WO2021250186A1 - 3d bioprinting method for forming a cell specific tissue construct - Google Patents

Abstract

The aim of the present invention is to provide a bioink that is easily printable, exhibits mechanical properties relevant for structural stability and pertinent for the final application, has good gelling capacity, is biodegradable, and comprises surface modifiable functional groups or cellular adhesion sequences, and has the capacity to sustain the maturation of the printed structure in time. The present invention accordingly relates to a method for forming a cell specific 3D cell or tissue construct with a desired biological function wherein the hydrogel support medium, the sequence of the said peptide, the ratio of the said peptide to the said extracellular matrix derived hydrogel and the polymerization conditions are selected with respect to cell specificity so as to obtain a 3D tissue construct with the desired biological function (such as promoting angiogenesis or neurogenesis) useful for tissue engineering, organ on a chip or in vitro tissue/organ model applications.

Description

3D BIOPRINTING METHOD FOR FORMING A CELL SPECIFIC TISSUE

CONSTRUCT

FIELD OF THE INVENTION:

The present invention relates a 3D bioprinting method and to hydrogel compositions for use as bioink gel formulations.

Specifically, the instant invention relates to a method for forming a 3D cell or tissue construct with a desired biological function useful for tissue engineering, organ on a chip or in vitro tissue/organ model applications.

BACKGROUND OF THE INVENTION

3D bioprinting has emerged as a technological approach to address unsolved question in tissue engineering and regenerative medicine [1, 2] Bioprinting technology offers the possibility for controlled deposition and patterning of cells, polymers, composites, and/or hydrogels to create structurally defined scaffolds. This approach takes advantage of rapid prototyping, assisted by computer-assisted design (CAD) and/or computer-assisted manufacturing (CAM) procedures, to build a cellularized scaffold with geometric control of its internal structure and external shape. The bioprinting of cell-laden biomaterials, termed bioinks, enables the deposition of cells encapsulated in a 3D construct and provides a procedure to develop complex synthetic biological systems and tissue-engineered constructs. However, the number of biomaterials that are bioprintable, enable high cell viability, are biocompatible and biodegradable, allow cells to migrate and maturate and permit defined customization remains limited.

Different technologies of bioprinting have been developed, including inkjet or microjet, microextrusion and laser-assisted bioprinting [2, 3] Different technologies enable spatial control over bioink deposition with inherent different resolutions, cell survival and speed. From the 3 main bioprinting technologies, microextrusion is the most common, due to its ease of operation and affordability. It allows to create, layer-by-layer, the deposition of a wide range of bioinks, where the viscosity of the chosen bioinks often play a key role in their printability [4] Usually, high viscosity biomaterials ( e.g . high concentrations or high molecular weight) provide better printability higher structural integrity to the printed geometry as they can easily support their own weight but upon chemical and/or physical gelation. Nonetheless, higher viscosity usually induces cell toxicity during bioprinting, due to the induced shear stress when extruded, and the tight matrix can also limit the mobility of encapsulated cells and their capacity to restructure their surrounding matrix. In this sense a huge effort has been dedicated to optimize at the same time the printability of a bioink while providing an optimal environment to live cells [5]

Various hydrogel compositions have been developed as bioinks for bioprinting [6, 7], including natural polymers like gelatin, type I collagen, hyaluronic acid, fibrin, agarose, alginate, or synthetic polymers such as polyethylene glycol, pluronics, among others [8] Different chemistries can be envisaged to achieve cross-linking of the bioprinted scaffold, one popular choice considers photoinitiated radical polymerization of (meth)acrylate groups, due to spatial and temporal control of reaction [8] Optimal bioink biomaterial properties include printability, mechanical properties, biodegradation, modifiable functional groups on the surface and post printing maturation. Also, extracellular matrix-derived polymers have inherent beneficial properties, like ease of degradation, better cell attachment, growth and proliferation [9] Such examples are collagen and hyaluronic acid.

Collagen, a major component of the extracellular matrix (ECM), is commonly used as bioink alone or in combination with other biomaterials [10-12], due to its excellent biocompatible properties [13] Nonetheless, the slow physical gelation of collagen and low mechanical bearing properties poses several limitations for 3D bioprinting. In this sense the combination with other biomaterials increases its extrusion properties in view of bioprinting, as for instance fibrin [14], alginate [11] or hyaluronic acid [15]

Hyaluronic acid (HA), also a natural ECM major component, is abundantly present in cartilage, skin and in connective tissues [16] Also it has shown relevant key functions that make it a key material for tissue engineering, namely wound healing properties [17], reduce inflammation [18], induce cell proliferation and migration [19], key role in embryogenesis and morphogenesis [20] and angiogenesis [21] Thanks to these inherent properties, HA is one of the prominent biomaterials which are used in 3D bioprinting alone or in combination with other biomaterials). Nonetheless, HA shows a weak post print stability and in this sense its chemical modification by methacrylation [22], thiolation [15], modification with phenolic hydroxyl moieties [23], among others, have shown improvements on the long terms stability and on the capacity to sustain both osteogenesis, stromal cell elongation or for the culture of chondrocytes. Poldervaart et al. [22] describe methacrylated hyaluronic acid hydrogels for 3D printing intended to promote osteogenicity.

Nonetheless, cell-specific tailored bioinks that can target specific functions have not yet been developed. Focusing on the field of tissue engineering and on tissue repair, the support of the angiogenesis process is widely accepted as key in order to attain tissue repair in most tissues. Additionally, more recent studies have shed light on the importance and implication of the innervation in tissue remodelling and repair [24] In this sense, the development of bioinks that can support both of these processes are strongly awaited in the field of biofabrication, bioprinting and tissue engineering.

The aim of the present invention is to provide a bioink that is easily printable, exhibits mechanical properties relevant for structural stability and pertinent for the final application, has good gelling capacity, is biodegradable, and comprises surface modifiable functional groups or cellular adhesion sequences, and has the capacity to sustain the maturation of the printed structure in time.

SUMMARY OF THE INVENTION:

The instant invention provides a method for forming a cell specific 3D cell or tissue construct with a desired biological function comprising the steps of: a) Providing a (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel support medium; b) Reacting said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel with a thiol flanked functionalized adhesion peptide to obtain a (meth) acrylate crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide, via a thiol-ene reaction; cl) Either combining said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide with a plurality of specific living cells to provide a printable bioink, dl) Printing the said bioink into a first layer of printed bioink with a defined design; el) Submitting the said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and fl) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, c2) Or printing said bioink into a first layer of printed bioink with a defined design; d2) Submitting said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and e2) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, and f2), adding to said printed (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide a plurality of specific living cells, g) Allowing the growth of the living cells in 3D-construct obtained thereby under suitable conditions until the desired biological function is achieved; wherein the hydrogel support medium, the sequence of the said peptide, the ratio of the said peptide to the said extracellular matrix derived hydrogel and the polymerization conditions are selected with respect to cell specificity so as to obtain a 3D tissue construct with the desired biological function.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides a method for forming a cell specific 3D cell or tissue construct with a desired biological function comprising the steps of: a) Providing a (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel support medium; b) Reacting said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel with a thiol flanked functionalized adhesion peptide to obtain a (meth) acrylate crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide, via a thiol-ene reaction; cl) Either, combining said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide with a plurality of specific living cells to provide a printable bioink, dl) Printing the said bioink into a first layer of printed bioink with a defined design; el) Submitting the said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and fl) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained c2) Or printing said bioink into a first layer of printed bioink with a defined design; d2) Submitting said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and e2) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, and f2), adding to said printed (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide a plurality of specific living cells, g) Allowing the growth of the living cells in 3D-construct obtained thereby under suitable conditions until the desired biological function is achieved; wherein the hydrogel support medium, the sequence of the said peptide, the ratio of the said peptide to the said extracellular matrix derived hydrogel and the polymerization conditions are selected with respect to cell specificity so as to obtain a 3D tissue construct with the desired biological function.

Accordingly in first alternative embodiment, the method of the invention is comprising the following steps: al) Providing a (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel support medium; bl) Reacting said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel with a thiol flanked functionalized adhesion peptide to obtain a (meth) acrylate crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide, via a thiol-ene reaction; cl) Combining said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide with a plurality of specific living cells to provide a printable bioink, dl) Printing the said bioink into a first layer of printed bioink with a defined design; el) Submitting the said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and fl) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, and gl) Allowing the growth of the living cells in 3D-construct obtained thereby under suitable conditions until the desired biological function is achieved;

Accordingly in a second alternative embodiment, the method of the invention is comprising the following steps: a2) Providing a (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel support medium; b2) Reacting said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel with a thiol flanked functionalized adhesion peptide to obtain a (meth) acrylate crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide, via a thiol-ene reaction c2) Printing said bioink into a first layer of printed bioink with a defined design; d2) Submitting said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and e2) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, and f2), adding to said printed (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide a plurality of specific living cells, and g2) Allowing the growth of the living cells in 3D-construct obtained thereby under suitable conditions until the desired biological function is achieved.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers, but not the exclusion of any other step or element or integer or group of elements or integers.

“Bioprinting or “3D bioprinting” is the process of creating cell patterns in a confined space using 3D printing technologies, where cell function and viability are preserved within the printed construct. Generally, 3D bioprinting utilizes the layer-by-layer method to deposit materials known as Bioinks to create tissue-like structures that are later used in medical and tissue engineering fields.

“Bioinks” consist on the combination of materials that mimic an extracellular matrix environment to support the adhesion, proliferation, and differentiation of living cells, and living cells. Layers of bioink are deposited, following a defined geometry, to create a cell laden scaffold.

The “storage modulus” (G’), also called “elastic modulus”, represents the elastic portion of the viscoelastic behavior of a hydrogel and corresponds to the amount of energy stored and released in each oscillation.

The “loss modulus” (G”) or viscous modulus characterizes the viscous portion of the viscoelastic behavior, which can be seen as the liquid-state behavior of the hydrogel sample. It characterizes the deformation energy lost (dissipated) through internal friction when the gel is flowing. Advantageously, G’ values exceed G” values over the entire frequency range, showing the elastic character of these materials. In the case of the present invention, gels may have G’ values comprised between 50 Pa and 250 kPa and G” values comprised between 5 Pa and 80 Pa, following photopolymerization

Advantageously, the hydrogel may comprise an aqueous solution. The aqueous solution may comprise a salt chosen in the group comprising lithium, sodium, potassium, rubidium, cesium, or ammonium chloride, preferably sodium chloride. The concentration of lithium, sodium, potassium, rubidium, cesium or ammonium chloride in the aqueous solution may be comprised between 0.5 to 1.5 wt.%, preferably is 0.9 wt. %.

Advantageously, the aqueous solution may be a 0.9% aqueous sodium chloride solution.

The term “biological function” can refer to the modulation, growth, and/or proliferation of at least one cell, such as a progenitor cell and/or differentiated cell, the modulation of the state of differentiation of at least one cell, and/or the induction of a pathway in at least one cell, which directs the cell to grow, proliferate, and/or differentiate along a desired pathway, e.g., leading to a desired cell phenotype, cell migration, angiogenesis, apoptosis, etc.

The term “macromer” can refer to any natural polymer or oligomer.

The term “peptide” refers to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules.

The term “cell” can refer to any progenitor cell, such as totipotent stem cells, pluripotent stem cells, and multipotent stem cells, as well as any of their lineage descendant cells, including more differentiated cells. The cells can derive from embryonic, fetal, or adult tissues. Exemplary progenitor cells can be selected from, but not restricted to, totipotent stem cells, induced pluripotent or progenitor stem cells, pluripotent stem cells, multipotent stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, neuronal stem cells, hematopoietic stem cells, pancreatic stem cells, cardiac stem cells, embryonic stem cells, embryonic germ cells, neural crest stem cells, kidney stem cells, hepatic stem cells, lung stem cells, hemangioblast cells, endothelial progenitor cells, and cancer stem cells.

Other cells are for example, fibroblasts, endothelial cells, nerve cells, schwan cells, mesenchymal stromal cells, primary or cell line cancer cells, hepatocytes, stellate cells, pericytes, macrophages, lymphocytes, chondrocytes, osteoblasts, osteoblasts, osteoclasts and epithelial cells.

Embodiments described herein relate to methods for three dimensional (3D) bioprinting of living cells to form scaffold-free 3D cell or tissue constructs. The 3D tissue constructs can be used in regenerative medicine, cell-based technologies, tissue engineering, and bioprinting applications. Unlike previous 3D bioprinting techniques which depend on external solid materials for structural maintenance or additional process for prefabrication of cell aggregates, the systems and methods described herein use a sheer thinning, crosslinkable, biocompatible hydrogel support medium to provide a structural support role for the printed cell constructs, allowing media provision, and long term culture. Precise maintenance of the structure, for example, mirroring an original computer aided design (CAD) file, can also be achieved even after maturation of the tissue by cell proliferation, differentiation, and extracellular matrix (ECM) production.

Different technologies of bioprinting have been developed, including inkjet or microjet, microextrusion and laser-assisted bioprinting [2, 3]

In a specific embodiment, in the method of the present invention, the printing step is performed through microextrusion

The hydrogel support medium can behave as a viscous fluid during printing and be resistant to flow before and after printing. For example, initially, the hydrogel support medium is in a flow-resistant or solid-like state before being printed with the bioink. The hydrogel support medium becomes fluidized under the increased shear stress caused by printing the bioink into the hydrogel support medium. Then, after the printing is finished the hydrogel support medium can be polymerized to form a solid-like stable support medium.

The extracellular matrix derived hydrogel is cytocompatible and, upon degradation produces substantially non-toxic products.

In a preferred embodiment, the living cells are added to the (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide prior to the printing of the bioink and the polymerization reaction is performed subsequently. In some embodiments, a culture medium in which the hydrogel support medium can be cultured can be provided after the bioink is printed and the hydrogel support medium is further crosslinked.

The culture medium can in addition include bioactive agents for promoting growth and/or differentiation of the cells of the printed bioink. For example, the culture medium can include a cell differentiation medium.

In some embodiments, the hydrogel support medium can include a plurality of hydrogel particles that include a plurality of crosslinkable biodegradable extracellular matrix derived polymer macromers. The hydrogel particles can have an average diameter of about 100 nm to about 100 pm. In particular embodiment, the crosslinkable biocompatible extracellular matrix derived hydrogel support medium of the invention comprised a combination of natural polymer such as hyaluronic acid and collagen.

The natural polymer (macromers) include a plurality of acrylated and/or methacrylated natural polymer macromers, preferably methacrylated polymers. The acrylated and/or methacrylated, polymer macromers extracellular matrix derived polymers are advantageously comprised of a combination of collagen and hyaluronic acid.

The degree of (meth) acrylation of the natural polymer macromers such as collagen and hyaluronic acid is advantageously comprised between 5 to 75%, preferably between 15 and 70%.

In particular embodiment, the degree of (meth) acrylation of collagen is advantageously comprised between 40 to 70% more preferably between 40 to 60%. As described in the experimental section, and the degree of methacrylation of collagen is 57% (+/- 15%)

In particular embodiment the degree of (meth) acrylation of hyaluronic acid is advantageously comprised between 10 to 30%. As described in the experimental section, the degree of methacrylation of hyaluronic acid is 20%

The ratio of collagen and hyaluronic acid is adjusted according to the desired properties of the bioink and hence the desired biological function of the 3D scaffold that is ultimately obtained. In sum, hyaluronic acid concentration plays a key role on the overall bioink viscosity and therefore in the printability of the final formulation. According to the concentration of hyaluronic acid used, the concentration of collagen, a major component of the extracellular matrix, is adjusted to enable optimal cell adhesion and survival.

The mass ratio, hyaluronic acid/ collagen, is advantageously comprised between 0.125 and 100, preferably between 5 and 20.

In a specific embodiment, the mass ratio, hyaluronic acid / collagen is 10 (as described in the experimental section)

Preferably, the concentration of collagen in said hydrogel in the range from 0.5 to 5 mg/mL more preferably between 1 and 2 mg/mL and the concentration of hyaluronic acid in said hydrogel is in the range from 0.5 to 50 mg/ml, more preferably between 10 and 20 mg/mL.

Both collagen and hyaluronic acid (CollMA and HAMA, respectively) are advantageously methacrylated, to form methacrylated collagen and methacrylated hyaluronic acid (CollMA and HAMA, respectively) and combined for instance with laminin-derived thiolated peptides. The thiol-ene reaction, between the acrylate groups of CollMA and HAMA, and the thiol moieties of the peptides is achieved before bioprinting. Then, subsequent radical polymerization, between the excess acrylate groups of the methacrylated polymers, of the composite hydrogel is accomplished following bioprinting. This approach enables to separate the optimization regarding printability, namely by changing the CollMA and HAMA concentrations and ratio and the cell active peptide sequence spatial density, drastically increasing the tunability of the developed bioinks and allowing to easily adapt to other peptide sequences or to create complex mixtures and/or gradients.

In a specific embodiment, the adhesion peptide is a laminin derived peptide.

Examples of said laminin derived peptides are the peptides containing the following sequences: YIGSR and IKVAV, that can be flanked by a spacer sequence (i.e. beta alanine or glycine repeats) and that can be flanked by terminal cysteines, at the amino and carboxyl terminal chains.

The density of said peptide within the said extracellular matrix is adjusted with respect to the desired properties of the bioink and hence the desired biological function of the 3D scaffold that is ultimately obtained.

Advantageously, the density inside the final composite hydrogel is comprised between 0.5 mg/mL and 10 mg/mL.

In some embodiments, the plurality of living cells can include progenitor cells, undifferentiated cells, differentiated cells, and/or cancer cells.

The cells may be selected from apical papilla stem cells, human bone marrow cells, human skin fibroblast, human gingival fibroblast, human umbilical vein cells, endothelial cells, neuronal cells, hematopoietic stem cells (FISC), human induced pluripotent stem cells (HiPSC) and mesenchymal stem cells (MFISC).

Advantageously, the gel may comprise a concentration of cells between 0.01 million to 100 million of cells/mL of gel, preferably between 0.05 and to 20 million of cells/mL of gel.

As described in the Experimental data, in the case of neurons the concentration of cell that can be used is 0.05 million of cells/ and for the endothelial the concentration of cell that can be used is 20 M/mL.

According to a first embodiment, the living cells comprise fibroblasts, preferably human skin fibroblasts and/or human umbilical vein endothelial cells.

Advantageously, the sequence of the peptide is YIGSR and the concentration of the peptide in the hydrogel comprised between 0.5 mg/ml and 6 mg/ml.

According to a second embodiment, the living cells are neurons, preferably primary sensory neurons. Advantageously, the sequence of the peptide is IKVAV and the concentration of the peptide in the hydrogel comprised between 0.5 mg/ml and 6 mg/ml.

The polymerization step is performed through crosslinking of the acrylate or methacrylate groups of the acrylated and/or methacrylated natural polymer macromer of the hydrogel particles.

The crosslinking is advantageously performed using UV light or visible light in the presence of adapted photoinitiators (see the review Lim KS et al “Fundamentals and Applications of Photo-Cross-Linking in Bioprinting” Chem. Rev. April 2020 https://doi.org/10.1021/acs.chemrev.9b00812). For example, acrylated and/or methacrylated natural polymer macromers of the hydrogel particles can be photocrosslinked with a photoinitiator that is provided in the hydrogel support medium. The hydrogel particles can be exposed to a light source at a wavelength and for a time to promote crosslinking of the acrylate groups of the polymers and form the photocrosslinked biodegradable hydrogel particles.

A photoinitiator can include any photo-initiator that can initiate or induce polymerization of the acrylate or methacrylate macromer. Examples of the photoinitiator can include camphor Quinone, benzoin methyl ether, 2-hydroxy-2-methyl- 1 -phenyl- 1-propanone, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, lithium phenyl-2, 4,6,- trimethylbenzoylphosphinate, benzoin ethyl ether, benzophenone, 9,10-anthraquinone, ethyl-4- N,N-dimethylaminobenzoate, diphenyliodonium chloride, roboflavin and derivatives thereof.

The polymerization conditions are preferably selected in order to obtain a polymerization degree between 70 and 98%.

According to one aspect , the invention provides a method for forming a 3D tissue construct for promoting angiogenesis, wherein the living cells comprise fibroblasts, preferably human skin fibroblasts and/or human umbilical vein endothelial cells and the sequence of the peptide is YIGSR at a concentration of 2 mg/mL.

According to a second aspect, the invention provides a method for forming a 3D tissue construct for promoting neurogenesis, wherein the living cells are neurons, preferably primary sensory neurons and the sequence of the peptide is IKVAV at a concentration of 2 mg/mL.

The printing step can be performed through several techniques as: inkjet, laser and extrusion-based bioprinting. Extrusion-based bioprinting (EBB) is part of the most widespread tools used in additive manufacturing, mainly due to its capabilities for building large volume constructs such as tissue or organ equivalents. This technique offers a large opportunity in terms of bioink as it can be adaptable to a wide range of materials. In some embodiments, the 3D printer is a bioprinter that can dispense the bioink from a cartridge or container in a specific pattern and at specific positions in the hydrogel support medium as directed by a computer aided design (CAD) software in order to form a specific cellular construct, tissue, or organ. In order to fabricate complex tissue constructs, the bioprinter deposits the bioink at precise speeds and in uniform amounts. In some embodiments, a cartridge containing the bioink comprises one dispensing orifice. In various other embodiments, a cartridge comprises from 1 to 100 or more dispensing orifices.

The hydrogel can further include a culture medium for modulating a function or characteristic of a cell and/or for promoting growth and/or differentiation of the cells of the printed bioink. For example, the culture medium can include a cell differentiation medium, such as an osteogenic differentiation media or chondrogenic differentiation media. The hydrogel support medium containing the printed bioink can be provided in the culture medium after crosslinking or before further crosslinking the hydrogel support medium to enhance the stability of the hydrogel support medium.

It will be appreciated that growth factors can be added to the medium to enhance or stimulate cell growth. Examples of growth factors include vascular endothelial growth factor, platelet-derived growth factor, brain derived growth factor, nerve growth factor, transforming growth factor, platelet-derived growth factor, insulin-like growth factor, acid fibroblast growth factor, basic fibroblast growth factor, epidermal growth factor, hepatocytic growth factor, keratinocyte growth factor, and bone morphogenic protein. It will also be appreciated that other agents, such as cytokines, hormones (e.g., parathyroid hormone, parathyroid hormone-related protein, hydrocortisone, thyroxine, insulin), fatty acids (e.g., Omega-3 fatty acids such as al8:3 linolenate), and/or vitamins (e.g., vitamin D), may also be added or removed from the culture medium to promote cell growth.

Advantageously, the gel may comprise between 0.1 and 10 % w/w concentration of hyaluronic acid and collagen, and between 90 and 99.9 % of an aqueous solution comprising lithium, sodium, potassium, rubidium, cesium or ammonium chloride

According to another aspect, the invention also provides a bioink comprising a biocompatible (meth) acrylated extracellular matrix optionally partially crosslinked with an adhesion peptide and a plurality of cells (as above describe), wherein the extracellular matrix is comprised of a combination of hyaluronic acid and collagen, and hyaluronic acid at a mass ratio of hyaluronic acid /collagen comprised between 0.125 and 100, preferably between 5 and 20 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - (A) Viscosity evaluation, in function of the shear rate (0.01-500 s ¹), of HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite, or HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite with 2 mg/mL of YIGSR peptide, or HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite with 2 mg/mL of IKVAV peptide. All bioinks were evaluated prior photopolymerization and at 25°C (Average ± SD, n=3). (B) Storage modulus (G’) of polymerized HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite, HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite with 2 mg/mL of YIGSR peptide, and HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite with 2 mg/mL of IKVAV. All composition contained 0.1 % (w/v) of LAP (Lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate) as photoinitiator. Evaluated following 2 min photopolymerization, measured at 37 °C (Aver±SD, n=3). Statistically significant differences between indicated groups was analyzed by the non-parametric Kruskal- Wallis test, followed by a Dunns post-test (NS denotes Non Significant).

Figure 2 - Fidelity of bioprinting struts composed of HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite, or HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite with 2 mg/mL of YIGSR peptide, using a 210 pm microextrusion tip diameter. The thickness of the bioprinted struts was evaluated by image analysis using ImageJ (Aver±SD, n=3). Statistically significant differences between indicated groups were analyzed by the non-parametric Mann- Whitney test (NS denotes Non Significant). The initial tip diameter is indicated in the correspondent graph using dotted line.

Figure 3 - (A) Quantification of percent viability of the live/dead assay with HUVECs cultured inside polymerized HAMA or HAMA /CollMA composite (a), at 24 hrs of culture (Aver±SD, n=4, ND denotes non-significant). (B) Metabolic activity of HUVECs when cultured inside HAMA /CollMA composite gels containing increased concentrations of the YIGSR peptide (0.25 - 2 mg/mL) at days 0, 3, 7 and 14 (b; Aver±SD, n=4, ND denotes non significant.

Figure 4 - Capillary-like structure maturation evaluation of human umbilical vein endothelial cells, in coculture with human skin fibroblasts, inside methacrylated hyaluronic acid (HAMA) and methacrylated type I collagen (CollMA) hydrogels containing increasing concentration of laminin derived peptides (i.e. YIGSR) for number of junctions (A), number of segments (B) and total segment length (C), at 7 and 14 days (Aver±SD, n=4, * and ** denotes p<0.05 and p<0.01, respectively). Figure 5 - (A) Metabolic activity of dorsal root ganglia (DRG) neurons cultured inside polymerized methacrylated hyaluronic acid (HAMA) and methacrylated collagen (CollMA) (at 1 and 0.1% (w/v), respectively) (HAMA/CollMA) and the composite with increased concentration of the IKVAV peptide (0, 1 and 2 mg/mL), at days 0, 3, 7, 14 and 21 (Aver±SD, n=4, ND denotes non-significant). Image analysis quantification (Neurite tracer, ImageJ) of the number of neurites (B), maximum neurite length (C) and neurite total length per neuron (D) (Aver±SD, n=6, ** and *** denotes p<0.01 and pO.001, respectively).

EXAMPLES

Materials and Methods

Hydrogels synthesis

Methacrylation of hyaluronic acid: The procedure was adapted from Poldervaart et al. [22] Briefly, one gram ofhyaluronic acid sodium salt from Streptococcus Equi (1.5-1.8 x 10E6 Da, Sigma-Aldrich, France) was dissolved in 75 mL of mQ grade water, overnight at room temperature. Then, 50 mL of dimethylformamide (Sigma-Aldrich, France) was added and the pH was adjusted to 8 using NaOH 1M. Then, 1.12 mL of methacrylic anhydride (Sigma- Aldrich) was added to the solution the pH was again adjusted to 8 and the solution was left under constant stirring overnight at room temperature. Then, we added 50 mL of NaCl 5M and the volume of the solution was adjusted to 500 mL with mQ grade water. The solution was filtered using a 0.2 pm filter and then dialyzed (12-14 kDa, Sigma-Aldrich) for five days against 5L mQ water and with 2 daily water changes. The obtained solution was then freeze-dried for 5 days, the polymer recovered and stored at -20 °C until further use. Hyaluronic acid methacrylate (HAMA) stock solution was prepared by dissolving the polymer at 2% (w/v) in DMEM HG (Gibco) and then stored at 4 °C until further use. Methacrylation degree was determined using nuclear magnetic resonance (NMR). Briefly, for 1H-NMR a 6% (w/v) solution of HAMA was prepared in D20 (Sigma-Aldrich) and analyzed at 25 °C on a Bruker Avance I NMR spectrometer operating at 400 MHz. 1H NMR spectra were recorded with an acquisition time of 4 sec, a relaxation delay of 2 and 64 scans. Methacrylate modification was determined by integration of the vinyl singlets relative to the ring ofhyaluronic acid.

Methacrylation of type I collagen: The procedure was adapted from Ravichandran et al. [27] Briefly, type I collagen from bovine skin (Sigma-Aldrich) was adjusted to pH 10 using 2N NaOH and kept on ice, under mild agitation. Then, methacrylic anhydride at a molar ratio of 5:1 (with respect to number of lysine amine groups in collagen) was added subsequently drop-wise and left to react for 4 hours. Then the obtained solution was dialyzed (12-14 kDa, Sigma- Aldrich) for five days against 5L mQ water and with 2 daily water changes. The obtained solution was then freeze-dried for 5 days, and the polymer recovered and stored at -20 °C until further use. The methacrylated type I collagen (CollMA) stock solution was prepared at 6 mg/mL in 0.02 M Acetic acid and stored at 4 °C until further use.

Methacrylation degree was determined using the Tri-nitro benzene sulfonic acid (TNBS) assay [28] Briefly, neutralized native collagen or CollMA were dissolved in 1 mL of carbonate buffer (0.1M, pH 8.5) at 0.1 mg/mL NaHC03 (pH 8.5). Then to 500 pL of collagen or CollMA solution, we added 250 pL of a 1:500 dilution, in carbonate buffer, of TNBSA solution (5% w/v, Sigma-Aldrich) and let to react for 2 hours at 37°C. The reaction was then stopped by the addition of 250pL of 10% (w/v) of SDS (Sigma-Aldrich) and 125pL of HC1 1M. The OD335 of both the blanc, collagen and CollMA was determined using a UV-Vis spectrophotometer (Nanodrop, ThermoFisher). The percentage of remaining free primary amines of CollMA was calculated in relation to the total amount of the pristine collagen. The degree of methacrylation was then determined using the loss of primary amines percentage loss (% DM).

Bioinks preparation

HAMA at 1% (w/v) was prepared by diluting HAMA 2% (w/v) using DMEM HG (Gibco) and mixed with 0.1 % (w/v) of the photonitiator tithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP, TCI Chemicals). CollMA 0.1% (w/v) was prepared by diluting the stock 0.6% (w/v) CollMA solution (dissolved in 0.02 M Acetic acid) with DMEM HG (Gibco), neutralizing with NaOH 0.1M and mixed with 0.1 % (w/v) of LAP photonitiator. The adhesion peptides Cys- (Beta-Ala)-Ile-Lys-Val-Ala-Val-(Beta-Ala)-Cys (IKVAV) and Cys-(Beta-Ala)-Tyr-Ile-Gly- Ser-Arg-(Beta-Ala)-Cys (YIGSR) (Biomatik, Canada) were dissolved at 25 mg/mL in acetic acid 0.02 M aliquoted and stored at -20 °C untill further use.

HAMA/CollMA, 1 and 0.1% (w/v) respectively, composite bioink was obtained by diluting and neutralizing the stock 0.6% (w/v) CollMA, then HAMA 2% (w/v) was added, the solution was mixed with 0.1 % (w/v) of LAP and the volume adjusted with DMEM HG. The peptide solution containing the laminin derived peptides was homogeneously added, at defined final concentrations, to the composite polymer solution prior use. All formulation were prepared fresh and kept protected from light. Rheological characterization

For viscosity determination, different formulations of the hydrogels were prepared, loaded in a Kinexus pro+ rheometer and rotational shear-viscosity measurements were performed in flow mode with shear rate ranging from 0.01 to 500 s ¹. Measurements were performed in triplicate and at 25 °C.

For storage and loss modulus determination, different formulations of the hydrogels were prepared, loaded and directly polymerized (a 2w, 365 nm UV source and an exposure time of 2 minutes) in a Kinexus pro+ rheometer (Malvern Instruments, UK), and stabilized at 37°C prior to the determination of the storage moduli (G’) and loss moduli (G”). Frequency sweeps were performed at a constant strain (0.1%) in the angular frequency range 0.1-100 rad/s. Measurements were performed in triplicate and at 37 °C.

Primary cell culture

Human umbilical vein endothelial cells (HUVECs) were isolated as previously described [29] HUVECS were cultured, under standard conditions, in IMDM (Gibco), 20% SVF + ECGS/H, until passage 9. As means to enable their visualization by fluorescent microscopy cells were stably transduced with lenti virus bearing the RFP gene, under the EFla promoter sequence (Vectalys, France), following the manufacturer’s instructions. Infection efficiency was evaluated by flow cytometry and was determined to be superior to 98% (data not shown). Human skin fibroblasts were isolated as previously described [30], and cultured, under standard conditions, in DMEM:F12 (Gibco), 20% (v/v) FBS, until passage 10.

Primary sensory neurons were obtained from dorsal root ganglion (DRG) from healthy 5-9 weeks old Wistar rats as previously described [31] Briefly, following animal sacrifice with C02, spinal columns were removed and opened from the caudal to the rostral in order to expose the DRGs. They were individually harvested and digested with 1 mg/mL Collagenase Type IV (Gibco®) for 2 h at 37 °C. Subsequently, digestion product was washed twice with culture medium supplemented with 2 % (v/v) B-27 Serum-Free Supplement® (B-27, Gibco®) and 1 % (v/v) Penicilin Streptomycin, and mechanically dissociated using fire-polished glass Pasteur pipettes (full diameter and ½ diameter). The cell suspension was finally washed three more times with medium and resuspended in complete culture medium. Nerve growth factor (NGF, 0.1 pg/mL, Sigma Aldrich) was added to the culture medium at 48 hrs of culture.

Viability evaluation Cell viability percentage was determined using the live-dead assay. Briefly, at defined time points samples were stained with calcein-AM (“live”, 1 pL/mL, Invitrogen) and ethidium homodimer (“dead”, 4 pL/mL, Invitrogen) for 45 min, following manufacturer’s instructions. Samples were then analysed by confocal laser-scanning microscopy (SPE7, Leica Microsystems). Image analysis of the percentage of viable cells was performed using ImageJ (n=4 replicates) by determining the total number of cells per field (cells positive for calcein + cells positive for ethidium homodimer) and determining the percentage of “live” cells (cells positive for calcein).

Cell imaging

The capacity of HUVEC cells to form capillary like structures in 3D was assessed by confocal imaging of RFP+ HUVEC cells at days 7 and 14, inside the different bioinks in 3D culture or after 3D bioprinting. For the 3D culture a 150 pm Z axis height was acquired using confocal microscopy. For the bioprinted structures a 500 pm Z axis height was acquired.

The capacity of DRG cells to extend neurites was assessed by confocal imaging following Calcein-AM (1 pL/mL, Invitrogen) staining for 45 minutes at day 14, inside the different bioinks in 3D culture or after 3D bioprinting. For the 3D culture a 150 pm Z axis height was acquired using confocal microscopy. For the bioprinted structures a 500 pm Z axis height was acquired.

Cell metabolic activity evaluation

Metabolic activity of ECS and DRGs, when cultured inside the hydrogels, was evaluated using a resazurin-based assay [32] Briefly, cells were seeded inside the different formulation hydrogels at 2 million cells/mL for ECS and 1 million/mL for DRGs, respectively, in a 96 well plate. 150 pL of culture medium containing resazurin (0.01 mg/mL) was added to each well and the microplate was incubated at 37 °C for 3 h. Subsequently, 100 pL of supernatant was transferred to another 96 wells microplate and fluorescence was measured (exc = 530 nm, em = 590 nm, Victor X3, Perkin Elmer). Metabolic activity was measured at days 0, 3, 7 and 14 for ECS and days 0, 3, 7, 14 and 21 for DRGs, respectively, (n=6 replicates) % metabolic activity was calculated in relation to day 0.

Bioprinting

The 3D Discovery bioprinting platform (RegenHU) was used in this work. It is composed of two independent pressure activated microextrusion heads and two microvalve heads, automated tip length and substrate height calibration and integrated ultraviolet (UV, 365 nm, 2W) photopolymerization diode. Also, the integrated computer assisted design software (BioCAD, RegenHU) enable to create the desired geometry of printing. The chosen geometry consisted on 5 parallel lines, spaced of 2.5 mm, and intercrossed with same geometry with a 90-degree angle rotation. The same geometry was repeated to a total of 4 layers. The geometry was adapted to print inside 6 well plate wells and repeated in series.

In the case of HUVEC+HSF bioprinting, a conic microextrusion tip of 0.21mm diameter was used. Both HUVECs and HSF were both loaded together inside the designated bioink (containing or not the YIGSR peptide), both at 10 million/mL. The ink was homogenized and loaded inside the bioprinting syringe. The bioprinting process consisted on the deposition of the first layer with a pressure of 0.008 MPa, speed of 140 mm/s and a distance to the substrate of 0.075 mm. Then the first layer was polymerized during 15 seconds using the UV source. The second perpendicular layer was then deposited using the same pressure and speed conditions at a distance of 0.1 mm of the first layer. The process was then repeated for a total of 4 layers, before adding 3 mL of cell culture media.

In the case of DRG bioprinting, a conic microextrusion tip of 0.41mm diameter was used. DRG were loaded inside the designated bioink (containing or not the IKVAV peptide), at 50 000 cells/mL. The ink was homogenized and loaded inside the bioprinting syringe. The bioprinting process consisted on the deposition of the first layer with a pressure of 0.005 MPa, speed of 160 mm/s and a distance to the substrate of 0.075 mm. Then the first layer was polymerized during 15 seconds using the UV source. The second perpendicular layer was then deposited using the same pressure and speed conditions at a distance of 0.1 mm of the first layer. The process was then repeated for a total of 4 layers, before adding 3 mL of cell culture media.

Image analysis

The capacity of HUVECs to form capillary -like structures in 3D culture was quantified by image analysis of confocal stacking images using the Angiogenesis Analyzer [33] plugin for Image! The software allowed to assess relevant parameters as number of junctions, number of segments and total segment length.

HUVEC evaluation upon 3D printing was achieved by 3D image analysis of the volumetric confocal acquisition, at day 14, and using the Imaris software (9.0, Bitplane, UK). The 3D segmentation of independent structures, inside the bioprinted matrix, were evaluated in terms of their length in the Z axis (Bounding Box length Z axis), as to further demonstrate the capacity of HUVECs to migrate inside the structure, and the volume of each one of these independent 3D structures (volume bounding box), as means to evaluate the capacity to interconnect and form a complex network.

The capacity of DRGs to extend neurites in 3D culture was quantified by image analysis of confocal stacking images using the NeuriteQuant [34] plugin for Image! The software allowed to assess relevant parameters as number of neurites per neuron, maximum neurite length and neurite total length/per neuron. DRG evaluation upon 3D printing was achieved by 3D image analysis of the volumetric confocal acquisition, at day 14, and using the Imaris software (9.0, Bitplane, UK). The 3D segmentation of independent structures, inside the bioprinted matrix, were evaluated by segmental analysis and allow to determine the dendrite length distribution for different compositions of bioink.

Statistical analysis

Using the Graphpad Prism 5.0 software, a D’Agostino and Pearson omnibus normality test was used in order to test if data obeyed to a Gaussian distribution. Statistically significant differences between several groups were analysed by the non-parametric Kruskal -Wallis test, followed by a Dunns post-test. The non-parametric Mann-Whitney test was used to compare two groups. A p value lower than 0.05 was considered to be statistically significant.

Results

Hydrogel Characterization

The chemical approach for the creation of a printable composite hydrogel was based on an initial methacrylation of the base polymers, namely hyaluronic acid and type I collagen, both natural components of the extracellular matrix. The inclusion of laminin-derived peptides was achieved by the inclusion of cysteine groups at both the extremities of the peptides, what permits to graft, via a thiol-ene UV initiated radical addition, the peptides to the methacrylated matrix. Also, the excess methacrylate moieties enables the photopolymerization of the matrix by radical polymerization via UV irradiation.

Chemical characterization of both CollMA and HAMA consisted on the quantification of acrylate groups introduced in the correspondent polymer backbone. CollMA showed a methacrylation degree was 57 ± 15 % (n=3), as determined by TNBS assay. For HAMA, NMR analysis by the integration of the vinyl singlets (1H each) relative to the ring of hyaluronic acid (10H) showed a degree of methacrylation of 20±1% (n=3). The following HAMA (1% (w/v)) and CollMA (0.1% (w/v)) formulations were selected based on previous unpublished results as a base for this study. The obtained results were based on the survival and proliferation of HUVECs and DRGs and on their printability character.

Shear-viscosity measurements (Figure 1A) were performed, prior photopolymerization, for HAMA (1% (w/v)) CollMA (0.1% (w/v)) alone or containing the highest peptide formulations (2 mg/mL of YIGSR or IKVAV). In the shear range tested, all formulations had similar viscosity values and showed a shear-thinning character. This fact is particularly relevant as shows that with the selected chemical peptide grafting approach one can achieve independent formulations, while maintaining viscosity unaltered.

Rheological characterization for polymerized methacrylated hyaluronic acid (HAMA) at 1% (w/v) show a G’ of 202±25 Pa (at 37°C), for methacrylated type 1 collagen (CollMA) at 0.1% (w/v) show a G’ of 114±12 Pa (at 37°C), for HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite show a G’ of 120±12 Pa (at 37°C), for HAMA(1% (w/v)) and CollMA (0.1% (w/v)) composite with 2 mg/mL of YIGSR peptide a G’ of 197±85 Pa (at 37°C), and for HAMA (1% (w/v)) and CollMA (0.1% (w/v)) composite with 2 mg/mL of IKVAV a G’ of 169±24 Pa (at 37°C). Based on the unique chemistry, we could establish that by using the optimized HAMA and CollMA ratios, and based on the peptide grafting via two thiols per molecule, we could establish bioinks viscosity and rheology independently of its peptide inclusion/concentration. This has great implications in terms of bioink optimization, as rheological properties can have great impact on cell maturation.

Both HAMA (1% (w/v)) and CollMA (0.1% (w/v)) based hydrogels showed modest G’ values, with no significant differences when the composite (HAMA/CollMA) was tested. Additionally, and as observed in Figure IB, the G’ values were at the same range for HAMA (1% (w/v)) and CollMA (0.1% (w/v)) alone or for the highest peptide formulations (2 mg/mL of YIGSR or IKVAV). In all formulations tested the storage modulus (G’) was in the range of 100 to 200 Pa.

Additionally, we have also evaluated bioink fidelity using the described bioprinting conditions. As observed in Figure 2 bioprinting strut dimensions show that the gel could respect the expected dimensions with an observable dispersion of 1.3x in relation to the expected dimensions and where no significant differences could be observed for the formulation with or without peptide. Additionally, the established conditions enabled to reach filament stability on air and to achieve the fabrication of complex structures, like a hollow tube (data not shown) or a parallelepiped (data not shown).

HUVEC culture in 3D gels

Based on previous results of the optimization of the concentration of HAMA in the behavior of encapsulated HUVEC cells (data not shown) the concentration of 1% (w/v) was established as the basis for this study. Also, it was previously show that the inclusion of CollMA could further support HUVECs migration. Here, and as observed in Figure 3A, it could be shown that in the present formulations and polymerization conditions that cells showed a percent viability of 67 and 77%, for HAMA and HAMA/CollMA, respectively, at 24 hours of culture. Based on this profile, the HAMA/CollMA formulation were selected in the subsequent studies. As means to evaluate the impact of the inclusion of the laminin-derived YIGSR peptide on HUVECs their metabolic activity progression up to 14 days of culture was then evaluated. As seen in Figure 3B, no significant differences could be observed for the different HAMA/CollMA formulations containing increased concentrations of the YIGSR peptide. Also, it could be seen a significant progression of the metabolic activity of all formulations towards time, up to 14 days.

The maturation of endothelial cells (HUVECs), when in coculture with HSF, inside the composite hydrogels containing different concentrations of the laminin-derived peptide YIGSR was then evaluated. For all formulations tested no significant maturation was observed at day 7. At day 14 of culture a significant increase on capillary-like structures was observed for the formulation containing the highest YIGSR concentration (2 mg/mL, Figure 4). The quantification of the capillary-like structure was achieved by image analysis (Angiogenesis Analyser, ImageJ) and as seen in Figure 4 A,B,C, a significant increase in terms of the number of junctions, number of segments and total segment length was observed at 14 days of culture and for the HAMA /CollMA + 2 mg/mL YIGSR peptide.

Bioprinting of HUVEC cells

The obtained results allowed the selection of the HAMA/CollMA + 2 mg/mL YIGSR peptide as optimal formulation for the maturation of HUVEC cells and was compared with the basal formulation, HAMA/CollMA. The chosen assisted computer design (CAD) consisted on 5 parallel lines, spaced of 2.5 mm, and intercrossed with same geometry with a 90 degree angle rotation. The same geometry was repeated to a total of 4 layers. Both the speed of printing (140 mm/s), the diameter of the tip (0.21 mm) and the distance between the tip and the substrate (0.075 mm) were controlled in order to attain a reproducible geometry. Additionally, the capacity of the composite hydrogel containing YIGSR was tested to further sustain the maturation of HUVECs in coculture with HSFs, following bioprinting and using the same CAD. Both composition permitted the microextrusion bioprinting of the scaffold containing both HUVECs and HSFs. Additionally, one could observe that the composition containing the YIGSR peptide could further support HUVECs maturation, particularly relevant at day 14.

At 14 days post printing, the HUVEC network was then evaluated by confocal microscopy on a thickness of 0.5 mm, and reconstructed using imaging analysis software (Imaris) in order to quantify its interconnectivity and dimensions. Software analysis enabled to identify and create a tridimensional box that includes each interconnected structure (bounding box). Then, the tridimensional dimensions of this bounding box give a quantification of the interconnection and distribution inside the bioprinted matrix. Both the thickness (Z axis length) and interconnection structure volume was shown to be significantly increased for the bioprinted structure containing the laminin-derived YIGSR peptide.

Dorsal root ganglia sensory neuron culture in 3D gels

Based on the previous results of the optimal bioink formulation for HUVECs the same base of HAMA/CollMA was established to which a new laminin-derived peptide, IKVAV was combined. Important to refer that the same YIGSR formulations were equally tested for DRG culture and were found no optimal (data not shown).

Again, and means to evaluate the impact of the inclusion of the laminin-derived IKVAV peptide on DRGs their viability at 7 days of culture and metabolic activity progression up to 21 days of culture were evaluated. No signs of cell mortality could be observed, for all formulations tested. Additionally, the metabolic activity follow-up, up to day 21, show no significant differences between formulations or a decrease in time (Figure 5A), indicating that in the use conditions the different composition could support the 3D culture of DRG neurons. Finally, based on image analysis we quantified the number of neurites, maximum neurite length and total neurite length per neuron (Figure 5B, C and D, respectively). We could observe a significant increase on all parameters for the highest peptide concentration, in relation with the formulation without peptide, and consistent with a dose response effect.

Bioprinting of dorsal root ganglia sensory neurons

The chosen CAD consisted on 5 parallel lines, spaced of 2.5 mm, and intercrossed with same geometry with a 90-degree angle rotation, as before. The same geometry was repeated to a total of 4 layers. Both the speed of printing (160 mm/s), the diameter of the tip (0.41 mm) and the distance between the tip and the substrate (0.075 mm) were controlled in order to attain a reproducible geometry. Live-dead assay staining assessed that the bioprinting process did not significantly affect the viability of the printed DRG neurons, as observe 14 days post printing, and the inclusion of the IKVAV peptide could improve neurite formation. As such, image analysis of calcein stained DRG neurons at 14 days post printing, was performed and the formation of dendrite processes was quantified using segment analysis with Imaris software. Dendrite length quantification could assert that for the formulation containing 2 mg/mL of IKVAV peptide a significant increase could be observed.

It is currently accepted that different matrix compositions have to be determined to individual cell types and for specific biological applications. In this sense, bioink formulations based on modular approaches considering at the same time the printability and rigidity (enabling to easily adapt components concentrations), while enabling to adjust for optimal cell adhesion sequence density or even on the combination or gradient of sequences, represents an added value in future developments in bioprinting. The approach follows this line of thought, enabling inherent modularity and tunability while respecting the aimed application simplicity.

Phage display technology and the rampant field of biotechnology have already identified a wide range of small peptide sequences with inherent biological activity [56] The simple approach of the invention enables to easily adapt to other bioactive peptide sequences, opening the field of application of such bioinks to a wide range of cell types and applications. The adaptation of these novel formulations to other major bioprinting technologies, namely inkjet or laser-assisted bioprinting, are currently being explored opening the range of applications of this approach.

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Claims

CLAIMS:

1. A method for forming a cell specific 3D cell or tissue construct with a desired biological function comprising the steps of: a) Providing a (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel support medium; b) Reacting said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel with a thiol flanked functionalized adhesion peptide to obtain a (meth) acrylate crosslinkable biocompatible extracellular matrix derived hydrogel partially crossbnked with said peptide, via a thiol-ene reaction; cl) Either, combining said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crossbnked with said peptide with a plurality of specific living cells to provide a printable bioink, dl) Printing the said bioink into a first layer of printed bioink with a defined design; el) Submitting the said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crossbnked bioink; and fl) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained c2) Or printing said bioink into a first layer of printed bioink with a defined design; d2) Submitting said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crossbnked bioink; and e2) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, and f2), adding to said printed (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crossbnked with said peptide a plurality of specific living cells, g) Allowing the growth of the living cells in 3D-construct obtained thereby under suitable conditions until the desired biological function is achieved; wherein the hydrogel support medium, the sequence of the said peptide, the ratio of the said peptide to the said extracellular matrix derived hydrogel and the polymerization conditions are selected with respect to cell specificity so as to obtain a 3D tissue construct with the desired biological function.

2. The method according to claim 1 comprising the following steps: al) Providing a (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel support medium; bl) Reacting said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel with a thiol flanked functionalized adhesion peptide to obtain a (meth) acrylate crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide, via a thiol-ene reaction; cl) Combining said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide with a plurality of specific living cells to provide a printable bioink, dl) Printing the said bioink into a first layer of printed bioink with a defined design; el) Submitting the said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and fl) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, and gl) Allowing the growth of the living cells in 3D-construct obtained thereby under suitable conditions until the desired biological function is achieved.

3. The method according to claim 1 comprising the following steps: a2) Providing a (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel support medium; b2) Reacting said (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel with a thiol flanked functionalized adhesion peptide to obtain a (meth) acrylate crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide, via a thiol-ene reaction c2) Printing said bioink into a first layer of printed bioink with a defined design; d2) Submitting said first layer of bioink to a polymerization reaction, under controlled conditions in order to provide a crosslinked bioink; and e2) Repeating the printing step and said polymerization step until a 3D construct with the desired shape is obtained, and f2), adding to said printed (meth) acrylated crosslinkable biocompatible extracellular matrix derived hydrogel partially crosslinked with said peptide a plurality of specific living cells, and g2) Allowing the growth of the living cells in 3D-construct obtained thereby under suitable conditions until the desired biological function is achieved.

4. A method according to anyone of the preceding claims, wherein the polymerization conditions are selected in order to obtain a polymerization degree between 70 and 98 %.

5. The method according to claim 1 to 5, wherein the crosslinkable biocompatible extracellular matrix derived hydrogel support medium is comprised of a combination of the natural polymer macromers such as hyaluronic acid and collagen at a mass ratio comprised between 0.125 and 100, preferably between 5 and 20.

6. The method according to claim 5, wherein the concentration of collagen in said hydrogel is in the range from 0.5 to 5 mg/mL and the concentration of hyaluronic acid in said hydrogel is in the range from 0.5 to 50 mg/mL.

7. A method according to claims 5 or 6, wherein the degree of (meth) acrylation of the natural polymer macromers is comprised between 5 to 75 %.

8. A method according to claim 7, wherein the degree of (meth) acrylation of collagen is comprised between 40 to 70%.

9. A method according to claim 7 or 8, wherein the degree of (meth) acrylation of hyaluronic acid is comprised between 10 to 30%.

10. The method according to anyone of the preceding claims, wherein the peptide is a laminin derived peptide.

11. The method according to anyone of the preceding claims, wherein the density of said peptide within the said (meth) acrylated extracellular matrix derived hydrogel is between 0.5 and 10 mg/mL.

12. The method according to anyone of the preceding claims, wherein the cells are progenitor cells selected from the list consisting of totipotent stem cells, induced pluripotent or progenitor stem cells, pluripotent stem cells, multipotent stem cells, mesenchymal stem cells (MSCs), hematopoietic stem cells, neuronal stem cells, hematopoietic stem cells, pancreatic stem cells, cardiac stem cells, embryonic stem cells, embryonic germ cells, neural crest stem cells, kidney stem cells, hepatic stem cells, lung stem cells, hemangioblast cells, endothelial progenitor cells, and cancer stem cells.

13. A method according to anyone of the preceding claims, for forming a 3D tissue construct for promoting angiogenesis, wherein the living cells comprise fibroblasts, preferably human skin fibroblasts and/or human umbilical vein endothelial cells and the sequence of the peptide is YIGSR at a concentration comprised between 0.5 mg/mL and 6 mg/mL.

14. A method according to anyone of the claims 1 to 11 for forming a 3D tissue construct for promoting neurogenesis, wherein the living cells are neurons, preferably primary sensory neurons and the sequence of the peptide is IKVAV at a concentration comprised between 0.5 mg/mL and 6 mg/mL.

15. A method according to anyone of the preceding claims wherein the printing step is performed through microextrusion.

16. A bioink comprising a biocompatible (meth) acrylated extracellular matrix optionally partially crosslinked with an adhesion peptide and a plurality of cells, wherein the extracellular matrix is comprised of a combination of hyaluronic acid and collagen, at a mass ratio of hyaluronic acid /collagen comprised between 0.125 and 100, preferably between 5 and 20

17. A scaffold obtained by printing a bioink according to anyone of method of claims 1 to 14.