Cryobioprinting formulation

Abstract

A bioink for cryobioprinting is described. The bioink comprises a decellularized extracellular matrix (dECM)-based hydrogel and a saccharide cryoprotectant. A method of making a 3D-printed biomaterial is also described, which includes depositing a bioink comprising a dECM-based hydrogel, cells, and a saccharide cryoprotectant from a 3D printer onto a freezing plate to form a frozen bioink filament; depositing additional bioink from a 3D printer in contact with a previously placed frozen bioink filament to form a 3D-printed biomaterial comprising a plurality of frozen bioink filaments; and removing the 3D-printed biomaterial from the freezing plate. 3D-printed biomaterials made according to the method are also described.

Description

CRYOBIOPRINTING FORMULATIONCONTINUING APPLICATION DATA

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 725,875, filed November 27, 2024, the disclosure of which is incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Number R56EB034702, awarded by the National Institutes of Health. The Government has certain rights in this invention.BACKGROUND

[0003] The use of biofabrication technologies to generate human tissues in vitro has revolutionized tissue engineering and in vitro disease modeling field. Amidst these, 3D bioprinting has been under the spotlight owing to its unique ability to create human tissue analogs with increasingly relevant structural complexity and size, in an automated and rapid mode. The synergetic combination of computer-aided design (CAD) models with medical imaging data (i.e., computer tomography (CT) and magnetic resonance imaging (MRI)) has significantly expanded 3D bioprinting applications allow one to produce more accurate, patient-specific models. Such progresses open new avenues to closely resemble native human intricate structures and hierarchy, being particularly relevant for tissue engineering, and to boost disease models' paradigm. Monteiro et al., ACS Appl Mater Interfaces, 16(42):56718-56729 (2024). In fact, the ability to produce such complex and architectural relevant cell-laden constructs has fostered an increased interest for the fabrication of storable tissue analogs capable of being cryopreserved and used on-demand.

[0004] 3D cryobioprinting, an automated manufacturing technique that seamlessly combines the traditional bioprinting with cryopreservation methods, has recently emerged as a highly valuable approach to shift the current biofabrication paradigm of print-to-use, allowing for the simultaneous fabrication-cryopreservation of complex 3D living models. Ravanbakhsh et al., Matter 5, 573-593 (2022). Such progress relies on the bioink deposition over a substrate, commonly a freezing plate or a cryogenic bath, at subzero temperatures, triggering bioink freezing on the fly. This advanced manufacturing technique offers a number of advantages over traditional bioprinting including extended shelf-life of living models, as well as enhanced bioink printability owing to instant freezing upon contact with the cryogenic printing bed contributing to improved structural fidelity of 3D constructs during and post- printing. Chen et al., Biomed Eng Lett 10, 453-479 (2020) Importantly, the freezing process enables the use of bioinks with close-to-liquid rheological features and without requiring any crosslinking methods during the fabrication process. Warburton et al., Transactions on Additive Manufacturing Meets Medicine 1, 3-15 (2019). Cryobioprinting also enables the design and fabricating of 3D living platforms exhibiting anisotropy and porosity, two valuable features for mimicking human tissues and assuring cell viability upon implantation. Choi et al., Journal of Industrial and Engineering Chemistry 67, 388-395 (2018).

[0005] Nevertheless, the freezing process inherent to cryobioprinting requires the careful design of bioinks integrating cryoprotectants in order to protect cells from ice crystal- mediated damage during freezing and thawing of 3D constructs. The identification of optimal cryoprotective agents (CPAs) for cryoprotective bioinks is thus crucial for maximizing cellular viability and tissue functionality. There are two categories of cryoprotectants, namely (i) non-permeable (e.g., saccharides, polyethylene oxide) and (ii) permeable (e.g., dimethyl sulfoxide (DMSO), glycerol), depending on the cell membrane permeability. Disaccharides (e.g., sucrose, trehalose, maltose, lactose) and trisaccharides (melezitose and raffinose) have been described to exhibit a superior impact on cell cryopreservation comparing to monosaccharides (i.e., glucose, galactose, and fructose). Murray, K.A. and Gibson, M.I., Nat Rev Chem 6, 579-593 (2022). Alternatively, thepotential of other permeable cryoprotectants such as glycerol has been explored, as it exhibits low cytotoxic behavior at high concentrations. Zhang et al.. Stem Cell Res Ther 13, 152 (2022).SUMMARY

[0006] Cryobioprinting enables simultaneous fabrication and cryopreservation of tissue analogs on the fly, surpassing the limitations of print-to-use biofabrication approaches. However, cryopreservation of living constructs is highly challenging, requiring optimal cryopreservation conditions tailored to specific cell types and hydrogel bioinks. To address this, we explore the formulation of a decellularized extracellular matrix (dECM)-based hydrogel bioink containing cryoprotective agents (CPAs) to generate shelf-ready tumorstroma pancreatic cancer models. Combinatorial screening of cryoprotective agents led to the discovery of a new melezitose-glycerol-dECM formulation that exhibited superior cryoprotective properties in both tumor and stroma compartments. Exometabolomics analysis revealed that cryopreserved constructs exhibit similar metabolic activity to nonfrozen counterparts 14 days, post-thawing. Cryobioprinted tumor-stroma models in dECM- CPA bioinks revealed increased viability post-thawing and suitable features for in vitro drug screening. Our optimized cryoprotective strategy opens new opportunities to explore virtually any type of tissue decellularized bioinks for cryobioprinting off-the-shelf living constructs for widespread drug-screening and beyond.BRIEF DESCRIPTION OF THE FIGURES

[0007] FIGS. 1A-1G provide graphs and images showing the characterization of porcine pancreas dECM. (A) Graphical representation of porcine pancreas decellularization process. Created in BioRender. Mano, J. (2025) (B-E) Quantitative analysis of DNA, native collagen, sulfated GAGs and HA in pancreas ECM and dECM. Data are presented as mean ± s.d., n=3 independent biological replicates, each replicate results from the decellularization of 3-5 porcine pancreas tissue. *p <0.05, **p <0.01***, p <0.001. (F) GO enrichment analysis of a total of 482 proteins shared between dECM and native ECM. Eachdot represents a significantly enriched biological process, plotted by fold enrichment on the x-axis. Dot size reflects the number of proteins associated with each GO term, while color indicates statistical significance as -logio(FDR). Enriched pathways include protein folding, catabolic and metabolic processes, and biosynthetic activities, highlighting a conserved and functionally active protein core retained across both ECM conditions. (G) Relative percentage of peptides found in pancreas dECM compared with the total amount of identified peptides. COL - collagen; LUM - lumican.

[0008] FIGS. 2A-2L provide graphs and images showing the evaluation of dECM- and dECM:HA-Tyr -based inks potential for cryo(bio)printing. (A,B) Assessment of ideal extrusion pressure and printing speed parameters, by using 22G nozzle, for dECM and dECM:HA-Tyr cryoprotective inks. Printing fidelity was evaluated comparing the fabricated grid models with CAD file. Shear flow curves displaying the shear-thinning profile of (C) dECM-based and (D) dECM:HA-based inks. Cryobioprinted 2D patterns and 3D structures through (E-H) dECM - glycerol - melezitose ink, and (I-L) dECM:HA-Tyr - glycerol - melezitose ink. Scale bars = 1 mm.

[0009] FIGS. 3A-3K provide graphs and images showing the physical characterization of dECM- and dECM:HA-based hydrogels. (A) Representative SEM images of (A) frozen dECM-based models at -20 °C, (B) cryopreserved dECM-glycerol- melezitose platform at - 80 °C for 24 h, (C) frozen dECM:HA-based models at -20 °C, (D) cryopreserved dECM:HA - glycerol- melezitose platform at -80 °C for 24 h. Scale bar = 20 pm. Representative fluorescence microscopy images showing the effect of freezing (-20 °C) and cryopreservation (-80 °C) on the microchannel sizes in (E) frozen dECM-based constructs, (F) cryopreserved dECM - glycerol - melezitose platform at -80 °C for 24 h, (G) frozen dECM:HA-based models, (H) cryopreserved dECM:HA - glycerol - melezitose platform at -80 °C for 24 h. dECM was prelabeled with albumin FITC (pseudo color : purple). Nonfrozen constructs were prepared as controls. Scale bar = 50 pm. (I) The corresponding quantification results of pore diameters at the different frozen hydrogels conditions, n=5 constructs per condition. Graphical representation of the elastic modulus for frozen andcryopreserved (J) dECM-based platforms and (K) dECM:HA-based models. Non-frozen dECM and dECM:HA hydrogels were used as controls. Data is presented as mean ± S.D., n = 3, *p<0.05, **p<0.01, ***p<0.001. Legend: cdECM: cryopreserved dECM hydrogels at - 20 °C; cdECM:HA : cryopreserved dECM:HA hydrogels at -20 °C, without the incorporation of cryoprotectants.

[0010] FIGS. 4A-4F provide graphs and images showing tumor and stromal cells viability in frozen dECM- and dECM:HA-based constructs, with different CPA formulations. The freezing plate was set at -20 °C. (A) Representative live / dead images of PANC-1 cells encapsulated in cryoprotective bioinks. 3D constructs were frozen in the freezing plate, photocrosslinked, and revived immediately. Scale bars = 50 pm. (B) Relative quantification of live area (%) in PANC-1 microtissues post- freezing at -20 °C, through live / dead images. Data is presented as mean ± S.D., n = 3. (C) Representative live / dead images of CAF cells encapsulated in cryoprotective bioinks. 3D constructs were frozen in the freezing plate, photocrosslinked and, and revived immediately. Scale bars = 50 pm. (D) Relative quantification of live area (%) in CAFs microtissues post-freezing at -20 °C, through live / dead images. Evaluation of cytoskeletal integrity and organization of cancer and stromal cells in frozen hydrogels at day 14 of culture. (E) Representative F-actin / nucleus staining of 3D platforms at different bioink compositions. Scale bars = 50 pm. (F) Close-up of CAFs in glycerol-melezitose frozen hydrogel. Red channel: F-actin; Blue channel: cell nuclei. Data is presented as mean ± S.D., n = 3, *p<0.05, **p<0.01, ***p<0.001.

[0011] FIGS. 5A-5E provide graphs and images showing tumor and stromal cells viability in cryopreserved dECM- and dECM:HA-based constructs, with different CPA formulations. Cell-laden constructs were produced setting the freezing plate at -20 °C, and after photocrosslinking they were transferred to -80 °C for 24 h. (A) Representative live / dead images of the PANC- 1 cells encapsulated in cryoprotective bioinks after revival at different time points of culture. Scale bars = 50 pm. (B) Relative quantification of live area (%) in PANC-1 microtissues post-cryopreservation at -80 °C, through live / dead images. Data is presented as mean ± S.D., n = 3. (C) Representative live / dead images of the CAF cellsencapsulated in cryoprotective bioinks after short-term cryopreservation. Scale bars = 50 pm. (D) Relative quantification of live area (%) in CAFs microtissues post-cryopreservation at -80 °C, through live / dead images. (E) Cytoskeletal characterization of cryopreserved models at day 14 of culture. Representative F-actin / nucleus staining of 3D cryopreserved platforms at different bioink compositions showed the preservation of cytoskeletal integrity post-thawing in glycerol-melezitose CPA formulation. Red channel: F-actin; Blue channel: cell nuclei. Scale bars = 50 pm. Data is presented as mean ± s.d., n = 3. *p<0.05, **p<0.01, ***p<0.001.

[0012] FIGS. 6A-6F provide graphs and images showing the characterization of cryobioprinted and cryopreserved tumor-stroma PDAC in vitro models. (A) Schematics of tumor-stroma models cryobioprinting. (B) Live / dead representative section of tumor-stroma model showing the tumor-stroma interface. Scale bars = 100 pm. (C) Representative Live / Dead confocal micrograph of tumor and stroma regions of non-frozen, cryobioprinted and cryopreserved (for 7 days) heterotypic 3D platforms, at day 14 of culture. Green channel: Calcein-AM, Red channel: PI. Scale bars = 50 pm. (D) Metabolic activity analysis in non-frozen, cryobioprinted and cryopreserved (for 7 days) tumor-stroma models, at day 1, 3, 7 and 14 of culture. Data is presented as mean ± s.d., n>3. Representative F-actin / nucleus staining of (E) cryobioprinted and (F) cryopreserved (for 24h) tumor-stroma 3D platforms. Red channel: F-actin; Blue channel: cell nuclei. Scale bar = 200 pm.

[0013] FIGS. 7A-7G provide graphs showing the (A) Metabolomic analysis of non-frozen, cryobioprinted and cryopreserved (for 24 h) tumor-stroma models at day 7 and 14 days of culture. Data is presented as mean ± S.D., n=5. *p<0.05, **p<0.01. KIC: a-ketoisocaproate, KIV: a- ketoisovalerate, KWV: a-keto- -methylvalerate. (B - E) ELISA-based quantification of TGF-P, SDF- 1 , MMP-9 and MMP-2, in non-frozen, cryobioprinted, and cryopreserved (for 7 days) models, during 14 days of culture. The concentration of the different factors was normalized to total protein concentration. Data is presented as mean ± S.D., n=3. (F) Quantification of protein concentration in cell culture medium of non-frozen, cryobioprinted, and cryopreserved models (for 7 days), after 14 days of culture. (G) Cellsviability following administration of Gemcitabine during 72 h, on non-frozen, cryobioprinted and cryopreserved (for 7 days) 3D models. Data is presented as mean ± S.D., n>3.DETAILED DESCRIPTION

[0014] A bioink for cryobioprinting is described. The bioink comprises a decellularized extracellular matrix (dECM)-based hydrogel and a saccharide cryoprotectant. A method of making a 3D-printed biomaterial is also described, which includes depositing a bioink comprising a dECM-based hydrogel, cells, and a saccharide cryoprotectant from a 3D printer onto a freezing plate to form a frozen bioink filament; depositing additional bioink from a 3D printer in contact with a previously placed frozen bioink filament to form a 3D- printed biomaterial comprising a plurality of frozen bioink filaments; and removing the 3D- printed biomaterial from the freezing plate. 3D-printed biomaterials made according to the method are also described.Definitions

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present specification, including definitions, will control.

[0016] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the application as a whole. For example, as used in this specification and the appended claims, the singular forms "a", "an" and "the" can include plural referents unless the content clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicate otherwise. The word "or" means any one member of a particular list and also includes any combination of members of that list. Further, all units, prefixes, and symbols may be denoted in its SI accepted form. The conjunctive phrase “and / or” indicates that either or both of the items referred to can be present.

[0017] The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method.

[0018] Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range. Throughout this disclosure, various aspects of this invention are presented in a range fomrat. It should be understood that the description in range format is merely for convenience and brevity and should not be constmed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges, fractions, and individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6, and decimals and fractions, for example, 1.2, 3.8, 11 / 2, and 43 / 4 This applies regardless of the breadth of the range.

[0019] As used herein, the term “about” means ±10% of the recited value.

[0020] As used herein, the terms “printing” and “bioprinting” are used interchangeably, and cover both normal 3D printing and printing that includes cells in the bioink.

[0021] A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. Furthermore, where the weigh percentage of the polymeric components of a hydrogel are provided, it can be assumed that the remaining weight percentage consists of water, cells, and other minor additives that may be present in the hydrogel unless the percentages of other components are specifically provided.

[0022] "Biocompatible" as used herein, refers to the capability of a material to be integrated into a biological system without harming or being rejected by the system. Examples of harm include inflammation, infection, fibrotic tissue formation, cell death, or thrombosis. The terms "biocompatible" and "biocompatibility" when used herein are art-recognized and mean that the material is neither itself toxic to a subject, nor degrades (if it degrades) at a rate that produces byproducts at toxic concentrations, does not cause prolonged inflammation or irritation, or does not induce more than a basal immune reaction in the host.

[0023] The term polymer, as used herein, also refers to polymer precursors that are crosslinked to form the polymer, unless it is specifically indicated that the polymers do not include polymer precursors.Cryobioinks

[0024] In one aspect, the invention provides a bioink for cryobioprinting. The bioink comprises a decellularized extracellular matrix (dECM)-based hydrogel and a saccharide cryoprotectant.

[0025] Extracellular matrix is a network of proteins and carbohydrates that provides structural support and regulates cellular function for tissues. The major components of extracellular matrix is collagen, fibronectin, and elastin, with proteoglycans, other glycoproteins, and minerals present in a lesser amount. ECM is primarily found in connective tissues, but is found within all other types of tissue as well. The two main types of extracellular matrix are interstitial matrix and basement membrane ECM. The composition of ECM can vary to some extent based on the type of tissue it is obtained from.

[0026] Extracellular matrix can be decellularized by removing cells from biological tissue include ECM to leave behind the ECM. Physical stress such as agitation or pressure, chemical methods such as the use of detergents, and / or enzymatic methods (e.g, proteases and / or nucleases) can be used to remove cells while preserving the ECM structure. See Shevchuk et al., Front Bioeng Biotechnol., 13:1621641 (2025). For example, in oneembodiment, dECM is obtained from ECM using a combination of proteinase and Triton- X-100 treatment, as described in further detail herein. An advantage of decellularized extracellular matrix (dECM) is that it can be re-cellularized with new cells to form a new tissue.

[0027] Decellularized ECM can then be converted into hydrogels using methods known to those skilled in the art. See Saldin et al., Acta Biomater., 49: 1-15. 10.1016 (2017), the disclosure of which is incorporated herein by reference. In addition to their excellent interactions with the in vivo milieu, biocompatibility, soft and elastic consistency, decellularized ECM (dECM) -derived hydrogels are superior to natural, synthetic, and composite hydrogels since they contain the original ECM components and mimic the structural and mechanical complexity of the native tissues. Boso et al., Materials (Basel), 13(11):2483 (2020).

[0028] Because of their biocompatibility, hydrogels are preferred for use in bioink formulations, since hydrogels are water-rich polymers that can hold considerable amounts of water and are benign to embedded cells. Hydrogels are polymeric networks with hydrophilic chains crosslinked either covalently or physically (via intra- and intermolecular attractions). Typically only a relatively small amount of the dECM-based hydrogel is required to form a bioink. In some embodiments, the bioink comprises from about 0.5% w / v to about 5% w / v of the dECM-based hydrogel, while in other embodiments the bioink comprises from about 1 % to about 2% w / v of the dECM-based hydrogel.

[0029] In some embodiments, the bioink can include another hydrogel. Biocompatible hydrogels include natural hydrogels and synthetic hydrogels. Examples of natural hydrogels include hyaluronic acid, hyaluronic acid derivatives (e.g., hyaluronic acid tyramine), silk (e.g., silk fibroin or silk sericin), collagen, gelatin, gelatin derivatives (e.g., gelatin methacryloyl) and alginate. Examples of synthetic hydrogels include polyethylene glycol derivatives and polyvinyl alcohol derivatives. Hydrogels can also include natural hydrogels that have been modified, such as methacrylated collagen. The hydrogel can either be aseparate polymer, or can be linked to the dECM. In some embodiments, the dECM-based hydrogel further comprises hyaluronic acid.

[0030] The bioink used to form the 3D-printed biomaterial includes a saccharide cryoprotectant. Cryoprotectants are compounds that protect the cells from the freezing temperatures used during formation and storage of the 3D-printed biomaterial. Saccharide cryoprotectants include disaccharides and trisaccharides. Examples of suitable disaccharides include D-(+)-trehalose, D-lactose, and sucrose, while examples of trisaccharides include D-(+)-raffinose and D-(+)-melezitose. In some embodiments, the cryoprotectant is D-(+)- melezitose (i.e., melezitose). The concentration of the cryoprotectant can range from about 1% w / v to about 20% w / v, with a concentration from about 5% w / v to about 15% w / v or about 8% w / v to 12% w / v in further embodiments. In some embodiments, an additional cryoprotectant such as dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol can be included. For example, in some embodiments glycerol is present in an amount from about 8% to about 12%. In a preferred embodiment, the saccharide cryoprotectant is melezitose and the cryoprotectant glycerol is also included.Methods of Making Three-Dimensional (3D)-Printed Biomaterial

[0031] Another aspect of the invention provides a method of making a 3D-printed biomaterial, comprising depositing a bioink comprising a dECM-based hydrogel, cells and a saccharide cryoprotectant from a 3D printer onto a freezing plate to form a frozen bioink filament; depositing additional bioink from a 3D printer in contact with a previously placed frozen bioink filament to form a 3D-printed biomaterial comprising a plurality of frozen bioink filaments; and removing the 3D-printed biomaterial from the freezing plate.

[0032] Three-dimensional printing (also known as additive manufacturing) is a method that takes information of the surface shape of a 3D object, models it using computer-aided design / computer-aided manufacturing (CAD / CAM) software, and then slices that model into multiple layers, creating a stacked cross-sectional version of the 3D object. This information is then fed into a 3D printer capable of fabricating the 3D object using anadditive or subtractive manufacturing process for constructing three-dimensional single or multi-layered structures disposed on a substrate. Examples of 3D printing methods include stereolithography, direct ink writing, viscous solution printing, and digital light processing.

[0033] Different types of 3D printers can be used to deposit the bioink onto the freezing plate. In some embodiments, depositing the bioink comprises extruding the bioink from, for example, a coaxial 3D printer. In some embodiments, the bioink is applied using an inject printing method (e.g., droplet-based printing) or a vat polymerization printing method, such as stereolithographic printing, digital light processing, or volumetric printing. Levato et al., Nature Reviews Methods Primers, 3, 47 (2023). In some embodiments, microfluidics- enhanced bioprinting can be used. See Chatinier et al., Biomicrofluidics, 15, 041304 (2021). Microfluidic systems contain channels on the micrometer-scale and can facilitate precise positioning of different materials.

[0034] In other embodiments, the bioink can be used with different types of 3D printing methods. In some embodiments, the bioink is applied by being extruded using an extrusion print-head. In other embodiments, the bioink is applied using a stereolithographic printing apparatus. In further embodiments, the bioink is applied using an inkjet / droplet method. The steps involved in carrying out these methods are known to those skilled in the art.

[0035] The 3D-printed biomaterial is typically crosslinked before the material is used. For example, the biomaterial can be crosslinked shortly after the 3D-printed biomaterial has been formed on the freezing plate, or the 3D-printed biomaterial can be crosslinked after being stored.

[0036] A variety of methods are available for crosslinking dECM-based hydrogels. In some embodiments, the dECM-based hydrogel is physically (e.g., ionically) cross-linked by calcium (e.g., CaCb) while in other embodiments the dECM-based hydrogel is cross-linked using microbial transglutaminase (mTG). In further embodiments, the hydrogel is crosslinked by exposure to ultraviolet or visible light and using a photoinitiator. The wavelength used will generally depend on the photoinitiator used. Examples of suitable wavelengthsinclude from about 200 nm to about 500 nm, from about 250 nm to about 450 nm, or from about 300 nm to about 400 nm.

[0037] In some embodiments, the frozen bioink filaments are formed in the vertical direction. Vertical, as used herein, refers to perpendicular to the freeze plate. The bioink filaments are formed by depositing bioink on the freeze plate, followed by further deposition of bioink on top of the bioink that has already been deposited, forming a column in the vertical direction. This can occur as a result of continuous extrusion of bioink onto a specific point of the freeze plate, while gradually raising the extrusion nozzle, or as a result of placing specific droplets of material, first on the freeze plate, and then on previously placed droplets.

[0038] The method of preparing a 3D-printed biomaterial includes depositing a bioink comprising a dECM-based hydrogel, cells, and a cryoprotectant from a 3D printer onto a freezing plate to form a frozen hydrogel filament. A freezing plate is a 3D printer print plate that has been modified so that it is chilled to a temperature that will freeze the hydrogel when the hydrogel is deposited onto the plate (or onto hydrogel previously deposited on the plate). The freezing plate can be chilled by placing it in proximity to a cold substance such as dry ice or liquid nitrogen, or using a refrigeration technique such as the use of a thermoelectric cooler (i.e., a solid-state heat pump). In some embodiments, the freezing plate is chilled to a temperature ranging from about 0 °C to about -200 °C, in further embodiments the freezing plate is chilled to a temperature ranging from about -5 °C to about -100 °C, or from about -5 °C to about -30 °C, while in yet further embodiments the freezing plate has a temperature ranging from about 0 °C to about -30 °C.

[0039] In addition to using a freezing plate, there are other methods that can be used to chill the bioink. In some embodiments the bioink is chilled (i.e., pre-chilled) to a temperature ranging from about 0 °C to about 10 °C before being deposited by the 3D printer, so that its temperature is reduced even before it contacts the freezing plate. Alternately, or in addition, it can also be useful to chill the compartment in which the 3D-printed biomaterial is formed. For example, air chilled to below 0 °C can be blown into the chamber and over the freezing plate to contact the bioink as it is deposited. For example, co-extruded liquid nitrogen canbe used together with, or as an alternative to the use of a freezing plate to freeze deposited bioink.

[0040] The method can be used to create 3D-printed biomaterials having a variety of different shapes. In some embodiments, the deposited bioink comprises a scaffold, while in other embodiments the extruded bioink comprises a tissue construct such as a blood vessel. Different methods of 3D-printing can be chosen that are better suited to providing the specific shape of interest.

[0041] In some embodiments, a plurality of frozen 3D-printed biomaterials are separately prepared and then combined to form a larger structure. This can be done to create shapes that might be difficult to prepare directly on a 3D printer, and / or to combine 3D-printed biomaterials made separately and having different characteristics, such as different porosity and / or different polymer composition. Different blocks (or segments) can be joined, for example, by placing frozen blocks adjacent to one another, allowing them to melt slightly, and then crosslinking the blocks so that they join together.

[0042] The method includes depositing a bioink comprising dECM, cells and a cryoprotectant. Viable cells that can be included in a 3D-printed object include prokaryotic and eukaryotic cells. Non-limiting examples of eukaryotic cells include mammalian cells such as myoblasts, fibroblasts, endothelial cells, and stem cells. Other living cells include embryonic stem cells, amniotic fluid stem cells, cartilage cells, bone cells, muscle cells, skin cells, pancreatic cells, kidney cells, nerve cells, liver cells, and the like. Viable cells are living cells.

[0043] In some embodiments, the cells included in the bioink comprise cancer cells. Cancer cells are cells that have developed through mutations in their DNA, causing them to grow and divide uncontrollably. Types of cancer cells include carcinomas, leukemias, lymphomas, sarcomas, and melanomas, depending on the tissue they originate from. Cancer cells include cultured cancer cell lines, and cancer cells associated with various types of tissues, such as lung, breast, colorectal, prostate, brain, skin, and blood cancers.

[0044] Standard cell culture techniques are typically used when handling the cells for the 3D- printed biomaterial. For example, a portion of or the entire printed article can be placed under standard cell culture conditions (e.g., temperature, pressure, nutrient concentrations, etc.) in order for the cells to remain viable. The 3D-printed biomaterial can comprise from about 1 x 101to about 1 x 109viable cells, or from about 1 x 102to about 1 x 108viable cells, or from about 1 x 103to about 1 x 107viable cells, or from about 1 x 104to about 1 x 107viable cells, or from about 1 x 105to about 1 x 107viable cells (all being cells per milliliter).

[0045] The hydrogel composition can also include serum to support the cells. A wide variety of serums for supporting cells (i.e., cell culture media) are known, and can vary depending on the type of cells. Examples of suitable serum include fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM), as well as chicken, caprine, equine, human, ovine, porcine, and rabbit serum. In some embodiments, the hydrogel composition includes from about 1% to about 10% v / v of serum.3D-printed Biomaterials

[0046] Another aspect of the invention provides a 3D-printed biomaterial. The biomaterial is made by depositing a bioink comprising dECM, cells and a cryoprotectant from a 3D printer onto a freezing plate to form a frozen bioink filament; depositing additional bioink comprising cells and a cryoprotectant from a 3D printer in contact with a previously placed frozen bioink filament to form a 3D-printed biomaterial comprising a plurality of frozen bioink filaments; and removing the 3D-printed biomaterial from the freezing plate. When initially prepared, the 3D-printed biomaterial is frozen, but it can later be thawed before use. However, in other embodiments, the 3D-printed biomaterial is kept frozen, for example when the biomaterial is kept in a freezer before use.

[0047] In some embodiments, the 3D-printed biomaterial comprises micropores. While not intending to be bound by theory, the inventors propose that micropores or interconnected microchannels are formed when ice crystals formed during the freezing process melt.Porosity of the 3D-printed biomaterial can be controlled by tuning different parameters in the cryobioprinting process, such as the freezing and cryopreservation temperatures, bioink viscosity, and biomaterial ink concentration. In some embodiments, the micropores have a pore size from about 10 pm to about 50 pm, in further embodiments the micropores have a pore size from about 10 pm to about 50 pm, while in yet further embodiments the micropores have a size from about 5 pm to about 25 pm.

[0048] In some embodiments, the 3D-printed biomaterial comprises microchannels. The microchannels can have a size ranging from about 10 pm to about 1000 pm, or in some embodiments, from about 50 pm to about 600 pm. In further embodiments, the microchannels form an anisotropic gradient. An anisotropic structure is one that has a different value when measured in different directions. For example, anisotropic 3D-printed biomaterial can include microchannels having a diameter that differs when measured at different points along the vertical direction, or when measured along the vertical direction versus the horizontal direction. In one embodiment, the microchannels along the bottom of the biomaterial have a diameter from about 50 pm to about 100 pm, while the microchannels along the top of the biomaterial have a diameter from about 400 pm to about 600 pm.

[0049] The 3D-printed biomaterials can have essentially any size and shape that can be obtained using a 3D-printer. For example, in some embodiments, the 3D-printed biomaterial comprises a tissue scaffold, spheroid, or organoid. In some embodiments, the 3D-printed biomaterial is shaped as an object intended for medical or pharmaceutical use, such as a tissue scaffold (e.g., an artificial transplant support). In some embodiments, the 3D biomaterial is a soft tissue construct (e.g., an organoid). In further embodiments, the 3D- printed biomaterial is a spheroid, which is a simpler 3D cell aggregate often consisting of a single cell type, which is often cultured as a free-floating aggregate. In some cases, the 3D biomaterial can be personalized for a specific subject by basing the 3D object on an image obtained from magnetic resonance imaging, computed tomography, or ultrasound. A wide variety of tissue engineering applications for 3D-printed biomaterials comprising hydrogelsare known to those skilled in the art. Advincula et al., MRS Commun., 11 (5):539-553 (2021).

[0050] In some embodiments, the 3D-printed biomaterial is a tissue construct. An example of a 3D-printed biomaterial tissue construct is neo-cartilage. In some embodiments, the 3D- printed biomaterial can be a soft tissue construct. Soft tissues connect and support other tissues and surround the organs in the body. They include muscles (e.g., the heart), fat, blood vessels, nerves, tendons, and tissues that surround the bones and joints. Examples of 3D soft tissue constructs include skin, musculoskeletal tissue, cardiac tissue, heart valve, liver, and neuronal tissue. In some embodiments, the 3D-printed biomaterial is a muscle tissue scaffold. The cells included in the tissue construct are preferably the type of cells normally found in the particular type of tissue, or precursor cells (e.g., stem cells) that will result in that particular type of tissue. The cells may be substantially uniformly distributed throughout the polymer, or they may be suspended within a part of the polymer. Examples of suitable cells include myoblasts, fibroblasts, endothelial cells, or stem cells.

[0051] In some embodiments, the 3D-printed biomaterial can be provided in the shape of an organ or tissue, such as circulatory organs. In some embodiments, the biomaterial comprises a hollow tube. A hollow tube can be used as a blood vessel, such as an artery or vein. The hollow tube comprises a cylinder having a hollow interior, an inner side facing the hollow interior, and an outer size facing the exterior of the cylinder. Blood vessels can have a variety of sizes, depending on the type of blood vessel, with a normal human aorta having a diameter of about 2 cm, while capillaries can have a diameter from about 2 to 12 pm. Accordingly, the blood vessels can have a diameter from about 2 pm to 2 cm, with noncapillary blood vessels having a diameter ranging from about 1 mm to about 2 cm.

[0052] In some embodiments, the 3D-printed biomaterial is a tumor model. The tumor models contemplated herein are 3D models similar to organoids that can be used to study cancer development and test new treatments. In some embodiments, the tumor model comprises a tumor region surrounded by a stoma compartment. Tumors are typically composed of cancer cells protected by a surrounding fibrotic stroma emulating the nativetumor microenvironment. Tumor stroma is the supportive tissue of a tumor, including connective tissue, blood vessels, and various cells like fibroblasts and immune cells, which plays a crucial role in cancer progression, metastasis, and resistance to therapy. In some embodiments, the tumor region comprises cancer cells embedded in crosslinked dECM, surrounded by a crosslinked dECM-hyaluronic acid-tyramine hydrogel comprising cancer- associated fibroblast cells. The tumor models can be used to model cancer found in a variety of different tissues, such as lung, breast, colorectal, prostate, brain, skin, and blood cancers. In one embodiment, the tumor model is a pancreatic ductal adenocarcinoma model.

[0053] The 3D-printed biomaterial can also include one or more additives. Non-limiting exemplary additives for the biomaterial include diluent synthetic polymers (e.g., polyethylene glycol, polypropylene glycol, poly(vinyl alcohol), poly(methacrylic acid)), drugs (e.g., antibiotics such as penicillin and streptomycin), cell nutrients (e.g., proteins, peptides, amino acids, vitamins, carbohydrates (e.g., starches, celluloses, glycogen), and minerals (e.g., calcium, magnesium, iron), synthetic or naturally occurring nucleic acids, absorbers to limit light penetration, inhibitors (e.g., scavengers and quenchers), refractive index modifiers (e.g., iodixanol), and nanocomposite components such as graphene or silica. The bioink formulation can comprise one or more additives in an amount of 0 wt % to about 25 wt % of the composition, based on total weight of the composition.

[0054] The 3D-printed biomaterial can be stored before use. To preserve the biomaterial, and cells embedded in the biomaterial, it is preferable to chill the 3D-printed biomaterial during storage. In some embodiments, the 3D-printed biomaterial is stored at 0 °C or less. In further embodiments, the 3D-printed biomaterial is stored at a temperature from -20 °C to about -80 °C. These temperatures can be achieved by, for example, storing the 3D-printed biomaterial in liquid nitrogen. Accordingly, in some embodiments, the 3D-printed biomaterial is frozen.

[0055] The present invention is illustrated by the following example. It is to be understood that the particular example, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.EXAMPLECryobioprinted Human Tumor Models with Shelf-Stable Programmability

[0056] Herein we explore 3D cryobioprinting potential to produce cryoprcscrvablc pancreatic ductal adenocarcinoma (PDAC) living constructs as shelf-ready preclinical drug-screening platforms. For that purpose, a tumor-stroma PDAC platform was designed to emulate native tumor composition and architecture, where cancer cells are protected by a surrounding fibrotic stroma emulating the native tumor microenvironment (TME). In particular, PDAC stroma is mainly populated by cancer-associated fibroblasts (CAFs) that are key orchestrators on PDAC TME, with a number of reports demonstrating their role on tumor growth, and abundant extracellular matrix (ECM) deposition, namely collagens and hyaluronic acid (HA), promoting tumor fibrosis and consequently leading to the inefficiency of current treatments. Chakkera et al., Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 1879, 189065 (2024). In order to resemble such a scenario, a 3D in vitro PDAC model was designed to include a tumor region comprising human pancreatic cancer cells embedded in a photocros slinkable porcine pancreas decellularized ECM (dECM). The tumor region is surrounded by a stroma compartment populated by CAFs embedded in a photocrosslinkable dECM:HA-tyramine (HA-Tyr) matrix aiming to resemble the native TME as close as possible. This approach fosters the production of more predictable and accurate preclinical tumor models for studying tumor-stroma interplay and for the development and screening of anti-tumor, anti-stroma therapies.

[0057] From this standpoint, the optimization of cytoprotective dECM and dECM:HA-based bioinks to generate cryobioprinted tumor-stroma PDAC platforms, that could be used for on-site or off-site applications, was conducted. dECM and dECM:HA-based bioinks were engineered using different permeable CPAs (i.e., DMSO or glycerol) and non-permeable CPAs (i.e., melezitose) combinations, in order to achieve the optimal cryoprotective bioinkformulation for tumor-associated cells culture and biofabricated PDAC in vitro models’ cryopreservation. Following bioinks compositional optimization, cryobioprinted 3D tumor models, which can be used immediately after printing or stored for on-demand purposes, were successfully fabricated and used as testing platforms for chemotherapeutics.Results

[0058] dECM-based Bioinks for Cryoprinting

[0059] Aiming to generate biomimetic 3D in vitro pancreatic cancer models, tumor-stroma cell types and pancreatic tissue-specific dECM were rationally combined to reproduce key tumor hallmarks in the fabricated constructs. We adapted a commercially available extrusion 3D bioprinter and engineered a new printing bed comprising a thermoelectric cooler (TEC), similarly to what was previously described. Luo et al., Advanced Materials 34, 2108931 (2022). This system setup allows one to precisely control the freezing plate temperature by simply adjusting the input voltage on the connected power supply.

[0060] In order to faithfully resemble native TME and tumor-stroma ECM stratification, two distinct cryoprotective bioinks were formulated: (i) tumor bioink comprising cancer cells and a photocrosslinkable dECM (1.5 %, w / v), intended to preserve the native tumor core matrix rich in collagens; and (ii) a stroma bioink were CAFs are supported by a dECM (1.5 %, w / v): HA-tyramine (HA-Tyr, 1 %, w / v) matrix, designed to simulate the high accumulation of HA in stroma compartment as found in vivo.

[0061] To generate pancreas dECM, porcine tissue was processed as already reported (FigureIA). Ferreira et al., Trends Biotechnol 38, 1-18 (2020). The efficiency of the decellularization process regarding DNA, native collagen, sulfated GAGs and HA content was examined (Figure 1B-E). Analysis of DNA content of both ECM and dECM indicated a decrease from 313.75 ± 22.1 1 ng. mg1dry tissue to 14.79 ± 7.61 ng. mg1dry tissue confirming the success of the cells’ removal during the decellularization process (FigureIB). Pancreatic dECM was also analyzed for the presence of collagens, GAGs and HA.Collagen quantification revealed the presence of native collagen in the decellularized tissue (Figure 1C), while a significant loss of sGAG and HA was confirmed (Figure ID, E).

[0062] Moreover, dECM was analyzed through liquid chromatography-mass spectrometry (LC-MS) to gain a more comprehensive understanding of its composition (Figure 1F,G). A total of 482 proteins were identified in both the dECM and the native ECM, representing a robust core set of matrix-associated proteins preserved across sample preparation methods. Gene ontology (GO) enrichment analysis of this shared proteome revealed several significantly overrepresented biological processes, indicating that these proteins are not randomly retained but functionally relevant. The most strongly enriched process was protein folding, with a fold enrichment of approximately 7.5 and the highest statistical significance (— logic False Discovery Rate (FDR)> 15). Moreover, proteomic analysis revealed that collagens represent the most abundant class of ECM proteins comprising the highest relative percentage of identified peptides (Figure 1G). This is consistent with previously reported results of decellularized pancreas tissue. Hoshiba, T., Materials 12, 1-16 (2019).

[0063] Optimization of cryobioprinting using dECM-bases bioinks

[0064] To explore cryoprotective formulations compatible with tissue-specific dECM-based bioinks, two different cryoprotective agent combinations were studied, namely DMSO (10%, w / v) - melezitose (12% w / v), a commonly used and characterized CPA cocktail for cryobioprinting (Weygant et al.. Aggregate, 5(5), e599 (2024)); and melezitose combined with glycerol (10% v / v), a permeable CPA reported to exhibit lower cytotoxicity and that remains to be explored in cryobioprinting applications, to the best of our knowledge.

[0065] To evaluate the printability and the fidelity of both dECM and dECM:-HA- - based inks, printing parameters such as the pressure and printing speed were evaluated. A 22G nozzle (ca. 410 pm) was used for cryoprinting as our preliminary analysis showed that it yielded a better printing performance than a 21G nozzle. The best printing fidelity was obtained at a printing pressure of 6 kPa and a printhead speed of 1 mm / s1for dECM inks, and a printing pressure of 9 kPa and printhead speed of 3 mm / s1for dECM:HA inks(Figures 2A, B). As expected, the inclusion of HA-Tyr required an increase in both printing parameters values, as the obtained formulation exhibits a higher viscosity (Figure 2B). Subsequently, viscometry assays were conducted to assess the influence of CPAs on inks extrudability. dECM and dECM:HA inks exhibited a non-Newtonian shear-thinning behavior as shear viscosity decreases with increasing shear rates (Figures 2C, D). Moreover, despite being widely explored as CPAs, the influence of cryoprotectants such as DMSO or glycerol on the shear-thinning behavior remains mostly unexplored. The results showed that glycerol increased viscosity of dECM inks requiring higher extrusion pressure during the bioprinting process.

[0066] Following printing parameter optimization, representative 2D and 3D structures were successfully cryoprinted from dECM (Figure 2E-H) and dECM:HA-based inks (Figure 21- L). These results demonstrate the feasibility of producing structurally complex and high- fidelity constructs using low-viscosity dECM-based inks under cryogenic conditions.

[0067] Impact of Cryoprinting on Physical Properties of Decellularized Hydrogels

[0068] Aiming to investigate the effects of freezing, cryopreservation, and bioink composition on the microstructure and porosity of dECM / dECM:HA -based hydrogels, constructs were prepared by setting the freezing plate temperature at -20 °C and using polydimethylsiloxane (PDMS) molds, followed by blue light mediated photocrosslinking and revival. After freezing, cryopreserved models were immediately transferred to -80 °C for 24 h prior to analysis. Platforms not subjected to the freezing and cryopreservation process were used as controls. Hydrogels’ microstructure was analyzed through scanning electron microscopy (SEM) (Figure 3A-D). For characterizing porosity, dECM was labelled with albumin-fluorescein isothiocyanate (albumin-FITC), and the generated hydrogels from different bioinks formulations were visualized under confocal microscopy (Figure 3E-H).

[0069] SEM imaging revealed the impact of freezing and ice templating on the hydrogels’ microstructure, with both frozen and cryopreserved conditions exhibiting micro-sized pores, a feature that was not observed for bulk dECM and dECM:HA models not exposed tosubzero temperatures. Moreover, cryopreserved dECM hydrogels (Figure 3B) showed larger pores and enhanced overall porosity, whereas this effect was less evident in cryopreserved dECM:HA platforms (Figure 3D), highlighting the role of macromolecular contents on ice crystals formation, and microstructure maintenance. Such results suggest that the addition of hyaluronic acid influences the freezing process and the resulting hydrogel microstructure.

[0070] Confocal imaging analysis evidenced the impact of CPA addition on the ice crystal formation and consequently on construct porosity (Figure 3E-H). Interestingly, while frozen hydrogels prepared with DMSO exhibited a relatively uniform porous network, glycerolbased scaffolds showed the formation of lamellar microchannels after freezing plate- mediated ice-templating process. In addition, we investigated the influence of HA inclusion on the polymer network. As expected, a general pattern was observed with a decrease on pore diameter for frozen dECM:HA hydrogel compared to dECM counterparts, emphasizing the role of polymer content and concentration in limiting ice crystal growth during freezing. Importantly, for both dECM and dECM:HA frozen hydrogel formulations, the addition of cryoprotectants led to a pore size decrease, with frozen dECM and dECM:HA hydrogels exhibiting pore diameter of 29.32 ± 17.70 pm and 21.07 ± 14.50 pm, respectively, while scaffolds prepared with melezitose and combined with DMSO or glycerol, exhibited pore sizes of about 15.60 ± 5.98 pm , 18.84 ± 11.17 pm for dECM- based hydrogels, respectively, and 10.54 ± 4.24 pm , 18.74 ± 8.01 pm for dECM:HA platforms, respectively (Figure 31).

[0071] To evaluate the cryopreservation influence on scaffolds porosity, frozen hydrogels were transferred to -80 °C for storage and subsequently analyzed after thawing. The confocal images assessment revealed that cryopreserved hydrogels (Figure 3F, H) demonstrated a slight decrease in average pore size, accompanied by an increase on the small pores' formation (Figure 3F, H, I). In fact, 3D models exposed to short-term preservation experienced a two-step freezing process, as they were initially frozen at -20 °C followed by cryopreservation at -80 °C. Herein, we have hypothesized that the initialfreezing at -20 °C enables the formation of larger ice crystals, while the subsequent freezing at -80 °C further induces additional nucleation of smaller ice crystals. In contrast, hydrogels frozen only at -20°C experience a single freezing process, which leads to the formation of larger ice crystals and, consequently, larger pores. Based on this, it is reasonable that pore size analysis showed a higher concentration of smaller pores for both dECM and dECM:HA cryopreserved models compared to the frozen counterparts.

[0072] In order to assess the impact of cryobioprinting and cryopreservation steps on the mechanical properties of dECM and dECM:HA hydrogels, time-sweep tests were performed. Both dECM and dECM:HA platforms fabricated on the freezing plate at -20 °C exhibited a lower elastic modulus (G’) compared to their non-frozen bulk hydrogels counterparts. Cryopreservation at -80 °C also caused a decrease on hydrogel stiffness compared to the frozen constructs. Such outcomes may be attributed to the void spaces produced during the cry opreservation and subsequent thawing. Moreover, as expected, dECM:HA models exhibited higher G’ values than dECM-only platforms, reinforcing the influence of HA incorporation on hybrid hydrogel stiffness.

[0073] Design of Cryoprotective Bioinks for Pancreatic Cancer Modeling

[0074] Cell-laden hydrogel precursors comprising different CPA formulations (i.e., glycerol- melezitose; DMSO-melezitose) were prepared to evaluate the suitability and cell-preserving capacity of dECM and dECM:HA-based bioinks for supporting PANC-1 cells and CAF cryobioprinting, respectively. Samples were produced using PDMS molds and a freezing plate temperature at -20 °C, followed by blue light-mediated photocrosslinking and immediate transference to culture medium. Live / dead viability assays were conducted at multiple time points to evaluate and identify the optimal CPA combination for preserving cell viability during cryobioprinting. Samples in the absence of cryoprotectants were used as negative controls.

[0075] The results demonstrated that both cell types exhibited significantly reduced viability in the absence of cryoprotectants, indicative of cell damage by cryoinjuries caused by icecrystals growth and osmotic stress, highlighting the importance of including CPAs in cryobioprinting and cry opreservation procedures (Figures 4A-D). In contrast, glycerolbased formulations exhibited higher post-thawing viability for both PANC-1 and CAFs- laden hydrogels, samples prepared with glycerol when compared to their DMSO-based counterparts. Similar outcomes were observed for living constructs formulated with glycerol-melezitose, demonstrating enhanced cell survival relative to those composed of DMSO-melezitose, further highlighting the improved cry oprot ective effect of glycerolbased formulations under the tested conditions (Figures 4A-D).

[0076] Cytoskeletal integrity and organization analysis showed that PANC-1 cells formed 3D aggregates, while CAFs displayed their characteristic elongated morphology by day 7 of culture in glycerol-melezitose bioinks, a signature that was not observed for cell-laden models prepared with DMSO (Figure 4E). Furthermore, the results showed that frozen constructs promoted a distinct CAFs alignment suggesting the presence of an aligned microstructure that induces cell orientation (Figure 4F).

[0077] To further investigate influence of the freezing plate temperature on CPA performance, stromal platforms were fabricated using both CPA formulations (glycerol- melezitose, and DMSO-melezitose), and setting the freezing plate temperature to -12 °C and -20 °C. Comparative analysis showed that the glycerol-melezitose CPA maintained its superior performance across conditions. Interestingly, CAF-laden hydrogels comprising DMSO-melezitose and frozen at -12 °C exhibited improved viability and an elongated phenotype, in contrast to those prepared at -20 °C, corroborating previous reported work. Ravanbakhsh et al., Matter 5, 573-593 (2022). However, at a freezing plate temperature of - 20 °C, and for the developed bioinks, tumor- associated cells exhibited increased viability and functionality with glycerol- melezitose CPA compared to DMSO counterparts. Overall, in this work the CPAs’ cocktail comprising glycerol and melezitose revealed to be effective for the preparation of both cryoprotective bioinks, demonstrating efficiency to minimize cell damage caused by the formation of ice crystals, thereby preserving cellular integrity andfunctionality. For that reason, the following assays were performed by using glycerol and melezitose as optimal cryoprotectants.

[0078] Aiming to validate the potential of the optimized cryoprotective bioinks for generating cryopreservable, storable living platforms, and to study the impact of short-term storage on cell viability, samples were prepared on the freezing plate at -20 °C, photocrosslinked, and then directly transferred to -80 °C for 24 h. After thawing, live / dead viability assays were performed to evaluate the ability to produce off-the-shelf PDAC platforms using the developed cryoprotective bioinks. Figure 5 shows representative live / dead images of cell-laden stored models at different time points. By using fluorescence images, the viability of PANC-1 and CAFs in their specific matrices was also determined at day 3, 7 and 14 of culture (Figure 5B, D). As expected, 3D cryopreserved constructs lacking the non-permeable trisaccharide showed lower cell viability, reinforcing its key role in protecting cells against cryoinjuries during freezing and thawing process and the importance of non-permeable saccharides inclusion on cryoprotective bioinks design. In contrast, PANC-1 and CAFs-laden models, prepared with the optimized glycerol-melezitose CPA maintained high viability, with both living platforms exhibiting cellular viability around 95% and 93%, respectively, at day 14 of culture, demonstrating the efficiency of the established cryoprotective bioinks for short-term storage. In addition, cytoskeletal structure analysis revealed the maintenance of the actin cytoskeleton in both PANC-1 cells and CAFs post-thawing, in the presence of glycerol-melezitose CPAs combination (Figure 5E). These findings indicate that the optimized CPAs not only support high post-thaw viability but also preserving PANC-1 and CAFs cytoskeletal integrity and architecture, being an indicative of cellular functionals maintenance.

[0079] Cryobioprinting tumor-stroma PDAC models for off-the-shelf availability

[0080] Pancreatic tumor-stroma models were fabricated via cryobioprinting using the previously optimized cryoprotective bioinks and a two cartridge bioprinter system. By setting the freezing plate temperature to -20 °C, a compartmentalized core-shell model was established, wherein the core tumor region (PANC-1 cells combined with dECMcryoprotective ink) is surrounded by the stroma compartment (CAFs embedded in dECM:HA cryoprotective ink), emulating the native spatial and cellular organization (Figure 6A). To evaluate the storage potential of the biofabricated constructs, 3D tumor platforms were photocrosslinked via blue light and cryopreserved at -80 °C for 24 h or 7 days. Non-frozen cast tumor-stroma platforms were used as control. Such an approach enables one to compare the effects of cryobioprinting and cryopreservation on the 3D tumor-stroma models’ viability and functionality.

[0081] To verify the successful spatial cellular organization, PANC-1 and CAFs were labeled with cell permeable dyes. Confocal imaging analysis confirmed the compartmentalization of the cell populations, with cancer cells encapsulated within astromal matrix, resembling the juxtatumoral position found in native malignancy, where the tumor core mass is enveloped by a desmoplastic and fibrotic stroma (Figure 6B).

[0082] Aiming to evaluate how the cryobioprinting and cryopreservation influenced the cellular viability and functionality of 3D tumor-stroma models, live / dead, cellular metabolic activity and phalloidin staining were performed in both control and cryopreserved platforms (Figure 6C-F). As evidenced by Live / Dead assays, both tumor and stromal cells remained viable up to 14 days of culture after cryobioprinting, cry opreservation and thawing (Figure 6C). While cryopreserved models initially exhibited lower metabolic activity compared to non-frozen controls, after 7 days of culture this difference was normalized, with no significant difference observed at day 7 and 14 days of culture (Figure 6D).

[0083] In addition, CAFs exhibited an elongated morphology after 14 days of culture in both cryobioprinted and cryopreserved constructs (Figure 6E, F). This morphology is indicative of activated CAFs exhibiting restored cytoskeletal organization suggesting migratory potential. Monteiro et al., Adv Funct Mater 34(6), 2305473 (2024).

[0084] Comparative exometabolomic analysis of conditioned media from non-frozen, cryobioprinted, and cryopreserved 3D PDAC models revealed no major differences in overall metabolic profiles across the different conditions (Figure 7A). Notably, the cellsconsistently consumed glucose, pyruvate, aspartate, and 2-hydroxyisobutyrate (2-HIB), while secreting lactate, alanine, glutamate, glycine, formate, and branched-chain a-keto acids (BCKAs: a-ketoisocaproate (KIC), a-ketoisovalerate (KIV), a-keto-P-methylvalerate (KWV). This metabolic signature reflects active glycolysis and robust lactate production, characteristic of tumor cell metabolism, alongside active amino acid turnover and anaplerotic flux into the TCA cycle. Slight differences between the cryobioprinted and conventionally cryopreserved platforms became apparent mainly at day 14 of culture, particularly in the consumption of glucose and pyruvate, and in the secretion levels of glutamate, alanine, and BCKAs. These variations may reflect subtle differences in metabolic adaptation following the cryopreservation process at -80 °C.

[0085] To evaluate the preservation of tumor-stroma interplay following cryobioprinting, cryopreservation and thawing, the secretion levels of key signaling molecules namely, transforming growth factor beta (TGF-P), stromal cell-derived factor 1 (SDF-1), and matrix remodeling enzymes (MMP-2, MMP-9) were quantified (Figure 7B-E), owing to their role on tumor-stroma interactions, PDAC progression and resistance. Quantitative analysis of 3D culture supernatants indicated that there were no statistically significant differences in the secretion levels of the analyzed factors between non-frozen, cryobioprinted and cryopreserved models. These results suggest that the paracrine signaling network within 3D PDAC TME are preserved, being also consistent with other indicators of functional stability, namely the metabolic activity and the unaltered total protein synthesis. Further, it was observed that total protein concentration in culture media after 14 days showed no significant differences between non-frozen, cryobioprinted, and cryopreserved PDAC models (Figure 7F). These findings are particularly interesting as they suggest that the overall cellular secretory activity and protein secretion remain consistent across all experimental conditions.

[0086] To investigate the screening functionality of both 3D- cryobioprinted and cryopreserved tumor-stroma models, a drug-screening assay was performed. From this standpoint, 3D heterotypic models were fabricated, photocrosslinked and cryopreserved.After tissue revival, tumor-stroma models were cultured for 14 days. As control, cast nonfrozen tumor-stroma models and 3D cryobioprinted platforms were produced and placed in culture.

[0087] 3D tumor models were exposed to Gemcitabine, a standard-of-care for PDAC, at day 14 of culture for 72 h. The metabolic activity analysis of cryobioprinted and cryopreserved models after chemotherapeutic administration revealed that tumor-associated cell sensitivity to Gemcitabine was not notably different than those in the non-frozen model (Figure 7D).Discussion

[0088] Aiming to generate physiomimetic tumor-stroma PDAC models emulating key tumor components, we initially formulated two cryoprolective tissue-specific bioinks: (i) a tumor bioink comprising PANC-1 pancreatic cancer cells and porcine pancreas dECM, and (ii) a stroma bioink combining pancreatic CAFs and dFCM:HA. Tissue specific decellularized matrices are recognized for their ability to retain key ECM components (e.g., collagens, laminin and fibronectin) and tissue-derived soluble factors, offering an unprecedent potential to reproduce the biochemical and biophysical cues found in tumor niche. Goh et al., Biomaterials 34, 6760-6772 (2013). However, during the decellularization process, there is still a significant loss of non-proteinaceous components, in particular glycosaminoglycans (GAGs). From this perspective, in the context of PDAC TME, hyaluronan exhibits a critical role in tumor stroma, being abundantly deposited in this region mainly by CAFs. Its accumulation has been strongly associated with stroma fibrosis, immune suppression, increased interstitial pressure and consequently the inefficiency of current treatments. For this reason, accurately modelling HA-rich PDAC stroma is paramount for bioengineering physiomimetic preclinical tumor platforms.

[0089] Given the prominent role of hyaluronan in PDAC TME, herein the stroma bioink was enriched with HA-Tyr to better emulate the in vivo scenario. Biochemical analysis of dECM confirmed a significant reduction of sGAGs and HA, underscoring the need for HA supplementation of dECM matrices to accurately emulate the fibrotic PDAC stroma. Incontrast, the preservation of collagen content supports the effective retention of these key matrix components in the dECM, confirming the efficacy of the followed decellularization protocol. Proteomic analysis further suggests a notable presence of chaperones and foldases, which may contribute to extracellular protein stability or reflect residual components from the secretory machinery. In addition, a large number of proteins were involved in catabolic processes, particularly those related to carboxylic acid, organic acid, and small molecule catabolism. These results imply that the ECM retains metabolic activity and may play a role in modulating extracellular biochemical environments through enzyme-mediated degradation or transformation. Other significantly enriched terms included peptide metabolic and biosynthetic processes, translation, and amide biosynthesis, suggesting that matrix-associated proteins also include biosynthetic and regulatory components. This may be indicative of vesicle entrapment, stable protein-matrix interactions, or structural incorporation into the ECM network.

[0090] Overall, the shared proteome reflects a biologically coherent and statistically robust group of proteins that likely represent essential matrix functions such as structural organization, remodeling capacity, and interaction with resident or infiltrating cells. The retention of this functional core across both dECM and ECM highlights its potential relevance in tissue engineering, scaffold design, and mechanistic ECM studies.

[0091] While conventional bioinks used in cryobioprinting to date are often based on single elements of tissues ECM such as gelatin or collagen, this study introduced a new class of cryoprotective bioinks composed of tissue-specific dECM matrices, offering a more realistic representation of the in vivo TME. Adamkiewicz, M. and Rubinsky, B., Cryobiology 71, 518-521 (2015). The optimization of these cryoprotective tissue-specific dECM-based bioinks is yet to be explored, and the results showed their potential to advance the fabrication of structurally and compositionally biomimetic human tissues analogs. The incorporation of glycerol as an alternative cryoprotectant revealed distinct rheological properties compared to the DMSO, as it increased the viscosity of the dECM-based inks and required a higher extrusion pressure.

[0092] One of the main challenges of 3D bioprinting relies on the complexity of printing low-viscosity materials in-air, such as dECM. Cryoprinting demonstrated to be an effective biofabrication technique to generate such tissue specific ECM-mimetic platforms with increased fidelity and complexity without requiring extensive optimization. Exploring cryogenic conditions to print such low viscous biomaterial inks increases the rigidity and strength of the printed layers, allowing to print complex structures taking advantage of the ink rapid solidification at subzero temperatures, surpassing the necessity of using supporting baths, commonly used in embedded bioprinting, opening new avenues to advance the discovery for more biomimetic ECM-like inks in a more viscosity independent manner.

[0093] Freezing and cryopreservation events have a significant impact on hydrogel physical properties, opening promising avenues for creating scaffolds with enhanced porosity comparing to the traditional bioprinted models. Loukelis et al., Gels 9, 103 (2023). Particularly, cryobioprinting emerged as an innovative technique that enables the fabrication of highly intricate structures with interconnected porosity through the ice-templating method. Such is particularly important to promote cell-cell / cell-ECM interactions while allowing nutrient and oxygen exchange. Herein, to analyze the influence of freezing and cryopreservation on the microstructure and porosity of tissue-specific-based models, 3D- cryobioprinted constructs were exposed to two steps, (i) the freezing step during cryobioprinting at which the freezing plate temperature is settled at -20 °C (frozen models), and then for shelf storage, (ii) cryopreservation at -80 °C for 24 h and 7 days (cryopreserved models), followed by thawing. In fact, our results demonstrated that the freezing and cryopreservation steps significantly altered the internal architecture of dECM-based platforms. As observed, the freezing step induced substantial microstructural changes compared to non-frozen control models, and the cryopreservation at -80 °C resulted in a higher number of smaller pores in cryopreserved models.

[0094] During the bioink freezing at subzero temperatures, ice crystals are formed and propagate in a directional mode according to the bioink deposition. Following crosslinking and thawing of constructs, the ice crystals give rise to aligned hollow microchannels. Luo etal., Advanced Materials 34, 2108931 (2022). Importantly, cryobioprinting using a freezing plate results in smaller and laminar micropores in the lower construct layers, while upper layers exhibit larger and oriented pores due to the temperature gradient across the height (z direction) and the slower freezing at higher temperatures.

[0095] Porosity of 3D constructs can be controlled by tuning different parameters in the cryobioprinting process, namely the freezing and cryopreservation temperatures as they affect the cooling rate and, consequently, the ice crystal growth that ultimately influence pore size in crosslinked living models. Higher freezing rates usually related to lower freezing temperatures, result in smaller ice crystals and pores, while slower freezing rates associated with higher freezing temperatures allow the formation of larger pores. Ahrens, D. Von et al., J Hematol Oncol 10, 1-8 (2017). Besides the printing parameters such as the nozzle size and printing speed, pore size is also dependent on the bioink composition. Increasing biomaterial ink concentration inversely affects pore diameter, with increased bioink viscosity limiting ice crystal growth, resulting in reduced pore size. The addition of cryoprotectants also has a major role in limiting ice crystal growth resulting in smaller pores. The latter is yet to be fully explored and is a major parameter in this technology, since it influences constructs porosity but also cell cryostability and viability. Murray, K.A. and Gibson, M.I., Nat Rev Chem 6, 579-593 (2022). Herein, the addition of HA to dECM seemed to limit ice crystal growth and yield constructs with reduced pore diameter. This effect is attributed to the higher viscosity and macromolecular density imparted by HA, which limits ice nucleation and crystals growth. Moreover, both DMSC) and glycerol exhibited distinct effects on 3D models microstructure. DMSO generated uniform porous networks, while the presence of glycerol led to more lamellar pore’s morphologies. Such differences may be attributed to their respective influence on water crystallization kinetics and viscosity modulation. In addition, both CPA formulations reduced overall pore size, further confirming their role in constraining ice crystals growth during freezing and cryopreservation.

[0096] 3D models’ mechanical analysis further suggested that the increased porosity introduced by the freezing and cryopreservation steps is correlated with decreased hydrogel stiffness. This effect may be attributed to the presence of void spaces formed during the freezing and thawing, which reduced the effective crosslinking density. As expected, hydrogels comprising HA exhibited a higher mechanical integrity, underscoring hyaluronan contribution.

[0097] The freezing and cryopreservation process inherent to cryobioprinting, requires the careful design of cryoprotective bioinks to protect cells and tissues from osmotic shock and ice crystals damage that affect cells membrane integrity and intracellular environment leading to cell death. Cryobioprinting storable tissues requires a “two-step” freezing protocol, (i) the freezing of the bioprinted construct at an initial subzero temperature during the bioink deposition over the cooled substrate, and then (ii) the cooling of the 3D model to the storage temperature. Finding optimal cryoprotectants is crucial to maximize cellular viability and tissue functionality during the freezing, cryopreservation and post-thawing process, by limiting ice crystal formation through the increasing of extracellular osmolarity and solute concentration. While non-permeable cryoprotectants work on extracellular environment by increasing the osmolarity and reducing the cell-water contact, permeable agents are able to cross the cells membrane preventing the intracellular ice crystals formation and cell damage by reducing the water melting point.

[0098] The glycerol-melezitose CPA combination provided superior cryoprotective effect for both cancer and stromal cells than the DMS O-based counterpart. These results are aligned with previous reports demonstrating that glycerol exhibited better cryoprotection performance while freezing adipocytes than the commonly used freezing medium comprising DMSO. Importantly, glycerol augmented cryoprotective effect was consistent for both dECM and dECM:IIA bioinks.

[0099] Constructs comprising glycerol-melezitose as the CPA supported the formation of PANC-1 aggregates as well as the acquisition of elongated, fibroblast-like morphology by CAFs, biological signatures generally associated with cellular functionality. In contrast,such features were not observed in DMSO-based models, reinforcing the superiority of glycerol-melezitose combination as a more biocompatible CPA under the tested cryogenic conditions. CAFs alignment observed in frozen constructs further highlights a key advantage of cryobioprinting, the creation of architecturally anisotropic matrices via ice templating emulating critical features found in native TME.

[0100] Our comparative study also revealed that CPAs performance is sensitive to the freezing temperature, as DMSO-melezitose combination resulted in significantly reduced viability at -20 °C, whereas cell survival and morphology were improved at -12 °C. In fact, previous research on cryobioprinting observed a reduction in cell viability when using DMSO and melezitose as cryoprotectants, with a temperature plate set at -20 °C (cellular viability was only ~ 60%, while cell viability was not significantly affected at a freezing temperature of -15 °C or above), suggesting that for low freezing temperatures the CPA combination studied may not be optimal.

[0101] From this scenario, it is needed to have in consideration that both freezing plate temperature and CPA optimization are highly dependent on the tissue-specific cell types and the developed bioink, and since no universal protocol can be followed yet, a balance between these two variants must be achieved to maximize cell viability and functionality of 3D- cryobioprinted constructs.

[0102] Short-term storage of 3D- cryobioprinted platforms was successfully achieved using optimized glycerol-melezitose cryoprotective bioinks. Remarkably, both tumor- associated cells exhibited increased viability up to 14 days of culture, demonstrating that the selected CPA formulation effectively mitigates cryoinjuries and maintains constructs integrity. The observed cryoprotective effect arises from the synergistic function of glycerol and melezitose cryoprotectants. As glycerol permeates cell membrane, it contributes for the reduction of intracellular ice formation, while both glycerol and melezitose exert osmotic protection extracellularly, limiting water flux and ice crystallization around the encapsulated cells during the cryobioprinting and cryopreservation promoting cell maintenance.

[0103] Additionally, the fluorescence micrographs revealed that F-actin was shown to be intact in both PANC-1 and CAF-laden constructs after thawing, highlighting the suitability of the optimized glycerol-melezitose CPA cocktail to maintain cellular integrity after the preservation process. In fact, previous reports demonstrated that during the cryopreservation and thawing process, actin cytoskeleton can be disrupted leading to impaired cellular functions. Kota et al., Cancer Lett 391, 38-49 (2017).

[0104] Aiming to explore the potential of cryobioprinting to generate off-the-shelf 3D PDAC models emulating as close as possible the human tumor, a compartmentalized tumorstroma platform was designed. In fact, pancreatic tumor resistance is largely attributed to the complex tumor microenvironment characterized by a dense and fibrotic stroma, mainly comprising CAFs responsible for the abundant ECM deposition that ultimately causes an increase on stromal stiffness. Besides supporting tumor growth, the produced desmoplastic stroma acts as physical barrier surrounding the core tumor mass hampering drug delivery to cancer cells and resulting in the inefficacy of current treatments. Growing efforts have been made on the discovery of new effective treatments targeting tumor immune system, stroma components and cancer cells. However, the lack of predictive and accurate preclinical tumor models has hindered the validation of anti-tumoral therapies, leading to a low success rate in translating preclinical outcomes to the clinical side. The ability of advanced manufacture techniques such as 3D bioprinting to generate complex, anatomically relevant living models has the potential to level-up preclinical tumor models research, enabling the fabrication of human tissue analogs with increased complexity and unprecedent biomimicry. Rodrigues et al., Sci Rep 11, 21517 (2021). From a more advanced perspective, the ability to generate cryopreserved tumor analogs that closely emulate the in vivo counlerparts open new avenues for the preclinical drug testing field. The stored preclinical platforms can be produced, cryopreserved and shipped worldwide for pharmaceutical companies or to the clinic, allowing them to evaluate new cancer therapies in more representative human tumors. Moreover, biofabricated 3D tumor models’ cryopreservation contributes to the acceleration and improvement of anti-cancer drug discovery process owing to the facility ofintegrating them in high-throughput screening, as a large number of living platforms can be produced at scale, stored and distributed on-demand.

[0105] This study demonstrates that cryobioprinting enables the fabrication and shortterm storage of anatomically organized 3D tumor-stroma platforms, maintaining cellular viability and tumor-stroma compartmentalization. The core-shell structure, with CAFs cultured in a peripheral shell around the core PANC-1 tumor mass, reflects the juxtatumoral organization observed in vivo, thereby enhancing the translational potential of the developed model. This spatial fidelity is particularly important for understanding tumorstroma interplay and the stroma barrier effects on immune escape and drug delivery.

[0106] Following cryopreservation and revival of cryobioprinted tumor-stroma models, both cancer and stromal cells retained high viability and metabolic activity. Immediately after thawing, a decrease in metabolic activity was observed as during the cryopreservation cells are subjected to osmotic stress remaining in a dormant state, and when revived they require time to recover and return to the basal metabolic activities, ultimately translated into a reduced adenosina trifosfato (ATP) production in the initial periods. Moreover, studies suggest that these processes can lead to structural and functional alterations in mitochondria causing oxidative stress. Gascard, P. and Tlsty, T.D., Genes Dev 30, 1002-1019 (2016). However, the fully metabolic recovery by day 7 confirms the efficacy of the cryoprotective system (i.e., glycerol-melezitose CPA formulation) and the ability of cells to resume normal function.

[0107] Importantly, CAFs maintained their elongated morphology after cryofabrication and storage, with phalloidin staining confirming intact cytoskeletal structures. Such features are critical for accurately mimicking stromal dynamics, which play a key role in tumor progression and resistance.

[0108] Metabolomic analysis showed that cryobioprinting preserved the metabolomic phenotype of PDAC models, with minor deviations observed following cryopreservation and revival. The observed uptake of glucose and pyruvate alongside the secretion of lactateand amino acid derivates, is consistent with enhanced glycolysis and lactate production, both hallmarks of cancer cells metabolism. Moreover, BCKAs secretion reflects active branched-chain amino acid catabolism, which plays a role in maintaining redox balance and supporting biosynthesis under nutrient-limited conditions. Overall, these findings indicate that key metabolic pathways supporting tumor growth and viability are maintained in cryobioprinted and cryopreserved living constructs, being comparable to those of the nonfrozen counterpart. This supports the feasibility of cryobioprinting to generate off-the-shelf platforms for off-site use.

[0109] PDAC TME exerts a critical role on tumor aggressiveness and in the inefficiency of current therapies. In this context, TGF-P is a central mediator on CAFs activation and tumor fibrosis. This factor stimulates CAFs to adopt a myofibroblastic phenotype, characterized by abundant ECM deposition, which ultimately impairs drug delivery, supports immune suppression and tumor invasion. TGF-P also promotes epithelial- to-mesenchymal transition, driving tumor resistance. SDF-1 is a chemokine abundantly secreted by CAFs that promotes pancreatic tumor cells migration and immune cell recruitment through CXCR4 signaling. High levels of SDF-1 are often associated with poor prognosis and resistance to therapeutics. From another perspective, metalloproteinases, especially MMP-2 and MMP-9 highly secreted in PDAC stroma, are responsible for ECM degradation facilitating tumor invasion in the surrounding tissues and metastasis. In this work, the maintenance of TGF- p, SDF-1, MMP-2, and MMP-9, and total protein secretion demonstrates that tumor-stroma paracrine signaling and protein secretory capacity is retained, suggesting that cryobioprinted and cryopreserved PDAC models faithfully retain the biological complexity of native tumors, including stromal activation, matrix remodeling, tumor-stroma interplay and invasive potential. Additionally, these results are highly consistent with the observed preserved metabolic profiles and high cell viability. This functional fidelity supports the potential use of cryobioprinted models for off-the-shelf applications.

[0110] From a more advanced perspective, the potential of cryobioprinted and cryopreserved models to function as preclinical off-the-shelf platforms was evaluated. In this study, the results indicated that the cryobioprinting and cryopreservation process did not strongly affect the functionality and chemotherapy response of tumor-stroma platforms. Overall, the cryobioprinting and cryopreservation of PDAC in vitro platforms represent a significant breakthrough for cancer research and screening of new therapies, as the results demonstrate that off-the-shelf models can potentially be used with confidence.

[0111] Overall, cryobioprinting emerged as a transformative advancement in biofabrication and 3D tissue engineering fields opening new avenues for the generation and storage of living tissues to be used on demand.

[0112] Nevertheless, cryobioprinting cell-laden constructs requires the careful addition of cryoprotective agents to the bioink to assure optimal cell functionality after fabrication, cryopreservation, and thawing. Pinpointing such optimal conditions in a bioink and in a cell-specific mode is paramount, as it opens new avenues for generating shelf-ready tissue analogs that can be shipped to the desired locations for on-demand clinical, pharmaceutical or academic applications. Such scenario could streamline experimental workflows and accelerate the transability of bioprinted models, allowing the screening of new therapeutics in tissue analogs independently of laboratory technical resources, expertise or operators.

[0113] The herein formulated bioink-cryoprotectant combinations using tissuespecific decellularized matrix has enabled the cryofabrication of pancreatic tumor models and the recapitulation of its microenvironment. The discovered combination of melezitose with glycerol ensured cellular viability after 3D tumor models cryobioprinting and cryopreservation, in comparison to the conventionally used CPAs (i.e., DMSO, melezitose), demonstrating the potential of this cryoprotective formulation to establish off-the-shelf tumor-stroma models. The cryopreserved 3D PDAC models also showed preclinical drug screening functionality, exhibiting no major differences in terms of drug response compared to non-cryopreserved controls. Our findings set the grounds for future evaluation of thepotential of dECM- and, dECM:HA- based bioinks for prolonged storage (months / years). Additionally, fabrication of tumor constructs with tissue-like cell densities could also be envisioned. Overall, the herein cryobioprinted tumor-stroma models represent a significant advancement in cancer research and drug screening, offering the ability to fabricate shelfready biomimetic tumor models.Concluding Remarks

[0114] Cryobioprinting represents a paradigm shift in tissue engineering and biofabrication of tissue analogs, surpassing traditional bioprinting limitations related to inks rheology, while unlocking the possibility to generate shelf-ready living constructs that retain their biofunctionality.

[0115] By enabling direct and rapid printing at subzero temperatures, the stabilization and maintenance of the bioprinted 3D architectures, as well as the cryopreservation of living constructs in a single, streamlined workflow, this technology advances how we conduct the fabrication, storage and development of 3D human tissues. Notably, cryobioprinting suppresses one of the major bottlenecks in cunent biofabrication practices, the inability to produce viable, storable functional constructs for off-site use.

[0116] Cryobioprinting unlocked the possibility to fabricate shelf-available, physiological relevant human tissues that can be stored and distributed globally, revived on demand, and integrated directly into drug discovery processes and pipelines. Such a possibility unveils a new era of standardized, reproducible, and scalable preclinical tumor platforms for cancer research and therapies screening or even the attainability of have ready-to-use tissues available to be used by pharmaceutical companies or hospitals, bioengineered using patient-specific cells and ECM for personalized treatment evaluation, without requiring specific expertise on biofabrication.

[0117] From another perspective, cryobioprinting surpasses constraints regarding bioink viscosity, enabling the use of low-viscosity biomaterials, such as dECM formulationsthat better emulative the native ECM. The inclusion of optimized cryoptrotective agents, while highly dependent of cell types, surrounding matrix and freezing temperatures, is critical for the preservation of cell viability and functionality, ensuring the design and establishment of physiomimetic, off-the-shelf models with translational relevance. Such advances pave the way for ready-to-use tissue analogs, future exploration of longer preservation periods, and the biofabrication of high cell density, complex living tissues.

[0118] Overall, cryobioprinting has the potential to turn complex and reliable tissue analogs broadly accessible to academic institutions, biotechnology companies, pharmaceuticals, and clinical settings, as ready-to-use biological tools. In this context it emerged not only as a technological biofabrication advance, but also as a crucial platform to level up scalable precision medicine and tissue engineering.METHODS

[0119] Pancreas Decellularization

[0120] Porcine pancreas decellularized extracellular matrix was obtained as previously described. Ferreira et al., Trends Biotechnol 38, 1-18 (2020). Briefly, after porcine pancreas tissue extensive washes performed with deionized cold water and all the large vessels, fat residues, connective tissue being removed, the remaining parenchyma was frozen at -80 °C until tissue processing. Following tissue thawing and cut into small sections, pancreas tissue was rinsed with lx PBS for 30 min and treated with 0.1 mM Gabexate Mesylate solution (Selleckchem, cat # S2101), for 30 min. After proteinase inhibition treatment, chopped tissue was decellularized in 1% (v / v) Triton-X 100 containing ammonium hydroxide (NFUOH, 0.1%, v / v) during 48 h at 4 °C, with solution changes at each 4-6 hours. Afterward, decellularized tissue was abundantly washed in D-Phosphate buffer saline ((D-PBS) without magnesium and calcium, Alfagene, Portugal) during 24 h to remove excess of detergents and immersed in a DNase I solution (1 M, 1 h, RT, Stem Cell Technologies, cat#07469). After DNase exposure, pancreatic tissue was immersed in a peracetic acid solution 1% (v / v) for 10 min and washed three times (5 min each) with D-PBS containing 1% (v / v) antibiotic / antimycotic (ATB, Thermo Fisher Scientific, cat #15240062). The obtained sterilized decellularized tissues were recovered by centrifugation and freeze-dried at -86 °C (Telstar LyoQuest), over a period of 14 days. Lyophilized dECM was milled to generate a fine powder for subsequent digestion. Reagents used for decellularization were of research grade and obtained from Merck-Sigma-Aldrich unless otherwise stated.

[0121] Tissue Digestion

[0122] Aiming to produce a dECM matrix to support cell culture, milled dECM (10 mg. ml.1) was digested in pepsin (pepsin from gastric mucosa, Merck-Sigma-Aldrich, cat #P6887; 1 mg. mL-1in 0.01 M HC1) for 72 h. Following tissue digestion, dECM was neutralized (pH 7.4) using IM NaOH, and the salt conditions were adjusted with 10X PBS. The obtained dECM solution was frozen, lyophilized, and stored at -80 °C until further use.

[0123] Hyaluronic Acid Chemical Modification

[0124] Hyaluronic acid tyramine (HA-Tyr) was synthesized as previously described. Ferreira et al., ibid. In brief, 600 mg hyaluronic acid (MW: -1.5 x 106-1.8xl06Da, Merk- Sigma-Aldrich, cat #53747) were dissolved in 2-morpholinoethane sulfonic acid (MES) buffer (0.1 M, pH 6.0) under magnetic stirring, at 25 °C. Afterward, 4-(4,6- Dimethoxydimethoxy-l ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM, 248 mg, purity > 95% by HPLC, cat #D2 19, Tokyo Chemical Industry (TCI), Japan) and tyramine (123 mg, purity > 98%, cat #T90344, Merck-Sigma-Aldrich) were both dissolved in 2 mL of MES buffer and drop wise added to the HA solution, and the reaction occurred for 72 h, at 25 °C, protected from light. The modified polymer was then dialyzed (MWCO: 6-8 kDa) for 5 days, at RT, by using 0.1 M NaCl for 2 days and double distilled deionized water as dialysate for 3 days. The purified polymer was freeze-dried (-86 °C), for 7 days, in the dark. HA degree of modification was determined through 1H NMR spectroscopy.

[0125] Cryoprotective Bioink Design

[0126] Pancreas dECM (1.5% w / v, in sterile D-PBS) and dECM 1.5% mixed with HA-Tyr (1% w / v, in sterile D-PBS) were used as the principal components of tumor and stroma bioinks, respectively. DMSO (10% w / v), glycerol (10% w / v) and D-(+)-melezitose (12% w / v) were used as cryoprotectants.

[0127] Freezing and Ccryopreservation of dECM, dECM:HA -Based Hydrogels

[0128] dECM and dECM:HA hydrogel precursor solutions were prepared by dissolving lyophilized digested dECM at 1.5% and dECM 1.5% : HA-Tyr 1% in a sterile DPBS solution containing DMSO or Glycerol 10%, D-(+)-melezitose 12%, and Ru / SPS (IxlO-3mg- mL-l / lxl0‘2mg- ml.1) photoinitiator (cat #5248, Advanced BioMatrix, Carlsbad, CA, USA). Cylindrical frozen hydrogels were produced by pipetting 100 pL of precursor solutions into polydimethylsiloxane (PDMS, Dow Corning, USA) molds with 8 mm diameter, over a thermoelectric cooler freezing plate set at -20 °C via adjusting the input voltage. After hydrogels freezing, and while they were still over the freezing plate, dECM, and dECM: HA platforms were photocrosslinked under blue light with a power of 3.2 W. cm-2(VALO Cordless curing light, Ultra-dent Products, Inc, USA). Afterwards, freezing hydrogels were placed in PBS for further physical analysis. To evaluate the effects of cryopreservation in hydrogels mechanically and structurally, photocrosslinked frozen hydrogels were immediately placed at -80 °C for 24 h, followed by transference to PBS.

[0129] Scanning Electron MicroscopySEM Analysis

[0130] Scanning electron microscopy (SEM) analysis was performed to evaluate the influence of the freezing and cryopreservation process as well as cryoprotectants role on dECM, dECM:HA platforms microarchitecture. Briefly, frozen and cryopreserved dECM, dECM:HA models were deposited on a metal stub containing an O.C.T. compound (Tissue - Tek, Sakura, Japan) and analyzed in a SU-3800 (Hitachi, Japan) at -20 °C, coupled with a standard SEM cooling stage ranging from -25 °C to 50 °C (Deben, UK).

[0131] Rheological Analysis

[0132] Cryoprotective inks rheological properties were characterized at 25 °C in a Kinexus lab+ rotational rheometer (Malvern Instruments, UK) equipped with a stainless- steel sandblasted parallel plate geometry and a solvent trap. Shear rate ramp tests (0.01-100 s’1) were conducted, and the viscosity was recorded to evaluate their shear-thinning profile. Unfrozen, frozen, and cryopreserved dECM and dECM:HA platforms (n = 3 for each condition) were prepared as previously described. The samples were analyzed under a control strain of 0.1%, at 1 Hz, by using a flat geometry with an 8 mm diameter. A single frequency oscillatory measurement was performed for 2 min and all measurements were performed at 37 °C. Results were recorded with the rSpace software and samples were compared related to the absence or presence of studied CPAs.

[0133] Cell Culture

[0134] Human pancreatic cancer cell line (PANC-1, ATCC CRL-1469) was cultured in Dulbecco’s Modified Eagle Medium-High Glucose (DMEM-HG, Alfagene, Carcavelos, Portugal) supplemented with sodium bicarbonate (3.7 g- L-1), 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% antibiotic / antimycotic. Human Pancreatic CAF-Stellate Cells (CAF08, Vitro Biopharma, USA) were cultured in MSC-GRO™ Pancreatic Stellate CAFs Maintenance Medium, supplemented with 1% antibiotic / antimycotic, as recommended by the manufacturer. Human cancer and stromal cells were cultured in cell culture-treated T175-flasks, and maintained under 5% CO2 atmosphere, at 37 °C. The medium was replaced every 2-3 days.

[0135] Evaluation of CPAs influence on cell-laden models freezing and cryopreservation

[0136] PANC-1 and CAFs samples were generated individually to evaluate the efficacy of different CPAs formulations on cell-laden platforms freezing and cryopreservation. PANC-1 :dECM and CAFs: dECM:HA hydrogel precursor solutions were prepared by dissolving the respective cell line at a cell density of 5xl06cell per mL of ink comprising the biomaterial, DMSO or glycerol 10 % (v / v), D-(+)-melezitose 12% (w / v),and Ru / SPS (IxlO3mg- mL-1 / lxlO2mg- mL-1) photoinitiator (cat #5248, Advanced BioMatrix, Carlsbad, CA, USA). The photoinitiator and the permeable CPAs were added to the mixture immediately before the samples production to limit the cell contact and cytotoxicity. Cylindrical frozen cell-laden hydrogels were produced by pipetting 100 pL of precursor solutions into polydimethylsiloxane (PDMS, Dow Corning, USA) molds with 8 mm diameter, over a thermoelectric cooler freezing plate set at -20 °C. After hydrogels freezing, and while they were still over the freezing plate, dECM and dECM:HA platforms were photocrosslinked under blue light with a power of 3.2 W. cm-2(VALO Cordless curing light, Ultra-dent Products, Inc, USA). Afterwards, freezing hydrogels extensively washed with cell culture medium to washout CPAs and were placed in culture for further analysis. To evaluate the cryopreservation potential and achieve optimized cryoprotective bioinks, photocrosslinked frozen hydrogels were immediately placed at -80 °C for 24h, followed by transference to culture medium and extensive washes to remove remaining CPAs. 3D models were maintained under 5% CO2 atmosphere, at 37 °C, until further analysis.

[0137] Cryobioprinting Pancreatic Tumor-Stroma Models

[0138] 3D cryobioprinting experiments were conducted on a CELLINK BIO X extrusion bioprinter loaded with two cartridges comprising the tumor bioink and the stroma bioink, respectively. A self-designed and manually assembled freezing plate was prepared as previously reported. The freezing plate temperature was set to -20 °C. Bioinks extrusion was performed at 25 °C and using a 22G nozzle. A cylinder of 0 10 mm and 1 mm height was cryobioprinted. The cylinder infill volume was produced by tumor bioink extrusion, and the outer layers were then cryobioprinted through the stroma bioink deposition. Tumor compartment cryobioprinting was performed setting the pressure to 6 kPa and the printing speed to 1 mm- s'1pressure. Afterwards, the second cartridge comprising the stroma bioink was extruded programming the printhead speed to 3 mm-s1and the pressure to 9 kPa. Immediately before printing, PANC-1 and CAFs cells were detached, centrifugated, and suspended in the respective cryoprotective bioinks, at a density of 5xl06and 10xl06cellsper mL, respectively. Besides the specific cell line and the studied CPAs, tumor bioink was composed of dECM 1.5% (w / v) while the stroma bioink was designed to include dECM 1.5% (w / v) and dECM 1.5% (w / v): HA-Tyr 1% (w / v). After cryobioprinting, the 3D constructs were photocrosslinked via blue light for 40 s (VALO Cordless curing light, Ultra-dent Products, Inc, USA). The samples were bioprinted on the freezing plate, photocrosslinked while they were still frozen, and then stored in -80 °C, 24 h and for 7 days. Alternatively, cryobioprinted models were immediately placed in culture after the cryobioprinting process. As control, owing to the inability the dECM-based bioinks in air, non-frozen models were produced through casting, following the same bioink composition and model dimensions of cryobioprinted constructs.

[0139] 3D Models Cell Viability Analysis

[0140] Live / dead cell assay was performed for viability assessment at specific time points. Briefly, 3D in vitro models were incubated with Calcein AM (5 pg / mL1) and propidium iodide PI (5 pg / mL1) (Thermo Fisher Scientific, USA), in PBS, at standard culture conditions (5% CO2 at 37 °C), for 30-45 min. After washing with PBS, the samples were observed under a scanning confocal microscope (Zeiss LSM 900, Carl Zeiss, Germany), equipped with a GaAsP detector and a 10x / 0.4 Plan- Apochromat objective. All data was processed in Zeiss Zen and Imaris Viewer software. The ratio of live cells to the total captured cells in representative regions of the samples (n=3) was determined through processing the fluorescent images using ImageJ.

[0141] Cell Tracking

[0142] Prior to being cryobioprinted, PANC-1 and CAFs were incubated with cell- permeable fluorescent probes, namely CellTracker™ Green CMFDA and DiD, respectively. In brief, the cells were incubated with each dye (5 pL per 1 x 106cells), at 37 °C, for 30 min (CAFs), and 1 h (PANC-1). Afterward, cryoprotective bioinks were prepared as previously described, and the tumor-stroma model cryobioprinted. Fluorescent livingconstructs were visualized in a scanning confocal microscope (Zeiss LSM 900, Carl Zeiss, Germany).

[0143] Cytoskeletal Proteins Labeling

[0144] The cellular distribution and cytoskeletal arrangement in cryobioprinted and cryopreserved tumor-stroma PDAC models was evaluated via F-actin / nuclei staining. Briefly, tumor-stroma living constructs were fixed with formaldehyde 4% (v / v) solution, 24 h, 4 °C. After extensive washing with PBS, the cells membrane was permeabilized with 0.1 % Triton X-100, 15 min, RT. Afterwards, cells were stained with Flash Phalloidin Red 594 (1 :40 (v / v), in PBS) in a humidified atmosphere, for 24 h, at RT. After wash three times with PBS, samples were stained with DAPI (4”,6-diamidino-2- phenylindole, dihydrochloride(DAPI) (1:250 (v / v)) and incubated for 30 min, at RT. Bioimaging was performed in a scanning confocal microscope (Zeiss LSM 900, Carl Zeiss, Germany).

[0145] Decellularization Efficacy

[0146] To evaluate the efficiency of decellularization, the DNA content was assessed on both ECM abd dECM samples after papain (Merck-Sigma-Aldrich, cat#76220), digestion at 65 °C for 4 h. Quant-iT PicoGreen dsDNA assay kit (Thermo Fisher Scientific, USA) was used according to the manufacturer’s instruction, and fluorescence was measured in a microplate reader (Synergy HTX, BioTek). The results were expressed in micrograms of DNA per milligram of dry tissue (pg mg-1of dry tissue).

[0147] dECM Biochemical Characterization

[0148] To evaluate sGAGs content samples of ECM and digested dECM were incubated with papain extraction reagent for 4 h at 65 °C. The sGAG content was quantified using the Blyscan GAG Assay Kit (Biocolor, UK) following the manufacturer’s instructions. The absorbance was measured at 4=650 nm using a microplate reader (SynergyHTX, BioTek, USA) and compared to standards. Results were expressed in micrograms of GAGs per milligram of dry tissue (pg mg-1of dry tissue).

[0149] Native collagen was quantified using the Sircol Insoluble Collagen Assay Kit (Biocolor, UK), following the manufacturer’s instructions. The absorbance was measured at A=556 nm using a microplate reader (Synergy HTX, BioTek, USA) and compared to standards. Results were expressed in micrograms of collagen per milligram of dry tissue (pg i ng1of dry tissue).

[0150] Hyaluronic acidHA was quantified using the Purple-Jelley Hyaluronan Assay (Biocolor, UK) following the manufacturer’s instructions. The absorbance was measured at =655 nm using a microplate reader (Synergy HTX, BioTek, USA) and compared to standards. Results were expressed in micrograms of hyaluronan per milligram of dry tissue (pg mg-1of dry tissue).

[0151] Proteomic Analysis of ECM and dECM Samples through LC-MS / MS

[0152] Pancreatic tissue specific ECM and dECM samples were freeze- dried and sent for proteomics analysis. Each sample was processed for proteomic analysis following the solid-phase-enhanced sample-preparation (SP3) protocol and enzymatically digested with trypsin / LysC as previously described.

[0153] Protein identification and quantitation was performed by nanoLC-MS / MS equipped with a Field Asymmetric Ion Mobility Spectrometry - FAIMS interface. This equipment is composed of a Vanquish Neo liquid chromatography system coupled to an Eclipse Tribrid Quadrupole, Orbitrap, Ion Trap mass spectrometer (Thermo Scientific, San lose, CA, USA). 250 nanograms of peptides of each sample were loaded onto a trapping cartridge (PepMap Neo C18, 300 pm xx 5 mm i.d., 174500, Thermo Scientific, Bremen, Germany). Next, the trap column was switched in-line to an Aurora Frontier XT 60 cm, 75 pm (AUR3-60075C18-XT) chromatographic separation column. A 116 min separation was achieved by mixing A: 0.1% FA and B: 100% ACN, 0.1% FA with the following gradientat a flow of 250 nL / min1: 2 min (0% B to 4% B), 20 min (4% B to 12% B), 65 min (12% B to 28% B), 1 1 min (28% B to 45% B), 2 min (45% B to 85 % B) and 16 min at 99% B. Subsequently, the column was equilibrated with 0% B. Data acquisition was controlled by Xcalibur 4.7 and Tune 4.1.4244 software (Thermo Scientific, Bremen, Germany).

[0154] MS results were obtained following a Data Dependent Acquisition - DDA procedure. MS acquisition was performed with the Orbitrap detector at 120, 000 resolution in positive mode, quadrupole isolation, scan range (m / z) 375-1,500, RF Lens 30%, standard AGC target, maximum injection time was set to auto, 1 microscan, data type profile and without source fragmentation. FAIMS mode: standard resolution, total carrier gas flow: static 4L / min, FAIMS CV: -45, -60 and -75 (cycle time, 1 s). Internal Mass calibration: Run-Start Easy-IC. Filters: MIPS, monoisotopic peak determination: peptide, charge state: 2-7, dynamic exclusion 30s, intensity threshold, 5.0e3. MS / MS data acquisition parameters: quadrapole isolation window 1 .8 (m / z), activation type: HCD (30% CE), detector: ion trap, IT scan rate: rapid, mass range: normal, scan range mode: auto, normalized AGC target 100%, maximum injection time: 35 ms, data type centroid.

[0155] The raw data was processed using the Proteome Discoverer 3.1.1.93 software (Thermo Scientific) and searched against the UniProt database for the Sus scrofa proteome (2024_04 with 46,174 entries). A common protein contaminant list from MaxQuant was also included in the analysis. The Sequest HT search engine was used to identify tryptic peptides. The ion mass tolerance was 10 ppm for precursor ions and 0.5 Da for fragment ions. The maximum allowed missing cleavage sites was set to two. Cysteine carbamidomethylation was defined as constant modification. Methionine oxidation, deamidation of glutamine and asparagine, peptide terminus glutamine to pyroglutamate, and protein N-terminus acetylation, Met-loss, and Met-loss+acetyl were defined as variable modifications. Peptide confidence was set to high. The processing node Percolator was enabled with the following settings: maximum delta Cn 0.05; target FDR (strict) was set to 0.01 and target FDR (relaxed) was set to 0.05, validation based on q-value. Protein label- free quantitation was performed with the Minora feature detector node at the processingstep. Precursor ions quantification was performed at the consensus step with the following parameters: inclusion of unique plus razor peptides, precursor abundance based on intensity, and normalization based on total peptide amount. For hypothesis testing, protein ratio calculation was pairwise ratio-based and a t-test (background based) hypothesis test was perfomied.

[0156] Protein Identification and Label-Free Quantification

[0157] Raw files were analyzed using MaxQuant (v2.1.0.0) with the Andromeda search engine against the Sus scrofa UniProtKB database (June 2025). Trypsin / P and chymotrypsin were used; up to two missed cleavages allowed. Modifications included carbamidomethylation (fixed) and oxidation / N-ethylmaleimide (variable). FDR was set to 1%. LFQ was enabled with a minimum ratio count of 2. Data were processed in Perseus (v2.0.7.0). LFQ intensities were log2-transformed. Only proteins with a score > 25, at least 2 unique peptide were retained.

[0158] NMR Metabolic Profiling of Medium Samples

[0159] Medium samples were collected from non-frozen, cryobioprinted, and cryopreserved for 24 h models (n = 5 replicates). Acellular media incubated under the same conditions were also collected.

[0160] To precipitate interfering proteins, 700 pL of methanol (pre-cooled to -80 °C) were added to 350 pL of each medium sample. The mixture was incubated for 30 min at -20 °C, followed by centrifugation at 13,000 x g for 20 min at 4 °C. The resulting supernatant (1 mL) was then dried using a vacuum concentrator (CentriVap, Labconco) and stored at -80 °C. For NMR analysis, the dried samples were reconstituted in 300 pL of deuterated PBS (100 mM, pH 7.4) containing 0.1 mM trimethylsilylpropanoic acid (TSP- 04), and transferred to 3 mm NMR tubes. NMR spectra were acquired on a Bruker Avance III HD 500 MHz spectrometer equipped with a 5 mm BBO CryoProbe Prodigy (Portuguese NMR Centre, University of Aveiro), operating at 500.13 MHz for 1H observation, at 298 K.One-dimensional 1H spectra were acquired with 32 k data points, a spectral width of 8012.82 Hz, a relaxation delay of 2 s, and 128 scans, using the ‘noesyprld’ pulse program (Bruker library). Spectral processing was performed using TopSpin 4.0.3 (Broker BioSpin, Rheinstetten, Germany) and included zero-filling to 64 k data points, phasing, baseline correction, and calibration to the TSP-d4 signal at 0 ppm. Metabolites were identified by matching the spectral data to reference spectra in BBIOREFCODE- 2-0-0 (Broker BioSpin, Rheinstetten, Germany) and Chenomx NMR Suite version 10.1 (Edmonton, AB, Canada). To quantify metabolic variations, selected signals were integrated using Amix software (version 3.9.15), and the differences relative to the acellular medium were calculated.

[0161] ELISA Assays

[0162] The quantification of soluble biomolecular markers secreted by the different 3D PDAC models (i.e., non-frozen, cryobioprinted and cryopreserved (for 24 h, and 7 days)) including: i) human TGF- / J1, ii) SDF-1, iii) MMP-2, and iv) MMP-9 was performed by ELISA. In brief, at predetermined time points (7 and 14 d) the culture medium (n = 3) of each condition was retrieved and stored at -80 °C. A proportion of 1 : 1 (medium of day 7: day 14) was prepared before ELISA assay. Human PDAC biomolecular markers quantification was performed by sandwich ELISA according to manufacturer’s instructions. Absorbance was determined by using a multimodal Synergy Hl'X microplate reader (BioTek Instruments, USA).

[0163] Total protein content Analysis

[0164] The total protein content in cell culture media was assessed using the BCA Protein Assay Kit following the manufacturer’s instructions were meticulously followed. The absorbance was measured at 562 nm using a microplate reader (Synergy HTX).

[0165] In vitro Drug Screening

[0166] In order to evaluate the screening functionality of cryobioprinted and cryopreserved 3D tumor-stroma platforms and the ability of the 3D constructs to be used as preclinical tumor platforms for drug-screening assays, living platforms were exposed to anti-cancer drug. Briefly, at day 14 of culture, 3D PDAC in vitro models were incubated with 125 pM of Gemcitabine, a standard therapy for pancreatic cancer, for 72 h. Cells viability was accessed by using the CellTiter-Glo 3D® Cell Viability Assay in accordance with the manufacturer instructions, with minor modifications. Prior to analysis, 3D in vitro models were mechanically disrupted and incubated with a mixture of culture medium and CellTiter-Glo® reagent (ratio of 1 : 1 (v / v)), as above mentioned. Luminescence was recorded in 96-well flat-bottom white plates by using a multi-modal Synergy HTX microplate reader (BioTek Instruments, Winooski, USA).Statistical Analysis

[0167] Statistical analysis was performed in GraphPad Prism 8TM Software. Oneway ANOVA test was used to evaluate differences among different groups. A value of p < 0.05 was deemed statistically significant.

[0168] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A bioink for cryobioprinting, wherein the bioink comprises a decellularized extracellular matrix (dECM)-based hydrogel and a saccharide cryoprotectant.

2. The bioink of claim 1, wherein the saccharide cryoprotectant is melezitose.

3. The bioink of claim 2, wherein the melezitose is present in an amount from about10% to about 15% v / v.

4. The bioink of claim 1 , wherein the bioink further comprises glycerol.

5. The bioink of claim 4, wherein the glycerol is present in an amount from about 8% to about 12%.

6. The bioink of claim 1, wherein the dECM-based hydrogel further comprises hyaluronic acid.

7. The bioink of claim 1, wherein the bioink comprises 1% to 2% w / v of the dECM- based hydrogel.

8. A method of making a 3D-printed biomaterial, comprising depositing a bioink comprising a dECM-based hydrogel, cells and a saccharide cryoprotectant from a 3D printer onto a freezing plate to form a frozen bioink filament; depositing additional bioink from a 3D printer in contact with a previously placed frozen bioink filament to form a 3D-printed biomaterial comprising a plurality of frozen bioink filaments; andremoving the 3D-printed biomaterial from the freezing plate.

9. The method of claim 8, wherein the 3D-printed biomaterial is crosslinked.

10. The method of claim 8, wherein the hydrogel is chilled to a temperature ranging from about 0 °C to about 10 °C before being deposited by the 3D printer.

11. The method of claim 8, wherein the dECM-based hydrogel further comprises hyaluronic acid.

12. The method of claim 8, wherein the freezing plate has a temperature ranging from about 0 °C to about -30 °C.

13. The method of claim 8, wherein the cryoprotectant further comprises glycerol.

14. The method of claim 8, wherein the saccharide cryoprotectant is melezitose.

15. A 3D-printed biomaterial, made according to the method of any one of claims 8 to 14.

16. The 3D-printed biomaterial of claim 15 wherein the 3D-printed biomaterial comprises micropores.

17. The 3D-printed biomaterial of claim 16, wherein the micropores have a pore size from about 10 pm to about 50 pm.

18. The 3D-printed biomaterial of claim 15 wherein the 3D-printed biomaterial comprises a tissue scaffold, spheroid, or organoid.1 . The 3D-printed biomaterial of claim 15, wherein the cells are cancer cells.

20. The 3D-printed biomaterial of claim 19, wherein the 3D-printed biomaterial is a tumor model.

21. The 3D-printed biomaterial of claim 20, wherein the tumor model comprises a tumor region surrounded by a stoma compartment.

22. The 3D-printed biomaterial of claim 20, wherein the tumor model is a pancreatic ductal adenocarcinoma model.

23. The 3D-printed biomaterial of claim 15, wherein the 3D-printed biomaterial is frozen.

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