Method for obtaining spheroids, and spheroids obtained

Abstract

The invention relates to a method for obtaining spheroids comprising the following steps: - taking a blood sample from an individual; - preparing a depleted blood serum from a sample; - culturing spheroids from human cells; characterised in that the spheroids are cultured in a reprogramming culture medium comprising from 30 to 60% by volume of the depleted blood serum. The invention will be applied in the field of precision medicine and in particular for the development of solutions for identifying and developing personalised drug molecules. More specifically, the invention will be applied in drug-induced hepatotoxicity tests and efficacy tests during drug development.

Description

[0001] PROCESS FOR OBTAINING SPHEROIDS AND SPHEROIDS OBTAINED

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method for obtaining spheroids and the spheroids obtained. The invention will find its application in the field of precision medicine and in particular for the development of solutions for the identification and development of personalized drug molecules. More specifically, the invention will find applications in drug-induced hepatotoxicity and efficacy tests during drug development.

[0004] STATE OF THE ART

[0005] When developing a new drug, controlling liver toxicity is a major challenge. During this phase, it is estimated that 20 to 40% of patients discontinue treatment due to hepatotoxicity observed during clinical trials.

[0006] It is well known that DILI (drug-induced liver injury) is a major challenge in drug development. Most compounds that have passed preclinical tests fail clinical trials due to liver toxicity. One reason for this failure is that existing preclinical models are unable to reproduce the inter-individual heterogeneity of drug response in a population.

[0007] There are several preclinical liver toxicity testing solutions.

[0008] First, the use of cell lines, which have the advantage of being easy to produce, use, and maintain at low cost. However, these cell lines are two-dimensional models that are of little relevance from a physiological point of view. The use of primary human hepatocytes (PHH) is also known. PHHs are prepared from human liver tissue. This preparation from human tissues allowed for preservation of the donor's genetic background. However, the quantity of tissue collected is limited, which requires pooling PPHs from several donors, thus resulting in the erasure of the contribution, if any, of the donor's genome in drug sensitivity. In addition, the collection is invasive with a major risk for the subject.

[0009] Finally, to move closer to precision medicine, organoids have been developed. Organoids are miniature organs grown in vitro. They are three-dimensional cellular structures that mimic the architecture and function of the entire organ. Organoids typically consist of a co-culture of cells that exhibit a higher order of self-assembly, allowing for an even better representation of complex in vivo cellular interactions and responses. Organoids are obtained from stem cells through a process of self-organization, promoted by a culture medium containing the appropriate growth and differentiation factors.

[0010] These organoids have the advantage of being physiologically relevant and being able to mimic the in vivo environment more closely than 2D cellular equivalents. However, their qualities are highly dependent on the starting stem cells. In addition, they are still particularly expensive to develop and use due to the complex culture conditions for their production and maintenance.

[0011] Spheroids have also been developed. Spheroids are three-dimensional (3D) cellular aggregates that can mimic tissues and microtumors. Unlike organoids, spheroids have a more simplified 3D structure, making them suitable for scalability and reproducibility.

[0012] These different solutions, although representing essential advances in the fields of cancer research and drug screening, do not allow for the representation of inter-individual variations found during clinical trials on a cohort of individuals.

[0013] There is therefore a need to propose a new solution for carrying out in vitro tests that are most representative of physiology and inter-individual variations while being easy to produce and implement.

[0014] SUMMARY

[0015] To achieve this objective, according to one embodiment, a method for obtaining spheroids is provided comprising the following steps:

[0016] • preparation of a depleted blood serum from a blood sample,

[0017] • culture of spheroids from human cells, characterized in that the culture of spheroids is carried out in a reprogramming culture medium comprising from 30 to 60% by volume of depleted blood serum.

[0018] Advantageously, the method comprises a step of determining a cohort of individuals representative of a study and the method is carried out for each individual of the cohort, that is to say that the preparation of a depleted blood serum from a blood sample and the culture of the spheroids are carried out for each individual of the cohort.

[0019] The method according to the invention makes it possible to obtain spheroids that are easy to handle while being specific to an individual. The spheroids obtained are called donor-dependent, because they are specific to an individual. The method according to the invention, and in particular the step of culturing the spheroids using the reprogramming culture medium comprising depleted blood serum, makes it possible to advantageously provide growth factors and other elements such as exosomes, cytokines, hormones and microbiotic residues useful for culturing the spheroids that are specific to an individual. The spheroids are also called reprogrammed spheroids in comparison to spheroids cultivated without the addition of the reprogramming culture medium.

[0020] The invention provides a method for obtaining a novel donor-dependent reprogrammed multicellular spheroid model that represents the inter-individual heterogeneity found in a cohort of individuals.

[0021] According to another aspect, the invention relates to a method of testing on spheroids obtained by the previous method.

[0022] In another aspect, the invention relates to a spheroid obtained by the method described above. In another aspect, the invention relates to a use of spheroids obtained by the method described above for evaluating the hepatotoxicity or efficacy of a molecule or composition. The method according to the invention does not involve the destruction of human embryos. Human primary cells and human cell lines do not comprise and are not obtained from embryonic stem cells.

[0023] The tests carried out on spheroids obtained by the present method are particularly representative of inter-individual differences and thus give in vitro results of greater reliability than the systems of the state of the art.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] The aims, objects, as well as the characteristics and advantages of the invention will emerge more clearly from the detailed description of an embodiment thereof which is illustrated by the following accompanying drawings in which:

[0026] Figure 1 shows pictures of unreprogrammed spheroids 100 and reprogrammed spheroids 200 after 1 day, 2 days and 3 days of culture according to Example 1.

[0027] Figure 2 represents a graph of percent cell viability versus log concentration of the drug bosentan on 100 unreprogrammed spheroids and 200 reprogrammed spheroids.

[0028] Figure 3 represents a PCA (Principal Component Analysis) type principal component plot after RNAseq sequencing of 100 non-reprogrammed spheroids and 200 reprogrammed spheroids.

[0029] Figure 4 represents an analysis of differentially expressed genes after RNAseq sequencing of non-reprogrammed spheroids 100 and reprogrammed spheroids 200. Figure 5 represents confocal microscopy images of bile canaliculi in a reprogrammed spheroid 200 from a 41-year-old woman.

[0030] Figure 6 represents confocal microscopy images of reprogrammed 200 spheroids from 6 different individuals showing hepatic stellate cell activation in a donor-dependent manner.

[0031] Figure 7 represents confocal microscopy images of reprogrammed 200 spheroids from 3 different individuals showing donor-dependent extracellular matrix (collagen) deposition.

[0032] Figure 8 represents an RNAseq transcriptomic analysis of 200 reprogrammed spheroids and 100 non-reprogrammed spheroids.

[0033] Figure 9 represents a “Gene Ontology” type analysis which makes it possible to structure, according to cellular functions, the genes which are differentially expressed in the reprogrammed spheroids 200 compared to the non-reprogrammed spheroids 100.

[0034] Figure 10 represents the effectiveness of the reprogrammed spheroids obtained in Example 1 in detecting drug-induced liver toxicity in comparison with existing in vitro and in vivo models.

[0035] Figure 11 shows the stratification of liver toxicity severity by donor sex and age. 200 reprogrammed spheroids are sensitive enough to perform this analysis.

[0036] Figure 12 represents the expression of genes at different stages of liver cancer in spheroids reprogrammed, or educated, according to the method of the invention.

[0037] Figure 13 compares the response of the spheroids reprogrammed, or educated, according to the method of the invention with the response during clinical trials documented in the literature for two anti-cancer drugs classically used in the treatment of liver cancer: sorafenib and cabozantinib.

[0038] Figure 14 illustrates a real case study to compare the results of anticancer efficacy prediction on reprogrammed, or educated, spheroids according to the method of the invention with the results actually obtained in clinical trials on the patient.

[0039] The drawings are given by way of example and are not limiting of the invention.

[0040] DETAILED DESCRIPTION

[0041] Before commencing a detailed review of embodiments of the invention, optional features which may possibly be used in combination or alternatively are set out below:

[0042] - According to one example, the preparation of the depleted blood serum comprises a phase of obtaining a blood serum and a phase of filtering the serum through a 0.45 pm filter;

[0043] - According to one example, the reprogramming culture medium is used for at least 3 days;

[0044] - According to one example, the human cells are derived from at least one human cell line or human primary cells; - According to one example, at least one cell line is chosen from the following cell lines: PANC-1, TWNT-1, THP-1, HepG2 or a mixture thereof;

[0045] - According to one example, the human primary cells are selected from the following human primary cells: primary human hepatocytes (PHH), primary liver cells, circulating cells or circulating tumor cells (CTC), peripheral blood mononuclear cells (PBMC) or a mixture thereof;

[0046] - According to one example, the method for testing the effect of a molecule or composition comprising obtaining spheroids comprises the following steps: o Preparation of a depleted blood serum from a blood sample, characterized in that the culture of spheroids from human cells is carried out in a reprogramming culture medium comprising from 30 to 60% by volume of the depleted blood serum, and that the method comprises a step of exposing the spheroids obtained in the previous step to a molecule or composition to be tested, the exposure of the spheroids being carried out with a reprogramming culture medium comprising from 30 to 60% by volume of the depleted blood serum.

[0047] Spheroids are aggregates of cells growing three-dimensionally in suspension and developing complex interactions between cells and advantageously with a three-dimensional matrix. Spheroids are therefore multi-cellular.

[0048] A cohort is a group of individuals selected for a special purpose, such as a preclinical study.

[0049] A blood sample is understood to mean at least one sample or a plurality of samples of a given volume of blood from a venous, capillary or arterial blood vessel. The blood is placed in one or more tubes. In the remainder of the description, the use of "a sample" or "the sample" is not limiting.

[0050] Blood serum is the fluid obtained after removing clotting factors from blood plasma. Blood serum is the supernatant fluid obtained after clotting and centrifuging blood in a "dry" tube, i.e., without a clotting inhibitor.

[0051] Serum is a mixture of hormones, growth and adhesion factors, buffering agents and other nutritional elements, exosomes and microbiotic residues.

[0052] According to the invention, depleted blood serum is understood to be blood serum from which a portion has been removed; it may optionally be referred to as depleted human blood.

[0053] The method according to the invention is intended for obtaining spheroids and more preferably donor-dependent and advantageously reprogrammed spheroids, also called educated. Reprogramming is understood to mean the education of the spheroid. By donor-dependent or donor-dependent is meant having a spheroid that is cultured from or with a biological element of the donor or an individual. In the present invention, the element is a depleted blood serum obtained from an individual and which is used to supplement the culture medium of the spheroid and form the reprogramming culture medium. The present method advantageously comprises a step of determining a cohort of individuals. This step is intended to define a group of individuals that will be studied.Preferably, individuals are selected according to the objective of a study on predefined criteria such as, for example, age, sex, the presence of one or more pathologies, the presence of one or more predefined markers.

[0054] The method according to the invention uses the blood of an individual to prepare a depleted blood serum. Preferably, the blood comes from a blood sample taken for each individual.

[0055] Blood sampling is carried out, for example, by venous blood sampling. Preferably, the volume of blood used, from the sample, is greater than or equal to 5 ml, preferably greater than or equal to 10 ml.

[0056] The method according to the invention advantageously comprises a step of preparing a depleted blood serum. This depleted serum is produced from a blood sample from an individual. According to a preferred embodiment, the blood sample is left to settle, for example at room temperature for at least 30 minutes up to 1 hour.

[0057] The decanted blood sample is advantageously centrifuged, for example at a speed of between 1000G and 2000G for 10 minutes. The upper translucent phase forming the blood serum is recovered.

[0058] According to one embodiment, the blood serum is filtered. Surprisingly, the filtration is carried out on a 0.45 pm mesh filter which ensures the depletion of the serum, but advantageously preserves the microbiota in particular. The serum obtained after filtration is called depleted blood serum. The depleted serum represents approximately 40% by volume of the blood sample. Depleted blood serum is serum from which a portion has been removed. The removed portion corresponds to the constituents of the serum which are larger than 0.45 pm and which therefore do not pass through the filter mesh.

[0059] The present method comprises the cultivation of spheroids. The cultivation of spheroids is carried out from advantageously human cells. The cells are for example derived from human primary cells such as primary human hepatocytes (PHH), primary human liver cells, circulating cells or circulating tumor cells (CTC) or PBMC for peripheral blood mononuclear cells or mixtures thereof. Alternatively, the cells are for example derived from advantageously immortalized and / or genetically modified and / or cancerous cell lines, for example the cell lines PANC-1, TWNT-1, THP-1, HepG2 or mixtures thereof.

[0060] According to a preferred possibility, mixtures are used in defined proportions, for example the proportions are of the type 100:10:1 or 10:1.

[0061] For example, human immortalized cell lines HepG2, TWNT-1 and THP-1 are used in the proportions (100:10:1).

[0062] For example, the human immortalized cell lines PANC-1, TWNT-1 and THP-1 are used in the proportions (100:10:1). For example, the human immortalized cell lines HepG2, TWNT-1 and PBMCs are used in the proportions (100:10:1).

[0063] For example, human immortalized cell lines PANC-1, TWNT-1 and PBMCs are used in the proportions (100:10:1).

[0064] Spheroid cultivation is typically carried out in culture plates, for example with a U-type bottom and low adhesion.

[0065] Spheroid culture can be performed in a static or microfluidic culture system. Advantageously, spheroid culture is performed with a culture medium that is supplemented with depleted blood serum forming a reprogramming culture medium. The reprogramming culture medium comprises 30 to 60% depleted blood serum by volume relative to the total volume of the reprogramming culture medium.

[0066] Advantageously, the use of depleted blood serum to supplement the spheroid culture medium allows for the production of donor-dependent spheroids, also referred to as reprogrammed spheroids.

[0067] According to a preferred possibility, the spheroid reprogramming culture medium is MammoCult basal, marketed by the company StemCell. Advantageously, the culture medium is supplemented by the addition of heparin, hydrocortisone, penicillin and streptomycin, amphotericin B and a mixture of broad-spectrum antibiotics, preferably Primocin marketed by the company Invivogen. Optionally, the MammoCult basal medium can be replaced by any type of cell culture media (for example DMEM, MEM or RPMI). Preferably, the reprogramming culture medium is used for at least 3 days for the culture of the spheroids. Preferably, the reprogramming medium is used for the entire culture of the spheroids and the maintenance of the spheroids as well. Advantageously, the reprogramming culture medium is also used during tests on the spheroids such as testing molecules or compositions.

[0068] Advantageously, the spheroids are reprogrammed in a static or microfluidic culture system for 3 days by the addition of 30 to 60% by volume of depleted human serum in a MammoCult Basal culture medium.

[0069] According to one aspect, the method according to the invention allows the obtaining of spheroids which can be used for in vitro tests, for example, in vitro tests for evaluating liver damage induced by drugs, chemicals.

[0070] This process advantageously makes it possible to analyze in vitro the toxicity or therapeutic efficacy produced by a synthetic chemical molecule or a natural molecule, on an organ or on a pathology.

[0071] Advantageously, in the method according to the invention, the serum is prepared from the blood of each individual, advantageously from a cohort, then depleted by filtration through a 0.45 μm mesh. The depleted serum is used at a level of 30 to 60% by volume in a culture medium to form a reprogramming culture medium to reprogram the spheroids for at least 3 days. After this step, the reprogrammed spheroids (donor dependent) can be used for example to perform a drug sensitivity analysis.

[0072] Alternatively, reprogrammed spheroids are used to analyze the toxicity or therapeutic efficacy induced by a synthetic or natural chemical molecule, preferably after exposure of 2 days up to 14 days advantageously at multiple concentrations. The toxicity or therapeutic efficacy is determined for example by measurements of cell viability, cell functions and cell structure.

[0073] EXAMPLES

[0074] Example 1: method for obtaining a spheroid according to the invention

[0075] A 10ml blood sample is taken from an individual in a dry tube. The blood is left to stand at room temperature for at least 30 minutes before being centrifuged at 1000G for 10 minutes. The translucent upper phase (the serum) is collected and then filtered through a filter with a 0.45 pm mesh to obtain the depleted serum which will be stored preferably at +4°C or at -20°C for storage beyond two weeks. For the culture of spheroids and in particular the reprogramming of spheroids according to the invention, HepG2 and TWNT-1 cells in the proportions 10:1 are cultured in U-type bottom plates with low adhesion with a reprogramming culture medium supplemented in volume to 50% of this depleted serum. The formation and reprogramming of the spheroids occur naturally for 3 days.

[0076] Example 2: characterization of spheroids obtained in example 1.

[0077] The spheroids obtained in Example 1 are characterized by phenotypic characterization in comparison with so-called non-reprogrammed spheroids 100 cultured only in a culture medium not supplemented with depleted blood serum. The results are visible in Figure 1. The reprogrammed spheroids 200 form more quickly, are denser and more compact than the non-reprogrammed spheroids 100.

[0078] In Figure 2, the cell viability of the reprogrammed spheroids 200 is compared to that of the non-reprogrammed spheroids 100 in response to a four-day exposure to a drug bosentan. The reprogrammed spheroids 200 are more resistant by the order of one Log, i.e., 10 times more resistant. Advantageously, this increase in resistance shows a change in the behavior of the spheroids after reprogramming, a behavior similar to human primary cells.

[0079] Figure 3 shows a PCA (Principal Component Analysis) type principal component graph after RNAseq sequencing of 100 non-reprogrammed spheroids and 200 reprogrammed spheroids. Advantageously, reprogramming the spheroids with the depleted donor serum modifies the overall signature of gene expression while preserving inter-individual heterogeneity.

[0080] In Figure 4, an analysis of differentially expressed genes after RNAseq sequencing of 100 non-reprogrammed spheroids and 200 reprogrammed spheroids. The transcriptomic profile of differentially expressed genes changes significantly, notably 40.5% of mRNAs undergo an increase while 59.5% undergo a reduction in expression after reprogramming.

[0081] The spheroids obtained in Example 1 are characterized structurally and functionally by confocal microscopy demonstrating that there is inter-individual heterogeneity after reprogramming as illustrated in Figures 5 to 7. In Figure 5, we can see spheroids obtained according to the method of Example 1 corresponding to a 41-year-old woman. We observe a formation of functional structures, the bile canaliculi.

[0082] In Figure 6, six reprogrammed spheroids are illustrated and demonstrate donor-dependent activation of hepatic stellate cells.

[0083] In Figure 7, three reprogrammed spheroids demonstrate donor-dependent extracellular matrix (collagen) deposition.

[0084] In Figure 8, a heatmap from RNAseq transcriptomic analysis of the reprogrammed spheroids obtained in Example 1 demonstrates a massive profile change with inter-individual heterogeneity within the group of 200 reprogrammed spheroids compared to 100 non-reprogrammed spheroids.

[0085] In Figure 9, a “Gene Ontology” type analysis which makes it possible to structure, according to cellular functions, the genes which are differentially expressed in the reprogrammed spheroids 200 compared to the non-reprogrammed spheroids 100. The change in gene expression concerns those involved in the cellular processes of biological and metabolic regulation, of the response to external stimuli, but also those which control enzymatic, transcriptomic and molecular transport activities.

[0086] Example 4: Use of spheroids obtained by the present method for the prediction of liver toxicity.

[0087] A comparative study of the effectiveness of the reprogrammed spheroids obtained in Example 1 to detect drug-induced liver toxicity is performed with existing in vitro and in vivo models. The results shown in Figure 10 demonstrate that donor-dependent reprogramming of spheroids can predict the clinical toxicity of these drugs with 100% reliability and sensitivity, a clear advantage over existing models.

[0088] Liver toxicity analysis revealed that the model could accurately predict clinical toxicity across a panel of FDA-approved drugs.

[0089] Reprogrammed spheroids obtained in Example 1 are used to stratify the severity of chemical-induced toxicity based on host risk factors such as age and sex. Reprogramming the spheroids in a donor-dependent manner confirms that the toxicity of some drugs is sex-dependent (e.g., bosentan) and others age-dependent (e.g., stavudine) as shown in Figure 11.

[0090] Example 5: Use of spheroids obtained by the present method for the stratification of liver cancer stages.

[0091] A comparative study of the effectiveness of educated, or so-called reprogrammed, spheroids obtained according to example 1, is carried out with the depleted serum of patients suffering from liver cancer to reproduce the molecular classification of the different stages of liver cancer.

[0092] The results of quantitative analysis of different genes by polymerase chain reaction (PCR) method on reprogrammed spheroids are illustrated in Figure 12. The expression of several genes is monitored for the different stages of liver cancers A, B, C. It was found that the expression levels of the different genes studied at the different stages of liver cancer correspond to the levels reported in the literature.

[0093] The educated spheroids faithfully reproduce a patient's molecular tumor classification.

[0094] The spheroids obtained according to the invention can therefore be validly used to stratify the stages of liver cancer in a patient.

[0095] Example 6: Use of spheroids obtained by the present method for predicting the effectiveness of a molecule.

[0096] A comparative study of the effectiveness of educated spheroids obtained according to Example 1 with depleted serum of a patient suffering from liver cancer with clinical results is carried out.

[0097] Sorafenib and cabozantinib, two drugs used to treat liver cancer, are used on educated spheroids obtained according to Example 1, with the depleted serum of a patient suffering from liver cancer.

[0098] The results obtained are compared with those obtained with clinical trials in a patient and reported in the literature. The results are illustrated in Figure 13.

[0099] The response to sorafenib and cabozantinib with educated spheroids is similar to those obtained in clinical trials.

[0100] Example 7: Use of spheroids obtained by the present method in comparison with a real case study.

[0101] Educated spheroids obtained according to Example 1 with the depleted serum of a 74-year-old male patient suffering from advanced stage C liver cancer are used in parallel in a case study detailed in Figure 14.

[0102] This study reveals the efficacy of educated spheroids in predicting personalized clinical responses to treatment with anticancer drugs such as sorafenib, cabozantinib, and lenvatinib in this patient.

[0103] The invention is not limited to the embodiments previously described and extends to all embodiments covered by the invention.

Claims

Claims 1. Method for obtaining spheroids comprising the following steps: • preparation of a depleted blood serum from a blood sample from an individual, • culture of spheroids from human cells, Characterized in that the spheroid culture is carried out in a reprogramming culture medium comprising 30 to 60% by volume of depleted blood serum.

2. Method according to the preceding claim in which the preparation of the depleted blood serum comprises a phase of obtaining a blood serum and a phase of filtering the serum through a 0.45 μm filter.

3. A method according to any preceding claim wherein the reprogramming culture medium is used for at least 3 days.

4. Method according to any one of the preceding claims in which the human cells are derived from at least one human cell line or from human primary cells.

5. Method according to the preceding claim in which at least one cell line is chosen from the following cell lines: PANC-1, TWNT-1, THP-1, HepG2 or a mixture thereof.

6. Method according to claim 4 wherein the human primary cells are chosen from the following human primary cells: primary human hepatocytes (PHH), primary liver cells, circulating cells or circulating tumor cells (CTC), peripheral blood mononuclear cells (PBMC) or a mixture thereof.

7. Method according to any one of the preceding claims in which the preparation of a depleted blood serum and the culture of the spheroids are carried out for each individual of a determined cohort of individuals representative of a study.

8. Method for testing the effect of a molecule or composition comprising obtaining spheroids comprising the following steps: • Preparation of a depleted blood serum from a blood sample from an individual, • culture of spheroids from human cells, Characterized in that the culture of spheroids from human cells is carried out in a reprogramming culture medium comprising from 30 to 60% by volume of the depleted blood serum, and that the method comprises a step of exposing the spheroids obtained in the previous step to a molecule or composition to be tested, the exposure of the spheroids being carried out with a reprogramming culture medium comprising from 30 to 60% by volume of the depleted blood serum.

9. Spheroid obtained by the method according to any one of claims 1 to 7.

10. Use of spheroids obtained by the method according to any one of claims 1 to 7 for evaluating the hepatotoxicity or the efficacy of a molecule or a composition.

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