WO2011098497A1

WO2011098497A1 - Zeolite membrane for cellular adhesion, growth and cultures and process for preparation thereof

Info

Publication number: WO2011098497A1
Application number: PCT/EP2011/051915
Authority: WO
Inventors: Palmira Tavolaro; Guglielmo Martino
Original assignee: Universita della Calabria
Current assignee: Universita della Calabria
Priority date: 2010-02-09
Filing date: 2011-02-09
Publication date: 2011-08-18
Anticipated expiration: 2012-08-09
Also published as: ITCS20100003A1; EP2533882A1

Abstract

The invention relates to an auto - supported zeolite membrane, its process for preparation and its application in cellular adhesions and cultures. Pore and cavity sizes of this zeolite membrane are in the range between 20 Å and 10 μm. The zeolite membrane preparation consists in pressing a microporous layer of calcined or ozonized or ground or as made pure structure zeolite crystals. To obtain an ideal antimicrobial and antimycotic scaffold the physico-chemical characteristics of the zeolite membrane surface and framework can be modulated. The applicability of this membrane to the cellular adhesion and growth is demonstrated.

Description

ZEOLITE MEMBRANE FOR CELLULAR ADHESION, GROWTH AND CULTURES AND PROCESS FOR PREPARATION THEREOF

FIELD OF THE INVENTION

The present invention relates to a novel zeolite membrane with a silicoaluminate crystalline structure containing alkali metal and/or alkaline earth characterized by the fact that is auto-supported, that it is formed by at least one, pure and homogeneous layer of zeolite crystals having a size inferior to 100 μιη and containing intercrystalline voids, pores and hollow cages in the range between 20 A and 10 μιη.

zeolite crystals are preformed and no chemically modified and they are smaller than 100 microns and are with or without a template species or are calcined. The physical- chemical superficial and internal characteristics of this membrane, with particular reference to the point of zero charge (PZC), are unique and modulate for treatment with acidic and basic solutions, ionic and/or organometallic and/or functionalizing species, and make it ideal for the application subject of this invention.

The present invention also covers the process for the preparation of this membrane that is characterized by the fact that includes the following steps: the zeolite layer is prepared using preformed and no chemically modified zeolite crystals (as-made and/or ozonized and/or calcined, pure and/or solid mixture and/or pre-treated with appropriate reagents and solvents and/or mixed with organometallic and/or inorganic species); a layer is spread with the desired thickness; the layer is first pressed with a pressure higher than 2 kg/cm² and then is subjected to a vacuum lower than 20 mm Hg.

In order to obtain a good quality membrane the preparation process can be repeated.

The zeolite membrane is indissoluble and non-disintegrable either by contact or by immersion in polar and/or non polar solvents and/or in a buffer solution.

The membrane covered by the present invention, because of its antimicrobial and / or antifungal characteristics is suitable as an excellent support for the cultivation of a single type or variety of cellular types and prokaryotic or eukaryotic, animals and / or vegetable , and aerobic or anaerobic, and differentiated or undifferentiated and / or modified cells. The zeolite membrane herein is suitable in cell separations and/or in special applications regarding the membranes or films uses as biomaterials for implantation tissue, organ and systems and/ or carrier for the introduction of peculiar cells inside the body. This zeolite membrane can also be used in bioreactors and in all continuous and/or cyclic processes for a practically unlimited life.

Still more particularly, the field of the present invention relates to zeolite membranes for cell cultures concerning crystalline MFI, FAU, BEA, LTA, MOR, and MEL type zeolite membrane.

The above briefly represents the field of industrial use of the invention, but this field is not limiting the scope thereof, as the zeolite membrane and the process, particularly as later described and claimed, can be advantageously used in any other equivalent field in which a zeolite membrane can be used to adhere, grow, differentiate and separate cells eventually delivering pure or mixed each other and/or ions during the anchorage step or in the later stages and/or to allow the removal of drugs and/or undesidered molecules and/or ions and/or pollutants through the membrane made in any form biologically active molecules such as nucleic acids and/or amino acids and/or proteins and/or enzymes and/or drugs and/or growth factors and/or vitamins and/or hormones and/or fatty acids or phospholipids.

BACKGROUND OF THE INVENTION

Zeolite membranes are formed by intergrowth crystals having a size in the range between a few nanometers up to several hundred microns, obtained from commercial materials and a usually inorganic, permanent support used to hold together the small crystals allowing them to close crystallize.

Zeolites are silica-alumina hydrate generally containing alkali or alkaline earth metals, such as sodium, potassium and calcium. These materials are characterized by the fact that their framework contains pores and channels wherein cations (Na +, K +, Ca2 +, etc..) are weakly to the crystal structure by electrostatic interactions and therefore easily exchangeable with other positive ions, present in solution. The peculiar characteristics of zeolites constitute the basis of their extensive use as molecular sieves, as industrial catalysts for conversion of hydrocarbons, as ion exchangers and adsorbents of impurities and pollutants. The synthetic zeolites play an important role because they can be synthesized in highly pure structures and they have great stability which make them suitable for applications in processes involving high temperatures and not suitable for m a t e r i a l s l i k e, for example, polymeric materials.

Zeolites include structures with a variable chemical composition obtained by the possible inclusion of aluminum or hetero-atoms such as boron, germanium, gallium, etc.. MFI-type zeolite, or named ZSM-5 (if prepared in the presence of aluminum atoms coordinated into crystalline framework) are for example described in U.S. 3,702,886 and 3,790,471 patents and can be easily identified by X-ray diffraction.

A characteristic XRD spectrum is shown for example in the 3,790,471 patent at the end of column 2 and the beginning of column 3.

The zeolite membranes are defined as membranes where selectivity is attributed to the microporous zeolite structure. The zeolite membranes are material in which the aforementioned characteristics of zeolite structures are enhanced by a membrane configuration making them better suited to the continuous transformation processes or the separation of fluid mixtures.

The preparation of zeolite membranes is described in numerous patents (e.g. U.S. 4,699,892, U.S. 5,100,596, U.S. 2008/0216650, U.S. 2009/0029845, EP 041658, EP 041659, EP 0416660, WO 92/13631 , WO 93/00155, WO 94/01209, U.S. 7,1 19,245). According to the classification reported in the Tavolaro and Drioli review (Tavolaro A. and Drioli, Adv. Mater., 1999) they can be grouped into auto-supported, composite and hybrid (mixed matrix) zeolite membranes. The preparation methods currently available are: hydrothermal, VPT and by conversion from the solid state. There are also methodological sub-classification such as the secondary crystallization, the pore- plugging, the i n s i t u a n d m i c r o w a v e crystallization. It is should be stressed that novel zeolite membranes prepared with innovative and economic systems will play increasingly important roles, given that global membrane demand of the commercial market will increase 8,6 times for year and that the commercial market for industrial membranes will hit the 10 billion dollars level in 2012 (World Membrane S eparation Technologies Market to 2012 , Freedonia) . In addition, an extraordinary contribution to the development of biomedical research is represented by the experimental simplified systems of cell and sub-cellular biological models. These tools are now indispensable in the field of basic and applied research as their crucial role; in fact, on the one hand, they are engaged in the development of experimental procedures for the regeneration of damaged tissues and on the other hand in the procedures dedicated to the handling and development of new therapeutic molecules to obtain the increasing substitution of the expensive and outdated in vivo animal experiments.

The cultivation of mammalian cells and tissues is a most used technique in cytology, in the physiopathology and genetic manipulation. The range of cell types that today we can grow in culture, already very large and still rapidly expanding, includes cells derived from several organs and tissues - bone, cartilage, liver, lung, breast, skin, bladder, kidney, nervous system, hypophysis - stem cells from embryos and adult tissues and various types of tumor cell lines. Only a few types of cells such as lymphocytes, can grow in a suspension, while the other types, called "anchorage- dependent" cells needed to stick to a surface to grow, such as fibroblasts, endothelial and epithelial cells.

Cell cultures realized using synthetic supports are perhaps the most promising among the biological models, as simplified with respect to the whole organism, but they can mimic the functions of the in vivo organ. These systems are highly reproducible, inexpensive and object of increasing experimental tests as they offer the possibility to regenerate different tissues reducing, at the same time, the percentuage of rejection and increasing the amount of available tissue.

It is known that today commercial flat polymeric membranes are used in cell cultures and tissue engineering [J. Gerlach, K. Klopper, H.H. Schauwecher, R. Tauber, C.H: Muller and E.S. Bucherl, Int J. Art Organs, 12, 788 (1989)].

It is also known that the physical-chemical and morphologic surface properties of the polymer (such as hydrophilicity, chemical composition, surface charge, porosity, stiffness and roughness) strongly influence the anchoring of mammalian cells and their functions [R. Singhwi, A. Kumar, G.P. Lopez, G.N. Stephanopoulos, D.I. Wang, D.I. Whitesides, D.E. Ingber, Science, 264, 696 (1994)]. The cell adhesion to the surface is a process that occurs in two stages and it consists in initial anchorage followed by the spreading of cells [F. Grinnell, "Cellular adhesiveness and extracellular substrates", Int Rev. Cytology, 53, 65 (1978)]. The first stage is characterized by weak interactions that cause small changes in cell morphology, while strong interactions occur in the second stage. The movements of cells and their cellular components that occur after adhesion are dependent on cell media and involve the pseudopodia formation as amoebae behavior.

Polymers such as polystyrene, polypropylene, polysulfone, polylactic acid, caprolactone have been used extensively to develop materials with a large surface area and a possible control of growth environmental in order to develop new production large-scale systems [U.S. 5,512,474]. Among the systems reported in the most recent literature there are non-woven fibers [as described by J. Aigner et al., "Cartilage tissue engineering with novel nonwoven structured Biomaterial based on hyaluronic acid benzyl ester," J. Biomed. Mater. Res, 42, 172 (1998); G.S. Bhat, "Nonwovens as three-dimensional textiles for composites," Mater. Manuf. Process, 10, 67 (1995); S.R. Bhatarai et al. "Novel biodegradable electrospun membrane: scaffold for tissue engineering," Biomaterials, 25, 2595 (2004)] or flat polymeric microfibrils [U.S. 2007/0238167] and three-dimensional media to improve the cell differentiation [Gong et al. , U. S . 2009/0068739 Al]. The idea relative to the use of zeolite surfaces like a support in cell culture suggested in WO 2009/005745. It should stress, however, that in this patent: a) the membrane prepared is a composite membrane is then not auto-supported, b) the zeolite membrane is made by hydrothermal synthesis.

The physical-chemical characteristics of membranes, used as a support for the cultivation of anchorage-dependent cells, are of utmost importance because they directly affect the interaction that can be established between the solid phase and the cell. In particular, the chemical reactivity of surfacial hydroxyl groups (Bronsted acid), the co-presence of vicinal acidic and basic groups (Bronsted acid and Lewis), the reactivity of bifunctional groups, the distribution of electrical charges, the PZC, the hydrophilicity, the distribution of pores, the oxygen permeability, and adsorptionand desorption performances and the geometry of the membrane play a crucial role in determining the biocompatibility and the functionality o f a membrane . Membranes and the scaffold widely used for cell culture, reported in the patents and in the free literature, are polymeric membranes and have several applicative problems. In particular, the chemical nature of the materials is not very suitable for long time or repeated treatments because the physical-chemical conditions used for cell culture provoke its rapid degradation and/or transformation, thus making them no longer reusable.

In addition, the polymeric materials used are known to be very sensitive to the presence of bacteria, and fungi that generally adhere to them and grow faster than culture cells. These cultures needed, therefore, as well as the addition of appropriate chemicals (such as antibiotics and antifungals), the very complex and expensive treatments and chemical environments monitored to ensure their sterility. It is know, finally, that symmetrical flat polymeric membranes have a low porosity and then a small area available for cell attachment.

Although today flat or tubular asymmetric polymer membranes are known, which have greater porosity and high surface area, they are always prepared from a polymer solution placed in contact with a non-solvent for the polymer liquid system but miscible with the solvent used to form the polymer solution. All the structures of these membranes are then produced by physical diffusive processes and they have an area available for cell adhesion characterized by low chemical variability and a homogeneous chemical reactivity in three dimensions. In fact, the preparation procedure involves the extraction of solvent from the non-solvent that diffuses from the bottom up until the top layer of membrane. This process involves the formation of gradually variable concentration layers until to a critical value that causes the liquid demixing of the two solutions: the first one solution causes the formation of porous layer through a mechanism of gelation and the second generates the cavities in the internal structure of t h e m e m b r a n e i n a l o w p o l y m e r i c c o n e entration condition. In contrast, zeolite membranes, although have physical-chemical unique characteristics such as surface hydrophilicity and the PZC, which are modulable at the synthesis time, have not yet been utilized in cell cultures. The applicability of these membranes, used mainly for gas separations, liquid or vapor and chemical reactions, has always been identified in selectivity due to the characteristics of the zeolite channel system, rather than its surface properties.

Furthermore, all samples of auto-supported zeolite membranes reported in the literature are extremely fragile and unsuitable for laboratory use and / or industrial. They have an inhomogeneous thickness and they are prepared by hydrothermal methods. The methods of preparation of zeolite membranes, so far reported, involving high costs caused by the need of high temperature synthesis, long time to prepare, expensive templates and / or reagents for the functionalization of the seed crystals and / or controlled porosity of supports. These disadvantages are worse by the preparation of inevitable cyclic repetitions of the reactions or pre-treatment or post-treatments for the elimination of formation defects.

It is know, finally, that among the main problems that still hinder the massive cultivation of mammalian cells on scaffolds and membranes there are the high costs, due to the essential and continuous quality controls, caused by the extreme sensitivity of these cells to the water impurities.

SUMMARY OF THE INVENTION

The present invention relates to a novel crystalline zeolite membrane made of alkali metal and/or alkaline-earth metal-containing silicoaluminate structure suitable for cell cultures. Moreover, the present invention regards the procedure for fabrication thereof and its application in cell cultures.

WO Patent 052803, issued to De Cola and Popovic, discloses a method to bind zeolite L (LTL structure) nanocrystals covalently with cells, the latter chemically modified using organic molecules such as affinity binding agents, spacers and protective chemical groups containing amino groups, azides, peptides, metal complexes, chelating ion metal sites loads, acid groups, aromatic, carbonyl derivatives, thiol groups, cyanate and thiocyanate, phosphonates and sulfonates, basic groups, halides, alkenes and alkynes, bioreceptors, sugars, lipids, oliginucleotides, antibodies and their derivatives, organosilanes, and so on. This method is suitable to make hybrid constructs binding cells with zeolite crystals, although these constructs can be obtained solely using zeolite crystals chemically modified by very expensive molecules. This drawback is combined with the difficulty to obtain a hybrid material of manageable size using a very complex, expensive chemical procedure. Moreover, another drawback is constituted by the fact that the hybrid material is obtained using a hydrothermal synthesis of Zeolite L crystals a n d i t s a n c h o r a g e t o t h e s u p p o r t . This invention not only maintains a low cost, but at the same time, enhances and improves the chemical and physiological performance of membranes already described in the state of the art and overcomes the membrane disadvantages mentioned above. Viability tests of the cells adhered to this zeolite membrane showed excellent results. Moreover, the present invention enables cellular penetration into the inorganic membrane and offers excellent and numerous sites of adhesion for focal points.

This zeolite membrane can be used in bioreactors and in all continuous and/or cyclic chemical transformation processes for unlimited life without support regeneration. In addition, possible fabrication defects and formation of mesopores, macropores or cracks at preparation time and/or at a later time, do not cause a sharp decrease of the membrane performance, but, on the contrary, were appropriately made to increase interaction site number with the biological material.

Intercrystalline spaces and hollow cages of the zeolite membrane are generated by the process wherein described. In particular, the first application of a pressure to the zeolite layer followed from the application of a decompression produces the intimate interaction between the single crystal and generates the zeolite membrane.

The adsorption capacity of a material is closely related to its surface area, and its porosity distribution. For this reason, the effect of experimental parameters of the procedure (which influence the thermodynamic processes related to the formation of spaces and hollow cages inside and on the crystal surface) on the porous characteristics of the prepared zeolite membrane were highlighted and related with to the gradient pressure applied. This characterization was performed by nitrogen adsorption at 77 K using a Micromeritics ASAP 2020 instrument equipped with a software for the characterization of meso and microporous solids. This instrument enables nitrogen adsorption isotherms to be obtained and, by processing them through appropriate mathematical models, surface area, pore volume and porosity distribution values. Different mathematical models, such as BET and Langmuir, have been applied. In addition, the influence of size, structure and chemical composition zeolite crystals on the presence and percentage of generated intercrystalline pores and hollow cages have been evaluated. In addition, the surface roughness was evaluated using Atomic Force Microscopy (AFM) and linked up on the crystal structures, sizes and morphologies used to prepare the zeolite membrane.

These characterization techniques are well known and widely used in the characterization of membrane materials. However, it has to be stressed that the presence of these structural features is strongly dependent on the specific zeolite structures and types, and experimental parameters used for the preparation of the crystals and membranes and that they are not restrictive or detrimental to the adhesion cell performances that are anchored on the surface and not are inside the pores prepared. The adhesion of biological material is then independent of the kinetic diameter, because the cells are seeded on the surface and do not require mechanical or chemical protection to the flowing culture media.

The use of pure, hydrophobic and nanocrystalline zeolite structures with crystal dimensions inferior to 100 x 40 x 40 nm has to be preferred to larger crystals, but it never causes deterioration in the performances of the made membranes.

Another purpose of this invention is to obtain an inorganic crystalline membrane support for cell cultures and separations to obtain better interaction control and to increase the working efficiency.

The zeolite membrane herein also enables the selective extraction of undesirable species (such as pollutants, toxic and poisonous ions and molecules) and is widely applicable in the removal of catabolites and/or cellular metabolism products. In addition, it is selective to the permeation of small molecules such as ammonia, urea or creatinine produced in metabolic processes.

This invention is also easily transformable in a solid support, since it has excellent antimicrobial and/or antifungal performances by simple treatment with for example, copper or silver salts. Moreover, it easily adsorbs drugs and molecules commonly used in the treatment of cancer (such as organometallic compounds of platinum or palladium, for example, salts of transition metals), allowing then a direct drug delivery to cells cultured.

The zeolite membrane having a crystalline porous surface, a silicoaluminate composition and a tensile strength higher than 4.75 kg/cm² for a membrane thickness greater than 50 microns achieves these and other aims, according to the invention. At the same time, the procedure herein described greatly improves the complex methodologies used to prepare autosupported zeolite membranes.

In summary, this peculiar zeolite membrane is:

Very stable in aqueous environment, apart from the ionic force and pH;

able to adsorb the pollutants of the culture medium;

capable to remove toxic ions and/undesirable molecules by ion exchange; antifungal and antimicrobial material;

able to immobilize and delivery molecules useful for the adhesion and/or growth and/or differentiation cells, like drugs;

a material having morphological, structural and physical-chemical properties that promote the cell adhesion, growth and differentiation and allow the modulation delivery of growth factors, cytokines and metabolites for the differentiation of progenitor cells; easy to prepare in different handy size and thickness for the necessary laboratory tests. In view of the importance of this material is a clear need to develop a zeolite membrane for that purpose. Of course, so that the zeolite membrane herein described can easily be commercialized, it has to be prepared from commercial pure products, using a simple preparation procedure characterized by simple, triable industrial process with a low production costs.

DETAILED DESCRIPTION

The zeolite membrane, a crystalline silicoaluminate structure containing alkali metal and / or alkaline earth metals, object of this invention is prepared with zeolite crystals smaller than 100 microns, with or without template agents or calcined. It is characterized by the fact that has an auto-supported structure, intercrystalline spaces, pores and cavities between 20 A and 10 μιη and is realized in different morphologies (flat, tubular, etc.) with surfaces and volumes from a few millimeters. This material also can be prepared in order to obtain physical-chemical characteristics suitable to the type of cells used. In particular, it is possible to produce a membrane with extremely high surfacial total area, a suitable hydrophilic or hydrophobic character, PZC and a suitable surface charge in order to improve its interactive capabilities.

The present invention relates to a zeolite membrane obtained from pure structure synthetic zeolite MFI, MOR, MEL, LTA, FAU and BEA nanocrystals or microcrystals as made or calcined. These structures can be isomorphously substituted by B, V, Fe, Ti, In, Ge, Ga, Cr, Co, Cu, Mn, Sn, P, Se, and W atoms, single or in combination with each other. Moreover, the crystals may be as made (ie, obtained from direct synthesis with template organic molecules) or calcined or grounded, or containing transition metals such as Ag, Au, Cu, Rh, Ru, Ir, Fe, Co, Cr, Cd, Zn and Sc, introduced by ion exchange processes. Finally, the crystals and the membrane can be impregnated or adsorbed or ion exchanged or bounded with transition metal complexes and / or organometallic compounds, mercaptans, polyols, phosphate, boronic acid, functionalizing molecules and biologically active molecules such as nucleic acids, amino acids, proteins, enzymes, drugs, growth factors, vitamins, hormones, fatty acids or phospholipids, alone or in combination each other, as well as isolated cells, microorganisms, bacteria, fungi and liposomes.

The present invention will now be better explained on the basis of examples. Example 1 : The zeolite membrane covered by this invention is prepared from MEL structure crystals obtained by hydrothermal synthesis in the alkaline environment. MEL structure zeolite crystals are arranged to form a homogeneous layer that is subjected to a forming pressure of about 25 Kg/cm in a suitable device. At the same time a vacuum is applied for longer than 3 minutes. The membrane thus obtained is without cracking, intact and manageable. XRD powder analysis can detect the presence of a pure structure zeolite membrane.

Example 2: The zeolite membrane covered by this invention is prepared from MEL structure nanocrystals obtained by hydrothermal synthesis in the alkaline environment. MEL structure crystals are grounded to obtain a homogeneous mixture that is subjected to a forming pressure of about 25 Kg/cm in a suitable device. After this treatment the membrane appears without cracking, intact and manageable. XRD powder analysis can detect the presence of a pure structure zeolite membrane.

Example 3 : MOR structure crystals are grounded to obtain a homogeneous mixture that is subjected to a forming pressure of about 2 Kg/cm in a suitable device. After this treatment the membrane appears without cracking, intact and manageable. XRD powder analysis can detect the presence of a structure zeolite membrane.

Example 4: MOR structure zeolite crystals are treated according to Example 1 and reduced to the form of thin membrane. Subsequently, this membrane is coated with crystals having a MFI structure and subjected to a pressure of 10 kg/cm to obtain a composite structure zeolite membrane due to the overlapped pure layers as revealed by X-ray diffraction analysis. Example 5: A dense, flat polymeric membrane polylactic acid (PLA) is inserted into a device made of steel and is coated with a mixture containing a pure crystalline zeolite layer. A pressure of 25 Kg/cm is applied then the membrane is placed inside an modified Morey-type autoclave for a VPT type hydrothermal synthesis for four days in a range of temperature comprising between 120 and 170 ° C in a saturated air steam. Example 6: The zeolite membrane in the flat membrane configuration is made according to Example 1, but overlapping two layers of structure of MFI and / or MOR and / or LTA or FAU crystals.

Example 7: The zeolite membrane in the flat membrane configuration is made according to Example 1 , but by overlapping multiple layers of MFI structure and / or MOR and / or LTA or FAU crystals.

Example 8 : The zeolite membrane in the flat membrane configuration is made according to Example 1, using s crystalline zeolite mixtures and / or reaction mixture dried and / or transition metal salts and / or lanthanide and / or actinides and / or elements of the first and second group, as well as amorphous silica and halide- containing compounds then a pressure of 25 kg/cm² is applied;

Example 9: The flat zeolite membrane is built according to Example 1 , using crystals already subjected to chemical treatments such as exchange (with metal ions and / or anions), reduction in the presence of reducing agents (gaseous and / or solution), impregnation with solutions containing the metal species and / or organic species functionalizing the zeolite surface, and / or protein (eg albumin), enzymes, drugs, amino acids, bifunctional molecules (hydrophilic / hydrophobic);

Example 10: The zeolite membrane surface is made according to the example 1 , using an inorganic layer of dust that is then covered with one or more layers of zeolite crystals pure and / or mixed according to the example 6 and 7.

The zeolite membrane thus obtained according to the description given in all the examples above and in Example 2 is an optimal environment for cells isolated for their anchorage, growth, maintenance of cell viability and metabolic functions. The comparison of the control biological parameters of this membrane object of this invention and the commercial supports, generally used for cell cultures showed that the zeolite membrane is the best currently available biocompatible support. The technical specifications of the present invention are described herein, provided that they are not limited to descriptions below.

According to a preferred embodiment of the invention, the membrane is formed of crystals of Silicalite-2, and still more preferably is a flat membrane of Silicalite-2 in substantially pure form of a disk with a diameter of 13 mm, a thickness of 3 mm and porosity above 50%.

The internal pores of the membrane according to the invention have an internal average diameter of some A, while the spaces, intercrystalline pores and hollow cages have a size between 20 A and 10 microns. This membrane is characterized by a SAR (silicon / aluminum ratio) infinite, since that it is a membrane-type silicic MEL structure. This membrane retains the structure of the zeolite crystals used with a slight increase in the zero point of charge (PZC) value up to 8.7, which is determined using crystalline aqueous suspensions containing different weight percentages (1%, 5%, 10% and 50%>). The determination of the acidity is always repeated after 24 hours in order to control the pH of the suspensions under equilibrium conditions. The PZC value of each membrane is strictly measured in order to check the acidic conditions for cell growth.

In particular, this membrane, characterized by means of X-ray diffraction (XRD, Philips PW 1730/10 X-ray diffractometer using Cuka radiation), showed a high crystallinity of not less than 95% of the crystallinity of the crystals used, and a conservation of zeolite structure without the formation of amorphous phases or other crystalline structures.

The atomic ratios, controlled by means of microanalysis (EDX), revealed constant composition compared to the original crystals showing a ratio Si / Na equal to 4.66. Light microscopy analysis reveals a uniform nano crystalline surface which is confirmed by the scanning electron microscope (SEM, Cambridge Instruments 360). Uniform morphology and few nanometer sized nanocrystals are interconnected to form a homogeneous surface. The gas permeability measurements, made using pure gases, have shown the successful formation of a zeolite membrane with high permeability and a flow constant over time. According to a preferred embodiment of the invention, the preparation of zeolite membrane is preferably made from synthetic zeolite crystals.

In particular, the process is characterized by the fact that the calcined nano-sized zeolite crystals, with a pure structure and a microporous framework, are subjected to a pressure greater than 1 atm, so are placed in intimate contact with each other. At the end of that process, the zeolite crystals are bonded to form a membrane from chemically homogeneous composition, desired thickness and shape, easy to handle, indissoluble and non-disintegrable by contact or immersion in polar or nonpolar pure or containing ions solvents, and in buffer solutions.

The application of the membrane of the invention regards its use for the cultivation of differentiated cells and / or undifferentiated and / or modified and / or used in cell separation and / or in bioreactors and / or biomaterials for implants. In particular, the application is described for human fibroblasts and human fetal endothelial cells. The fibroblasts strain used is the NHDF from human fetus and bought by ICTC of Genoa. The cells are maintained in complete medium DMEM (Dulbecco's Modified Eagle Medium) enriched to 10% (v / v) FCS (fetal calf serum), penicillin (50 IU / mL), streptomycin (50 mg / mL), L-glutamine (2 mM) and sodium pyruvate (1 mM).

The cells are grown in an incubator with humidified air atmosphere at 37 ° C, and 5% C0₂.

The cells were counted (counting chamber or cell counter) and plated at a final

5 2

concentration of 4 x 10 cellule/mm on the zeolite membrane. The material was kept in multi-well plates with four slots for use at the same time for control, for observations of morphology and vitality to the fluorescence microscope and SEM studies. Every 48 hours the cell viability has been tested.

The culture medium has been removed and replaced with fresh medium when the indicator signaled its consumption. The indicator has been used or not depending on the optical method used after the assay.

The primary lines of HUVEC (Human Umbilical Vein Endothelial Cells) endothelial cells that we used are produced by the collaboration of three entities Genoa: (1) the Department of Obstetrics and Gynecology of San Martino (2) the complex structure of 1ST Molecular Oncology (3) the 1ST cell bank, core facility.

The primary cultures of endothelial cells are tested up to the 7th passage of culture to ensure that cellular senescence cannot interfere with the search results and guarantees the reproducibility of results. The cells were expanded with divisions 1 :2 or 1 :3 when they reached at least 80% confluence. The cells are cultured in a humidified incubator at 37 ° C, and 5% C0₂.

For each milliliter of suspension are added 5 ml of culture medium with full indicator. The cell suspension is centrifuged and the pellet suspended again in the same medium. The cells are counted (counting chamber or cell counter) and plated at a final concentration of 150.000 cells / 3 ml medium on the zeolite membrane.

Evaluation of the density of cell population by fluorescence microscopy (acridine test of orange).

All cultured cells were stained with acridine orange in PBS to assess their cell viability. The count has been carried out on an average of nine squares of the Burker chamber. The green cells, in which the dye failed to bind to damaged nucleic acids giving the nuclei and / or the color orange to the cytoplasm, are living and they have intact their DNA.

Determination of cell viability evaluated as mitochondrial activity by spectrophotometric analysis (MTT test).

Cell viability evaluated like mitochondrial activity has been quantified by measuring the dehydrogenase activity of cultured cells, using the l-(4,5-dimethylthiazol-2-yl) -3,5- diphenylformazan (MTT) assay. This test is based on the ability of living cells to convert the compound soluble MTT in the insoluble formazan salt. The quantity of formazan obtained has been considered to be proportional to the number of living cells. In particular, for each sample of cells seeded onto zeolite material, cells have been incubated in 1 mL of MTT (0.5 mg / mL) for 1 hour. Subsequently, the medium was removed and the cells have been treated with 1 mL of dimethyl sulfoxide (DMSO) to solubilize the formazan salt. The spectrophotometric analysis has been performed at 540 nm in order to evidence the characteristic absorbance band.

Sample analysis

Morphological, histological and structural observations of all zeolite and cellular samples, were carried out before of the seeding step and after the growth. A phase- contrast and polarizing Zeiss and an inverted fluorescence Olympus photomicroscopes and FESEM microscope have been used for the characterizations.

Samples prepared for SEM observation were fixed in a 3% solution of glutaraldehyde in cacodylate buffer, pH 7.4, post-fixed in 1% solution of osmium tetroxide, progressively dehydrated in ethanol, dried in Critycal point dryer and coated with a thin layer of gold.

The zeolite membrane with a crystalline structure silicoalluminatica containing alkali metal and / or alkaline earth metals, biocompatible and feasible in many shapes and thicknesses, as described here, has been a great support for membership and inorganic growth of various cell types so as to establish a unique opportunity, not only in the field of transplant medicine, the biotechnology research and pathophysiology, but also in its understanding of normal physiological processes. The process of the invention is simple, inexpensive and innovative. In fact, the zeolite membranes reported so far are crystalline structures formed by crystals intercresciuti ranging in size from a few nanometers up to several hundred microns. They are obtained from commercial materials and support, usually inorganic, used to hold together the crystals allowing them to develop and crystallize neighbors. Their preparation, hydrothermal often is long and complex and may require the use of molecules as expensive templanti or bifunctional molecules (for the synthesis of secondary) and long reaction times or heating to high temperatures (for the synthesis in situ) . The high performance potential are then easily undone by the presence of defects formation and costly preparations and pre-treatment or post-treatments make them, in fact, hardly applicable to large-scale industrial processes.

On the contrary, in the speedy and inexpensive procedure described here, defects formation and the creation of mesopores, macropores or cracks in preparing and / or at a later time does not cause a sharp decrease of the applicability of the membranes, but were specially designed to increase the sites of interaction with biological material. The zeolite membrane of the invention, the unique chemical and physical characteristics, offers the opportunity to give directly reactive molecules (such as proteins, enzymes and / or drugs) to cells in culture by ensuring the integrity biostability.

In such an invention is exploited the peculiar characteristics of ion-exchange of zeolites, which include structures with a variable chemical composition for the possible inclusion of aluminum or hetero-atoms (such as boron, germanium, gallium, etc..) Wide at the base of their use as molecular sieves, such as industrial catalysts for conversion of hydrocarbons, such as ion exchangers, adsorbents such as impurities and pollutants.

Checks on the morphology, integrity and on the anchorage of cells adhering to the material covered by the patent showed excellent cell growth, and excellent cell viability by showing that the new membrane has the ability not only to promote cell growth but to improve the biocompatibility of the system.

This zeolite membrane, the characteristics of antimicrobial and / or antifungal agents, for simple treatment with copper or silver, can be used in bioreactors and in all processes of continuous and / or cyclic for a practically unlimited.

It also readily adsorbs substances such as drugs commonly used in the treatment of cancer (such as organometallic compounds of platinum or palladium, for example, salts of transition metals), allowing a direct administration to cells in culture.

BRIEF DESCRIPTION OF FIGURES

Fig. 1. Scanning electron microphoto graph of the MOR zeolite membrane surface.

Fig: 2. FESEM microphoto graph of the MEL zeolite membrane surface.

Fig. 3. Scanning electron microphoto graph of the cross-section of a MFI zeolite membrane.

Fig. 4. Scanning electron microphoto graph of the cross-section of a MEL zeolite membrane.

Fig. 5. XRD pattern of the FAU zeolite membrane prepared. Fig. 6. Scanning electron microphotograph of the FAU zeolite membrane surface covered with the adhered fibroblast cells.

Fig. 7. Scanning electron microphotograph of the cross-section of the MEL zeolite membrane covered with the adhered fibroblast cells.

Fig. 8. Scanning electron microphotograph of the FAU zeolite membrane surface covered with the adhered fibroblast cells.

Fig. 9. Scanning electron microphotograph of the cross-section of the MFI zeolite membrane covered with the adhered fibroblast cells.

Fig. 10. Scanning electron microphotograph of the MEL zeolite membrane surface covered with the adhered HUVEC cells.

Fig. 11. Fluorescence microscopy image of fibroblasts incubated using the MEL zeolite membrane after 5 culture days (Acridine orange).

Fig. 12. Fluorescence microscopy image of fibroblasts incubated using the MOR zeolite membrane after 3 culture days (Acridine orange).

Fig. 13. Cellular viability versus day of culture using zeolite membranes. Fig. 14. Cellular density versus day of culture using zeolite membranes.

Claims

1. A membrane for cellular adhesions, growth and cultures comprising a zeolite membrane characterized by the fact that this membrane is auto-supported, that it is formed by at least one, pure and homogeneous layer of zeolite crystals having a size inferior to 100 μιη and containing intercrystalline voids, pores and hollow cages in the range between 20 A and 10 μιη.

2. A membrane for cellular adhesions, growth and cultures according to claim 1 characterized by the fact that zeolite crystals are preformed and no chemically modified and that they have a crystalline silicoaluminate MFI, MOR, BE A, MEL, FAU, LTL and LTA type structure.

3. The process for the preparation of a membrane for cellular adhesions, growth and cultures characterized by the fact that comprises the following steps: a zeolite layer is prepared using preformed and no chemically modified zeolite; a layer is spread with the desired thickness; the layer is first pressed with a pressure higher than 2 kg/cm² and then is subjected to a vacuum lower than 20 mm Hg.

4. The process for the preparation of a membrane according to claim 3 characterized by the fact that the crystals used to form the zeolite layer having a crystalline silicoaluminate structure contain template and are ozonized or calcined by heating in a oven up to 800 °C.

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