WO2016194011A1

WO2016194011A1 - Method for preparing cellularized constructs based on heat-sensitive hydro-gels

Info

Publication number: WO2016194011A1
Application number: PCT/IT2016/000142
Authority: WO
Inventors: Valeria Chiono; Susanna SARTORI; Monica BOFFITO; Emilia GIOFFREDI; Marcella Trombetta; Pamela MOZETIC; Alberto Rainer; Sara Maria GIANNITELLI
Original assignee: Politecnico Di Torino; Universita' Campus Bio-Medico Di Roma
Priority date: 2015-06-05
Filing date: 2016-06-01
Publication date: 2016-12-08

Abstract

A method is described for preparing cellularized constructs through quick prototyping comprising the following steps: (A) preparing a polymeric solution comprising one or more natural and/or synthetic polymers in a culture medium at a temperature included between 1°C and 20°C; (B) dispersing cells inside the polymeric solution; (C) gelling the polymeric dispersion containing said cells by increasing the temperature to a value included between 36°C and 38 °C in order to obtain a cellularized hydro-gel; (D) extruding the cellularized hydro-gel through a suitable nozzle on a thermostated support at a temperature included between 36°C and 38 °C.

Description

METHOD FOR PREPARING CELLULARI ZED CONSTRUCTS BASED ON HEAT- SENSITIVE HYDRO-GELS

The present invention is related to the sector of tissue engineering and refers to a method for preparing cellularized constructs based on hydro- gels by using a quick prototyping technique. In particular, the invention deals with preparing cellularized constructs based on heat-sensitive hydro-gels, which are particularly stable under physiologic conditions of the human body.

Tissue engineering identifies procedures for regenerating human body tissues by seeding cells on structures (scaffolds) made of suitable materials and features and their cultivation in suitable reactors (bio-reactors) till the scaffold is colonized and a new tissue is produced (through deposition of an Extra Cellular Matrix, ECM, by the cells) . The scaffold is generally made of biodegradable or bio-re-absorbable materials. Both terms, even if not equivalent, define the phenomena for which the scaffold, after a certain period passed in contact with the biologic environment, is subjected to chemical modifications which brings about its progressive "disappearance".

Recently, the tissue engineering has more and more frequently used quick prototyping techniques, in order to obtain two- or three-dimensional cellularized scaffolds having a controlled geometry and incorporating cells inside the obtained structure.

Quick prototyping is an innovative technology which makes it possible to produce, in a few hours and without the use of tools, objects with an even complex three-dimensional geometry, based on a "file" containing information about their geometry. In case of applications in the biomedical sector, quick prototyping is also designated with the terms "bio-printing" or "cell printing", in order to point out a process for generating structures with controlled geometry incorporating cells, wherein cell functionality and vitality are preserved inside the printed construct (scaffold) . Through such technique, "printed" tissues and organs can be made available for their implant on a patient. The "cell printing" technique uses cells and hydro-gels as printing materials, which, when mixed together in suitable amounts, form the so-called bio-ink.

A 3D printer is equipped with one or more heads which depose the bio-ink one layer at a time, embedding the cells into the polymeric hydro-gel which operates as support. The three-dimensional object is obtained based on a computerized model, prepared with dedicated software, which is then "printed", deposing one layer of material above the other in the desired shape. The object can be made of a single material or of a combination of mutually different materials: whichever the adopted solution, there is always a progressive addition, one layer above the other, of new material modelled in the desired shape through the relative movement between delivery nozzle and motored piece-holder table, together with material dispensing. This latter one can occur, for example, by extruding the material shaped as filaments organized according to the desired geometry, conveying the bulk of material through one or more holes (nozzle) with suitable shape and sizes.

Several "cell printing" techniques are known, in order to make two- or three-dimensional cellularized scaffolds having a controlled structure and incorporating cells inside the filaments obtained by extrusion. Such cellularized scaffolds can be obtained starting from suspensions of cells in aqueous solutions based on hydro-gels precursors, both of natural and of synthetic origin. Hydro-gels are three-dimensional polymeric structures composed of hydrophilic homopolymers or copolymers, made insoluble in water through cross- linking (chemical or physical cross-linking) , which guarantee the shape stability of the structure.

Known "cell printing" techniques which are referred to implied first of all the preparation of a dispersion of cells in a solution based on hydro- gels precursors, the followings extrusion of the cellular dispersion through a suitable nozzle and its deposition in filaments with controlled geometry on the deposition substrate. The construct is afterwards converted into a cellularized hydro- gel through a cross-linking process which can be of the physical or chemical type, through:

1) use of cross-linking agents, for example salts, for a ionic cross-linking, use of low-molecular- weigh chemical agents (forming covalent links) and unsaturated cross-linkers in combination with a initiator and a physical irradiation stimulation (photo-cross-linking with formation of covalent links ) ;

2) variation of physical conditions, such as pH and temperature, in order to make a physical cross- linking;

3) a combination of the two previous methodologies.

The disadvantages of the above method, which provides for the use of a cross-linking to transform the printed cellularized construct into cellularized hydro-gel, can be summarized as follows :

a) low reproducibility, control and resolution of the structure of individual filaments deposited by extrusion, due both to the low viscosity of the deposited fluid and to the necessary time for its gelling following its deposition, with consequent difficulty in checking the final geometry;

b) possibility of a volumetric shrinkage following the cross-linking with physical and/or chemical mechanism of the deposited material on the deposition substrate, with consequent difficulty in controlling the final geometry;

c) use of chemical reagents and/or ultraviolet (UV) radiations which can have a cyto-toxic effect on embedded cells;

d) need of a secondary material to help depositing many layers, in order to obtain a three-dimensional geometry. Such material must afterwards be removed, implying a further working step for preparing the cellularized construct.

Some patent applications have recently been filed, whose subjects are methods for preparing hydro-gel-based constructs, embedding and not embedding cells.

WO 2010/030964 discloses a method to obtain three-dimensional multi-layer, cellularized or non- cellularized hydro-gels. The method is based on the nebulization of a thin layer of cross-linker on a substrate; on the following deposition of an hydro- gel precursor for its partial cross-linking; on the nebulisation of another thin layer of cross-linker to complete the cross-linking; finally, on the repetition of the above described processes for the desired number of times. The method for embedding the cells between the various hydro-gel layers provides for nebulising a thin layer of cross- linker on a substrate; afterwards depositing an hydro-gel precursor for its partial cross-linking; depositing a layer of cells; depositing a layer of hydro-gel precursor; nebulising another thin layer of cross-linker to complete the cross-linking; finally, repeating the above described processes for the desired number of times. Such description does not deal with a "cell printing" method like in the present patent application.

WO 2010/060080 disclosed necessary method and materials to obtain three-dimensional cellularized constructs with smooth muscle cells. In compliance with the description, drops of collagen solution which embed smooth muscle cells are deposited inside the scaffold under construction. The scaffold can be based on collagen, hyaluronic acid, fibrin and kitosan, synthetic polymers (poly glycol acid PGA, poly caprolactone PCL, poly (lactic acid) PLA) and carbon nanotubes. Such method does not use heat-sensitive hydro-gels and is based on the cellular printing technique as drops of a cellular dispersion in a collagen solution.

WO 2011/119607 defines the composition of a polymeric ink for "quick prototyping" based on a polymer added with a photo-polymerizing monomer or a monomer having photo-polymerizing functionality. Ink can include a cross-linking agent, a photo- initiator and water as solvent. The polymer is solubilized in water at a concentration grater than 5-100 times with respect to a critical concentration (namely the transition concentration from a diluted to a semi-diluted solution) . Viscous-elastic ink can include a photo- polymerizing monomer, such as acryl-amide or hydroxyl ethyl methacrylate for inks based on poly acryl-amide (pAM) or poly ( 2-hydroxyl ethyl methacrylate (pHEMA) . After extruding the viscous- elastic ink into filaments which are the structure layer, the filament is cross-linked by irradiation. The obtained structures are non-cellularized . It is then possible to seed the cells after having produced the structures. Such patent application does not deal with a "cell printing" method. Moreover, the material is extruded as a viscous- elastic solution and not as a gel, with the inconvenience that the final structure must necessarily be cross-linked by irradiation. US 2012/0282448 discloses a method for preparing three-dimensional structures through quick prototyping starting from a complex ink containing various ingredients: a compound cross- linkable with a cationic mechanism, a cationic photo-initiator, a compound cross-linkable with a radical mechanism, a radical photo-initiator and a gelling material. The gelling material is used as semi-solid gel and is extruded in combination with the other materials. The gelling material is then used to avoid the structure collapse after its deposition and before its chemical cross-linking. Such patent application does not deal with a "cell printing" method, and provides for a chemical cross-linking of the structure, after its deposition by extrusion.

US 2014/0179822 proposes an hydro-gel for printing three-dimensional constructs. Hydro-gel is composed of a three-block ABA polymer in combination with a second three-block Ama-B-Ama polymer, where Ama is a polymer based on A units functionalized with methacrylate functionality (ma) . The obtained material is heat-sensitive and photo-cross-linkable and is solubilised in de- ionized water. The solution is afterwards extruded through the nozzle of a quick prototyping machine on a substrate heated above the gelling point. Under the selected process conditions, the sol-gel transition quickly occurs. Afterwards, the material is stabilized by irradiation (photo-cross-linking) . Also in this case, the above listed inconveniences can occur, related to the stabilization of hydro- gels due to photo-induced cross-linking.

Scientific and patent literature has also included examples of cellularized constructs with geometry capable of being controlled and reproduced, obtained by extruding heat-sensitive hydro-gels with gel-sol transition upon increasing the temperature, due to a different procedure from previously shown techniques. In this case, a cellular suspension is prepared in a solution of the material and partially or completely gelled through a decrease of the temperature below the gel-sol transition point. Hydro-gel or semi-hydro- gel is afterwards extruded in the desired geometry. Since the gel-sol transition temperature is lower than the physiologic temperature (37°C), the construct, before being brought to the physiologic temperature, must necessarily be stabilized through a cross-linking process. Such method has been solely applied to gelatine-based polymers of natural origin.

An example of this methodology is given in EP

2679699, which proposes a process and an apparatus for obtaining cellularized scaffolds starting from hydro-gels. The process consists in preparing a cellular suspension in a solution based on a mixture of the "first hydro-gel precursor" (component capable of gelling when the temperature decreases and having a transition temperature from solution phase to gel phase included between 10°C and 30 °C) and of the "second hydro-gel precursor" (chemically cross-linkable component) . The material is extruded as a solution or as a gel on a substrate with temperature lower than the gelling point of the first component, in order to stabilize the shape of the extruded structure. The construct is then chemically cross-linked, possibly by immersion in a medium containing the cross-linking agent. During the cross-linking process, in general, only the second component of the mixture is cross-linked. The first component of the system, therefore, has the function of ensuring the structure stability in short times, while the second component is the structural material of the construct. When the temperature of the final cellularized construct is taken to the physiologic value of 37 °C, the first component usually is quickly solubilised and moved away from the system. However, the use of heat-sensitive hydro-gels with gel-sol transition upon increasing the temperature, as disclosed in EP 2679699, has several negative aspects, such as:

1) the need of a cooling for the temporary physical gelling of the construct, which implies the use of a refrigerating system combined with the quick prototyping apparatus, with consequent increase of production costs;

2) the use of reagents for chemical cross- linking, which can impair the cellular viability. Without a chemical cross-linking, the heat- sensitive hydro-gel is not stable at a physiologic temperature (37°C);

3) moreover, the use of natural hydro-gels, even is cross-linked, is associated with problems of scarce stability in an aqueous solvent. Natural cross-linked hydro-gels, in fact, usually have a high swelling degree and a relatively short decay time (as an average 1-2 weeks for gelatine-based hydro-gels) .

US-A1-2015/084232 discloses a prior art method for producing a scaffold which includes cells.

EP-A1-2679669 discloses a prior art process for preparing three-dimensional hydro-gel objects comprising living cells.

O-A2-2005/016114 discloses a prior art method of manufacturing hydro-gels tissue scaffolds loaded with cells.

In view of all disadvantages related to the above described procedure, there is the need of providing an innovative method for the controlled preparation of cellularized constructs based on hydro-gels, through a quick prototyping technique.

The Applicant has discovered the chance of preparing cellularized constructs based on hydro- gels having high stability, using heat-sensitive hydro-gels having a transition from the sol phase to the gel phase upon increasing the temperature.

These and other advantages of the invention, which will result from the following description, are obtained with a method as claimed in claim 1.

Preferred embodiments and non-trivial variations of the present invention are the subject matter of the dependent claims.

It is intended that all enclosed claims are an integral part of the present description.

Therefore, the present invention deals with a method for preparing cellularized constructs through quick prototyping comprising the following steps:

A) preparing a polymeric solution comprising one or more natural and/or synthetic polymers in a culture medium, at a temperature included between 1°C and 20°C, preferably between 1°C and 5°C;

B) dispersing cells inside the polymeric solution prepared in step A) ;

C) gelling the polymeric dispersion containing the cells through an increase of the temperature up to a value included between

36°C and 38°C to obtain a cellularized hydro- gel;

D) extruding the cellularized hydro-gel through a printing nozzle on a thermostated support at a temperature included between 36 °C and 38 °C according to a controlled geometry, in order to obtain three-dimensional cellularized constructs;

wherein the polymeric solution prepared in step A) has a transition from the "sol" phase to the "gel" phase upon increasing the temperature.

The method for preparing cellularized constructs according to the present invention uses a polymeric dispersion as heat-sensitive hydro-gel, having a transition from the "sol" phase to the "gel" phase which is performed through heating, and occurs at a temperature included between 20 °C and 30°C.

The chemical nature of the biocompatible and biodegradable hydro-gel material consists in a natural or synthetic polymer, or in a mixture of natural polymers or of synthetic polymers, or in a mixture of natural and synthetic polymers, possibly embedding one or more pharmaceuticals for promoting the cellular survival (for example, anti-oxidizing and/or anti-inflammatory pharmaceuticals) and/or other agents (for example, inorganic calcium phosphate nanoparticles in case of applications in bone regeneration) .

The synthetic polymers which can be used in step A) of the preparation of the polymeric solution are preferably chosen among poloxamers, polyurethanes and polyester-based amphiphilic copolymers (for example, block copolymers composed of poly (caprolactone) and of poly (ethylene glycol) units) . Among natural polymers, kitosan and gelatine can be preferably used.

As stated, step A) of the invention consists in preparing a polymeric solution comprising one or more polymers in a culture medium. The culture medium is a saline solution, generally isotonic and at physiologic pH, enriched with nutrient substances for the cells, such as for example amino-acids, proteins (among which hormones) and, optionally, colorimetric indicators of pH and antibiotics, which is used for the in vitro culture of cells. Such solution is usually referred to as "culture medium" and can be purchases ready made, or can be prepared in a laboratory. There are several types of culture mediums according to the type of cell that has to be embedded, which are differentiated for the type of uses nutritive substances. Therefore, once having selected the type of cells that have to be embedded in the polymeric dispersion, the most appropriated culture medium for them will be used.

Moreover, the polymeric solution prepared in step A) of the inventive method can optionally comprise one or more bioactive molecules or pharmaceuticals suitable for promoting a particular cellular response.

Once having obtained the polymeric solution in the culture medium, the dispersion of the cells to be embedded in the polymeric solution is performed, namely step B) of the method of the present invention. The obtained cellular dispersion is then subjected to gelling through a temperature increase, such as to take the dispersion to its gel status. The operating temperature is then increases from a starting value, suitable to prepare the polymeric solution (step A) included between 1°C and 20°C, preferably between 1°C and 5°C, till the value provided in step C) , namely between 36°C and 38 °C, thereby determining the gelling of the cellular dispersion obtained in step B) .

The necessary time to obtain the gelling of the polymeric dispersion comprising the cells (step C) is generally included between 1 and 30 minutes, preferably between 5 and 15 minutes.

Finally, step D) of the invention is performed, namely extruding the cellularized hydro- gel on a thermostated support at a temperature included between 36°C and 38 °C, according to a controlled geometry. The thermostat support is composed of a plate for the cellular culture, which is kept at a temperature around 37 °C through a suitable heating system (for example, due to contact with a metallic support heated through resistances) . Keeping the temperature at a value as much as possible near 37 °C is above all functional to the viability of cells dispersed inside the hydro-gel to be printed.

Generally, step (D) of extrusion printing is repeated for a number n of times for depositing n two-dimensional layers, in order to obtain a three- dimensional cellularized construct according to the desired geometry.

Through the method proposed by the present invention, it is possible to obtain three- dimensional constructs layer by layer, without using other materials for helping the overlapping of many layers. The cellularized construct obtained through extrusion faithfully reproduced the starting computerized model and makes it possible to obtain three-dimensional structures with a simple and quick processs, without using additional materials .

Moreover, the cellularized construct obtained with the method of the invention remains stable in a physiologic medium at 37 °C for some days, even without the use of cross-linking agents (its stability depends on the chemical nature of the used material) . The cells remain viable during the process for gelling and producing the cellularized constructs as described above.

The proposed system is based on heat-sensitive hydro-gels with sol-gel transition upon increasing the temperature, since heat-sensitive hydro-gels having an inverse behaviour (sol-gel transition upon decreasing the temperature) , if processed according to a similar method, show various critical points. Firstly, the cells should ideally be embedded in a polymeric solution prepared at high temperature (>37°C), and such methodology could result aggressive for the cells. Moreover, at such temperature, it would not be possible to embed into the system heat-sensitive pharmaceuticals or growth factors. For such reason, the method of the invention specifically deals with heat-sensitive hydro-gels with sol-gel transition upon increasing the temperature.

With respect to traditional "cell printing" techniques, the main advantages of the claimed method consist in the following aspects:

1) control of obtained geometry, capability of the structures to be reproduced and correspondence between cellularized construct and starting computerized model, due to the high material viscosity in the gel phase and its reologic characteristics (shear thinning) , which lower its viscosity in the extrusion step;

2) possibility of obtaining 3D structures without using additional materials which allow overlapping the different layers embedding the cells. This implies a reduction of the preparation time for the construct and its related preparation costs. Moreover, the step of moving away the material which helps overlapping the layers is generally critical for the construct stability. Last but not least, the lack of an auxiliary material is an advantage for a future use of the method in printing the constructs directly onto the patient body (an example is given by the artificial skin case) ;

3) possibility of obtaining stable constructs in the long term, without a cross-linking agent. The use of a cross-linking process is anyway optional and compatible with the method of the present invention, and allows further extending the stability of the cellularized construct in a physiologic medium over 2 weeks;

4) possibility of embedding bioactive molecules, such as pharmaceuticals and growth factors, given the mild conditions used in the method of the invention for preparing cellularized constructs (physiologic temperature; aqueous solvents; absence of cross-linkers) ;

5) possibility of directly printing on the most exposed patient tissues (for example, on skin injuries) , due to the physiologic conditions of the printing process and the simple and quick technique being used; 6) lower costs with respect to some prior art systems, due to the absence of a refrigerating system;

7 ) greater cellular survival, due to the mild process conditions: the low shearing forces in the extrusion step do not negatively affect the cellular viability;

8) high resolution of extruded cellularized structures: the diameter of the extruded structures can drop down to 50 microns, being 200 microns the preferred value in order not to induce cellular stress. Moreover, with respect to printing of constructs in a liquid phase, due to the greater viscosity of the extruded fluid, it is possible to obtain fibres with a circular section, avoiding to flatten the filament on the printing plate. The circularity parameter computed through ImageJ software on scaffold sections with 200-μπι fibres is equal to 0.9986 +/- 0.0011.

The method of the invention finds application in the biomedical field within tissue engineering, mainly for making models of human/animal tissues for in vitro studies and for making cellularized structures for reconstructing tissues/organs with in vitro or in vivo techniques.

Another field of application is the possibility of performing a printing of the construct directly onto the injuried tissue (for example, injuried skin) . In such application, it is possible to manufacture "customized" cellularized constructs with a geometry suitable for the end use and the patient needs, due to a quick method which minimizes the handling of the biologic component (cells) .

The described method could be particularly advantageous for treating severe burns. Burnt patients, in fact, can be subjected to deadly infections unless timely treated (as an average within two weeks) . The current treatment consists in implanting heterologous skin (skin graft) , but this treatment is painful and implies the formation of scars. The direct printing technique onto the patient has the advantage of being able to be timely applied, avoiding the danger of having potentially deadly infections, and is a promise for the regeneration of a completely functional skin.

Claims

1. Method for preparing cellularized constructs through quick prototyping comprising the following steps :

A) preparing a polymeric solution comprising one or more natural and/or synthetic polymers in a culture medium at a temperature included between 1°C and 20°C;

B) dispersing cells inside the polymeric solution prepared in step A) ;

C) gelling the polymeric dispersion containing said cells increasing the temperature up to a value included between 36°C and 38°C to obtain a cellularized hydro-gel, the gelling step C) requiring a time included between 1 and 30 minutes;

D) extruding the cellularized hydro-gel through a printing nozzle on a thermostated support at a temperature included between 36°C and 38 °C, said polymeric solution prepared in step A) being subjected to a transition from the "sol" phase to the "gel" phase which occurs upon increasing the temperature, said transition from the "sol" phase to the "gel" phase occuring at a temperature included between 20°C and 30°C.

2. Method according to claim 1, characterized in that said synthetic polymers are chosen among poloxamers, polyurethanes and polyester-based amphiphilic copolymers.

3. Method according to claim 1, characterized in that said natural polymers are chosen among kitosan and gelatine.

4. Method according to claim 1, characterized in that the preparation of the polymeric solution in step A) occurs at a temperature included between 1°C and 5°C.

5. Method according to any one of the previous claims, characterized in that said culture medium is an isotonic saline solution and at physiologic pH enriched with nutrient substances for the cells.

6. Method according to any one of the previous claims, wherein said polymeric solution of step A) comprises one or more pharmaceuticals suitable for promoting the cellular survival.

7. Method according to claim 1, characterized in that said thermostated support of step D) is a plate for the cellular culture heated at a temperature included between 36°C and 38°C.

8. Method according to claim 1, characterized in that step (D) is repeated for a number n of times for depositing n bidimensional layers to obtain a three-dimensional cellularized construct according to a desired geometry.