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    • 专利摘要:
    Method to evaluate the cytotoxicity of chemical substances. The present invention relates to a method for in vitro evaluation of the cytotoxicity of chemical products, especially those containing water-immiscible volatile substances, preferably those chemicals that are used in the biosanitary field. By means of this method, said products are brought into direct contact with the cell and/or tissue cultures in vitro and a non-volatile substance is deposited on the volatile substance under study, which prevents the evaporation of the latter and makes it possible to control the exposure time of the product. Cell or tissue cultures to the products under study. In addition, the method allows to recover the study substance. This method allows to reliably and reproducibly determine the biocompatibility and biosecurity of these chemical products, preferably volatile, before conducting in vivo studies and their use in clinical practice. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2644987A1
    申请号:ES201630708
    申请日:2016-05-30
    公开日:2017-12-01
    发明作者:Girish Kumar SRIVASTAVA；Mª Luz ALONSO ALONSO；Iván FERNÁNDEZ BUENO；Mª Teresa GARCÍA GUTIÉRREZ；Rosa Mª COCO MARTÍN；José Carlos PASTOR JIMENO
    申请人:Universidad de Valladolid；
    IPC主号:
    专利说明:

    The present invention falls within the field of health and cytotoxicology, more specifically within the methods of evaluation of biosecurity and biocompatibility of chemical products, especially, although not exclusively, of those containing highly volatile compounds, which are preferably used in clinical practice because they are included in biosanitary products. STATE OF THE TECHNIQUE
    Chemical sanitary products are widely used in clinical practice. These products are direct, or indirect, contact with the human body and react with the environment and with the areas of the body where they are applied. Although they may have adequate mechanical, physical and chemical properties for certain medical applications, they must show that they also have good biocompatibility, and therefore demonstrate that they are safe for use in clinical practice (Council Directive 93/42 / EEC of 14 June 1993, related to medical devices), since during their use they may come into direct or indirect contact with cells and tissues of the human body.
    Thus, the design of a strategy to evaluate the biocompatibility and safety of the application of a medical device is always a topic of interest that concerns the certifying authorities of any country. International standards for medical devices (ISO 10993) provide a series of guidelines in this regard. Following these standards, medical devices undergo a series of rigorous biocompatibility tests, which include, among others, cytotoxicity analysis, sensitization, intradermal irradiation and acute systemic toxicity.
    Cytotoxicity studies are a first indicator of biocompatibility, and therefore, of the level of safety of the medical device (Li, W., Zhou, J. & Xu, Y. 2015, Biomedical Reports, vol. 3, no. 5, pp. 617-620). The results of this first study mark the fate of the medical device: if further research is necessary, if modifications are necessary, or if it is discarded in the initial manufacturing phase, before starting studies with experimental animals (Fricker, S. 1994, Toxicology in Vitro, vol. 8, no. 4, pp. 879-881). A good cytotoxicity test is one that follows standard protocols, has high sensitivity and produces quantitative and comparable data quickly to be evaluated. Current advances in science and technology provide several types of tests for cytotoxicity studies, many of which are described in the ISO standards already mentioned. Following the recommendations of the ISO standards, biocompatibility can be tested worldwide, and therefore, the level of safety of medical devices.
    ISO 10993-5 recommends three types of cytotoxicity tests: the use of a sample extract, direct contact or indirect contact. So far the method of dilution of extracts and indirect contact have been the most commonly accepted, since they can be applied to a wide variety of sanitary chemicals and detect extractable and leachable toxins. The extraction conditions (time and temperature) depend on the physicochemical characteristics of the medical device and the extraction vehicle. On the contrary, the direct contact method makes it possible to detect weaker levels of cytotoxicity and of non-removable or leachable compounds, due to their high sensitivity.
    Mammalian cell cell cultures are common in in vitro cytotoxicity studies; more specifically, established cell lines obtained from recognized repositories have been used. ISO standards recommend the CCL 1 cell lines (clone 929 of the NCTC; mouse fibroblasts), CCL 163 (clone A31 of the Balb / 3T3; mouse fibroblasts), CCL 171 (MRC-5; human fibroblasts), CCL 75 (WI-38; human fibroblasts), CCL 81 (Vero; non-human primate epithelial cells) and CCL 10 [BHK-21 (C-13); hamster fibroblasts] and V-79 379A (hamster fibroblasts) of the American Type Culture Collection (ISO 10993-5: 2009). However, primary cell and tissue cultures obtained directly from living tissues are also recommended for analysis in which a specific sensitivity is required, provided that the reproducibility and accuracy of the cellular and tissue response against The samples studied. The choice of cell and tissue cultures, the methodology followed for the preparation of the study samples, the type of cytotoxicity test and the analysis methods, may vary depending on the standards of the certifying authorities of the different countries.
    Some manufactured polymers, such as perfluoro-n-octane, perfluorodecalin or sodium hyaluronate (viscoelastic solution) among others, are used as medical devices. Perfluoro-n-octane, also known as octadecafluorooctane, is a liquid fluorocarbon, a perfluorinated derivative of octane hydrocarbon. Its molecular formula is C8F18. It has a molar mass of 438.06 g / mol, a melting point of -13 ° F (-25 ° C) and a density of 1.77 g / cm³. This medical device is very popular and widely accepted as an intraoperative mechanical tool in vitreoretinal surgery in patients with retinal detachment, penetrating eye trauma, giant retinal tear (s), proliferating vitreoretinopathy (VRP) or cataract surgery complications. Perfluoro-n-carbon acts as a manipulator of the retina in vitreoretinal surgery or is used, for example, to refloat dislocated lenses to the vitreous cavity. Several companies in the health market market this product with different names such as Perfluoron®, Okta-line ™, Arcotane (ARCADOPHTA®), Ala®Octa, F-Octane (FLUORON®) and Bio Octane ™ among others.
    The adequate synthesis and purification of liquid perfluoro-n-octanes are always unavoidable issues for the companies that manufacture and market them. Different techniques have been used, such as electrochemical fluorination for the synthesis of raw materials and distillation and filtration for purification. The biocompatibility of the raw materials and their safety are evaluated before packing them in syringes and vials of 5 or 7 milliliters and marketing them for ophthalmic use. Cytotoxicity studies are carried out in accordance with the guidelines of the ISO standard (UNE-EN ISO 10993-5: 2009 Biological evaluation of medical devices, Part 5: In vitro cytotoxicity tests and UNE-EN ISO 10993-12: 2007 Biological evaluation of medical devices, Part 12: Preparation of samples and reference materials). Tests with the method of dilution of extracts by indirect contact can be applied in most cases.
    The use of the NCTC 929 clone cell line [L cell, L-929, mouse subcutaneous connective tissue, derived from strain L] (ATCC® CCL-1 ™) is common in performing such tests. The standard protocols for the evaluation of cell proliferation are used to determine the cytotoxicity of the test samples of liquid perfluoro-n-octanes. The study samples are extracted by agitation with culture medium under certain conditions of time and temperature, observed to detect anomalies (clarity, color, absence of foreign materials) and finally exposed to a cell culture of the L929 line. On the other hand, in the case of the agarose diffusion method, the study samples or extracts thereof are placed on top of an agarose mixture to then diffuse into the cell culture. In these tests it is considered that the cytotoxic components of perfluoro-n-octane, the medical device, would be separated in the extract or filtered through the agarose.
    However, the recent cases of blindness after vitreoretinal surgery alerted the certifying / sanitary authorities of Europe, Asia and South America, which notified the health authorities of the Autonomous Communities and the general public about the products health systems containing perfluoro-n-octane (Spanish Agency for Medicines and Health Products. Web. Information note Ref. PS, 19/2015. Information on incidents related to the product ALA OCTA (perfluoroctane), used in retinal surgery; Swiss Agency for Therapeutic Products, Recalls and other field safety corrective actions (FSCA) -Archive, 12/21/2915; Institute of Public Health of Chile (ISP) 08/27/2013. Withdrawal of the liquid perfluoroctane medical device manufactured by Meran Tip , Turkey and distributed by Falc Chile Ltda .; Department of Health, The Government of Hong Kong Special Administrative Region 12/17/2015. and Alert: Arcadophta SARL Arcotane 5ml). These alerts come from an investigation of medical devices containing perfluoro-noctane, especially focused on the search for failures in the current methods used to detect cytotoxicity in commercialized perfluoro-n-octane batches.
    As previously indicated, perfluoro-n-octane is an example of a substance of high molecular weight, volatile and insoluble in water. Being insoluble in water makes it necessary to use another more suitable extraction vehicle, which is not known, in which the toxic components released are soluble. The preparation of the samples of the compound under study, by stirring with culture medium under certain conditions, cannot confirm the presence of water-insoluble toxic components in the products. The method in which the extract of this sample is used would fail to determine cytotoxicity for this reason. On the other hand, the volatile nature makes it difficult to regulate the exposure time of the samples of compound under study on cell and tissue cultures, and more in a highly volatile product such as perfluoron-octane. The indirect contact test would also fail for this reason. In addition, the cellular response would be different compared to different stimuli. For example, in a study published in 2015 (Zhongguo Yi Liao Qi Xie Za Zhi., 2015 Mar; 39 (3): 212-5, In Vitro Cytotoxicity Study of Nickel Ion) cell lines L929, h9c2 (2-1) , 293 [HEK-293], hFOB1.19, THLE-3, H9 and IM-9 were tested against medical devices containing nickel, and the results confirmed that the L929 line cells were the cells that best tolerated exposure to nickel (less sensitive). Therefore, the cellular response obtained in this type of experiments to determine cytotoxicity is also dependent on the type of cell used.
    Therefore, taking into account the clinical problems caused by the toxicity of substances such as the one described above and the difficulties for the toxicological analysis of volatile compounds insoluble in water, due to the impossibility of performing an adequate cytotoxicity test, it becomes necessary to design a new strategy to assess in a sensitive and reliable way the biocompatibility, and therefore the safety, of chemical sanitary products that may contain volatile or water-insoluble substances or non-removable or leachable compounds before conducting in vivo studies and their use in clinical practice. DESCRIPTION OF THE INVENTION
    The present invention provides a method that allows to evaluate the cytotoxicity of the chemical substances used in the production of sanitary products, in particular of the volatile compounds immiscible in water, which are more complicated to analyze. The method allows to evaluate its cytotoxicities in a reliable, reproducible, controlled manner and with high sensitivity.
    When the toxic effects of highly volatile substances are evaluated in vitro by means of a direct contact study with cell cultures, it is difficult to control the actual exposure time, which makes it impossible to carry out an adequate, reliable and reproducible toxicological study. In addition, when said substances are immiscible in water, the diffusion of the toxic compounds present therein into the cell or tissue culture medium is hindered. The method proposed by the present invention represents a solution to these problems, since it incorporates the use of a non-volatile substance, preferably an aqueous medium, located on the volatile compound under study, thus preventing the latter from evaporating and therefore , allowing to control the exposure time of the cells or tissue to the volatile chemicals studied. This makes it possible to reliably and reproducibly determine the biocompatibility, as well as the biosecurity, of these volatile chemicals before conducting in vivo studies and their use in clinical practice, that is, before contact with the patient. In addition, this method provides a direct contact during a controlled exposure time between cell and / or tissue culture and the volatile compound, which allows to detect cytotoxicity levels with high sensitivity, including the detection of cytotoxicity associated with compounds that present toxicity. mild. The invention allows, by the described method, to evaluate the biocompatibility and safety, and therefore the toxicity, of a large number of medical devices that come into contact with patients during clinical practice, providing useful, concrete and tangible results.
    The advantages associated with the method proposed by the present invention are, therefore, the following:
    - It allows direct contact between the volatile compound and the cells and / or tissues, which implies that there is no interference with any other factor, as it does in indirect contact tests or in dilution methods of extracts, where others are involved. factors such as the extraction vehicles, the agar layer on the cell layer, etc., which may be affecting cell proliferation or tissue structure in culture and, consequently, the test results.
    - It prevents the volatile substance under study from evaporating during the test, thus allowing to regulate and control its exposure time to cell or tissue cultures.
    - It allows to clearly confirm which compound is the one that is in direct contact with the cells and tissues since, due to the insoluble nature of the volatile compound and its difference in molecular weight with the aqueous medium with which it is in contact, they can be easily visualized the different layers of the system.
    - It allows the collection of substances that have come into contact with cells and tissues after the desired exposure time for further studies.
    Thus, this method facilitates testing volatile chemicals, preferably sanitary products, generating sufficient guarantees to evaluate their biocompatibility and safety.
    Therefore, one aspect of the invention relates to a method for evaluating the cytotoxicity of chemical compounds, preferably volatile, comprising:
    to. deposit a chemical compound, preferably volatile, to study on a cell or tissue culture in vitro,
    b. depositing a non-volatile aqueous medium on the chemical compound, preferably volatile, deposited in step (a),
    C. incubate the cell or tissue culture obtained after step (b), and
    d. evaluate the effect produced by the presence of the chemical compound, preferably volatile, deposited on the culture;
    where the chemical compound, preferably volatile, deposited is insoluble in water and of high molecular weight.
    From now on, this method will be referred to as the "method of the invention".
    The term "volatile compounds", "VOCs" or "VOCs" refers to any organic compound (containing carbon) that evaporates easily into the atmosphere at room temperature. They are chemical substances that contain carbon and one or more of the following elements, but not limited to: hydrogen, halogens, oxygen, sulfur, phosphorus, silicon or nitrogen, except for carbon oxides and inorganic carbonates and bicarbonates. On the other hand, volatile compounds easily convert into vapors or gases and can also pose a health problem since they can produce, for example, an irritating effect on the eyes and respiratory tract, which can trigger asthmatic reactions. Its main characteristics are volatility, fat solubility, flammability and sometimes toxicity. Some examples of these compounds are, but are not limited to, perfluoro-n-octane, perfluorodecalin, sodium hyaluronate, toluene, formaldehyde, butane, propane, xylene, butyl alcohol, methyl ethyl ketone, acetone, ethylene glycol, isoprene, pinene, limonene, benzene, nitrobenzene , chlorobenzene, perchlorethylene (or tetrachlorethylene), trichlorethylene, hydroxypropylmethylcellulose, silicone oil, etc., which can be tested using the method of the invention.
    The chemical compound, preferably volatile, referred to in the present invention is a water insoluble and high molecular weight compound. The term "high molecular weight" refers to the compound, preferably volatile, having a molecular weight greater than that of the non-volatile aqueous medium of step (b). That is, the compound, preferably volatile, referred to in the present invention is not capable of floating in an aqueous medium, preferably in the non-volatile aqueous medium employed in the method of the present invention that is deposited above it. Thus, the compound, preferably volatile, to be studied remains during the method of the invention below the non-volatile aqueous medium deposited in step (b), that is, it does not float thereon. And in turn, said non-volatile aqueous medium will prevent evaporation of the compound, preferably volatile, located below. This enables direct and permanent contact (during the desired exposure time) of the compound, preferably volatile, with the cell or tissue culture. The method of the present invention therefore allows the cells or tissues in culture to be in direct contact with the samples of compound, preferably volatile, for a certain time of exposure. If the sample of compound, preferably volatile, is toxic or contains leachable toxic components, it will directly affect these cultures and their effect can be evaluated later.
    The term "cell culture" refers to a culture of eukaryotic or prokaryotic cells, preferably eukaryotes, more preferably mammalian (for example human, feline, canine, bovine, rodent or pig), even more preferably human. The three types of cells: cells derived from ectoderm, mesoderm and endoderm, can be used in the method of the invention. The preferred cell lines for the cell culture of step (a) are those recommended by ISO standards (ISO 10993-5: 2009), for example, but not limited to, the CCL 1 cell lines (clone L929 of the NCTC; fibroblasts of mouse), CCL 163 (clone A31 of Balb / 3T3; mouse fibroblasts), CCL 171 (MRC-5; human fibroblasts), CCL 75 (WI-38; human fibroblasts), CCL 81 (Vero; primate epithelial cells non-human) or CCL 10 [BHK-21 (C-13); hamster fibroblasts] and V-79 379A (hamster fibroblasts) of the American Type Culture Collection. However, primary cell and tissue cultures obtained directly from living tissues are also recommended to perform the method of the invention.
    The term "tissue culture" includes from organ culture to the culture of isolated tissues. All types of tissues: tissues derived from ectoderm, mesoderm and endoderm, can be used in the method of the invention, for example, without limiting ourselves, epithelial tissue, eye tissue, muscle tissue, nerve tissue, bone tissue, connective tissue, connective tissue, etc.
    Preferably the cells and tissues used in the cell or tissue culture of step (a) are cells and tissues from the area of the body in which the medical device is intended to be applied or with which said medical device will be in contact. The evaluation of medical devices on the types of cells and tissues where they are routinely applied in clinical practice provides more guarantees about their biocompatibility, and therefore, about their safety.
    In a preferred embodiment of the method of the invention, the cell or tissue culture comprises neurorretin explants and / or retinal pigment epithelial cells (RPE). In a more preferred embodiment, neurorretin explants and retinal pigment epithelium cells are of human origin. In an even more preferred embodiment, the EPR cells are cells of the ARPE-19 cell line (ATCC CRL-2302), which is a cell line generated spontaneously from human EPR. These cells form a stable monolayer that shows morphological and functional polarity. ARPE-19 cells express specific EPR markers, such as CRALBP and RPE-65, and exhibit morphological polarization when seeded in Transwell-COL filters coated with laminin and in a culture medium with a low serum concentration. These cells form tight junctions in monolayers with transepithelial resistance. Likewise, other sources of EPR cells and other retinal cells in culture, such as, but not limited to, photoreceptors, ganglionics, Müller, etc., and of not only modified human origin, but also fresh and other animals, may also be used. in the embodiment of the method of the invention.
    Preferably, the neuroretin explants referred to in the present invention are explants obtained under laboratory conditions from living tissue.
    The term "neurorretin explants" refers to a neurorretin tissue, that is, a tissue obtained from the innermost layer, in relation to the position of the pigmentary epithelium, of the two parts of the retina. Neuroretin is the layer of the retina from which the nerve impulse caused by photons starts. It consists of nine layers, which from outside to inside are: layer of cones and rods, external limitation, external nuclear, external plexiform, internal nuclear, internal plexiform, ganglion cells, nerve fibers and internal limitation. Neuroretine explants can be grown in the presence of culture media and conditions known in the technical field for explaining eye tissue explants. Thus, the culture medium may comprise, for example, but not limited to, fetal bovine (FBS) or human serum, antibiotics, antifungals, growth factors, etc. The base medium that can be used in the culture medium could be any of those known in the state of the art for tissue culture in vitro, such as, but not limited to, Neurobasal A medium.
    The "pigment epithelium of the retina" or "EPR" is the layer of pigmented cells located on the outside of the retina that interacts closely with the photoreceptor cells (cones and rods) in maintaining visual function. It is firmly anchored to the underlying choroid by Bruch's membrane. The retinal pigmentary epithelium is composed of a layer of hexagonal cells that are densely packed with pigment granules. Seen in section, each cell consists of a non-pigmented outer part in which a large and oval shaped core is located and a pigmented inner portion that extends a series of straight filiform processes between the rods. It serves as a limiting factor of the transport that maintains the environment of the retina, providing small molecules such as amino acids, ascorbic acid and D-glucose, while representing a narrow barrier for substances carried by the choroid's blood. The retinal pigment epithelium also has the function of phagocytosis of the external segments of the photoreceptor cells and regeneration of the photopigment.
    "Retinal pigment epithelium cells" or "EPR cells" means any cell type present in said epithelium, preferably epithelial cells. EPR cells can be cultured in the presence of culture media and conditions known in the technical field for epithelial cell culture. Thus, the culture medium may comprise, for example, but not limited to, fetal bovine (FBS) or human serum, antibiotics, antifungals, growth factors, etc. The base medium that can be used in the culture medium could be any of those known in the state of the art for in vitro cell culture, such as, but not limited to, basal medium "Eagle", CRCM-30, CMRL -1066, “Dulbecco's Modified Eagle's Medium” (DMEM), “Eagle´s Minimum Essential Medium” (EMEM), “Fischer's Medium”, “Glasgow Minimum Essential Medium”, Ham's F-10, Ham's F-12 (F12), “High Cell Density Medium”, “Iscove's Modified Dulbecco's Medium”, Leibovitz's L-15, McCoy's 5A, medium 199, “Minimum Essential Medium Eagle”, “Alpha Minimum Essential Medium”, CnT20, NCTC 109, NCTC 135, RPMI-1640 , "William's Medium E", Waymouth's MB 75211, Waymouth's MB 70511, "Keratinocyte serum-free medium" (KSFM), or any combination thereof. Preferably, the culture medium comprises a DMEM / F12 base medium, supplemented or not with FBS and in the presence of antibiotics and antifungals. In addition, the culture conditions can be, for example, but not limited to, standard culture conditions, that is, in the presence of between 5 and 10% CO2, between 36 and 38 ° C and renewing the medium every 48 to 72h. Preferably, said culture is carried out as described below in the examples of the present invention.
    In vitro cell or tissue culture in the method of the present invention is preferably carried out, as indicated by the guidelines of the ISO standards (UNE-EN ISO 10993), more preferably following the culture plate preparation protocol.
    In the method of the invention a product, preferably an aqueous, non-volatile medium is deposited on the compound, preferably volatile, to be studied to prevent the latter from evaporating and disappearing before the end of the exposure time.
    The term "non-volatile aqueous medium" refers to a liquid medium without the ability to evaporate easily into the atmosphere at room temperature, which in turn prevents the evaporation of the preferably volatile compound to be studied once deposited thereon. The compound, preferably volatile, to be studied is insoluble in said medium. This medium will preferably be water or any aqueous medium in which the compound, preferably volatile, to be studied does not dissolve. The non-volatile aqueous medium of the present invention does not contain elements or components that may compromise cell or tissue viability or growth or that may affect the physicochemical properties of the compound, preferably volatile, object of study. That is, the components comprised in the non-volatile aqueous medium of the present invention will be safe for both cell or tissue culture and for the compound, preferably volatile, to be studied. Said aqueous medium preferably has an alkaline character. In another preferred embodiment of the method of the invention, the non-volatile aqueous medium of step (b) is a culture medium with the same or similar composition as that used in cell or tissue culture of step (a) of the method.
    The amount of compound, preferably volatile, to be studied and non-volatile aqueous medium depend on the surface of the support or the volume of the container in which the cells or tissues are being grown, and also it should be taken into account that the amount thereof It should not generate a weight / pressure that will crush the cells or tissues in culture. To avoid this problem, negative controls such as those described below will preferably be used in the method of the invention.
    In another preferred embodiment of the method of the invention, the culture of step (a) is a cell culture, the amount of compound, preferably volatile, deposited in step
    (a) is 80 µl or any other amount that can cover the layer of cells in culture and the amount of nonvolatile aqueous medium deposited in step (b) is 100 µl or any other amount that can cover the layer of compound deposited in the step (a).
    In another preferred embodiment of the method of the invention, the culture of step (a) is a tissue culture, the amount of compound, preferably volatile, deposited in step (a) is 500 µl or any other amount that can cover the layer of tissue in culture and the amount of nonvolatile aqueous medium deposited in step (b) is 500 µl or any other amount that can cover the layer of compound deposited in step (a).
    Incubation of the cell or tissue culture of step (b) in the presence of the compound, preferably volatile, and of the non-volatile aqueous medium, as described in step (c) of the method of the present invention, is performed during an exposure time determined (desired and preferably based on the time during which the compound will be in contact with cells and tissues in the organism, preferably human, during actual clinical practice) and preferably in the presence of appropriate conditions to allow cell growth or proliferation or tissue. Said conditions and culture time will depend on the cell or tissue type chosen for the culture. Those skilled in the art will recognize said conditions and incubation time applicable in each case, although preferably the incubation of step (c) is carried out for at least 30 minutes.
    In a particular embodiment of the method of the invention, for the cell culture of the passage
    (a) the cells are seeded in a multiwell plate and the cells of the second column are destined on the right and the second column on the left as controls to assess the quality of the analysis. In accordance with the guidelines of the ISO standard (UNE-EN ISO 10993-5: 2009 Biological evaluation of medical devices, Part 5: In vitro cytotoxicity tests; Annex C: MTT toxicity test) the average value of the optical density ( OD570) of the control groups of the second column on the right and left should be ≥ 0.2 and the difference between the average value OD570 of the controls of the second column on the right and left should not be greater than 15% of the total average of both control columns.
    Thus, in another preferred embodiment, the method of the invention is performed on a multiwell plate in which at least two columns of the plate comprise control cell or tissue cultures. In a more preferred embodiment, the average value of the optical density (OD570) of the control cell cultures of the two columns is at least 0.2. In an even more preferred embodiment, the difference between the OD570 average value of the control cell cultures of the two columns is equal to or less than 15% of the total average of both columns. In the case of control tissue cultures, fresh tissue from the day of isolation and preparation of the experiments is preferably applied to evaluate the quality of the analysis.
    Said control cultures comprise cells of the same cell type and the same origin as the cells grown in step (a) of the method of the present invention, or tissue of the same type and same origin as the tissue grown in step (a) of the method of The present invention. Said cells and tissue of the control cultures are incubated under the same conditions of time, temperature, pH, light / dark cycles, humidity, CO2 concentration, composition of culture medium, etc., as the culture under study of the step ( a), but said control cultures are not exposed to the compound, preferably volatile, added in step (a). The control cultures will also be processed in the same manner as the cultures of step (a) after the incubation of step (c).
    In a more preferred embodiment, the control cultures comprise cells or tissues not exposed to any compound (blank), cells or tissues exposed to a non-toxic compound of molecular weight equal to that of the compound, preferably volatile, deposited for study (negative control) or cells or tissues exposed to a toxic control compound (positive control). The negative control comprises a non-toxic compound of molecular weight equal to that of the compound, preferably volatile, to be studied to help determine that the effects on the cells or tissues are due to the compound, preferably volatile, object of study and not to damage caused by crushing due to the weight / pressure exerted by the deposited substances.
    In the ISO standard (UNE-EN ISO 10993-12: 2007 Biological evaluation of medical devices, Part 12: Preparation of samples and reference materials) it is mentioned that materials whose effects have been shown to be reproducible can be considered positive controls ( reproducible cytotoxic effect) and negative controls (reproducible non-cytotoxic effect). In this way the state of the cell or tissue culture, that is, the response of the cells or tissues to the stimulus, can be determined to perform the evaluation by direct contact. In an even more preferred embodiment, the toxic control compound is selected from the list, mentioned in the ISO standard, which consists of: PVC-org. Sn, SPU-ZDEC, SPU-ZBEC, natural latex and polyurethane or phenol. However, any other chemical that generates reproducible results can be applied. In a particular embodiment, the toxic control compound is phenol.
    Prior to step (a) of the method, the cells and tissues under study as well as the controls can be cultured in a multiwell plate, preferably for at least 7 days or until the average optical density value (OD570) of cultures of at least 0.2 in the case of cells, and then the culture medium is removed and incubated again with incomplete medium (without serum but with antibiotics and antifungals) under standard culture conditions for preferably 24 hours to Synchronize the cell cycle. In the case of tissues, isolation is done and fresh tissue is preferably prepared on the same day as the start of the experiments. After this culture phase, the compound, preferably volatile, is deposited on the culture of each well (with the exception of the controls) so that it is in direct contact with the cells or tissues, as described in step (a ) of the method of the invention. Next, the non-volatile aqueous medium (preferably culture medium) is deposited on the samples of compound, preferably volatile, deposited and the samples are incubated for, preferably, at least 30 minutes. This 30-minute exposure time may vary by adjusting to the time during which the compound under study would be in contact with cells and tissues in the body, preferably human, during actual clinical practice.
    The toxicity data of the samples studied are always comparable with the data of the cultures not exposed to any compound (blank), preferably following the following formula (for the case of cells) or table 1 (for the case of tissues) .
    In the case of cell culture;
    = 100 × OD570e
    .%
    570�
    According to UNE-EN ISO 10993-5 "C.2.5 Data analysis"; OD570e is the average value of the optical density measured in the test samples and OD570b is the average value of the optical density measured in the targets.
    Thus, in an even more preferred embodiment, the cytotoxicity of the cell culture studied is compared with the cytotoxicity of the control cultures comprising cells not exposed to any compound following the formula:
    ∗
    5 Via%. =
    �
    OD570e being the average value of optical density in the cell culture studied and OD570b the average value of optical density in the control culture comprising cells not exposed to any compound.
    10 And in the case of tissue culture;
    Subjective qualitative graduation of tissue / cell modifications in neuroretin explants. Table configured from the Spanish Standard UNE-EN ISO 10993
    15 5: 2009 “Table 1 - Qualitative morphological evaluation of the cytotoxicity of the extracts”.
    Grade DegenerationCrop conditions
    0 AnyAbsence of degenerative changes in neurorretin explants
    one LightPresence of slight degenerative changes, such as: partial absence of the external segments of the photoreceptors and initiation of tissue vacuolization
    2 MildPresence of mild degenerative changes, such as: fragmentation and loss of the external segments of the photoreceptors; edema of the internal segments of the photoreceptors; rosette formation of degeneration; reduction of the number of nuclei in the nuclear layers; alteration of the plexiform layers; presence of pycnotic nuclei; and tissue vacuolization
    3 ModeratePresence of moderate degenerative changes, such as: absence of the internal segments of the photoreceptors; alteration and reduction of plexiform layers; loss of retinal parenchyma; disorganization of the retinal structure; reduced
    number of cell nuclei and picnosis present; and marked tissue vacuolization throughout the retinal parenchyma
    4 SeverePresence of severe degenerative changes, such as: total loss of retinal structure; marked loss of cell nuclei; and marked loss of retinal parenchyma
    For "the evaluation of the effect produced by the presence of the compound, preferably volatile, deposited on the culture" one or 5 several parameters associated with the determination of cell viability, growth, proliferation and / or survival can be detected or quantified in said culture. associated to the determination of the damage in the morphology of the cells or in the anatomical structure or morphology of the tissue in culture. The measurement of these parameters is preferably performed following the standard cytotoxicity protocols described in the ISO standards (UNE-EN ISO 10993-5: 2009 Biological evaluation of medical devices, Part 5: In vitro cytotoxicity tests; UNE-EN ISO 10993-5: 2009 "Table 1-Qualitative morphological evaluation of the cytotoxicity of extracts"). In the case of cell cultures, an MTT cytotoxicity test can be carried out, for example, but not limited to following the guidelines of the ISO standards where cell growth will be evaluated (UNE-EN ISO 10993-5: 2009 Biological evaluation of medical devices, Part 5: In vitro cytotoxicity tests; Annex C: MTT toxicity test). In addition, the damage produced can be evaluated by checking the morphology of the cells in cultures by microscopy during the time of cell growth. A significant change in the appearance of the crop can be detected compared to the control crop. In the case of tissue cultures, you can
    20 Perform, for example but not limited to, sample fixation, processing and staining with toluidine blue and subsequent visual evaluation of tissue morphology under a microscope.
    In a more preferred embodiment of the method of the invention, in step (d) cell proliferation and / or survival or tissue morphology is evaluated.
    25 When in step (d) significant changes in cell proliferation and / or survival or in tissue morphology are detected with respect to the "control" culture not exposed to the compound, preferably volatile, added in step (a) of the method of the invention, said compound, preferably volatile, is classified as cytotoxic. When in step (d) no significant changes in cell proliferation and / or survival or tissue morphology are detected with respect to the "control" culture not exposed to the compound, preferably volatile, added in step (a) of the method of the invention, said compound, preferably volatile, is classified as non-cytotoxic.
    In another preferred embodiment of the method of the invention, this comprises an optional additional step (c`) after the incubation step (c) in which the cultures are washed, preferably with PBS or culture medium, to remove the Compound residues, preferably volatile, are added fresh culture medium and incubated again for a certain time, preferably for at least 24 hours in the case of cells in culture or for at least 72 hours in the case of tissues in culture, under conditions cultivation standard After this additional step (c`), the effect of the compound, preferably volatile, is evaluated as described in step (d).
    In another preferred embodiment of the method of the invention, this further comprises a step (e) in which the compound, preferably volatile, deposited in step (a) is recovered. The insolubility of the compound, preferably volatile, in aqueous media facilitates a clear labeling of the evaluated samples of said compound in contact with the cells. The sample of compound, preferably volatile, is insoluble in the culture medium, which helps to clearly and visibly mark the layer with the sample of the product under study. Once the compound, preferably volatile, is recovered and collected in step (e), it can be reused in new studies and analyzes.
    Volatile compounds are usually found in industrial sectors, such as, but not limited to, the steel, plastics and rubber industry, footwear, graphic arts, paints, varnishes, lacquers, solvents, repellents, glues, dispersants , degreasers, cleaning products, flavorings, perfumery and aerosols, food, wood, cosmetics, fracking, and pharmaceutical, this being the most relevant with respect to the invention presented here. On the other hand, also included, but not limited to, the products used as electrical, thermal or acoustic insulators and semi-finished plastic materials, in the form of sheets, plates or bars, as well as rubber, gutta-percha, rubber, asbestos, mica and products of these materials not included in other classes, products of semi-finished plastic materials, materials used for caulking, closing with tow and insulating, non-metallic flexible tubes, etc. Without ruling out volatile products that are released as a result of burning fossil fuels, such as gasoline, coal or natural gas, or burning wood. Thus, the volatile compounds evaluated in the method of the present invention can be any of those present in said industry sectors, or in any other sector in which volatile compounds are also employed or released.
    In another preferred embodiment of the method of the invention, the compound, preferably volatile, deposited in step (a) is a compound used in the manufacture of medical devices or medical devices.
    The term "medical device" refers to any instrument used in the biosanitary or clinical setting, both in medicine and veterinary medicine, which is in direct or indirect contact with the patient. This includes any instrument, apparatus, device, biomedical equipment, reagent for diagnosis or supervision or other similar or related article, used alone or in combination, including its components, parts and accessories involved in its correct application, intended by the manufacturer for use in humans or non-human animals, preferably in humans. This term includes the devices used for the treatment, relief, diagnosis, prognosis, prevention, supervision, monitoring or monitoring, investigation and / or surgery of the patient, including the devices intended for the replacement, modification or support of the anatomical structure of a tissue or of a physiological process or the devices used in the regulation of conception. Thus, prostheses or implantable devices in the human or animal body intended for the treatment of some pathological condition or for tissue regeneration are included within this term.
    Some examples in the context of the present invention are, but not limited to:
    - Chemical medical devices for medical use: dietary substances for medical use, baby food; plasters, dressing material; material for filling teeth and for dental molds; disinfectants; products for the destruction of harmful animals; fungicides, herbicides.
    - Apparatus and instruments for diagnostic or therapeutic use (surgical or medical, including those for use in dentistry and veterinary medicine), coated with chemicals that may come into contact with skin or mucous membranes, eyes and artificial teeth; orthopedic items; as well as suture material, syringes, needles, gauze, probes, bandages, gloves, especially those used in surgery, pacemakers, bone matrix implants, prostheses, contact lenses, etc.
    - As well as any medical device or medical device that contains chemicals that may come into contact with cells and animal tissue, preferably human.
    In a more preferred embodiment, the medical devices referred to in the present invention are used in surgery, even more preferably in ophthalmic surgery.
    The term "ophthalmologic surgery" refers to any surgical technique carried out in any ocular structure, such as but not limited to, refractive surgery, vitreoretinal surgery, glaucoma surgery, cataract treatment surgery, among others. Vitreoretinal surgery is an ocular intervention to treat diseases that affect the vitreous and the retina. This type of disease or trauma causes a detachment of the retina, that is, a tear of the retina and accumulation of fluid in the vitreous cavity under the retina. Thus, vitreoretinal surgery is applied, but not limited to, patients with retinal detachment, penetrating eye trauma, giant retinal tear / s, proliferating vitreoretinopathy (VRP) or cataract surgery complications.
    In another preferred embodiment of the method of the invention, the volatile compound is a manufactured polymer.
    The term "manufactured polymer" refers to polymers formed from natural or synthetic polymers. On the other hand, polymers are macromolecules formed by the union of smaller molecules called monomers. Some of the natural polymers from which manufactured polymers are generated are starch, cellulose or silk and some of the synthetic polymers from which manufactured polymers are generated are nylon, polyethylene or bakelite. Examples of manufactured polymers are, but are not limited to, perfluoro-n-octane, perfluorodecalin or sodium hyaluronate (viscoelastic solution) among others, which are used as medical devices.
    In a more preferred embodiment, the volatile compound is perfluoro-n-octane.
    The term "perfluoro-n-octane" or "octadecafluorooctane" refers to liquid fluorocarbon, a perfluorinated derivative of octane hydrocarbon, whose molecular formula is C8F18, with a molar mass of 438.06 g / mol, a melting point of - 13ºF (-25ºC) and a density of 1.77 g / cm3. This polymer is applied in ophthalmology, particularly in vitreoretinal surgery, acting as a retinal manipulator due to both its good physical properties and its good adhesion to the retina. Therefore, this polymer comes into contact with the patient's eye cells and tissues during vitreoretinal surgery, therefore it is necessary to assess its toxicity. The pigment epithelium of the retina is one of the layers of the retina that is exposed to perfluoro-noctane during such surgery, in the event that ruptures in the neuroretin occur. Therefore, in the method of the invention preferably EPR cells of human origin are used, since they are considered the most appropriate cells for the evaluation of the cytotoxicity of this volatile chemical. In vitreoretinal surgery, neuroretin is also exposed to perfluoro-n-octane, therefore, neuroretin explants are also appropriate for the evaluation of the cytotoxicity of this type of medical device by the method described in the present invention. Several companies in the market market this perfluoro-n-octane product with different names such as Perfluoron®, Okta-line ™, Arcotane (ARCADOPHTA®), Ala®Octa, F-Octane (FLUORON®) and Bio Octane ™ among others.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    DESCRIPTION OF THE FIGURES Fig. 1. Design diagram of the method of the invention. 1) Culture plate, 2) cells
    or tissues 3) culture medium, 4) sample to be tested.
    Fig. 2. Culture plates showing the method of the invention designed to perform the evaluation by direct contact. A) perfluoro-n study sample
    octane in a well of a 96-well plate. B) culture medium on a perfluoro-n-octane study sample in a well of a 96-well plate. C) culture medium on a perfluoro-n-octane study sample in a well of a 12-well plate. D) culture medium in a well of a 12-well plate.
    Fig. 3. The volatile perfluoro-n-octane sample (lower layer) was collected after the exposure time on the cell culture or on the tissue section.
    Fig. 4. Proliferation of cell culture of ARPE-19 (A) and L929 (B) cells. Cell cultures were in contact with toxic and non-toxic perfluoro-n-octane (PFO) samples for 30 minutes. They were incubated with culture medium for 24 hours under standard culture conditions of 37 ° C and 5% CO2. Next, an MTT test was performed following guidelines of the ISO standard, the results obtained are presented in this graph. The percentage of cell viability is represented on the Y axis, and the different treatments are represented on the X axis.
    Fig. 5. Morphology of neurorretin explants. The neurorretin explants were in contact with toxic and non-toxic perfluoro-n-octane samples for 30 minutes. They were incubated with culture medium for 72 hours under standard culture conditions of 37 ° C and 5% CO2. Next, the morphology of each neurorretin explant was analyzed by a toluidine blue stain and the results are presented following the guidelines of the ISO standard. It is shown: the morphology of neurorretin explants in time 0 without incubation with culture medium, the morphology of neurorretin explants not exposed to perfluoro-n-octane and incubated for 72 hours, the morphology of neuroretin explants exposed to non-toxic samples of perfluoro-n-octane and incubated for 72 hours and the morphology of neuroretin explants exposed to toxic perfluoro-n-octane samples and incubated for 72 hours. EXAMPLES
    Next, the invention will be illustrated by tests carried out by the inventors that demonstrate the effectiveness of the described method for evaluating the cytotoxicity of chemical compounds, preferably volatile, particularly those comprised in medical devices, on ARPE-19 cell cultures, L929 and neurorretin explants.
    Example 1. Evaluation of the proliferation of a retinal pigment epithelial cell culture (ARPE19 cell line) treated with toxic and non-toxic perfluoro-noctane samples.
    The cell line was obtained from the American Tissue Culture Collection (ATCC). After thawing, the cells were cultured in culture medium [(Dulbecco's Modified Eagle Medium and F12 Nutrient Mixture (Ham)] supplemented with 10% fetal bovine serum and 1% antibiotic and antifungal (100 U / ml Penicillin, 100 μg / ml of Streptomycin and 0.25 μg / ml of Amphotericin B) The cell culture was maintained in 75 cm2 bottles under standard culture conditions of 37 ° C and 5% CO2, renewing the culture medium every 2 days and by subculturing the cells when they reached a confluence of 80-90% When the cells reached said confluence, they were trypsinized and counted using trypan blue staining, then seeded in a 96-well plate at an optimum density 1x104 cells / well and incubated for 7 days with complete medium under standard conditions of 37 ° C and 5% CO2, renewing the culture medium every two days, then the complete culture medium was removed and the cells were incubated with incomplete culture medium (without serum but with 1% antibiotic and antifungal) for 24 hours under standard culture conditions, in order to synchronize the cell cycle. Subsequently, 80 µl of the toxic and non-toxic perfluoro-n-octane samples were deposited on the cell culture in the corresponding wells, to expose the cells directly to the perfluoro-n-octane. Then 100 µl of culture medium was deposited on the perfluoro-n-octane samples in the wells with the samples, and 180 µl of culture medium in the unexposed wells (Fig. 1). Cells were incubated for 30 minutes and, after this time, perfluoro-n-octane samples and culture medium were removed from each well. The cells were washed twice with PBS (it is also possible to do it with culture medium) and again incubated with fresh culture medium for 24 hours under standard conditions. Subsequently, cell growth in each well was determined following the MTT cytotoxicity test protocol, in accordance with the recommendations of the ISO standard [UNE-EN ISO 10993-5: 2009 Biological evaluation of medical devices, Part 5: Cytotoxicity tests in vitro; Annex C: MTT toxicity test].
    1.1. Results The results showed that perfluoro-n-octane samples are unable to evaporate when culture medium is deposited on them (Figure 2B and C). After the exposure time, the samples could be collected for later analysis (Figure 3). The cell culture exposed to the non-toxic sample has a viability greater than 70% when compared to the viability of the unexposed cell culture (100%) [(Figure 4A; first column (cells not exposed to the sample), second and third columns (cells exposed to non-toxic samples)] Thus, it is confirmed that the perfluoro-n-octane sample does not crush the cell culture (Figure 4 A; second and third columns) when an amount of 80 µl of the product is deposited The cell culture exposed to the toxic samples presents a viability of less than 70% when compared with the non-exposed cell culture and with the non-toxic samples (Figure 4 A; fourth and fifth columns). The data analysis also confirms that the average of the OD570 of the unexposed white group is ≥ 0.2 after ≥ 7 days of culture, and that the difference of the average of the OD570 of the right and left white groups does not differ by more than 15% of the average value of all targets s This confirms that the experiments comply with ISO standards.
    Example 2. Evaluation of the proliferation of a fibroblast cell culture (L929 cell line) treated with toxic and non-toxic perfluoro-n-octane samples.
    The L929 cell line was obtained from the American Tissue Culture Collection (ATCC). After thawing, the cells were cultured in culture medium [(Eagle's Minimum Essential Medium (EMEM) (ATCC® 30-2003 ™)] supplemented with 15% fetal bovine serum and 1% antibiotic and antifungal (100 U / ml of Penicillin, 100 μg / ml of Streptomycin and 0.25 μg / ml of Amphotericin B) The cell culture was maintained in 75 cm2 bottles under standard culture conditions of 37 ° C and 5% CO2, renewing the culture medium every 2 days and subculturing the cells when they reached a confluence of 80-90% When the cells reached that confluence, they were trypsinized and counted using trypan blue staining, then seeded in a 96-well plate at an optimum density of 1x104 cells / well and incubated for 1 day with complete medium under standard conditions of 37 ° C and 5% CO2, then the complete culture medium was removed and the cells were incubated with incomplete culture medium (no serum but co n 1% antibiotic and antifungal) for 24 hours under standard culture conditions, in order to synchronize the cell cycle. Subsequently, 80 µl of the toxic and non-toxic perfluoro-n-octane samples were deposited on the cell culture in the corresponding wells, to expose the cells directly to the perfluoro-n-octane. Then 100 µl of culture medium was deposited on the perfluoro-n-octane samples in the wells with the samples, and 180 µl of culture medium in the unexposed wells. Cells were incubated for 30 minutes and, after this time, perfluoro-n-octane samples and culture medium were removed from each well. The cells were washed twice with PBS (it is also possible to do it with culture medium) and again incubated with fresh culture medium for 24 hours under standard conditions. Subsequently, cell growth in each well was determined following the MTT cytotoxicity test protocol, in accordance with the recommendations of the ISO standard [UNE-EN ISO 10993-5: 2009 Biological evaluation of medical devices, Part 5: Cytotoxicity tests in vitro; Annex C: MTT toxicity test].
    2.1. Results
    The results showed that perfluoro-n-octane samples are unable to evaporate when culture medium is deposited on them (Figure 2B and C). After the exposure time, the samples could be collected for later analysis (Figure 3). The cell culture exposed to the non-toxic sample has a viability greater than 70% when compared to the viability of the unexposed cell culture (100%) [(Figure 4B; first column, cells not exposed to the sample), second and third columns (cells exposed to non-toxic samples)]. Thus, it is confirmed that the perfluoro-n-octane sample does not crush the cell culture (Figure 4B; second and third columns) when an amount of 80 µl of the product is deposited. The cell culture exposed to the toxic samples has a viability of less than 70% when compared with the non-exposed cell culture and with the non-toxic samples (Figure 4B; fourth and fifth columns). The analysis of the data also confirms that the mean of the OD570 of the unexposed white group is ≥ 0.2 after ≥1 day of culture, and that the difference of the average of the OD570 of the right and left white groups does not differs by more than 15% from the average value of all whites. This confirms that the experiments comply with ISO standards.
    Example 3. Evaluation of alterations in the structure of neuroretin explants treated with toxic and non-toxic perfluoro-n-octane samples.
    The porcine eyeballs were dissected within two hours after enucleation to obtain the neuroretin explants. The explants were placed in a 12-well plate, and then 500 µl of the toxic and non-toxic perfluoro-n-octane samples were deposited on them, thus exposing the neuroretins directly to the medical device. 500 µl of culture medium (Neurobasal A medium supplemented with 10% fetal bovine serum, 1% antibiotic and antifungal, 1% L-Glutamine and 2% B-27) were deposited on the perfluoro-n- samples octane to prevent evaporation. 1 ml of culture medium was deposited on the neuroretin explants for the group of explants not exposed to perfluoro-n-octane. After 30 minutes, the perfluoro-n-octane and culture medium samples were removed from each well. The neuroretin explants were washed with fresh culture medium and transferred to a 12-well plate with Transwell® membranes. Neuroretin explants were deposited on said Transwell® membranes so that the photoreceptor layer was in contact with the membrane. Each well was filled with culture medium so that it reached the explants but they were not able to float. The plates with the explants were incubated for 72 hours under standard conditions of 37 ° C and 5% CO2 by renewing the culture medium daily. At the same time (0 hours), one of the explants obtained was fixed and stored to be processed and to observe the morphological changes in the neurorretin explants without being cultured. After 72 hours of culture, the neuroretin explants were fixed and stored for processing. The neurorretin explanes are processed and stained with toluidine blue. Finally, they were observed under a microscope to assess their morphology. The results are shown in figure 5.
    3.1. Results
    The results showed that perfluoro-n-octane samples are not able to evaporate when culture medium is deposited on the product (Figure 2B and C). After the exposure time, the samples could be collected for later analysis (Figure 3). Neuroretine explants show good morphology at zero time (Figure 5). The neurorretin explants that were exposed to the non-toxic perfluoro-n-octane samples show a morphology similar to that observed in the case of unexplained explants at 72 hours of culture (Figure 5). However, neurorretin explants exposed to toxic perfluoro-n-octane samples show deteriorated morphology at 72 hours of culture (Figure 5). Thus, it is confirmed that perfluoro-n-octane samples do not crush neurorretin explants and the exposure time of explants to perfluoro-n-octane samples can be regulated. The experiments comply with ISO standards.
    Thus, taking into account the results obtained in the previous examples, it can be concluded that:
    5 -The designed method does not allow perfluoro-n-octane, which is a highly volatile substance, to evaporate.
    - The designed method allows to regulate the exposure time of perfluoro-n-octane to cell and tissue cultures.
    10 -The designed method is able to detect the toxic effect of perfluoron-octane samples on EPR cell cultures and on neuroretin explant cultures.
    - The designed method facilitates the collection of perfluoro-n-octane samples after the exposure time for further analysis.
    权利要求:
    Claims (18)
    [1]
    1. Method for evaluating the cytotoxicity of chemical compounds, preferably volatile, comprising:
    to. Deposit a chemical compound, preferably volatile, on a cell or tissue culture in vitro,
    b.  Deposit a non-volatile aqueous medium on the compound deposited in step (a),
    C.  Incubate the cell or tissue culture obtained after step (b), and
    d.  Evaluate the effect produced by the presence of the compound deposited on the crop;
    where the deposited compound is insoluble in water and of high molecular weight.
    [2]
    2. The method according to claim 1, wherein the deposited compound is a compound used in the manufacture of medical devices or medical devices.
    [3]
    3. The method according to claim 2, wherein the medical devices are used in ophthalmic surgery.
    [4]
    Four. The method according to any one of claims 1 to 3, wherein the volatile compound is a manufactured polymer.
    [5]
    5. The method according to any of claims 1 to 4, wherein the volatile compound is perfluoro-n-octane.
    [6]
    6. The method according to any one of claims 1 to 5, wherein the cell or tissue culture comprises neurorretin explants and / or retinal pigment epithelium cells.
    [7]
    7. The method according to claim 6, wherein the neurorretin explants and retinal pigment epithelium cells are of human origin.
    [8]
    8. The method according to any one of claims 1 to 7, wherein the non-volatile aqueous medium of step (b) is a culture medium with the same composition as that used in the cell or tissue culture of step (a).
    [9]
    9. The method according to any of claims 1 to 8, wherein the step incubation
    (c) is carried out for at least 30 minutes.
    [10]
    10. The method according to any one of claims 1 to 9, wherein in step (d) cell proliferation and / or survival or tissue morphology is evaluated.
    [11]
    eleven. The method according to any one of claims 1 to 10, further comprising a step (e) in which the compound deposited in step (a) is recovered.
    [12]
    12. The method according to any one of claims 1 to 11, wherein said method is carried out in a multiwell plate in which at least two columns of the plate comprise control cell or tissue cultures.
    [13]
    13. The method according to claim 12, wherein the average value of the optical density (OD570) of the control cell cultures of the two columns is at least 0.2.
    [14]
    14. The method according to claim 13, wherein the difference between the average OD570 value of the control cell cultures of the two columns is equal to or less than 15% of the total average of both columns.
    [15]
    fifteen. The method according to any of claims 12 to 14, wherein the control cultures comprise cells or tissue not exposed to any compound, cells or tissue exposed to a non-toxic compound of equal molecular weight to that of the deposited compound to be studied or cells or tissue exposed to a toxic control compound.
    [16]
    16. The method according to claim 15, wherein the toxic control compound is selected from the list consisting of: PVC-org. Sn, SPU-ZDEC, SPU-ZBEC, natural latex and polyurethane or phenol.
    [17]
    17. The method according to any of claims 15 or 16, wherein the cytotoxicity of the cell culture studied is compared with the cytotoxicity of the control cultures comprising cells not exposed to any compound following the formula:
    ∗
    Viab.% =
    �
    OD570e being the average value of optical density in the cell culture studied and OD570b the average value of optical density in the control culture comprising cells not exposed to any compound.
    [18]
    18. The method according to any one of claims 1 to 17, wherein the amount of compound deposited in step (a) covers the layer of cells or tissue in culture and the amount of non-volatile aqueous medium deposited in step (b) covers the layer of compound deposited in step (a).
    Fig. 1
    Biological, physical and chemical analysis
    Fig. 2 Fig. 3 Fig. 4 (A) Fig. 4 (B) Fig. 5
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    同族专利:
    公开号 | 公开日
    WO2017207845A1|2017-12-07|
    ES2859424T3|2021-10-04|
    EP3467118A4|2020-03-04|
    ES2644987B1|2018-10-15|
    EP3467118B1|2020-12-02|
    EP3467118A1|2019-04-10|
    引用文献:
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    IT201900003605A1|2019-03-12|2020-09-12|Al Chi Mi A S R L|METHOD FOR PERFORMING AN IN VITRO CYTOTOXICITY TEST IN OPHTHALMIC FIELD|
    WO2021094588A1|2019-11-15|2021-05-20|Vitrolife Sweden Aktiebolag|Use of perfluoro-n-octane for piezo-mediated intracytoplasmic sperm injection|
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