A SYNTHETIC HYDROGEL AND ITS USE FOR IMMUNOTHERAPY AND 3D-PRINTING

Abstract

The present invention discloses a hydrogel comprising a functionalized PEG multi-arm star polymer covalently combined with maleimide-functionalized low molecular weight heparin, further comprising at least one positively charged immune molecule, a method for producing this hydrogel and its use in T cell culture for immunotherapies. Furthermore, the invention relates to a bioink and its use in 3D-(bio)-printing.

Claims

1. A hydrogel comprising a functionalized PEG multi-arm star polymer covalently combined with heparin, further comprising at least one positively charged immune molecule.

2. The hydrogel according to claim 1 wherein the functionalized PEG multi-arm star polymer is a functionalized PEG four-arm star polymer.

3. The hydrogel according to any preceding claim wherein the functionalized PEG multi-arm star polymer is a thiol-PEG multi-arm star polymer.

4. The hydrogel according to any preceding claim wherein the functionalized PEG multi-arm star polymer is a thiol-PEG four-arm star polymer of formula: ##STR00002##

5. The hydrogel according to any preceding claim wherein the concentration of the functionalized PEG multi-arm star polymer ranges between 2% wt to 10% wt on the total weight percentage of hydrogel.

6. The hydrogel according to any preceding claim wherein the heparin is low molecular weight heparin.

7. The hydrogel according to claim 6 wherein the heparin is maleimide-low molecular weight heparin.

8. The hydrogel has a median pore size between 5 to 105 μm, preferably is 55 μm.

9. The hydrogel according to any preceding claims wherein the positively charged immune molecule is a cytokine and/or a cell adhesion molecule.

10. The hydrogel according to claim 9 wherein the cytokine is a chemokine selected from the list consisting of: CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28 and any combination thereof.

11. The hydrogel according to claim 10 wherein the chemokine is CCL21 and/or CCL19.

12. The hydrogel according to any one of the claims 9 to 11 wherein the concentration of cytokine ranges between 0.1 ng/mL to 250 ng/mL, preferably is 100 ng/mL.

13. The hydrogel according to claim 9 wherein the cell adhesion molecule is a intercellular adhesion molecule selected from the list consisting of: ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5 and any combination thereof.

14. The hydrogel according to claim 13 wherein the cell adhesion molecule is ICAM-1.

15. The hydrogel according to claim 13 or 14 wherein the concentration of cell adhesion molecule ranges between 1 μg/mL and 50 μg/mL, preferably is 5 μg/mL.

16. The hydrogel according to any preceding claims wherein the positively charged immune molecule is surface-immobilized or in suspension.

17. A composition comprising the hydrogel according to any one of claims 1 to 16.

18. A hydrogel according to any one of claims 1 to 16 or a composition according to claim 17 for use as a medicament.

19. The hydrogel according to claim 18 for use in immunotherapy treatment, preferably in cancer treatment and autoimmune diseases.

20. Use of a hydrogel according to any one of claims 1 to 16 or a composition according to claim 17 in cell culture, preferably for T cell culture.

21. The use according to claim 20 wherein T cell is a CD4+ T cell.

22. A bioink comprising a hydrogel according to any one of claims 1 to 16 or a composition according to claim 17.

23. Use of the bioink according to claim 22 in 3D printing, preferably in 3D bio-printing.

24. A method for producing a hydrogel according to any one of claims 1 to 16 comprising the following steps: (a) mixing a solution of functionalized PEG multi-arm star polymer with another solution of heparin, both solutions in buffer solution, (b) incubating the solution obtained in step (a) at between 15° C. to 45° C. to gelify, and (c) loading the hydrogel obtained in step (b) with at least one positively charged immune molecule.

Description

DESCRIPTION OF THE DRAWINGS

[0165] FIG. 1. Spectrum of the low molecular weight heparin functionalized with maleimide (peak circled) after an overnight reaction.

[0166] FIG. 2. Scheme of a PEG-Hep hydrogel formation through a maleimide-thiol reaction resulting in a covalent crosslink and gelation.

[0167] FIG. 3. A) Strain sweeps and B) frequency sweeps of 6% wt, 4% wt and 3% wt of PEG-Hep hydrogels.

[0168] FIG. 4. Time sweeps of 6% wt, 4% wt and 3% wt PEG-Hep hydrogels.

[0169] FIG. 5. A) SEM images of the surface and section of the samples studied at different compositions of PEG (6% wt, 4% wt and 3% wt). B) Pore size evaluation of 6% wt, 4% wt and 3% wt PEG-Hep hydrogels. The statistical significance was determined by the non-parametric Kruskal Wallis ANOVA test (*** p<0.001).

[0170] FIG. 6. A) Lateral and B) top views of an overall 3% PEG-Hep hydrogel of 1 cm of diameter obtained by X-ray microtomography. C) and D) show the same perspectives but of a small zone of the hydrogel used to analyzed its porosity of 3.5 mm of diameter and 500 μm of height.

[0171] FIG. 7. Porosity analysis of 3% wt PEG-Hep samples studied by microtomography (N(samples)=2) and the Gaussian fitting performed.

[0172] FIG. 8. GFP loading curve of 6% wt, 4% wt and 3% wt of PEG-Hep hydrogels. The statistical significance was determined by the Mann-Whitney U test (*** p<0.001).

[0173] FIG. 9. A) Normalized proliferation analysis of CD4+ T cells 5 days after seeding in unloaded PEG-Hep hydrogels (Ndonors=6). Statistical significance was determined by the Mann-Whitney U test (*p<0.05, **p<0.01). B) Diagram of the resulting CFSE fluorescence peaks of a representative data point.

[0174] FIG. 10. Normalized proliferation analysis of CD4+ T cells 6 days after seeding in suspension (positive control) and with different concentrations of CCL21 (Ndonors=5, with a minimum of Ndonors/condition=4).

[0175] FIG. 11. A) Scheme of the experiment performed to study the adhesion of CCL21 onto gold substrates using an indium titanium oxide (ITO) substrate decorated with a quasi hexagonal array of gold nanoparticles (AuNPs) functionalized with CCL21 and further immunostained (Alexa Fluor 488). B) SEM image of the ITO substrate with the quasi-hexagonal array of (AuNPs). C) Fluorescence image of the surface area decorated with AuNPs, functionalized with CCL21, immunostained with human anti-CCL21 and the secondary antibody with mouse anti-human Alexa 488, showing signal only on its lower half, where de AuNPs are present.

[0176] FIG. 12. Normalized proliferation analysis of CD4+ T cells 6 days after seeding in suspension (positive control) and with different concentrations of CCL21 fixed to Au surfaces (Ndonors=8). Statistical significance was determined by the Mann-Whitney U test (** p<0.01).

[0177] FIG. 13. Normalized replication, expansion, and proliferation indexes 6 days after seeding CD4+ T cells in PEG-Hep hydrogels loaded with 100 ng/mL of CCL21 and unloaded (Ndonors=18, with a minimum of Ndonors/condition=6). Statistical significance was determined by the Mann-Whitney U test (*p<0.05, **p<0.01, ***p<0.001).

[0178] FIG. 14. A) Normalized proliferation indexes 6 days after seeding CD4+ T cells in PEG-Hep hydrogels loaded with 100 ng/mL of CCL21 (Ndonors=6). Statistical significance was determined by the Mann-Whitney U test (*p<0.05, **p<0.01). B) Diagram of the resulting CFSE fluorescence peaks of a representative data point.

[0179] FIG. 15. Differentiation analysis of CD4+ T cells 5 days after seeding (Ndonors=10, with a minimum of Ndonors/condition=4). Percentage of A) naïve (TN), B) central memory (TCM), and C) effector memory (TEM). Representative dot plot graphs of cells in a D) negative control, E) positive control, F) 3% wt PEG-Hep hydrogel, and G) 3% wt PEG-Hep hydrogel loaded with a solution of 100 ng/mL of CCL21. Statistical significance was determined by the Mann-Whitney U test (*p<0.05).

[0180] FIG. 16. Normalized proliferation analysis of CD4+ T cells 6 days after seeding with different concentrations of CCL19 in suspension (Ndonors=8). Statistical significance was determined by the Mann-Whitney U test (**p<0.01, ***p<0.001).

[0181] FIG. 17. Normalized proliferation indexes 6 days after seeding CD4+ T cells in PEG-Hep hydrogels loaded with 100 ng/mL and 20 ng/mL of CCL19 (Ndonors=6). Statistical significance was determined by the Mann-Whitney U test (*p<0.05, **p<0.01).

[0182] FIG. 18. A) Normalized proliferation indexes 6 days after seeding CD4+ T cells in PEG-Hep hydrogels loaded with 100 ng/mL of CCL21 and with 100 ng/mL of CCL19 in solution (Ndonors=6). Statistical significance was determined by the Mann-Whitney U test (*p<0.05, **p<0.01). B) Diagram of the resulting CFSE fluorescence peaks of a representative data point.

[0183] FIG. 19. Schematic images of the scaffold designed to 3D print structures.

[0184] FIG. 20. Microscope images of the resulting scaffolds printed with a 3% wt PEG-Hep hydrogel after A) 3.5 hours of gelation, B) one day at room temperature, and C) one day incubated at 37° C. D) Microscope images of scaffolds printed with 3% wt PEG-Hep hydrogels made in DMEM media instead of PBS.

[0185] FIG. 21. Normalized proliferation analysis of CD4+ T cells seeded in unloaded printed PEG-Hep hydrogels of 4 and 6 layers of height, 6 days after seeding (Ndonors=6). Statistical significance was determined by the Mann-Whitney U test (*p<0.05, **p<0.01).

[0186] FIG. 22. Differentiation analysis of CD4+ T cells (Ndonors=6). Percentage of A) naïve (TN), B) central memory (TCM), and C) effector memory (TEM) for 3D structures consisting of 4 layers. Percentage of D) naïve (TN), E) central memory (TCM), and F) effector memory (TEM) for 3D structures consisting of 6 layers.

[0187] FIG. 23. A) Normalized proliferation indexes 6 days after seeding CD4+ T cells in PEG-Hep hydrogels loaded with 100 ng/mL of CCL21 (Ndonors=6). Statistical significance was determined by the Mann-Whitney U test (*p<0.05, **p<0.01). B) Diagram of the resulting CFSE fluorescence peaks of a representative data point.

[0188] FIG. 24. Normalized proliferation analysis of CD4+ T cells seeded in unloaded hydrogels and loaded PEG-Hep hydrogels with 1, 5, and 50 μg/ml 6 days after seeding (Ndonors 2). Statistical significance was determined by the Mann-Whitney U test (**p<0.01).

EXAMPLES

[0189] Materials and Methods

[0190] 1. Chemical Techniques: Synthesis of a Peg-Hep Hydrogel

[0191] 1.1. Materials

[0192] Green fluorescence protein (GFP) was synthesized as described in Unzueta, U. et al, Int. J. Nanomed. 7, 4533-4544 (2012). Low molecular weight heparin was purchased from Fisher Scientific (Fisher BioReagents, Spain). Boc-L-phenylalanine from Merk, Germany, and poly(ethylene oxide), 4-arm, thiol terminated (Mn=10000 g/mol), N-(2-aminoethyl) maleimide trifluoroacetate salt (AEM), 1-hydroxybenzotriazole hydrate (HOBT), N-(3-dimethylamino-propyl)-N′-ethylcarbodiimide hydrochloride (EDC HCl), 2-(N-morpholino) ethanesulfonic acid (MES), and the rest of the products not otherwise specified were purchased from Sigma-Aldrich (USA).

[0193] 1.2. Synthesis of Heparin Functionalized with Maleimide

[0194] The functionalization of heparin with maleimide was based in a method described in Nie, T., et al 2009, Acta Biomater. 5, 865-875. Briefly, the inventors added 0.02 mmol of heparin in a solution of MES with HOBT (0.15 mmol), AEM (0.08 mmol), and EDC HCl (0.11 mmol) and left it overnight. The product was purified by dialysis (MWCO 1000) against water, lyophilized, and characterized by proton nuclear magnetic resonance (1H-NMR) spectroscopy.

[0195] 1.3. PEG-Hep Hydrogel Formation

[0196] To prepare PEG-Hep hydrogels, the inventors mixed a solution of 4-arm thiolated PEG (PEG-SH) with a solution of maleimide-functionalized low molecular weight heparin (Hep-Mal) in a proportion of 1:1.5, both in PBS. The inventors used different concentrations in weight of PEG: 6% wt, 4% wt, and 3% wt, to obtain different types of hydrogels. Once the solutions were mixed, they were kept in the incubator at 37° C. during at least 1 h to form the hydrogel. Negative controls consisting of solutions of only one of the reactants at relevant concentrations confirmed that gelification and therefore hydrogel formation were caused by the reaction between the thiol groups of the 4-arm PEG and the maleimide-functionalized heparin.

[0197] 2. Physical Techniques

[0198] 2.1. Rheology

[0199] The inventors used the small-amplitude oscillatory shear (SAOS) technique to characterize the linear-viscoelastic regime (LVE) of the hydrogels using a Rheometer HAAKE RheoStress RS600 (Thermo Fisher Scientific, USA). The experiments performed were strain sweeps, frequency sweeps, and time sweeps at 37° C. to calibrate the range of pressure and frequency where the hydrogels maintain their viscoelastic behavior, characterize the gelification process, and achieve the value of the gel equilibrium shear modulus (Ge).

[0200] 2.2. Scanning Electron Microscopy (SEM)

[0201] Although this technique is typically used in dry samples, a special protocol at the vacuum chamber was used, slowly decreasing the pressure and temperature, which enabled to image the structure of the hydrated hydrogels. The equipment used by the inventors was a FEI Quanta 650F Environmental scanning electron microscope.

[0202] 2.3. Microtomography

[0203] Microtomography is an X-ray 3D imaging technique with a high resolution that allows the visualization of the internal structure of a sample. In this case, the inventors used a skyscan 1272 high-resolution micro computed tomography (Bruker).

[0204] 2.4. Preparation of Surfaces with AuNPs for CCL21 Immobilization.

[0205] The inventors prepared nanostructures by block copolymer micellar lithography (BCML) as described in Guasch, J., et al 2016, Chem. Mater. 28, 1806-1815. BCML consists of dissolving an amphiphilic block copolymer in an apolar solvent to create reverse micelles, which can be loaded with a metallic precursor. The inventors dissolved polystyrene (x)-2-vinylpyridine (y) (PS(106)-P2VP(75) (Polymer Source Inc., Canada) in a dry solution of o-toluene at room temperature and stirred for 24 h. Then, they added gold (III) chloride trihydrate (Sigma-Aldrich, USA) to the block copolymer micellar solution and stirred for 48 h. The inorganic metal complex compound is dissolved into the polar micellar cores, where it loses a proton that binds to the nitrogen atom of the P2VP chain, stabilizing the micelles.

[0206] The inventors dipped-coated commercial indium titanium oxide (ITO)-coated glass substrates (20 mm×15 mm Ossila Ltd, UK) with the loaded gold micellar solution at a constant velocity of 110 mm/min and then plasma treated the sample with oxygen plasma (150 W, 0.15 mbar, 45 min) using a microwave gas plasma system (210 PVA TePla, Germany) to obtain quasi-hexagonally ordered AuNPs with lateral interparticle distances of 68±20 nm. After that, the inventors passivated the ITO surface with PEG-silane (Prochimia, Poland), milliQ water, and triethylamine (Sigma Aldrich, USA) in toluene overnight at 80° C. Then, they functionalized it with CCL21 (Sigma Aldrich, USA) during 1 h at room temperature by means of the cysteine groups of the cytokine.

[0207] 2.5. Preparation of Au Surfaces. Gold Evaporation

[0208] The inventors studied the function of CCL21 and its effect in T cell proliferation in suspension and immobilized on planar Au surfaces to analyze the influence of fixing this cytokine on CD4+ T cell proliferation. The inventors incubated it with CCL21 during 1 h at the desired concentration.

[0209] 2.6. 3D Printing with PEG-Hep Hydrogel

[0210] For these experiments, the inventors used 3% wt PEG-Hep hydrogels as ink. To make the 3D scaffolds, the inventors mixed sterilized solutions of PEG-SH and Hep-Mal a day before the impression in a sterile syringe adequate for the printing, and incubated the mixture at room temperature. After that, the inventors placed the syringe in the printer with a tip TIP27GA TT 008″ NAT, which showed to be adequate for the printing of this material at a pressure of 1.2 bar, and an impression speed of 15 mm/s in a 24WP.

[0211] 2.7. Loading Capacity

[0212] The inventors loaded 3% wt PEG-Hep hydrogels with solutions at different concentrations of GFP during 1 h. After that, the inventors removed the supernatant and washed the hydrogels with PBS. The inventors measured the fluorescence of the hydrogels with a Victor 3 Multioption plate reader (Perkin Elmer, USA).

[0213] 3. Biological Techniques

[0214] 3.1. CD4+ T Cell Isolation and Purification

[0215] The inventors obtained the primary human CD4+ T cells through a purification process of buffy coats of healthy adult donors, obtained from “Banc de Sang i Teixits” (Barcelona, Spain) after the approval of the “Ethics Committee on Animal and Human Experimentation” of the Autonomous University of Barcelona (No. 3511). The buffy coat is the fraction of an anticoagulated blood sample that contains the white blood cells and platelets. The inventors worked under a flow hood and used of sterile tools and materials.

[0216] To obtain the CD4+ T cells, they first purified the PBMCs by density gradient centrifugation using Ficoll. Briefly, the inventors diluted blood from the buffy coat with pre-warmed PBS with 2 mM of EDTA in a proportion of 1:4. Then, they added the Ficoll and centrifuged the mixture during 20 min at 300 g. Once the sample was centrifuged, the “white” phase between the supernatant (plasma) and the Ficoll was collected and washed with PBS with 2 mM of EDTA. Afterwards, the inventors counted the achieved cells and used a CD4+ T cell isolation kit purchased from Miltenyi Biotec S. L. (Germany) to obtain the CD4+ T cells, following the instructions of the manufacturer.

[0217] This kit contains a Biotin-Antibody cocktail with antibodies against CD8, CD14, CD15, CD16, CD19, CD36, CD56, CD123, TCR γ/δ, and CD235a (Glycophorin A), and the inventors used it to label non-CD4+ cells, i.e., CD8+ T cells, monocytes, neutrophils, eosinophils, B cells, dendritic cells, NK cells, granulocytes, γ/δ T cells, or erythroid cells. Then, they used the second component of the kit, which is a CD4+ T Cell MicroBead Cocktail that consists of magnetic microbeads conjugated with monoclonal anti-biotin to target all non-CD4+ T cells. This suspension was added on an LS column that is able to retain the targeted cells. Finally, the inventors counted the resulting cells and analysed their purity by flow cytometry.

[0218] 3.2. CD4+ T Cell Purity Assay

[0219] To determine the CD4+ T cell purity, the inventors incubated the cells with antihuman CD3 FITC and antihuman CD4 PE (Immunotools GmbH, Germany) during 30 min at 0° C. Then, they washed the samples in PBS with 0.1% of FBS and analyzed them through flow cytometry. For experiments, the inventors only used samples that were at least 90% positive for both CD3+ and CD4+ (usually CD3+CD4+ T cells >95%). Viability was constantly above 80% (usually viability >90%).

[0220] 3.3. CSFE Staining and Proliferation Assay

[0221] To study the proliferation of CD4+ T cells, they were stained with a CellTrace CFSE cell proliferation kit provided by Thermo Fisher Scientific (USA), before seeding, following the instructions of the manufacturer.

[0222] 3.4. Cell Culture and Seeding

[0223] The inventors seeded cells on 96 well plates (WP), except for the 3D printing experiments, which required 24WP given the characteristics of the printer (3D Discovery printer, RegenHU Biosystem Architects (Switzerland)). The inventors used the culture media Roswell Park Memorial Institute (RPMI) medium with 10% FBS and 1% penicillin/streptomycin. The inventors used a cell seeding concentration of a million cells/mL in all the cases, except for the proliferation studies with 3D printed scaffolds, when they used a concentration of 5.Math.105 cells/mL. They induced the activation of cells by adding Dynabeads (Thermo Fisher, USA) in a 1:1 ratio, as suggested by the manufacturer. Positive controls were done by seeding the cells in suspension, as well as negative controls, which did not include Dynabeads. For the loading experiments with a positively charged immune molecule, the hydrogels were incubated with such a molecule (CCL21, CCL19 or ICAM-1) during 1 h. Afterwards, the supernatant was removed and the cells were seeded.

[0224] 3.5. Immunostaining of CCL21 in Glass Surfaces Functionalized with Gold Nanoparticles (Au NPs)

[0225] To prove that CCL21 binds to gold (Au), the inventors incubated recombinant CCL21 on the desired substrates in a PBS solution for 1 h at room temperature. The chosen substrates were glass surfaces half-functionalized with gold nanoparticles (Au NPs), with the objective of clearly see the difference between the part with and without Au in the same sample. After the incubation, the inventors washed the surfaces and performed a staining protocol. For that, the inventors incubated the substrates with a solution of primary antibody mouse anti-human CCL21 (Invitrogen, USA) in PBS for 1 h at room temperature, which was afterwards washed. After that, the inventors added a second antibody, goat anti-mouse Alexa 488 (Invitrogen, USA), which binds the first antibody and provides fluorescence, in a PBS with 1% of BSA solution. The incubation time was again of 1 h at room temperature under the dark. Finally, the inventors washed the samples imaged them with a fluorescence microscope (Olympus BX51, Japan).

[0226] Results

[0227] 1. Peg-Hep Hydrogels for CD4+ T Cell Culture

[0228] The inventors have studied the PEG-Hep hydrogels with the objective of mimicking the physicochemical properties of the SLOs, based on the properties of both PEG and Hep. On one hand, PEG is responsible to imitate the physical 3D structure of the LNs, due to its specific structural and mechanical properties, which can be easily regulated thanks to its synthetic nature. On the other hand, the heparin is resembling the function of the heparan sulphates naturally present in the ECM, acting as molecular sinks, storage sites, or presentation platforms to bind growth factors and chemokines. The inventors have fully characterized and used PEG-Hep hydrogels as 3D scaffolds for CD4+ T cell activation, expansion, and differentiation, to study its further application into immunotherapy treatments.

[0229] More specifically, the inventors have synthetized, designed, and characterized PEG-Hep hydrogels with different stiffness, porosities, and loading capacities, in order to achieve the desired properties to mimic the ECM of SLOs, and have used said hydrogels for CD4+ T cell culture under different conditions, studying the resulting changes observed in proliferation and in the phenotypes achieved in comparison with the suspension cultures.

[0230] 1.1. Synthetic PEG-Hep Hydrogels

[0231] First, the inventors optimized the protocol of functionalization of heparin with maleimide by leaving the reaction overnight. The results were analyzed by proton nuclear magnetic resonance (H-RMN) spectroscopy (FIG. 1). As shown in the spectrum, there is a peak at 6.83 ppm, corresponding to the protons of the maleimide linked to the heparin, which is slightly shifted from the reported peaks of the free maleimide ring at 6.86 ppm.

[0232] Once the heparin was fully functionalized, the inventors studied the hydrogel formation. For that, a solution of a 4-arm thiolated PEG (PEG-SH) was mixed with a solution of functionalized heparin in a proportion of 1:1.5, in PBS. As mentioned before, hydrogels with different mechanical properties can be obtained by varying the percentage of PEG. Thus, the inventors prepared hydrogels with the 3% wt, 4% wt, and 6% wt of PEG, all of them with the same proportion of PEG:Hep (1:1.5). PEG-Hep hydrogels were formed through a maleimide-thiol reaction between the maleimide of the functionalized heparin, and the thiols of the PEG, which results in a covalent crosslink and the consequent gelation (FIG. 2).

[0233] 1.2. Structural and Mechanical Properties of PEG-Hep Hydrogels

[0234] To optimize the resulting hydrogels for immune cell culture, the inventors performed various experiments, and characterized their physical properties (e.g. stiffness, porosity, gelification time, and pore interconnectivity). Rheology was the technique used to characterize the gelification time and stiffness of the hydrogels. SEM was used to observe the structure of the hydrogel in the sections and surface. Finally, x-ray microtomography was used to obtain 3D images of the scaffold and study their pore interconnectivity.

[0235] 1.2.1. Rheology

[0236] The main rheological technique to characterize hydrogels is small-amplitude oscillatory shear (SAOS). SAOS is used to identify the equilibrium shear modulus of a gel (Ge), which is the ratio of stress and strain in a balanced state where the material supports stress without deformation. To determine the Ge of a gel, the storage modulus (G′) and the loss modulus (G″) are measured in the LVE limit, and the limiting value of G′ at low frequency is identified as the shear modulus.

[0237] This technique requires a trial-and-error approach to find the appropriate values of strain and frequency of each gel. Thus, the measurements require strain sweeps, frequency sweeps, and time sweeps at 37° C. to calibrate the range of pressure and frequency where the hydrogels maintain their viscoelastic behavior. In strain sweeps, G′ and G″ are measured at a fixed frequency, while the pressure applied to the hydrogel increases within the range of interest. For frequency sweeps, the procedure is quite similar but, in this case, the pressure applied is constant and is the frequency which is changed. These two experiments show the values of pressure and frequency where the hydrogels maintain their viscoelastic behavior, and which are applied to the time sweep, where the changing variable is the time.

[0238] To perform these experiments, the inventors prepared hydrogels with different percentages of PEG (3% wt, 4% wt, and 6% wt). Strain sweeps were performed at 37° C. and a constant frequency of 1.0 Hz, while the pressure was conducted from 1 Pa to 150 Pa on fully formed hydrogels (after optimizing the range where the hydrogels maintain their viscoelastic behavior). Then, frequency sweeps were performed from 0.01 Hz to 10 Hz at a constant strength of 50 Pa (FIG. 3).

[0239] All hydrogels showed linear behavior of G′ from a strength of 10 Pa to 100 Pa. More interestingly, samples of 3% wt show a G′ lower than those of 4% wt and 6% wt, i.e. they can accumulate less energy in their structure without causing deformation. Thus, hydrogels with less amount of PEG are softer than those with higher amounts of PEG. The frequency sweeps were linear from 0.01 Hz to 1 Hz. Similarly, lower G′ values were obtained for the 3% wt than the 4% wt and 6% wt PEG-Hep hydrogels. The values of the equilibrium shear modulus (Ge) achieved were 4.5±0.2 KPa for the 6% wt PEG-Hep hydrogel, 3.1±0.1 KPa in the case of 4% wt PEG-Hep, and 1.1±0.1 KPa for the 3% wt PEG-Hep. G″ was too small to be measured for fully formed hydrogels. It is worth noting that these values of Ge correspond to fully formed and completely hydrated hydrogels. This is important because this is the state in which hydrogels were applied to cell culture.

[0240] The inventors performed the time sweeps for the characterization of the gelification process, at 50 Pa and 0.1 Hz, values in which PEG-Hep hydrogels showed to maintain their viscoelastic behavior, with the rheometer at 37° C. (FIG. 4). The 6% wt and 4% wt PEG-Hep hydrogels were stabilized after ca. 200 min, at a storage modulus of 8.2±0.5 KPa, while the 3% wt PEG-Hep hydrogel required 240 min at a storage modulus of 2.7±0.2 KPa. The inventors analyzed the gelification process and the properties of the hydrogel when this process ends, without the addition of water or any other possible treatment. This means that, without the hydration process, the PEG-Hep hydrogels were more compact and stiff. Although this experiment was useful to characterize the gelification process, the stiffness values relevant for immune cell culture are the ones reported above, i.e. the ones obtained after hydrogel hydration in PBS.

[0241] 1.2.2. SEM

[0242] After the rheological characterization, the inventors studied the pore size of fully formed and hydrated PEGHep hydrogels at different percentages of PEG (6% wt, 4% wt, and 3% wt) by SEM imaging of their surface and section (FIG. 5).

[0243] The median pore size of the 6% wt PEG-Hep hydrogels resulted of 20 μm with a porosity range of 5-50 μm, which increased to 40 μm for the 4% wt hydrogels with a range of 20-75 μm, and to 55 μm with a range of 25-105 μm for the 3% wt PEG-Hep hydrogels. These results show that the lower the amount of PEG present in the sample, the higher the porosity of the hydrogel. In order to apply PEG-Hep hydrogels to immune cell culture, the inventors chose the 3% wt PEG-Hep hydrogel due to its porosity and mechanical properties. For a deeper study of this hydrogel, the inventors measured the interconnectivity of its pores through 3D x-ray microtomography.

[0244] 1.2.3. X-Ray Microtomography

[0245] In addition to the previous characterization, the inventors freeze-dried the 3% wt PEG-Hep hydrogel and lyophilized it in order to obtain the dried 3D structure to characterize it by X-ray microtomography. The images achieved support the data obtained by SEM and provide high quality images and videos of the internal structure of the hydrogels where the interconnectivity of the pores can be seen (FIG. 6A, B, C y D). Moreover, the percentage of the different pore sizes measured by this technique was represented and fitted to a Gaussian model (R2=0,998; FIG. 7). From this analysis the inventors extracted that most of the pores measured around 70 μm, in agreement with the data obtained by SEM, where the median pore size observed was of 55 μm.

[0246] 1.3. Biofunctionalization of PEG-Hep Hydrogels: Loading Capacity

[0247] To confirm the ability of PEG-Hep hydrogels to attract positively charged molecules to their negatively charged heparin units, the inventors incubated different concentrations of GFP, which was used as a positively charged protein model (estimated charge of +5.6 mV), with the hydrogels during 1 h to observe the dependency of the loading capacity of the hydrogel with the concentration of positively charged protein. The fluorescence was maintained by the hydrogel after rinsing, indicating that the GFP was retained inside (FIG. 8). The 3% wt of PEG-Hep hydrogel showed the highest retention of GFP, which suggest that these hydrogels have a better pore interconnectivity than the rest, so that the GFP solution can easily reach the whole hydrogel.

[0248] 1.4. Unloaded PEG-Hep Hydrogels Applied to CD4+ T Cell Culture

[0249] Once the PEG-Hep hydrogels of different PEG percentages (6% wt, 4% wt, and 3% wt) were fully characterized, the inventors chose the 3% wt PEG-Hep hydrogels for CD4+ T cell culture, given their mechanical properties, higher porosity and interconnectivity as well as loading capacity. The inventors analyzed T cell proliferation through CFSE staining and flow cytometry. Thus, the inventors calculated the expansion, replication, and proliferation indexes 5 and/or 6 days after seeding. The expansion and replication indexes determine the fold-expansion of the overall culture and that of the responding cells, respectively, whereas the proliferation index is equal to the number of divisions that cells from the original population have undergone divided by the number of divided cells.

[0250] The inventors normalized results of the unloaded 3% wt PEG-Hep hydrogels used as a scaffold for CD4+ T cell culture to the positive control (FIG. 9A). The median of the normalized replication index obtained was 1.25, i.e. an improvement of a 25% was achieved, whereas the expansion and proliferation indexes showed a median of 1.1 and 1.05, respectively. All three parameters showed statistically significant increases compared to the positive controls, which corresponded to cultures in suspension with Dynabeads. The strongest difference was observed for the replication index, which indicates that the responding cells that get activated in the synthetic hydrogels proliferate more than the activated cells in suspension. A representative graph of the peaks of fluorescence obtained in the flow cytometer after culturing is also shown (FIG. 9B). This graph shows the displacement of the CFSE fluorescence peaks to the left in the positive control and sample compared to the negative control, indicating the new generations of cells obtained.

[0251] These results indicate, not only that PEG-Hep hydrogels do not show any cytotoxicity for the cells, but also that the 3D physical effect of the hydrogel, even without the introduction of any chemical stimuli, already causes an improvement in CD4+ T cell proliferation.

[0252] 1.5. Study of Different Chemical Stimuli to Introduce into PEG-Hep Hydrogels

[0253] Once the beneficial effect of unloaded PEG-Hep hydrogels was observed, the inventors studied the effect of different chemical stimuli through the introduction of biomolecules into the hydrogels. For the introduction of such chemical stimuli, the inventors used the already proved capacity of the heparin present in the hydrogels for anchoring positively charged molecules by electrostatically interactions, mimicking the natural function of the heparin sulphates present in the ECM of the LNs.

[0254] The inventors studied the positively charged molecules associated with immune cell activation and expansion in suspension and fixed in 2D and 3D systems to observe their effect on the proliferation and differentiation of CD4+ T cells. The inventors chose the cytokines CCL21 and CCL19 as well as the cell adhesion molecule ICAM-1.

[0255] 1.5.1. CCL21

[0256] Chemokine (C-C motif) ligand 21 (CCL21) is a small cytokine involved in the activation process of the immune system. It plays an important role in costimulating the expansion of CD4+ and CD8+ T cells and inducing Th1 polarization. It is highly expressed in the endothelium of lymphatic vessels and SLOs and interacts with T cells and mature DCs which express the chemokine receptor CCR7.

[0257] Initially, the inventors chose the concentrations 1 ng/mL, 20 ng/mL, and 100 ng/mL for experiments in suspension (FIG. 10). The median values for the replication index for 100 ng/mL, 20 ng/mL, and 1 ng/mL were 1.01, 0.95, and 0.96, respectively, i.e. very similar to the positive controls. The inventors observed the same tendency for the expansion index, with median values of 0.99, 1.02, and 0.99, and the proliferation index with values of 1.05, 1.12, and 1.00 for 100 ng/mL, 20 ng/mL, and 1n g/mL, respectively. No significant differences could be observed among the different CCL21 concentrations in suspension.

[0258] In the next steps, the inventors tested the capacity of CCL21 to effect T cell proliferation when immobilized. To analyze the effect of such immobilization in a well-defined system, they used planar Au surfaces.

[0259] With this objective, they first corroborated the capacity of immobilizing the cytokine CCL21 through its cysteine residues. For that, the inventors used a surface that was functionalized with a quasi-hexagonal pattern of AuNPs only on its lower part by dip-coating. The inventors used block copolymer micellar lithography (BCML) to prepare a Au-loaded micellar solution by dissolving an amphiphilic block copolymer in an apolar solvent to create reverse micelles. The inventors dipped-coated commercial ITO-coated glass substrates with the loaded Au micellar solution obtaining AuNPs with a lateral interparticle distance of 68±20 nm (FIG. 11B).

[0260] The inventors passivated these surfaces with PEG overnight and incubated with CCL21 during 1 h. After the incubation, they performed an immunostaining (FIG. 11C) using human anti-CCL21 as primary antibody and mouse anti-human Alexa 488 as secondary antibody to observe through fluorescence where was the cytokine retained. A scheme of the experiment performed can be seen in FIG. 11A, wherein fluorescence could only be detected in the part of the surfaces decorated with AuNPs, proving that CCL21 was fixed on their surface.

[0261] After verifying that CCL21 binds to Au, the inventors prepared 2D Au surfaces functionalized with CCL21, and used them for cell culture. They tested different concentrations of CCL21 to find the optimum amount of cytokine needed to obtain CD4+ T cell proliferation of relevant phenotypes. Elevated concentrations of CCL21 were avoided given their potentially inhibitory effect.

[0262] Thus, the inventors immobilized CCL21 on Au surfaces at concentrations of 1 ng/mL and 20 ng/mL (FIG. 12), which resulted in significant changes (**p<0.01) for the proliferation and expansion indexes. Specifically, the median values for the expansion index were of 1.1 and 1.16 for the concentrations of 20 ng/mL and 1 ng/mL respectively, showing an improvement of 10% and 16% in comparison with the positive control. Similarly, the proliferation index increased to 1.06 and 1.08 for each concentration. The replication index also showed a slight tendency to increase with median values of 1.06 for 20 ng/mL and 1.1 for 1 ng/mL.

[0263] These results confirmed that CCL21 increases CD4+ T cell proliferation when fixed. Accordingly, the inventors loaded this cytokine into PEG-Hep hydrogels. The inventors incubated different concentrations of CCL21 in PEG-Hep hydrogels and identified 100 ng/mL as the concentration which led to the highest expansion results in contrast with the CCL21-immobilized on Au surfaces (FIG. 13). This difference was probably caused by the increase in total area available when moving from 2D to 3D biomaterials. As shown in FIG. 14, all the proliferation indexes were improved when the hydrogel was loaded with CCL21 in comparison with the unloaded hydrogel. For the hydrogels with 100 ng/mL of CCL21, a 30% increase in the replication index was obtained in comparison with the positive control (1.3 of median value), while the unloaded hydrogel showed a 15% (1.15 of median value) of improvement. The proliferation and expansion indexes showed less pronounced increases with average median values of 1.05 and 1.06, respectively (FIG. 14A). Again, the strongest difference was observed for the replication index. A representative graph of the peaks of fluorescence obtained in the flow cytometer after culturing is also shown (FIG. 14B).

[0264] Once verified that PEG-Hep hydrogels functionalized with CCL21 increase CD4+ T cell proliferation, the inventors performed differentiation assays 5 days after seeding to determine the phenotype of the resulting T cells. The changes in the surface of CD4+ T cells caused by their activation result in different phenotypes, which were analyzed by flow cytometry. These phenotypes were naïve (TN; CD45RO−/CD62L+), central memory (TCM; CD45RO+/CD62L+), and effector memory (TEM; CD45RO+/CD62L−).

[0265] CD45 is a transmembrane protein tyrosine phosphatase (receptor type C) expressed on the cell surface of human leukocytes. The isoforms of CD45 are associated to different differentiation stages and are commonly used as markers to identify different types of immune cells. More specifically, CD45RA is associated with cells that have not encounter yet a matching antigen, and they are therefore naive. In contrast, once a naive cell gets activated, it expresses the isoform CD45RO, which is preserved in memory T cells. Consequently, TN are CD45RA+ and CD45RO−, whereas TEM are CD45RA- and CD45RO+. The TCM are a special type of cells, which have already encounter a matching antigen and thus are CD45RO+, but preserve a high potential of reproduction. These cells are in an “intermediate” differentiation state and some studies have suggested their higher potential to eliminate tumors compared to other cell types. This capacity has been explained by their higher capacity to proliferate than memory cells, i.e. the presence of more immune cells per cancer cell, and their higher specificity than naive cells, which are cells that have also a high proliferation capacity.

[0266] CD62L, also named L-selectin is a type I transmembrane cell adhesion molecule expressed on most circulating leukocytes, including neutrophils. L-selectin is one of three family members: L-, E- and P-selectin. Each selectin is defined according to the cell type in which it was first characterized (L=lymphocyte, E=endothelial cell, P=platelet). TN cells express CD62L because they need to enter SLOs to encounter their antigen. TCM, which have already encountered antigen are CD62L+ because they still need to localize in SLOs, where they reside ready to proliferate upon re-encountering their specific antigen. TEM do not express CD62L, as they circulate in the periphery and have immediate effector functions upon re-encountering their specific antigen.

[0267] The percentages of CD4+ T cells that express CD45RO and CD62L prior to stimulation (negative control) are submitted to the intrinsic donor variability. They mostly showed a TN phenotype with a median value of 53%, whereas the TEM and TCM phenotypes were found in lower percentages, 12% and 32%, respectively.

[0268] After stimulation, the median value of TN cells decreased to 4% in suspension (positive control). Both unloaded and loaded (100 ng/mL of CCL21) hydrogels exhibited a decrease of this phenotype, being the median values 14% and 11% respectively. Consequently, the TEM and TCM phenotypes increased in comparison with the negative control. Specifically, the median values for the TCM phenotype of the positive control, unloaded, and loaded hydrogels were 68%, 51%, and 47%, respectively. For the TEM phenotype, the median values raised to 26% for T cells in suspension, 21% for unloaded hydrogels, and 33% for hydrogels with 100 ng/mL of CCL21, showing an increase of effector cells for those seeded in cytokine loaded hydrogels. These results point out that PEG-Hep hydrogels can be used to modify the resulting phenotype of T cells and different chemical inputs can be used to achieve diverse differentiation pathways.

[0269] Finally, representative dot plots of the negative control, positive control, unloaded and loaded hydrogels are shown (FIG. 15D-G), wherein the resulting activated populations of cells evolve differently in suspension and in the hydrogel, although both come from the same initial population of cells.

[0270] After analyzing these results, the inventors concluded that CCL21 improves CD4+ T cell proliferation and tune differentiation, increasing the total amount of effector T cells.

[0271] 1.5.2. CCL19

[0272] CCL19 is a cytokine of the same family of CCL21, which acts as a potent inducer of T cell proliferation in DC-T cell co-cultures although only with activated DCs. This cytokine interacts with the CCR7 receptor such as CCL21. However, slight conformational changes in CCR7 following binding of the two different chemokines results in differential T cell signaling.

[0273] The inventors used the same concentrations for CCL19 than the previously employed in solution for CCL21, 100 ng/mL, 20 ng/mL, and 1 ng/mL. After 6 days of culture they measured the proliferation results. In this case the highest increase of the proliferation parameters was observed for the concentration of 1 ng/mL, with median values of 1.19, 1.20, and 1.06 for the replication, expansion, and proliferation indexes, respectively.

[0274] Although no statistical changes were observed for the concentrations of 100 ng/mL and 20 ng/mL of CCL19 in suspension, the inventors observed a tendency to increase the proliferation of CD4+ T cells when a concentration of 1 ng/mL was employed. In contrast with the results observed for CCL21, where no significant differences were observed, CCL19 as an obligate soluble chemokine that it is, showed its influence in suspension for the concentration of 1 ng/mL (FIG. 16).

[0275] Subsequently, the inventors loaded different concentrations of CCL19 in PEG-Hep hydrogels to study the differences between having CCL19 in solution and anchored to the hydrogels (FIG. 17). All the proliferation indexes showed an improved tendency in comparison with the positive control. The replication index showed an increase of 50% and 56% for unloaded hydrogels and loaded with 100 ng/mL of CCL19, respectively (1.5 and 1.53 of median value). A lower increase of 38% for hydrogels loaded with 20 ng/mL of CCL19 was obtained. A similar tendency was observed for the expansion index, with median values of 1.35, 1.47, and 1.34 for unloaded, 100 ng/mL and 20 ng/mL of CCL19 respectively. Finally, the achieved proliferation indexes median values were 1.23, 1.20, and 1.14 respectively.

[0276] 1.5.3. CCL21 and CCL19

[0277] Once having studied CCL21 and CCL19 separately, the inventors used both cytokines with the objective of mimicking the natural environment of the LNs and maximizing the proliferation results. In this case, they loaded CCL21 in the hydrogel during 1 h and added CCL19 in solution with the media [CCL21(h) CCL19(s)], both with concentrations of 100 ng/mL. The inventors measured proliferation 6 days after seeding (FIG. 18).

[0278] These hydrogels with both cytokines duplicated the replication index (2.00 of median value) of the positive control and showed also an improve compared to the unloaded hydrogel, which had a median value of 1.80. The expansion index was also improved, obtaining median values of 1.50 for the hydrogel with cytokines and 1.13 for the unloaded hydrogel. Finally, the proliferation index showed similar median values, 1.21 and 1.20 respectively, although higher statistical difference was observed when both cytokines were employed (FIG. 18A). A representative graph of the peaks of fluorescence obtained in the flow cytometer after culturing is also shown (FIG. 18B). These results show the benefits of mimicking the natural environment of cells with both cytokines, which resulted in higher proliferation rates than the hydrogels with only one of the cytokines or none.

[0279] 1.5.4. ICAM-1

[0280] Finally, the inventors used a different type of positively charged immune molecule, the cell adhesion molecule ICAM-1, a key molecule in immune-mediated and inflammatory processes functioning as an important co-stimulatory signal for the activation of T cells.

[0281] The inventors used different concentrations of ICAM-1, 1 μL/mL, 5 μL/mL and 50 μL/mL, to load the hydrogels before cell seeding (FIG. 24). After 6 days, the proliferation index showed the median values of 1.22, 1.20, 1.25 and 1.15, whereas the expansion index exhibited median values of 1.36, 1.28, 1.40 and 1.25 for the unloaded 3% wt hydrogel as well as ICAM-1 (1 μL/mL), ICAM-1 (5 μL/mL), and ICAM-1 (50 μL/mL) loaded hydrogels, respectively. Finally, median values of 1.44, 1.43, 1.53 and 1.36 were observed for the unloaded 3% wt hydrogel and ICAM-1 (1 ICAM-1 (5 μL/mL) and ICAM-1 (50 μL/mL) loaded hydrogels, respectively.

[0282] Although the use of a hydrogel improves the state of the art expansion system (Dynabeads in suspension), no statistical changes were observed for the concentrations of 1 μL/mL, 5 μL/mL, and 50 μL/mL of ICAM-1 in suspension. Nevertheless, the inventors observed a slight increase of 2.3% for the proliferation index, 2.9% for the expansion index, and 6.1% for the replication index, when compared with the unloaded 3% wt hydrogel. These results might be explained by the lower estimated net charge of ICAM-1 (+4.8) compared to the one of CCL21 (+17.0), which will have a weaker interaction with the hydrogel that could result in a less efficient cellular response.

[0283] 1.6. Conclusions

[0284] PEG-Hep hydrogels were synthesized, optimized, characterized, and loaded with different biomolecules to mimic the LNs. Hydrogels of different percentages of PEG were efficiently developed (6% wt, 4% wt, and 3% wt). The higher the PEG concentration, the higher the stiffness, the smaller the pore size and the interconnectivity. 3% wt PEG-Hep hydrogels showed an increase in the proliferation of CD4+ T cells and an influence on the resulting phenotypes, even without the addition of any chemical stimuli. CCL21, a positively charged immune molecule of the cytokine family, further induced CD4+ T cell proliferation when anchored to the PEG-Hep hydrogels. The highest proliferation parameters were achieved through the combination of two cytokines, CCL21 loaded to the hydrogels and CCL19 added in solution, mimicking the LNs. Moreover, the cell adhesion molecule ICAM-1 was also evaluated to demonstrate the versatility of the platform to introduce different types of positively charged immune molecules. This system could be further improved to fabricate artificial LNs, which are expected to overcome the limitations of current immunotherapies such as producing large amounts of T cells with therapeutic phenotypes.

[0285] 2. PEG-Hep Hydrogels for 3D Printing

[0286] 3D printing is a technique that consists of producing 3D objects with precisely designed geometries in a layer by layer approach that can be used in biomedicine. For example, cell-laden 3D constructs can be built, which include cells as printable materials, to be used for implants in regenerative medicine or artificial tissues can be created with the objective of replicating the structures of native tissues.

[0287] Due to the beneficial effects showed by PEG-Hep hydrogels for T cell expansion above mentioned, the inventors analyzed these hydrogels as bioink for 3D printing, by optimizing the gelification process of PEG-Hep hydrogels and adjusting to the 3D printer requirements.

[0288] 2.1. PEG-Hep Hydrogels as Bioink for 3D Printing

[0289] To use the PEG-Hep hydrogels as bioink, the inventors optimized the gelification process to obtain well defined pre-designed 3D scaffolds for cell culture in a 3D Discovery printer from RegenHU Biosystem Architects (Switzerland). The inventors chose a design consisting of a grid with a separation of 1.5 mm between its lines and of 4 or 6 layers of height (FIG. 19).

[0290] With this purpose, the inventors prepared 3% wt PEG-Hep pre-hydrogels in PBS by mixing the solutions with both reagents (PEG and Hep) and heated them up to 37° C. Afterwards, the resulting mixture was analyzed as bioink for 3D printing at different times. The inventors could performed the first printings after 3.5 h (after 3 h, the mixture was still too liquid), when the sample had enough consistency.

[0291] The resulting scaffolds had though a very low rigidity and no differentiated lines were achieved in the printed grid (FIG. 20A). To improve that, samples were stored overnight under two conditions, at room temperature and in the incubator at 37° C., and the experiment was repeated after 24 h of gelation. The material stored at room temperature showed optimal properties for its printing, obtaining quite well defined scaffolds (FIG. 20B). Nevertheless, samples stored at 37° C. got very dried, making the printing heterogeneous and difficult, and the resulting scaffolds were not adequate for cell culture (FIG. 20C). Additionally, different pressures of extrusion and tips for the printing were tested. After the corresponding optimization experiments, it was found that the optimum extrusion pressure was of 1.2 bar by using a conic tip with an inner diameter of 27 G (0.3606 mm).

[0292] Moreover, the inventors prepared PEG-Hep hydrogels for cell-laden experiments. Unexpectedly, the gelation was immediate when cell medium was used and the resulting gel could be properly printed (FIG. 20D). Thus, cells can be introduced inside the hydrogel through printing.

[0293] 2.2. PEG-Hep Printed Scaffolds for CD4+ T Cell Expansion

[0294] Once the inventors demonstrated that 3% wt PEG-Hep hydrogels can be used as bioink and the protocol was optimized, they printed 3D layered structures to observe their effect on CD4+ T cell culturing and ensure their (bio)compatibility. The inventors printed scaffolds of 4 and 6 layers in WP.

[0295] The inventors first evaluated unloaded printed hydrogels and after they incubated the scaffolds with 100 ng/mL of CCL21. Specifically, they cultured unloaded printed scaffolds of 4 and 6 layers with CD4+ T cells during 6 days and afterwards, they measured the replication, expansion, and proliferation indexes by flow cytometry and compared with the ones obtained for cells seeded in suspension (positive control). The inventors normalized the results to the positive control.

[0296] The PEG-Hep scaffold printed with a height of 4 layers exhibited a slight tendency to increase the proliferation parameters in comparison with the positive control, obtaining normalized values of 1.03 for the replication and proliferation indexes. The only significant change obtained was in the proliferation index. On the other hand, the PEG-Hep scaffolds printed with 6 layers of height showed stronger proliferation improvements. Specifically, the replication and proliferation indexes increased a 7% (1.07 as a result of their normalized value) and the expansion index a 4% (FIG. 21). It can therefore be concluded that the presence of a printed 3D scaffold improves T cell proliferation and the higher the structure, the more the cells reproduce.

[0297] To determine if the phenotype of the resulting T cells was affected, the inventors performed a differentiation assay with scaffolds of 4 and 6 layers. The resulting CD4+ T cell populations were classified in naïve (TN; CD45RO−/CD62L+), central memory (TCM; CD45RO+/CD62L+), and effector memory (TEM; CD45RO+/CD62L−) 5 days after seeding (FIG. 22).

[0298] In the 4 layers scaffolds (FIG. 22A-C), the inventors observed a statistically significant increase of the percentage of TCM, which is a phenotype that has been associated with successful clinical outcomes. Namely, the median value of TCM raised from the 45% of the negative control and the 61% of the positive control to the 66% for the 4 layer printed hydrogels (FIG. 22B). On the other hand, the TEM median values augmented for CD4+ T cells activated in suspension from a 14% of the negative control to a 30%, while this increase was lower for cells seeded in the printed hydrogels, with a median value of 23% (FIG. 22C). Naive cells decreased from a 39% of the inactivated cells to a 5% and 6% of cells activated in suspension and in hydrogels, respectively (FIG. 22A). However, the inventors observed no significant changes between cells activated in suspension or using a scaffold in this population. The 6 layer scaffolds showed the same tendency. Specifically, the TCM (FIG. 22E) and TEM (FIG. 22F) phenotypes increased, while the TN decreased (FIG. 22D). However, but the achieved differences were less pronounced, especially for the TCM.

[0299] As previously done for non-printed hydrogels, the inventors incubated a solution of 100 ng/mL of CCL21 during 1 h prior to cell seeding to decorate the printed scaffolds and study its effect on CD4+ T cell proliferation (FIG. 23). The replication index improved a 3% and 10% (with median values of 1.03 and 1.10) when using unloaded and loaded printed hydrogels, respectively, in comparison with the positive control. The expansion index showed no significant changes for the unloaded gels, slightly increasing its median value from 1.00 to 1.06 with the addition of the cytokine. Finally, the same tendency was obtained for the proliferation index with median values of 1.05 and 1.08 for unloaded and loaded hydrogels, respectively (FIG. 23A). The CSFE diagram (FIG. 23B) shows representative plots for each sample. Once again, all the proliferation indexes improved when using the hydrogel loaded with CCL21 in comparison with the unloaded hydrogel and the positive control.

[0300] 2.3. Conclusions

[0301] After an adequate optimization of the gelification process, 3% wt PEG-Hep hydrogels can be used as an ink for 3D printing. Thus, 3D scaffolds were obtained with PEG and Hep diluted in both, PBS and media, opening the way to a wide range of applications, including cell-laden experiments. The proliferation of CD4+ T cells increased when cells were incubated in printed scaffolds, observing higher rates for scaffolds of 6 layers in comparison with 4 layer scaffolds, as expected by the presence of a larger amount of material. Additionally, these scaffolds also resulted in an increase of the percentage of TCM cells obtained after 5 days of incubation, which is known to be phenotype associated with effectiveness in immunotherapies. Finally, it is also worth mentioning that the addition of CCL21 as a chemical stimulus to the printed hydrogels, resulted in an increased proliferation of CD4+ T cells as observed for non-printed hydrogels.