SILK FIBROIN BIOINKS AND RELATED USE FOR 3D BIOPRINTING

Abstract

The present invention relates to silk fibroin bioinks specifically formulated for use in 3D bioprinting and the production of ex-vivo models capable of supporting hematopoiesis and the production of platelets and blood cells.

Claims

1. A bioink formulation comprising: a silk fibroin in an amount of 0.5%-35% w/v; a gelatin or derivatives thereof in an amount of 5-20% w/v; an alginic acid or derivatives thereof in an amount of 0.5-2% w/v; a glucose or analogue thereof in an amount of 3-4% w/v; a controlled density solution in an amount of 1-3% w/v; a buffer in an amount of 4-6% w/v; and a saline buffer solution up to 100% w/v; wherein the gelatin and alginic acid or derivatives thereof are present in a reciprocal ratio ranging from about 10:1 to about 20:1; wherein the silk fibroin and gelatin are present in a reciprocal ratio ranging from about 1:5 to about 1:0.5; wherein said glucose or analogue thereof is selected from the group consisting of disaccharides, stereoisomers, isomers, epimers, alditols or acids of glucose, precursors or products deriving from the glucose metabolism, capable of entering the cell via glucose transporters to be part of the glycolysis cycle; suitable for extrusion with hematopoietic lineage cells.

2. The bioink formulation according to claim 1, further comprising a cell component selected from the group consisting of progenitor cells of the myeloid or lymphoid lineage, stem cells or differentiated cells of the hematopoietic lineage selected from the group consisting of monocytes, macrophages, granulocytes, neutrophils, basophils, eosinophils, red cells, megacaryocytes, platelets, dendritic cells, T lymphocytes, B lymphocytes, NK cells, fibroblasts, mesenchymal stem cells, endothelial cells.

3. The bioink formulation according to claim 1, wherein said cells are present at a concentration within the range of 0.5-4?10.sup.6 cells/ml, preferably 1?10.sup.6 cells/ml.

4. The bioink formulation according to claim 1, wherein the gelatin is type A or type B.

5. The bioink formulation according to claim 1, wherein the derivatives of alginic acid are salts with alkaline and alkaline earth metals selected from the group consisting of sodium alginate, calcium alginate, magnesium alginate, potassium alginate and alginate methacrylate.

6. The bioink formulation according to claim 1, wherein the controlled density solution is a defined density solution selected from the group consisting of colloidal silica, copolymers, hydrophilic polysaccharides or synthetic high-molecular-weight polymers.

7. The bioink formulation according to claim 1, wherein the buffer is organic or mineral selected from the group consisting of: HEPES, MES, ADA, ACES, PIPES, MOPSO, Bis-6Tris Propane, BES, MOPS, TES, DIPSO, MOBS, TAPSO, HEPPSO, POPSO, EPPS (HEPPS), Tricine, Gly-Gly, Bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS and CABS.

8. The bioink formulation according to claim 1, further comprising at least one of a fluorescent marker, a contrast agent, a luminescent agent, a radiopaque agent, a radioactive element or combinations thereof.

9. The bioink formulation according to claim 1, wherein the formulation is sterile.

10. The bioink formulation according to claim 1, further characterized in that it is at a temperature of 37? C.

11. A method of bioprinting comprising applying of a bioink formulation according to claim 1.

12. A method for the production of scaffolds comprising progenitor cells of the myeloid or lymphoid lineage, stem cells or differentiated cells of the hematopoietic lineage comprising applying the bioink formulation of claim 1.

13. A method for the production of a 3D scaffold, comprising: a) bioprinting of the bioink formulation according to claim 1 through a nozzle having a caliber ranging from 18 G to 27 G at an extrusion pressure within the range of 5-200 kPa and a printing rate within the range of 5-1,000 mm/min on a substrate to obtain a mono- or multi-layer 3D scaffold; b) soaking the 3D scaffold obtained according to step a) in a reticulating solution; c) adding the cell culture medium to the scaffold and optionally growth factors to promote cell differentiation.

14. The method for the production of a 3D scaffold according to claim 13, wherein the extrusion pressure ranges from 5 kPa to 20 kPa and the printing rate is within the range of 400-1,000 mm/min.

15. A scaffold obtained by a bioprinting method of the bioink formulation according to claim 1.

16. A scaffold obtained by a bioprinting method of the bioink formulation according to claim 13.

17. The scaffold according to claim 15, wherein the bioink formulation further comprises one or more molecules selected from the group consisting of: components of the extracellular matrix such as proteoglycans, hyaluronic acid, collagens, elastin, fibronectin, fibrin, fibrinogen, laminin, thrombospondin; growth factors, cytokines and chemokins, preferably hematopoietic, such as interleukins, CSF-1, G-CSF, GM-CSF, SCF, FLT3-L, TPO, EPO, SDF-1?; TGF-?1, TNF?, VEGF, FGF, Notch ligands, WNT, Angiopoietin-1, BMP, IGF-2 and fragments or variants thereof; antigens, conjugated or unconjugated antibodies, or their fragments such as CDRs or epitopes; hormones, such as insulin, glucagon, triiodothyronine, thyroxine, or their antagonists; drugs or prodrugs, such as TPO mimetics, TPO receptor agonists, tyrosine kinase receptor agonists, tyrosine kinase receptor inhibitors, Rho kinase inhibitors (ROCK), kinase inhibitors, aryl hydrocarbon receptor antagonists, chemotherapeutic agents; nucleic acids, such as DNA, RNA, siRNA, RNAi and microRNA, plasmids, lentiviruses, CRISPR; enzymes and/or intermediates of enzymatic reactions; and toxins.

18. A method for dissolving the scaffold obtained from a bioprinting method of the bioink formulation according to claim 1, wherein the method comprises preparing the scaffold by means of the method of claim 13, and further comprises using a dissolution solution of the scaffold comprising sodium citrate, collagenase, alginate lyase and a source of glucose.

19. The method according to claim 18, wherein the dissolution solution of the scaffold comprises: sodium citrate in an amount of 15 mM collagenase in an amount of 10 U/mL alginate lyase in an amount of 5 U/mL 5 mM glucose a saline buffer solution.

20. A method of supporting hematopoiesis or for the production of platelets and blood cells comprising using the scaffold according to claim 15 as an ex vivo model.

21. A method of supporting in vivo hematopoiesis or the production of platelets and blood cells comprising using the scaffold of claim 15 as a surgical implant.

Description

[0086] The present invention will now be described for illustrative but non-limiting purposes, according to a preferred embodiment with particular reference to the attached figures, in which:

[0087] FIG. 1 shows the comparison between the characteristics (Printing, Crosslinking and Maintenance) of the formulation B (S+G+A) tested in comparison with the variants S+G or S+A, and reveals the clearly superior profile of the formulation comprising silk fibroin, gelatin and alginate (S+G+A).

[0088] FIG. 2 illustrates the graphs relating to the parameters of the bioprinting process (panel A) with the silk fibroin ink (SGA) of the invention relating to the extrusion pressure and ink deposition rate (panel B); size of the nozzle, extrusion pressure and ink deposition rate (panel C); impact of the presence of fibroin in the sample (SGA) vs sample (GA) (panels D-E); viscosity of the ink SGA (panel F); analysis of the fibroin fibril formation by optical or fluorescence microscopy (panel G).

[0089] FIG. 3 shows images of the bioprinting process (panels A-B) with the bioink formulation according to the invention and the diffusion capacity of biocompatible dyes in the culture medium (panel C).

[0090] FIG. 4, panel A shows confocal microscopy images of the scaffold obtained by bioprinting using the bioink of the invention to which CD34.sup.+ hematopoietic stem cells (1?10.sup.6 cells/ml) are added. Panel B shows the z-stack confocal microscopy images after 24 hours post-printing showing a cell survival of about 90%. Panel C shows images showing the morphological change observed in cells that begin to enlarge and become polyploid during culture. Panel D shows a significant increase in the cell diameter, a typical feature of megakaryocyte differentiation and maturation, after 14 days of culture. Panel E shows the images following the marking of the actin cytoskeleton with TRITC-phalloidin which highlights the presence of a network of proplatelets within the ink.

[0091] FIG. 5, shows the image of the customized culture chamber for microscopy viewing. The presence can be noted of a microscope glass at the bottom of the chamber. The space for the ink deposition and the subsequent culture of the scaffold and the wells for depositing the liquid reservoir for the maintenance of the humidified chamber, can be seen. 3D visualization of the culture obtained by real-time confocal microscopy shows a platelet-producing megakaryocyte. In addition, numerous platelets released into the ink are visible at the end of the culture. The megakaryocytes were pre-marked with an anti-CD61 antibody conjugated with fluorescein isothiocyanate before being included in the bioink and printed.

[0092] FIG. 6, panel A shows the result of the activation of a chemiluminescent reaction in a bioink functionalized with horseradish peroxidase (HRP). Panel B shows the colorimetric reaction developed in the HRP functionalized bioink and incubated with 2 or 16 mM lactate in the presence of lactate oxidase. Lactate-free incubation was used as a negative control. Panel C shows the colorimetric reaction activated by the lactate released by the same cells grown inside the 3D scaffold.

[0093] FIG. 7, panel A shows images of the dissolution process of the bioprinted scaffold with the formulation according to the invention. Dissolution was carried out after 2 weeks of culture. Released megakaryocytes were characterized by immunofluorescence microscopy after marking of the CD61 surface marker. Panel B shows the cyto-fluorometric flow analysis of the marking of membrane integrins CD41a and CD42a in cells recovered after dissolution of the scaffold. Panel C shows the result of an electrophoretic run of a protein lysate obtained from cells recovered after dissolution of the scaffold. The protein lysate obtained from cells grown in a classical culture plate was used for comparison. The Western blotting assay of the same protein lysates reveals the expression of the thrombopoietin receptor (TPO) comparable between cells grown under classic 2D culture conditions and cells obtained after ink dissolution.

[0094] The following non-limiting examples are now provided for a better illustration of the invention, in which different formulations of silk fibroin-based bioink were tested and compared, evaluating the quality and compatibility with the cells in the scaffolds obtained by 3D bioprinting.

EXAMPLE 1: PREPARATION OF FIBROIN-BASED BIOINK FORMULATIONS

Preparation of Silk Fibroin

[0095] The aqueous solution of silk fibroin was obtained from silkworm cocoons of the Bomby mori species deprived of the worms. The cocoons were boiled for 10, 20, 30, 40, 50 or 60 minutes in a solution of Na.sub.2CO.sub.3 in a weight/volume (w/v) ratio of 10 g/4 L. By varying the boiling time fibers having different molecular weights can be obtained. The fibers are rinsed for 20 minutes three times in ultra-pure water and dried overnight. The dry fibers were solubilized for 4 hours at 60? C. in LiBr. The solubilized silk solution was subjected to dialysis with distilled water using a dialysis cassette with a threshold of 3.5 kDa for three days and changing the water for a minimum of eight times. The silk solution was finally centrifuged at a minimum rate of 2,000 rpm for 10-15 minutes to remove larger particles and stored at 4? C. The concentration of the silk solution was determined by drying a known volume of the solution overnight at 60? C. and weighing the remaining solids. When necessary, the fibroin solution was concentrated in a dialysis cassette for 24 hours in a 20% PEG suspension.

Silk Fibroin-Based Bioink Formulations

[0096] Various Formulations were Tested:
Formulation A: Silk fibroin ink A was prepared with a concentrated silk fibroin solution (35% w/v) modified with a saline buffer solution (2 mM NaCl, 6 mM KCl, 1 mM MgCl.sub.2, 10 mM glucose, 10 mM HEPES) at pH 7.4 and gently sonicated by 3 pulses of 5 seconds at an amplitude of 20%. The ink is stored at 4? C. for up to one month after preparation.

[0097] This composition made it possible to carry out the 3D printing of a single-layer structure.

Formulation B (EMS according to the invention): The silk fibroin-based ink B was prepared with an 8% w/v silk fibroin solution. The solution was heated to 45? C. Type A gelatin (15% w/v) and sterile alginic acid (1% w/v) were incorporated in a 15:1 ratio, progressively under gentle magnetic stirring until complete dissolution, in order to respectively ensure the printability and cross-linking of the final form. A concentrated saline buffer solution is then slowly added to reach a final concentration of 2 mM NaCl, 6 mM KCl, 1 mM MgCl.sub.2, Glucose was added to a final concentration of 10 mM. HEPES was added to a final concentration of 10 mM. Percoll? was added to a final concentration of 2.5%. The pH was adjusted to 7.4. The solution was centrifuged for 3 minutes at 300?g at room temperature and stored at 4? C. for up to two months after preparation. The ink was heated to 37? C. before the cells were incorporated in the composition.

[0098] The printing efficiency was verified by comparing the composition with respect to the SG formulation (8% w/v silk fibroin, 15% w/v type A gelatin in saline solution) or SA (8% w/v silk fibroin, sterile alginic acid (1% w/v) in saline solution). Only with the SGA composition was it possible to 3D print a multi-layer structure (up to 20 layers) with stable junctions between the layers which, after crosslinking, remain intact even after repeated stretching and rewinding.

Formulation C: This formulation is specific for 3D printing in a gel bath. The silk fibroin-based ink B was prepared with an 8% w/v silk fibroin solution. The solution was heated to 45? C. Sterile alginic acid (8% w/v) was gradually incorporated under gentle magnetic stirring until complete dissolution, in order to respectively ensure printability and crosslinking of the final form. A concentrated saline buffer solution is then slowly added to reach a final concentration of 2 mM NaCl, 6 mM KCl, 1 mM MgCl.sub.2. Glucose was added to a final concentration of 10 mM. HEPES was added to a final concentration of 10 mM. The pH was adjusted to 7.4. The preparation was gently sonicated by 3 5-second pulses at an amplitude of 20%.

[0099] The solution was centrifuged for 3 minutes at 300?g at room temperature and stored at 4? C. for up to one month after preparation. The ink was heated to 37? C. before being added to the cells.

[0100] This composition made it possible to 3D print a multi-layer structure (up to 10 layers) having stable junctions between the layers that remain intact even after repeated stretching and rewinding.

[0101] All of the above formulations were tested in combination with different biomolecules, such as for example: [0102] components of the extracellular matrix (fibronectin, collagen, laminin); [0103] growth factors, preferably hematopoietic (TGF-?1, VEGF); [0104] cytokines and chemokines (TPO, GM-CSF, EPO, SDF-1?); [0105] drugs (TPO mimetics, ROCK inhibitors, kinase inhibitors); [0106] conjugated or unconjugated antibodies; [0107] enzymes and/or substrates and intermediates of enzymatic reactions; [0108] toxins.

[0109] All of the silk fibroin-based inks were sterilized by UV radiation prior to testing the cell cultures.

EXAMPLE 2: USE OF INK FORMULATIONS IN A BIOPRINTING PROCESS

Bioprinting Process

[0110] 3D models were produced using CAD software, processed with Slic3r or Cura to obtain the gCode which is then read by the bioprinter.

Different printing protocols were tested: [0111] A) Formulation A was loaded into a 3 ml cartridge fitted with a 27 G nozzle and kept at 30? C. Printing was carried out directly in cell culture plates keeping the printer base at 37? C. The extrusion pressure was set at 5 kPa and the printing rate at 5 mm/s. The resulting scaffold was dried and sterilized by UV light before carrying out the cell culture tests. The structure is stable for at least 6 months at room temperature and for at least two weeks of culture at 37? C. and 5% CO.sub.2. [0112] B) Formulation B was loaded into a 3 ml cartridge fitted with an 18 G, 20 G or 22 G nozzle and kept at 37? C. in the printer. The printer base was kept at 16-20? C. to allow the gelatin to solidify ensuring the stability of the 3D structure during the extrusion process. The extrusion pressure was set from 8 to 20 kPa and the printing rate from 6 to 12 mm/s. The scaffold was then immersed for a minimum of 10 minutes in a crosslinking solution containing 0.1 M CaCl.sub.2) dissolved in a buffer solution containing 2 mM NaCl, 6 mM KCl, 1 mM MgCl.sub.2, 5 mM glucose, 10 mM HEPES, under conditions sterile at room temperature, and finally immersed in a cell culture medium. During culture at 37? C. and 5% CO.sub.2, a fraction of the gelatin is immediately released into the medium, but the structure is kept stable for at least one month of culture. [0113] C) Formulation C was bioprinted in a gel bath that supports ink deposition during the printing process. The bath consisted of an agarose mixture enriched with 0.1 M CaCl.sub.2) to ensure immediate crosslinking. The bioink was loaded into a 3 ml cartridge equipped with a 25 G nozzle and printed at an extrusion pressure of 20 kPa and at a printing rate of 8 mm/s. After 10 minutes of incubation at 37? C., the plate was immersed in an 0.1 M CaCl.sub.2) solution. The printed form was finally recovered and immersed in a cell culture medium. The structure was kept stable for up to two weeks of culture.

[0114] In all of the formulations tested, the removal of silk fibroin from the ink components negatively affects the duration of the final scaffold, demonstrating that fibroin plays a fundamental role in giving total stability to the structure (FIG. 1).

[0115] FIG. 1 shows the comparison between the printing performance of formulation B in the presence of silk, gelatin and alginate (SGA) compared with silk and alginate (SA) or silk and gelatin (SG). The analysis reveals the clearly superior profile in terms of printability, crosslinking and duration of the composition only in the SGA formulation, including silk fibroin, gelatin and alginate.

Printability and Mechanical Properties of the Bioink Formulations Tested

[0116] The printability of the ink was evaluated using a polyvalent bioprinter equipped with a thermoregulated head with a 3 ml piston cartridge. Stainless steel cylindrical or plastic conical nozzles were used

[0117] The tests were carried out by printing a square layer of 30?30?1 mm with a progressive rate from 360 mm/min to 720 mm/min and using an extrusion pressure of 8 kPa, 12 kPa, 16 kPa or 20 kPa. The printing lines were observed by bright-field microscopy using an Olympus IX53 (Olympus Deutschland GmbH, Hamburg, Germany) with a 4? objective for measuring the diameter. Measurements of the rheological properties were conducted using a HR-2 TA rotational rheometer with a plate diameter of 20 mm. The compression tests were conducted with an HR-2 TA rheometer. Hydrogels of formulation B were produced by distributing the fibroin inks in a 3D mould and then crosslinking for 10 minutes with an 0.1 M CaCl.sub.2 solution. The disc produced had a diameter of ?8 mm and a height of ?3 mm. The samples were loaded between the parallel stainless steel plates. The upper plate was then lowered until a compression force sufficient for ensuring contact was exerted.

Characteristics of the Bioprinted Scaffold

[0118] The overall morphology of the printed structure was observed by confocal microscopy (FV10i, Olympus, Germany) using the autofluorescence of silk under UV light or by marking with fluorescent dyes. The degradation rate of the bioprinted construct was determined by quantifying the weight reduction under culture conditions (37? C., 5% CO.sub.2). In short, the freshly printed samples were weighed at the beginning and incubated in DMEM at 37? C. for 7 days. At predetermined times, the samples of the bioprinted construct were dried and weighed.

Cell Sources

[0119] In order to test the efficiency and biocompatibility of silk fibroin inks, experiments were conducted with different cell sources, including human leukemia cell lines, hematopoietic stem cells, mature blood cell progenitors (i.e., megakaryocytes) and induced pluripotent stem cells: [0120] 1. Human leukemic cell lines: the samples were maintained in Dulbecco's modified Eagle medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine and 10% ng/mL GM-CSF. The cells were marked with CFSE fluorescent dye and combined with silk fibroin-based bioinks (formulation B) at a concentration of 0.5-4?10.sup.6 cells/ml. After printing multiple layers, the scaffolds were crosslinked and cultured for 2 weeks, with a change of medium/cytokines every 2 hours. The starting concentration was set at 1?10.sup.6 cells/ml. [0121] The cells however retained the capacity of proliferating which was assessed by counting the increasing number of cells/?m.sup.2 by 3D reconstruction of the printed scaffold in the culture period. [0122] 2. Hematopoietic stem cells: human umbilical cord blood or peripheral blood hematopoietic progenitors were collected after issuing informed consent, according to the ethical requirements and principles of the Declaration of Helsinki. CD34.sup.+ or CD45.sup.+ hematopoietic stem cells were isolated by selection with immunomagnetic beads. The hematopoietic stem cells were marked with the CFSE dye and mixed with fibroin-based inks (formulation B) at a concentration of 1?10.sup.6 cells/ml. After printing multiple layers, the scaffolds were crosslinked and cultured for 2 weeks, with a change of medium/cytokines every 3 days. Alternatively, the cells were genetically engineered to express the GFP marker. [0123] 3. Mature blood cell progenitors: hematopoietic stem cells were cultured in a classical liquid medium containing cell-line specific cytokines. Once differentiated, the cells were mixed with the fibroin-based inks (formulation B) at a concentration of 1?10.sup.6 cells/ml. After printing multiple layers, the scaffolds were crosslinked and cultured for 1-2 weeks, with a change of medium/cytokins every 3 days. [0124] In the presence of thrombopoietin, the cells showed the formation of an invasive network of proplatelets, precursors of platelets, inside the ink, thus demonstrating that the 3D microenvironment provided is compatible with the functionality of blood cells. [0125] 4. Induced pluripotent stem cells: the samples derive from hematopoietic stem cell progenitors and differentiation is induced in vitro in the presence of a cocktail of cytokines.

[0126] Under all the conditions tested, the cell vitality after bioprinting was evaluated by the Live/Dead assay. The maximum intensity projection of five single random Z-stack fields, for each specimen under the microscope, was chosen for the calculations. The results demonstrate a cell survival of the printing process of 90% or more.

Results

Mechanical Properties and Printability of Silk Fibroin-Based Ink

[0127] The silk fibroin ink (formulation B) was printed at 37? C. and tested at different rates and using different nozzles (FIG. 2, panels A-B). For cell printing, the variable parameters were the size of the nozzle, the extrusion pressure and the deposition rate of the ink (FIG. 2, panel C). The amplitude of the variation of ink deposition is directly related to the variation of the rate and pressure, and shows a better resolution at 8-16 kPa in the printing-rate range of 480-720 mm/min.

[0128] Different configurations can therefore be produced depending on the desired final print resolution (FIG. 2, panel C).

[0129] By comparing the ink in the presence of silk fibroin/gelatin/alginate (SGA) or gelatin/alginate (GA), it was observed that the 3D form was maintained only with SGA. The weight of both scaffolds fluctuated after 7 days and the phenomenon is related to the dispersion of the gelatin fraction at 37? C. in the medium. The silk fibroin scaffold deposition however held a better form and was easier to manipulate (FIG. 2, panels D-E).

[0130] In order to study the viscosity variations of the silk fibroin-based bioink that occur with printing, analyses were carried out using a rheometer. The analysis of the dependence of the modules on the temperature of the silk fibroin ink shows that the solution has viscoelastic properties with a critical gelling point in the temperature range of 28-30? C. The dependence of viscosity on the shear stress of the silk fibroin ink was measured at different temperatures and a stable behaviour was found below 30? C. The rigidity analysis was carried out by measuring the frequency sweeps of the silk fibroin ink after ionic crosslinking at 37? C. The storage modules were less than 1 kPa. The viscosity of the silk fibroin-based ink was studied in relation to the sudden switch of the shear stress, demonstrating a thixotropic behaviour. Furthermore, it takes at least 10 minutes to return to the initial viscosity and the process is completely reversible (FIG. 2, panel F).

Characteristics of 3D Post-Print Constructs

[0131] SGA-type silk fibroin ink (formulation B) was loaded into the printer at 37? C. under sterile conditions and a base square scaffold of 20?20?3 mm with 15-20% internal reticular filling was printed using a 22 G nozzle, an extrusion pressure of 10 kPa and a printing rate of 10 mm/sec. The print bed was set at 16? C. to allow a fast solidification of the ink after deposition on the plate. The scaffold was finally crosslinked with calcium ions for 15 minutes and subsequently immersed in culture medium to be kept under cell culture conditions (37? C., 5% CO.sub.2). Upon optical microscopy, the printed forms were transparent and showed some fibrillar structures (FIG. 2, panel G). The observation of a scaffold marked with Hoechst fluorescent dye in confocal microscopy showed the typical fibrillar organization of the polymerized silk fibroin (FIG. 2, panel G).

[0132] As the diffusion coefficients of nutrients in the materials of 3D printed scaffolds are generally extremely low, the composition of the ink can have an important impact on their distribution within the printed scaffolds. The bioinks according to the invention (SGAformulation B) allow the addition of a wide range of additives, including, but not limited to, cytokines, ECM (e.g. fibronectin), small molecules (e.g. ADP, ATP), fluorescent probes of intracellular compartments (e.g. cytoskeleton, organelles), biocompatible dyes, antibodies and enzymes to provide 3D printed scaffolds with additional functions. The dispersion of biocompatible dyes in the culture medium, for example, demonstrated a high retention of molecules within the silk fibroin ink (FIG. 3, panel C).

[0133] The enzymatic activity of horseradish peroxidase integrated in a printed scaffold significantly allowed the emission of chemiluminescence in the presence of enzymatic substrates. Another advantage of the ink formulations according to the invention is that their use avoids the use of vulcanization methods of the printed scaffold that could reduce the biological activity of the additives integrated therein and/or of the cells themselves.

Cell Vitality and Differentiation within 3D Constructs

[0134] Bioprinting is a process that can lead cells to a condition of stress or even death. The pneumatic pressure applied in the cartridge and the rheological changes that occur inside the nozzle are parameters (among others) that have a significant impact on cell vitality.

[0135] The CD34.sup.+ hematopoietic stem cells were combined with silk fibroin ink at a concentration of 1?10.sup.6 cells/ml and bioprinted following the settings described above (FIG. 4, panel A, i, ii).

[0136] Observation using confocal microscopy showed that the cells were well distributed within the scaffold and in a three-dimensional space (FIG. 4, panel A, iii). 24 hours after post-printing, the scaffolds were marked with cell vitality/death probes and observed with z-stack confocal microscopy showing approximately 90% survival (FIG. 4, panel B). The cells were kept in culture for 14 days during which the medium/cytokine was changed every 3 days. The scaffolds were observed by live microscopy at different times or fixed in a solution of 5% formaldehyde and 0.1 M CaCl.sub.2 to effect marking of the cytoskeleton. After 7 days of culture, the cells are still homogeneously distributed in the scaffold and begin to enlarge and become polyploid (FIG. 4, panel C). A significant increase in the cell diameter, a typical feature of megakaryocyte differentiation and maturation, was observed after 14 days of culture (FIG. 4, panel D). Furthermore, the marking of the actin cytoskeleton with TRITC-phalloidin revealed the presence of a network of platelets within the printed structure, thus demonstrating that the 3D microenvironment provided is compatible with the function of megakaryocytes (FIG. 4, panel E). Another noteworthy element is that the removal of silk fibroin from the components of the ink tested prevents long-term cell vitality and the culture of megakaryocytes, which are not capable of being differentiated to form proplatelets. All of these results show that the bioink formulation and bioprinting conditions are favourable for studying hematopoiesis and the formation of platelet.

In Situ Analysis of the Bioprinted Cells:

[0137] The advantage of using fibroin for bioprinting is the transparency that allows easy optical accessibility for viewing in confocal microscopy and for detecting the signal of enzymatic reactions activated in the bioink itself.

[0138] The possibility of visualizing the culture in real-time was tested by bioprinting CD61.sup.+ hematopoietic progenitors pre-marked with antibody directed against the marker, conjugated with fluorochrome. The cells were combined with silk fibroin ink at a concentration of 1?10.sup.6 cells/ml and bioprinted following the settings described above. For visualization in confocal microscopy, the scaffold was printed inside a sterile culture chamber with a glass bottom for microscopy (FIG. 5). 7 days after post-printing, the chamber was placed on the microscope stand and held at 37? C. for a further 48 hours. Confocal reconstruction of the 3D scaffold shows the phases of the platelet production, from the elongation of the pseudopods to the release of platelets within the bioink, demonstrating the capacity of the scaffold of promoting and supporting the differentiation and function of hematopoietic cells (FIG. 5). An advantage of printing pre-marked cells is the possibility of proceeding with the direct visualization of the sample without the need for adding additional reagents.

[0139] A further bioactivity test was performed by incorporating horseradish peroxidase (HRP) into the ink to verify the stability and functionality of the enzyme in the printed tissue. The formulation was printed at 37? C. and kept in culture for 2 weeks. Incubation with luminol and hydrogen peroxide triggered a chemiluminescence reaction. The measurement of the variation in intensity of the light emission was detected only by the HRP-functionalized bioink, with respect to the control bioink (HRP-free formulation B) (FIG. 6A).

[0140] The HRP activity was also tested through a colorimetric reaction. The B+HRP formulation was added to lactate and incubated in a solution containing lactate oxidase, DHBS and 4-AAP. The resulting reaction produces pyruvate and hydrogen peroxide. The latter reacts with HRP causing a change in the colour of the printed scaffold, proportional to the quantity of lactate present in the scaffold (FIG. 6B). In order to validate the capacity of the B+HRP formulation of revealing the presence of molecules released by the cells cultured in the scaffold, CD61.sup.+ cells were added to the ink at a concentration of 1?10.sup.6 cells/ml and bioprinted following the settings described above. After 7 days of culture the scaffold was immersed in a solution containing lactate oxidase, DHBS and 4-AAP. The staining reaction takes place within a few minutes (FIG. 6). When the cells are printed in the presence of lactate dehydrogenase inhibitor, the amount of lactate detectable decreases significantly reducing the colorimetric signal. These results extend the usefulness of adopting bioink not only as a biological stabilizer of the enzymatic activity of the molecules added to the formulation for carrying out chemical and enzymatic assays but also for detecting multi-analytes produced by the hematopoietic cells.

Dissolution of the Scaffold and Recovery of Bioprinted Cells:

[0141] 3D cultured hematopoietic cells can be used for biochemical and molecular studies. Furthermore, there is increasing interest in the possibility of using the cells in functional assays or for clinical use. For this purpose, the cells must be able to be recovered from the bioprinted scaffold for subsequent uses.

[0142] CD61.sup.+ cells were combined with silk fibroin ink at a concentration of 1?10.sup.6 cells/ml and bio-printed following the settings described above. After 7 days of culture the scaffold was immersed in a dissolution solution (according to the invention) (FIG. 7A). The cells were recovered in liquid solution and analyzed by immunofluorescence microscopy (FIG. 7A) and flow cytometry (FIG. 7B). The cell lysate of the same cells was subjected to an electrophoretic run. The staining of the bands using Coomassie blue reveals a protein abundance comparable to that of a sample of cells obtained from a classic 2D culture, demonstrating efficiency in the recovery of the sample. The protein sample can be used for subsequent biochemical analyses (e.g. Western blotting) (FIG. 7C).

EXAMPLE 3

Use of the Ink Formulations for Real-Time Visualization of In Vitro Platelet Production

[0143] In order to test the possibility of viewing the bioprinted cells in real-time without fixing or manipulating the 3D printed scaffold, customized culture chambers having the same shape as the scaffold were created. Both of the 3D models of the scaffold and the culture chamber were created using CAD software. The perfusion chamber was printed using a biocompatible resin by means of the stereolithography technique. The bottom of the chamber was sealed using a confocal microscopy glass (0.17 mm) (FIG. 5). Formulation B of the bioink was printed inside the chamber. Before being injected into the bioink, the hematopoietic progenitors were marked with anti-CD61 antibody conjugated with a fluorescent marker. After bioprinting the cells in formulation B, the scaffold was crosslinked and subsequently incubated in a culture medium. Visualization of the cells during the production of the platelets can take place at different times according to the experimental requirements, by removing the culture medium and positioning the chamber directly on the stand of the confocal microscope, maintained at 37? C. During visualization, the maintenance of the pH of the scaffold is ensured by the presence of the buffer system (HEPES). The humidified environment is guaranteed by the presence of a liquid reservoir in the chamber itself.

EXAMPLE 4: USE OF THE INK FORMULATIONS FOR THE ENZYMATIC DOSAGE OF LACTATE RELEASED BY THE CELLS DURING CULTURE

[0144] In order to test the bioactivity of the bioink, the possibility of functionalizing formulation B with the horseradish peroxidase (HRP) enzyme was tested. For this purpose, 110 U/mL of HRP were added to formulation B. The B+HRP formulation was printed and, after crosslinking, incubated for 14 days in a culture medium.

[0145] The bioactivity was evaluated after several days of culture by activating a reaction leading to chemiluminescence in the presence of luminol and hydrogen peroxide. The chemiluminescent signal was acquired using a chemiluminescence detector. In all the conditions tested, the enzymatic activity was detectable.

[0146] The B+HRP formulation was also tested in the presence of hematopoietic progenitors for dosing molecules released during the 3D culture by the cells themselves, without the need for manipulating the scaffold or resoring to the preparation of enzymatic assays of the culture mediums. In particular, the release of lactate, a product of glycolysis, indicative of an active cellular metabolism and whose variation can be indicative of alterations in the platelet production, was analyzed. For this purpose, the reagents: 20 U/mL of lactate oxidase; 600 ng/mL of sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS), 100 ng/mL of 4-aminoantipyrine (4-AAP) were added to the B+HRP formulation. Alternatively, the reagents can be added directly to the culture medium and diffused in the bioink at the time of the assay. In both cases, the bioactivity of the preparation was confirmed by the activation of a colorimetric reaction following incubation with lactate (2 or 16 mM). Lactate-free incubation was used as a negative control. The presence of increasing the concentrations of lactate released by the cells was detected during the culture of the bioprinted 3D scaffold. The specificity of the reaction was confirmed by treating the sample with the lactate dehydrogenase inhibitor.

EXAMPLE 5

[0147] Dissolution of the Bioprinted Scaffold and Use of the Cells Recovered from 3D Culture

[0148] In order to test the possibility of recovering the cells from the bioink after 3D culture, and using them for functional, biochemical or molecular studies, hematopoietic progenitors were bioprinted with formulation B.

[0149] After 7 days of culture, the scaffold was incubated in a dissolution solution (according to the invention), at 37? C. for 20 minutes.

[0150] The cells were then centrifuged at 1,200 rpm for 10 minutes, washed in a physiological solution and used in the experimental assays.

[0151] The morphological parameters and the expression of surface antigens of the megakaryocyte lineage were evaluated by immunofluorescence microscopy and flow cytometry after incubation with specific antibodies. The protein lysate was dosed by spectrophotometry, tested by an electrophoretic run in 8% polyacrylamide gel and visualized by marking with Coomassie Blue or Western blotting.

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