PL235678B1 - Optimized culture medium for neuron cultures, particularly for the primary cortical and thalamic neuron cultures - Google Patents

Abstract

The subject of the application is an optimized medium for growing neurons, preferably primary cortical and thalamic neurons, which contains lactate at a concentration of about 125 µM to about 6 mM and glucose at a concentration of about 3.0 to about 12.5 mM. The application also includes the use of a neuron culture medium and a method for growing neurons using an optimized neuron culture medium.

Description

Description of the invention

TECHNICAL FIELD

The invention relates to an optimized medium for the cultivation of primary cortical and thalamic neurons, its use for the cultivation of primary cortical and thalamic neurons, and a method of culturing primary cortical and thalamic neurons using the optimized neuronal culture medium of the invention.

STATE OF THE ART

Commercially available media for the cultivation of neurons are known. The available media is intended both for the cultivation of pre- and post-natal cells, including neurons derived from the hippocampus or cortex, and other regions of the brain, including the following mentioned by the trade name: Neurobasal® (Thermo Scientific), NbActiv4 (BrainBits), Primary Neuron Growth Medium (PNGM, Lonza), BrainPhys ™ Neuronal Basal Medium (Stemcell) and NeuroCult ™ Neuronal Basal Medium (Stemcell).

Neurobasal® [Brewer et al., 1993] is a medium based on DMEM medium intended for rapidly dividing cells (including cancer origin and cell lines immortalized in vitro), so it is not surprising that this medium is not optimal for survival and neuronal differentiation. Currently, the vast majority of scientists around the world cultivate neurons in the Neurobasal (Thermo Scientific) medium, which for many years has been treated as the so-called The "gold standard". However, Neurobasal is not an optimal medium for the cultivation of neurons as it has been shown to have excitotoxic effects on neurons in primary cultures [Hogins et al., 2011; Maggioni et al., 2015], which involves a pathological process in which neurons are damaged and killed by glutamate and similar chemicals. In addition, high glucose concentration in the medium causes a toxic effect on neurons in primary cultures [Russell et al., 2002; Peng et al., 2016]. Therefore, there is a need to create a new medium with an optimal composition that would allow for effective primary cultivation of neurons under physiological conditions that do not cause negative excitoxic and glucotoxic effects.

A known medium for the cultivation of neurons is NbActiv4 (BrainBits). NbActiv4 medium (BrainBits) is a modification of the medium Neurobasal (Thermo Scientific) composition. The nutrient solution for Neurobasal is additionally supplemented with cholesterol, creatine and estrogen. This modification increases the density of neuronal synapses and the number of neurotransmitter receptors in cultured neurons, which increases the number of spike potentials recorded. This medium has the following disadvantages: it is based on the formulation of the Neurobasal medium and still contains i.a. non-physiological amount of glucose (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC239354).

Other known neuronal culture media are NeuroCult ™ Neuronal Basal Medium (StemCell) and BrainPhys ™ Neuronal Medium (StemCell) (see http://www.pnas.org/conent / 112/20 / E2725.long). The composition of BrainPhys is similar to the cerebrospinal fluid. Has, among others reduced glucose content to a value of 2.5 mM. The NeuroCult and BrainPhys media have the following disadvantages: the most important of which is that primary neuronal culture requires two types of media - one to establish a culture (NeuroCult, which is a high toxic glucose concentration medium) and the other BrainPhys to maintain the culture for a longer period than 5 days.

DISCLOSURE OF THE INVENTION

In view of the described prior art, the object of the present invention is to overcome the indicated disadvantages and to provide a new optimized medium for the cultivation of neurons, especially primary cortical and thalamic neurons, and its use for the cultivation of primary cortical and thalamic neurons and a culture method using it.

The essence of the present invention is thus based on the unexpected finding that adding lactate to the medium suitable for the culture of neurons at a concentration of about 125 μM to about 6 mM, preferably about 125 μM, while lowering the glucose concentration to concentrations in the range of about 3.0 to about 12.5 mM, preferably up to about 3 mM, leads to an optimized neuron culture medium that provides the physiological conditions for maintaining the culture of neurons, especially primary cortical and thalamic neurons, and increases their viability compared to current high concentration solutions glucose.

Unexpectedly, it turned out that by adding lactate to the media suitable for the cultivation of neurons at a concentration of about 125 μM to about 6 mM while lowering the glucose concentration to a concentration of about 3.0 to about 12.5 mM, i.e. in the concentration ranges indicated for the optimized

When using the medium according to the invention, a synergistic effect (see Fig. 9) of the interaction of these two factors is observed, not observed for the action of adding lactate alone or only of lowering the glucose concentration. Such a synergistic effect of lactate and glucose interaction over certain concentration ranges has not been known or suggested in the art.

It is obvious that the optimized medium for the culture of neurons contains other nutrients in the concentration enabling the growth and development of neurons, including proteins, amino acids, lipids, in particular fats, waxes, sterols (including cholesterol), fat-soluble vitamins (A, D, E, K), monoacylglycerols, diacylglycerols, phospholipids, sugars, vitamins, micronutrients and macronutrients, in addition, it may advantageously contain stabilizing agents, buffering agents, antibacterial agents, antiviral agents, antifungal agents, dyes, vital factors, growth activators, osmolytes, etc.

The other ingredients and agents of the optimized medium for neuronal culture are preferably selected from glycine, alanine, arginine, asparagine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, glutamic acid, glutamine, choline chloride, calcium pantothenate, folic acid, niacin amide, pyridoxal hydrochloride, riboflavin, thiamine, thiamine hydrochloride, aspartic acid, vitamin B12, inositol, NaCI, KCI, MgSO4, CuSO4, FeSO4, NaH2PO4, CaCb, Fe (NO3) 3x9H2O, MgCb, KCI, NaHCO3, NaCI, NaH2PO4-H2O, ZnSO4-7H2O, HEPES, Na2SeO3, HEPES, biotin, alpha-tocopherol, alpha-tocopherol acetate, vitamin A, albumin, catalase, insulin, superoxide dismutase, corticosterone transfer , galactose, ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine, B-27 (ThermoScientific) supplement, SM1 (StemCell) supplement, IGA1000 + NSF-1 (Lonza) supplement, nutrients post-culture from astrocytes, phenol red, sodium pyruvate or mixtures thereof. A person skilled in the art is able to determine the appropriate components and concentrations of these components, for example based on the concentrations and components present in the currently known neuronal culture media NeuroCult ™ Neuronal Basal Medium (StemCell), BrainPhys ™ Neuronal Medium (StemCell), Neurobasal (ThermoScientific), PNGM (Lonza) by adjusting the glucose concentration as appropriate (eg Neurobasal A (Thermo Scientific) or BrainPhys (StemCell) as appropriate) and adding lactate.

The optimized medium for the cultivation of primary cortical and thalamic neurons according to the invention contains the optimal amount of glucose for the growth of neurons at a concentration identical to the conditions occurring naturally in the brain and lactate, which is used by nerve cells as an alternative energy source or signaling molecule, which act synergistically with each other. .

The optimized medium for the cultivation of primary cortical and thalamic neurons according to the invention increases the survival of neurons in relation to the previous known solutions, which allows for efficient cultivation of neurons. The optimized medium for the cultivation of primary cortical and thalamic neurons according to the invention makes it possible to carry out primary cultures and experiments on neurons under near-physiological conditions. Providing the physiological conditions for maintaining the primary neuron culture and increasing their viability in relation to the existing solutions based on high glucose concentration in the media, prevents the side effect occurring when using the media known in the art for culture, i.e. glucotoxicity, i.e. the toxic effect of glucose after chronic exposure to its high concentration. In addition, the medium contains a component that has not been used so far in other solutions - lactate, which is an alternative to glucose and pyruvate energy source for neurons or a signaling molecule. Known media for the cultivation of primary cortical and thalamic neurons do not contain lactate.

The invention therefore relates to an optimized medium for the cultivation of primary cortical and thalamic neurons both in the stage of establishing the primary culture of cortical or thalamic neurons and in the stage of maintaining the primary culture of cortical or thalamic neurons, which contains lactate in a concentration from 125 μΜ to 6 mM, glucose in a concentration from 3 0 to 12.5 mM.

In a preferred optimized neuronal culture medium, the lactate is in the form of sodium lactate and the glucose is in the form of D-glucose.

A preferred optimized medium for the cultivation of primary cortical and thalamic neurons contains sodium lactate at a concentration of 125 µM, D-glucose at a concentration of 3 mM.

A preferred optimized medium for the cultivation of primary cortical and thalamic neurons further comprises components selected from glycine, alanine, arginine, asparagine, cysteine, histidine,

Isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, glutamic acid, glutamine, choline chloride, calcium pantothenate, folic acid, niacin amide, pyridoxal hydrochloride, thiamin, riboflavin thiamine hydrochloride, aspartic acid, vitamin B12, inositol, NaCI, KCI, MgSO4, CuSO4, FeSO4, NaH2PO4, CaCb, Fe (NO3) 3x9H2O, MgCb, KCI, NaHCO3, NaCI, NaH2PO4-H2O, ZnSO4-7SeO3, NaH2PO4-H2O, ZnSO4-7SH2O, NaH2PO4 , biotin, alpha-tocopherol, alpha-tocopherol acetate, vitamin A, albumin, catalase, insulin, superoxide dismutase, transferrin, corticosterone, galactose, ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine, phenol red , sodium pyruvate or mixtures thereof.

A preferred optimized medium for the cultivation of primary cortical and thalamic neurons comprises glycine 0.4 mM; L-alanine 0.02247191 mM; L-arginine hydrochloride 0.39810428 mM; L-asparagine 0.0055333334 mM; L-cysteine 0.2603306 mM; 0.2 mM L-histidine hydrochloride; L-isoleucine 0.8015267 mM; L-leucine 0.8015267 mM; L-lysine hydrochloride 0.7978142 mM; L-methionine 0.20134228 mM; 0.4 mM L-phenylalanine; L-proline 0.06747826 mM; 0.4 mM L-serine; L-threonine 0.7983193 mM; L-tryptophan 0.078431375 mM; L-tyrosine, 0.39779004 mM; L-valine 0.8034188 mM; glutamic acid 0.0125 mM; L-glutamine 2.0 mM; choline chloride 0.028571429 mM; calcium pantothenate 0.008385744 mM; folic acid 0.009070295 mM; niacin amide 0.032786883 mM; pyridoxal hydrochloride 0.019607844 mM; riboflavin 0.0010638298 mM; thiamine hydrochloride 0.011869436 mM; vitamin B12 5.0184503E-6 mM; inositol 0.04 mM; CaCl2 1.8018018 mM; Fe (NOs) sx9H2O 2.4752476E-4 mM; MgCl2 0.8136842 mM; KCI 5.3333335 mM; NaHCOs 26,190,475 mM; NaCl 51.724136 mM; NaH2PO4-H2O 0.9057971 mM; ZnSO4-7H2O 6.736111E-4mM; D-glucose 3.125 mM; HEPES 10.92437 mM; phenol red 0.021519661 mM; sodium pyruvate 0.22727273 mM; sodium lactate 0.125 mM.

The optimized medium is preferably supplemented with astrocyte culture medium or a non-serum supplement, for example selected from the B-27 supplement (ThermoScientific), SM1 supplement (StemCell), IGA-1000 + NSF-1 supplement (Lonza).

The invention also relates to the use of an optimized medium according to the invention for the cultivation of primary cortical or thalamic neurons, the optimized medium containing lactate at a concentration of 125 μM to 6 mM, glucose at a concentration of 3.0 to 12.5 mM, wherein the optimized medium is used both for establishing primary culture of cortical or thalamic neurons, and for maintaining primary culture of cortical or thalamic neurons.

In a preferred use of an optimized medium for the cultivation of primary cortical or thalamic neurons, the optimized medium comprises sodium lactate at a concentration of 125 µM, D-glucose at a concentration of 3 mM.

In a preferred use of an optimized medium for the cultivation of primary cortical or thalamic neurons, the optimized medium further comprises components selected from glycine, alanine, arginine, asparagine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine. , valine, glutamic acid, glutamine, choline chloride, calcium pantothenate, folic acid, niacin amide, pyridoxal hydrochloride, riboflavin, thiamine, thiamine hydrochloride, aspartic acid, vitamin B12, inositol, NaCI, KCI, MgSO4, CuSO4, FeSO4, NaH2PO4, NaH2PO4, CaCI2, Fe (NO3) 3x9H2O, MgCI2, KCI, NaHCO3, NaCI, NaH2PO4-H2O, ZnSO4-7H2O, Na2SeO3, HEPES, biotin, alpha-tocopherol, alpha-tocopherol acetate, vitamin A, albumin, catalase, insulin, superoxide dismutase , transferrin, corticosterone, galactose, ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine, phenol red, sos pyruvate many or mixtures thereof.

In a preferred use of an optimized medium for the cultivation of primary cortical or thalamic neurons, the optimized medium comprises glycine 0.4 mM; L-alanine 0.02247191 mM; L-arginine hydrochloride 0.39810428 mM; L-asparagine 0.0055333334 mM; L-cysteine 0.2603306 mM; 0.2 mM L-histidine hydrochloride; L-isoleucine 0.8015267 mM; L-leucine 0.8015267 mM; L-lysine hydrochloride 0.7978142 mM; L-methionine 0.20134228 mM; 0.4 mM L-phenylalanine; L-proline 0.06747826 mM; 0.4 mM L-serine; L-threonine 0.7983193 mM; L-tryptophan 0.078431375 mM; L-tyrosine, 0.39779004 mM; L-valine 0.8034188 mM; glutamic acid 0.0125 mM; L-glutamine 2.0 mM; choline chloride 0.028571429 mM; calcium pantothenate 0.008385744 mM; folic acid 0.009070295 mM; niacin amide 0.032786883 mM; pyridoxal hydrochloride 0.019607844 mM; riboflavin 0.0010638298 mM; thiamine hydrochloride 0.011869436 mM; vitamin B12 5.0184503E-6 mM; inositol 0.04 mM; CaCl2 1.8018018 mM; Fe (NO3) 3x9H2O 2.4752476E-4 mM; MgCl2 0.8136842 mM; KCI 5.3333335 mM; NaHCO3 26, 190 475 mM; NaCl 51.724136 mM; NaH2PO4-H2O 0.9057971 mM; ZnSO4-7H2O 6.736111E-4mM;

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D-glucose 3.125 mM; HEPES 10.92437 mM; phenol red 0.021519661 mM; sodium pyruvate 0.22727273 mM; sodium lactate 0.125 mM.

In a preferred use of an optimized medium for the cultivation of primary cortical or thalamic neurons, a fraction of the volume of the culture medium is exchanged during cultivation for an optimized medium as used in the application described. More preferably, in the use of an optimized medium for the cultivation of primary cortical or thalamic neurons, the replacement of a part of the volume of the optimized medium occurs after 34 days from the day of culture start, preferably the step of replacing the optimized medium is repeated many times every 3-4 days.

More preferably, when using an optimized medium for the cultivation of primary cortical or thalamic neurons, the optimized medium is replaced in an amount from about 1/3 to about 2/3 of the volume of the culture medium, more preferably half the volume of the culture medium is replaced with the optimized medium. as used in the application described.

The invention also relates to a method of culturing primary cortical and thalamic neurons, which comprises the steps of:

a) a primary culture of cortical or thalamic neurons is assumed on an optimized lactate and glucose-containing medium,

b) the primary culture of cortical or thalamic neurons is maintained on an optimized lactate and glucose-containing medium, where the optimized medium used in step a) and b) contains lactate in a concentration from 125 μΜ to 6 mM, glucose in a concentration from 3.0 to 12, 5 mm.

In a preferred method of culturing primary cortical and thalamic neurons, the optimized medium used in step a) and / or b) comprises sodium lactate at a concentration of 125 µM, D-glucose at a concentration of 3 mM.

In a preferred method of culturing primary cortical and thalamic neurons, the optimized medium used in step a) and / or b) further comprises components selected from glycine, alanine, arginine, asparagine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, glutamic acid, glutamine, choline chloride, calcium pantothenate, folic acid, niacin amide, pyridoxal hydrochloride, riboflavin, thiamine, thiamine hydrochloride, aspartic acid, vitamin B12, inositol, NaCl, KCI , CuSO4, FeSO4, NaH2PO4, CaCI2, Fe (NO3) 3x9H2O, MgCl2, KCI, NaHCO3, NaCI, NaH2PO4-H2O, ZnSO4-7H2O, Na2SeO3, HEPES, biotin, alpha-tocopherol, alpha-tocopherol acetate, vitamin A, albumin , catalase, insulin, superoxide dismutase, transferrin, corticosterone, galactose, ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine, phenol red, sodium pyruvate or mixtures thereof.

In a preferred method of culturing primary cortical and thalamic neurons, the optimized medium used in step a) and / or b) comprises glycine 0.4 mM; L-alanine 0.02247191 mM; L-arginine hydrochloride 0.39810428 mM; L-asparagine 0.0055333334 mM; L-cysteine 0.2603306 mM; 0.2 mM L-histidine hydrochloride; L-isoleucine 0.8015267 mM; L-leucine 0.8015267 mM; L-lysine hydrochloride 0.7978142 mM; L-methionine 0.20134228 mM; 0.4 mM L-phenylalanine; L-proline 0.06747826 mM; 0.4 mM L-serine; L-threonine 0.7983193 mM; L-tryptophan 0.078431375 mM; L-tyrosine, 0.39779004 mM; L-valine 0.8034188 mM; glutamic acid 0.0125 mM; L-glutamine 2.0 mM; choline chloride 0.028571429 mM; calcium pantothenate 0.008385744 mM; folic acid 0.009070295 mM; niacin amide 0.032786883 mM; pyridoxal hydrochloride 0.019607844 mM; riboflavin 0.0010638298 mM; thiamine hydrochloride 0.011869436 mM; vitamin B12 5.0184503E-6 mM; inositol 0.04 mM; CaCl2 1.8018018 mM; Fe (NO3) 3x9H2O 2.4752476E-4 mM; MgCl2 0.8136842 mM; KCI 5.3333335 mM; NaHCO3

26,190,475 mM; NaCl 51.724136 mM; NaH2PO4-H2O 0.9057971 mM; ZnSO4-7H2O 6.736111E-4mM; D-glucose 3.125 mM; HEPES 10.92437 mM; phenol red 0.021519661 mM; sodium pyruvate 0.22727273 mM; sodium lactate 0.125 mM.

In a preferred method of culturing primary cortical and thalamic neurons, the method further comprises at least one step of replacing a portion of the volume of the culturing medium with an optimized medium as used in the culturing method described above.

More preferably, in the method of culturing primary cortical and thalamic neurons, the replacement of a part of the optimized medium volume takes place 3-4 days after the culture start, preferably the optimized medium replacement step is repeated many times every 3-4 days.

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More preferably, in the method of culturing primary cortical and thalamic neurons, about 1/3 to about 2/3 of the volume of the culture medium is replaced, more preferably half the volume of the culture medium is replaced with an optimized medium as used in the culture method defined above.

The inventors developed a new formulation of an optimized medium for the cultivation of primary cortical and thalamic neurons. The newly developed medium, hereinafter referred to as the medium according to the invention, allows for the efficient cultivation of neurons. It enables primary cultures and experiments on neurons in conditions similar to physiological, which reduces the risk of obtaining research artifacts, for example caused by stress and non-physiological conditions. The medium according to the invention, for example in contrast to the previously used media for the cultivation of neurons, for example Neurobasal medium, does not exert an excitotoxic and glucotoxic effect. The use of the medium according to the invention reduces the neuronal mortality in primary culture, which has been proven by a significant decrease in the content of the active form of caspase 3, which is a marker of apoptosis - programmed cell death (see Fig. 1).

The medium according to the invention increases the survival and viability of neurons in culture compared to current standards (see Fig. 2).

The effect of better viability was achieved by lowering the glucose concentration below 12.5 mM which prevents the glucotoxic effect (see Fig. 7). Most of the media available on the market contain far too high a toxic concentration of glucose, ie 25 mM.

Lactate was used as an additional component, not previously used in this type of media, as an additional source of energy or as a signaling molecule for nerve cells. The addition of lactate to the culture medium positively influenced the viability of the neurons (see Fig. 8). This component is not present in commercially available media and it is physiologically present in the brain. The addition of lactate to any commercially available high glucose medium results in a slight increase in neuronal viability (see Fig. 6). Surprisingly, it turned out that the simultaneous lowering of the glucose concentration in the medium and the addition of lactate not used so far gives a neuronal medium formulation which, through the synergistic interaction of these two factors (see Fig. 9), makes the culture conditions more similar to the physiological conditions in the brain . The balanced composition of the energy substrates in the medium according to the invention enables optimal cultivation and research on neurons.

Neurons grown in the medium according to the invention show a morphology that is more in line with the physiological state, for example they show a denser dendritic tree compared to the cultivation on current standard media (see Fig. 3).

Moreover, the neurons grown in the medium according to the invention show a higher content of neurofilament, an axon marker, suggesting a better axon growth compared to standard conditions (see Fig. 4).

The use of the medium according to the invention for the cultivation of neurons has the advantage that it provides more physiological conditions for the cultivation of neurons and a greater viability of the neurons is observed as compared to the prior art. In order to ensure optimal cultivation conditions and to maintain a constant concentration of nutrients and energy substrates, similarly to the media known from the state of the art, e.g. Neurobasal, it is recommended according to the cultivation method according to the invention to cyclically replace a part of the medium, preferably from about 1/3 to about 2/3, more preferably replacement of about half the volume of the medium every 3-4 days, due to the consumption of energy substrates and nutrients (see Fig. 5).

SHORT DESCRIPTION OF THE DRAWING FIGURES

For a better understanding of the invention, it is illustrated in the exemplary embodiments and in the accompanying drawing figures, in which:

Fig. 1. Shows the results of a western blot analysis of the active form of caspase 3 (marker of apoptosis) in neurons from a 14-day primary culture of rat cortical neurons. Gapdh - glyceraldehyde-3-phosphate dehydrogenase (control protein).

Fig. 2. Shows the results of neuronal viability in primary culture of rat cortical neurons measured on culture day 14 using the Celltiter-Blue assay (Promega). * p <0.05 vs. Neurobasal.

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Figure 3. Shows the results of neuronal morphology analysis from a 10-day primary culture of rat cortical neurons A. - Dendrite number analysis B. - Dendrite length analysis C. - Sholl D analysis - microscopic photo showing neuronal morphology (or neuronal morphology image).

Figure 4. Shows the results of western blot analysis of the content of neurofilament (axonal marker) in neurons from a 14-day primary culture of rat cortical neurons. NF-M - 140-160 kDa neurofilament; Gapdh - glyceraldehyde-3-phosphate dehydrogenase (control protein).

Fig. 5. Shows the results of the determination of glucose concentration in medium X - medium according to the invention and in Neurobasal medium during primary culture of rat cortical neurons. D0, D1, D4, D7, D10, D14 - breeding day 0, 1.4, 7, 10, 14, respectively.

Fig. 6. Shows the results of neuronal viability in primary culture of rat cortical neurons grown in high-glucose media (25 mM) from various suppliers: Neurobasal (ThermoScientific), NeuroCult (StemCell), PNGM (Lonza). Lactate added at a concentration of 125 μΜ. Viability was measured on culture day 14 using the Celltiter-Blue assay (Promega). * p <0.05 vs. PNGM.

Fig. 7. Shows the results of neuronal viability in primary culture of rat cortical neurons cultured in medium containing various glucose concentrations ranging from 1.56 mM to 25 mM. Viability was measured on culture day 14 using the Celltiter-Blue assay (Promega). * p <0.05 vs. 25 mM glucose (Neurobasal).

Figure 8. Shows the results of neuronal viability in primary culture of rat cortical neurons cultured in media containing a constant glucose concentration of 3 mM and a variable concentration of lactate ranging from 0 to 20 mM. Viability was measured on culture day 14 using the CelltiterBlue assay (Promega). * p <0.05 vs. 25 mM glucose (Neurobasal).

Fig. 9. Shows the effect of the synergistic effect of lactate addition and reduced glucose concentration on neuronal viability in primary culture of rat cortical neurons measured on culture day 14 using the Celltiter-Blue assay (Promega). * p <0.05 vs. 25 mM glucose; #p <0.05 vs. 3 mM glucose.

METHODS OF CARRYING OUT THE INVENTION

The following examples are provided merely to illustrate the invention and to explain its various aspects, not to limit it, and should not be construed as being within the scope of the invention as defined in the appended claims.

In the following examples, unless otherwise indicated, standard materials and methods known to those skilled in the art were used or the recommendations of the manufacturers for specific materials and methods were used.

All references and publications cited are hereby incorporated by reference in their entirety.

EXAMPLES

Example 1. Preparation of optimized medium for the cultivation of neurons

The inventive medium in the form of the preferred Medium X was obtained by mixing loose or liquid components to obtain the appropriate final molar concentrations with water of molecular purity and filter sterilization (pore width 0.22 μM from VWR) under sterile conditions (in a laminar chamber) .

As control media, Neurobasal and Neurobasal-A (Thermo Scientific), NeuroCult (StemCell), PNGM (Lonza) were used, supplemented or not, depending on the specific experiment, with L-glutamine, L-glutamic acid, D-glucose, sodium lactate, sodium pyruvate. and supplements B27 (Thermo Scientific) or SM1 (StemCell) or GA-1000 + NSF-1 (Lonza).

In order to obtain the final Medium X used in the experiments, the medium with the composition listed in Table 1 was additionally supplemented with the non-serum B-27 supplement (ThermoScientific) according to the manufacturer's instructions in the ratio of 1:50 supplement / medium (v / v), which the supplement contains: Na ₂ SeOs, HEPES, biotin, alpha-tocopherol, alpha-tocopherol acetate, vitamin A, albumin, catalase, insulin, superoxide dismutase, transferrin, corticosterone, galactose, ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine.

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Table 1 Composition of the inventive medium for the cultivation of neurons (Medium X without non-serum supplement).

concentration [mM] Glycine 0.4 L-Alanine 0.02247191 L-Arginine Hydrochloride 0.39810428 L-Asparagine 0.0055333334 L-Cysteine 0.2603306 L-Histidine Hydrochloride 0.2 L-Isoleucine 0.8015267 L-Leucine 0.8015267 L-Lysine Hydrochloride 0.7978142 L-Methionine 0.20134228 L-Phenylalanine 0.4 L-proline 0.06747826 L-Cheeses on 0.4 L-Threonine 0.79831934 L-T rptophan 0.078431375 L-Tyrosine 0.39779004 L-Valine 0.8034188 Glutamic acid 0.0125 L-Glutamine 2.0 Choline chloride 0.028571429 Calcium pantothenate 0.008385744 Folic acid 0.009070295 Niacin amide 0.032786883 Pyridoxal Hydrochloride 0.019607844 Ribotlavin 0.0010638298 Thiamine Hydrochloride 0.011869436 Vitamin B12 5.0184503E-6 inositol 0.04 CaCl2 1,8018018

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Fe (NO3) 39H2O 2.4752476E-4 MgCl2 0.8136842 KOI 5.3333335 NaHCO3 26,190475 NaCI 51.724136 NaH2PO4-H2O 0.9057971 ZnSO4-7H2O 6.736111E-4 D-Glucose 3.125 HEPES 10.92437 Phenol red 0.021519661 Sodium pyruvate 0.22727273 Sodium lactate 0.125

Example 2. Method of culturing neurons

A pregnant female Wistar rat was subjected to gas euthanasia and to death by dislocation of the cervical vertebrae. Fetuses of E18.5 embryonic age were extracted in order to isolate parts of the cortex and thalamus. The isolated cortex and thalamic fragments were washed in cold HBSS buffer (Sigma-Aldrich) and subjected to enzymatic digestion with 1.25% trypsin (Thermo Scientific) at 37 ° C for 10 min. The tissue was then dissociated in HBSS solution (Sigma-Aldrich) mechanically with a pipette. The number of released nerve cells in suspension was counted using a Burker chamber, and then suspended in culture medium and seeded in a volume of 500 μΙ in the number of 200 thousand. cell / well of 24-well culture adherent plates coated with poly-D-lysine (Corning). Primary culture of cortical or thalamic neurons was carried out in an incubator at 37 ° C and 5% CO ₂ for 2 weeks. Every 3-4 days% of the culture medium was replaced with fresh. Cultures were carried out in the medium according to the invention with the composition defined in the given experiment, and / or in media known from the state of the art.

Example 3. Western-blot analysis of the apoptosis marker (caspase 3) in neurons from primary culture and western-blot analysis of the content of the axonal growth marker (neurofilament)

Cortical or thalamic neurons in a primary culture grown on Medium X (prepared according to Example 1) and grown on Neurobasal were rinsed once with PBS buffer, then harvested using a scraper and incubated on ice for 30 minutes in cold lysis buffer. The lysates were then centrifuged. The protein-containing supernatant was transferred to a new tube and its protein content was determined by the Bradford method.

The electrophoretic separation of proteins (50 µg protein / sample) was performed on a polyacrylamide gel under denaturing conditions using a 10% separation gel and a 5% thickening gel [the PageRuler Plus Prestained Protein Ladder (Thermo Scientific) protein size marker was used].

The separated proteins were transferred to a nitrocellulose membrane (GE Healthcare) and incubated in the presence of the appropriate 1: 1000 anti-casp3 (Cell Signaling), 1: 500 anti-NFMH (ProteinTech), 1: 40,000 anti-GAPDH (Sigma-Aldrich) antibody. ] followed by a 11-row horseradish peroxidase conjugated antibody [1: 10000 anti-mouse-HRP and anti-rabbit-HRP (Sigma-Aldrich)]. Protein detection was performed with chemilinescence reagents. The signal from the membrane was visualized using an Amersham Imager600 RGB instrument (GE Healthcare).

The results of western-blot analysis of the active form of caspase 3 in neurons from a 14-day-old primary culture of rat cortical neurons showed a decrease in the content of this apoptosis marker in neurons cultured in the medium according to the invention (Fig. 1). This proves a lower mortality rate

Of neurons grown in the medium of the invention. The results of western-blot analysis of neurofilament content in neurons from 14-day primary culture of rat cortical neurons showed an increase in the content of axonal growth marker in the lysate of neurons cultured in Medium X (Fig. 4), which may indicate a better condition of axons of neurons cultured in Medium X.

P r o x l a d 4. Determination of the viability of neurons 4.1.

The survival of cortical or thalamic neurons in primary culture grown in Medium X and Neurobasal medium was determined using the CellTiter-Blue Cell Viability Assay (Promega). An indicator of cell viability in this assay is the ability of metabolically active cells to reduce resazurin (dark blue) to resorufin (pink). The amount of resorufin produced is proportional to the number of living cells. For this, complete culture medium containing the CellTiter-Blue reagent was added to the cells. After 3 hours of incubation at 37 ° C, the absorbance was measured at 620 nm with a reference wavelength of 690 nm using a Bio-tek microplate reader. Cell viability is presented as the difference in the relative absorbance value measured at 620 nm and 690 nm taking into account the blank value. In this and the following examples, the obtained and presented results were statistically analyzed for significance using the "One-way Anova" test.

The result of neuronal viability measurement in primary culture of rat cortical neurons measured on culture day 14 using the Celltiter-Blue assay (Promega) showed increased survival of neurons cultured in Medium X (Fig. 2).

4.2.

The survival of cortical neurons in primary culture was determined using CellTiter-Blue Cell Viability Assay (Promega) after a period of 14 days of cultures as described above. The nutrients were supplemented according to the manufacturers' recommendations.

a) Various lactate media

125 gM sodium lactate was added to commercially available high-glucose media (25 mM glucose) including Neurobasal (Thermo Scientific), NeuroCult (StemCell), and PNGM (Lonza). In these media, the primary culture of cortical neurons was established and carried out. On the 14th day of cultivation, the viability was measured. Neurons cultured in high-glucose lactate-supplemented media showed a slight trend of increased viability as compared to neurons cultured in identical lactate-free media (Fig. 6).

b) Varying glucose levels

Neurobasal-A glucose-depleted medium (Thermo Scientific) was supplemented with sodium pyruvate at 0.277 mM and various glucose concentrations ranging from 1.56 mM to 25 mM. In these media, the primary culture of cortical neurons was established and carried out. On the 14th day of cultivation, the viability was measured. Neurons cultured in glucose-reduced media (i.e., in the range 1.56-12.5 mM) showed an increase in viability compared to neurons cultured in standard high-glucose medium, ie at a glucose concentration of 25 mM (Fig. 7).

c) Constant glucose concentration and variable lactate concentration

Neurobasal-A medium (Thermo Scientific) was supplemented with B27 supplement (Thermo Scientific), sodium pyruvate at 0.277 mM, D-glucose at 25 and 3 mM, and sodium lactate at 125 gM to determine the effect of glucose and lactate on neuronal viability. In these media, the primary culture of cortical neurons was established and carried out. On the 14th day of cultivation, the viability was measured. Neurons cultured in lactate-supplemented media in the concentration range of 0.63 gM to 6 mM showed an increase in viability compared to neurons cultured in lactate-free media (Fig. 8).

Neurons cultured with only 3 mM lactate or 6 mM lactate without glucose had very low survival and showed 10-20% viability (results not shown in the graph).

Example 5. Comparative analysis of the morphology of neurons from primary culture of cortical neurons

Cortical neurons cultured on slides coated with poly-D-lysine and laminin (Corning) in Neurobasal and Medium X were transfected on day 7 of culture with the expression plasmid containing the GFP green fluorescent protein using Lipofectamine 2000 reagent (Thermo Fisher). Three days after transfection, cells were rinsed with PBS solution and incubated in 4% paraformaldehyde for 10 minutes at room temperature. Then, the fixed cells were washed with a PBS solution and the nuclei were stained with the fluorescent dye DAPI (Thermo Fisher), and then

The material was sealed with VectaShield Fluorescent Mounting Medium (Vector Laboratories). The microscopic photos were taken using a confocal microscope and their morphology was assessed, including the total length of dendrites, the number of dendrites and the morphology of the dendritic tree (the so-called Scholl analysis).

The results of the neuronal morphology analysis from the 10-day primary culture of rat cortical neurons showed an increased number of dendrites (Fig. 3 A), length of dendrites (Fig. 3 B) and a more extensive dendritic tree of neurons grown in Medium X (Fig. 3 C, D) .

Example 6. Determination of glucose concentration in the medium during primary culture of cortical neurons

Part of the culture medium was collected during the primary culture of cortical or thalamic neurons. 10 µl of medium was mixed with 1 ml of glucose oxidase reagent containing glucose oxidase and peroxidase (Biosystems). The enzyme reaction was carried out at room temperature for 10 minutes, and then the absorbance of the color product of the enzyme reaction, ie quinoimine, was measured at a wavelength of 500 nm in a cuvette spectrophotometer (Eppendorf biophotometer D30). Glucose concentration was determined from the absorbance value measured at 500 nm with the blank value and the standard kit supplied by the manufacturer (Fig. 5).

Example 7. Demonstration of the synergistic effect of lactate and reduced glucose levels on the viability of neurons

Neurobasal-A medium (Thermo Scientific) was supplemented with B27 supplement (Thermo Scientific), sodium pyruvate at 0.277 mM, D-glucose at 3 mM, and various sodium lactate concentrations ranging from 0 to 20 mM. In these media, the primary culture of cortical neurons was established and carried out. On the 14th day of cultivation, the viability was measured. Neurons cultured in a standard medium with a glucose concentration of 25 mM with lactate and neurons grown in a medium with a low concentration of glucose showed an increase in viability compared to neurons cultured in a standard high-glucose medium, i.e. at a concentration of 25 mM (Fig. 9). However, unexpectedly, the combination of low glucose concentration (i.e., in the range of 3.0-12.5 mM) and the addition of lactate resulted in a synergistic effect of increasing neuronal viability, greater than that of the other study groups and greater than the additive glucose-lowering effect and lactate supplementation (Fig. 9).

Summary:

The optimized culture medium for neurons according to the invention contains an amount of glucose, optimal for the growth of neurons, in a concentration similar to the conditions naturally occurring in the brain. In addition, the medium contains a component that has not been used so far in other solutions - lactate, which is an alternative (to glucose and pyruvate) source of energy for neurons or acting as a signaling molecule. Unexpectedly, it turned out that by adding lactate to the media suitable for the cultivation of neurons at a concentration of 125 μM to 6 mM with the simultaneous reduction of the glucose concentration to a concentration of 3.0-12.5 mM, a synergistic effect of the interaction of these two factors for the growth of neurons was observed. The medium according to the invention provides physiological conditions for maintaining primary neuron cultures and increasing their viability compared to the previous solutions based on high glucose concentration in the media (25 mM), the side effect of which was the so-called glucotoxicity, i.e. the toxic effect of glucose after chronic exposure to its high concentration. Without being bound by any theory according to hypothesis about the exchange of lactate between astrocytes and neurons, it is lactate that is the primary source of energy for neurons, supplied in the brain by astrocytes to neurons. The addition of lactate to the medium simulates the naturally occurring composition of nutrients available to neurons under the physiological conditions of the brain. The current products (nutrients) available on the market do not contain lactate. So far, the synergistic effect of lactate and glucose interaction in certain concentration ranges has not been known and has not been known or suggested in the art.

Literature:

Brewer GJ, Torricelli JR, Evege EK, Price PJ. Optimized survival of hippocampal neurons in B27supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 1993 Aug 1; 35 (5): 567-76. PubMed PMID: 8377226;

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Hogins J, Crawford DC, Zorumski CF, Mennerick S. Excitotoxicity triggered by Neurobasal culture medium. PLoS One. 2011; 6 (9): e25633. doi: 10.1371 / joumal.pone.0025633. Epub 2011 Sep 28. PubMed PMID: 21980512; PubMed Central PMCID: PMC3182245;

Maggioni D, Monfrini M, Ravasi M, Tredici G, Scuteri A. Neurobasal medium toxicity on mature cortical neurons. Neuroreport. 2015 Apr 15; 26 (6): 320-4. doi: 10.1097 / WNR.0000000000000343. PubMed PMID: 25756909;

Peng Y, Liu J, Shi L, Tang Y, Gao D, Long J, Liu J. Mitochondrial dysfunction precedes depression of AMPK / AKT signaling in insulin resistance induced by high glucose in primary cortical neurons. J Neurochem. 2016 Jun; 137 (5): 701-13. doi: 10.1111 / jnc. 13563. Epub 2016 Apr 18. PubMed PMID: 26926143.

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Claims (18)

Patent claims A method of culturing primary cortical and thalamic neurons, characterized by the steps of: a) a primary culture of cortical or thalamic neurons is assumed on an optimized lactate and glucose-containing medium, b) the primary culture of cortical or thalamic neurons is maintained on an optimized lactate and glucose-containing medium, the optimized medium used in step a) and b) contains lactate from 125 μ 6 to 6 mM, glucose from 3.0 to 12 .5 mm. 2. A method of culturing primary cortical and thalamic neurons according to claim 1. The method of claim 1, wherein the optimized medium used in step a) and / or b) comprises sodium lactate at a concentration of 125 µM, D-glucose at a concentration of 3 mM. 3. The method of culturing primary cortical and thalamic neurons according to claim 1. A method according to claim 1-2, characterized in that the optimized medium used in step a) and / or b) further comprises components selected from glycine, alanine, arginine, asparagine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine , threonine, tryptophan, tyrosine, valine, glutamic acid, glutamine, choline chloride, calcium pantothenate, folic acid, niacin amide, pyridoxal hydrochloride, riboflavin, thiamine, thiamine hydrochloride, aspartic acid, vitamin B12, inositol, NaCI, KCI, MgSO4 CuSO4, FeSO4, NaH2PO4, CaCl2, Fe (NO3) ₃ x9H ₂ O, MgCl2, KCI, NaHCO3, NaCI, NaH ₂ PO4-H ₂ O, ZnSO4-7H2O, Na ₂ SeO3, HEPES, biotin, alpha-tocopherol, acetate alpha-tocopherol, vitamin A, albumin, catalase, insulin, superoxide dismutase, transferrin, corticosterone, galactose, ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine, phenol red, sodium pyruvate or mixtures thereof. 4. The method of culturing primary cortical and thalamic neurons according to claim 1. The method according to the method of 1-3, characterized in that the optimized medium used in step a) and / or b) comprises glycine 0.4 mM; L-alanine 0.02247191 mM; L-arginine hydrochloride 0.39810428 mM; L-asparagine 0.0055333334 mM; L-cysteine 0.2603306 mM; 0.2 mM L-histidine hydrochloride; L-isoleucine 0.8015267 mM; L-leucine 0.8015267 mM; L-lysine hydrochloride 0.7978142 mM; L-methionine 0.20134228 mM; 0.4 mM L-phenylalanine; L-proline 0.06747826 mM; 0.4 mM L-serine; L-threonine 0.7983193 mM; L-tryptophan 0.078431375 mM; L-tyrosine, 0.39779004 mM; L-valine 0.8034188 mM; glutamic acid 0.0125 mM; L-glutamine 2.0 mM; choline chloride 0.028571429 mM; calcium pantothenate 0.008385744 mM; folic acid 0.009070295 mM; niacin amide 0.032786883 mM; pyridoxal hydrochloride 0.019607844 mM; riboflavin 0.0010638298 mM; thiamine hydrochloride 0.011869436 mM; vitamin B12 5.0184503E-6 mM; inositol 0.04 mM; CaCl2 1.8018018 mM; Fe (NO3) 39H2O 2.4752476E-4mM; MgCl2 0.8136842 mM; KCI 5.3333335 mM; NaHCO3 26, 190 475 mM; NaCl 51.724136 mM; NaH ₂ PO ₄ -H ₂ O 0.9057971 mM; ZnSO4-7H2O 6.736111E-4mM; D-glucose 3.125 mM; HEPES 10.92437 mM; phenol red 0.021519661 mM; sodium pyruvate 0.22727273 mM; sodium lactate 0.125 mM. PL 235 678 B1 5. The method of culturing primary cortical and thalamic neurons according to claim 1. The method of claim 1, further comprising at least one step of replacing a portion of the volume of the culturing medium with an optimized medium as used in the culturing method of any of claims 1 to 4. 1-4. 6. A method for culturing primary cortical and thalamic neurons according to claim 1. 5. The method of claim 5, characterized in that the replacement of a part of the volume of the optimized medium takes place after 3-4 days from the day of starting the culture, preferably the step of replacing the optimized medium is repeated many times every 3-4 days. 7. A method for culturing primary cortical and thalamic neurons according to claim 1. 5-6, characterized in that about 1/3 to about 2/3 of the volume of the culture medium is replaced, more preferably half the volume of the culture medium is replaced with an optimized medium as used in the culture method according to claims 1-6. 4. 8. Optimized medium for the cultivation of primary cortical and thalamic neurons both in the stage of establishing primary culture of cortical or thalamic neurons and in the stage of maintaining primary culture of cortical or thalamic neurons, characterized by the fact that it contains lactate in a concentration of 125 μΜ to 6 mM, glucose from 3.0 to 12.5 mM. 9. Optimized medium for the cultivation of primary cortical and thalamic neurons according to claim 1, 8. The composition of claim 8, wherein the concentration of sodium lactate is 125 µM, D-glucose is 3 mM. 10. Optimized medium for the cultivation of primary cortical and thalamic neurons according to claim 1. 8-9, characterized in that it further comprises ingredients selected from glycine, alanine, arginine, asparagine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, glutamic acid, glutamine, choline chloride, calcium pantothenate, folic acid, niacin amide, pyridoxal hydrochloride, riboflavin, thiamine, thiamine hydrochloride, aspartic acid, vitamin B12, inositol, NaCI, KCI, MgSO4, CuSO4, FeSO4, NaH2PO4, CaCl2, Fe (NO3) 3x9H2O, MgCb, KCI, NaHCO3, NaCI, NaH2PO4-H2O, ZnSO4-7H2O, Na2SeO3, HEPES, biotin, alpha-tocopherol, alpha-tocopherol acetate, vitamin A, albumin, catalase, insulin, superoxide dismutase, transferrin, galactose, corticosterone , ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine, phenol red, sodium pyruvate or mixtures thereof. 11. Optimized medium for the cultivation of primary cortical and thalamic neurons according to claim 1. 8-10, characterized in that the glycine is 0.4mM; L-alanine 0.02247191 mM; L-arginine hydrochloride 0.39810428 mM; L-asparagine 0.0055333334 mM; L-cysteine 0.2603306 mM; 0.2 mM L-histidine hydrochloride; L-isoleucine 0.8015267 mM; L-leucine 0.8015267 mM; L-lysine hydrochloride 0.7978142 mM; L-methionine 0.20134228 mM; 0.4 mM L-phenylalanine; L-proline 0.06747826 mM; 0.4 mM L-serine; L-threonine 0.7983193 mM; L-tryptophan 0.078431375 mM; L-tyrosine, 0.39779004 mM; L-valine 0.8034188 mM; glutamic acid 0.0125 mM; L-glutamine 2.0 mM; choline chloride 0.028571429 mM; calcium pantothenate 0.008385744 mM; folic acid 0.009070295 mM; niacin amide 0.032786883 mM; pyridoxal hydrochloride 0.019607844 mM; riboflavin 0.0010638298 mM; thiamine hydrochloride 0.011869436 mM; vitamin B12 5.0184503E-6 mM; inositol 0.04 mM; CaCl2 1.8018018 mM; Fe (NO3) 3x9H2O 2.4752476E-4 mM; MgCl2 0.8136842 mM; KCI 5.3333335 mM; NaHCO3 26, 190 475 mM; NaCl 51.724136 mM; NaH2PO4-H2O 0.9057971 mM; ZnSO4-7H2O 6.736111E-4mM; D-glucose 3.125 mM; HEPES 10.92437 mM; phenol red 0.021519661 mM; sodium pyruvate 0.22727273 mM; sodium lactate 0.125 mM. 12. Use of an optimized medium for the cultivation of primary cortical or thalamic neurons, wherein the optimized medium contains lactate from 125 μM to 6 mM, glucose from 3.0 to 12.5 mM, and wherein the optimized medium is used for both establishing a primary culture of cortical or thalamic neurons and maintaining primary culture of cortical or thalamic neurons. Use of an optimized medium for the cultivation of primary cortical or thalamic neurons according to claim 1, 12. The method of claim 12, wherein the optimized medium comprises sodium lactate at a concentration of 125 µM, D-glucose at a concentration of 3 mM. PL 235 678 B1 14. Use of an optimized medium for the cultivation of primary cortical or thalamic neurons according to claim 1. 12-13, characterized in that the optimized medium further comprises components selected from glycine, alanine, arginine, asparagine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, acid glutamine, glutamine, choline chloride, calcium pantothenate, folic acid, niacin amide, pyridoxal hydrochloride, riboflavin, thiamine, thiamine hydrochloride, aspartic acid, vitamin B12, inositol, NaCI, KCI, MgSO4, CuSO4, FeSO4, NaH2PO4, CaCI2 ( NO3) 3x9H2O, MgCb, KCI, NaHCOs, NaCI, NaH2PO4-H2O, ZnSO4-7H2O, Na2SeO3, HEPES, biotin, alpha-tocopherol, alpha-tocopherol acetate, vitamin A, albumin, catalase, insulin, superoxide dismutase, corticosterone transferrin , galactose, ethanolamine, glutathione, carnitine, linoleic acid, linolenic acid, progesterone, putrescine, triiodothyronine, phenol red, sodium pyruvate or mixtures thereof. 15. Use of an optimized medium for the cultivation of primary cortical or thalamic neurons according to claim 1. 12. The method of claim 12-14, wherein the optimized medium comprises glycine 0.4 mM; L-alanine 0.02247191 mM; L-arginine hydrochloride 0.39810428 mM; L-asparagine 0.0055333334 mM; L-cysteine 0.2603306 mM; 0.2 mM L-histidine hydrochloride; L-isoleucine 0.8015267 mM; L-leucine 0.8015267 mM; L-lysine hydrochloride 0.7978142 mM; L-methionine 0.20134228 mM; 0.4 mM L-phenylalanine; L-proline 0.06747826 mM; 0.4 mM L-serine; L-threonine 0.7983193 mM; L-tryptophan 0.078431375 mM; L-tyrosine, 0.39779004 mM; L-valine 0.8034188 mM; glutamic acid 0.0125 mM; L-glutamine 2.0 mM; choline chloride 0.028571429 mM; calcium pantothenate 0.008385744 mM; folic acid 0.009070295 mM; niacin amide 0.032786883 mM; pyridoxal hydrochloride 0.019607844 mM; riboflavin 0.0010638298 mM; thiamine hydrochloride 0.011869436 mM; vitamin B12 5.0184503E-6 mM; inositol 0.04 mM; CaCb 1.8018018 mM; Fe (NOs) sx9H2O 2.4752476E-4 mM; MgCl2 0.8136842 mM; KCI 5.3333335 mM; NaHCOs 26,190,475 mM; NaCl 51.724136 mM; NaH2PO4-H2O 0.9057971 mM; ZnSO4-7H2O 6.736111E-4mM; D-glucose 3.125 mM; HEPES 10.92437 mM; phenol red 0.021519661 mM; sodium pyruvate 0.22727273 mM; sodium lactate 0.125 mM. 16. Use of an optimized medium for the cultivation of primary cortical or thalamic neurons according to claim 1. The method of claim 12-15, characterized in that, during the cultivation, a fraction of the volume of the cultivated medium is exchanged for an optimized medium as used in the application defined in any of claims 12 to 15. 12-15. 17. Use of an optimized medium for the cultivation of primary cortical or thalamic neurons according to claim 1. The method of claim 16, characterized in that the replacement of a part of the volume of the optimized medium takes place after 3-4 days from the day of starting the culture, preferably the step of replacing the optimized medium is repeated many times every 3-4 days. 18. Use of an optimized medium for the cultivation of primary cortical or thalamic neurons according to claim 1, 16-17, characterized in that the optimized medium is replaced in an amount from about 1/3 to about 2/3 of the volume of the medium from the cultivation, more preferably half the volume of the medium on which the culture is grown is replaced by the optimized medium as used in the application. as defined in claim 12-15.