RU2661105C2 - Method for producing spheroids of heparg cells in a medium without dimethyl sulphoxide - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely to production of spheroids of HepaRG cells expressing cytochromes P450 3A4, 2B6, 2C19, 2C9. Method comprises culturing HepaRG cells in a complete William's E culture medium supplemented with L-glutamine, 10 % fetal bovine serum, 5 mcg/ml recombinant human insulin, 5x10^-5 M hydrocortisone hemisuccinate, 1 % penicillin/streptomycin, in CO₂-incubator at 37 °C, 95 % air, 5 % CO₂ and relative humidity of 98 %, induction. Method further includes transfer to 96-well plates with low adhesion and a U-shaped bottom to form spheroids and the subsequent incubation of the spheroids in serum-free William's E nutrient medium supplemented with L-glutamine, 1x Insulin-Transferrin-Selenium, 1 mcg/ml bovine serum albumin, 5x10^-5 M hydrocortisone hemisuccinate, 1x interchangeable amino acids, 1 % penicillin or streptomycin at 37 °C, 95 % air, 5 % CO₂ and a relative humidity of 98 %.

EFFECT: invention allows to obtain spheroids of HepaRG cells expressing cytochromes P450 3A4, 2B6, 2C19, 2C9, to study the metabolism and toxicity of pharmaceuticals.

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Description

The invention relates to biotechnology and can be used to obtain differentiated HepaRG cells in the composition of spheroids with a high level of expression of the main cytochromes P450 as a model of the human liver, in particular, for preclinical evaluation of biotransformation and hepatotoxicity of drugs.

S. et al. Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. // Drug Metab. Dispos. 2010. Vol. 38, №3. P. 516-525].HepaRG cells have the unique ability to differentiate in vitro and acquire the basic characteristics of mature human hepatocytes, including the expression of cytochromes of the P450 family, phase II metabolism enzymes, transporters, and nuclear receptors [

S. et al. Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. // Drug Metab. Dispos. 2010. Vol. 38, No. 3. P. 516-525].

S. et al. Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. // Drug Metab. Dispos. 2010. Vol. 38, №3. P. 516-525; LeCluyse E.L. et al. Organotypic liver culture models: meeting current challenges in toxicity testing. // Crit. Rev. Toxicol. 2012. Vol. 42, №6. P. 501-548].To differentiate and achieve optimal expression of metabolic enzymes, HepaRG cells are cultured in high density (in a monolayer, 2D) in the presence of DMSO (1-2% DMSO). Under such conditions (in the presence of DMSO), for example, the main liver cytochrome Sur3A4 does not respond to standard inducers, phenobarbital and rifampicin, and when DMSO is canceled, a significant decrease in the activity and expression of cytochromes occurs. Therefore, the selection of cultivation conditions is necessary to create an accurate model containing functional hepatocytes [

S. et al. Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. // Drug Metab. Dispos. 2010. Vol. 38, No. 3. P. 516-525; LeCluyse EL et al. Organotypic liver culture models: meeting current challenges in toxicity testing. // Crit. Rev. Toxicol. 2012. Vol. 42, No. 6. P. 501-548].

One approach to solving this problem is to form organotypic three-dimensional (3D) models. These models most reflect the structural organization of the tissue (intercellular contacts, cell polarity, etc.). In addition, biochemical, metabolic, and signaling pathways in cells as part of such models are most active compared to 2D models [LeCluyse E.L. et al. Organotypic liver culture models: meeting current challenges in toxicity testing. // Crit. Rev. Toxicol. 2012. Vol. 42, No. 6. P. 501-548; Takahashi Y. et al. Three-dimensional (3D) spheroid cultures improve the metabolic gene expression profiles of HepaRG cells. // Biosci. Rep. 2015]. Several approaches or a combination of these are used to maintain and maintain the functional activity of 3D liver models for a long time: 1) spheroids are cultured in media with a mixture of factors, hormones, antioxidants without the addition of animal serum / components (Biopredic, Life Technologies, PromoCell); 2) include nonparenchymal liver cells (vascular endothelial cells, stellate cells, Kupffer cells, etc.) in the composition of spheroids, capable of forming an extracellular matrix and secreting growth factors, chemokines (Insphero); 3) use extracellular matrices and microbioreactors with nutrient circulation [Bao J. et al. Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. // Cell Transplant. 2013. Vol. 22, No. 2. P. 299-308; Wang Z. et al. HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening. // J. Appl. Toxicol. 2015. Vol. 35, No. 8. P. 909-917; Materne E.-M., Tonevitsky A.G., Marx U. Chip-based liver equivalents for toxicity testing - organotypicalness versus cost-efficient high throughput. // Lab Chip. 2013. Vol. 13, No. 18. P. 3481-3495; Press A.T. et al. Cell type-specific delivery of short interfering RNAs by dye-functionalized theranostic nanoparticles. // Nat. Commun. 2014. Vol. 5. P. 5565.]. The protocols for the formation of 3D models of the liver and methods for maintaining their functional activity are diverse and depend on the type of cells and their origin (primary human or animal hepatocytes, HepaRG cells, etc.), as well as on tasks (analysis of hepatotoxicity of drugs, creation of a fibrosis model diabetes, etc.).

US 7456018 B2 describes a human hepatoma line and culture conditions. The invention provides a set of cells obtained from cloned cancer cells of a human liver. The presented cell culture is suitable for use in diagnostic, therapeutic and prophylactic purposes, for the study of viral diseases. The HepaRG cell line is derived from hepatitis and is an example of a cell line that has all the characteristics of the lines, can be naturally infected with parasites, viruses, has morphological characteristics corresponding to liver cells, and has all the biological functions of the hepatocyte.

Cells were isolated from a liver tumor taken from a patient suffering from hepatocarcinoma and viral hepatitis C.

The cultivation process includes:

- the phase of cell proliferation in a culture medium containing corticosteroids in a non-toxic concentration, which contributes to the optimal differentiation of normal human hepatocytes;

- further, having reached complete confluence, in the phase of cell differentiation the addition of DMSO in an amount sufficient to induce differentiation.

The patent describes the use of various culture media to obtain, maintain, proliferate, differentiate, transfect, and line infection. The culture medium should contain at least one corticosteroid, mainly hydrocortisone hemisuccinate in a non-toxic concentration, which contributes to the optimal differentiation of normal human hepatocytes in order to maintain the stability of the cell population. For the cultivation of HepaRG cells, Williams E medium is used, with 5 μg / ml insulin, 100 u / ml penicillin, 100 μg / ml streptomycin, 5.10 - 7 M hemisuccinate hydrocortisone, 2 mM / ml L-glutamine and 10% FCS.

HepaRG cell karyotypes should be analyzed after 8 passages. To change the morphology of cells during differentiation, a medium is used, which is prepared from the main medium containing only 5 mg / l of insulin and 10% FCS, 2% DMSO and 5 * 10-5 M hydrocortisone hemisuccinate. When cultured after 2-4 days, the medium causes the death of more than 90% of the cells and the morphology of the surviving cells changes significantly.

During the proliferation phase, HepaRG cells form a homogeneous population with an epithelial phenotype (lack of regular structural organization). After complete confluence is achieved, the cells undergo significant morphological changes. After four weeks, the number of granular hepatocytes increases. The addition of DMSO in two weeks induces complete morphological differentiation - the cells are organized into trabeculae, the results are comparable to those obtained in primary culture of normal hepatocytes that form tubule-like structures.

The patent shows the expression values of the main genes of the HepaRG cell line. Moreover, by comparison, correlated expression levels were established in human hepatocytes and HepaRG cells.

HepaRG cells have enzyme activity associated with detoxification at a level almost equivalent to normal human hepatocytes, with the exception of dextromethorphan demethylase, which is slightly less active.

The disadvantage of this cultivation technique in the presence of DMSO is the lack of induction of the main liver cytochrome Sur3A4 with the addition of phenobarbital and rifampicin, as well as the absence of three-dimensional structural organization.

Patent EP 2421954 A1 (WO 2010123357 A1) describes an invention comprising the culture of a human hepatocyte cell line and its use in the bio-artificial liver system (BAL). These systems are used to treat patients suffering from liver failure, which allows you to temporarily compensate for the loss of hepatocellular function and contain a bioreactor with functional liver cells. The main difficulty is obtaining cells with a wide functional spectrum of metabolism from recently isolated human hepatocytes suitable for successful clinical use in BAL. The authors of the present invention managed to develop a line of human hepatocytes with a wide spectrum of metabolism and functionality.

As a culture using HepaRG, taken from the national collection of cultures of microorganisms of the Pasteur Institute, (No. 1-2652). Cells are cultured as 3D formations. In the proliferation phase, hepatocytes are cultured in a medium preferably containing serum, hormones, growth factors and antibiotics. In the differentiation phase, hepatocytes are cultured in a medium preferably containing at least 0.5 mM glutamate, serum, hormones, growth factors and antibiotics. The cell differentiation phase involves culturing cells in a culture medium not containing DMSO.

At the proliferation stage, cells are cultured as a monolayer or suspension, then in the form of three-dimensional models.

The patent describes a culture medium. Differentiated cells have a liver-specific metabolic activity, characterized by the following metabolic parameters:

- the rate of elimination of ammonia should be at least 0.05 μmol / h / mg protein;

- the urea production rate should be at least 0.003 μmol / h / mg protein;

- the production rate of apoAl should be at least 0.02 ng / h / mg protein;

- the rate of consumption of lactate should be at least 0.02 μmol / h / mg protein.

- albumin production rate should be at least 1 ng / h / mg protein.

The level of CPS expression in differentiated cells should be no more than 20%, preferably more than 40% of the expression level of 18S rRNA; the level of GS expression is more than 250%, preferably more than 300%, the level of CYP3A4 expression should exceed 70%, preferably more than 100%.

The technical problem to which the developed technical solution is directed is to develop a method for differentiating HepaRG cells to obtain a human liver model in the form of spheroids of HepaRG cells with a high level of expression of the main P450 cytochromes.

The technical result obtained by the implementation of the developed method consists in the possibility of studying the metabolism and toxicity of pharmaceuticals in HepaRG cells.

To achieve the technical result, it is proposed to use the method of differentiation and cultivation without the addition of DMSO and serum.

HepaRG cells were cultured in complete culture medium (PPS): William's E supplemented with L-glutamine (Gibco, USA), 10% fetal bovine serum (HyClone, Thermo Scientific, USA), 5 μg / ml recombinant human insulin (Gibco, USA ), 5 × 10 ^-5 M hydrocortisone hemisuccinate (Sigma, USA), 1% penicillin / streptomycin (Gibco, USA), in a CO2 incubator (37 ° C, 95% air, 5% CO ₂ , relative humidity 98%) .

After the cells reach a confluent monolayer, 2% DMSO is added to the PPS to induce differentiation. 14 days after the start of differentiation, spheroids consisting of 5000 HepaRG cells are formed in PPS without DMSO;

Spheroids are formed in 96-well plates with low adhesion and a U-shaped bottom for 5 days.

After 5 days of formation, spheroids are transferred to serum-free culture medium and incubated for another day. Serum-free culture media (BPS) include: William's E supplemented with L-glutamine (Gibco, USA), 1x Insulin-Transferrin-Selenium (ITS-G, Thermo Scientific, USA), 1 μg / ml bovine serum albumin (BSA, Sigma, Germany), 5 × 10 ^-5 M hydrocortisone hemisuccinate (Sigma, USA), 1 x essential amino acids (NEAA, Thermo Scientific, USA), 1% penicillin / streptomycin (Gibco, USA) [PromoCell (Hepatocyte Medium), Life Technologies (Hepatocyte Maintenance Supplement Pack)] [Bao J. et al. Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. // Cell Transplant. 2013. Vol. 22, No. 2. P. 299-308; Wang Z. et al. HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening. // J. Appl. Toxicol. 2015. Vol. 35, No. 8. P. 909-917].

The invention is illustrated by drawings.

In FIG. Figure 1 presents the results of a study of the expression level of cytochrome 3A4 under various conditions of differentiation and cultivation in serum-free nutrient medium (BPS) and serum-containing medium (PPS). In particular, in FIG. Figure 1 presents an analysis of the expression level of cytochrome 3A4 (Sur3A4) in undifferentiated HepaRG cells cultured in a monolayer (2D), in differentiated for 2 weeks in a medium with 2% DMSO HepaRG (2D) cells, in cells composed of spheroids formed over 5 days in a tablet with low adhesion in PPS without DMSO, with 2% DMSO or 2% fibroblasts, and in cells composed of spheroids after transferring them to BPS (without DMSO and without serum) for a day. The change in Cyp3A4 expression was calculated relative to the level of expression in differentiated HepaRG cells.

In FIG. 2 presents the results of a study of the level of expression of cytochromes under various conditions of differentiation and cultivation in serum-free nutrient medium (BPS) and serum-containing medium (PPS). In particular, FIG. Figure 2 shows the change in gene expression in HepaRG cells during the formation of spheroids in PPS without DMSO, with 2% DMSO or 2% fibroblasts (section 2) and further transfer of the formed spheroids per day to BPS (section 3) relative to HepaRG cells differentiated for 2 weeks.

The invention is illustrated by examples of specific performance.

Example 1. The study of hepatotoxicity of xenobiotics.

HepaRG cells were cultured in complete nutrient medium (PPS): William's E supplemented with L-glutamine (Gibco, USA), 10% fetal bovine serum (HyClone, Thermo Scientific, USA), 5 μg / ml recombinant human insulin (Gibco, USA ), 5 × 10 ^-5 M hydrocortisone hemisuccinate (Sigma, USA), 1% penicillin / streptomycin (Gibco, USA), in a CO ₂ incubator (37 ° C, 95% air, 5% CO2, relative humidity 98%) .

After the cells reached a confluent monolayer, 2% DMSO was introduced into the PPS to induce differentiation. 14 days after the start of differentiation, spheroids consisting of 5,000 HepaRG cells were formed in PPS without DMSO.

Spheroids were formed in 96-well plates with low adhesion and a U-shaped bottom for 5 days.

After 5 days of formation, spheroids were transferred to serum-free culture medium and incubated for another day. Serum-free culture media (BPS) include: William's E supplemented with L-glutamine (Gibco, USA), 1x Insulin-Transferrin-Selenium (ITS-G, Thermo Scientific, USA), 1 μg / ml bovine serum albumin (BSA, Sigma, Germany), 5 × 10 ^-5 M hydrocortisone hemisuccinate (Sigma, USA), 1 x essential amino acids (NEAA, Thermo Scientific, USA), 1% penicillin / streptomycin (Gibco, USA) [PromoCell (Hepatocyte Medium), Life Technologies (Hepatocyte Maintenance Supplement Pack)] [Bao J. et al. Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. // Cell Transplant. 2013. Vol. 22, No. 2. P. 299-308; Wang Z. et al. HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening. // J. Appl. Toxicol. 2015. Vol. 35, No. 8. P. 909-917].

The obtained spheroids were cultured in a closed cycle of nutrient medium circulation using the Homunculus microbioreactor (ICBM) [Samatov T.R. et al. Modeling the metastatic cascade by in vitro microfluidic platforms. // Prog. Histochem. Cytochem. 2015. Vol. 49, No. 4. P. 21-29].

Xenobiotics (acetaminophen, metformin and isoniazid) were incubated for 48 h in a serum-free culture medium consisting of William's E (Gibco, USA), 2 mM L-glutamine (Gibco, USA), 1 mg / ml human serum albumin ( Sigma, USA), nutritional supplement consisting of insulin, transferrin and selenium (5.5 μg / ml insulin, Gibco, USA), non-essential amino acids (Gibco, USA), 50 μM hemisuccinate hydrocortisone (Sigma, USA), 1% penicillin / streptomycin (Gibco, USA).

Cell viability was assessed by the ability of living cells to convert soluble yellow 3- (4,5-dimethylthiazol-2-yl) -2,5-tetrazolium bromide (MTT) into insoluble purple-blue intracellular crystals of MTT-formazan (MTT-f), as described in [Burmistrova et al., 2013].

DNA breaks were detected cytofluorometrically using the TUNEL (terminal deoxyuridine tagging of the ends) Click-iT® Plus TUNEL Assay (Life Technologies, USA) according to the manufacturer's protocol.

The experimental determination of lactate concentrations in samples of culture media was carried out spectrophotometrically using the Lactate Dry-fast test system (Sentinel Diagnostics, Italy) according to the manufacturer's protocol.

Example 2. The study of the biotransformation of drugs in vitro on the example of two test drugs, warfarin and dasatinib.

HepaRG cells were cultured in complete nutrient medium (PPS): William's E supplemented with L-glutamine (Gibco, USA), 10% fetal bovine serum (HyClone, Thermo Scientific, USA), 5 μg / ml recombinant human insulin (Gibco, USA ), 5 × 10 ^-5 M hydrocortisone hemisuccinate (Sigma, USA), 1% penicillin / streptomycin (Gibco, USA), in a CO ₂ incubator (37 ° C, 95% air, 5% CO2, relative humidity 98%) .

After the cells reached a confluent monolayer, 2% DMSO was introduced into the PPS to induce differentiation. 14 days after the start of differentiation, spheroids consisting of 5,000 HepaRG cells were formed in PPS without DMSO.

Spheroids were formed in 96-well plates with low adhesion and a U-shaped bottom for 5 days.

After 5 days of formation, spheroids were transferred to serum-free culture medium and incubated for another day. Serum-free culture media (BPS) include: William's E supplemented with L-glutamine (Gibco, USA), 1x Insulin-Transferrin-Selenium (ITS-G, Thermo Scientific, USA), 1 μg / ml bovine serum albumin (BSA, Sigma, Germany), 5 × 10 ^-5 M hydrocortisone hemisuccinate (Sigma, USA), 1 x essential amino acids (NEAA, Thermo Scientific, USA), 1% penicillin / streptomycin (Gibco, USA) [PromoCell (Hepatocyte Medium), Life Technologies (Hepatocyte Maintenance Supplement Pack)] [Bao J. et al. Serum-free medium and mesenchymal stromal cells enhance functionality and stabilize integrity of rat hepatocyte spheroids. // Cell Transplant. 2013. Vol. 22, No. 2. P. 299-308; Wang Z. et al. HepaRG culture in tethered spheroids as an in vitro three-dimensional model for drug safety screening. // J. Appl. Toxicol. 2015. Vol. 35, No. 8. P. 909-917].

The obtained spheroids were cultured in a closed cycle of nutrient medium circulation using the Homunculus microbioreactor (ICBM) [Samatov T.R. et al. Modeling the metastatic cascade by in vitro microfluidic platforms. // Prog. Histochem. Cytochem. 2015. Vol. 49, No. 4. P. 21-29].

Claims (4)

1. A method of producing spheroids of HepaRG cells expressing cytochromes P450 3A4, 2B6, 2C19, 2C9, comprising cultivating HepaRG cells in William's E complete culture medium supplemented with L-glutamine, 10% fetal bovine serum, 5 μg / ml recombinant human insulin, 5x10 ^-5 M hydrocortisone hemisuccinate, 1% penicillin / streptomycin in a CO ₂ incubator at 37 ° C, 95% air, 5% CO ₂ and 98% relative humidity, induction, transfer to 96-well plates with low adhesion and U-shaped bottom to form spheroids and subsequent incubation of spheroids rotochnoy medium William's E supplemented with L-glutamine, 1x Insulin-Transferrin-Selenium, 1 ug / ml bovine serum albumin, 5x10 ^-5 M hydrocortisone hemisuccinate, 1x nonessential amino acids, 1% penicillin and streptomycin at 37 ° C, 95% air , 5% CO ₂ and relative humidity 98%. 2. The method according to claim 1, characterized in that when the cells reach a confluent monolayer to induce differentiation, 2% DMSO is added to the PPS and cultured for 14 days. 3. The method according to claim 1, characterized in that the spheroids are formed in tablets for 5 days. 4. The method according to claim 1, characterized in that the incubation in serum-free culture medium is carried out for 1 day in a CO ₂ incubator.

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