WO2011098497A1 - Membrane de zéolite pour l'adhésion cellulaire, la croissance et des cultures de cellules et procédé pour la préparation de celle-ci - Google Patents

Membrane de zéolite pour l'adhésion cellulaire, la croissance et des cultures de cellules et procédé pour la préparation de celle-ci Download PDF

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WO2011098497A1
WO2011098497A1 PCT/EP2011/051915 EP2011051915W WO2011098497A1 WO 2011098497 A1 WO2011098497 A1 WO 2011098497A1 EP 2011051915 W EP2011051915 W EP 2011051915W WO 2011098497 A1 WO2011098497 A1 WO 2011098497A1
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membrane
zeolite
zeolite membrane
cells
crystals
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PCT/EP2011/051915
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English (en)
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Palmira Tavolaro
Guglielmo Martino
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Universita' Della Calabria
Tavolaro, Adalgisa
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Priority to EP11706773A priority Critical patent/EP2533882A1/fr
Publication of WO2011098497A1 publication Critical patent/WO2011098497A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/02—Inorganic material
    • B01D71/028—Molecular sieves
    • B01D71/0281—Zeolites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0039—Inorganic membrane manufacture
    • B01D67/0041—Inorganic membrane manufacture by agglomeration of particles in the dry state
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0081—After-treatment of organic or inorganic membranes
    • B01D67/0088—Physical treatment with compounds, e.g. swelling, coating or impregnation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/10—Specific pressure applied
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/48—Antimicrobial properties
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates

Definitions

  • 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 ⁇ and containing intercrystalline voids, pores and hollow cages in the range between 20 A and 10 ⁇ .
  • 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/cm 2 and then is subjected to a vacuum lower than 20 mm Hg.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cations Na +, K +, Ca2 +, etc..
  • 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
  • ZSM-5 if prepared in the presence of aluminum atoms coordinated into crystalline framework
  • U.S. 3,702,886 and 3,790,471 patents can be easily identified by X-ray diffraction.
  • 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.
  • zeolite membranes 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) .
  • 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 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.
  • 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.
  • 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.
  • 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 are polymeric membranes and have several applicative problems.
  • 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.
  • 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.
  • asymmetric polymer membranes 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 o n e entration condition.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • drugs and molecules commonly used in the treatment of cancer such as organometallic compounds of platinum or palladium, for example, salts of transition metals
  • the zeolite membrane having a crystalline porous surface, a silicoaluminate composition and a tensile strength higher than 4.75 kg/cm 2 for a membrane thickness greater than 50 microns achieves these and other aims, according to the invention.
  • the procedure herein described greatly improves the complex methodologies used to prepare autosupported zeolite membranes.
  • this peculiar zeolite membrane is:
  • zeolite membrane for that purpose.
  • 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.
  • 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 ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PLA polymeric membrane polylactic acid
  • 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/cm 2 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);
  • 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.
  • 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%>).
  • PZC zero point of charge
  • 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.
  • 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.
  • XRD X-ray diffraction
  • 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.
  • the preparation of zeolite membrane is preferably made from synthetic zeolite crystals.
  • 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.
  • 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.
  • 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).
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • penicillin 50 IU / mL
  • streptomycin 50 mg / mL
  • L-glutamine 2 mM
  • sodium pyruvate 1 mM
  • the cells are grown in an incubator with humidified air atmosphere at 37 ° C, and 5% C0 2 .
  • the cells were counted (counting chamber or cell counter) and plated at a final
  • 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.
  • HUVEC Human Umbilical Vein Endothelial Cells
  • 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% C0 2 .
  • 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.
  • MTT l-(4,5-dimethylthiazol-2-yl) -3,5- diphenylformazan
  • 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.
  • cells have been incubated in 1 mL of MTT (0.5 mg / mL) for 1 hour.
  • DMSO dimethyl sulfoxide
  • 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.
  • 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.
  • zeolite membrane of the invention offers the opportunity to give directly reactive molecules (such as proteins, enzymes and / or drugs) to cells in culture by ensuring the integrity biostability.
  • 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.
  • aluminum or hetero-atoms such as boron, germanium, gallium, etc..
  • 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.
  • Fig. 1 Scanning electron microphoto graph of the MOR 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.

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  • Chemical & Material Sciences (AREA)
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  • Inorganic Chemistry (AREA)
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  • Silicates, Zeolites, And Molecular Sieves (AREA)
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Abstract

L'invention concerne une membrane de zéolite autosoutenue, son procédé de préparation et son application dans des adhésions cellulaires et des cultures de cellules. Les tailles des pores et des cavités de cette membrane de zéolite se situent dans la plage comprise entre 20 Å et 10 μm. La préparation de la membrane de zéolite consiste à presser une couche microporeuse de cristaux de zéolite calcinés ou ozonisés ou broyés ou de structure pure telle quelle. Pour obtenir un échafaudage antimicrobien et antimycotique idéal, les caractéristiques physico-chimiques de la surface et de la structure de la membrane de zéolite peuvent être modulées. L'applicabilité de cette membrane à l'adhésion et la croissance cellulaires est démontrée.
PCT/EP2011/051915 2010-02-09 2011-02-09 Membrane de zéolite pour l'adhésion cellulaire, la croissance et des cultures de cellules et procédé pour la préparation de celle-ci WO2011098497A1 (fr)

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EP11706773A EP2533882A1 (fr) 2010-02-09 2011-02-09 Membrane de zéolite pour l'adhésion cellulaire, la croissance et des cultures de cellules et procédé pour la préparation de celle-ci

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ITCS2010A000003 2010-02-09
IT000003A ITCS20100003A1 (it) 2010-02-09 2010-02-09 Membrana zeolitica per adesioni e colture di cellule, procedimento per la preparazione ed applicazione.

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