WO2024252292A1 - Modèle de peau bio-imprimée, procédé d'impression tridimensionnelle associé et dispositif de perfusion directe équipé dudit modèle - Google Patents

Modèle de peau bio-imprimée, procédé d'impression tridimensionnelle associé et dispositif de perfusion directe équipé dudit modèle Download PDF

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Publication number
WO2024252292A1
WO2024252292A1 PCT/IB2024/055478 IB2024055478W WO2024252292A1 WO 2024252292 A1 WO2024252292 A1 WO 2024252292A1 IB 2024055478 W IB2024055478 W IB 2024055478W WO 2024252292 A1 WO2024252292 A1 WO 2024252292A1
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fact
skin
fibroblasts
model
endothelial cells
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Lorenzo Maria VISENTIN
Federico MAGGIOTTO
Elisa CIMETTA
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Bio System Lab Srl
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Bio System Lab Srl
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Priority to EP24737819.3A priority Critical patent/EP4724558A1/fr
Priority to CN202480038381.4A priority patent/CN121693559A/zh
Publication of WO2024252292A1 publication Critical patent/WO2024252292A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture

Definitions

  • the present invention relates to a bio-printed skin model, a related three-dimensional printing process and a direct perfusion device provided with said model.
  • the skin is the largest organ in the human body, accounting for about 16% of the body weight, and plays a key role in protecting the body from the external environment. Its layered structure, composed of epidermis, dermis and subcutis, is essential for its functions as a protective, thermoregulation and homeostasis physical barrier.
  • 3D bioprinting is becoming increasingly popular in clinical and research settings.
  • 3D bioprinting technology enables customized deposition of cells embedded in a biomaterial (bioink) according to pre-processed digital models depending on the final performance required.
  • Two-dimensional skin models consist of a single two-dimensional layer of cells or of a few overlapping cell sheets (Suhail, S. et al., Biotechnology Journal vol. 14 (2019)).
  • Two- dimensional models are the most stable and easiest to use and are employed for culturing or co-culturing keratinocytes and immune cells.
  • Another known type of skin model is three-dimensional skin models. These have a separate three-dimensional structuring and subdivision of the various layers of the skin (epidermis, dermis and subcutaneous layer). These structures allow the formation of tight cell-cell bonds and intracellular junctions allowing exchanges of molecules, gases and nutrients between them and maintaining the structural integrity of the skin and the functionality thereof. In addition, the formation of the stratum comeum reduces the rate of drug diffusion and its bioavailability, mimicking the barrier function of human skin (Polini, A. et al., Drug Discovery, vol. 9, 335-352 (2014)).
  • On-chip models are micro-fluidic devices with micrometer-sized housing chambers for dynamic cell culture in order to mimic the physiology of a tissue or of an organ (Bhatia, S. N. & Ingber, D. E., Nature Biotechnology, vol. 32, 760-772 (2014)).
  • the improved control of the cellular microenvironment and the ability to apply physical or chemical stimuli to the tissue inside help to recreate physiology more accurately than in a static and traditional 3D culture.
  • the application of these stimuli leads to changes in cell behaviors, with improved cell differentiation, better cell-cell and cell-matrix interactions and cell morphologies.
  • on-chip models involve the use of porous microchannel-dividing substrates, allowing the study of tissue barrier functions and simulating tissue-tissue interfaces (Kim, H. J. et al., Lab Chip, 12, 2165-2174 (2012)).
  • vascular canals that effectively simulate the vascular network present in human skin.
  • the wall of vascular canals greatly affects the exchange of substances between tissues and blood, thus creating a barrier effect.
  • two-dimensional skin models have a structural simplicity, characterized by a single layer or meager layers of cells, which makes this model unsuccessful in recreating the three-dimensional structural complexity and the cell-cell and cell-matrix interactions that exist in a body or parts of a body at both physiological and biological levels, thus limiting the accuracy in being able to predict the complicated effects of a drug, resulting from the cellular metabolism of the aforementioned skin.
  • Also absent in two-dimensional cell cultures is the barrier effect in the stratum corneum that is created in 3D structures and better mimics slowing the diffusion of molecules and active ingredients. It follows that two-dimensional skin models cannot be considered representative of the chemical, physical and biological dynamics involved within the same sample obtained from vivo or in vivo.
  • three-dimensional skin models of known type have major limitations related to various aspects, such as the lack of vasculature for nutrient supply, oxygen, waste removal or nutrient concentration gradient, leukocyte trafficking and transdermal penetration of drugs into the bloodstream, the weak barrier properties and the lack/shortage of skin adnexa (sweat glands, hair follicles) and, finally, the inability to offer precise control over spatial-temporal chemical gradients and over physical environmental factors (temperature, mechanical forces, gases), making the sampling complicated of luminal contents for the analysis of drug adsorption, distribution, elimination and toxicity (ADMET) and the collection of cellular components at specific locations for extended biological analysis.
  • ADMET drug adsorption, distribution, elimination and toxicity
  • the main aim of the present invention is to devise a bio-printed skin model, a related three-dimensional printing process and a direct perfusion device provided with said model which allow effectively simulating the human skin both structurally and functionally.
  • Another object of the present invention is to devise a bio-printed skin model, a related three-dimensional printing process and a direct perfusion device provided with said model which allow in vitro tests to be performed that are representative of real human skin behavior.
  • a further object of the present invention is to devise a bio-printed skin model, a related three-dimensional printing process and a direct perfusion device provided with said model which allow avoiding the need for additional in vivo tests.
  • Another object of the present invention is to devise a bio-printed skin model, a related three-dimensional printing process and a direct perfusion device provided with said model which allow the aforementioned drawbacks of the prior art to be overcome within the framework of a simple, rational, easy and effective to use as well as cost- effective solution.
  • Figure 1 is a top view schematic representation of the bio-printed skin model according to the invention.
  • Figure 2 is a schematic perspective representation of the bio-printed skin model
  • Figure 3 is a perspective view of a perfusion support provided with bio-printed skin models
  • Figure 4 is an exploded view of a direct perfusion device according to the invention.
  • Figure 5 is a perspective view of a tank of a direct perfusion device
  • Figure 6 is a schematic representation from above of a direct perfusion device
  • Figures 7-9 represent characterization graphs of polymerizable materials according to the invention.
  • Figures 10-12 represent characterization graphs of the bio-printed skin model.
  • reference numeral 1 globally denotes a bioprinted skin model.
  • the skin model 1 comprises: at least one dermal layer 2 comprising at least a first polymer matrix and a mixture of fibroblasts and endothelial cells and configured to simulate a human dermal tissue; at least one epidermal layer 3 comprising at least one mixture of keratinocytes and configured to simulate a human epidermal tissue; wherein the dermal layer 2 comprises at least one inner duct 4 formed by fibroblasts and endothelial cells, configured to simulate a vascular channel of human skin and provided with at least two end stretches communicating with the outside and connectable to a system for active perfusion of substances.
  • This model is made by means of a three-dimensional printing process, as will be better described later in the disclosure, thus ensuring the fabrication of a cellular construct in three dimensions, in a fast, accurate, standardized and low-cost manner, and allowing controlled deposition of the biomaterials used in the process.
  • This model features the same layered structure of epidermis and dermis found in tissue in vivo, thus ensuring the formation of tight intracellular bonds and junctions so that molecules, gases and nutrients can be exchanged between them.
  • fibroblasts, endothelial cells and keratinocytes are live cells, and the inner duct 4 can be perfused with a working solution, which may consist of nutrients, oxygen or other substances intended to be transferred to the cells themselves for their sustenance.
  • the working solution may also contain active substances, such as drugs or other molecules, in order to carry out bioavailability, bioequivalence tests and, in general, ADME (Absorption, Distribution, Metabolism, Elimination) tests.
  • the inner duct 4 which simulates an actual blood vessel found in human skin.
  • the inner duct 4 in fact, is not only a channel formed in the dermal layer 2 but also has a coating wall formed by live cells.
  • the use of fibroblasts in combination with the endothelial cells makes it possible to improve the biomechanical properties of the inner duct 4.
  • This skin model 1 therefore, is able to simulate the barrier effect which is normally present in blood vessels and that regulates the exchange of substances between blood and tissues by bringing oxygen, therefore, to the layers 2, 3, thus allowing the removal of waste substances or the formation of a nutrient concentration gradient, regulating leukocyte trafficking and transdermal penetration of drugs into the bloodstream.
  • the dermal layer 2 also comprises the polymer matrix that has a sustaining function for fibroblasts and endothelial cells.
  • the polymer matrix comprises photopolymerized methacrylate gelatin.
  • Methacrylate gelatin also abbreviated as “GelMA”
  • GelMA is a gelatin-based hydrogel functionalized with methacrylate groups that cross-link in the presence of a photoinitiator.
  • GelMA is a bioink widely used in bioprinting processes due to its biocompatibility, no-cytotoxicity and the presence of arginylglycylaspartic acid (RGD) for integrin binding and matrix metalloproteinase-sensitive groups for cell adhesion and migration (Bova, L. et al., Macromol Biosc (2022)).
  • RGD arginylglycylaspartic acid
  • the epidermal layer 3 in turn comprises a polymer matrix and the keratinocyte mixture.
  • the polymer matrix of the dermal layer 2 will be referred to as the “first polymer matrix” while the polymer matrix of the epidermal layer 3 will be referred to as the “second polymer matrix”.
  • the second polymer matrix also comprises photopolymerized methacrylate gelatin.
  • the dermal layer 2 and the epidermal layer 3 comprise GelMA at two different concentrations. This allows a stiffness gradient to be established between the two layers 2, 3, so as to mimic the viscoelastic properties of the tissue in vivo.
  • the first polymer matrix comprises methacrylate gelatin in saline buffer, in a concentration comprised between 5% and 10% m/V.
  • the first polymer matrix comprises methacrylate gelatin in saline buffer, in a concentration of 8% m/V.
  • the second polymer matrix comprises methacrylate gelatin in saline buffer, in a concentration comprised between 12% and 18% m/V.
  • the second polymer matrix comprises methacrylate gelatin in saline buffer, in a concentration of 15% m/V.
  • the second polymer matrix is denser and imparts greater rigidity to the epidermal layer 3.
  • the layers 2, 3 also have live cells dispersed in the relevant polymer matrices.
  • fibroblasts belong to the BJ cell line, i.e., neonatal preputial fibroblasts. It cannot, however, be ruled out that fibroblasts belong to a different cell line, e.g., to the HDF cell line, i.e., primary dermal fibroblasts.
  • Endothelial cells are derived from the HUVEC cell line, i.e., endothelial cells of the human umbilical cord.
  • Keratinocytes are derived from the HEK cell line, i.e., human epidermal keratinocytes.
  • the dermal layer 2 comprises the first polymer matrix and the mixture of fibroblasts and endothelial cells in a ratio comprised between 60:40 and 80:20. Preferably, in a ratio of 70:30.
  • fibroblasts are present in a cell density comprised between 1 million cells/mL and 2 million cells/mL and endothelial cells in a cell density comprised between 0.25 million cells/mL and 0.75 million cells/mL with respect to the first polymer matrix.
  • keratinocytes are present in a cell density comprised between 5 million cells/mL and 10 million cells/mL.
  • the cell density and the ratio of cells to their respective polymer matrices were appropriately selected in order to obtain a good cell population while imparting sufficient rigidity to the relevant layers 2, 3.
  • the dermal layer 2 has a thickness comprised between 0.7 mm and 1.3 mm.
  • the inner duct 4 comprises fibroblasts and endothelial cells in a ratio comprised between 15:85 and 35:65. Preferably, in a ratio of 30:70.
  • the inner duct 4 comprises a cross section having a characteristic dimension comprised between 0.3 mm and 0.8 mm. Specifically, if the inner duct 4 has a circular cross-section, the characteristic dimension is represented by the diameter of the cross-section, while if the inner duct 4 has a rectangular cross-section, the characteristic dimension is represented by the length of one of the sides of the cross-section.
  • the epidermal layer 3 comprises the keratinocyte mixture alone.
  • keratinocytes are present in a cell density comprised between 5 million cells/mL and 10 million cells/mL.
  • the present invention also relates to a three-dimensional printing process for the production of skin models.
  • the process according to the invention first comprises a phase of supply of a three- dimensional bioprinter and of a three-dimensional digital model of vascularized skin.
  • the three-dimensional bioprinter is of the type of an extrusion printer and is provided with at least three independent print heads mounted on a three-axis movement system.
  • the bioprinter has a temperature control system for controlling the print bed and the print heads.
  • the three-dimensional digital model has been designed using digital drawing programs of known type.
  • the process also comprises a phase of supply of: at least one bioink comprising a mixture of a polymerizable material and live cells selected from the list comprising: fibroblasts and endothelial cells; and at least one sacrificial bioink.
  • the process then comprises a phase of three-dimensional printing of the bioinks according to the digital model to obtain at least a first layer made in the first bioink and at least one track made in the sacrificial bioink, developing within the first layer and provided with at least two end stretches communicating with the outside.
  • the process comprises a phase of polymerization of the polymerizable material to obtain at least one dermal layer 2 comprising the polymer matrix and the mixture of fibroblasts and endothelial cells and within which the track develops.
  • the process then involves a phase of distribution of at least one mixture of keratinocytes on top of the first layer to obtain at least one epidermal layer 3.
  • the process comprises the phases of: removal of the track to obtain a through cavity; coating of the through cavity with fibroblasts and endothelial cells to obtain at least one inner duct 4 developing in the dermal layer 2 and configured to simulate a vascular canal of human skin; and cell growth carried out by means of direct perfusion of a culture medium through the inner duct 4 to obtain a skin model 1.
  • the phase of distribution is in turn carried out by means of three-dimensional printing.
  • the phase of supply involves the supply of a first bioink comprising a mixture of a first polymerizable material, fibroblasts and endothelial cells intended for making the dermal layer 2 and of a second bioink comprising a mixture of a second polymerizable material and keratinocytes intended for making the epidermal layer 3.
  • the sacrificial bioink is intended to make a through cavity within the dermal layer 2.
  • the first and the second bioinks comprise methacrylate gelatin that has not yet been polymerized, that is, in the form of a hydrogel suitable for extrusion.
  • Methacrylate gelatin can be synthesized using the protocol developed by Shirahama et al. (Sci Rep, 6, (2016)), as will be better described later in this disclosure.
  • Each of the first bioink and the second bioink also comprises a photo-initiating agent.
  • the photo-initiating agent is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.
  • Each of the first bioink and the second bioink also comprises cell lines in the quali- quantitative compositions described above.
  • the sacrificial bioink comprises a thermo-gelling polymer.
  • the sacrificial bioink is poloxamer 407.
  • Poloxamer 407 is a synthetic copolymer with a peculiar thermo-reversibility, that is, it is in the liquid state at temperatures below 20 °C and is in gel form at higher temperatures.
  • the printing phase is advantageously carried out at a temperature comprised between 20°C and 30°C.
  • the process then comprises the phase of three-dimensional printing of bioinks according to the digital model.
  • this phase makes it possible to achieve: at least a first layer made in the first bioink; at least one track made in the sacrificial bioink, developing within the first layer and provided with at least two end stretches communicating with the outside; and at least one second layer made in the second bioink and placed on top of the first layer.
  • the process involves a polymerization phase of the polymerizable materials to obtain: at least one dermal layer 2 comprising the first polymer matrix and the mixture of fibroblasts and endothelial cells and within which the track is developed; and at least one epidermal layer 3 comprising the second polymer matrix and the keratinocyte mixture.
  • the first polymerizable material generates the first polymer matrix and the second polymerizable material generates the second polymer matrix.
  • the first polymerizable material is a solution of methacrylate gelatin in saline buffer in a concentration comprised between 5% and 10% m/V. Preferably, in a concentration of 8% m/V.
  • the second polymerizable material is a solution of methacrylate gelatin in saline buffer in a concentration comprised between 12% and 18% m/V. Preferably, in a concentration of 15% m/V.
  • Polymerizable materials also comprise lithium phenyl-2,4,6- trimethylbenzoylphosphinate in a concentration of 0.1% m/V.
  • the polymerization phase is carried out by means of irradiation of the bioinks with electromagnetic radiation.
  • Electromagnetic radiation is selected from the list comprising: UV radiation and IR radiation.
  • the polymerization phase is performed by exposure to UV light (405 nm) for a time comprised between 1 min and 2 min.
  • the polymerization of the first layer and of the second layer is carried out simultaneously.
  • the process then comprises a phase of removal of the track to obtain a through cavity.
  • the removal phase comprises a cooling step of the track at a temperature of less than 20°C. Below this temperature, in fact, the sacrificial bioink is in the liquid phase and can be easily removed.
  • the phase of removal also comprises a washing step of the through cavity by means of a washing liquid.
  • the washing liquid can be a buffer solution, such as e.g. a phosphate buffer.
  • a sacrificial bioink allows the formation of the through cavity and, later, of the inner duct 4, without jeopardizing in any way the structural integrity of the through cavity itself and allowing perfusion to actively stimulate the rooting of the endothelial cells and of the fibroblasts to form the coating wall of the inner duct 4.
  • the process subsequently comprises a phase of coating the through cavity with fibroblasts and endothelial cells to obtain the inner duct 4 developing in the dermal layer 2 and configured to simulate a vascular canal of human skin.
  • the coating phase comprises a step of injecting a cell culture comprising fibroblasts and endothelial cells into the through cavity and a step of incubating the cell culture into the through cavity.
  • the cell culture comprises fibroblasts and endothelial cells in a mutual ratio comprised between 15:85 and 35:65 and at a total density comprised between 7 million cells/mL and 13 million cells/mL.
  • the cell culture incubation step is performed by cyclically flipping the layers 2, 3. By doing so, the cell culture cyclically coats the through cavity so as to promote even cell deposition.
  • the process comprises a cell growth phase carried out by means of direct perfusion of a culture medium through the inner duct 4 to obtain the skin model 1.
  • the culture medium is composed of equal parts of the culture media of the single cell lines.
  • the growth phase is carried out by means of a direct perfusion device 5.
  • Direct perfusion devices allow tissue models to be perfused continuously with solutions containing nutrients.
  • the skin model is contained in a device designed to ensure continuous perfusion of nutrients through the vascular canal.
  • the printing phase is carried out on a perfusion support 6 for direct perfusion devices 5.
  • the skin model 1 is made directly on a direct perfusion device.
  • the process also comprises a phase of attaching at least the first polymerizable material to the perfusion support 6.
  • the first polymerizable material comprises nucleophilic functional groups, in this case represented by the amine groups of the lysine residues of gelatin, and the attaching phase comprises a step of functionalization of the perfusion support 6 with electrophilic functional groups, performed prior to the printing phase.
  • the perfusion support 6 is of the type of a microscope slide made of biocompatible material.
  • the perfusion support 6 is provided with at least one housing seat 7 intended to receive the polymerizable materials and with at least one inlet duct 8 and at least one outlet duct 9 for a working fluid, connected to the housing seat 7 in a fluid-operated manner.
  • the perfusion support 6 is made of polydimethylsiloxane (PDMS), with a crosslinker-to-silicone ratio of 1 :20, through a replica molding technique and molded on a glass microscope slide.
  • PDMS polydimethylsiloxane
  • the functionalization step then involves activating the surface of the perfusion support 6 made of polydimethylsiloxane with electrophilic functional groups, specifically free carbonyl groups.
  • the free carbonyl groups establish covalent bonds with the amine functional groups of the gelatin, thus ensuring adhesion of the biomaterial to the microscope slide perfusion support.
  • this process differs from what previously described in that the phase of distribution is carried out by deposition of a mixture of keratinocytes in a culture medium above the first layer.
  • the phase of distribution is carried out after the phase of polymerization of the polymerizable material.
  • the epidermal layer 3 acquires a degree of rigidity similar to that of the human epidermis and to that which would be obtained in accordance with the first embodiment, by means of the second polymer matrix.
  • this invention also relates to a skin model 1 obtainable by the process according to one or more of the previously described embodiments.
  • this invention also relates to a direct perfusion device 5 for bio-printed skin models.
  • the direct perfusion device 5 comprises at least one perfusion support 6 provided with at least one housing seat 7 and with at least one skin model 1 according to one or more of the above-described embodiments, arranged in the housing seat 7, and with at least one inlet duct 8 and with at least one outlet duct 9 for a working fluid, connected to the inner duct 4 in a fluid-operated manner.
  • each of the inlet duct 8 and the outlet duct 9 is connected to an end stretch of the inner duct 4.
  • the skin model 1 is, therefore, directly bio-molded in the housing seat 7.
  • the skin model 1 is attached to the housing seat 7 by means of covalent bonds.
  • the direct perfusion device 5 also comprises an adjustable compression unit 10 adapted to contain the perfusion support 6.
  • the direct perfusion device 5 also comprises a tank 11 adapted to contain the culture medium and connected to the perfusion support 6 through the adjustable compression unit 10.
  • the direct perfusion device 5 also comprises at least one pumping unit 12 positioned between the tank 11 and the perfusion support 6 for the movement of fluids.
  • the flow of cell medium through the vascular canal was established by connecting the skin model 1 to a perfusion circuit.
  • the perfusion support 6, arranged within the adjustable compression unit 10 is connected to the pumping unit 12 and to the tank 11 through gas-permeable silicone tubing with an inner diameter of 0.51 mm.
  • the perfusion support 6 is fitted within an adjustable compression unit ( Figure 4).
  • This element has a dual purpose: to compress the PDMS to seal the skin model 1, thus preventing possible leakage of fluid, and to connect the perfusion support 6 to the tank 11 of the culture medium and to the pumping unit 12. Specifically, compression is carried out through butterfly screws, thereby moving the upper block of the adjustable compression unit 10 to the required height.
  • the adjustable compression unit 10 is connected to the perfusion support 6 through adapters that fit snugly into the inlet and outlet ends of the culture medium present in the adjustable compression unit, thus limiting pressure drops in the stretch.
  • the set of the adjustable compression unit 10 and the perfusion support 6 is connected to the pumping unit 12 and to the tank 11 of the culture medium through adapters into which the fluid movement tubing is directly fitted.
  • the culture medium is collected in the tank 11 and then recirculated to the skin models 1 in a closed loop.
  • the tank 11 of the culture medium is capable of collecting up to 35 mL of medium, and the connection to the other elements of the loop is ensured by the presence of adapters that allow direct insertion of the fluid-carrying tubing.
  • the tank 11 is designed so that up to ten skin models, i.e., two perfusion supports 6, can be connected in parallel.
  • the fluid is taken from the tank 11 and is pumped through the skin models 1 using a peristaltic pump.
  • the flow rate of the culture medium is changed during the experiment: the first two days of culture is kept at 15 pL/min, then increased to 30 pL/min for the following two days and finally raised to 45 pL/min until the tissue matures.
  • the direct perfusion device 5 allows the skin model to be maintained in active perfusion through the continuous supply of nutrients and oxygen via the inner duct 4, without the need to use porous membranes to recreate the contact membrane between fluid and cellular material, and without having to separate the various layers since the perfect physiology of the structures allows the transit of nutrients (via diffusion), within the cellular membranes.
  • the wall of the inner duct 4, coated with endothelial cells mimics the biophysical and biological characteristics of the vascular system in vivo.
  • GelMA is chemically synthesized by functionalizing gelatin with methacrylic anhydride to enable photo cross-linking of the biomaterial.
  • the key parameter to be monitored for GelMA synthesis is the degree of functionalization (DoF, or degree of substitution), which represents the percentage of lysine functionalized with methacrylate groups.
  • DoF was determined for each synthesized batch using proton magnetic resonance analysis (H-NMR).
  • a 10% (w/v) solution of GelMA with a degree of functionalization (DoF) of 70% is prepared by initially dissolving a type A gelatin (-300 blooms from pig skin) in a 0.25 M carbonate-bicarbonate buffer (CB buffer) (sodium carbonate and anhydrous sodium carbonate in IxPBS (phosphate buffer solution) for 20 min at 40°C and constant stirring (800 rpm).
  • CB buffer carbonate-bicarbonate buffer
  • IxPBS phosphate buffer solution
  • the pH is adjusted to 9.2-9.4 by adding HC1 (hydrochloric acid, 37%).
  • HC1 hydrochloric acid, 37%)
  • H-NMR Proton nuclear magnetic resonance
  • DoF Degree of functionalization
  • Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0.1% m/v) is dissolved in warm IxPBS (40°C) for 20 min with constant stirring (800 rpm), then the solution is filtered (0.22 pm filters) into a falcon containing lyophilized and weighed GelMA. The mixture is kept at 37°C and stirred intermittently until GelMA is completely dissolved. Before use, the ink is centrifuged to remove air bubbles (3500 rpm, 5 min). The sacrificial bioink is composed of a solution of Pluronic F-127 (powder) in cold IxPBS (4°C). Intermittent mixing and stirring result in a clear solution.
  • SR Swelling capacity
  • Young’s modulus was derived from indentation data obtained with an atomic force microscope (Park Instruments XE-Bio AFM (South Korea)) provided with an inverted microscope (Nikon Eclipse Ti). Force-displacement curves were obtained using PPP-CONTSCR-10 pyramidal tips mounted on SisN4 cantilevers with a nominal elastic constant of 0.2 N m' 1 (NanoSensors, Neuchatel, Switzerland). The elastic constants of cantilevers were calibrated by the manufacturer before use. Before each test, AFM photodetector sensitivity (optical lever sensitivity) was calculated by measuring the slope of the force-distance curve acquired on a silicon standard. All experiments were performed at room temperature, in a fluid environment (DPBS).
  • DPBS fluid environment
  • Indentation curves were acquired by approaching the sample surface at a rate of 3 pm s' 1 and producing an indentation with a depth of 3 pm. At least six force curves were recorded at different locations for a minimum of three samples per condition. Young’s modulus was calculated by applying a Hertz model fit to each individual force-distance curve, assuming a Poisson’s ratio of 0.5. The evaluation of Young’s modulus allows the mechanical properties of the polymer matrices to be determined and the similarities/differences thereof to real human tissues to be assessed.
  • D Diffusion coefficient
  • the diffusion coefficient was obtained by fluorescence recovery after photobleaching (FRAP) assay.
  • the assays were performed with dextran of different molecular weights (4kDa, 70kDa, 250kDa) to measure the diffusion coefficient for a wider range of molecules.
  • 100 pL of the first polymerizable material (GelMa 8%) and of the second polymerizable material (GelMa 15%) were cast and polymerized, incubated overnight in 4-kDa, 70-kDa, and 250-kDa dextran, and observed with a confocal microscope (ZEISS LSM 800).
  • Printability analysis was evaluated following the printability parameter (Pr) introduced by Ouyang et al. (Biofabrication, 8, (2016)). The calculation was based on the equation that determines the circularity of a closed area:
  • Pr 1 when the area enclosed by the printed filaments is a perfect square.
  • Bioinks were printed and tested under different conditions to optimize the printing parameters and multi-material printing protocol to reproduce the desired model.
  • Gelatin-based hydrogels are highly temperature-dependent and identifying the optimal temperature value during the printing phase is critical to achieve a smooth, well- defined filament.
  • different values were set in the temperature-controlled print head and monitored the aforementioned printability parameter.
  • Bioinks were loaded into the extrusion syringes and mounted in the temperature-controlled print heads of an extrusion bioprinter.
  • hydrogels were printed in single-layer grids with square gaps between the filaments; calculation of the perimeter and of the area of the gaps provides the printability parameter, Pr.
  • the print bed was set at 22°C throughout the process to promote the formation of the physical GelMA gel.
  • BJ cells preputial neonatal fibroblasts
  • EMEM Eagle’s Minimum Essential Medium
  • FBS Fetal Bovine Serum
  • MEM Minimum Essential Medium
  • P/S Penicillin / Streptomycin
  • HEK human epidermal keratinocytes
  • the cell lines were then added to the polymerizable materials in a 1 :25 ratio of cells suspended in medium to GelMA so as not to alter the properties of the hydrogel.
  • the cell densities used for the dermis were 1.5 million cells/mL of first polymerizable material for fibroblasts and 0.5 million cells/mL of first polymerizable material for endothelial cells.
  • the cell density used for the epidermis is 7 million cells/mL of second polymerizable material for keratinocytes.
  • Functionalization involves activation of the surface by plasma treatment and, then, vapors of (3 -aminopropyl)tri ethoxy silane (APTES) are bonded to the activated surface (2h, vacuum gas phase).
  • APTES is an amino-silane mainly used as a dispersion agent; it can attach an amine group to functional silane for bio-conjugation.
  • 0.5% glutaraldehyde is added to bind to the amine groups (Ih, liquid phase).
  • the free carbonyl group of the glutaraldehyde is then involved in forming the bond with the amine functional groups of the gelatin, thus ensuring adhesion of the biomaterial to the glass microscope slide.
  • the bioinks are transferred into extrusion syringes and mounted on the bioprinter.
  • the first bioink and the second bioink were mounted on temperature-controlled print heads with 25G conical nozzles, while the sacrificial bioink did not require temperature control and was printed through a 27G conical nozzle.
  • the printing speed was set at 5 mm s' 1 .
  • the first layer and the second layer (when provided) were exposed to UV light (LED, 405 nm) for 90s to cross-link the structure.
  • Inner duct endothelialization was performed following the post-seeding method after the printing process. Once the sacrificial bioink was washed from the through channel, HUVEC and BJ were injected at a total density of 10 million cells/mL medium in a ratio of 70:30%, respectively. The structure was then kept at 37°C for 4 hours, turning it upside down every 30 minutes, before gently washing the unattached cells with warm medium. Finally, the structure was connected to the perfusion circuit and incubated at 37°C and 5% CO2.
  • Figure 11 shows the graph of cell viability assessment at day 2 and day 7 (with p value ⁇ 0.001).
  • Figure 12 shows an image of the inner duct wall: (i) portion of the vascular canal at day 14 of perfusion, (ii) complete formation of the vascular lumen.
  • Vascularized structures were fixed and stained before being analyzed by confocal microscopy.
  • the constructs were first washed with IxPBS (3 cycles, 5 minutes each) and fixed in paraformaldehyde (PF A, 4% v/v) at 4°C for 4 hours. After further washing with IxPBS (3 cycles, 5 min each), they were permeabilized with PBS-T (Triton X-100 0.1% in IxPBS) for 30 min. F-actin (1 :250, 45 min) and DAPI (1 :500, 15 min) staining were preceded by a rinse step with IxPBS (3 cycles, 5 min each). Confocal microscopy was performed on a confocal microscope (ZEISS LSM 800), using spectral lasers at 561 nm and 455 nm.
  • bio-printed skin model the related three-dimensional printing process and the direct perfusion device provided with said model according to the present invention allow for the effective simulation of human skin both structurally and functionally.
  • bio-printed skin model the related three-dimensional printing process and the direct perfusion device allow in vitro tests to be performed that are representative of real human skin behavior.
  • this bio-printed skin model, the related three-dimensional printing process and the direct perfusion device make it possible to drastically reduce and thus largely replace the performance of additional in vivo tests.

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Abstract

Le modèle de peau bio-imprimée (1) comporte : - au moins une couche dermique (2) comportant au moins une matrice polymère et un mélange de fibroblastes et de cellules endothéliales et conçue pour simuler un tissu dermique humain ; - au moins une couche épidermique (3) comportant au moins un mélange de kératinocytes et conçue pour simuler un tissu épidermique humain ; la couche dermique (2) comportant au moins un conduit interne (4) constitué de fibroblastes et de cellules endothéliales, conçu pour simuler un canal vasculaire de la peau humaine et pourvu d'au moins deux tronçons d'extrémité communiquant avec l'extérieur et pouvant être reliés à un système de perfusion active de substances.
PCT/IB2024/055478 2023-06-06 2024-06-05 Modèle de peau bio-imprimée, procédé d'impression tridimensionnelle associé et dispositif de perfusion directe équipé dudit modèle Ceased WO2024252292A1 (fr)

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EP24737819.3A EP4724558A1 (fr) 2023-06-06 2024-06-05 Modèle de peau bio-imprimée, procédé d'impression tridimensionnelle associé et dispositif de perfusion directe équipé dudit modèle
CN202480038381.4A CN121693559A (zh) 2023-06-06 2024-06-05 生物打印的皮肤模型、相关的三维打印方法和具备所述模型的直接灌注装置

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IT102023000011436A IT202300011436A1 (it) 2023-06-06 2023-06-06 Modello di pelle biostampato, relativo processo di stampa tridimensionale e dispositivo di perfusione diretta provvisto di tale modello
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