POPULATION OF AMNIOCYTES HAVING PHAGOCYTIC ACTIVITY AGAINST PATHOLOGICAL CELLS, PROCESS FOR THE PREPARATION THEREOF AND USES THEREOF IN THE MEDICAL FIELD

Abstract

The present invention relates to a population of amniocytes having phagocytic activity against pathological cells, such as tumour cells or the cells of other cellular diseases, such as, for example, proliferative vitreoretinopathy, the process for the preparation thereof and uses thereof in the medical field.

Claims

1. A population of amniotic cells having phagocytic activity against pathological cells.

2. A pharmaceutical composition comprising or consisting of the population of amniotic cells according to claim 1, as an active ingredient, together with one or more excipients and/or adjuvants.

3. The population of amniotic cells according to claim 1, for use as a medicament.

4. The population of amniotic cells according to claim 1, for use in the treatment of proliferative vitreoretinopathy.

5. The population of amniotic cells according to claim 1, for use in the treatment of tumours.

6. A process for preparing the population of amniotic cells according to claim 1, said process comprising or consisting in a step of co-culturing amniotic cells with pathological cells for a period of time equal to at least the time that elapses until the start of phagocytic activity on the part of the amniotic cells.

7. The process according to claim 6, wherein the amniotic cells are co-cultured with the pathological cells in a ratio of at least 100 amniotic cells to every 10 pathological cells.

8. The process according to claim 6, wherein said pathological cells are selected from the group consisting of hyalocytes from a vitreous with proliferative vitreoretinopathy or tumour cells.

Description

[0027] The present invention will now be described, by way of non-limiting illustration, with particular reference to several illustrative examples and the figures in the appended drawings, in which:

[0028] FIG. 1 shows an amniocyte observed under a transmission electron microscope; one may note the jagged cytoplasmic membrane, rich in protuberances; inside the cell, the cellular organelles and the evident nucleus.

[0029] FIG. 2 shows an amniocyte observed under a transmission electron microscope; the nucleus with the characteristic kidney shape and chromatin arranged in dumps (light areas) are evident; the nuclear membrane is also clearly evident.

[0030] FIG. 3 shows hyalocytes of the vitreous body obtained from an excised eyeball. The elongated cell at the centre is a Muller cell, an occasionally found element originating from the retina; granules of melanin can be observed.

[0031] FIG. 4 shows the presence of amniocytes and hyalocytes in culture after 3 hours.

[0032] FIG. 5 shows the production of hyaluronic gel by healthy hyalocytes and the amniocytes that surround some altered hyalocytes after 24 hours.

[0033] FIG. 6 shows the production of a hyaluronic matrix and the presence of amniocytes which engulf some pathological hyalocytes after 36 hours.

[0034] FIG. 7 shows that the amniocytes in co-culture with hyalocytes express CD68 like in the controls; plate obtained after 12 hours of culture, as an immunohistochemical control.

[0035] FIG. 8 shows cells with small and prominent nuclei (hyalocytes) and rounded, elongated cells with an irregular surface (amniocytes); plate obtained after 24 hours of culture; haematoxylin and eosin staining.

[0036] FIG. 9 shows that the altered hyalocytes are brought together by the amniocytes prior to phagocytosis; plate obtained after 36 hours of culture; haematoxylin and eosin staining.

[0037] FIG. 10 shows amniocytes with a cytoplasm that is extremely rich in vacuoles after phagocytosis, the production of hyaluronic gel and vitreal matrix, as in a normal vitreous; plate obtained after 72 hours of culture; haematoxylin and eosin staining.

[0038] FIG. 11 shows hyalocytes immersed in hyaluronic matrix and melanin pigments. Disappearance of the altered hyalocytes. Plate obtained after 96 hours of culture. Haematoxylin and eosin staining.

[0039] FIG. 12 shows the co-culture in which the immunohistochemical staining reveals the CD68+ amniocytes and altered vitreal hyalocytes.

[0040] FIG. 13 shows the co-culture in which the immunohistochemical staining reveals the CD68+ amniocytes and the normal vitreal hyalocytes are barely visible.

[0041] FIG. 14 shows MCF-7 cells in culture; the cells have reached confluence and are ready to be co-cultured.

[0042] FIG. 15 shows the co-culture after 24 hours, with amniocytes that are elongated or rounded, but have evident extensions of the cytoplasmic membrane, stretching toward the MCF-7 cells, characterised by a particularly prominent round nucleus in which the nucleolus can be easily seen.

[0043] FIG. 16 shows round cells of a bright yellow colour, with nucleus and nucleolus (MCF-7), and elongated amniocytes stretching towards the tumour cells after 12 hours.

[0044] FIG. 17 shows that, after 24 hours of co-culture, the amniocytes enter into contact with the tumour cells.

[0045] FIG. 18 shows that, after 36 hours, the cytoplasm of the amniocytes appears rich in numerous granules of a bright white colour, like the one characterising the tumour cells.

[0046] FIG. 19 shows an amniocyte with its cytoplasm completely filled with bright white material, after 48 hours of co-culture.

[0047] FIG. 20 shows the MCF-7 cells that are inside the amniocyte, or confluent amniocytes, after 72 hours of co-culture.

[0048] FIG. 21 shows that, after 96 hours of co-culture, the amniocytes show a cytoplasm rich in small, bright white granules and the rounded shape that characterises them when they are in culture prior to phagocytosis. Confluent amniocytes can be noted on the right.

[0049] FIG. 22 shows, after 96 hours, rounded amniocytes and one in the process of changing morphology from elongated to rounded. In both images the cytoplasm contains bright white granules.

[0050] FIG. 23 shows SH-SY5Y tumour cells in culture, at confluence. Note the characteristic spindle-like appearance, the clearly evident nucleus and long neurites.

[0051] FIG. 24 shows a co-culture of amniocytes that are both round and spindle-shaped in appearance after 38 hours. The small spindle-shaped tumour cells appear to be surrounded by amniocytes.

[0052] FIG. 25: after 72 hours, no tumour cells are visible. Some amniocytes form a syncytium. A few scattered nuclei belonging to the tumour cells remain in the medium.

EXAMPLE 1: METHOD FOR PREPARING A POPULATION OF AMNIOTIC CELLS HAVING PHAGOCYTIC ACTIVITY ACCORDING TO THE PRESENT INVENTION AGAINST PATHOLOGICAL CELLS

[0053] The amniocytes are obtained from discarded amniotic fluid culture specimens from prenatal diagnosis, said amniotic fluid being collected through amniocentesis and cultured for three weeks for diagnostic purposes.

[0054] The amniocytes are cultured for 3 days in Dulbecco's Modified Eagle Medium (DMEM) in order to enable the cells to better adapt and above all to restore their physiological parameters. The 3-day culture sometimes requires a 1- or 2-day extension with a change of culture medium at 24 hours each day. This is because in the discarded cultures from the prenatal diagnosis the amniocytes are in a state of metabolic impoverishment, due to the fact that the culture medium has not been changed, keeping alive cells from which diagnostic responses have already been obtained being no longer necessary.

[0055] Before being revitalised, the amniocytes contained in the culture flasks are subjected to trypsinization, that is, separated from one another and placed in a water and glycerol gradient and centrifuged at 500 rpm for about 30 minutes to eliminate all the fibroblasts that could prejudice the outcome of the co-culture. In fact, fibroblasts reproduce at an extremely fast rate and reach confluence, i.e. come to occupy the entire culture plate, in just a few days, especially if nourished with a daily change of culture medium.

[0056] The band of cells corresponding to the amniocytes is highlighted with a blue lamp. The cells are immediately placed into culture in a specific medium (TopGrow, by the company Resnova) and left to adapt until they reach a sufficient number, generally about 200,000, or until the cells reach confluence. The number of amniocytes can vary based on the quantity of cells originally present in the discarded cultures and the recovery capacity of the amniocytes. However, a sufficiently large number of cells is generally reached, such as to enable the collection of about 100 amniocytes to be co-cultured with about 10 pathological cells. Amniocytes are few in number in the cultures obtained from amniocentesis and their ability to divide is extremely slow, so they require constant and abundant nourishment.

[0057] The amniocyte culture can be preserved by freezing, for example at 80 C., or else it can be used directly to prepare amniocytes with phagocytic activity by co-culturing the amniocytes with pathological cells.

[0058] During co-culture, daily observation is fundamental in order to observe when the amniocytes begin phagocytising the pathological cells.

EXAMPLE 2: IN VITRO STUDY ON THE PHAGOCYTIC ACTIVITY OF THE AMNIOCYTES ACCORDING TO THE PRESENT INVENTION AGAINST PATHOLOGICAL CELLS OF PROLIFERATIVE VITREORETINOPATHY

[0059] About 100 amniocytes obtained according to example 1 were cultured with about 10 hyalocytes from a pathological vitreous excised due to vitreoretinopathy.

[0060] The vitreous was subjected to centrifugation (3,000 rpm for 30-40 minutes) in order to recover the hyalocytes, which, being in an extremely small number, are difficult to be isolated. Once concentrated on the bottom of the test tube, the hyalocytes are immediately introduced into the same culture well (TopGrow culture medium by Resnova) in which the amniocytes have already been placed and the culture is left at a controlled temperature of 36 until the time of observation, about 24 hours later. The number of cultures carried out per case is never less than 3 wells, because it has to be foreseen that some cells, amniocytes and/or hyalocytes, for reasons that cannot always be explained, may not find ideal conditions for growth and it is thus preferable to have a sufficiently large number of cultures (FIG. 4).

[0061] The results obtained from the co-cultures of amniocytes and hyalocytes brought to light some interesting aspects, which confirm not only the macrophage-like activity of the amniocytes, as proven by their positivity to CD68, but also that during vitreoretinopathy the vitreous contains altered hyalocytes within it (see comparison between FIGS. 3 and 8). The pathological hyalocytes are characterised by having a thickened cell membrane and a rounded appearance differing from the characteristic cell morphology with a light-coloured cytoplasm containing few organelles. After just 24 hours of co-culture, one witnesses a phenomenon of the amniocytes encircling the vitreal cells (FIG. 5). Encircling is undoubtedly followed by a phenomenon of phagocytosis of vitreal cells by the amniocytes, which tend to become elongated almost as if to prepare to embrace the cells to be phagocytised. In a total time of about 72 hours, and even more so after 96 hours, in the culture it is possible to observe a resumption of the activity of hyaluronic acid matrix synthesis by healthy hyalocytes that had been present in the culture but were overwhelmed by the pathological ones (FIG. 6-11). The vitreous body is a closed structure that acts like a lens, and its opacification creates blindness. Irrespective of what triggered the disease, pathological hyalocytes also cease producing hyaluronic acid, a fundamental medium in which these cells are immersed and from which they derive, or at least are assumed to derive, nourishment. The production of this matrix in vitro is a sign of resumption of the normal activity of healthy hyalocytes, which, in the event of pathology, are overwhelmed by pathological cells. The phagocytic activity of the amniocytes is directed not only at pathological vitreal cells, but also at the melanin granules that may be found in the midst of the hyalocytes when they are isolated.

[0062] A further immunohistochemical test was carried out to verify whether the amniocytes maintained their macrophagic status at a more advanced stage of co-culture, FIGS. 12 and 13.

[0063] Based on the experiments performed on about 90 vitreous bodies excised due to vitreoretinopathy and the results obtained, it appears evident that the amniocytes are capable of eliminating pathological hyalocytes within the vitreous body, cleaning the body itself of cells that, due to various causes, destroy the transparency of this important lens of the organ of vision.

EXAMPLE 3: IN VITRO STUDY ON THE PHAGOCYTIC ACTIVITY OF THE AMNIOCYTES ACCORDING TO THE PRESENT INVENTION AGAINST PATHOLOGICAL CELLS OF BREAST CANCER

[0064] Approximately 100 amniocytes obtained as per example 1 were cultured (TopGrow culture medium) with about 10 malignant human breast tumour cells of the MCF-7 cell line (Altogen Labs).

[0065] MCF-7 cells are cells that are kept frozen at 80 C. and not thawed until the time of experimentation. At least 3-4 days of adaptation in an incubator are necessary and the cells must reach confluence, i.e. they must occupy the whole surface of the culture plate (diameter of about 3 cm) before they can be collected (FIG. 14).

[0066] On reaching confluence, the MCF-7 cells are counted and 10 are introduced onto the culture plate, where they will come into contact with the amniocytes, present on the plate in a number of 100. The number of cultures per experiment is never less than 3 wells, because it has to be foreseen that some cells, amniocytes and/or MCF-7, but almost always amniocytes, for reasons that cannot always be explained, may not find ideal conditions for growth and it is thus preferable to have a sufficiently large number of cultures.

[0067] From the results obtained, it appears clear that the amniocytes, on contact with the MCF-7 cells, are capable of picking up a signal of a foreign cell and activating a series of morphological changes. The cells become elongated, almost as if to suggest a sort of diapedesis, or cellular movement, which is already known in macrophages, in order to reach the enemy cells (FIG. 15). The change in the configuration of the amniocyte shows the ability of these cells to surround tumour cells (FIG. 16) and then engulf them. Subsequently, no whole tumour cell is any longer visible, only bright yellow tumour cell fragments inside the cytoplasm of the amniocytes (FIGS. 17-22).

[0068] It is interesting to note that the amniocytes join together in a syncytium, that is, they unite their cytoplasm while maintaining every nucleus intact (FIGS. 20 and 21). This phenomenon was not observed in the co-cultures with hyalocytes. The phenomenon is part of the process of phagocytosis carried out by macrophages. The amniocytes that join together in a syncytium can be considered activated and hence capable of undertaking this same action every time they are together with MCF-7 cells, cells of the malignant human breast tumour cell line, which are estrogen receptor-negative.

EXAMPLE 4: IN VITRO STUDY ON THE PHAGOCYTIC ACTIVITY OF THE AMNIOCYTES ACCORDING TO THE PRESENT INVENTION AGAINST PATHOLOGICAL CELLS OF NEUROBLASTOMA

[0069] Approximately 100 amniocytes obtained as per example 1 were cultured (TopGrow culture medium) with about 10 cells of the SH-SY5Y neuroblastoma cell line (SH-SY5Y human cell line, SIGMA-ALDRICH).

[0070] The original cell line, called SK-NSH, is the line from which the subclone SH-SY5Y derives. These cells propagate by mitosis and show long neurites, which stretch out in the areas surrounding the cells. During the process of division they tend to form clusters, which clearly reveal their origin as tumour cells (FIG. 23).

[0071] SH-SY5Y cells are cells that are kept frozen at 80 C. and not thawed until the time of experimentation. At least 3-4 days of adaptation in an incubator are necessary and the cells must reach confluence, i.e. they must occupy the whole surface of the culture plate (diameter of about 3 cm) before they can be collected.

[0072] On reaching confluence, the SH-SY5Y cells are counted and 10 are introduced onto the culture plate, where they will come into contact with the amniocytes, present on the plate in a number of 100. The number of cultures per experiment is never less than 3 wells, because it has to be foreseen that some cells, amniocytes and/or SH-SY5Y, but almost always amniocytes, for reasons that cannot always be explained, may not find ideal conditions for growth and it is thus preferable to have a sufficiently large number of cultures.

[0073] SH-SY5Y tumour cells, extremely malignant, appear to succumb to amniocytes, so much so that after 72 hours of co-culture it is no longer possible to see any presence. It was not possible to document the events in sequence, since, despite their high capacity for multiplication, the SH-SY5Y cells are phagocytised by the amniocytes in just a few hours (FIG. 24). FIG. 25 shows the amniocytes in a syncytium or isolated after 72 hours of co-culture; their cytoplasm appears swollen with granules, an appearance that is more than evident if compared with the cytoplasm of the amniocytes in FIG. 24, which is lighter and devoid of granulation.