GB2146523A - Preparation for stimulating growth of blood vessels prepared from aminochorionic tissue and macrophages - Google Patents

Abstract

A preparation for stimulating growth of blood vessels is produced by culturing aminochorionic tissue, such as human amniochorion, in a culture medium and then using amniochorionic tissue conditioned culture medium, or a reconstituted lyophilisate thereof, for culture of macrophages e.g. bone marrow cells, followed by removal of the macrophage cells therefrom. The resulting macrophage conditioned medium exhibits higher angiogenic activity in the chick chorioallantoic membrane assay than the amniochorionic tissue conditioned medium. This macrophage conditioned medium can be lyophilised for ease of storage and reconstituted by disolution of the lyophilisate in sterile distilled water. It can also be incorporated in wound dressings or ointments.

Description

SPECIFICATION Preparations and method This invention relates to preparations which are useful for stimulting growth of blood vessels.

For satisfactory wound healing it is necessary for the body to grow new blood vessels. Research into the mechanism by which the body generates new blood vessels suggests that tissue cells called macrophages, known as monocytes in the blood, release a so-called blood vessel growth factor (or angiogenic factor) which in turn acts on a type of cell known as endothelium, which is present in the tissue, and causes such endothelial cells to join together to form a blood vessel.

As the body ages its ability to grow new blood vessels decreases. This can be ascribed to a decrease in the ability of blood monocytes and tissue macrophages to release the blood vessel growth factor. Thus a fairly common condition, particularly amongst elderly women, is the development of chronic venous ulcers, particularly on the lower part of the leg.

Many types of cells have been studied in an attempt to produce cell free extracts containing blood vessel growth factor. For example, U.S.-A-4273871 discloses investigations by Tolbert et al. of numerous normal human cell lines for the production of blood vessel growth factor. However, most such cell lines exhibited relatively poor angiogenic activity, as measured by the chorioallantoic membrane assay of Auerback eta!., Devel. Biol., Vol.41, pages 392 to 394(1974) and of Folkman, Cancer Res., Vol.34, pages 110 to 114 (1976).

Alternatively Tolbert etna!. found such cell lines difficult to maintain in culture.

The use of human amnion tissue in wound healing has been studied on a number of occasions, at least since 1910. a review of their use appeared in Obstetrics and Gynaecology Annual, Vol. (1982) at pages 31 to 58 under the title "A Review of the Role of Amniotic Membranes in Surgical Practice" by Richard N.

Matthews, W. Page Faulk and John P. Bennett.

Suitable sterile human amnion tissue is usually obtained from Caesarean deliveries. Although this source can produce sufficient material for research purposes, it has become clear that it would be desirable to culture human amnion tissue in order to study further its wound healing properties. A description of culturing human amnion has appeared in an article by H. Burgos and W. Page Faulk, in British Journal of Obstetrics and Gynaecology (1981), Vol. 88, pages 294 to 300. A further development of this was the production of a wound healing composition comprising or derived from a culture medium in which amnion has been cultured: see G.B.-A-21 10531. This described the culturing of choriion-supported amnion in a medium supplemented with 10% foetal calf serum or newly born calf serum.This culture medium was centrifuged to remove any free amnion cells and was then lyophilised and sterilised by gamma-irradiation from a 90Cs source. When required for use a wound healing preparation could be derived from this lyophilisate by reconstitution by addition of sterile distilled water.

Although the preparations of G.B.-A-2110531 show considerable promise in promoting wound healing, the production of such preparations involves use of biological material of non-human origin, i.e. foetal or newly born calf serum, which of necessity introduce xenotypic entities into the preparation.

There is accordingly a need to provide improved preparations whih can encourage the growth of blood vessels for use, inter alia, in assisting healing of wounds.

The present invention seeks to provide a novel preparation which exhibits significantly improved activity in stimulating growth of blood vessels, as well as a method of producing same.

According to the present invention there is provided a preparation for stimulating growth of blood vessels comprising or derived from a culture medium in which macrophages have been cultured, the culture medium comprising or being derived from a medium in which amniochorionic tissue has previously been cultured.

The invention further provides a method of producing a preparation for use in stimulating growth of blood vessels comprising: (a) culturing amniochorionictissue in a culture medium, (b) separating the resulting medium from amniochorionic tissue, (c) culturing macrophages in a medium comprising or derived from the medium of step (b), and (d) separating resulting medium from macrophages.

By the term "amniochorionic tissue" there is meant tissue found in the amniochorion, that is to say the laminar structure consisting of the amnion and chorion.

Preferably the amniochorionic tissue comprises amniochorion tissue. Amnion or chorion tissue dissected from the amniochorion is also suitable. Preferably the tissue is obtained under sterile conditions.

The amniochorionic tissue is preferably human tissue. Such tissue can be obtained, for example from a Caesarean delivery. However, animal tissue can alternatively be used, for example bovine amniochorionic tissue.

The amniochorionic tissue may be cultured in any suitable tissue culture medium. The tissue culture medium is preferably buffered to a pH in the range of from about 7.2 to about 7.4 Examples of suitable tissue culture media are described, for example, by H.J. Morton, In Vitro 6,89-108(1970). Such media contain, in various combinations, known essential amino acids, mineral salts, carbon sources such as carbohydrates, for example glucose, and vitamins. Particularly when culturing human amniochorionic tissue, the culture medium is preferably free from biiological materials containing xenotypic entities.Hence in contrast to the procedure described in G.B.-A-21 10531, when culturing human amniochorionic tissue, it is preferred that no foetal calf serum or newly born calf serum or any other material containing xenotypic entities is added to the culture medium.

The aminochorionic tissue is preferably incubated in an oxygen containing atmosphere, even more preferably one that contains also a small concentration of CO2. The CO2 concentration should preferably not exceed about 5% and may be, for example, about 2%.

The amniochorionictissue may be cultured in one or more culture media. To avoid contamination of the final preparation the initial culture medium can be discarded and only a subsequent culture medium used in the production of the preparation of the invention. Culturing may continue, for example, for a period of up to 10 days.

After culturing has been completed, the amniochorionic tissue conditioned medium (ACCM) is usually separated from the amniochorionic tissue and then centrifuged to remove free cells. The resulting medium can be used for the next stage of the process, i.e. macrophage culture, or may be lyophilised until required and then reconstituted by adding sterile distilled or de-ionised water. The amniochorionictissue can be used for further culturing.

In the next stage, macrophages are cultured on a medium comprising or derived from a medium in which amniochorionic tissue has previously been cultured. The culture medium used in this stage may thus comprise a medium on which the amniochorionic tissue has been cultured or may comprise a reconstituted form of a lyophilised culture medium on which amniochorionic tissue has previously been cultured. The culture medium used for culturing the macrophages may further comprise added nutrients, mineral salts and/or vitamins, such as are conventionally used in culture media. Again, it is preferred to formulate this culture medium without adding any xenotypic entities.

The macrophages preferably comprise bone marrow cells (pro-macrophages). Such macrophages may be derived from human sources or from an animal source, such as bovine bone marrow or mouse bone marrow. The macrophages have preferably been obtained under sterile conditions.

Culturing of the macrophages in the conditioned medium is preferably effected under similar conditions to those used for culturing the arnniochorionic tissue. Sterile conditions should be maintained throughout.

After completion of culturing, the resulting culture medium (which may be conveniently described as macrophage conditioned medium (MCM)) may be separated from free cells by centrifugation. The resulting preparation may be used as prepared or lyophilised for convenience of storage. Prior to use or storage the preparation may be sterilised, for example by gamma-irradiation from a suitable gamma-ray source such as a 90Cs source. The lyophilised preparation can be reconstituted using sterile distilled or de-ionised water.

The macrophage conditioned medium may be used as prepared or as reconstituted from the lyophilisate.

Alternatively it can be subjected to gel filtration chromatography on a molecular sieve of relatively large pore size, such as Sephacryl 300, with a view to removal of extraneous materials, and then, if desired, lyophilised.

The invention further contemplates a surgical dressing comprising factors derived from macrophages cultured in a medium comprising or derived from a medium in which amniochorionic tissue has previously been cultured. Such a dressing may comprise a carrier material that has bveen impregnated with a liquid medium comprising a preparation according to the invention and then dried. A material treated in this way can be used as the wound-facing layer of a surgical dressing. An antiseptic or antibiotic can also be incorporated in the dressing in any convenient manner.

It is further contemplated to provide ointments comprising factors present in a medium used for culturing macrophages, which medium comprises or is derived from a medium in which amniochorionic tissue has previously been cultured. Such an ointment may take the form of a water-based cream. Suitable recipes for such an ointment can be found, for example, in the British Pharmacopoeia, 1980, pges 696 to 702 and include recipes for emulsifying ointment, macrogol ointment or paraffin ointment. The prepartion of the invention can alternatively, if desired, be absorbed in the form of an aqueous medium into a hydrophilic gel based, for example, on poly-2-hydroxyethyl acrylate, gelatin or the polymers described in GB-A-1524899. An antiseptic may be included in an ointment according to the invention.The inclusion of an antiseptic in the ointment may enable the dose of gamma-ray radiation, which may be required for sterilisation, to be reduced.

The ointment may be applied directly to the wound bed and should preferably be covered with a non-adherent wound dressing such as tulle gras dressing. If the non-adherent dressing chosen is not absorbent, it may be desirable to cover it with a conventional absorbent material, such as pulped cellulose.

This, in turn, may be covered with a conventional occlusive film. The occlusive film may be secured by adhesive tape.

A dressing comprising a non-adherent layer coated with ointment, an absorbent layer and an occlusive film may be pre-packed in a sterile envelope.

The invention further provides a method of treating a wound as an aid to healing said wound, comprising applying to the wound a preparation according to the invention. The preparation may for example be applied alone, or in or on a suitable carrier. Examples of suitable carriers are the dressing and the ointment described above. Alternatively, the wound may be continuously or intermittently irrigated with the preparation in the form of an aqueous solution, as in the method described by Westaby etna!. in Annals R.C.S. Eng. (1981) 63 at pages 353 to 356.

The invention Is further described in the following example.

Example Human amniochorion membranes were collected aseptically from electrive Caesarean sections in otherwise healthy women at term. The membranes were immediately immersed in ice-cold Dulbecco's phosphate buffered saline (PBS), (Flow Laboratories, Puteau, France), and transported to the laboratory where they were cleaned of blood clots and decidual tissue and washed in two changes of PBS at 50C.

Whole amniochorion was cut into pieces approximately 5 x 5cm which were separated into two batches and cultured in separate 245 x 245 x 20 mm polystyrene dishes (Screening plaque, Nunc, Roskilde, Denmark) in 400 ml of the Dutch modification of RPMI 1640 (Flow) to which were added: 10 mM sodium bicarbonate (Flow), 2 mM L-glutamine (Flow), gentamycin (Unilabo, Levallois-Perret, France) 80 mgil, and fungizon (Squibb, Neuilly sur Seine, France) 2 mg/l (this medium is hereafter referred to as "complete culture medium"). The membrane culture was maintained at 37"C in humid air containing 2% added CO2, as long as the buffering capacity of the medium permitted, which was usually about 10 days. After culture, the amniochorion conditioned medium (ACCM) was collected and lyophilised for storage.

As a quantitative test for each batch of ACCM a colony forming unit (CFU) culture assay in methyl-cellulose semi-solid medium was carried out as described by Iscove petal., J. Cell Physiol. 83 (1974) pages 309 to 320.

In this assay, 100,000 human or mouse bone marrow cells contained in 1 ml of semi-solid medium were plated in 35 mm tissue culture dishes. The medium contained 20% ACCM, 20% alpha medium, and 40% methyl-cellulose stock solution. The stock solution contained 2% essential amino acids solution (Eurobio), 1% non-essential amino acids solution (Eurobio), and 0.4% L-asparagine (10 mgil, Merck-Clevenot, Nogent sur Marne, France). The plates were incubated at 37"C in a humid oxygen-containing atmosphere with 6% CO2 for 7 (mouse cells) or 14 (human cells) days, and the number of colonies (more than 50 cells) was counted. The results are shown in Table 1.

TABLE 1 Colony stimulating activity of A CCM* ACCM Human BMC* Mouse BMC* Day of Number Colonies Colonies Culture 1 31 t 5 52 t 3 1 - 3 2 34 4 1,2,3-4 3 23 -+9 9 1,2,3-4 4 80+12 1,2,3-5 5 88+18 1,2,3-6 6 104+11 1,3,3-6 7+A 80 " 7 1-3 7 8 87 -C 6 4-6 8+1 122+7 1-3 8 2 93 + 14 1 - 10 91 88+4 1-10 9 2 213+9 1-10 10 1 223 -F 23 1 - 10 * ACCM is amniochorion conditioned medium. BCM means bone marrow cells + Lettered subscripts are ACCM from different times; numbered subscripts are from different batches of same amniochorion.

To prepare macrophage conditioned medium (MCM) 750,000 MBM cells in 5 ml of media were seeded into 35 mm culture dishes containing a culture medium to which was added 20% ACCM. MBM cells (500,000) were seeded into 35 mm culture dishes in 3 ml medium. The control dishes received 20% FCS and the experimental dishes received 20% ACCM. Two populations of cells were visible in both the experimental and control dishes after 24 hours. These consisted of large attached cells and smaller ones which were either unattached or loosely attached.After 48 hours, the attached cells in the experimental dishes appeared noticeably enlarged in comparison with their counterparts in the control dishes, and by 72 hours small colonies of large, round or spreading cells appered in experimental dishes, and by the sixth day there was a vigorous growth of colonies in the experimental dishes, and in the control dishes there were no colonies at all.

When charcoal particles were added into the dishes whih contained proliferating MBM cells, more than 90% of the cells were observed to phagocytose these particles suggesting that the proliferating cells were macrophages.

The macrophage conditioned medium (MCM) was decanted from the cells and centrifuged to remove free cells. Upon testing MCM and ACCM for angiogenic activity by the chorioallantoic membrane assay of Auerback, loc. cit., and of Folkman, loc. cit., MCM was found to exhibit significantly greater activity than ACCM.

The MCM was lyophilised and could be stored at low temperature until required.

Upon reconstituting MCM by adding sterile distilled water to the lyophilisate it exhibited similar activity in the chorioallantoic membrane assay to that of freshly prepared, non lyophilised MCM.

Claims (26)

1. A preparation for stimulating growth of blood vessels comprising or derived from a culture medium in which macrophages have been cultured, the culture medium comprising or being derived from a medium in which amniochorionictissue has previously been cultured.

2. A preparation according to claim 1, in which the macrophages are human bone marrow cells.

3. A preparation according to claim 1 or claim 2, in which the amniochorionic tissue is human amniochorion.

4. A preparation according to any one of claims 1 to 3, in which the culture medium in which the amniochorionic tissue has been cultured is free from xenotypic entities.

5. A preparation according to claim 4, in which the culture medium in which the macrophages are cultured is free from xenotypic entities.

6. A preparation according to any one of claims 1 to 5, in which the amniochorionic tissue is cultured in a culture medium having a pH of from about 7.2 to about 7.4.

7. A preparation according to any one of claims 1 to 6, in which the macrophages are cultured in a culture medium having a pH of from about 7.2 to about 7.4.

8. A preparation according to any one of claims 1 to 7, in which the culture medium in which the amniochorionictissue is cultured is centrifuged in order to remove free cells therefrom prior to contact with the macrophages.

9. A preparation according to any one of claims 1 to 8, in the form of a lyophilisate.

10. A preparation according to any one of claims 1 to 9, which has been subjected to sterilisation by gamma-ray radiation.

11. A preparation according to claim 1, substantially as herein described with particular reference to the Example.

12. A method of producing a preparation for use in stimulating growth by blood vessels comprising: (a) culturing amniochorionic tissue in a culture medium, (b) separating the resulting medium from amniochorionictissue, (c) culturing macrophages in a medium comprising or derived from the medium of step (b), and (d) separating resulting medium from macrophages.

13. A method according to claim 12, in which the macrophages are human bone marrow cells.

14. A method according to claim 12 or claim 13, in which the amniochorionic tissue is human amniochorion.

15. A method according to any one of claims 12 to 14, in which the culture medium of step (a) is free from xenotypic entities.

16. A method according to claim 15, in which the culture medium of step (c) is free from xenotypic entities.

17. A method according to any one of claims 12 to 16, in which the culture medium of step (a) has a pH of from about 7.2 to about 7.4.

18. A method according to any one of claims 12 to 17, in which the culture medium of step (c) has a pH of from about 7.2 to about 7.4.

19. A method according to any one of claims 12 to 18, in which the separation step (b) includes a centrifugation step.

20. A method according to any one of dims 12 to 19, in which the separation step (d) includes a centrifugation step.

21. A method according to any one of claims 12 to 20, in which the medium from step (d), possibly following further purification, is lyophilised.

22. A method according to any one of claims 12 to 21, in which the medium from step (d), possibly following further purification, is sterilised by gamma-ray irradiation.

23. A method according to claim 12, substantially as herein described with particular reference to the Example.

24. A preparation made by a method according to any one of claims 12 to 23.

25. A surgical dressing including a preparation according to any one of claims 1 to 11 and 24.

26. An ointment including a preparation according to any one of claims 1 to 11 and 24.