Serum-free culture medium for the culture of human monocytic cells and method using the same

Abstract

The invention relates to a serum-free culture medium suitable for the culture of human monocytic cells, preferably THP-1 cells, to the use of said medium for cultivating human monocytic cells and to a method for cultivating human monocytic cells using the same.

Description

[0001] SERUM-FREE CULTURE MEDIUM FOR THE CULTURE OF HUMAN MONOCYTIC CELLS AND METHOD USING THE SAME

[0002] FIELD OF THE INVENTION

[0003] The invention belongs to the technical field of culture media for cell culture.

[0004] In particular, the invention relates to a serum-free culture medium suitable for the culture of human monocytic cells, to the use of said medium for cultivating human monocytic cells and to a method for cultivating human monocytic cells using the same.

[0005] TECHNICAL BACKGROUND

[0006] In life sciences, cell culture has been used to study many aspects of cell biology, toxicology or cancer biology. For the last decades and since its first use in 1958, Fetal Bovine Serum (FBS) has been used to maintain cell cultures. However, due to the scientific drawbacks and its ethically debatable production method, the importance of replacing FBS but also any other animal-derived laboratory reagents has already reached regulatory authorities. Hence, the European Centre for the Validation of Alternative Methods (ECVAM) recommended the use of non-animal serum substitutes of all animal-derived supplements. In addition, one can observe in all sectors of the society that animal-free products (vegan) are in demand. During the last years, in vitro cell culture models that enable to study the effects of respiratory toxicants on the alveolar region of the lung have been developed.

[0007] As an example, international application WO 2018 / 122219 a three- dimensional in vitro model able to mimic the alveolar surface of the lungs in order to assess the respiratory sensitization potential of inhalable products, chemicals as well as particles. This three-dimensional in vitro model is made of different types of cells, i.e. alveolar type II epithelial cells, endothelial cells, dendritic-like cells and macrophage-like cells, these latter being able to participate to defense mechanisms by ingesting foreign materials by phagocytosis. In this model, the macrophage-like cells are human monocytic cells, namely THP-1 cells, differentiated with PMA (phorbol-12-myristate-13-acetate).

[0008] Currently, this model still implies the use of animal derived components including FBS for their maintenance and use while they were developed to replace animal testing and experimentation. In particular, the growth of the human monocytic cells present in this model is usually performed in a basal culture medium supplemented with FBS and other supplements. As examples, one can mention R.PMI medium (Roswell Park Memorial Institute medium) and DMEM (Dulbecco's Modified Eagle's Medium), these media being supplemented, among other supplements, with about 10% (v / v) FBS.

[0009] FBS was first introduced in the 1950s and is used as a universal supplement to facilitate cell growth, attachment, and proliferation in cell culture media. As a complex mixture of an undefined composition of biomolecules, with an estimated 1800 proteins and >4000 metabolites, FBS has substantial batch-to-batch variation. Undefined and variable components can lead to unexpected and undesired interactions in studies and could result in discrepancies in data from in vitro studies that use FBS as a cell culture supplement. In addition, the process often used for obtaining FBS involves inserting a large-gauge needle into the heart of an unborn calf when their mother is slaughtered, causing the unborn calf fetus to suffocate or bleed to death. Thus, in addition to scientific issues, animal welfare concerns exist.

[0010] To address these scientific, ethical, and legal challenges, researchers are replacing FBS. Transitioning cells to serum-free media requires the identification of appropriate media formulations.

[0011] Early attempts to grow cells in FBS-free media, mostly hormone- supplemented media, were performed in the 90's but they were not successful. Since then, several different serum-free media have been developed where the media are supplemented with a high number of essential components of plant origin and / or with recombinant products but only 10-20% of these strategies appeared to be successful (Pazos P. et al, ALTEX21, 2, 2004, pages 67-72).

[0012] In addition, cultivating media for growing cells must be adapted to the different cell lines to be grown because a medium suitable for cultivating a given cell line is not systematically suitable for cultivating another cell line.

[0013] There are certain culture media on the market that are serum-free. However, these media are not specifically adapted to the culture of human monocytic cells, even less to the culture of THP-1 cells, and thus they do not give complete satisfaction insofar as they do not allow the cells to propagate and / or to retain their full ability to differentiate into macrophages under certain conditions. In addition, these media are not always free from any other animal-derived components so that they may contain e.g., bovine pituitary extract, human- derived components such as human serum, human serum albumin, or human transferrin.

[0014] Therefore, there is still a need for medium suitable for the culture of human monocytic cells that is serum-free and in which this type of cells could be grown, in particular in conditions allowing their further differentiation into macrophages when needed.

[0015] The inventors set out themselves to solve this technical problem and developed the culture medium, which is the subject of the invention described below.

[0016] OBJECTS AND SUMMARY

[0017] A first object of the present invention is a serum-free culture medium suitable for the culture of human monocytic cells comprising a basal serum-free growth medium, wherein said basal serum-free growth medium is supplemented with at least the following supplements: a) a mixture of amino acids, said mixture comprising at least L-cysteine, and L- carnosine, b) a mixture of lipids, c) nicotinamide or at least one derivative thereof, d) at least one early-acting cytokine, e) one or more divalent cations, f) insulin g) transferrin h) at least one selenite salt and i) at least one thyroid hormone.

[0018] A second object of the present invention is the use of said serum-free culture medium for cultivating human monocytic cells, preferably THP-1 cells.

[0019] A third object of the present invention is a method for cultivating human monocytic cells, comprising the steps of: i) providing a serum-free culture medium as defined according to the first object of the present invention, and ii) propagating or maintaining said human monocytic cells in said serum-free culture medium to form a cell culture.

[0020] DEFINITIONS

[0021] In the following disclosure a serum-free medium does not contain human or animal serum but may contain discrete proteins or bulk proteins fractions (e.g., human or animal tissue or plant extracts).

[0022] Nicotinamide is a form of vitamin B3 found in food. Nicotinamide is also known as niacinamide or pyridinecarboxamide, nicotinic acid amide or vitamin PP or nicotinic amide or vitamin B3.

[0023] In the sense of the present invention "cultivating" in relation to human monocytic cells, and in particular to THP-1 cells, encompasses both their maintenance and growth in a favorable environment after cells of interest have been isolated from living tissue.

[0024] According to the invention, the terms "basal serum-free growth medium" designate the basic growth medium in which the supplements a) to i) are further added to produce the serum-free culture medium according to the invention.

[0025] The terms "basal medium" means any culture medium classically used to grow human monocytic cells, such as THP-1 cells, in particular R.PMI medium. A basal medium is usually supplemented with FBS.

[0026] In the sense of the present invention, a recombinant product encompasses any product produced by cells including, but not limited to, proteins, peptides, antibody molecules, RNA, DNA, antibiotics and amino acids.

[0027] DETAILED DESCRIPTION

[0028] The first object of the present invention is a serum-free culture medium suitable for the culture of human monocytic cells comprising a basal serum-free growth medium, wherein said basal serum-free growth medium is supplemented with at least the following supplements: a) a mixture of amino acids, said mixture comprising at least L-cysteine, and L- carnosine, b) a mixture of lipids, c) nicotinamide or at least one derivative thereof, d) at least one early-acting cytokine, e) one or more divalent cations, f) insulin g) transferrin h) at least one selenite salt and i) at least one thyroid hormone.

[0029] According to a preferred embodiment of the first object of the present invention, the human monocytic cells are chosen among THP-1 cells and U-937 cells, THP-1 cells being even more preferred.

[0030] THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte / dendritic cells / macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocytes and macrophage activities.

[0031] U-937 is a cell line derived from malignant cells of a pleural effusion of 37- year-old Caucasian male with diffuse histiocytic lymphoma. It is one of only a few human lines still expressing many of the monocytic like characteristics exhibited by cells of histiocytic origin. This cell line can be used in tumourigenicity studies.

[0032] According to an embodiment, the total concentration of said mixture of amino acids in the serum-free culture medium is from about 0.5 to about 25 mM.

[0033] The concentration of L-cysteine in the serum-free culture medium is preferably from about 0.05 mM to about 5 mM.

[0034] The concentration of L-carnosine in the serum-free culture medium is preferably from about 0.05 mM to about 20 mM.

[0035] Any mixture of lipids preferably comprising cholesterol and non-animal fatty acids can be used according to the invention. According to a particular and preferred embodiment of the invention, the mixture of lipids is in a liquid form and comprises cholesterol and one or several non-animal fatty acids selected in the group comprising arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid and stearic acid. The concentration of said mixture of lipids in the serum-free culture medium is preferably from about 0.5 to 20 mb L.

[0036] Among nicotinamide derivatives, one can mention isonicotinamide and nicotinamide riboside.

[0037] According to a particular and preferred embodiment of the present invention, said serum-free culture medium comprises nicotinamide.

[0038] The concentration of nicotinamide or of at least one derivative thereof in the serum-free culture medium is preferably from about 0.01 to 2.0 g / mL.

[0039] The at least one early-acting cytokine may be selected from the group consisting of stem cell factor, FLT-3 ligand, interleukin-5, interleukin-6, thrombopoietin, interleukin-3, and a mixture thereof. According to a preferred embodiment of the present invention, said early-acting cytokine is stem cell factor.

[0040] The concentration of the least one early-acting cytokine in the serum-free culture medium is preferably from about 0.4 to 40 ng / L.

[0041] According to a particular embodiment of the invention, divalent cations are selected in the group consisting of Ca2+, Zn2+, Mg2+, Mn2+, Fe2+, Cu2+, and any mixtures thereof. Among these divalent cations, Ca2+, Zn2+, and mixtures thereof are preferred.

[0042] The concentration of divalent cations in the serum-free culture medium is preferably from about 0.3 to 300 pM.

[0043] In addition to divalent cations, the serum-free culture medium according to the invention may further comprise monovalent cations such as for example K+, Na+, and / or trivalent cations such as for example Fe3+.

[0044] The divalent cations, and optionally monovalent and / or trivalent cations, may be present in the serum-free culture medium according to the invention in the form of salt, such as chloride, sulfate, nitrate, bicarbonate, and phosphate.

[0045] According to a preferred embodiment of the invention, the serum-free culture medium comprises a mixture of calcium chloride and zinc sulfate.

[0046] According to a preferred embodiment of the invention, insulin is a recombinant human insulin and in particular, a recombinant human insulin derived from the yeast Pichia pastoris. The concentration of insulin in the serum-free culture medium is preferably from about 0.001 to about 0.1 mg / mL and more preferably is equal to about 0.01 mg / mL.

[0047] According to a preferred embodiment of the invention, transferrin is a recombinant human transferrin, and in particular, a recombinant human transferrin derived from Oryza sativa, which is a variety of rice.

[0048] The concentration of transferrin in the serum-free culture medium is preferably from about 0.00055 to about 0.055 mg / mL and more preferably is equal to about 0.0055 mg / mL.

[0049] The at least one selenite salt is preferably sodium selenite.

[0050] The concentration of the at least one selenite salt in the serum-free culture medium is preferably from about 0.0005 to about 0.05 pg / mL, and more preferably is equal to 0.005 pg / mL.

[0051] According to a preferred embodiment, the at least one thyroid hormone is selected in the group consisting of 3,3',5-triiodi-L-thyronine and thyroxine (also known as T4); 3,3',5-triiodi-L-thyronine being particularly preferred. These thyroid hormones are from synthetic origin.

[0052] The concentration of the at least one thyroid hormone in the serum-free culture medium is preferably from about 0.001 to about 1.0 pM.

[0053] The basal serum-free (BSF) growth medium may be chosen among classical liquid basal media that are usually used to grow human monocytic cells and comprising basic components and essential nutrients such as a buffer, salts, amino acids other than L-cysteine and L-carnosine, sugars, such as for example glucose, vitamins, growth factors, trace elements, etc... As some examples, one can mention minimal essential medium (MEM), R.PMI medium, Dulbecco's modified Eagle's medium (DMEM), DMEM-F12, and Iscove's Modified Dulbecco's Medium (IMDM).

[0054] According to a particular and preferred embodiment of the present invention, the basal serum-free growth medium is a minimal essential medium further comprising at least one of the following ingredients: sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and a pH indicator such as phenol red for example. As a specific example, the basal serum-free growth medium is the commercial medium sold under the trade name Opti-MEM ® by Gibco.

[0055] A very particular and preferred serum-free culture medium according to the invention comprises the following supplements: a) L-cysteine in a concentration of about 0.5 mM and L-carnosine in a concentration of about 7.5 mM, b) a mixture of lipids in a concentration of about 10 mL / L, c) nicotinamide in a concentration of about 0.001 g / mL, d) stem cell factor in a concentration of about 3.3 mg / mL, e) CaC and ZnSC , each in a concentration of about 0.3 mM, f) recombinant human insulin in a concentration of about 0.01 mg / mL, g) recombinant human transferrin in a concentration of about 0.0055 mg / mL, h) sodium selenite in a concentration of about 0.005 pg / mL, and i) 3,3',5-triiodi-L-thyronine in a concentration of 1000 pM.

[0056] The serum-free culture medium according to the invention may be prepared according to the techniques well known by one skilled in the art consisting generally in adding the different components a) to i) into a basal serum-free growth medium in the appropriate amounts or concentrations.

[0057] Thanks to the serum-free culture medium according to the invention which has a perfectly defined chemical composition, it is now possible to grow and maintain human monocytic cells in reliable reproducible conditions, providing increased control of cellular fate. The serum-free culture medium according to the invention has the additional advantage of preserving the intrinsic characteristics of human monocytic cells cultured in it, in particular their capacity to be transitioned into macrophages and express cell surface markers characteristic for monocytes and macrophages.

[0058] A second object of the present invention is therefore the use of a serum-free culture medium as defined according to the first object of the present invention, for cultivating human monocytic cells, preferably TH P-1 cells.

[0059] Finally, a third object of the present invention is a method for cultivating human monocytic cells, comprising the steps of: i) providing a serum-free culture medium as defined according to the first object of the present invention, and ii) propagating or maintaining said human monocytic cells in said serum-free culture medium to form a cell culture.

[0060] According to a preferred embodiment of this third object, said human monocytic cells are THP-1 cells.

[0061] Step ii) is preferably carried at a temperature of about 37°C in an incubator with CO2 (5%) in a controlled humidified environment.

[0062] Prior to step ii), said human monocytic cells are preferably transitioned to said serum-free culture medium by first cultivating said cells in said basal growth medium supplemented with FBS and then progressively subcultured in the same basal growth medium comprising decreasing amounts of FBS. The transitional of said cells may for example be carried out according to the following protocol:

[0063] - cultivation of the cells in a basal medium, e.g. R.PMI, + 10%FBS,

[0064] - subcultivation of the cells in the basal serum-free growth medium of the invention (e.g. without any supplement a) to i)) + 10% FBS,

[0065] - subcultivation of the cells in the basal serum-free growth medium of the invention (e.g. without any supplement a) to i)) + 5% FBS,

[0066] - subcultivation of the cells in the basal serum-free growth medium of the invention (e.g. without any supplement a) to i)) + 2% FBS,

[0067] - subcultivation of the cells in the basal serum-free growth medium of the invention (e.g. without any supplement a) to i)) + 1% FBS,

[0068] - subcultivation of the cells in the basal serum-free growth medium of the invention (e.g. without any supplement a) to i)) + 0.5% FBS, and

[0069] - subcultivation of the cells in the serum-free culture medium according to the invention, e.g. in the basal serum-free growth medium of the invention comprising at least supplements a) to b).

[0070] At the end of step ii); the cell culture can be maintained at 37°C or cryopreserved.

[0071] It is also possible to differentiate the cells into macrophages. Therefore, according to another particular and preferred embodiment of the third object of the invention, said method comprises a further step of differentiating said human monocytic cells into macrophages.

[0072] The differentiation of said human monocytic cells into macrophages can be carried out according to the techniques known in the art, for example by contacting said cells with phorbol 12-myristate 13-acetate or with 1,25-dihydroxyvitamin D3.

[0073] The following examples are provided by way of example and cannot be interpreted as limiting the scope of the present invention.

[0074] BRIEF DESCRIPTION OF THE DRAWINGS

[0075] - Figure 1 is a graph showing the percentage of differentiated THP-1 cells expressing CDllb marker for macrophages when grown in a classical R.PMI + 10% FBS medium not forming part of the invention (black bars), or in a FBS-free medium according to the present invention (Medium 1, bars with chart patterns) or in a commercial FBS-free cultivating medium sold under the trade name X-VIVO 10 ® (white bars) before (monocytes) and after differentiation into macrophages with phorbol 12-myristate 13-acetate.

[0076] - Figure 2 is a graph showing the percentage of THP-1 cells expressing CD40 marker for monocytes when grown in a classical R.PMI + 10% FBS medium not forming part of the invention (black bars), or in a FBS-free medium according to the present invention (Medium 1, bars with chart patterns) or in a commercial FBS-free cultivating medium sold under the trade name X-VIVO 10 ® (white bars) before (monocytes) and after differentiation into macrophages with phorbol 12- myristate 13-acetate.

[0077] - Figure 3 is a graph showing the percentage of THP-1 cells expressing HLA- DR / DQ / DP markers (for monocytes / DCs) when grown in a classical R.PMI + 10% FBS medium not forming part of the invention (black bars), or in a FBS-free medium according to the present invention (Medium 1, bars with chart patterns) or in a commercial FBS-free cultivating medium sold under the trade name X-VIVO 10 ® (white bars) before (monocytes) and after differentiation into macrophages with phorbol 12-myristate 13-acetate EXAMPLES

[0078] The following materials have been used in the examples:

[0079] - Basal serum-free growth medium sold under the trade name Opti-Mem ®, Gibco;

[0080] - RPMI 1640 medium with GlutaMAX ® (ref. 72400-021, Gibco),

[0081] - Fetal bovine serum (FBS) superior, heat inactivated (Ref. S0615, Merck),

[0082] - Ultra pure sterile water,

[0083] - Penicillin-streptomycin (pen / strep) (10.000 U / mL), Gibco;

[0084] - L-cysteine (L-cyst) (Merck, ref. C7352-25G);

[0085] - L-carnosine (L-carn) (Merck, ref. C9625-5G);

[0086] - Lipid mixture (Merck, ref. L0288-100ML);

[0087] - Nicotinamide (Merck, ref. N0636-100G);

[0088] - Stem cell factor (Merck, ref. S7901-10UG);

[0089] - 3,3',5-Triiodo-L-thyronine (L-Thy) (Merck, ref. T2877-100MG);

[0090] - CaCI2(Merck, ref. C5670-100G);

[0091] - ZnSO4(Merck, ref. I3146-5ML);

[0092] - Mixture of insulin, transferrin and selenium (ITS) (Merck, ref. I3146-5ML); this product contains 1.0 mg / mL recombinant human insulin, 0.55 mg / mL human transferrin (substantially iron-free), and 0.5 pg / mL sodium selenite;

[0093] - Dimethyl sulfoxide (DMSO) (Merck);

[0094] - TH P-1 cells (ATCC),

[0095] - Fetal Bovine Serum (FBS) Superior (Ref. : S0615, Merck),

[0096] - Phosphate Buffered Saline (PBS, Ref. 14190 Gibco),

[0097] - Animal recombinant trypsin sold under the trade name TrypLE® (Gibco),

[0098] - Freezing media sold under the trade name ProFreeze® (Ref BP12-769E, Lonza Bioscience),

[0099] - Blue dead cell stain sold under trade name SytoxBIue ® (Ref. SI 1348, Thermo Fisher Scientific),

[0100] - Anti-HLA-DR, DP, DQ-VioBright 515, human, Clone REA332 (Miltenyi),

[0101] - CDllb Antibody, anti-human, Vio® Bright B515, Clone REA713 (Miltenyi),

[0102] - CD40 Antibody, anti-human, PE-Vio® 615, Clone REA733 (Miltenyi),

[0103] - MACS Comp Bead Kit, anti-REA (Miltenyi),

[0104] - REA Control Antibody (S), human IgGl, PE-Vio® 615, Clone REA293 (Miltenyi),

[0105] - REA Control Antibody (S), human IgGl, APC, Clone REA293 (Miltenyi), - REA Control Antibody (S), human IgGl, Vio® Bright B515, Clone REA293 (Miltenyi),

[0106] - REA Control Antibody (S), human IgGl, APC-Vio® 770, Clone REA293 (Miltenyi),

[0107] - MACS BSA Stock Solution (Miltenyi),

[0108] - AutoMACS® Rinsing Solution (Miltenyi),

[0109] - Commercial FBS-free cultivating medium sold under the trade name X-VIVO 10 ® (Lonza Bioscience),

[0110] - Phorbol 12-myristate 13-acetate (PMA) (Merck).

[0111] These products were stored according to the recommendations of the suppliers before use.

[0112] EXAMPLE 1: Preparation of a serum-free culture medium according to the invention (Medium 1)

[0113] A serum-free culture medium according to the invention (Medium 1) was prepared in the following conditions.

[0114] 1.1 Composition of the serum-free culture medium

[0115] Opti-MEM ® basal serum-free grow medium was supplemented with pen / strep (1 vol. %) and different supplements as described in the following table 1 :

[0116] TABLE 1

[0117] 1.2. Preparation of Medium 1

[0118] When needed, each supplement has first been reconstituted as follows: Stem Cell Factor stock solution

[0119] A 10 |jg / mL stock solution was prepared by adding 1 mL of sterile water to 10 pg of stem cell factor.

[0120] ITS / Lipid Mixture / ZnSO4

[0121] These supplements were already received from the manufacturer as a liquid and thus simply stored at 4°C (ITS and Lipid Mixture) or at room temperature (ZnSO4).

[0122] The product ITS has been used at a dilution of 1 / 100 with sterile water, so that the concentration of its three components in Medium 1 was 0.01 mg / mL recombinant human insulin, 0.0055 mg / mL human transferrin and 0.005 pg / mL sodium selenite.

[0123] L-cvsteine stock solution

[0124] A 25 mg / mL stock solution was prepared by adding 25 mg of L-cysteine in a tube and then adding 1 mL of sterile water.

[0125] L-carnosine stock solution

[0126] A 200 mg / mL stock solution was prepared by adding 5 g of L-carnosine in a flask and then adding 25 mL of sterile water. stock solution

[0127] A 100 mg / mL stock solution was prepared by adding 100 mg of CaC in a tube and then adding 1 mL of sterile water.

[0128] Nicotinamide stock solution

[0129] A 50 mg / mL stock solution was prepared by adding 50 mg of Nicotinamide in a tube and then adding 1 mL of sterile water.

[0130] 3,3',5-Triiodo-L-thyronine stock solution (L-Thv stock solution)

[0131] A 42 mg / mL stock solution of 3,3',5-Triiodo-L-thyronine in DMSO was prepared. 1.3 Preparation of Opti-MEM ® basal serum-free growth medium at 1 vol. % pen / strep

[0132] 5 mL of pen / strep were added in 495 mL of Opti-MEM ® medium. The 500 mL of the Opti-MEM ® basal serum-free growth medium at 1 vol. % pen / strep thus obtained were conditioned in a bottle.

[0133] 1.4 Intermediate dilutions of the supplements

[0134] 3,3',5-Triiodo-L-thyronine intermediate solution

[0135] Dilution 1 / 1000 in Opti-MEM ® medium with 1 vol. % pen / strep: 1 pL of the L-Thy stock solution prepared here above in example 1, step 1.2 wad added to 999 pL of Opti-MEM ® at 1 vol. % pen / strep as prepared here above at step 1.3.

[0136] ZnSO4 intermediate solution

[0137] Dilution 1 / 1000 in Opti-MEM ® medium with 1 vol. % pen / strep: 1 pL of the ZnSO4 solution as received from the supplier wad added to 999 pL of Opti-MEM ® at 1 vol. % pen / strep as prepared here above at step 1.3.

[0138] 1.5 Preparation of Medium 1 according to the invention

[0139] 500 mL of Medium 1 were prepared according to the following protocol :

[0140] - 17.7 mL of Opti-MEM ® medium at 1 vol. % pen / strep as prepared here above at step 1.3 were removed from the 500 mL bottle.

[0141] - the following supplements were then added into the 500 mL bottle containing the Opti-MEM ® medium at 1 vol. % pen / strep according to the details given in the following table 2:

[0142] TABLE 2

[0143] The bottle was then stored at 4°C until use. EXAMPLE 2: Transition of THP-1 cells to serum-free conditions

[0144] In the following example, THP-1 cells have been progressively adapted to grow in fetal-bovine serum (FBS)-free conditions (Medium 1 as prepared here above in example 1).

[0145] 2.1 Materials and method

[0146] 2.1.. k _Growth_ cond i tions

[0147] Incubator with 5% CO2 controlled humidified environment at 37°C.

[0148] 2.1..2^ _Com plete cel L cujtu re _m_e_dj u m_with _FBS_ (CCMH FBS)

[0149] THP-1 cells also were cultivated in standard R.PMI medium + 10% FBS (as standard cultivating conditions), 1% (vol.) Pen / Strep and 0.05 mM p- mercaptoethanol.

[0150] 2.1..3^ _Cell_cou nting

[0151] Cells were centrifuged from 180 to 300g for 5 minutes and resuspended in fresh culture medium to be counted. 1 million cells should be seeded in 30 mL of medium in T75 and placed in the incubator at 37°C, 5% CO2 under humidified atmosphere.

[0152] 2.1..4 _FBS_fre_e transition

[0153] The THP-1 cells have been first cultivated in normal medium (R.PMI) supplemented with 10% FBS and then subcultivated in Medium 1 supplemented with decreasing quantities of FBS as shown in the following Table 3 and according to the subsequent protocol :

[0154] TABLE 3 Protocol

[0155] Step 1 : Grow THP-1 cells in CCM-BSF medium

[0156] Step 2: Recover THP-1 cells in a 50 mL falcon tube, centrifuge at 300g for 5 min and resuspend the pellet in Opti-MEM ®. Cells are counted and seeded in a new flask containing 30 mL of Medium 1 and 3 mL of FBS (10%). Place the flask into the incubator.

[0157] Step 3: Recover THP-1 cells in a 50 mL falcon tube, centrifuge at 300g for 5 min and resuspend the pellet in Opti-MEM ®. Cells are counted and seeded in a new flask containing 30 mL of Opti-MEM ® and 1.5 mL of FBS (5%). Place the flask into the incubator.

[0158] Step 4: Recover THP-1 cells in a 50 mL falcon tube, centrifuge at 300g for 5 min and resuspend the pellet in Opti-MEM ®. Cells are counted and seeded in a new flask containing 30 mL of Opti-MEM ® and 0.6 mL of FBS (2%). Place the flask into the incubator.

[0159] Step 5: Recover THP-1 cells in a 50 mL falcon tube, centrifuge at 300g for 5 min and resuspend the pellet in Opti-MEM ®. Cells are counted and seeded in a new flask containing 30 mL of Opti-MEM ® and 0.3 mL of FBS (1%). Place the flask into the incubator.

[0160] Step 6: Recover THP-1 cells in a 50 mL falcon tube, centrifuge at 300g for 5 min and resuspend the pellet in Opti-MEM ®. Cells are counted and seeded in a new flask containing 30 mL of Opti-MEM ® and 0.150 mL of FBS (0.5%). Place the flask into the incubator.

[0161] Step 7: Recover THP-1 cells in a 50 mL falcon tube, centrifuge at 300g for 5 min and resuspend the pellet in Medium 1. Cells are counted and seeded in a new flask containing 30 mL of Medium 1. Place the flask into the incubator.

[0162] After step 7, THP-1 cells were adapted to the Medium 1 according to the invention. Cells are passaged for 3 further passages. At the last passage, cells are multiplied. At this stage, cells can be either used for further experiments or frozen.

[0163] 2_. k 5^ _Freez i ng. pjptpcpJ

[0164] THP-1 cells adapted to medium 1 conditions can be frozen using a solution of freezing medium prepared with 85% (v / v) of ProFreeze® and 15% (v / v) of DMSO according to the following protocol: - Aliquot 500 pL (2 x 106cells) to the appropriate number of cryovials.

[0165] - Add 500 pL of freezing medium

[0166] - Transfer the vials to a freezing container and freeze the cells at -80°C overnight.

[0167] - Store the cells in liquid nitrogen for long periods.

[0168] 2.1.. Ek _Tha wi ng. protocol

[0169] After freezing and before use, THP-1 cells can be thawed according to the following protocol :

[0170] - Prepare the T75 flask by adding 30 mL of medium 1.

[0171] - Remove the aliquot of cells from liquid nitrogen.

[0172] - Thaw the vial

[0173] - Gently pipette the cell solution in and out several times to remove any remaining ice crystals in the cryovial.

[0174] - Transfer the cells from the cryovial to the flask and place it in the incubator.

[0175] - Keep the cells for 3 passages before starting any experiment.

[0176] EXAMPLE 3: Functionality assessment of THP-1 cells adapted to grow in Medium 1

[0177] The differentiation of THP-1 cells into macrophages in a key function of this cell line.

[0178] In this example, experiments have been carried out to verify that THP-1 adapted to grow in a serum-free culture medium, e.g. in Medium 1, were still able to differentiate into macrophages.

[0179] THP-1 cells were differentiated using Phorbol Myristate Acetate (PMA), thus they were detached using the recombinant trypsin TrypLE® and stained for specific markers:

[0180] - specific marker for macrophages: CDllb, HLA A, B, C and CD40

[0181] - specific markers for dendritic cells and monocytes: major histocompatibility complex (MHC) type II (CD4, HLA-DR, HLA-DR, DP, DO and CD35). Undifferentiated THP-1 cells were also stained for these markers as negative control.

[0182] The expression of the markers was measured using flow cytometry. In addition, the viability of the THP-1 cells was checked by using Sytox blue staining.

[0183] 3.1 Materials and method

[0184] 3, 1_ Pre pa ration _of sta in. i ng. buffe r

[0185] A solution containing phosphate-buffered saline (PBS), pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA was prepared by diluting MACS® BSA Stock Solution 1 :20 with autoMACS® Rinsing Solution. The resulting staining buffer was kept at 2-8°C before use.

[0186] 3, k 2_Panels_of antibodies

[0187] Panel 1 :

[0188] - HLA-DR APC

[0189] - MHCI - PE vio615

[0190] - HLA-DR / DP / DO VB515

[0191] - CD4-APC vio770

[0192] Panel 2:

[0193] - CD35 APC

[0194] - CD40 - PE vio615

[0195] - CDllb VB515

[0196] 3, k 3_ E g uj m ent

[0197] - Incubator with CO2 (5%) controlled humidified environment

[0198] - 70% Ethanol

[0199] - Pipettes and tips: 0.5-10, 5-200, 30-300, and 100-1000 pL

[0200] - Multichannel pipettes 300 pL

[0201] - Eppendorf tubes: 1.5 and 5 mL

[0202] - V-shaped 96-well plate (ref. 353263, Falcon)

[0203] - V-shaped reservoir (ref 613-1175, VWR)

[0204] 3, 1..4_CeJL cultu re _a_nd .plating

[0205] THP-1 cells were cultivated in standard RPMI medium + 10% FBS (as standard cultivating conditions), in Medium 1 as prepared above in example 1 according to the present invention and also, in a comparative purpose, in the commercial FBS-free medium sold under the trade name X-VIVO 10 ®.

[0206] The R.PMI + 10% FBS culture medium has been prepared by removing 57.5 mL of R.PMI 1640 with GlutaMAX® from a 500 mL bottle of this medium and transferring 10 mL in a Falcon tube Add 7 pL of commercial solution of p- mercaptoethanol into 10 mL R.PMI to get a working solution of p-mercaptoethanol in R.PMI (lOmM). Add 2.5 mL of p-mercaptoethanol working solution to get 0.05 mM p-mercaptoethanol in the 500 mL bottle of R.PMI medium and then 50 mL of FBS (10%) + 5 mL of penicillin / streptomycin. Mix by inversion.

[0207] Cell plating was performed by seeding 0.5 million of THP-1 cells in 2.2 mL of cultivating medium (R.PMI + 10% FBS or Medium 1 or X-VIVO 10® medium).

[0208] 3, k 5_CelL differentiation, into jpagroghages

[0209] The cell differentiation has been carried out with the phorbol 12-myristate 13-acetate (PMA) stock solution at 10 mg / mL. Thereafter a PMA working solution has been prepared by diluting the stock solution 1 / 1000 in PBS. The PMA working solution has been prepared extemporaneously.

[0210] The THP-1 cells were differentiated into macrophage-like cells (MO-THP-l) using 11 pL PMA working solution + 2.2 mL of cultivating medium (R.PMI + 10% FBS or Medium 1 or X-VIVO-10® medium) + 0.5 million THP-1 cells per well of a 6 well-plate.

[0211] 48 hours after PMA stimulation, the cultivating medium were and add 2.2 mL of fresh new medium for 4 days.

[0212] 3, k 6_CelL sta in. i ng

[0213] The macrophages / differentiated THP-1 cells were recovered from the 6-well plates. The surnatant was transferred in a 5 mL Eppendorf tube, centrifuged at 300g for 5 min.

[0214] The wells were washed with 2 mL PBS, aspirated and 2 mL of TryPLE were added for 10 to 30 minutes at 37°C (check detachment every 10 minutes under microscope). The cells were recovered with 2 mL of cultivating medium in 5 mL Eppendorf tubes. The tubes were centrifuged at 300 g for 5 min to recover the cells and washed with PBS two times. The pellet was resuspended in 400 pL of staining buffer. The cell suspension was well homogenized and the cells were distributed in 4 wells of the V-shape 96 well plate according to a predetermined plate layout:

[0215] - 100 pL in unmarked well

[0216] - 100 pL in isotype well

[0217] - 100 pL in panel 1 antibodies

[0218] - 100 pL in panel 2 antibodies.

[0219] Aliquots for isotype controls and antibodies were also prepared as follows:

[0220] For panel 1 antibodies mix:

[0221] - 2 pL of anti HLA-DR APC

[0222] - 2 pL of anti MHCI - PE vio615

[0223] - 2 pL of anti HLA-DR / DP / DO VB515

[0224] - 2 pL of anti CD4-APC vio770.

[0225] For panel 2 antibodies mix:

[0226] - 2 pL of anti CD35 APC

[0227] - 2 pL of anti CD40 - PE vio615

[0228] - 2 pL of anti CDllb VB515

[0229] For isotypes mix:

[0230] - 2 pL of REA control APC

[0231] - 2 pL of REA control PE vio615

[0232] - 2 pL of REA control VB515

[0233] - 2 pL of REA control APC vio770

[0234] The plates were then gently mixed, sealed with aluminum foil and incubated for 10 minutes at 4°C.

[0235] After the incubation time, cells were washed with 150 pL PBS. The plates were centrifuged at 300g for 5 min and the supernatant discarded by firmly inverting the plate.

[0236] A solution of SytoxBIue in PBS 1 : 1000 was prepared and 200 pL / well were added (isotype and antibody wells). 200 pL / well of PBS in unmarked wells were also added.

[0237] The plates were incubated 5 minutes at room temperature into the dark. Each sample was measured using a multicolor flow cytometer (BD FACSCelesta ®).

[0238] 3.2 Results

[0239] The results are reported in Figures 1 to 3 annexed.

[0240] These results show that the percentage of THP-1 cells grown and differentiated in Medium 1 according to the present invention expressing CDllb marker (for macrophages) is lower than the ones grown in a classical medium RPMI + 10% FBS but is superior than the ones grown in the commercial FBS-free serum X-VIVO 10 ® (Figure 1). In addition, the percentage of THP-1 cells grown in Medium 1 according to the invention expressing CD40 marker (for monocytes / DCs) is significantly higher as compared to the same cells grown in RPMI + 10% FBS and in the FBS-free commercial medium X-VIVO 10 ® (figure 2). Finally, the percentage of THP-1 cells grown in Medium 1 according to the present invention expressing HLA-DR / DQ / DP markers (for monocytes / DCs) is significantly higher as compared to the same cells grown in RPMI + 10% FBS and in the FBS-free commercial medium X-VIVO 10 ® (figure 3).

[0241] As demonstrated in the present example, the serum-free culture medium according to the present invention enables the growth of human monocytic cells in satisfying conditions and represents a good alternative to classical cultivating medium supplemented with fetal bovine serum. In addition, the monocytic cells grown in the FBS-free medium according to the invention retain their ability to be differentiated into macrophages.

Claims

CLAIMS1. A serum-free culture medium suitable for the culture of human monocytic cells comprising a basal serum-free growth medium, wherein said basal serum-free growth medium is supplemented with at least the following supplements: a) a mixture of amino acids, said mixture comprising at least L-cysteine, and L- carnosine, b) a mixture of lipids, c) nicotinamide or at least one derivative thereof, d) at least one early-acting cytokine, e) one or more divalent cations, f) insulin g) transferrin h) at least one selenite salt and i) at least one thyroid hormone.

2. The serum-free culture medium according to claim 1, wherein said human monocytic cells are THP-1 cells.

3. The serum-free culture medium according to claim 1 or 2, wherein said mixture of lipids is in a liquid form and comprises cholesterol and one or several non-animal fatty acids selected in the group comprising arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid and stearic acid4. The serum-free culture medium according to any one of claims 1 to 3, wherein said early-acting cytokine is selected from the group consisting of stem cell factor, FLT-3 ligand, interleukin-5, interleukin-6, thrombopoietin, interleukin- 3, and a mixture thereof.

5. The serum-free culture medium according to any one of claims 1 to 4, wherein said early-acting cytokine is stem cell factor.6 The serum-free culture medium according to any one of claims 1 to 5, wherein said divalent cations are selected in the group consisting of Ca2+, Zn2+, Mg2+, Mn2+, Fe2+, Cu2+, and any mixtures thereof.

7. The serum-free culture medium according to any one of claims 1 to 6, wherein the concentration of said mixture of amino acids is from 0.5 to 25 mM.

8. The serum-free culture medium according to any one of claims 1 to 7, wherein the concentration of L-cysteine is from 0.05 to 5 mM.

9. The serum-free culture medium according to any one of claims 1 to 8, wherein the concentration of L-carnosine is from 0.5 to 20 mM.

10. The serum-free culture medium according to any one of claims 1 to 9, wherein the at least one thyroid hormone is selected in the group consisting of 3,3',5-triiodi-L-thyronine and thyroxine.

11. The serum-free culture medium according to any one of claims 1 to 10, wherein the concentration of the at least one thyroid is from 0.001 to 1.0 pM.

12. The serum-free culture medium according to any one of claims 1 to 11, wherein the concentration of said mixture of lipids is from 0.5 to 20 mb / L.

13. The serum-free culture medium according to any one of claims 1 to 12, wherein the concentration of nicotinamide or of at least one derivative thereof is from 0.01 to 2 g / mL.

14. The serum-free culture medium according to any one of claims 1 to 13, wherein the concentration of said early-acting cytokine is from 0.4 to 40 ng / L.

15. The serum-free culture medium according to any one of claims 1 to 14 wherein the concentration of divalent cations is from 0.3 to 300 pM.

16. The serum-free culture medium according to any one of claims 1 to 15 wherein the concentration of insulin is from 0.001 to 0.1 mg / mL.

17. The serum-free culture medium according to any one of claims 1 to 16 wherein the concentration of transferrin is from 0.00055 to 0.055 mg / mL.

18. The serum-free culture medium according to any one of claims 1 to 17 wherein the concentration of the at least one selenite salt is from 0.0005 to 0.05 pg / mL.

19. The serum-free culture medium according to any one of claims 1 to 18 wherein said basal serum-free growth medium comprises a buffer, salts, amino acids other than L-cysteine and L-carnosine, sugars, vitamins, growth factors and trace elements.

20. The serum-free culture medium according to any one of claims 1 to 19 wherein it comprises the following supplements: a) L-cysteine in a concentration of about 0.5 mM and L-carnosine in a concentration of about 7.5 mM, b) a mixture of lipids in a concentration of about 10 mL / L, c) nicotinamide in a concentration of about 0.001 g / mL, d) stem cell factor in a concentration of about 3.3 mg / mL, e) CaC and ZnSC , each in a concentration of about 0.3 mM, f) recombinant human insulin in a concentration of about 0.01 mg / mL, g) recombinant human transferrin in a concentration of about 0.0055 mg / mL, h) sodium selenite in a concentration of about 0.005 pg / mL, and i) 3,3',5-triiodi-L-thyronine in a concentration of 1000 pM.

21. Use of a serum-free culture medium as defined in any one of claims 1 to 20 for cultivating human monocytic cells, preferably TH P-1 cells.

22. A method for cultivating human monocytic cells, comprising the steps of: i) providing a serum-free culture medium as defined in any one of claims 1 to 20, and ii) propagating or maintaining said human monocytic cells in said serum-free culture medium to form a cell culture.

23. The method according to claim 22, wherein said human monocytic cells are THP-1 cells.

24. The method according to claim 22 or 23, wherein said method comprises a further step of differentiating said human monocytic cells into macrophages.

Images