ITMI20082037A1 - HYDROGEL SUITABLE FOR CONTAINING AND CARRYING CELLS - Google Patents

Description

Description of the patent for industrial invention entitled:

"HYDROGEL SUITABLE TO CONTAIN AND VEHICLE CELLS" POLITECNICO DI MILANO

The present invention relates to a hydrogel with a double interpenetrating polymeric matrix formed by a polymer of acrylic or carbomeric acid, a natural polymer, a cross-linking agent, said hydrogel containing cells resting on its external surface or housed within the three-dimensional network formed from said polymeric matrix.

BACKGROUND OF THE INVENTION

In the treatment of many diseases that lead to the degeneration of specific organs, the only possible therapeutic alternative is often represented by organ transplantation. Where possible, however, it is associated with a high degree of risk for the patient, since complications are possible such as the transmission of pathologies from donor to recipient or rejection of the organ itself, combined with the chronic shortage of available organs. Furthermore, this type of approach cannot be applied to all areas of the body subject to degenerative pathologies. In fact, to date there is no possibility of applying transplantation therapies in many degenerative pathologies such as, in some cases, those affecting some districts with connective tissues, those of bone tissue, dermis and epidermis, as well as of the system central nervous.

An emblematic and well known case is that of pathologies related to myocardial infarction. In fact, they are a common cause of death and decrease in the quality of life all over the world. Following a heart attack, the myocardium suffers more or less extensive damage that leads to the loss of functionality of the heart muscle and the formation of necrotizing and non-functional tissue. When death is an avoided consequence, chronic heart failure, at different levels of severity, is a certain prospect. At present, effective pharmacological treatments are not yet available for the most serious stages of this disease and therefore transplantation is often the last therapeutic option but unfortunately it is not always punishable. For less severe cases, the treatments available only allow a maintenance of the clinical status quo. In fact, no therapies are available that lead to significant improvement and recovery for the myocardium [see in this regard the monograph J.O. Mudd, D.A. Kass, Nature, volume 451: pp. 919-928, 2008]. The possibility of injecting specific healthy cells into the infarcted areas in order to induce their regeneration is emerging as the way with the greatest chance of success [as is evident for example in the following monographs: M.N. Giraud et al., Tissue Eng 13 (8): 1825-1836; 2008. V.F.M. Segers, R.T. Lee, Nature 451: 937-942; 2008. S.J. Dimmeler et al., Arterioscl. Thromb. Vasc. Biol. 28: 208-216, 2008].

The lower level of cellular specialization in tissues with a strong connective component represents a perhaps better situation in some ways since there are some possibilities of creating artificial two- and three-dimensional structures capable of hosting and carrying specific cells for the functional reintegration of damaged structures. These artificial devices, also known as scaffolds, represent one of the pillars of tissue engineering but are certainly not free from problems [see for example what is reported in the monograph C.H. Evans et al., Tissue Engineering, Volume 13 (8), pp. 1987-1993, 2007]. In this context, a well-known type of damage concerns those affecting the cartilages, especially the articular ones. Cartilage tissue is a particular type of connective tissue characterized by remarkable qualities of resistance and elasticity, which plays a structural support role within the body. It is a particular type of connective tissue and, as such, consists of cells dispersed in an abundant gelatinous extracellular matrix, rich in fibers (responsible for elasticity) and amorphous substance of protein origin. The articular cartilages are those that line the surfaces of the joints and the ones most known to be prone to trauma are those of the knee. Certainly these are not fatal diseases but they are annoying and can reduce the quality of life and be disabling. The surgical techniques developed essentially aim to reduce the defect by eliminating the injured area but this exposes the joint to greater friction and greater wear over time. There are no substantially effective drug therapies. Regenerative medicine is therefore seen as the frontier for the treatment of these pathologies, especially those of traumatic origin. Carrying chondrocytes and keeping them alive in situ is the basic setting of therapies under development which are mainly based on two similar approaches: a) to bring the cells through the use of solid planar matrices of the shape of the defect to be integrated, b ) bring the cells through gels that are injected until the defect to be integrated is filled. In both cases, the solutions favor the use of biodegradable materials in order to better promote integration [as demonstrated for example in the following monographs: Jorgensen C et al., Best Practice & Research In Clinical Rheumatology 22 (2), Pgg 269-284, 2008. Grayson WL et al., Trends In Biotechnology, (26) 4, Pgg. 181-189, 2008].

Similarly, degenerative pathologies affecting the bone tissue can result in particularly severe structural fragility and the main therapies are pharmacological ones which, however, do not always guarantee efficacy and are not without serious side effects. However, the tissue engineering sector is also bringing new perspectives to bone regeneration [as can be seen from the observations reported in the following work: Waese EYL, Kandel RR, Stanford WL, Skeletal Radiology 37 (7), Pgg. 601-608, 2008]. In this context the fundamental role is played by the mechanical properties of the materials used for the realization of the scaffolds, which are the most usable devices [as reported in numerous monographic works among which we can mention the following: Stevens MM, Materials Today 11 (5) , Pgg. 18-25, 2008. Barrere F et al., Materials Science & Engineering R-Reports, 59 (1-6), Pgg. 38-71, 2008]. For the sake of completeness, it should also be remembered that in the bone, tendon and cartilage regenerative field there are numerous studies that see the use of high porosity scaffolds imbued with hydrogel-based matrices containing the cells in turn [see in this regard, for for example, the extensive bibliography cited in US patent US6171610].

On a general level, briefly, the scientific literature basically identifies four possible ways for tissue regeneration:

a) the stimulation of endogenous autonomous regeneration mechanisms - an approach based on still not solid foundations and with procedures not yet well identified, on the whole currently considered unpromising; b) the transplantation of isolated cells directly into the damaged site - empirical approach based on a good functional principle but which brings with it numerous disadvantages and problems such as high cell loss and consequent low integration with adjacent tissue;

c) tissue transplantation generated in vitro (also called graft) - empirical approach based on a good tissue principle but with considerable complexity since the graft must integrate better with the adjacent tissue, be able to withstand any mechanical stresses to which it must be subjected once in vivo and possibly have an adequate bioabsorbability characteristic with the target tissue;

d) the use of advanced micro and nano structured materials that are able to host specific cell populations providing a compatible and favorable environment and that can subsequently be used as a vehicle for implantation - a futuristic approach based on rational principles but which still needs a full understanding of all the phenomena involved.

Whatever the approach pursued, the procedures available today often allow the achievement of only a partial reconstruction and often not even that. The reasons for this limited success are many and mainly to be sought in the cellular aspects and in the lack of adequate physical support for the cells thus transplanted, or injected, in the time lapse between implantation and integration with adjacent tissues. . Furthermore, in the absence of adequate support permeable to nutrients, the cells are unable to supply themselves with the nutrients themselves necessary for their survival.

The previous reasons are leading the research to move towards the development of support matrices that allow the transport of cells, increasing their survival and integration with the target tissue, in order to give rise to a new functional and histologically integrated tissue. with adjacent structures.

In light of the above, in addition to cells, an essential element in tissue engineering is therefore represented by the materials used, among these the hydrogels play a leading role being particularly useful as systems for supporting and conveying cells and cell populations. In some cases the requests for mechanical functionality make the use of scaffolds essential but very often the target tissues require to be flooded with a cellular flow that is stable and allows its reintegration and this can be easily achieved with the use of special hydrogels. . As mentioned, in some cases it is also possible to use mixed solutions in which solid scaffolds are soaked in hydrogels containing or supporting the cells.

Hydrogels are three-dimensional polymeric structures capable of swelling by retaining water, or other liquids, inside them. The polymer chains are linked together through intermolecular bonds which can be of different nature. Hydrogels can be produced in numerous ways, one of which consists in the reaction of one or more monomers or thanks to the association of hydrogen bridges or strong van der Waals interactions between polymer chains. Depending on the components used, the hydrogels can be biodegradable or biostable, biocompatible or cytotoxic. Many biocompatible, biodegradable and non-biodegradable hydrogels are gaining considerable attention from the scientific-technical world thanks to their intrinsic characteristics that make them particularly suitable and ideal for many applications in the biomedical field.

From a strictly chemical point of view, hydrogels can be classified in different ways, based on their method of preparation, their distributed ionic charge or their physical structure. Regarding the method of preparation, hydrogels can be classified as homopolymer, copolymer, multipolymer or interpenetrating polymer. The former consist of polymeric networks linked together by a monomer unit which must be hydrophilic. The latter consist of two comonomers, at least one of which must be hydrophilic. The multipolymers are obtained starting from three or more inter-reactive comonomers, of which at least one is hydrophilic. Finally, the interpenetrating polymers are obtained by swelling a first network around which a three-dimensional structure is subsequently formed.

The chemical and physical properties of the hydrogels therefore make them particularly suitable for use in the biomedical and pharmaceutical fields. In particular, their biocompatibility is the first fundamental requirement for such applications. Their hydrophilicity can guarantee excellent characteristics in terms of permeability to gases and other substances, also allowing the controlled release of active ingredients and supporting the presence of cell populations within them or on their surface. One of the first applications developed, still massively widespread today, is related to the manufacture of contact lenses where hydrogels are used thanks to their good mechanical stability, favorable refractive index and high oxygen permeability. Other applications, such as those described in this application, include the use of hydrogels as bio-adhesive artificial materials, artificial membranes, joint cartilages, artificial skin, materials for maxillofacial reconstruction and vocal cords.

It should be emphasized that hydrogels are also widely used as a support for in vitro cell growth in drug screening, drug development assay, pharmacological efficacy or toxicity applications or for other electrophysiological or biomechanical characterizations. Some of these assays are conducted through devices also known as bioreactors.

STATE OF THE TECHNIQUE

The technical-scientific literature available regarding materials, including hydrogels, and their use in the regenerative field is very vast and in this broad panorama, hydrogels are known per se and are often described as means for controlled release. of drugs (drug-delivery) and as supports for tissue engineering [as can be seen from the monographic reviews reported below by way of example: J.D. Kretlow et al., Advanced Drug Delivery Reviews 59 pp.

263–273, 2007. L. Yu et al., Tutorial Review on Chemical Society Reviews, 2008].

The preparation of various anionic gels for controlled drug release was described in 1986 by Hsu et al. in Pharmaceutical Research (1996) vol. 13 pp. 1865-1870. In particular, the preparation of a two-component hydrophilic gel consisting of a formulation of agarose and carbomer is described. This particular gel is prepared by mixing an aqueous carbomer dispersion with an agarose dispersion preheated to a certain temperature. In particular, however, neither the addition of cross-linking compounds, capable of modifying the mechanical properties of the polymer at the base of the hydrogel, nor the addition of buffering substances capable of neutralizing the acidity that is released during the gel formation process due to cross-linking reactions, nor the addition of elasticizing compounds. Furthermore, no mention is made of the possibility of applying this gel to the engineering of living tissues, nor does it provide for other specific applications other than the release of drugs. Moreover, the formulation described does not contain references to the addition of culture media and / or factors or active ingredients suitable for providing the cells with the necessary nourishment and modulating their action and activity.

European patent EP929323 describes a biostable (and therefore non-biodegradable) polymeric hydrogel for therapeutic use, consisting of a copolymer of (a) an N-substituted acrylamide or methacrylamide, (b) a crosslinking agent and (c) copolymerizable material, usable to rebuild damaged brain or spinal tissue.

The European patent EP1206254 describes a three-dimensional matrix based on absorbable fibers loaded with specific drugs, in which cells are grown in vitro and then implanted in vivo.

Patent US6171610 is also cited, where formulations obtained by mixing biocompatible substances and their applications extended to all possible cell populations and in particular to central nervous tissue are described. Neither the addition of cross-linking compounds, capable of modifying the mechanical properties of the polymers at the base of the hydrogel, nor the addition of buffering substances capable of neutralizing the acidity that is released during the gel formation process are mentioned. and during the subsequent degradation, nor the addition of elasticizing compounds. In the case of mixtures between agarose and carbomer, for example, the use of cross-linking agents and acidity buffers is not only essential for controlling the gelation but also for obtaining an adequate matrix to host the specific selected cells and to convey them keeping them vital and thus guaranteeing their survival and therapeutic efficacy once implanted in situ. It is still necessary to specify that the polymeric matrix, obtained in gel form, will be all the more effective in allowing the support, vitality and cell proliferation within it, the more the conformation of the structure matrix of the gel itself adapts to that of the extracellular matrix. that cells expect.

In summary, the previous analysis shows that hydrogels mainly based on carbomer and agarose are not described, whose mechanical properties, and consequently also their biodegradability, are modulated by the addition of cross-linking compounds such as propylene glycol, glycerol and triethyl amine, containing cellular nutrients, specific factors, active ingredients and / or drugs and used as means of supporting and conveying cells for tissue regeneration and reconstruction applications.

DESCRIPTION OF THE INVENTION

The purpose of the present invention is to provide a polymeric matrix based on biodegradable compounds that can be used in its swollen state as a device for housing cells of different types for the regeneration and / or reconstruction and / or replacement of various damaged living tissues, dead or no longer functional.

This purpose is achieved through a double interpenetrating polymeric matrix hydrogel formed by an acrylic acid polymer or a carbomer, a natural polymer selected from agarose, chitosan, dextran or a glycosaminoglycan, a crosslinking agent selected from glycerol, propylene glycol, ethylene , triethylamine or a mixture thereof and optionally an elasticizing agent selected from alginic acid and its salts, any type of collagen, fibrin, fibrinogen and hyaluronic acid or a derivative thereof, such as for example gelatin, laminin, or a mixture of said agents , said hydrogel containing cells resting on its external surface or housed inside the three-dimensional polymeric matrix.

Another aspect of the present invention relates to the use of said hydrogel for the preparation of a cellular implant for the regeneration or reconstitution or replacement of damaged or dead or removed or non-functioning tissues, even if only partially. It is in fact possible through the present invention to improve and cure the tissue in the area of said implant by supplying cells and controlling their proliferation, infiltration and integration with the tissues surrounding the implant area itself.

Another object of the present invention is to provide a means for tissue regeneration in different parts of the body through the use of a biodegradable polymeric matrix which is particularly advantageous both from a clinical and economic point of view for patients suffering from degenerative pathologies, traumatic outcomes, of resections or other pathologies.

Another object of the present invention is to provide a means for regenerating various living tissues with characteristics such that it can be easily positioned in the target area through traditional or minimally invasive surgical techniques and which can be easily adapted to the size of the implantation site.

Another aspect of the present invention is a method for preparing said hydrogel, which comprises the preparation of an aqueous dispersion containing (% by weight) from 0.01% to 5% of acrylic polymer or of a carbomer, from 0.01% with 5% of natural polymer, from 0.1% to 40% of cross-linking agent and possibly from 0.01% to 5% of elasticizing agent, at a temperature of 20-90 ° C and for a time ranging from 10 minutes seconds at 240 minutes, depending on the degree of crosslinking to be obtained and the polymers used, and any subsequent cooling to a temperature between 37 ° C and room temperature. A base capable of neutralizing the acid that develops during the reaction can be added to said mixture of polymers, preferably selected from triethylamine and ammonia. The weight ratios between the various components can be varied in the ranges defined above, thus being able to modulate the size of the meshes of the polymeric network according to the cell type to be conveyed to make the matrix more suitable for each specific population. Preferably the polymers and all the reagents used meet the purity and sterility criteria required for medical, biological, biomedical and pharmaceutical applications.

It is another purpose of the present invention to provide a new polymeric mixture with the appropriate chemical-physical characteristics so that living cells can be mixed inside it, guaranteeing them the possibility of surviving and proliferating.

Another aspect of the invention is therefore a method for making a two-dimensional or three-dimensional bio-hybrid device that can be used for housing and conveying cells for the regeneration of living tissues within the human body. A further fundamental feature is that this device degrades with a predetermined and compatible kinetics with the integration between the conveyed cells and the target tissue.

Another aspect of the present invention relates to a method for preparing a device based on said cell-containing hydrogel. These cells can be taken from fresh tissue or kept in culture detached from the support (for example Petri dish or other), possibly washed and subsequently added to the mixture of polymers, cross-linking agent and eventual elasticizing agent during the gelation process, preferably in the phase of cooling down. To keep the largest number of cells trapped in the hydrogel matrix in a viable state and under growth conditions, it is preferable to add a suitable culture medium to the aqueous dispersion of the polymers before gelling. Alternatively, the cells trapped in the polymer matrix can be supplied with nutrients and factors necessary for their growth by contacting or immersing the hydrogel in a suitable culture medium. Furthermore, during the preparation of the gel, drugs and / or active ingredients and / or specific factors and / or other substances can be added that are able to modulate the specific action of the cells, also based on the target tissue to be regenerated, and which they are gradually released from the polymer matrix once the implant has been introduced into the target tissue.

Another aspect of the present invention is the use of a hydrogel that has a high capacity to swell, to incorporate a mixture mainly based on water and a culture medium possibly added with factors and / or other active ingredients, said mixture being suitable to provide the cells hosted with the necessary nourishment so that they can survive and proliferate within the hydrogel itself.

Another aspect of the present invention is the use of a suitable culture medium aimed at improving the vital conditions of the cells once encapsulated in the gel. This medium can be used directly for making the gel with or without the addition of a saline buffer solution.

Different types of cells can be used for the preparation of said bio-hybrid device according to the invention, such as adult, embryonic, stem, wild-type, engineered, primary or immortalized (line) cells.

In an embodiment of the present invention, the hydrogel possesses adequate chemical-physical characteristics to be produced in the form of a thin film by means of one of the methods known to the state of the art such as, by way of non-exhaustive example, the spincoating from melt with subsequent cooling, the dip-coating from melt with subsequent cooling or the sol-gel. The hydrogel thus obtained has a thickness in the order of the size of the cells to be housed (from a few micrometers to a few tenths of a millimeter).

Another aspect of the present invention relates to the use of said hydrogel produced in the form of a thin film as a support for cell growth in vitro in applications of drug screening, assay for drug development, pharmacological efficacy or toxicity. This support is made as a thin film on which or within which single cells or cell populations, even different ones, are deposited and grown. This support is also realized as a connection substrate between cells and measuring devices specially made to monitor the cellular activity of single cells or cell populations during different types of tests ranging from monitoring the conductivity of signals (for example for excitable cells such as the cardiac or nervous ones), to drug efficacy tests. This thin film of polymeric hydrogel must be specially designed and manufactured in order to allow the passage of electrical and chemical signals. The high porosity of the hydrogel structure combined with the presence of electrolytes in the aqueous solution allows to obtain high values of electrical conductivity (comparable to those measured in the solution itself) and ion diffusion.

In a preferred embodiment of the invention, the hydrogel is obtained starting from a mixture containing carbopol, agarose, glycerol and propylene glycol, in the presence of triethylamine, dispersed in a suitable aqueous medium with the addition of a mixture of nutrients such as glucose, essential amino acids, proteins, trace elements, mineral salts, vitamins and bactericidal antibiotics. This hydrogel containing the cells can be placed for convenience inside wells suitable for cell cultures. The cells encapsulated in the three-dimensional matrix remain viable and continue to grow and aggregate, as has been observed in the course of experiments in which the morphology, viability and cell proliferation within a hydrogel obtained as described above was evaluated.

In another preferred embodiment of the invention, the hydrogel is obtained from a mixture containing carbopol, agarose, glycerol and propylene glycol, in the presence of triethylamine, dispersed in a suitable aqueous medium. This hydrogel is inside a hollow circular support and subjected to rotation at such a speed as to allow obtaining a thin and regular film of the same once cooled. Subsequently on this hydrogel film, once removed from the support and placed in a microelectronic device, a liquid dispersion containing cells is deposited. Similarly, the cells can be positioned directly inside the mixture and then produced with the described method a thin film containing the cells themselves.

A further aspect of the invention relates to the use of a hydrogel as described above, in any of its embodiments, for the preparation of an implantation of cells for the regeneration or reconstitution, integral or partial, of damaged or diseased tissues or removed, in particular muscle, myocardial, connective, bone, tendon or ligamentous, hepatic, renal, corneal, dermal or epidermal tissue, articular cartilage.

EXAMPLES

The following examples illustrate the invention in greater detail. EXAMPLE 1 - Preparation of the hydrogel with two different possible concentrations and with myoblast-type cells (C2C12 line) to be inserted inside

The procedure described below leads to the formation of a hydrogel with a percentage of carbomer of 0.5% and of agarose of 0.5% or 0.25% and with myoblasts inside.

Reagents required for the preparation of 100 mL of gel with neuronal cells:

- Carbomer 974 P (CAS 151687-96-6, Fagron, Bufa, The Netherlands):

0.5 g

- Agarose (CAS 9012-36-6, Invitrogen Corp., Carlsbad, USA): (0.5 g or 0.25 g)

- Propylene glycol (CAS 504-63-2, Sigma-Aldrich, Germany):

30 mL

- Glycerol (CAS 56-81-5, Merck Chemicals, Germany): 1 mL

- Triethylamine (CAS 121-44-8, high purity preparation, Sigma-Aldrich, Germany): 0.5 mL

- PBS: just enough

- Cells: C2C12 (myoblasts)

- Culture medium: DMEM (Dulbecco's Modified Eagle's Medium, Sigma-Aldrich Germany)

Operative procedure for cell preparation. This procedure begins with the following operations of thawing the cells from the cryopreservative solution, to be carried out entirely under a sterile class II hood:

1. take the necessary amount of cryopreserved cells in their criovial;

2. take 10 mL of culture medium and transfer it to a 50 mL tube keeping it at T = 37 ° C;

3. take 1 mL of medium from the tube and transfer it to the criovial containing the cryopreserved cells and mix with the pipette gently to dilute the cryopreservant in contact with the cells in order to obtain complete thawing;

4. Once complete thawing has been achieved, transfer the entire solution with the cell suspension from the cryovial to the tube containing the medium and continue to suspend the cells again in the tube with the culture medium;

5. transfer the tube with the cells into the centrifuge and centrifuge at 2000 rpm for 5 minutes;

6. remove the culture medium from the tube, leaving the cells at the bottom, then add 1 mL of fresh culture medium and resuspend the cells;

7. perform the cell count (for example with a Neubauer chamber) and then take a volume of suspension that contains the number of cells to be seeded in the flask and transfer it to it; in this case for a concentration of 100,000 units / mL;

8. leave the flask inside a special cell incubator for the time necessary for the cells to reach sufficient confluence.

Operating procedure for the preparation of the gel, under a class II sterile hood:

1. in a Becker pyrex weigh the carbomer and add the respective quantity of PBS by stirring strongly (1400 rpm, with stir bar) for 30 minutes at room temperature (T = 25 ° C); 2. add glycerol and propylene glycol, stirring at the same speed for a further 20 minutes, always at room temperature;

leave the suspension without shaking for 60 minutes at room temperature;

To neutralize the pH of the solution (if necessary) add q.b. a 98% solution of triethylamine in water, stirring slowly for 10 minutes under slow stirring (250 rpm, with stir bar);

hexare and add the powdered agarose based on the previously set concentration and leave under stirring (250 rpm, with stir bar) for 5 minutes at room temperature;

heat the solution with electromagnetic radiation (500W) for about 2 minutes until reaching a temperature of about 80 ° C;

extracted the solution, in the cooling phase, at about 40 ° C, add the previously prepared solution containing the cells, in a volumetric ratio of 1: 1, keeping the new solution thus obtained in slight rotation;

detect from the final solution of gel and cells the quantity necessary to fill the wells from cell cultures used which will be filled for 50% of their total volume from the final solution of gel and cells (applicable standard wells are those of the "multiwell" type plates from 48 wells each), fill the remaining volume with the culture medium suitable for the type of cells in use (supernatant medium);

9. to maintain the gel with the cells inside, it is necessary to place the samples in a special cell culture incubator and replace the supernatant medium every 24 hours with a new one. Operating procedure for cell activity assays whose purpose is to evaluate the morphology, viability and cell proliferation of cells that have been encapsulated within a hydrogel constituted as described in the previous procedures.

In order to evaluate cell viability, on a daily basis perform the MTT (Sigma-Aldrich, Germany) and Alamar Blue ™ (AbD Serotec, Great Britain) assays on the samples prepared according to the procedures described above. A visible comparison relating to cell morphology, on the other hand, can be obtained by optical and / or scanning electron microscopy.

A confirmation of the state of viability and cell proliferation can be obtained by performing the aforementioned assays on the cells once they have been extracted from the gel. This operation is configured as destructive for the gel but not significantly for the cells and taking care to design a sufficiently large experimental set it can be performed at different times. This extraction can be obtained with the following operating procedure, to be performed under a class II sterile hood:

1. recover the supernatant of the wells containing the samples and transfer it to a 50 ml tube and bring it to 30 ml by adding q.b. of solution of PBS and SSPE 1X (Sigma-Aldrich, Germany) and carry out a first centrifugation at 2000 rpm for 5 minutes;

2. at the end of centrifugation, remove the supernatant and suspend again with 30 mL of the previously described 1X PBS / SSPE solution to which 0.5 mL of 1X trypsin-EDTA solution (Sigma-Aldrich, Germany; EDTA = Ethylene Diamine Triacetic must be added Acid, ethylene-diamino-triacetic acid);

in the wells where the samples are located, add 0.5 ml of the previously described solution of PBS / SSPE 1X to which 0.5 ml of the previously described trypsin-EDTA 1X solution must be added and place in a suitable incubator for at least 5 minutes;

after incubation, recover the entire contents of the wells and transfer it to the same 50 mL tube previously used, then perform a second centrifugation at 2000 rpm for 5 minutes;

at the end of the centrifugation, recover the supernatant in a second 50 mL tube and suspend again what remained in the first tube with 20 mL of the previously described solution of PBS / SSPE 1X to which 1 mL of the previously described trypsin-EDTA 1X solution must be added;

perform a third centrifugation of both 50 mL tubes at 2000 rpm for 5 minutes;

at the end of the centrifugation, remove the supernatant from both tubes, suspend what in both tubes with 20 mL of the previously described 1X PBS / SSPE solution;

combine the contents of the two tubes into a single tube and subject it to a fourth centrifugation at 2000 rpm for 5 minutes;

9. from the end of centrifugation, remove the supernatant and suspend in a suitable medium;

10. remove the suspension from the tube and transfer it to the appropriate flask for storage in an appropriate incubator.

EXAMPLE 2 - preparation of the hydrogel with two different possible concentrations and with cells of the P19 line, phenotype CL6 (i.e. contractile cardiomyocytes of embryonic stem derivation) to be inserted inside it and with a specific culture medium for this population Proceed as in the procedures described in the previous example where Alpha-MEM (Invitrogen, USA) is used instead of the DMEM culture medium.

EXAMPLE 3 - preparation of the hydrogel with two different possible concentrations and with cells of the N9 line (microglia) to be inserted inside it and with a specific culture medium for this population Proceed as in the procedures described in the previous example nr 1, where instead of the DMEM culture medium, the following medium is used, obtained as a solution specifically designed to favor these cells inside the gel; quantity of reagents proportioned for 20 mL of solution:

- PBS (Sigma, Germany) 18.790 mL - B27 (GIBCO, Invitrogen, USA) 400 µL - Insulin-Transferrin-Selenium-G Supplement, 100X

(GIBCO, Invitrogen, USA) [dilution 1: 100] 10 µL - D - (+) Glucose @ 45% (Sigma G8769, Germany) 80 µL - L – Glutamine (Sigma G7513, Germany) 200 µL - Penicillin-Streptomycin ( Sigma P0781, Germany) 60 µL - Sodium pyruvate (Sigma S8636, Germany) 200 µL - MEM Vitamins 100X (Sigma M6895, Germany) 20 µL - MEM Aminoacids 50X (Sigma M5550, Germany) 20 µL - MEM Non-Aminoacids 100X NEAA ( Sigma M7145, Germany) 20 µL - HEPES Buffer (Sigma H0887, Germany) 200 µL The reagents are then added in sodium bicarbonate and the solution is diluted 1: 100 and taken in aliquots of what is necessary.

EXAMPLE 4 - preparation of the hydrogel with two different possible concentrations, with an elasticizing agent and cells of type L929 (fibroblasts) to be inserted inside

Proceed as in the procedures described in the previous example no. 1 where, however, the PBS used for the preparation of the gel is added with 0.5% by weight of gelatin (Sigma-Aldrich, Germany).

EXAMPLE 5 - preparation of hydrogel with two different possible concentrations, with the addition of specific factors for cell populations of the central nervous system or for myocardial cells

Proceed as in the procedures described in the previous example no. 1 where, however, the culture medium is that described in example no. 3 and is added with carnitine (alcar) in a concentration range between 30 and 100 millimolar to better favor the cell populations derived from the central nervous system, or alternatively the said culture medium is added with vitamin D in one of its variants (such as cholecalciferol by way of non-exhaustive example) at a concentration of about 0.0004 g / ml to better favor cardiomyocytic cell populations.

Claims (25)

CLAIMS 1. Hydrogel with double interpenetrating polymeric matrix formed by a polymer of acrylic or carbomeric acid, a natural polymer selected from agarose, chitosan, dextran, a polysaccharide or a glycosaminoglycan, a cross-linking agent selected from glycerol, propylene glycol, ethylene, triethylamine or a mixture thereof, called hydrogel containing cells resting on its external surface or housed inside the three-dimensional lattice formed by said polymeric matrix. 2. Hydrogel according to claim 1, wherein said polymeric matrix further contains an elasticizing agent selected from alginic acid and its salts, collagen or its derivatives, gelatin, fibrin, fibrinogen, hyaluronic acid and its derivatives, laminin or a mixture of the aforesaid agents . 3. Hydrogel according to claims 1 or 2, wherein said polymeric matrix comprises from 0.01% to 5% of acrylic or carbomeric acid polymer, from 0.01% to 5% of natural polymer, from 0.1% to 40% of crosslinking agent, and optionally from 0.01% to 5% of elasticizing agent, where the aforesaid% are by weight. 4. Hydrogel according to claims 1 and 2, wherein said acrylic polymer is carbopol, said natural polymer is agarose, said crosslinking agent is a mixture of glycerol, propylene glycol and triethylamine and said elasticizing agent is gelatin. 5. Hydrogel according to claims 1-4, wherein said cells are adult, stem, embryonic, wild-type, engineered, primary or immortalized (line) cells. 6. Hydrogel according to claims 1-5, further comprising nutrients and / or growth factors for cells, preferably selected from the following list: saline buffer, B27 (GIBCO, Invitrogen, USA), Insulin-Transferrin-Selenium-G Supplement, D - (+) Glucose, L – Glutamine, Penicillin-Streptomycin, Sodium pyruvate, MEM Vitamins 100X, MEM Aminoacids 50X, MEM Non-Aminoacids 100X NEAA, HEPES Buffer, bicarbonate, carnitine (Alcar), vitamin D, l-arginine. 7. Hydrogel according to claim 1-6, further comprising one or more drugs and / or factors and / or active ingredients capable of modulating the action of the cells and / or keeping them vital and / or stimulating their metabolism and / or to stimulate their growth and development and / or to stimulate their differentiation and / or to stimulate their interaction and mutual interconnectivity. 8. Hydrogel according to the preceding claims, in the form of a thin film suitable for supporting cells for use in measuring devices specially made for monitoring various parameters of cellular activity. 9. Hydrogel according to the preceding claims, in the form of a set of thin films arranged on several superimposed layers and suitable for supporting and hosting different populations of cells in order to obtain an advanced polyfunctional structure (Multi Layer Systems). 10. Method for the preparation of a hydrogel according to the preceding claims, which comprises the preparation of a mixture containing (% by weight) from 0.01% to 5% of acrylic polymer, from 0.01% to 5% of natural polymer , from 0.1% to 40% of cross-linking agent in an aqueous medium, at a temperature between 20 ° C and 90 ° C, for a time ranging from 10 minutes seconds to 240 minutes, and any subsequent cooling until to reach a temperature between room temperature and 37 ° C. 11. Method according to claim 10, which further comprises the addition of a base capable of neutralizing the acid which develops during the reaction and the addition of suitable salts or saline solutions or buffer solutions aimed at controlling the pH. . The method according to claim 11, wherein said base is triethylamine and the buffer solution is PBS. Method according to claims 10-12, wherein the aqueous mixture is added or replaced with a culture medium suitable for maintaining the cells in vital and growth conditions containing the nutrients or growth factors according to claim 6. 14. Method according to claim 8 or 13, in which one or more drugs and / or factors and / or active ingredients capable of modulating the action of the cells and / or keeping them viable are added to the aqueous mixture or culture medium and / or to stimulate its metabolism and / or to stimulate its growth and development and / or to stimulate its differentiation and / or to stimulate its interaction and mutual interconnectivity. 15. Use of a hydrogel according to claims 1-9 for the preparation of a cell implant for regeneration or reconstitution, integral or partial, of damaged or diseased or removed tissues. 16. Use according to claim 15, where said tissue is muscular or myocardial. 17. Use according to claim 15, where said tissue is connective. 18. Use according to claim 15, wherein said tissue is an articular cartilage. 19. Use according to claim 15, where said tissue is bone. 20. Use according to claim 15, where said tissue is tendon or ligamentous. 21. Use according to claim 15, where said tissue is corneal. Use according to claim 15, where said tissue is dermis or epidermis. 23. Use according to claim 15, where said tissue is hepatic or renal. 24. Use of a hydrogel according to claims 1-9 as an interface between cells and electronic devices. Use of a hydrogel according to claim 8, where different cell activity parameters are monitored during drug screening tests or drug development tests or drug efficacy tests or toxicity tests. Milan, November 17, 2008