Method of dehydration of extracellular matrix and particles formed therefrom

Abstract

To provide a method for extracellular matrix (ECM) particle formation.SOLUTION: A method for ECM particle formation includes the steps of: providing one or more portions of perfusion decellularized mammalian organ comprising extracellular matrix (ECM); dehydrating the one or more ECM portions at a temperature from about 1°C to about 30°C; and subjecting the dehydrated ECM portions to milling, thereby making a population of particles, wherein at least 90% of the particles are less than about 2 mm in size, and the population has a moisture content of less than about 10%.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Application No. 62 / 789,218, filed January 7, 2019, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] background Three-dimensional (3D) printing is becoming a popular technique to fabricate scaffolds and devices for tissue engineering. This is due to 3D printing's ability to offer patient-specific designs, high structural complexity, and rapid, on-demand manufacturing at low cost. One of the major bottlenecks limiting the widespread adoption of 3D printing in biomanufacturing is the lack of variety in "bio-based inks."

[0003] While many printable materials have impressive properties for in vitro applications, implantable biomaterials require specific characteristics based on both physiological conditions and interactions with the body, making them more challenging to develop. In general, printable biomaterials must (1) be printable, (2) be biocompatible, (3) have appropriate mechanical properties, (4) have good degradation kinetics, (5) form safe degradation byproducts, and (6) exhibit biomimicry to tissue. How each of these requirements is met varies slightly depending on which printing method is used and the planned end use of the device. Furthermore, many of these characteristics can work against each other. For example, in bone tissue, it is desirable to have a hard material for osteoblast development and load bearing, but this may slow or not degrade. Soft materials can be printable and quickly biodegrade, but their ability to be handled and applied to certain tissue types can be a concern. Most 3D printed constructs are used in bone or cartilage applications because most printed biomaterials have an inherent stiffness outside of the hydrogel system that mimics the natural stiffness of these tissues. Summary of the Invention

[0004] Abstract The present disclosure provides methods of dehydrating decellularized extracellular matrix from a mammalian organ, e.g., a porcine or human organ, e.g., liver, pancreas, kidney, lung, spleen or heart, including portions thereof having dimensions of, e.g., about 10 x Y inches, 5 x Y inches, 2 x Y inches, 1 x Y inches, 0.5 x Y inches, 0.25 x Y inches or 0.1 x Y inches, where Y can be 0.1-1 inch, 0.5-5 inches, 0.2-1 inch, 1-5 inches or 1-10 inches, without the application of heat, e.g., dehydration is performed at a temperature of less than about 75°F, less than about 70°F, less than about 68°F, less than about 25°C, less than about 22°C or less than about 19°C. In one embodiment, the portion can be compressed prior to dehydration. For example, the thickness of a portion of decellularized extracellular matrix derived from a mammalian organ can be compressed by at least 0.01%, 0.5%, 1%, 5%, 10%, 20%, 50%, 90% or more, hi one embodiment, the dehydrated portion maintains most of the composition of the original organ after dehydration at ambient temperature and optional compression to remove water from the decellularized organ portion. The dehydrated material may then be subjected to milling, e.g., freeze milling, to provide a population of particles ranging in size from about 0.001-0.005 mm up to about 10 mm, including about 0.01 mm to about 0.05 mm, about 0.05 mm to about 0.1 mm, 0.1 mm to about 5 mm, about 0.25 mm to about 0.5 mm, about 0.4 mm to about 4 mm, about 1 mm to about 2 mm, about 0.5 mm to about 1.5 mm, about 1.5 mm to about 3 mm, or about 3 mm to about 4 mm, which may be obtained through milling or milling and sizing, e.g., using a sieve or other size separation device or method. The original composition of the whole organ extracellular matrix, in the dehydrated and milled extracellular matrix particles, provides a structure for preparing organ-specific inks for use in printing 3D structures that enable therapy or aid in the function of the organ. The inks can be prepared by sizing the dehydrated particles before combining in a solvent, such as an aqueous solvent, such as water or phosphate buffered saline (PBS), or by subjecting the dehydrated particles to chemical or enzymatic digestion.Bioprinting of the ink can be done through any method, including but not limited to thermal inkjet bioprinting, piezo inkjet bioprinting, pneumatic extrusion bioprinting, mechanical extrusion bioprinting, or laser-assisted bioprinting. Thus, the particles can be used in methods including but not limited to ex vivo cell assays or compositions useful in the preparation of tissue engineering constructs for in vivo use, such as printed structures based on perfused decellularized ECM that are implanted for in vivo remodeling or combined with cells to provide functional implants. [The present invention 1001] Providing one or more portions of a perfused decellularized mammalian organ comprising an extracellular matrix (ECM); Dehydrating one or more ECM portions at ambient temperature; and subjecting the dehydrated ECM portion to milling, thereby providing a population of ECM particles. A method for forming ECM particles, comprising: [The present invention 1002] The method of claim 1001, wherein the portion is compressed before dehydration. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the portion or portions are expanded with a gas prior to dehydration. [The present invention 1004] 1004. The method of claim 1003, wherein the perfused decellularized mammalian organ comprising said extracellular matrix is ​​inflated with gas prior to providing one or more portions thereof. [The present invention 1005] The method according to any one of claims 1001 to 1004, wherein said ECM is liver, heart, lung or kidney ECM. [The present invention 1006] The method of claim 1003, wherein said ECM is porcine or human. [The present invention 1007] The method according to any one of claims 1001 to 1006, wherein the portion is dehydrated at a temperature of about 1°C to about 30°C. [The present invention 1008] The method of any of claims 1001 to 1007, wherein the portion is placed in a physiologically compatible solution before dehydration. [The present invention 1009] The method of claim 1008, wherein said solution comprises water or PBS. [The present invention 1010] The method of any of claims 1001-1009, wherein said milling produces a population in which at least 90% of the particles are less than about 2 mm in size. [The present invention 1011] The method of any of claims 1001-1009, wherein said milling produces a population in which at least 90% of the particles are less than about 0.15 mm in size. [The present invention 1012] The method of any of claims 1001 to 1009, further comprising the step of separating said population of particles by size. [The present invention 1013] The method of claim 1012, wherein said separation is performed by sieving, by subjecting said population to acoustic energy, by subjecting said population to density separation, or by using a fluid. [The present invention 1014] The method of any of claims 1001 to 1013, further comprising a step of subjecting the particles to enzymatic digestion. [The present invention 1015] The method of claim 1014, wherein the enzymatically digested particles comprise high molecular weight collagen. [The present invention 1016] The method of any one of claims 1001 to 1015, wherein the population has a water content of less than about 10%. [The present invention 1017] dehydrating the ECM-containing perfused decellularized mammalian organ or one or more portions thereof in a planar configuration at ambient temperature; and subjecting the dehydrated portion to freeze-milling, thereby providing a population of ECM particles. A method for forming ECM particles, comprising: [The present invention 1018] The method of the present invention 1017, further comprising compressing said portion. [The present invention 1019] The method of any one of claims 1017 to 1018, wherein the ECM is a liver ECM. [The present invention 1020] The method according to any one of claims 1017 to 1019, wherein the portion is dehydrated at a temperature of about 1°C to about 30°C. [The present invention 1021] The method of any of claims 1017 to 1020, wherein said milling produces a population in which at least 90% of the particles are less than about 2 mm in size. [The present invention 1022] The method of any one of claims 1017 to 1021, wherein said milling produces a population in which at least 90% of the particles are less than about 0.15 mm in size. [The present invention 1023] The method of any of claims 1017 to 1022, further comprising the step of separating the population of particles by size. [The present invention 1024] The method of any of claims 1017 to 1023, further comprising a step of subjecting the particles to enzymatic digestion. [The present invention 1025] The method of any one of claims 1017 to 1024, wherein the population has a water content of less than about 10%. [The present invention 1026] A population of particles produced by any one of the methods of the present inventions 1001 to 1025. [The present invention 1027] A population of the present invention 1026, wherein at least 95% of the particles are less than about 2 mm in size. [The present invention 1028] A population of the present invention 1026, wherein at least 95% of the particles are less than about 0.15 mm in size. [The present invention 1029] A gel comprising the population of 1026, 1027 or 1028 of the present invention. [The present invention 1030] The gel of claim 1029, further comprising mammalian cells. [The present invention 1031] The gel of the present invention, wherein the mammalian cells are human cells. [The present invention 1032] Use of the population of 1026, 1027 or 1028 or the gel of 1029, 1030 or 1031. [The present invention 1033] Use of the mass or gel of the present invention 1033 for 3D printing. [The present invention 1034] Use of the population of invention 1026, 1027 or 1028 for treating wounds. [The present invention 1035] Providing a bio-ink composition comprising the population of invention 1026, 1027 or 1028 or the gel of invention 1029, 1030 or 1031; and Applying the bio-ink composition to form a 3D structure. and a method of using bioinks for 3D printing, comprising: [The present invention 1036] The method of claim 1035, wherein the bio-ink composition further comprises cells. [The present invention 1037] The method of any one of claims 1035 to 1036, wherein the bioink composition further comprises an isolated protein or glycoprotein. [The present invention 1038] A 3D structure prepared by any one of the methods of the present invention 1035 to 1037. [The present invention 1039] 1038. A construct of the present invention comprising a cell. [Brief description of the drawings]

[0005] [Figure 1] Moisture content analysis. [Diagram 2] Particle size analysis. [Diagram 3]Gel showing the size of products of perfused decellularized liver extracellular matrix (dehydrated ECM; dECM) dried in a non-destructive manner (e.g., at ambient temperature). 1) MW marker (Biorad); 2) 1 mg / mL, 10 μL; 3) 1 mg / mL, 10 μL; 4) 2.5 mg / mL; 5) 2.5 mg / mL; 6) 5 mg / mL; 7) 5 mg / mL; 8) 10 mg / mL; 9) 10 mg / mL; 10) 1 mg / mL, 10 μL; 11) 1 mg / mL, 10 μL; 12) 2.5 mg / mL; 13) 2.5 mg / mL; 14) 5 mg / mL; 15) 5 mg / mL. Lanes 2-9, no NaOH; lanes 10-15, NaOH added. Even-numbered lanes contain samples where the reaction was performed at room temperature, and odd-numbered lanes contain samples where the reaction was performed at 4 °C. [Figure 4] Examples of drying and freeze milling of perfused decellularized matrices. [Diagram 5] Example of preparation and storage of dECM liver particles. [Figure 6] Moisture content analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description The present disclosure provides a decellularized particle product prepared by dehydration of a decellularized whole organ, e.g., a perfused decellularized whole organ. Many dehydration methods use high temperatures to achieve liquid removal. The method of the present invention uses selection or sizing of the decellularized material, optional compression, followed by spacing, e.g., in a laminar flow hood, for a defined time. In one embodiment, dehydration reduces the water content in the product to less than 10%, e.g., less than about 9%, 8%, 7%, or 5%. In one embodiment, compression and dehydration reduces the water content in the product to less than 8%, e.g., less than about 7%, 6%, 5%, or 4%. The ambient temperature of the dehydration process is non-destructive to any extracellular matrix components, thereby producing a product with potentially higher efficacy. This material can then be freeze-milled into a particulate powder that can be used as particulate in many embodiments, including but not limited to 3D printing, or can be dissolved into a liquid for gel formation by digestion into a solution that can be printed through reorganization by temperature, chemical crosslinking, UV crosslinking or other means, including but not limited to protease digestion, and can be dissolved into a liquid for use as an ink in 3D printing. The particles can also have direct therapeutic capabilities, such as direct application to wounds, filling voids, intramuscular injection, tunnel wounds and fistulas.

[0007] Source of decellularized ECM In one embodiment, the method of decellularizing a mammalian organ or tissue comprises cannulating the organ or tissue. The vessels, ducts and / or cavities of the organ or tissue can be cannulated using methods and materials known in the art. The next step in decellularizing an organ or tissue is to perfuse the cannulated organ or tissue with cell disruption media. Perfusion through the organ can be multidirectional (e.g., antegrade and retrograde).

[0008] Langendorff perfusion of the heart is a common technique in the art as physiological perfusion (also known as four-chamber working mode perfusion). See, for example, Dehnert, The Isolated Perfused Warm-Blooded Heart According to Langendorff, In Methods in Experimental Physiology and Pharmacology: Biological Measurement Techniques V. Biomesstechnik-Verlag March GmbH, West Germany, 1988. Briefly, in Langendorff perfusion, the aorta is cannulated and connected to a reservoir containing a cytolysis medium. The cytolysis medium can be delivered retrogradely through the artery either at a constant flow rate delivered, for example, by infusion or a roller pump, or by constant hydrostatic pressure. In both instances, the aortic valve is forced closed and the perfusion fluid is directed to the coronary ostia (thereby perfusing all the ventricles of the heart) and then drains into the right atrium via the coronary sinus. In the perfusion mode of operation, a second cannula can be connected to the left atrium and perfusion can be switched from retrograde to antegrade.Methods of perfusing other organs or tissues, including the lungs, liver, pancreas, spleen, kidneys, brain and limbs, are known in the art.

[0009] To decellularize an organ or tissue, one or more cell disruption media may be used. The cell disruption media usually includes at least one detergent, such as SDS, PEG, or Triton X. The cell disruption media may include water to be osmotically incompatible with the cells. Alternatively, the cell disruption media may include a buffer (e.g., PBS) for osmotically compatibility with the cells. The cell disruption media may also include an enzyme, such as, but not limited to, one or more collagenases, one or more dispases, one or more DNases, or a protease, such as trypsin. In some examples, the cell disruption media may also or alternatively include one or more enzyme inhibitors (e.g., protease inhibitors, nuclease inhibitors, and / or collagenase inhibitors).

[0010] In certain embodiments, cannulated organ or tissue can be perfused with two different cell disruption media successively.For example, the first cell disruption media can include an anionic surfactant, such as SDS, and the second cell disruption media can include an ionic surfactant, such as Triton X-100.After perfusion with at least one cell disruption media, cannulated organ or tissue can be perfused with, for example, a washing solution and / or a solution that includes one or more enzymes, such as those disclosed herein.

[0011] Alternating the direction of perfusion (e.g., antegrade and retrograde) may help effectively decellularize the entire organ or tissue. The decellularization described herein essentially decellularizes the organ from its inside out, with little damage to the ECM. The organ or tissue may be decellularized at an appropriate temperature between 4 and 40°C. Depending on the size and weight of the organ or tissue and the individual surfactants and surfactant concentrations in the cell disruption medium, the organ or tissue is typically perfused with the cell disruption medium for about 2 to about 12 hours per gram of solid organ or tissue. Including washing, the organ may be perfused for up to about 12 to about 72 hours per gram of tissue. Perfusion is typically adjusted to physiological conditions, including pulsatile flow rate and pressure.

[0012] A decellularized organ or tissue essentially consists of the extracellular matrix (ECM) components of all or most of the regions of the organ or tissue, including the ECM components of the vascular tree. The ECM components may include any or all of the following: fibronectin, fibrillin, laminin, elastin, members of the collagen family (e.g., collagen I, III, and IV), glycosaminoglycans, ground substance, reticular fibers, and thrombospondin, which may remain organized as defined structures, such as basal lamina. Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and nuclei in histological sections using standard histological staining procedures. Preferably, but not necessarily, remaining cellular debris is also removed from the decellularized organ or tissue.

[0013] The morphology and structure of the ECM (before dehydration) can be examined visually and / or histologically. For example, the basement membrane on the outer surface of a solid organ or within the vasculature of an organ or tissue should not be removed or significantly damaged by decellularization. In addition, the fibrils of the ECM should be similar to or not significantly altered from that of a non-decellularized organ or tissue. Unless otherwise indicated, decellularization as used herein refers to perfusion decellularization, and unless otherwise indicated, the decellularized organs or matrices referred to herein are performed using perfusion decellularization as described herein. The perfusion decellularization described herein can be compared to immersion decellularization as described, for example, in U.S. Patent Nos. 6,753,181 and 6,376,244. Either method can be used as a source of ECM for the methods disclosed herein.

[0014] Thus, decellularization of solid organs removes most or all of the cellular components while substantially maintaining the extracellular matrix (ECM) and vascular bed. Mammals from which solid organs can be obtained include, but are not limited to, rodents, pigs, rabbits, cows, sheep, dogs, and humans. The organs and tissues used in the methods described herein can be cadaveric, or fetal, neonatal, or adult. Solid organs referred to herein include, but are not limited to, the heart, liver, lungs, skeletal muscle, brain, pancreas, spleen, kidneys, stomach, uterus, and bladder. In one embodiment, a solid organ refers to an organ with a "substantially closed" vascular system. A "substantially closed" vascular system in the context of an organ means that during perfusion with liquid, it is assumed that the majority of the liquid is contained within the solid organ and does not leak out of the solid organ, and that major vessels are cannulated, ligated, or otherwise restricted. Despite having a "substantially closed" vascular system, many of the solid organs listed above have defined "inflow" and "outflow" vessels that are useful for introducing and moving fluids through the organ during perfusion.

[0015] In addition to the solid organs described above, other types of vascularized organs or tissues can be decellularized using the methods disclosed herein, such as all or portions of joints (e.g., knee, shoulder, hip, or vertebrae), trachea, skin, mesentery or intestine, small and large intestine, esophagus, ovary, penis, testes, spinal cord, or single or branched vessels. Additionally, the methods disclosed herein can also be used to decellularize non-vascularized (or relatively non-vascularized) tissues, such as cartilage or cornea.

[0016] The decellularized organs or tissues described herein (e.g., heart or liver) or any portion thereof (e.g., aortic valve, mitral valve, pulmonary valve, tricuspid valve, pulmonary vein, pulmonary artery, coronary vessels, septum, right atrium, left atrium, right ventricle, left ventricle, or liver lobes) may be used as a source of ECM in methods of dehydrating and forming ECM-based particles.

[0017] Exemplary Methods Prior to dehydration, the decellularized material may be processed in various batches and stored for pooling and later processing. In one embodiment, the method involves incubation of the compacted decellularized whole organ material in a pH-neutral sterilant, such as a 500 ppm peracetic acid (PAA) bath (5-10 L) for about 5 minutes under gentle agitation. Other sterilants may also be used, including but not limited to lactic acid, alcohol, hydrogen peroxide, hypochlorite, carbon dioxide, sodium dichloroisocyanurate, chloramine-T, etc. In one embodiment, this is followed by a 5 minute deionized water bath (5-10 L) under gentle agitation. After sterilization and washing, the material may be aseptically packaged using deionized water or other physiologically compatible solution to prevent the material from drying out. The packages are then stored at 4°C. After accumulating several lots of material, the material may be pooled and used to manufacture other classes of products.

[0018] Thus, in one embodiment, the process results in a material produced by perfusion decellularization of a mammalian organ or portion thereof and gentle compression of any organ. The material may be stored at 4° C. until dehydrated. For dehydration, in one embodiment, the material is sized into approximately 1″ x 1″ strips and then placed in a laminar flow hood (LFH) in a sterile environment. The strips may be left in the LFH used for a period of approximately 24-48 hours or until they appear sufficiently dehydrated. Complete dehydration may be determined by a visually distinct color change in the material. After drying and processing, the material may be analyzed for water content.

[0019] After dehydration, the dried decellularized ECM can then be milled, e.g., freeze-milled, to produce a fine ECM powder (having particles) and optionally sized, which can be packaged, used aseptically, or sterilized through a variety of methods, including e-beam or gamma irradiation. The powdered ECM can then be further processed through enzymatic digestion to provide an increased surface area that allows for printable inks for 3D printing.

[0020] Extrusion-Based 3D Printing Methods The particles generated by dehydration and sizing can be used in extrusion-based 3D printing methods, such as fused deposition modeling (FDM) and direct ink writing (DIW), to fabricate devices and scaffolds for tissue engineering. In these methods, ink is ejected through a nozzle as a viscous liquid following a predefined path determined by a computer model that builds the 3D object layer by layer. Solvent-cast direct writing inks contain hydrogels that maintain their structure after extrusion.

[0021] Inkjet printing Inkjet printing allows for the deposition of individual droplets of very small volume (1-100 picoliters) from a nozzle onto a printing surface with the aim of forming a construct after solidification. To accelerate this printing process, multi-nozzle inkjet printheads containing hundreds of individual nozzles have been developed. Based on the droplet generation mechanism, inkjet printers are broadly divided into two groups: continuous inkjet (CIJ) printing and drop-on-demand (DOD) inkjet printing. In CIJ printing, a continuous stream of droplets (diameter about 100 μm) is generated and unused ink is recycled. In DOD inkjet printing, individual droplets (diameter range 25-50 μm) are generated when needed. DOD type printers are commonly used in tissue engineering applications. The capability of inkjet printing is the spatial resolution, e.g., the placement of picoliters of droplets containing ECM particles with high positional precision (about 10 μm in xy axis). The most important properties of the ink are viscosity and surface tension. The viscosity of the ink should be appropriately low, typically below 10 cP (mPa s) under high shear rates between 1 x 105 and 1 x 106-1. Surface tension determines the shape of the droplets ejected from the nozzle and on the substrate. Surface tension values ​​for inks generally range from 28 to 350 mN m-1. The resolution and precision of the printed object are determined by the interactions between adjacent droplets (coalescence) and between individual droplets and the substrate (e.g., surface tension and wetting). The liquid-to-solid phase transition (i.e., solvent evaporation, temperature-controlled transition, or gelation of the precursor solution) controls the final shape and size of the printed object.

[0022] Laser-assisted 3D bioprinting Laser-assisted 3D bioprinting (LAB) is a non-contact, nozzle-free printing process that sends a laser pulse through a "ribbon" containing a bioink containing, for example, ECM particles as described herein. The ribbon is supported by a titanium or gold layer that can absorb and then transfer energy to the ribbon. The bioink and optionally cells are suspended at the bottom of the ribbon and, when ablated by a laser pulse, create a high-pressure bubble that ultimately propels individual droplets to a receiving substrate located just beyond the ribbon. This process is repeated to create functional 3D structures. LAB has demonstrated high retention of phenotype and cell viability after printing.

[0023] Stereolithography Stereolithography (SLA) bioprinting utilizes photopolymerization, a process in which UV light or a laser is sent patternwise down the path of a photopolymerizable liquid polymer, thereby crosslinking the polymer into hardened layers. As each layer is polymerized, the print platform is lowered further into the polymer solution, allowing multiple cycles to form a 3D structure.

[0024] Hydrogel Ink Hydrogels are three-dimensional polymer networks with the ability to retain large amounts of water, providing a microenvironment with tunable organization, degradability and functionalization. Hydrogel inks can be referred to as bioinks when they contain cells and / or biochemical molecules, such as ECM components. Inkjet, light-assisted and extrusion-based 3D printing systems are the most common methods for hydrogel printing. The classical approach to designing hydrogel inks is to prepare a polymer solution that immediately forms a network after printing. This network can be physically or chemically crosslinked in response to external stimuli, such as temperature, light or ion concentration. The main advantage of physically crosslinked hydrogels is the absence of chemical agents, which reduces the toxicity of the material. On the other hand, chemically crosslinked hydrogels are prepared through the formation of covalent bonds, and the resulting hydrogels have a higher resistance to mechanical forces, which usually cause a larger volume change than physically crosslinked networks.

[0025] The physicochemical properties and gelation method of the hydrogels formed containing the ECM can be tuned through chemical, physical and / or enzymatic mechanisms or can be adjusted by thermal / pH sensitivity. ECM-based bioinks can be prepared, for example, as a solution (e.g., 3%), remain in solution at or below 15° C., gel within 30 minutes at 37° C., and be pH adjusted to physiological pH.

[0026] Exemplary Aspects In one embodiment, the disclosure provides a method of forming ECM particles. The method includes providing one or more portions of a decellularized mammalian organ comprising ECM; dehydrating the one or more portions at ambient temperature (e.g., in the absence of heat) and optionally under positive air pressure; and subjecting the dehydrated portions to milling, thereby providing a population of particles of ECM. In one embodiment, the portions are compressed prior to dehydration. In one embodiment, the ECM is liver ECM. In one embodiment, the liver ECM is porcine or human liver ECM. In one embodiment, the portions are dehydrated at a temperature of about 1° C. to about 30° C. In one embodiment, the portions are dehydrated at a temperature of about 4° C. to about 25° C. In one embodiment, the portions are dehydrated at a temperature of about 15° C. to about 25° C. In one embodiment, the portions are dehydrated at a temperature of about 20° C. to about 25° C. In one embodiment, the portions are dehydrated at a temperature of about 10° C. to about 20° C. In one embodiment, the portion is placed in a biologically compatible solution prior to dehydration, e.g., the solution comprises water or PBS. In one embodiment, milling produces a population in which at least 90% of the particles are less than about 2 mm in size. In one embodiment, milling produces a population in which at least 90% of the particles are less than about 0.15 mm in size. In one embodiment, the method further comprises separating the population of particles by size. In one embodiment, separation is performed by sieving. In one embodiment, the method further comprises subjecting the particles to enzymatic digestion. In one embodiment, the enzymatically digested particles comprise high molecular weight collagen, e.g., at least 150 kDa. In one embodiment, the population has a moisture content of less than about 10%, less than 5%, less than 2%, or less than 1%.

[0027] The population of particles prepared by the method can be used alone or in mixtures, such as to form gels, such as hydrogels or bioinks. The gels or inks can be used to form structures using 3D printing processes.

[0028] The present invention will be further illustrated by the following non-limiting examples. EXAMPLES

[0029] Example 1 The dried and blended perfused decellularized liver samples were sized in a laminar flow hood. Different sized pieces of decellularized liver ECM (MM or MD), e.g., 0.5" x 0.5" or 1" x 1" pieces (approximately 15 grams), were placed in polycarbonate grinding vials. The contents of the vials were subjected to grinding in a SPEX Cryogrinder. Two different protocols were used. One included 4 grind phases, 10 CPS, 5 min pre-cooling, 2 min cooling. The other protocol included 2 grind phases, 10 CPS, 5 min pre-cooling, 2 min cooling. Both products were sieved through a 2 mm sieve for particle size analysis. The 4 grind phase product had 144 mg >2.0 mm (1.1%) and 13,051 mg <2.0 mm (98.9%). The products of the 2-grind phase had 233 mg of >2.0 mm (2.2%) and 10,228 mg of <2.0 mm (97.8%). Each product (approximately 1500 mg) was packaged in a non-sterile 5 mL amber glass vial and sealed with a butyl stopper and aluminum seal. Water content analysis was performed (70 °C). Table 1 shows the data for two different samples for each protocol and original source (MM or MD, both liver-derived perfused decellularized ECM).

[0030] [Table 1]

[0031] The data in Table 1 show that dehydration in a laminar flow hood at ambient temperature resulted in <10% moisture content.

[0032] Example 2 The data in Figure 1 show that compression and dehydration of the seven sample types of Example 1 does not remove high molecular weight collagen (e.g., collagen > 150 kDa) from the ECM. Figure 2 shows the particle size analysis for the seven sample types of Example 1.

[0033] To determine how enzymatic digestion alters structure, 26.2 mg pepsin (Sigma), 26.2 mL HO, and 262 microliters of 1N HCl were mixed. 500 microliter reactions of 1 mg / mL pepsin in 10 mM HCl containing 1, 2.5, 5, and 10 mg of freeze-milled, decellularized, dehydrated ECM particles were split into two tubes and incubated at room temperature or 4°C for approximately 20 minutes, after which the reaction was stopped by adding 1N NaOH (55 microliters) to half of the sample. Samples were diluted 1:1 in sample buffer and run on a 7.5% SDS-PAGE at 180 V for 35 minutes (Figure 3).

[0034] FIG. 5 provides an overview of the steps in one embodiment of the method.

[0035] Example 3 Test conditions: Six vials of freeze-milled liver particles were packaged and divided into three groups (Table 2 summarizes Example 1). Control: Baseline MC% Positive control: Vials were left open during 72 hours of incubation at 37°C in a humidified environment. Test: The vials were kept sealed during 72 hours of incubation at 37°C in a humidified environment. TIFF2025026955000002.tif28128

[0036] [Table 2]

[0037] Example 4 In one embodiment, one or more portions of a dehydrated decellularized mammalian organ comprising extracellular matrix (ECM) are subjected to milling, thereby providing a population of particles of ECM. In one embodiment, the portion is compressed prior to dehydration. In one embodiment, the ECM is liver ECM, such as porcine or human liver ECM. In one embodiment, the portion is dehydrated at a temperature of about 1° C. to about 30° C. In one embodiment, the portion is placed in a physiologically compatible solution prior to dehydration, e.g., the solution comprises water or PBS. In one embodiment, milling produces a population in which at least 90% of the particles are less than about 2 mm in size and optionally greater than about 0.01 mm in size. In one embodiment, milling produces a population in which at least 90% of the particles are less than about 0.15 mm in size and optionally greater than about 0.01 mm in size. In one embodiment, the method further comprises the step of separating the population of particles by size. In one embodiment, separation is performed by sieving, by subjecting the population to acoustic energy, by subjecting the population to density separation, or by applying a fluid. In one embodiment, the method further comprises subjecting the particles to enzymatic digestion. In one embodiment, the enzymatically digested particles comprise high molecular weight collagen. In one embodiment, the population has a water content of less than about 10%.

[0038] In one embodiment, prior to dehydration, the extracellular matrix is ​​expanded with gas. In one embodiment, the expanded extracellular matrix has a height of about 0.3 cm to about 0.5 cm, including about 0.4 cm, greater than about 0.2 cm and up to about 0.6 cm, greater than about 0.2 cm and up to about 0.6 cm, greater than about 0.3 cm and up to about 0.5 cm, greater than about 0.4 ... In one embodiment, the gas-filled decellularized extracellular matrix of an organ or tissue has a shape, size, or volume of about 25% to 125%, e.g., about 50% to about 150%, or about 75% to about 110%, of that of the corresponding original organ or tissue. Active introduction of a gas, e.g., steam (gas with particles or droplets), into the decellularized extracellular matrix of an organ or tissue through its native vasculature or any other conduit, e.g., duct or void, provides a gas-filled decellularized extracellular matrix of the organ or tissue matrix that is expanded compared to its non-expanded shape, and in some embodiments has the original shape of the original (cellularized) organ or tissue before decellularization. The shape of the gas-filled decellularized extracellular matrix, e.g., one filled with air, is retained as a result of the gas being trapped within the tissue or organ and filling the space originally occupied by the cells. In one embodiment, the gas comprises ordinary air, CO2, argon, nitrogen, or oxygen, or any combination thereof.

[0039] In one embodiment, the dehydrated planar structure of a perfused decellularized mammalian organ containing ECM, or one or more portions thereof, is subjected to freeze milling, thereby providing a population of particles of ECM. In one embodiment, the planar structure or one or more portions are compressed. In one embodiment, the ECM is liver ECM. In one embodiment, the planar structure or one or more portions are dehydrated at a temperature of about 1° C. to about 30° C. In one embodiment, the milling produces a population in which at least 90% of the particles are less than about 2 mm in size. In one embodiment, the milling produces a population in which at least 90% of the particles are less than about 0.15 mm in size. In one embodiment, the method further comprises the step of separating the population of particles by size. In one embodiment, the method further comprises the step of subjecting the particles to enzymatic digestion. In one embodiment, the population has a water content of less than about 10%.

[0040] The population of particles produced by the method, for example, where at least 95% of the particles are less than about 2 mm in size or less than about 0.15 mm in size, or a gel containing the population, can be used in 3D printing or in wound treatment. In one embodiment, the population or gel is used for a bioink for 3D printing. In one embodiment, the 3D printed structure is used for functional testing of embedded cells, for example, cells can be added after the 3D printed structure is formed. In one embodiment, the population or gel is injected or added to or mixed with a different bioink to form a suspended ECM particle composite ink. For example, the different bioink can include cells, proteins, such as one or more growth factors, or other scaffold materials, such as agarose, gelatin, Pluronics (poloxamer), alginate, collagen, hyaluronic acid, fibrin, silk, or cellulose.

[0041] In one embodiment, dehydration or freeze milling of the perfused decellularized organ part results in a moisture content of about 5-20%, depending on environmental conditions, among other things.

[0042] In one embodiment, the overall size of the freeze-milled particles ranges from 0.02 mm to about 4 mm, depending on the conditions used during freeze-milling.

[0043] In one embodiment, particles of freeze-milled organs are added to 3D printing inks for printing 3D structures comprising whole organ-derived ECM.

[0044] In one embodiment, the freeze-milled organ particles are digested with various enzymes to form a solution that can be used as an ink in 3D printing. In one embodiment, the enzyme digestion results in an ECM size of less than 2 mm, such as less than 1.8, less than 1.6, less than 1.4, less than 1.2, or less than 1.0 mm.

[0045] In one embodiment, a 3D printed construct based on a whole organ part can be implanted into a mammal.

[0046] All publications, patents and patent applications are incorporated herein by reference. In the above detailed description, the invention has been described in connection with certain preferred embodiments thereof, and although numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that further embodiments may be derived from the invention, and that the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

1. providing one or more portions of a perfused decellularized mammalian organ comprising extracellular matrix (ECM); expanding the one or more ECM portions with a gas to produce one or more gas-expanded ECM portions that are expanded compared to the non-expanded shape and have the original shape of the mammalian organ before decellularization; dehydrating the one or more gas-expanded ECM portions in a temperature range of about 1 °C to about 30 °C; and subjecting the one or more dehydrated and gas-expanded ECM portions to milling to thereby produce a population of ECM particles, wherein the population has a water content of less than about 10% A method for forming ECM particles, comprising the steps of:

2. The method of claim 1, wherein the one or more ECM portions are compressed before dehydration.

3. The method of claim 1, wherein the gas occupies the space within the ECM that was originally occupied by cells.

4. The method of claim 1, wherein the ECM is an ECM of the liver, heart, lung, or kidney.

5. The method of claim 1, wherein the ECM is porcine ECM or human ECM.

6. The method of claim 1, wherein the one or more gas-expanded ECM portions are dehydrated in a temperature range of about 15 °C to about 25 °C.

7. The method of claim 1, wherein the one or more ECM portions are placed in a physiologically compatible solution before dehydration.

8. The method of claim 7, wherein the solution comprises water or PBS.

9. The method of claim 1, wherein the milling produces a population of ECM particles wherein at least 90% of the particles are less than about 2 mm in size or at least 90% of the particles are less than about 0.15 mm in size.

10. The method of claim 1, further comprising the step of separating the population of ECM particles by size.

11. The method of claim 10, wherein the separation is performed by sieving, subjecting the population to acoustic energy, subjecting the population to density separation, or using a fluid.

12. The method of claim 1, wherein the milling comprises cryogenic milling.

13. A population of ECM particles produced by the method of claim 1, wherein at least 95% of the particles are less than about 2 mm or less than about 0.15 mm in size.

14. A gel comprising the population of ECM particles of claim 13.

15. The gel according to claim 14, further comprising mammalian cells.

16. A step of providing a bioink composition comprising the population of ECM particles according to claim 13; and A step of applying the bioink composition to form a 3D structure A method of using a bioink for 3D printing, comprising.

17. The method according to claim 16, wherein the bioink composition further comprises cells.

18. The method according to claim 16, wherein the bioink composition further comprises an isolated protein or glycoprotein.

19. A step of providing one or more portions of a mammalian organ that is expanded with a gas and perfusion decellularized and contains an extracellular matrix (ECM), wherein the gas occupies the space within the ECM that was originally occupied by cells, and the one or more gas-expanded ECM portions are expanded compared to the non-expanded shape and have the original shape of the mammalian organ before decellularization; A step of dehydrating the one or more gas-expanded ECM portions in a temperature range of about 1 °C to about 30 °C; and A step of subjecting the one or more dehydrated gas-expanded ECM portions to milling to thereby generate a population of ECM particles, wherein the population has a water content of less than about 10%. A method for forming ECM particles, comprising.

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