0027] The chip may provide a gas exchange of up to 7 xio-8moles of oxygen per hour in the cell chamber, most preferably 4.3 xio-8moles of oxygen per hour.

[0028] By limiting the gas exchange of the cells with the atmosphere, the chip may enable what is called auto-hypoxia herein: When the cells are in the chip, they are subject to a limited gas exchange with the atmosphere. Thus, the natural respiration of the cells may cause a reduction of the oxygen concentration in the volume around the cells. Concomitantly, the concentration of CO2 may rise. This may in turn give rise to a reduced pH-value. However, a buffer may be used to regulate the pH value as appropriate.

[0029] The scaffold may be a hydrogel, e.g. a hydrogel comprising collagen (herein also: collagen hydrogel), e.g. a hydrogel comprising collagen type I (herein also: collagen I hydrogel), and / or a synthetic analogue with similar biophysical functions and / or chemical nature as a collagen hydrogel or a collagen type I hydrogel.

[0030] The collagen hydrogel may comprise collagen types native to connective tissue, e.g. collagen I. The collagen hydrogel may, additionally or alternatively, comprise collagen III, collagen VI, vimentin and / or fibronectin. Preferably, the collagen hydrogel is a primary extract from tissues or recombinantly produced collagen type I hydrogel of human, plant or animal-origin at a concentration from 0.1 mg / mL to 100 mg / mL that can be polymerized by physical means, e.g. pH shift, temperature change and / or photo-crosslinking.

[0031] The present invention and disclosure also relate to a drug-screening method, comprising: a) inducing a fibrotic state in a first plurality of cells in cell culture and / or ex-vivo by applying the method as described hereinabove and / or hereinbelow; b) subjecting the first plurality of cells resulting from step a) to a drug of interest, e.g. by adding the drug of interest to the cell culture medium; c) optionally determining one or more effects of the drug on the first plurality of cells.

[0032] Determining the one or more effects of the drug on the first plurality of cells (i.e. step c)) may include comparing at least one property as determined for the first plurality of cells before step b) with said at least one property as determined for the first plurality of cells after step b) in order to determine the one or more effects of the drug. Alternatively or additionally, step c) may include comparing at least one property as determined for the first plurality of cells after step b) with said at least one property as determined for a control plurality of cells that is not subjected to step b) and or step a) in order to determine the one or more effects of the drug.

[0033] "Cells resulting from step [XY] are used for [YZ]" as used herein may have the meaning that the cells are treated as specified in step [XY] and then directly used for [YZ], Additionally or alternatively, it may include that the cells are treated as specified in step [XY], treated according to a further protocol, and only then used for [YZ],

[0034] The drug screening method may further comprise: d) inducing a fibrotic state in a second plurality of cells in cell culture by applying the method described hereinabove and / or hereinbelow with a lower amount and / or concentration of the drug than in step b), preferably in the absence of the drug; e) regenerating the second plurality of cells as described hereinabove and / or hereinbelow; f) optionally comparing at least one property as determined for the first plurality of cells resulting from step b) with said at least one property as determined for the regenerated second plurality of cells resulting from step e) in order to determine the one or more effects of the drug.

[0035] The at least one property of step f) may be the same or partially the same properties as in step c). Alternatively, the at least one property of step f) may be different from the properties in step c). The at least one property may be determined after the beginning of step b), i.e. while the cells are subjected to the drug and / or after subjecting the cells to the drug has been finished.

[0036] The drug screening method may further comprise: g) subjecting the regenerated second plurality of cells resulting from step e) to the drug of interest, e.g. by adding the drug of interest to the cell culture medium; h) comparing at least one property as determined for the first plurality of cells resulting from step b) with said at least one property as determined for the regenerated second plurality of cells resulting from step g) in order to determine the one or more effects of the drug.

[0037] The at least one property of step h) may be the same or partially the same properties as in step f). Alternatively, the at least one property of step h) may be different from the properties in step f). The at least one property of step h) may be determined for the first plurality of cells after the beginning of steps b) and g), i.e. while the first plurality of cells are subjected to the drug and / or after subjecting the first plurality of cells to the drug has been finished. The at least one property of step h) may be determined for the second plurality of cells after the beginning of steps b) and g), i.e. while the second plurality of cells are subjected to the drug and / or after subjecting the second plurality of cells to the drug has been finished.

[0038] The at least one property may be determined based on the whole, or essentially the whole, respective plurality of cells, based on a sub-plurality of the respective plurality of cells, and / or on single cell level. The at least one property may include one or a combination of one or more mRNA expression levels, RNA methylation levels, protein secretion levels, intracellular protein expression levels, and extracellular protein levels.

[0039] In the drug-screening method o step b) may be performed while maintaining the application of the method of inducing a fibrotic state to the first plurality of cells; or o the application of the method of inducing a fibrotic state to the first plurality of cells may be stopped before, upon starting, or during step b).

[0040] The invention is further described with reference to the figures, which provide a more thorough description of the invention with reference to non-limiting examples. The examples shall not limit the scope of the invention, which is solely defined by the appended claims and their equivalents. The figures show:

[0041] Fig. 1 A) A two-dimensional schematic view of a fibrosis-biochip in layer-by-layer top views and B) isometric, side and top views of a fibrosis-biochip comprising two individual tissue units in an object slide format as design variant.

[0042] Fig. 2 A) The time-resolved effect of the fibrosis biochip in the absence of any pro- inflammatory medium additives on pro-fibrotic qPCR derived gene expression of ACTA2 gene for a variety of stromal fibroblast-like cells including primary human fibroblast-like synoviocytes (FLS), stromal cells from the stromal vascular fraction of the infrapatellar fat pad (IPFP) of the knee joint (HFib), human dermal fibroblasts isolated from skin tissue (HDF), as well as equine adipose-tissue-derived stromal cells (ASC) isolated from subcutaneous adipose tissue biopsies.

[0043] B) Comparison of ACTA2 expression in 2D culture, micromass culture, and chip culture of HDF cells after 7 days of cultivation.

[0044] C) Reversal of a pro-fibrotic / -myofibrogenic environment of the fibrosis biochip when treated with 15 pM SB 431542 inhibitor targeting the activin receptorlike kinase (ALK) receptor (TGFb receptor family) after exposure for 36 h starting at day 8 post-seeding.

[0045] Fig. 3 A) Partial pressure time-curves of the hypoxic cell environment within the biochip based on natural cell respiration in comparison to acellular PureCol hydrogels using integrated opto-chemical oxygen sensor spots. B) Long-term monitoring of the generation of a hypoxic environment of organoids over a culture duration of 8 days in the absence of fluid perfusion (-FSS) compared to acellular hydrogels (n=4-5). C) Comparison of oxygen levels of non-perfused biochip cultures compared to conventional 3D micromass and 2D monolayer cultures (n=4).

[0046] Fig. 4 A) Impact of pump-less fluid perfusion (1 Hz at maximal tilting angle of a tilting plate) on an establishment of a hypoxic pro-fibrotic environment in comparison to acellular and non-perfused IPFP fibrosis biochips comprising stromal cells isolated from a vascular stromal fraction of the IPFP of osteoarthritic (OA) patient biopsies (n=4). B) Time-resolved relative gene expression of pro- myofibrotic ACTA2 mRNA marker for adipose tissue-cell containing fibrotic biochip organoids additionally stimulated with 5 ng / mL TGF- 1 cytokine in comparison to non-perfused treated biochip samples over 96 h period starting at day 11 post-seeding. Data was normalized vs. SDHA as the housekeeping gene.

[0047] Fig. 5 A) Representative light microscopy images of a pneumatic actuator structure in xz plane (lateral view) for actuation pressures ranging from 0.5 to 3 bars applied pressure. Dark meniscus shows the deflected membrane, dashed line indicates the bottom surface of the membrane at 0.0 bar applied pressure (i.e. nondeflected state). "Ceiling" indicates the membrane in the non-deflected state. Scale bars indicate 500 pm. B) Hydrogel height (top) as depending on the pressure applied to the pressure chamber of a chip as shown in Fig. 1; pressure applied to the pressure chamber of a chip as shown in Fig. 1 with corresponding actuated distance (bottom) of the center of the membrane adjacent the pressure chamber as measured with fluorescence imaging. C) Microscopy images of xy-plane (lateral) cell displacement during pneumatic actuation along the z-axis (i.e. top-bottom axis) of the chip at an actuation pressure of 0 mbar (left image) and 50 mbar (right image) for the chip of Fig. 1; The two microscopy images show top views of fluorescently-labeled cells in a hydrogel scaffold in a cell chamber, the scale bar representing 150 pm; The left image shows the nondeflected state, the right image shows the deflected state at 50 mbar actuation pressure with the white lines on each cell indicating the xy-plane cell trajectory; Bottom: Characterization of deflection of n=25 fluorescently-labeled fibroblast cells for an on-chip actuation pressure from 25 mbar to 250 mbar as measured in the pressure line to the chip. The graph shows deflection data as extracted from microscopy images. D) Impact of a 1 Hz actuation routine at 200 mbar pressure for up to 4 hours on day 11 post-seeding to demonstrate the modulatory and regenerative effect of biomechanical actuation on the myofibrotic expression marker ACTA2 (actuation is indicated with "+L" for 'loading'). A non-loaded TGF- control is shown for comparison. Data was normalized vs. GAPDH as the housekeeping gene.

[0048] Fig. 6 Gene expression analysis of the myofibrotic expression response of the marker ACTA2 for 5 ng / mL TGF-|31 cytokine or galectin 3 (GAL3) -stimulated fibrotic IPFP organoids in comparison to anti-myofibrotic treatments including commercially available pooled human platelet lysate ELAREM™ (hPL) and home-brew single donor platelet lysate at a 30% v / v concentration in complete culture medium after 36 h of treatment. Data is expressed as fold-change for mean value of n=2 individual replicates relative to the TGF-pi-treated sample group.

[0049] Figure 1A) shows a two-dimensional schematic view of a fibrosis-biochip (herein also referred to as a "chip") 100 in layer-by-layer top view. The layers 1-12 correspond to cross sections through the chip 100. The chip 100 may be constructed from a number of layers 1- 12, e.g. of PDMS (short for polydimethylsiloxane) or other silicone copolymers such as methylhydrosiloxane-dimethylsiloxane, that are attached to one another to assemble the chip 100 having the desired 3D structure, for example the one shown in Fig. 1. However, the chip 100 may be fabricated in any suitable manner, e.g. from a single piece of material, 3D printing, etc. Figure IB) shows isometric, side and top views of the chip 100.

[0050] As shown, the chip 100 may comprise two individual tissue units 110 on an object slide format, but this is not limiting. Each tissue unit 110 may be configured for cell culture and manipulation.

[0051] Each tissue unit 110 may comprise two medium reservoirs 112, one or more microchannels 114, a cell chamber 116, a pressure chamber 118 adjacent the cell chamber 116, and a flexible membrane 120 separating the cell chamber 116 from the pressure chamber 118. The chip 100 may be used with the cell chamber 116 arranged below the pressure chamber 118. The one or more microchannels 114 may fluidly connect the cell chamber 116 with the medium reservoirs 112 and may be configured to enable fluid exchange between the medium reservoirs 112 and the cell chamber 116. The chip 100 may comprise a microchannel structure 122 configured to supply fluid, e.g. liquid or gas, to the pressure chamber 118 to pressurize the pressure chamber 118. The chip 100 may comprise a pressurization inlet 123 configured to connect the chip 100, and ultimately the microchannel structure 122 and the pressure chamber 118 to a fluid supply (not shown), e.g. compressed air connection in a building, a pump, etc. The pressure chamber 118 may be configured such that the membrane 120 bulges towards and into the cell chamber when the pressure chamber 118 is pressurized. An object in the cell chamber 116 may thus be compressed and / or deformed, depending on the pressure in the pressure chamber 118. In other words, the cell chamber 116, medium reservoirs 112 and one or more microchannels 114 may be fluidly separated from the pressure chamber 118 and the microchannel structure 118.

[0052] In an exemplary embodiment the chip 100 comprises a bottom layer 1 that may be a layer without any openings, cutouts and / or structures. Layer 2 comprises the lower parts of the cell chambers 116 of the two tissue units 110. They are cutouts in the otherwise closed layer 2. Layers 3-6 comprise the remaining part of the cell chambers 116 as well as the medium reservoirs 112 and the one or more microchannels 114 as cutouts. As the layers 3-6 may be, as shown, identical, reference signs are only provided in one layer. Layers 7 and 9 are each a membrane layer, which may differ in material and other, e.g. mechanical, properties from the other layers. Between the membrane layers 7 and 9 there is a pressure layer 8 including the cut outs for the pressure chambers 118 and the microchannel structures 122 in fluid connection with the corresponding pressure chamber 118. The membrane layers below and above the pressure layer 8 define and seal the pressure chamber 118 and the microchannel structures 122 in addition to the pressure layer 8. The membrane layer 7 separates the cell chambers 116 from the corresponding pressure chamber 118 and thus provides the membrane 120 that separates the pressure chamber 118 from the cell chamber 116. The region of the membrane layer 7 that is the membrane 120 (i.e. the actuator) is marked as the hatched region in the left tissue unit in Fig. 1A. The hatched region is not cutout. The right tissue unit 110 is shown without the hatched region. The microchannel structure 122 is shown to be shared by both tiss ue units 110, i.e. the tissue units 110 connected by a common microchannel structure 122 (in this case two tissue units 110) may be configured for simultaneous and / or collective pressurization, but this is not limiting, i.e. each tissue unit 110 may comprise an individual microchannel structure 122 and be configured to be pressurized individually. With more than two tissue units 110, any combination of connected or nonconnected tissue units 110 is encompassed.

[0053] The chip 100 may further comprise a suitable number of interface layers 10-12 which cover the upper membrane layer 9.

[0054] All layers above the layers including the cell chamber 116 preferably include an access port 124 to the cell chamber, which allows supply of material to the cell chamber 116. In Fig. 1, this is the case for layers 7-12. However, a lateral access is also contemplated.

[0055] All layers from the lowest layer including the medium reservoirs 112 to the top may comprise cut outs for defining the medium reservoirs 112. However, other configurations with, e.g., lateral access to the medium reservoir 112 are contemplated. The interface layers 10-12 may provide additional stability to the system and / or additional volume to the medium reservoirs 112. Since the interface layers may, as shown, be identical, reference signs are only provided for one of them.

[0056] The chip 100 may be configured for housing a sample in the cell chamber 116. The chip 100 may be configured for being loaded with a sample via the access port 124, e.g. by introducing the sample with a pipette via the access port 124. For example, a sample may comprise several components, such as a liquid (e.g. water), cells, and further components configured for gelation. After introduction into the cell chamber 116, the sample may form a hydrogel including the cells. The hydrogel including the cells may be deformed by pressurizing the pressure chamber 118 such that the membrane 120 bulges to an extent that the membrane deforms the hydrogel including the cells. This applies equally to other types of samples, e.g. other scaffolds.

[0057] The medium reservoirs 118 may be filled with medium and / or other components. Via the one or more microchannels 114, the medium and / or other components may enter the cell chamber 116 and also be present in the cell chamber 116. Thus, nutrients and / or oxygen may enter the cell chamber 116 and also be present in the cell chamber 116.

[0058] Figure 1 shows exemplary and preferred sizes of the individual structures of the chip 100 (note that for reasons of readability the values may be given in the context of one of the tissue chambers 110 only but may equally apply to the other tissue chamber 110). For example, the cell chamber 116 may have an essentially cylindrical shape, with the cylinder axis oriented from top to bottom of the chip 100. Deviations from a cylinder may occur, e.g., at the connection to the one or more microfluidic channels 114 and or the access 124. The cylinder may have a diameter of 4 mm and a height of 2.5 mm in the non-pressurized state. Pressurization may reduce the volume of the cell chamber 116.

[0059] The medium reservoirs 112 may each have an essentially cylindrical shape, with the cylinder axis oriented from top to bottom of the chip 100. Deviations from a cylinder may occur, e.g., at the connection to the one or more microfluidic channels 114. The cylinder may have a diameter of about 10 mm and a height of about 10 mm, for example.

[0060] The use of the terms "top" and "bottom" is merely to provide relative relationships of locations of certain features. Although they correspond to a preferred orientation of the chip during its use, these terms shall not be understood as limiting. For example, the direction topbottom may be perpendicular to an outer surface of the bottom layer 1. With reference to Fig. 1, layer 12 may be the top-most layer and layer 1 may be the bottom-most layer.

[0061] The chip 100 may provide a gas exchange in the cell chamber of up to 7 xio-8moles of oxygen per hour in the cell chamber, most preferably 4.3 xio-8moles of oxygen per hour. By limiting the gas exchange of the cells with the atmosphere, the chip may enable auto-hypoxia and / or increased carbon dioxide concentration and / or reduced pH-value in the cell chamber without active chemical or physical gas supply mechanisms driven by an oxygen generating and / or oxygen scavenging compounds, and / or pressurized oxygen, nitrogen, CO2 or air from a gas generating active mechanism (e.g., a pressurized gas tank or liquid nitrogen gas phase exchange) or other equipment e.g. by pressures swing adsorption and / or membrane and / or electrolysis-based gas generating approaches.

[0062] The chip 100 may be configured such that medium in the medium reservoirs 118 may be exchanged, e.g. via a pumping system (not shown) or via pipetting. Exchanging the medium in the medium reservoirs 118 may cause a flow and / or exchange of medium in the cell chamber 116 as well, which may provide new nutrients to the cells and / or regenerate the pH-value, carbon dioxide concentration and / or oxygen concentration.

[0063] Further description of useful chips and their production may be found in Rothbauer et al.

[0064] 2021 (10.1039 / D1LC00130B)1and Purtscher et al 2021 (10.1039 / D0LC01056A)2which are incorporated herein by reference in their entirety, but particularly chapters 'Microfabrication' (Page 3) and 'Chip design and fabrication' (Page 2), respectively.

[0065] Example

[0066] 1. Establishment of a multifunctional fibrosis biochip to model pro-myofibrotic mechanisms in fibro-proliferative disorders

[0067] 1.1 Methods

[0068] 1.1.1 Cell Isolation

[0069] Tissue specimens of various origins including equine subcutaneous adipose tissue, human OA- patient-derived IPFP tissues and synovia were separated from surrounding tissue, washed with PBS (DPBS (lx) Dulbecco's Phosphate Buffered Saline, [-] CaCb [-] MgCb, REF 14190-094, Lot. 2156414), and minced into small fragments. The tissue fragments were outgrown in a T- 25 flask (TPP®, Tissue Culture Flask 25, Growth area 25 cm2) with complete culture medium (alpha MEM with 10% FCS and 1% Anti / Anti) and incubated at 37 °C. After 7 days, the medium was aspirated, followed by a biweekly medium replacement until 80-90% cell confluence.

[0070] 1.1.2 Cell Culture & Organoid preparation

[0071] Primary cells were maintained in a T-75 flask (TPP®, Tissue Culture Flask 25, Growth area 75 cm2) at 37°C and 5% CO2 using Dulbecco's Modified Eagle's Medium (MEM alpha containing phenol red) supplemented with 1% Penstrep (Anti / Anti; 5 mL), 500 pL Amphotericin B was initially sterile filtered with the TPP™ Vacuum Filtration System "Rapid"- Filtermax™ (500 mL, Product-Nr. 99500). Cells were subpassaged using enzymatic protocols for cell subcultivation using TrypILE express IX solution (Gibco).

[0072] After pelleting, on-chip organoids were generated at a cell density of 150,000 cells per 60 pL PureCol EZ Collagen type 1 hydrogel at a final matrix concentration of 4.5 mg / mL, as shown in Table 1. The gel was incubated for 60 min at 37°C in a cell incubator to polymerize the hydrogel via thermo-gelation. Thermo-gelation may be understood as inducing gelation at an elevated temperature above 25 °C, most preferably under humidified atmosphere and pH-buffered conditions (i.e., active bi-carbonate or HEPES buffer systems). The medium volume of 400 pL per chip unit was replaced weekly.

[0073] Table 1 Volumes and concentrations of the PureCol EZ hydrogel mix.

[0074] 1.1.3 Chip fabrication and device operation

[0075] The process of building a chip was divided into a few steps as described previously3and above. The chip was designed using AutoDesk AUTOCAD MECHANICAL 2022 software and exported as .dxf file. Xurograhic structuring of PDMS foil of 500 pm thickness (MVQ Silicones, Germany) was performed after protection-film removal using optimized parameters (Cutting force: 80, Cutting Speed: 19, Overlap Cutting: 0, Quality: Normal). After clearing of cut-out PDMS, layers were bonded in layer-by-layer fashion using air plasma activation (high power, 450 Torr; Harrick Plasma) and initially annealed for 5 minutes in an 80°C convection oven which was repeated for each layer. The assembled chip device was finally post-processes for proper bonding overnight at 80°C. The result was a chip as described above and as shown in Fig. 1.

[0076] PureCol EZ stock solution (~5 mg / mL) and cell suspension (1,500,000 cells in 60 pl volume of lOx cell stock solution) were mixed in 1:10 volumetric ratio by pipetting 20-25 times for homogenization prior to injection of 45 pL to load the fibrosis-on-a-chip device via the access port. Some of the samples in the chips were treated at day 10 with one or a combination of 15 pM SB 431542 compound (Bio-Techne Austria), 5 ng / mL recombinant human TGF-|31 (Bio-Techne Austria), ELAREM™ Perform-FD (Human Platelet Lysate, Fibrinogen-depleted, PL BioScience) or home-brew single donor platelet lysate generated from standard protocols (2x freeze-thaw cycle with down spinning of fibrin precipitate) from platelet concentrates. The ELAREM™ Perform-FD cell culture supplement was centrifuged for 10 minutes at 3000 rpm (Awel® Centrifugation CF 108-GR, SL-750 Swing-Out Rotor, Radius of rotor 202.8 mm, 2044 x g) as suggested by the manufacturer's instructions. Supplements including hPL (as treatment modalities), TGF-|31 as well as SB 431542 were added from stock solutions on the day of experiment to the complete aMEM culture medium. Individual biochips were placed in sterile QuadriPerm ™ cassettes for routine cultivation.

[0077] Cell culture in a scaffold (here in a hydrogel) in a chip is considered a 3D cell culture technique.

[0078] 1.1.4 2D cell culture

[0079] To create 2D cultured controls, 1 mL of cell-containing medium adjusted to a respective 20% cell surface coverage was placed on the surface of a 24-well cell-culture-treated well plate and cultivated analogously using 1 mL complete biochip medium in accordance to the biochip cultivation protocols for up to 7 days of culture.

[0080] 1.1.5 Micromass cell culture

[0081] To create micromass 3D cultures of the sample cell hydrogel solution mixtures, a drop of 45 pL of cell-containing hydrogel was placed in the center of a 48-well cell-culture-treated well plate and cultivated analogously using 500 mL complete biochip medium in accordance to the biochip cultivation protocols for up to 14 days of culture. Micromass cell culture in a scaffold (here in a hydrogel) is considered a 3D cell culture technique.

[0082] 1.1.6 Treatment of 2D and 3D cultures

[0083] To treat the specimens with cytokines prior to biomechanical stimuli, 10% of the cell culture medium was aspirated from any fibroblast model and replaced with a lOx stock solution of TGF-pi containing stock at 50 ng / mL concentration to produce a final concentration of 5 ng / mL.

[0084] 1.1.7 RNA Extraction & Reverse Transcription (cDNA)

[0085] To extract RNA from samples, the innuPREP RNA Mini Kit 2.0 was utilized. For 2D cell cultures, excess medium was removed, and cells were rinsed with 500 pL of lxPBS. The PBS was then aspirated, and cells lysed using 350 pL of lysis buffer, which was pipetted in the well and transferred to a 1.5 mL Eppendorf tube. In 3D cultures, fibrin clots were cut from the chip and soaked in lxPBS for 45 minutes to cleanse phenol red, then transferred to a tube with 350 pL of lysis buffer containing 1% P-Mercaptoethanol. Following the manufacturers' standard protocols, both 2D and 3D cultures underwent the same RNA isolation, cDNA synthesis, and qPCR procedures. RNA isolation continued with the lysate being applied to spin filter D. An equal volume (350 pL) of 70% ethanol solution was added to the flow through, and thorough mixing was ensured by pipetting up and down multiple times. Next, spin filter R was placed into a new receiver tube, and the solution was transferred on top of it. Centrifugation was carried out for 2 minutes at 11,000 g. The receiver tube containing the flowthrough was discarded, and spin filter R was transferred to a new receiver tube. For the subsequent steps, 500 pL of high salt washing solution (HS) was added, and centrifugation was performed at 11,000 g for 2 minutes. The receiver tube along with the flowthrough was discarded, and spin filter R was again placed into a new receiver tube. Following this, 700 pL of low salt washing solution (LS) was added, and centrifugation was conducted for 2 minutes at 11,000 g. The flowthrough was discarded along with the receiver tube, and spin filter R was transferred once more to a new receiver tube. To remove all traces of liquid, the filter was dried by centrifugation at 11,000 g for 3 minutes. After discarding the receiver tube, spin filter R was placed into an elution tube. Here, 30 pL of Rnase-free water was added, and the mixture was incubated for 1 minute at room temperature. Subsequently, centrifugation was carried out at 11,000 g for 1 minute. The elution tube containing the RNA was placed on ice. For the determination of the RNA concentration, 1 pL of the RNA sample was transferred onto a spectrophotometer (Nanodrop 2000c, Thermo Scientific), and the RNA concentration was quantified prior to qPCR mRNa analysis.

[0086] Table 2. Master mix composition for cDNA synthesis (one reaction)

[0087] _ Component

[0088] Molecular grade H2O 4.2 lOx RT Buffer 2.0 25x dNTP Mix 0.8 lOx RT Random Primers 2.0 MultiScribe™ Reverse Transcriptase 1.0

[0089] Total 10 RNA sample 10 Final volume 20

[0090] 1.1.8 Real-Time / quantitative Polymerase Chain Reaction (qPCR)

[0091] After synthesizing complementary DNA (cDNA), we used quantitative polymerase chain reaction (qPCR) to measure gene expression levels relative to a control gene. Initially, we prepared a master mix on ice, comprising molecular grade water, SYBR Green (PowerTrack SYBR Green Master Mix, Applied Biosystems™, #A46109), and specific forward and reverse primers, following the volume ratios listed in Table 3. We employed both equine and human primers, as detailed in Table 4. For each test, the SDHA gene (encoding the succinate dehydrogenase complex flavoprotein subunit A) served as the reference or housekeeping gene. Next, we added 19 pL of the master mix into each section of a 96-well plate (MicroAmp® Fast 96-Well Reaction Plate (0.1 mL), Applied Biosystems™, #4346907). We then added 1 pL of the cDNA sample to each well. After sealing the plate with PCR foil and performing a brief spin-down using a PCR plate spinner (VWR™ international, #521-1648), we placed the plate in a PCR cycler and initiated the specified PCR program. This program included three phases: denaturation at 95 °C, annealing at 60 °C, and elongation at 60 °C. Table 3. qPCR master mix for one reaction

[0092] Component Molecular grade H2O 5.8 SYBR Green MM 10

[0093] Forward primer : 1.6

[0094] Reverse primer 1.6

[0095] Total : 19 cDNA sample 1 Final volume 20

[0096] Table 4 Human and equine primer sequences.

[0097] The gene ACTA2 encodes the protein a-Smooth Muscle Actin (a-SMA), a hallmark of myofibroblast differentiation and a direct target of TGF-|3 signaling. The expression of ACTA2 / a-SMA is upregulated in response to TGF-|3, indicating the differentiation of fibroblasts to myofibroblasts, which play a critical role in the fibrotic remodeling of tissues. Myofibroblasts, characterized by a-SMA expression, are responsible for the excessive deposition of ECM proteins, leading to the pathological stiffening of tissues observed in fibrosis. Thus, both TGF-|3 signaling and ACTA2 expression are central to the fibrogenic process, making them significant targets for therapeutic interventions aimed at treating fibrotic diseases. SDHA gene was used as a housekeeping reference gene for the calculation of the delta CT values.

[0098] 1.1.9 Fluid mechanical actuation and compressive loading of fibrosis biochips

[0099] Pneumatic actuation of the integrated actuators (i.e. the pressure chamber / membrane) was driven by a compressor and a P-switch control and a connector station using OXYgen control software (Fluigent) for a 1Hz actuation frequency. Each solenoid valve of the p-switch was connected via standard pneumatic tubing and a tube connector to the pressurization inlet of the fibrosis biochip. Thus, the pressure chambers of the chips could be actuated, i.e. pressurized or depressurized to cause a deflection of the membranes and thus of the hydrogels including the cells in the cell chambers.

[0100] For subjecting the hydrogels comprising the cells to medium flow (which is also referred to as fluid actuation), one set of chips was placed on a tilting plate rocker (PMR-30 Mini Rocker- Shaker, Grant-bio) which was set to 1 Hz tilting speed to create fluid flow and / or shear forces inside the chips. All cassettes containing chips (loaded and control specimens) were closed with parafilm on three sides from day 7 on to minimize evaporation and create a comparable setting between static and loaded with perfused chips actuated on the tilting plate.

[0101] 1.1.10 Opto-chemical oxygen monitoring

[0102] To determine oxygen concentrations in the microplate settings (2D cultures and 3D micromass cultures) and in the chips, dye-loaded particles (i.e., OXNANO, Pyroscience) were used together with a one-channel light detection system (Fiber-Optic Oxygen Meter Piccolo , Pyroscience) to obtain a quantitative readout of oxygen levels.

[0103] On the day prior to cell experiments, chips were spotted with 2 pL of an aqueous oxygen sensor solution in the center of the cell chamber as previously described.4Additionally, a 24 well plate was spotted in the same way and in this case the dots were placed in the center of the well. All sensor spots were allowed to dry in the laminar flow hood over night

[0104] 1.1.11 Cell deflection analysis by ImageJ image analysis

[0105] Pressurizing the pressure chambers had the effect that the hydrogels comprising the cells in the corresponding cell chambers were deformed. This is also referred to as the loaded state. Deformation took place because the height of each cell chamber was reduced due to the bulging of the membranes of the chips. Since each chip was oriented such that the pressure chamber was located above the cell chamber, the deformation included a reduction of height (i.e., in the top-bottom direction) of the hydrogel and an increase of the diameter of the hydrogel, the diameter being measured perpendicular to the height.

[0106] The effect of such deformation of the hydrogels was analyzed as follows.

[0107] Videos were acquired of cell regions in the non-loaded state that were transferred to loaded states by pressurizing the pressure chambers. The cell regions were imaged from the bottom with an 1X83 automated epifluorescence microscope (Olympus) equipped with a Hamamatsu flash orca 4.0 camera and 10-40 x dry objectives. ImageJ software was used to analyze the deformation. Representative frames from before the deformation and in the deformed state were loaded into ImageJ software, and, using the 'merge channels' function, the two frames representing the non-loaded state and the loaded (i.e. deformed) state were merged into a single frame. Distances between cell positions in the non-loaded (green channel) and loaded states (red channel) were manually measured with the line tool and the 'measure' function using manually identified center-to-center distances for n=25 cells per gel in 2 different gels.

[0108] Results are shown in Fig. 5 (discussion see below).

[0109] 1.1.12 Visualization & Statistical analysis

[0110] For data visualization and statistical analysis GraphPad Prism (Version 1) was performed using one-way ANOVA in combination with Tukeys' post-hoc test where applicable. 1.2 Results & Discussions

[0111] Figure 2A shows that the present invention can gradually increase the ACTA2 mRNA expression over a period of 11 days (n=2 for each cell type) and demonstrate its feasibility for a variety of different stromal cell types including musculoskeletal fat pad fibroblasts (Hfib) of osteoarthritic (OA) origin, OA-patient derived fibroblast-like synoviocytes (FLS), human dermal fibroblasts derived from pooled human foreskin (HDF) as well as equine adipose-tissue derived stromal cells (ASCs) from healthy non-OA donor origin. Figure 2A shows the time-resolved effect of the method in the fibrosis biochip in the absence of any pro-inflammatory medium additives on pro-fibrotic qPCR derived gene expression of ACTA2 gene for a variety of fibroblast-like stromal cells including primary human fibroblast-like synoviocytes (FLS), stromal cells from the stromal vascular fraction of the infrapatellar fat pad (IPFP) of the knee joint (Hfib), human dermal fibroblasts isolated from skin tissue (HDF), as well as equine adipose-tissue-derived stromal cells (ASC) isolated from subcutaneous adipose tissue biopsies. The data was normalized vs. the gene expression values of samples harvested after 4 days of on-chip cultivation. Hfib, FLS and HDF cells demonstrate a pronounced increase to values up to more than 300%. In contrast, ACTA2 gene expression remained unchanged for biochip cultivation for 11 days indicating the different, non-myofibrotic response of adipose-derived stem cells.

[0112] Figure 2B shows a comparison of ACTA2 expression in 2D culture, micromass culture, and chip culture of HDF cells within 7 days of culture. A pronounced effect of an approximately 5-fold increase of ACTA2 expression as compared to the 2D culture and the micromass experiments was observed for the method according to the present invention as performed in the chip.

[0113] To demonstrate that the process relating to the increase in gene expression is relating to TGF- beta receptor family activity, it was next demonstrated that treatment of the biochip fibrosis models (i.e. the scaffold comprising the cells) with the activin receptor-like kinase (ALK) receptor (TGFb receptor family) inhibitor SB 431542 at a concentration of 15 pM from day 8 to 11 significantly down regulated the myofibrotic marker ACTA2 (a-SMA). Figure 2C shows the reversal of the pro-fibrotic / -myofibrogenic state when treated with 15 pM SB 431542 inhibitor targeting the activin receptor-like kinase (ALK) receptor (TGFb receptor family) after exposure for 36 h and exposure start at day 8 (see the protocol section above). The data was normalized to the untreated control (the control was subjected to chip-induced fibrotic state but was not treated with SB 431542 inhibitor). ASC-containing biochip specimens also reacted to the TGFb receptor inhibition with a decrease of ACTA2 expression, although their ACTA2 was not influenced by the biochip treatment (as shown in Fig. 2A).

[0114] The situation regarding oxygen is shown in Figure 3.

[0115] Figure 3 A shows for a cell density of 150,000 fibroblast cells of Hoffa adipose-tissue origin per 60 pL hydrogel that the natural cell respiration can produce a slightly hypoxic environment within the biochip based on natural respiration of crowded scaffolds (here hydrogels). Chip and hydrogel are as described above. The measurement with integrated opto-chemical oxygen sensors started at 24 h of cultivation using a pre-dried spot within the hydrogel compartment of the chip prior to cell seeding. The slight peak at the second measurement point is due to initial sensor drift and equilibration caused by sample cooling to room temperature during the time-resolved measurements. For comparison, control samples having acellular PureCol hydrogels (acellular hydrogels are free from cells) were used. Longterm monitoring with the integrated opto-chemical oxygen sensors as shown in Figure 3B revealed a significant reduction of oxygen levels from normoxia (20-21%) down to approx. 19% 02 as early as day 1, which stayed at constant levels over the next two weeks for the samples with 150,000 cells per 60 pL hydrogel. The control samples showed a constant oxygen level of 20-21%. Data shows a comparison of oxygen levels of biochip cultures (cells, concentration, hydrogel, etc.) compared to conventional 3D micromass and 2D monolayer cultures (n=4 for each cell culture type). All of these samples were non-perfused, i.e. the samples were not subjected to fluid flow except for medium exchange at day 7 post-seeding. Oxygen was measured as explained above from day 8 to 11 for 15 minutes each interval relative to an acellular control biochip. As can be derived from Fig. 3C, the micromass samples and 2D culture samples remained at an ambient oxygen level, whereas the sensors in the biochip samples reported a reduced oxygen concentration of approximately 3 percentage points in the negative direction relative to donor-paired 2D control samples. This demonstrates again the slightly hypoxic environment of the biochips according to one aspect of the present invention.

[0116] The experiments for which results are shown in Figure 4A investigated whether the application of pulsatile fluid flow at a physiological frequency of 1 Hz, which mimics human gait patterns, could reverse the pro-fibrotic effect of the fibrosis-on-a-chip. The application of 24 h of fluid flow of approx. 2 dyn / cm2at 1 Hz frequency could significantly recover normoxia of the HFib fibrosis model (see Fig. 4A), which was confirmed by gene expression analysis in more detail (as shown in Figure 4B) for a sample stimulated with fluid flow (indicated FSS in the graphs) and further stimulated by 5 ng / mL TGF-|31. 24 hours of fluid perfusion could significantly decrease gene expression levels of ACTA2. The gene expression levels of ACTA2 was even more attenuated after 36 hours of fluid perfusion, namely to levels observed with biochip models that were treated with 15 pM ALK4 inhibitor SB 431542 (see also Fig. 2C). Contrarily, both untrained control specimens (i.e. without pulsatile fluid flow) remained at increased levels of ACTA2 indicating the generation of a myo-fibrotic tissue model, i.e. a fibrotic state.

[0117] Figure 5 shows the effect of compressive loading of scaffolds (here hydrogels) comprising cells, e.g. at 1Hz. This was again investigated with a chip as described above. Figure 5A shows the basic characterizations of the actuation principle that was used to apply a cyclic biomechanic stretching motion or deformation to the on-chip hydrogel construct by pressurizing the pressure chamber (see representative images of the deflected membrane). Figure 5B (top graph) shows the dependency of the height of a hydrogel in the cell chamber from the pressure applied to the pressure chamber for pressures up to 200 mbar. Figure 5B (bottom graph) shows the actuator deflection depending on the applied pressure in the pressure chamber. The distance was measured at the membrane interface post-actuation relative to either the initial z-position of i) the non-deflected hydrogel top relative to its bottom layer or ii) the actuation layer at the hydrogel interface relative to its un-actuated z-position, respectively. As can be seen, the relationship between the height of the deflected hydrogel and the actuator deflection is linear and indirect proportional.

[0118] Figure 5C shows two microscopy images showing top views of cells in a hydrogel scaffold in a cell chamber, the scale bar representing 150 pm. The upper image shows the non-deflected state, the lower image shows the deflected state at an actuation pressure of 50 mbar for the chip as shown in Fig. 1. The graph shows deflection data as extracted from microscopy images of the cells that were acquired at different actuation pressures. The deflection of n=25 fluorescently labelled fibroblast cells for an on-chip actuation pressure (i.e. in the pressure chamber) from 25 mbar to 250 mbar was manually extracted as described above. For example, 40 pm lateral cell deflection for an actuation pressure of 200 mbar could be observed.

[0119] The protocol of 1 Hz actuation frequency at 200 mbar pressure in the pressure chamber was next applied to fibrosis biochip models generated with HFibs in hydrogel scaffolds for up to 4 hours at day 11 post-seeding. The result is shown in Fig. 5D, a TGF- control is shown for comparison. The results demonstrate the modulatory effect of mechanical actuation on the myofibrotic expression marker ACTA2, which was significantly downregulated when applying the loading protocol for 4 hours. It was observed that the mechanical loading routine of 4 hours could attenuate the marker expression in similar magnitude as 2-4 days of fluid perfusion.

[0120] To demonstrate that the developed model can be used for drug screening studies according to the methods of the present invention, samples were subjected to the method according to the present invention to stimulate a fibrotic state, and subjected to different substances. The effects of the different substances were determined via mRNA expression of ACTA2. Particularly, the following protocol was applied: After maturation for 11 days, fibrotic biochip samples containing adipose-derived fibroblasts were treated with a variety of anti-fibrotic agents including 30% hPL containing complete culture medium of either home-brew or commercial origins for the following 36 h in the presence or absence of 5 ng / mL TGF-|31 or 50 pg / mL GAL3 as pro-fibrotic co-stimulus. Fig. 6 shows that the expression levels of myofibrotic marker ACTA2 for 5 ng / mLTGF-pi cytokine or galectin 3 (GAL3) -stimulated HFib models could be significantly reversed by anti-fibrotic treatment regimens including commercially available pooled human platelet lysate ELAREM™ (hPL) and home-brew single donor platelet lysate at a 30% v / v concentration in complete culture medium after 36 h of treatment as described in the material section.

[0121] Conclusions

[0122] The innovative biomimetic biomechanical fibrosis-on-a-chip model disclosed herein was developed to promote a stimulative niche to model fibro-proliferative disorders under dynamic mechanobiological parameters including hypoxia, fluid perfusion as well as dynamic compressive loading. The developed biomechanical system, firstly, allows precise in situ formation and maturation of patient-derived stromal organoids using primary cells (i.e., fibroblasts or fibroblast-like cells) under varying actuation frequencies and loading intensities. Secondly, in situ oxygen sensing was implemented to develop a slightly hypoxic environment via stromal cell crowding that stimulates a natural pro-fibrotic environment of local tissue fibrosis that promotes myofibrogenic stroma cell differentiation in the absence of a stimulative fibrotic compound such as transforming growth factor beta (TGF-R1). Thirdly, it was shown that, when applying fluid actuation as well as multi-axial pneumatic loading, the fibrosis model can be used to investigate pro- and anti-fibrotic treatments and stimulants under more relevant conditions. Lastly, it was demonstrated that the developed fibrosis-on- a-chip system may be employed in a screening study on the anti-fibrotic effects of platelet lysates on a pathological model of infrapatellar fat pad fibrosis. BIBLIOGRAPHY:

[0123] 1) M. Rothbauer, R. A. Byrne, S. Schobesberger, I. Olmos Calvo, A. Fischer, E. I. Reihs, S. Spitz, B. Bachmann, F. Sevelda, J. Holinka, W. Holnthoner, H. Redl, S. Toegel, R. Windhager, H. P. Kiener and P. Ertl, Lab Chip, 2021, 21, 4128 DOI: 10.1039 / D1LC00130B

[0124] 2) M. Purtscher, M. Rothbauer, S. R. A. Kratz, A. Bailey, P. Lieberzeit and P. Ertl, Lab Chip, 2021, 21, 1364 DOI: 10.1039 / D0LC01056A

[0125] 3) D. A. Ferreira, M. Rothbauer, J. P. Conde, P. Ertl, C. Oliveira and P. L. Granja, Adv. Sci., 2021, 8, 2003273

[0126] 4) H. Zirath, S. Spitz, D. Roth, T. Schellhorn, M. Rothbauer, B. Muller, M. Walch, J. Kaur, A. Wbrle, Y. Kohl, T. Mayr and P. Ertl, Lab Chip, 2021, 21, 4237 DOI: 10.1039 / D1LC00528F

[0127] List of Reference signs:

[0128] 1-12 layers

[0129] 100 chip

[0130] 110 Tissue unit

[0131] 112 medium reservoir

[0132] 114 microchannel

[0133] 116 cell chamber

[0134] 118 pressure chamber

[0135] 120 membrane

[0136] 122 microchannel structure

[0137] 124 access port

Claims

Claims1. Method of regulating a fibrotic state in cells in cell culture, the cells preferably being fibroblasts, fibrocytes, fibroblast-like cells and / or progenitors of fibroblasts, the method including inducing a fibrotic state in the cells by performing the cell culture under the following conditions: o culturing the cells in a scaffold comprising the cells at a density of from 3000 to 6000 cells / mm3, preferably 3400 cells / mm3cells, for 4 hours to 14 days, wherein the scaffold comprising the fibroblasts is cultured in cell culture medium; and / or o culturing the fibroblasts at hypoxic conditions, preferably at an oxygen concentration of less than 21%, more preferably at an oxygen concentration of 20% to 1%, more preferably at an oxygen concentration of 19 % to 1%; most preferably at an oxygen concentration of 18 % to 1%; and / or o culturing the fibroblasts at starving conditions.

2. The method according to claim 1, wherein performing the cell culture is carried out under the following conditions: o substantially in absence of external mechanical and / or hydraulic deformation and / or compression of the scaffold comprising the cells; and / or o substantially in absence of externally applied medium flow through the scaffold comprising the cells.

3. The method according to claim 1, wherein performing the cell culture is carried out under the following conditions: subjecting the scaffold comprising the cells to liquid flow that allows fibrotic state formation either as a periodical stimulus or as a stimulus pattern comprising several intervals with intermittent resting periods per day, for example up to 50 pl / s through an area of 3.6 mm2, up to 0.42 dyn / cm2, at a frequency from 0.1 to 2 Hz, preferably at an actuation frequency of 0.5 Hz to 2 Hz, more preferably 1 Hz.

4. The method according to any one of the preceding claims, wherein the scaffold comprising the cells is subject to liquid exchange and / or supplementation with fresh cultivation medium and / or nutrients essentially only for cell culture medium exchange with time intervals of 1 hour to 14 days, preferably 6 hours to 10 days, more preferably 48 hours to 8days, most preferably 7 days to allow fibrotic state formation.

5. The method according to any one of the preceding claims, wherein the cell culture medium comprises less than 100 pg / ml of pro-inflammatory and / or pro-fibrotic additives, most preferably no pro-inflammatory additives that are known as contributing to inducing a fibrotic state; optionally wherein the cell culture medium comprises less than 100 pg / ml TGF-pi, PDGF, IL-6, ILlp or TNF-a.

6. The method according to any one of the preceding claims, wherein the cells comprise one or a combination of the following cell types: o Fibroblasts o Fibrocyteso Fibroblast progenitors o Pericytes o Myogenic progenitor cells o Myofibroblasts o Adipose-tissue-derived fibroblast-like cells o Adipose-tissue-derived stem cells o Adipose-tissue-derived myeloid or lymphoid cells o Bone-marrow-derived stem cells o Synovium-tissue-derived fibroblast like cells o Synovium-tissue-derived myeloid or lymphoid cells o Synovium-tissue-derived endothelial cells o Synovium-tissue-derived stem cells o Dermal-tissue-derived myeloid or lymphoid cells o Dermal-tissue-derived endothelial cells o Dermal-tissue-derived stem cells o Fibroblast-like and / or fibroblast cells derived from meniscus, discus, tendon or ligament tissues.

7. The method according to any one of the preceding claims, wherein the scaffold comprising the cells is provided in a cell chamber of a chip, the chip being configured to apply optional deformation and / or compression and / or optional fluid shear to the scaffold comprising the cells in the cell chamber, preferably to regenerate and / or reverse the fibrotic state of the cells.

8. The method according to the preceding claim, wherein the chip comprises a pressure chamber adjacent to the cell chamber and separated from the cell chamber by a flexible membrane, the pressure chamber being configured to be pressurized by adding fluid to the pressure chamber; wherein pressurizing the pressure chamber results in application of compression and / or deformation to the scaffold comprising the cells via the membrane.

9. The method according to any one of the preceding claims, wherein the scaffold is a hydrogel, preferably a hydrogel comprising collagen, more preferably a hydrogel comprising collagen type I, and / or a synthetic analogue with similar biophysical functions and / or chemical nature as a collagen hydrogel or a collagen type I hydrogel.

10. The method according to any one of the preceding claims, wherein a deformation and / or compression of the scaffold is applied to regenerate and / or reverse the fibrotic state of the cells: such that it results in reducing a height of the scaffold up to 50%, preferably 10% to 30%, more preferably 15%; and / or at a frequency between 0.1 and 2 Hz, preferably at a frequency of 0.5 Hz or more, more preferably 1 Hz; and / orfor a duration of 15 minutes up to 4 hours per pressurization interval, and up to 3 pressurization intervals per day.

11. The method according to any one of the preceding claims, wherein the scaffold comprising the cells is subject to liquid flow to regenerate and / or reverse the fibrotic state either as a periodical stimulus or as a stimulus pattern comprising of several intervals with intermittent resting periods per day, preferably: up to 500 pl / s, e.g. more than 50 pl / s and up to 500 pL / s, through an area of 3.6 mm2, up to 4.2 dyn / cm2(0.42 Pa), at a frequency between 0.1 and 2 Hz, preferably at an actuation frequency of 0.5 Hz to 2 Hz, more preferably 1 Hz.

12. A drug-screening method, comprising: a) inducing a fibrotic state in a first plurality of cells in cell culture by applying the method according to any one of claims 1-9; b) subjecting the first plurality of cells resulting from step a) to a drug of interest, e.g. by adding the drug of interest to the cell culture medium; c) optionally determining the effect of the drug on the first plurality of cells.

13. The drug screening method according to the preceding claim, further comprising: d) inducing a fibrotic state in a second plurality of cells in cell culture by applying the method according to any one of claims 1-9 with a lower amount and / or concentration of the drug than in step b), preferably in the absence of the drug; e) regenerating the second plurality of cells according to any one of claims 10-11; f) optionally comparing at least one property of the first plurality of cells resulting from step b) with said at least one property of the regenerated second plurality of cells resulting from step e) to determine the effect of the drug.

14. The drug screening method according to the preceding claim, further comprising: g) subjecting the regenerated second plurality of cells resulting from step e) to the drug of interest, preferably by adding the drug of interest to the cell culture medium; h) comparing at least one property as determined for the first plurality of cells resulting from step b) with said at least one property as determined for the regenerated second plurality of cells resulting from step g) in order to determine the one or more effects of the drug.

15. The drug-screening method according to any one of claims 12-14, wherein step b) is performed while maintaining the application of the method of inducing a fibrotic state according to any one of claims 1-9 to the first plurality of cells; or the application of the method of inducing a fibrotic state according to any one of claims 1-9 to the first plurality of cells is stopped before, upon starting, or during step b).

Images