FR3145363A1 - Process for obtaining spheroids and spheroids obtained - Google Patents

Abstract

Process for obtaining spheroids and spheroids obtained The invention relates to a method for obtaining spheroids comprising the following steps: taking blood from an individual, preparing a depleted blood serum from a sample, culturing spheroids from human cells, characterized in that the culture of spheroids is carried out in a reprogramming culture medium comprising 30 to 60% by volume of depleted blood serum. 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 during drug-induced hepatotoxicity testing and effectiveness during drug development.

Description

Method for obtaining spheroids and spheroids obtained

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 during drug-induced hepatotoxicity tests and efficacy during drug development.

STATE OF THE ART

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

It is well known that DILI (drug-induced liver injury) is a major challenge in drug development. Most compounds that have passed preclinical testing fail in 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.

There are several preclinical testing solutions for liver toxicity.

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. PHH are prepared from human liver tissues. This preparation from human tissues allowed for the preservation of the donor's genetic background. However, the quantity of tissues collected is limited, which requires grouping PPHs from several donors, thus resulting in the erasure of the contribution, if any, of the donor's genome in the sensitivity to a drug. In addition, the collection is invasive with a major risk for the subject.

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 self-organization process, promoted by a culture medium containing the appropriate growth and differentiation factors.

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.

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.

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

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

SUMMARY

• prélèvement sanguin sur un individu,
• préparation d’un sérum sanguin appauvri à partir du prélèvement sanguin,
• culture de sphéroïdes à partir de cellules humaines,

caractérisé en ce que la culture de sphéroïdes est réalisée dans un milieu de culture de reprogrammation comprenant de 30 à 60% en volume du sérum sanguin appauvri.To achieve this objective, according to one embodiment, a method for obtaining spheroids is provided comprising the following steps:

• blood sample from an individual,
• preparation of depleted blood serum from the blood sample,
• culture of spheroids from human cells,

characterized in that the spheroid culture is carried out in a reprogramming culture medium comprising from 30 to 60% by volume of the depleted blood serum.

Advantageously, the method comprises determining a cohort of individuals representative of a study and the method is carried out for each individual in the cohort, i.e. a blood sample, the preparation of a depleted blood serum and the culture of spheroids are carried out for each individual in the cohort.

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 by the reprogramming culture medium comprising an impoverished blood serum, makes it possible to advantageously provide growth factors and other elements such as exosomes, cytokines, hormones and microbiotic residues useful for the culture of the spheroids that are specific to an individual. The spheroids are also called reprogrammed spheroids in comparison with spheroids cultured without the addition of the reprogramming culture medium.

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.

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

According to another aspect, the invention relates to a spheroid obtained by the method described above.

According to another aspect, the invention relates to a use of spheroids obtained by the method described above for evaluating the hepatotoxicity or the efficacy of a molecule or a composition.

The method according to the invention does not involve the destruction of human embryos. The human primary cells and human cell lines do not comprise and are not obtained from embryonic stem cells.

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 better reliability than the systems of the state of the art.

BRIEF DESCRIPTION OF THE FIGURES

The aims, objects, as well as the features and advantages of the invention will become more apparent from the detailed description of one embodiment thereof which is illustrated by the following accompanying drawings in which:

There shows photos of unreprogrammed spheroids 100 and reprogrammed spheroids 200 after 1 day, 2 days and 3 days of culture according to example 1.

There represents a graph of percent cell viability versus log concentration of drug bosentan on non-reprogrammed spheroids 100 and reprogrammed spheroids 200.

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

There represents an analysis of differentially expressed genes after RNAseq sequencing of 100 non-reprogrammed spheroids and 200 reprogrammed spheroids.

There depicts confocal microscopy images of bile canaliculi in a reprogrammed 200 spheroid from a 41-year-old woman.

There represents confocal microscopy images of reprogrammed 200 spheroids from 6 different individuals showing activation of hepatic stellate cells in a donor-dependent manner.

There represents confocal microscopy images of reprogrammed 200 spheroids from 3 different individuals showing donor-dependent extracellular matrix (collagen) deposition.

There represents a transcriptomic analysis by RNAseq of 200 reprogrammed spheroids and 100 non-reprogrammed spheroids.

There 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.

There represents the effectiveness of the reprogrammed spheroids obtained in Example 1 to detect drug-induced liver toxicity in comparison with existing in vitro and in vivo models.

There represents the stratification of liver toxicity severity grades based on donor gender and age. The reprogrammed 200 spheroids are sensitive enough to perform this analysis.

The drawings are given as examples and are not limiting of the invention.

DETAILED DESCRIPTION

Before beginning a detailed review of embodiments of the invention, optional features which may optionally be used in combination or alternatively are set out below:

- 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 0.45µm filter;

- In one example, the reprogramming culture medium is used for at least 3 days;

- In one example, the human cells are derived from at least one human cell line or from human primary cells;

- According to one example, at least one cell line is selected from the following cell lines: PANC-1, TWNT-1, THP-1, HepG2 or a mixture thereof;

- 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;

• Prélèvement sanguin d’un individu,
• Préparation d’un sérum sanguin appauvri à partir du prélèvement sanguin, caractérisé en ce que la culture de sphéroïdes à partir de cellules humaines est réalisée dans un milieu de culture de reprogrammation comprenant de 30 à 60% en volume du sérum sanguin appauvri, et que le procédé comprend une étape d’exposition des sphéroïdes obtenus à l’étape précédente à une molécule ou une composition à tester, l’exposition des sphéroïdes étant réalisée avec un milieu de culture de reprogrammation comprenant de 30 à 60% en volume du sérum sanguin appauvri.

– according to one example, the method of testing the effect of a molecule or composition comprising obtaining spheroids comprises the following steps:

• Blood sample taken from an individual,
• Preparation of a depleted blood serum from the 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.

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.

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

Blood sampling means at least one sampling or a plurality of samplings of a determined 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 sampling” or “the sampling” is not limiting.

Blood serum is the fluid obtained after removal of 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.

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

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

The method according to the invention is intended for obtaining spheroids and more preferably spheroids dependent on the donor and advantageously reprogrammed.

By donor dependent or donor dependent is meant 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 spheroid culture medium 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 who will be studied. Preferably, the individuals are selected according to an 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.

The method according to the invention uses the blood of an individual to prepare an impoverished blood serum. Preferably, a blood sample is taken for each individual.

The blood sample is, for example, taken by venous blood sampling. Preferably, the volume of blood collected, in one or more samples, is greater than or equal to 5 ml, preferably greater than or equal to 10 ml.

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 to 1 hour.

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.

According to one embodiment, the blood serum is filtered. Surprisingly, the filtration is carried out on a 0.45 µm mesh filter which ensures the depletion of the serum, but advantageously preserves in particular the microbiota. 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 a serum from which a portion has been removed. The removed portion corresponds to the constituents of the serum which are larger than 0.45 µm and which therefore do not pass through the filter mesh.

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. According to another possibility, 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.

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.

As an example, human immortalized cell lines HepG2, TWNT-1 and THP-1 are used in the proportions (100:10:1).

As an example, human immortalized cell lines PANC-1, TWNT-1 and THP-1 are used in the proportions (100:10:1).

As an example, human immortalized cell lines HepG2, TWNT-1 and PBMCs are used in the proportions (100:10:1).

As an example, human immortalized cell lines PANC-1, TWNT-1 and PBMCs are used in the proportions (100:10:1).

The culture of spheroids is classically carried out in culture plates, for example with a U-type bottom and low adhesion.

Spheroid culture can be done in a static or microfluidic culture system.

Advantageously, the culture of the spheroids is carried out with a culture medium which is supplemented with the depleted blood serum forming a reprogramming culture medium. The reprogramming culture medium comprises from 30 to 60% of depleted blood serum by volume relative to the total volume of the reprogramming culture medium.

Advantageously, the use of depleted blood serum to supplement the spheroid culture medium allows the production of donor-dependent spheroids, also known as reprogrammed spheroids.

According to a preferred possibility, the culture medium for reprogramming the spheroids is the basal MammoCult, 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 basal MammoCult 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 molecule or composition tests.

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 blood in a MammoCult Basal culture medium.

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.

This process advantageously allows the analysis of the toxicity or therapeutic efficacy produced by a synthetic chemical molecule or a natural molecule, on an organ or on a pathology.

Advantageously, in the method according to the invention, the serum is prepared from the blood of each individual and 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.

According to one possibility, the reprogrammed spheroids are used to analyze the toxicity or therapeutic efficacy induced by a synthetic or natural chemical molecule, preferably after an 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.

EXAMPLES

Example 1: Process for obtaining a spheroid according to the invention

A 10 ml blood sample is taken from an individual in a dry tube. The blood is left to rest at room temperature for at least 30 min before being centrifuged at 1000 G for 10 min. The translucent upper phase (the serum) is collected and then filtered through a filter with a 0.45 µm 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 the spheroids and in particular the reprogramming of the 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 occurs naturally for 3 days.

Example 2: characterization of spheroids obtained in example 1.

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 supplanted with depleted blood serum. The results are visible at . Reprogrammed 200 spheroids form faster, are denser and more compact than non-reprogrammed 100 spheroids.

In , 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.

In , a PCA (Principal Component Analysis) type principal component graph is shown after RNAseq sequencing of the non-reprogrammed spheroids 100 and the reprogrammed spheroids 200. Advantageously, reprogramming the spheroids with the impoverished serum of the donors modifies the global signature of gene expression while preserving inter-individual heterogeneity.

In , an analysis of differentially expressed genes after RNAseq sequencing of non-reprogrammed spheroids 100 and reprogrammed spheroids 200. The transcriptomic profile of differentially expressed genes changes significantly with 40.5% of mRNAs undergoing an increase while 59.5% undergoing a reduction in expression after reprogramming.

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

In , six reprogrammed spheroids are illustrated and demonstrate donor-dependent activation of hepatic stellate cells.

In , three reprogrammed spheroids demonstrate donor-dependent extracellular matrix (collagen) deposition.

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

In , a "Gene Ontology" type analysis that allows to structure, according to cellular functions, the genes that 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 that control enzymatic, transcriptomic and molecular transport activities.

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

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

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

In , the use of the reprogrammed spheroids obtained in Example 1 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).

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

Claims (10)

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. 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. A method according to any preceding claim wherein the reprogramming culture medium is used for at least 3 days. A method according to any preceding claim wherein the human cells are derived from at least one human cell line or from human primary cells. 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. The method of claim 4 wherein 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. A method according to any preceding claim, wherein 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. 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. Spheroid obtained by the method according to any one of claims 1 to 7. 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.