WO2016198699A1

WO2016198699A1 - Method for grafting a heartcardiac cell onto the chorioallantoic membrane of a fertilized egg

Info

Publication number: WO2016198699A1
Application number: PCT/EP2016/063534
Authority: WO
Inventors: Philippe NIRDÉ; Sylvain Richard; Amanda FINAN-MARCHI
Original assignee: Universite De Montpellier; Centre National De La Recherche Scientifique (Cnrs); Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2015-06-12
Filing date: 2016-06-13
Publication date: 2016-12-15
Also published as: FR3037338B1; FR3037338A1

Abstract

The present invention relates to a method for grafting at least one heartcardiac cell, or at least one cell capable of being differentiated into a heartcardiac cell, onto the vascularized chorioallantoic membrane of a fertilized egg. The present invention also relates to a method for characterizing the activity of a compound vis-à-viswith respect to heartcardiac tissue using a fertilized egg, on the chorioallantoic membrane of onto which at least one heartcardiac cell, or at least one cell capable of being differentiated into a heartcardiac cell, has been grafted. The present invention also relates to a kit including a fertilized egg whereon onto which at least one heartcardiac cell, or at least one cell capable of being differentiated into a heartcardiac cell, has been grafted.

Description

HEART CELL GRAFT METHOD

ON THE CHORIALLANTOID MEMBRANE OF EGG FECONDE

The present invention is in the medical field, and more particularly in the field of cardiology.

The subject of the present invention is indeed a method of grafting on the vascularized chorioallantoic membrane of a fertilized egg of at least one cardiac cell or of at least one cell capable of differentiating into a cardiac cell. The present invention in particular

A method of grafting a fragment of cardiac tissue to the vascularized chorioallantoic membrane of a fertilized egg is described. The subject of the present invention is also a method for characterizing the activity of a compound with respect to cardiac tissue using a fertilized egg on the chorioallantoic membrane from which at least one cardiac cell has been grafted. at least one cell capable of differentiating into a cardiac cell, or a fragment of tissue

Heart. The present invention also relates to a kit comprising a fertilized egg on which has been grafted at least one cardiac cell, or a cell capable of differentiating into a cardiac cell, or a fragment of cardiac tissue.

Cardiology to date lacks intermediate experimental models between in vitro experimentation (isolated cells, cardiomyocyte culture, organotypic transplantation) and

In-vivo experimentation (heart transplantation on mammals, including humans). There is therefore a real need to have new models of in vivo experimentation.

The inventors have now developed a method of transplanting at least one cardiac cell, or a cell capable of differentiating into a cardiac cell or a fragment of cardiac tissue on a vascularized chorioallantoic membrane of a fertilized egg, said method

Comprising providing, on said fertilized egg, a microlysis on the surface of the chorioallantoic membrane (MCA) of the egg, contacting said isolated cell or tissue fragment with said MCA at said level; microlesion, and the incubation of the egg so treated for at least 48 hours in a thermostatically controlled chamber and under suitable conditions, so as to allow the transplantation of said isolated cell or said cardiac tissue fragment on the MCA and the

Vascularization of the cardiac tissue fragment.

The transplantation of cells onto chorioallantoic membranes of fertilized eggs under in-ovo conditions (in the shell) dates from 1912 (Murphy J., ROUS P., 1912). Much more recently, some tumor cells have been grafted in ovo (Deryugina, E. I and Quigley P., 2008). It is only more recently that culture out of the egg (ex-ovo) has been used (Dunn B.E. and Boone M.A.,

1976). The chorioallantoic membrane is also widely used for the study of anti-angiogenic activity of molecules (Ribatti D., 2008). Eggs are also used for vaccine production for the Goodpasture man, Woodruff and Buddingh, 1931). However, for more than a hundred years, the date of the first in ovo transplant publication, no team had the idea of grafting heart fragments, more particularly mammalian heart cells, mammalian heart cell clusters or mammalian heart fragments on CAM as an alternative to animal testing.

A method according to the invention of grafting at least one cardiac cell or at least one cell capable of differentiating into a cardiac cell, or a fragment of cardiac tissue on the chorioallantoic membrane of a fertilized egg, is located half way. path between the two techniques mentioned above, providing by the vascularization of the graft a reliable alternative to test new therapeutic routes safely before passing to the clinical stage.

A method according to the invention makes it possible in particular to graft myocardial fragments onto the chorioallantoic membrane (MCA) of a fertilized egg: once implanted, these grafts spontaneously vascularize and return to their pulsatile function after 2 to 5 days. A method according to the invention also makes it possible to graft isolated neonatal and adult cardiac cells, or cells capable of differentiating into cardiac cells, on the chorioallantoic membrane (MCA) of a fertilized egg. After transplantation, isolated heart cells can form cardiac tissue. Similarly, after transplantation, isolated cells capable of differentiating into cardiac cells differentiate and can form cardiac tissue. The heart tissue thus formed is vascularized and acquires a pulsatile function. It is then possible to test, by morphological or biological analysis, the effects of different treatments on these implanted cardiac tissues. This model avoids the use of laboratory animals and allows direct visual observation of implants, thus creating an alternative to commonly used in vivo grafting techniques. This model is usable for human cells or tissues, hence its great interest, indeed the inventors have shown that there is no species barrier opposing the transplantation of mammalian tissues. It therefore represents a reliable alternative model between the test tube (cardiac cell culture or organotypic transplant) and the transplantation of whole organs on pigs or large primates, while exempting itself from the legal constraints related to animal experimentation. The inventors have improved the culture technique out of the egg to obtain viabilities comparable to those of in-ovo cultures.

Unlike tumor cells, which are highly proliferative, cardiac cells are highly differentiated and specialized cells with, at the adult stage and in the state of knowledge, a proliferative capacity almost non-existent. The chorioallantoic membrane makes it possible to graft fragments of cardiac tissues capable of revascularizing after implantation and spontaneously recovering a pulsatile function. The culture ex ovo allows a high accessibility to MCA, favoring the direct dynamic observation of grafts and quantitative morphological studies (echo DOPPLER) and biological that no other existing model proposes. The injection of therapeutic products into the omphalomesternal vein of the egg makes it possible to study both the pharmacological properties, the stability and the safety.

In addition, the other existing models have the major disadvantage of producing cardiac cells with modified (electro) physiological properties, making it difficult to interpret the data obtained on long-term cultures. A method according to the invention also makes it possible to solve various technical difficulties encountered during the cultivation of cells, such as precursors of cardiac cells, in particular concerning the cellular phenotype, the full functionality of these cells and the organization of functional cardiac tissue. .

The use of fertilized eggs, authorized by the Food and Drug Administration (FDA, guideline HFD-240, HFM-40, HFZ-200, June 2006. Guidance for Industry), does not enter the legislation of laboratory animals Article R214-90 of the Rural Code and Maritime Fisheries) and is therefore exempt from the heavy constraints of animal testing.

The invention, inexpensive to implement (1.50 euros / chicken egg / experiment), allows the implantation of two grafts at least per egg,

The main advantages of a method according to the invention are: legally the MCA is not considered as an animal model, the model is non-immune during the first 18 days of development, the micro-environment is antiseptic, direct observations and dynamics are possible, in fact the organization of isolated cells into a functional, beating and / or contractile tissue is rapid. This model allows relatively long-term studies, especially to observe substances whose passage and / or accumulation in a living organism is slow, but also in the short term, for acute phenomena, such as for example observing the effect drugs, or study mechanisms and biological signaling pathways. In addition, MCA is resistant enough to receive isolated cells or tissue fragments, even after removal of the ectodermal membrane.

It is important to proceed at the right moment of the development of the egg to a sufficient lesion to promote implantation of the graft without this lesion becoming unacceptable, which would fail the transplant. In particular, in a particular aspect of a method according to the invention of grafting at least one cardiac cell, said cell being part of cardiac tissue consisting of undissociated cells, it is important to carry out said microlysis at a stage of egg development of at least 11 days, so that the vascularization of MCA is sufficient to allow grafted heart tissue to become pulsatile and / or contractile.

Thus, when the graft involves contacting one or a few cells, microlesion can be carried out on a fertilized egg of at least 6 days. When the graft consists in bringing into contact a cluster of dissociated cells, the microlesion can be carried out on a fertilized egg of at least 7 or 8 days.

When the graft consists in bringing into contact a cluster of undissociated cells, such as a fragment of cardiac tissue, the microlesion can be carried out on a fertilized egg of at least 11 days.

Moreover, if the size of the chicken egg only allows the implantation of small fragments, the size of the implants is however sufficient to carry out physiological studies there. However, to carry out laboratory tests, such as the faculty of new therapies to repair or regenerate the heart muscle after infarction, or the safety of new molecules, the invention is a significant advance in a field today. lack of alternative techniques. It will be readily apparent to those skilled in the art that the process of transplanting isolated cells or a fragment of cardiac tissue onto the chorioallantoic membrane of a fertilized egg according to the invention can be carried out on a larger egg. a chicken egg, especially on an ostrich egg.

The invention will now be described in detail with the aid of examples intended to illustrate it without limiting it, various modifications that may be made without departing from the general scope of the invention. The present invention relates to a method of grafting at least one cardiac cell, or at least one cell capable of differentiating into cardiac cells, on the vascularized chorioallantoic membrane of a fertilized egg, said method comprising the implementation of following steps:

- culture in ex ovo conditions of the fertilized egg until a development stage of at least 6 days,

the production of a microlysis on the surface of the chorioallantoic membrane (MCA) of the fertilized egg,

contacting at least one cardiac cell, or at least one cell capable of differentiating into cardiac cells, and said MCA at the level of said microlesion, and incubating the fertilized egg thus treated for at least 48 hours in a thermostatically controlled chamber, at a temperature between 36 ° C and 39 ° C and with a degree of hygrometry between 80% and 90%.

In a method according to the invention, the grafting of at least one cardiac cell, or at least one cell capable of differentiating into cardiac cells, designates the implantation of said cell, or graft, on the vascularized chorioallantoic membrane. a fertilized egg.

The term "fertilized egg" also refers to a fertilized egg. The term "chorioallantoic membrane" is an anatomical term that refers to the extraembryonic membrane that results from the fusion of the allantoic vesicle membrane and the Von Baer serosal membrane (chorion). The chorioallantoic membrane comprises three layers, or layers: the ectoderm, attached to the shell membrane, the mesoderm, enriched in blood vessels and the endoderm, in contact with the allantoid cavity.

By "ex ovo conditions" is meant a fertilized egg, grown completely out of its shell after transfer of the egg 48 hours after the beginning of the incubation in a concave container, preferably polystyrene weighing cups. According to a particular embodiment, said concave container whose lid is manufactured by putting face-to-face 2 cups connected by adhesive tape, a bit like a mold.

In a particular aspect of a method of transplanting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell on the vascularized chorioallantoic membrane of a fertilized egg, said cardiac cell is a cardiac cell. isolated. According to a more particular aspect of a method according to the invention, said isolated cardiac cell is a cardiomyocyte.

According to a particular aspect of a method of transplanting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell on the vascularized MCA of a fertilized egg, said cell capable of differentiating into a cell cardiac is an isolated cell. By "cell capable of differentiating into a cardiac cell" is meant an undifferentiated cell, a totipotent cell, a multipotent cell, a stem cell, an "Induced Pluripotent Cell" (iPS) cell, as obtained according to a method described in EP 1 970 446), a precursor cell of a cardiac cell or a progenitor cell of a cardiac cell. Those skilled in the technical field of cardiology and cardiac cells will easily determine the phenotypic and / or genotypic criteria for selecting an isolated cell capable of differentiating into a cardiac cell, appreciated in particular by the expression of specific protein markers. or the characteristic contractile function of the cardiac cell. The skilled person skilled in the technical field of cardiology and cardiac cells will also determine the culture conditions and the addition of differentiation factors, to lead to the differentiation of said cell into a cardiac cell. Phenotypic and / or genotypic criteria for selecting an isolated cell capable of differentiating into a cardiac cell, appreciated in particular by the expression of specific protein markers or the characteristic contractile function of the cardiac cell.

According to a more particular aspect, the invention therefore relates to a process characterized in that it comprises the following steps: - the culture, in ex ovo conditions, of the fertilized egg until a stage of development of the egg of at least 6 days,

the production of a microlysis on the surface of chorioallantoic membrane (MCA) of Pouf,

contacting at least one isolated cardiac cell and said MCA, at said microlesion, and

incubating the egg thus treated for at least 48 hours in a thermostatically controlled chamber at a temperature of between 36 ° C. and 39 ° C., preferably between 36.5 ° C. and 38.5 ° C., preferably between 37 ° C. and 37 ° C., ° C and 38 ° C and more preferably at 37.8 ° C and with a degree of hygrometry between 80% and 90% and more preferably 84%.

According to another aspect more particular, the invention therefore relates to a method characterized in that it comprises the following steps:

- the culture, in ex ovo conditions, of the fertilized egg until a stage of development of the egg of at least 6 days,

the production of a microlysis on the surface of the chorioallantoic membrane (MCA) of the egg,

contacting at least one isolated cell capable of differentiating into a cardiac cell and said MCA, at the level of said microlesion, and

In one embodiment, the culture, in ex ovo conditions, of the fertilized egg is made up to an egg development stage of at least 7 days.

In one embodiment, the culture, in ex ovo conditions, of the fertilized egg is made up to an egg development stage of at least 8 days.

In another particular aspect of a method of transplanting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell on the vascularized MCA of a fertilized egg, said cardiac cell is a cell. cardiac tissue fragment.

According to this particular aspect, the subject of the invention is a method characterized in that it comprises the following steps:

- the culture in ex ovo conditions of the fertilized egg until a stage of development of the egg of at least 9 days,

the realization of a microlesion on the surface of the MCA of the fertilized egg, contacting a fragment of cardiac tissue and said MCA at the level of said microlesion, and

incubation of the fertilized egg thus treated for at least 48 hours in a thermostatically controlled chamber, at a temperature of between 36 ° C. and 39 ° C. and with a degree of hygrometry of between 80% and 90%.

In one embodiment, the culture, in ex ovo conditions, of the fertilized egg is made up to an egg development stage of at least 10 days.

In one embodiment, the culture, under ex ovo conditions, of the fertilized egg is made up to an egg development stage of at least 11 days.

The expression "an egg development stage of at least 9 days" means an egg that has been cultured for at least nine days after fertilization. This expression includes an egg having reached a developmental stage of nine days (D9), ten days (D10), eleven days (D11), twelve days (D12), thirteen days (D11), fourteen days (D14), fifteen days (D15), sixteen days (D16), seventeen days (D17) or eighteen days (D18).

The expression "an egg development stage of at least 10 days" means an egg that has been cultured for at least 10 days after fertilization. This expression includes an egg having reached a developmental stage of ten days (D10), eleven days (D11), twelve days (D12), thirteen days (D11), fourteen days (D14), fifteen days (D15), sixteen days (D16), seventeen days (D17) or eighteen days (D18).

The expression "an egg development stage of at least 11 days" means an egg that has been cultured for at least nine days after fertilization. This expression includes an egg having reached a developmental stage of eleven days (D1), twelve days (D12), thirteen days (D1), fourteen days (D14), fifteen days (D15), sixteen days (D16), seventeen days (day seven) or eighteen days (day eight).

The expression "an egg development stage of at least 6 days" means an egg that has been cultured for at least six days after fertilization. This expression includes an egg having reached a developmental stage of six days (D6), seven days (D7), eight days (D8), nine days (D9), ten days (D10), eleven days (D11), twelve days. days (D12), thirteen days (D1 3), fourteen days (D14), fifteen days (D15), sixteen days (D16), seventeen days (D17), or eighteen days (D18). The expression "an egg development stage of at least 11 days" means an egg that has been cultured for at least eleven days after fertilization. This expression includes an egg that has reached a developmental stage of eleven days (J1l), twelve days (D12), thirteen days (D1-3), fourteen days (D14), fifteen days (D15), sixteen days (D16), seventeen days (D17) or eighteen days (D18). In a particular aspect, in a method for grafting on the MCA of a fertilized egg according to the invention, the fertilized egg is cultivated under ex ovo conditions until a development stage of at least 10 days and from plus 12 days, either D10, D11 or D12. Vascular density and vascular pressure at days J10 to J12 is favorable for graft revascularization in the case of cardiac tissue.

In a more particular aspect, the present invention relates to a method of grafting a cardiac tissue fragment to vascularized MCA of a fertilized egg, said cardiac tissue fragment consisting of a cardiac tissue biopsy, and in one aspect more particularly, a fragment of cardiac tissue also comprising pericardial tissue

In a method of grafting on the vascularized MCA of a fertilized egg, according to the invention, the culture, in ex ovo conditions, of the fertilized egg is carried out under standard conditions, namely:

the temperature is preferably between 36 ° C and 39 ° C, preferably between 36.5 ° C and 38.5 ° C, preferably between 37 ° C and 38 ° C and more preferably 37.7 ° C. the degree of hygrometry is preferably between 30% and 90%, preferably between 80% and 85%, and more preferably 84%.

In a method of grafting at least one cell onto the vascularized MCA of a fertilized egg, according to the invention, the incubation of the egg after completion of the microlesion for at least 48 hours in a thermostatically controlled chamber and under conditions adapted allows the grafting of said fragment of cardiac tissue on the MCA and the vascularization of said fragment.

The method for grafting on the MCA of a fertilized egg according to the invention comprises the application of a microlesion in the chosen implantation zone, said microlesion corresponding to a superficial lesion capable of causing micro-haemorrhage and / or to a small lesion.

In a method of grafting a cardiac tissue fragment on the MCA of a fertilized egg, according to the invention, the size of said microlesion is slightly greater than the size of the graft, preferably the size of the graft + 1 mm . The size of the microlysis is chosen so that the microlysis does not cause death by hemorrhage of the embryonated egg, and depends on the egg and the vascularization density of the MCA. The surface area of the microlysis applied is between 0.5 cm ² and 16 cm ^2. Preferably, the application is followed and / or is concomitant with the absorption of blood flowing out due to the lesion of the microorganisms. capillaries.

According to a particular aspect of a grafting method on the MCA of a fertilized egg, according to the invention, said microlesion is performed by removing the ectodermal leaf from the fertilized egg in the chosen implantation zone.

According to a more particular aspect of a method of grafting on the MCA of a fertilized egg, according to the invention, the microlesion is carried out by removing the ectodermal leaf from the fertilized egg, according to one of the following methods: - The ectodermal sheet is removed using a flexible paper: said flexible paper is able to match the irregularities of the surface of the MCA. According to a particular aspect of a method according to the invention, the ectodermal sheet is removed by applying to the MCA an absorbent paper of the "blotting paper" type, the application of flexible paper has the advantage of absorbing the

5 blood flowing during the micro-haemorrhage created,

- The ectodermal sheet is removed using an abrasive object: said abrasive object is able to create the abrasion of the ectodermal sheet by scraping. According to a particular aspect of a method according to the invention, the ectodermal sheet is removed by contacting the MCA with an abrasive object, and by scraping, said abrasive object is preferably chosen from: a needle tip

10 for a syringe, a glass rod and a cone tip for a pipette,

the ectodermal sheet is removed using an absorbent substance capable of sticking to the surface of the MCA, the ectodermic layer is then removed by rolling. According to a particular aspect of a method according to the invention, the ectodermal sheet is removed with an absorbent substance, and preferably a cotton-spike tip.

According to a particular aspect of a method of grafting on the MCA of a fertilized egg, according to the invention, said microlysis is carried out by removing the ectodermal leaf from the fertilized egg, in the chosen implantation zone, concomitantly. or immediately followed by a step allowing the absorption of the blood that has elapsed resulting from the microhemorrhage induced by the removal of the ectodermal leaflet. Preferably, blood absorption is achieved by the application of a substance

Absorbent, preferably using blotting paper, or using a single channel pipette, without touching the MCA.

In another particular aspect of a method of grafting on MCA of a fertilized egg, according to the invention, wherein a fragment of cardiac tissue is grafted, said heart tissue is applied to the MCA so that the tissue cardiac itself is in contact with the MCA and the pericardium is not in contact with the MCA. Such an arrangement has the technical effect of promoting vascularization of the graft.

In a method according to the invention, the egg is kept at a constant temperature by incubation in a thermostatically controlled chamber, in which the internal temperature is preferably between 36 ° C and 39 ° C, preferably between 36.5 ° C and 39 ° C. C. and 38.5 ° C., preferably between 37 ° C. and 38 ° C. and preferably 37.7 ° C.

In a method according to the invention of grafting a fragment of cardiac tissue on a vascularized MCA of a fertilized egg, the egg is incubated in a thermostatically controlled chamber, in which the degree of hygrometry is preferably between 80%. and 90%, more preferably 84%.

According to another particular aspect, the subject of the present invention is a method of grafting on the MCA of a fertilized egg, characterized in that during all the manipulations carried out outside the thermostatically controlled enclosure, the egg is placed on a support heating. The inventors have indeed shown that it is important that the temperature of the egg is kept constant, not only during the incubation periods but also during manipulations taking place outside the thermostated chamber. The skilled person wishing to maintain constant temperature of the egg during handling may choose a heating medium to maintain the temperature of the egg to an optimal value. According to a particular aspect of a method according to the invention, during handling the egg is placed on a heating blanket.

In a particular aspect, in a method of transplanting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell on the MCA of a fertilized egg, according to the invention, said fertilized egg is a bird's egg, said bird's egg is preferably chosen from: a chicken egg and an ostrich egg.

According to another particular aspect, in a method of grafting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell on the MCA of a fertilized egg, according to the invention, the cardiac cell the cell capable of differentiating into a cardiac cell or the cardiac tissue fragment is from a vertebrate animal, preferably said vertebrate animal is selected from: 1) birds, preferably chickens, and 2) mammals, preferably monkeys, pigs, mice, rats and humans.

In particular, the cardiac cell, the cell capable of differentiating into a cardiac cell or the cardiac tissue fragment originates from an animal of a species different from the species from which the fertilized egg originates. Typically, the cardiac cell, the cell capable of differentiating into a cardiac cell or the cardiac tissue fragment is from a vertebrate animal selected from mammals; more particularly selected from the group consisting of monkeys, pigs, mice, rats and humans.

According to another particular aspect, in a method of grafting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell on the MCA of a fertilized egg, according to the invention, said cardiac cell said cell capable of differentiating into a cardiac cell or said cardiac tissue fragment is healthy, free from known pathology. According to another particular aspect, in a method of grafting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell on the MCA of a fertilized egg, according to the invention, said cardiac cell said cell capable of differentiating into a cardiac cell or said cardiac tissue fragment is afflicted with a pathology, preferably a cardiac pathology.

The present invention also relates to a method for transplanting at least one cell capable of differentiating into a cardiac cell, in a fertilized egg, said method comprising the implementation of the following steps: - culture in ex ovo conditions of the fertilized egg until a development stage of at least 6 days,

injecting at least one cell capable of differentiating into a cardiac cell in the vascular system of the fertilized egg, more particularly at the level of the vascular system of the chorioallantoic membrane (MCA) of the fertilized egg, and

the incubation of the fertilized egg thus treated for at least 24 hours, particularly at least 48 hours in a thermostatically controlled chamber, at a temperature of between 36 ° C. and 39 ° C. and with a degree of hygrometry of between 80% and 90%; %.

The injection into the vascular system of the fertilized egg can be performed intravenously or intra-arterially.

By "cell capable of differentiating into a cardiac cell" is meant an undifferentiated cell, a totipotent cell, a multipotent cell, a stem cell, an "Induced Pluripotent Cell" (iPS) cell, as obtained according to a method described in EP 1 970 446), a precursor cell of a cardiac cell or a progenitor cell of a cardiac cell. The injection can be performed by any suitable instrument known to those skilled in the art, such as a micropipette for example.

The expression "an egg development stage of at least 6 days" means an egg that has been cultured for at least six days after fertilization. This expression includes an egg having reached a developmental stage of six days (D6), seven days (D7), eight days (D8), nine days (D9), ten days (D10), eleven days (D11), twelve days (D12), thirteen days (D13), fourteen days (D14), fifteen days (D15), sixteen days (D16), seventeen days (D17), or eighteen days (D18).

In a particular embodiment, the cell (s) capable of differentiating into a cardiac cell may be labeled with a fluorescent dye or marker, thus making it possible to follow their migration in the vascular system of the fertilized egg, ie of the embryo. .

The conditions of ex ovo culture and incubation of the fertilized egg treated according to this embodiment are the same as those indicated above for the other processes according to the invention in which the cells are deposited following the microlesion of the MCA.

This particular object of the invention comprising the injection of cells capable of differentiating into a cardiac cell at the level of the vascular system of the fertilized egg has many advantages over the simple deposition of cells at the level of the MCA.

The inventors have indeed discovered, and surprisingly, that such cardiac progenitor cells (cells capable of differentiating into a cardiac cell) after their injection into the vascular system will circulate in said vascular system and will be able to go nestle and to fix and then to differentiate at the level of the cardiovascular tissue and the heart of the embryo and will be able to be inserted into said integrated cardiac and vascular system of the embryo in formation. These cells can thus be found in the complete differentiated cardiovascular system of the organism after hatching. Such a method according to this variant of the invention allows the insertion, observation and study of the evolution of cardiac progenitor cells within a complex and integrated system in a complete environment involving the various metabolic interactions of such a system.

This allows the study of the differentiation and maturation of cardiac progenitor cells in a complex integrated medium and opens new avenues for studying the physiology of cardiac development.

In addition, the labeling of the cells injected according to this method according to the invention facilitates the monitoring of their displacement, their fixation, their differentiation and their integration into the tissues of the fertilized egg and the embryo. According to another particular aspect, the present invention also relates to a method for characterizing the activity of a compound with respect to cardiac tissue, said method comprising the following steps:

Obtaining a fertilized egg grafted with at least one cardiac cell, or at least one cell capable of differentiating into cardiac tissue, on the MCA of a fertilized egg, said method comprising the implementation of the following steps: the culture in ex ovo conditions of the fertilized egg until a development stage of at least 8 days, the realization of a microlesion on the surface of the MCA of the fertilized egg, the bringing into contact of at least a cardiac cell, or at least one cell capable of differentiating into cardiac cells, and said MCA at said microlesion, and incubating the fertilized egg thus treated for at least 48 hours in a thermostatically controlled chamber, until 'to formation of cardiac tissue,

Injecting the compound into said fertilized egg grafted, preferably by intravenous injection,

• observation of said cardiac tissue,

The characterization of the activity of said compound.

According to another particular aspect, the present invention also relates to a method for characterizing the activity of a compound with respect to cardiac tissue, said method comprising the following steps:

Obtaining a fertilized egg grafted with a fragment of cardiac tissue on the MCA of a fertilized egg, said method comprising the implementation of the following steps: the cultivation in ex-ovo conditions of the fertilized egg to a stage of development from at least 9 days, performing a surface microlysis of the MCA of the fertilized egg, contacting a fragment of cardiac tissue and said MCA at said microlesion, and incubating the egg fertilized and treated for at least 48 hours in a thermostatically controlled chamber,

• observation of said cardiac tissue,

The characterization of the activity of said compound.

The inventors have indeed shown that a compound could be injected into a fertilized egg on which heart tissue was grafted, obtained in particular by a process according to the invention, and that it was possible to observe the activity of said compound. with respect to said fragment, and from this observation to characterize the activity of said compound vis-à-vis the cardiac tissue.

The cardiac activity can in particular be characterized according to the following parameters:

presence or absence of biochemical markers, such as proteins specifically expressed by heart cells,

presence or absence of biological or functional markers, in particular of contractile activity, automatic activity, global electrical activity or cellular electrical activity, such as the presence of an action potential, in particular, mapping cellular electrical activity in the tissue formed, analysis of the biological activity by fluorescent indicators (calcium, potential, oxidative stress, for example).

According to a more particular aspect, the present invention also relates to a method for characterizing the activity of a compound with respect to cardiac tissue, said method comprising the following steps:

a) obtaining a fertilized egg grafted with a cardiac cell, a cell capable of differentiating into a cardiac cell, heart tissue obtained by a method of transplanting at least one cell or a fragment of cardiac tissue , in particular according to the invention, b) injecting the compound into said fertilized egg grafted, preferably by intravenous injection,

c) observing said cardiac tissue,

d) comparing the results of the observation of step c) with the results of the observation of cardiac tissue of a fertilized egg grafted with cardiac tissue that did not receive said compound, and

e) the characterization of the activity of said compound. In fact, the comparison of the activity of the cardiac tissue having been grafted onto an egg to which the compound has been administered with the activity of the cardiac tissue grafted onto an egg which has not received the compound, or cardiac tissue of reference, makes it possible to characterize the properties of said compound.

According to a more particular aspect, the present invention also relates to a method of characterizing the activity of a compound with respect to the cardiac tissue, in which the cardiac tissue is observed by an intravital method chosen from preferably among: Doppler ultrasound and / or microscopic observation, imaging methods using fluorescent probes or other method for measuring contraction and rhythm. In a method according to the invention, the Doppler ultrasound is preferably performed with a high frequency probe, the use of a Doppler ultrasound with a 33 MHz probe had never been used for objects as small as the grafts as used in the present invention. According to another embodiment of the invention, the microscopic observation is preferably performed by means of a fluorescence optical microscope. Those skilled in the art will easily understand the value of using an intravital method and will choose from among the existing methods that which seems most appropriate according to the nature of the desired characterization.

According to another more particular aspect, the present invention also relates to a method for characterizing the activity of a compound with respect to the cardiac tissue, in which the cardiac tissue is observed by an invasive analytical method, preferably selected from histological analysis and / or biochemical analysis. In this case, one skilled in the art will choose from the existing methods that which will appear most appropriate according to the nature of the desired characterization.

Grafted heart cells, or grafted tissues, respond to cardiotonic stimuli, such as intravascular injections (or local applications) of compounds such as epinephrine. This demonstrates that the experimental model obtained according to the methods of the invention represents a faithful reflection of the behavior of a cardiac tissue in vivo. It is thus possible to modulate the physiology of the grafted tissues by an external agent, which demonstrates that the tissues thus obtained constitute faithful models of an integrated cardiac system and allow the study of various compounds. The subject of the invention is also the use of a grafted fertilized egg, obtained by a process according to the invention, for characterizing the cardiac toxicity of a compound.

The subject of the invention is also the use of a grafted fertilized egg, obtained by a method according to the invention, for characterizing the activity of a compound with respect to the cardiac tissue.

The subject of the invention is also a kit comprising a grafted fertilized egg obtained, or obtainable, by a process comprising the following steps: - obtaining a fertilized egg under ex ovo conditions, and incubating the fertilized egg for at least 9 days,

contacting said tissue fragment and said MCA at said microlesion, and

incubating the egg thus treated for at least 48 hours in a thermostatically controlled chamber at a temperature of between 36 ° C. and 39 ° C., preferably between 36.5 ° C. and 38.5 ° C., preferably between 37 ° C. and 37 ° C., ° C and 38 ° C and more preferably at 37.8 ° C and with a degree of hygrometry between 80% and 90%>, preferably 84%.

The subject of the invention is also a kit comprising a grafted fertilized egg obtained by a process according to the invention.

Other features and advantages of the invention appear in the following examples and figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Ex ovo system photographs.

Figures 2A and 2B: Photographs of fully revascularized graft in diastole and systole states. Figure 2A: systole, Figure 2B: diastole.

Figure 3: Very late vascularization of the partially infarcted graft. The revascularized portion becomes pulsatile.

Figure 4: Photograph of an injection of the nanoemulsion into the veins of the fertilized egg.

Figure 5: Evidence of the existence of Dil + murine cardiac progenitor cells on the CAM 48 hours after transplantation, as well as their contribution to the manufacture of a blood vessel. Figure 6: (A) Fluorescent marker positive cells Dil (red) at the transplant site revealed in tissue frozen in OCT (20x). Representative images of Dil + cells recruited from the embryonic heart of the host (B, C, 40x). The arrows indicate the Dil + cells. Figure 7: Ultrasound of a mammalian cardiac xenograft.

EXAMPLES

Example 1

The fertilized eggs are incubated under standard conditions of temperature (37.5 ° C) and humidity (60%) for two days and then transferred out of their shells into sterile polystyrene weighing dishes (ex-ovo culture). After a total of 12 days of incubation at 37 ° C. and 84% humidity, the grafts can be performed as follows: Donors: After opening the rib cage of adult mice or rats, the hearts are clamped, removed and transferred to a DEM-glucose culture medium maintained at 30 ° C. The atria are removed and the ventricles cut either frontally, sagittally or transversely, depending on the experiments envisaged.

Recipients: The 12-day-old fertilized eggs are kept outside the incubator at a constant temperature of 38 ° C by means of heated blankets, in order to avoid the death of the embryo by hypothermia. The chorioallantoic membrane is delicately injured on ashless Whatman paper to create microhemorrhages that will facilitate the re-vascularization of the cardiac fragment. The grafts are then deposited on these lesioned areas, with the pericardial surface on the outside, and the implantation site chosen so that the implant, which will move during the growth of the chicken embryo, remains visible at the end of the experiment.

After the transplant, the egg is incubated for 48 hours without manipulation to promote neovascularization of the graft.

At the end of this period, the eggs are observed every day with a binocular magnifying glass. Grafts that have successfully implanted themselves on CAM have an autonomous rhythm of about 60 beats / min. Those whose pulsations are not apparent are not eliminated because a late vascularization is possible with this model.

At the end of the first four independent experiments, 12 implants out of 18 were revascularized and 8 of them returned to their pulsatile function at the latest 5 days after implantation. It should be noted that, under the experimental conditions tested, one of the experiments with 3 implants was performed on day 8 and gave no positive result.

Example 2

Fertilized eggs from White Leghorn Chicken were received from Haas Hatchery (Hass Poultry Products, Kaltenhouse - France), and immediately cleaned with cold distilled water, soaked on a sheet of folded 4 sided paper (1 face per cleaned egg) then disinfected with a solution of 0.2% aqueous chlorhexidine (Gilbert, France) soaked on a 4 folded sheet of paper, before being stored at 12-13 ° C until use, in a ventilated refrigerator whose Airflow arrives directly on a container filled with water and not directly on stored eggs.

Incubation begins when the eggs have been placed in an egg incubator (Fiem

MG140 -200, Guanzate - Italy) at 37.5 ° C and 60% humidity, equipped with automatic tilting racks and an automatic ultrasonic cold humidifier (Necchi N7600, Bremed - Italy, or equivalent compatible with the automation of the incubator).

After 42 hours of incubation (Hamburger - Hamilton stage 11), the eggs were transferred to weighing dishes (89 x 89 x 25) mm sterilized with alcohol (Dominique Dutcher SAS, Brumath - France), the lid of which was made with an inverted cup, held with a ribbon transparent adhesive on one edge, and placed in an incubator at 37.5 ° C and 80% humidity, without mechanical air circulation. Humidity is ensured by the presence on the heating floor of the incubator of a container (170 x 260 x 25) mm filled with water. Freshly opened eggs are not handled at all for 48 hours as soon as they are incubated in the incubator.

Survival under these conditions varies from 53 to 75% at day 14 after incubation (it is at most 90%>) depending on the initial quality of the eggs.

1. Implementation on the MCA:

Tissue fragment implants can only be performed when the MCA is richly vascularized and the vascular pressure is sufficient to irrigate the implant that will be placed on it, that is, not before D10-J11 . Unlike cell suspensions of implants that can be achieved from the ^7th day of egg development.

The date of implantation of fragments is a compromise between "not being too late" to benefit from a longer possible period of transplant (until D18) "but not too early" to allow the vascularization of implants to achieve.

On the day of implantation, the implant is first prepared before being implanted on the MCA at the last moment.

The neonatal rat heart (1-2 days after birth), or chicken embryo, is extracted and incubated in DMEM (or equivalent) + heparin (20 U / ml) at 37 ° C if implantation. immediate or 4 ° C if transport to the place of implantation. Without heparin in DMEM, the percentage of revascularized graft is lower. The heart can also be massaged between the fingers to remove red blood cells and figured elements from the blood that is there and that can lead to a thrombus unfavorable to revascularization of the graft.

The atria are removed using thin forceps, and the ventricles cut into small dice about 1.5 mm by side with a scalpel (blade # 24).

The fragments will be implanted surfaces sectioned towards the MCA.

The implantation zone of the MCA is chosen according to its rich vascularity. The ectodermal leaflet is removed:

1 / with the aid of a "blotting" paper, flexible enough to match the irregularities of the surface of the MCA, and sufficiently thick to be able to absorb the micro-hemorrhage that will result from the removal of the ectodermal leaf,

21 or using a cotton swab that will be rolled over the area to be implanted until microhemorrhage of the MCA.

The implant is gently placed on the hemorrhagic area, areas cut to the MCA. However, the intensity of the bleeding is not measurable, but it is one of the parameters related to the success of the vascularization of the implant. Once implanted, the egg is returned to the incubator as gently as possible (not to move the newly implanted fragment) and the egg left unmoved or even opened for 48 hours.

In order to minimize the mortality of fertilized eggs during long periods of observation or manipulation, weighing cups were placed on the electric blankets for remote-controlled car models (GTTW AC740, GT-Power Tech Co., Ltd.; , China) set at 40 ° C.

2. Graft observation:

Chicken eggs grafted with cardiac tissue fragments grafted onto the MCA were kept in culture for 8 days.

Doppler ultrasound analyzes showed functional vascularization in the grafted tissue, even non-pulsatile tissue, which had intra-tissue vessels. The functionality of allografts and xenografts was recorded on video.

After nine independent experiments, the percentage of vascularized cardiac fragments is 80% and the percentage of pulsatile fragments is close to 50%.

Conclusion: Cardiac tissue xenografting on MCA appears to be a promising model for testing the effect of active agents injected systematically on a healthy graft (toxic effect) but also on pathological grafts (therapeutic effect), but also as model of ischemia-reperfusion and revascularization, but also as a model for repair or regeneration of heart tissue, and finally as a model for the development of a biological pacemaker. The other advantage of cardiac tissue xenografting on CAM, especially compared to existing in vitro devices of the cells is to present a model where the heart tissue vascularizes thus approaching a physiological situation which is very different from a classical in vitro culture. It should be noted that for more than 100 years that MCA fertilized chicken eggs is used, there is never, to the knowledge of the inventors, published work on heart xenografts.

Example 3

Fertilized eggs are incubated under standard conditions of temperature (37.5 ° C) and humidity (60%>) for two days and then transferred out of their shells into sterile polystyrene weighing dishes (ex-ovo culture). .

The fertilized eggs aged 8 days or 15 days are kept outside the incubator at a constant temperature of 38 ° C by means of heated blankets, in order to avoid the death of the embryo by hypothermia. The chorioallantoic membrane is delicately damaged.

Cardiomyocytes isolated from rats or from mice, obtained by enzymatic isolation, are then delicately deposited on these injured areas, and the implantation site chosen so that the implant, which will move during the growth of the chicken embryo, remains visible at the end of the experiment.

After the transplant, the eggs are incubated for 48 hours, without manipulation. At the end of this period, the eggs are observed every day with a binocular magnifying glass. The pulsations are apparent, demonstrating the organization of cardiomyocytes into true cardiac tissue. Cell pulses can be synchronous or asynchronous

Example 4

Murine cardiac progenitor cells c-ki + were labeled with the fluorescent dye, Dil. About 250,000 cells were transplanted into rings on the surface of the CAM of 8-day-old eggs. Some cells have borrowed the vascular system of the embryo. Forty-eight hours after transplantation, a spheroid with fluorescent cells (Dil +) could be visualized in the ring. Vascularization appeared in all transplants. The contribution of Dil + cells to the formation of a blood vessel was evident, as shown in Figure 5, in 2/3 of the grafts. One-third of these transplants were viable until day 17. At the end of the experiment, the graft and heart of the host embryo were frozen in OCT for histological analysis. The graft area clearly showed the presence of cells labeled with Dil (Figure 6). Half of the hearts analyzed also demonstrated the presence of Dil + cells in the host embryo heart suggesting recruitment of transplanted cardiac progenitor stem cells into this tissue (Figure 6 and 7).

BIBLIOGRAPHIC REFERENCES

Murphy. J.B. and Rous P. (1912) "The Behavior of Chicken Sarcoma Implanted in the Developing Embryo." J. Exp. Med. 15 (2); 119-132.

Deryugina, E.I. and Quigley J.P. (2008) "Chick Embryo Chorioallantoic Membrane Model Systems to Study and Visualize Human Tumor Cell Metastasis" Histochemistry and Cell Biology 130 (6), 1119-1.

Dunn, B.E. and Boone M.A. (1976). "Growth of the Embryo Chick In vitro Poultry" Science. 55 1067-1071.

Ribatti D. (2008). "Chick embryo chorioallantoic membrane as a useful tool to study angiogenesis" Int. Rev. Cell. Mol. Biol. 270: 191-224.

Goodpasture E.W.A.M., Woodruff and Buddingh G. J. (1931) "The Cultivation of Vaccine and Other Viruses in the Chorioallantoic Membrane of Chick Embryos" Science 74: 371-372.

Claims

A method of grafting at least one cardiac cell, or at least one cell capable of differentiating into a cardiac cell, onto the vascularised chorioallantoic membrane of a fertilized egg, said method comprising the steps of:

- the culture in ex ovo conditions of the fertilized egg until a stage of development of the egg of at least 6 days,

contacting at least one cardiac cell, or at least one cell capable of differentiating into cardiac cells, and said MCA, at the level of said microlesion, and

the incubation of the fertilized egg thus treated for at least 48 hours in a thermostatically controlled chamber, at a temperature of between 36 ° C. and 39 ° C. and a degree of hygrometry of between 80% and 90%.

2. Method according to claim 1, characterized in that said cardiac cell is an isolated cardiac cell or a cardiac cell of a cardiac tissue fragment.

3. Method according to claim 1 or 2, characterized in that it comprises the following steps:

the production of a micro-lesion on the surface of the MCA of the fertilized egg,

contacting a fragment of cardiac tissue and said MCA at the level of said microlesion, and

4. Method according to one of claims 1 to 3, characterized in that the microlesion is performed by removing the ectodermal layer of the fertilized egg, in the selected implantation area.

5. Method according to one of claims 1 to 4, wherein the ectodermal sheet is removed by one of the following methods:

- Removal using a flexible paper capable of marrying the irregularities of the surface of the MCA, preferably blotting paper, removal with the aid of an abrasive object, preferably capable of creating the abrasion of the ectodermal sheet by scraping, said abrasive object being preferably chosen from: a metal needle tip for a syringe, a glass rod and a cone tip for pipette,

- Removal with an absorbent substance, able to stick to the surface of the MCA and remove by rolling the ectodermal sheet, preferably a tip cotton swab.

A method of grafting at least one cell capable of differentiating into a cardiac cell in a fertilized egg, said method comprising the implementation of the following steps:

7. Method according to one of claims 1 to 6, characterized in that said cardiac cell or said cell capable of differentiating into a cardiac cell comes from a vertebrate animal chosen from: birds, preferably chickens, and mammals preferably monkeys, pigs, mice, rats and humans.

A method of characterizing the activity of a compound against cardiac tissue, said method comprising the steps of:

a) obtaining a fertilized egg grafted with cardiac tissue, obtained by a process according to one of claims 1 to 6,

b) injecting the compound into said fertilized egg grafted, preferably by intravenous injection,

c) observing said cardiac tissue,

d) comparing the results of the observation of step c) with the results of the observation of cardiac tissue of a fertilized egg grafted with cardiac tissue and not having received said compound, and

e) the characterization of the activity of said compound.

The method of claim 8, wherein the heart tissue is observed by an observation method selected from: an intravital method, preferably Doppler echo and / or microscopy, imaging methods using fluorescent probes or another method for measuring contraction and rhythm, and an invasive analytical method, preferably histological and / or biochemical analysis.

10. Use of a transplanted fertilized egg, obtained by a method according to one of claims 1 to 6, for characterizing the cardiac toxicity of a compound or for characterizing the activity of a compound vis-à-vis cardiac tissue.

11. Kit comprising a grafted fertilized egg obtained by a method according to one of claims 1 to 6.