WO2011018485A1

WO2011018485A1 - Medium for preserving organs, biological tissue, or living cells

Info

Publication number: WO2011018485A1
Application number: PCT/EP2010/061713
Authority: WO
Inventors: Quang Tran
Original assignee: Laboratoires Eurobio
Priority date: 2009-08-11
Filing date: 2010-08-11
Publication date: 2011-02-17
Also published as: FR2949118A1

Abstract

The present invention relates to a novel medium for preserving organs, biological tissue, or living cells, in particular live human corneas, including natamycin as an antifungal agent, to the use of such a medium, as well as to a preservation method using such a medium.

Description

ENVIRONMENT FOR PRESERVING ORGANS, BIOLOGICAL TISSUES OR

LIVING CELLS

The present invention relates to the preservation of living cells. More specifically, the subject of the invention is a new cell culture medium, in particular of living human corneas, the use of such a medium for the preservation of organs, biological tissues or living cells, as well as a preservation process.

In the field of organ transplants, when an organ is removed from a donor, in order to be grafted onto a recipient, it is necessary to keep the organ in a preservation medium able to maintain its vitality for good taking the graft. In fact, often, different environments are needed.

Corneal transplantation involves replacing a sick cornea with a healthy cornea taken from a deceased donor. This ophthalmic surgery technique is well developed and is practiced in many clinics and hospitals in France. The cornea grafts (transfiratory keratoplasty) allow many patients to recover good vision while they were carriers of an opacity of the cornea that made them, if not blind, at least visually impaired. These transplants are now performed with over 85% success. However, these results depend on the eye diseases that led to the transplant.

In the case of human corneas in particular, it is generally used:

a transport medium for transporting the corneas from the sampling site to the culture center, on the one hand, and the culture center to the transplant site, on the other hand,

- a conservation environment. The preservation takes place, most often, at + 4 ° C or + 31 ° C, according to the techniques. This medium must guarantee optimal preservation of cell viability in the medium term, ie approximately 4 to 5 weeks, maximum safety in terms of quality (endothelial density control), sterility (bacteriological and virological controls), and

- a deturgescence medium, used about 24 to 72 hours before the transplant, to reduce the thickness of the cornea and make it transparent.

There are two techniques for keeping grafts for transplant: - hypothermic storage at + 4 ° C keeps the cornea for a week. This technique is mainly used in the United States.

- The organo-culture, mainly used in Europe, allows storage at +31 ⁰ C for a longer period of about 5 weeks. This culture allows a high bacteriological safety of the grafts, since they are treated for two weeks and at the end of this period is eliminated the grafts carrying resistant germs. This time is also used to investigate the causes of death and receive blood test results. The precise study of the corneal endothelium makes it possible to be sure that the graft will remain transparent after the graft thanks to the good functioning of the endothelial cells which have a role of pumps (deturgence of the stroma).

However, it has been shown that the presence of filamentous fungi can be harmful and prevent the transplantation of the organ, tissue or living cells. In particular, it has been shown that the presence of filamentous fungi during a corneal transplant could be responsible for infection and subsequent complications, and could in particular be responsible for keratitis. Unfortunately, these filamentous fungi are not necessarily detected at the time of transplantation.

There is therefore a real need to develop a preservation medium to eliminate the possible filamentous fungi before performing a transplant of organ, tissue or living cells, without said medium has toxic effect.

Some commercially available preservation media do not include antifungals. Others use Amphotericin B as antifungal.

However, it has been shown that Amphotericin B is highly toxic (CHANG et al., Cornea 1995, WANG et al., Journal of Cancer Pharmacology and Therapeutics, 1996), especially with respect to endothelial cells.

Thus, the quality of a corneal graft, measured in particular by evaluation of its cell density (in practice, only the grafts having endothelial cell densities greater than 2000 cells per mm 2 are considered as capable of being grafted), is strongly reduced when Amphotericin B is used. In addition, although Amphotericin B is fungicidal on yeast, it is only fungistatic on filamentous fungi, ie Amphotericin B blocks the growth of filamentous fungi, without eliminating them. . Thus, once the transplanted cornea is achieved, the filamentous fungi can start to grow again.

In this context, the present invention aims to provide a new cell culture medium preserving the viability of living cells, while having a high fungicidal power.

Surprisingly, the Applicant has shown that Natamycin has a high fungicidal power without exhibiting toxicity towards the cell culture.

Thus, the present invention relates to a cell culture medium comprising Natamycin as antifungal.

The term "cell culture medium" means a medium for transporting, preserving and / or deturging organs, biological tissues or living cells.

Thus, the cell culture medium according to the present invention may be indifferently a transport medium, a preservation medium and / or a deturgescence medium.

Natamycin, also called Pimaricin or E235, is an antifungal of formula (I) below:

NH _{; (])}

Preferably, the medium according to the present invention does not comprise Amphotericin B. Preferably, the medium according to the present invention is a medium free of any other antifungal compound.

Such a limitation to a single antifungal makes it possible to limit the possible interferences between different antifungals and between antifungals and antibiotics. However, the Applicant has shown that the use of Natamycin alone has the antifungal properties necessary for the production of a cell culture medium, in particular an effective preservation medium.

Preferably, the medium according to the present invention further comprises at least one compound as an antibiotic. Such a medium has the advantage of allowing the removal of bacteria therefrom.

Preferably, the at least one compound as an antibiotic is selected from antibiotics of the aminoglycoside family and the beta lactam family.

For the purposes of the present invention, the aminoglycoside family comprises antibiotics selected from the group consisting of: amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin and tobramycin.

Within the meaning of the present invention, is included in the family of beta lactam antibiotic any which contains a β-lactam ring in its molecular structure. By way of example of beta lactamines, mention may be made of penicillins, cephalosporins, monobactams, carbapenems and β-lactamase inhibitors.

Natamycin may be present in the medium or as a stable individual solution for supplementing a medium. Preferably, Natamycin is present in the medium according to the present invention, which makes it possible to ensure a better stability thereof.

Preferably, the Natamycin used in the cell culture medium according to the present invention is a liquid Natamycin. The medium according to the present invention may be suitable for preservation according to a hypothermic technique or an organoculture medium.

The term "hypothermic technique" is understood to mean a preservation technique carried out between +4 and + 6 ° C., preferably at about + 5 ° C. and allowing the preservation of organs, biological tissues or living cells, especially living human corneas, for about a week.

Preferably, the medium according to the present invention is an organoculture medium.

The term "organoculture" means a preservation technique carried out between +28 and + 34 ° C., preferably at about +31 ^° C., which is the physiological temperature of the cornea.

Conventionally, maintaining at a temperature of about +31 ^° C. is provided by means of thermostatically controlled enclosures such as ovens.

Organoculture helps maintain the viability of corneal tissue, especially the endothelium, by stimulating metabolic activities.

Conservation in organoculture allows:

microbiological safety far superior to all other preservation techniques, extending the shelf life (from 1 week to 1 month). This delay offers sufficient time for the realization of serologies on the donor and even offers the possibility of more complex examinations (PCR);

exchanges between banks at room temperature;

to make available graft stocks for emergency situations and thus better planning interventions.

A last viability check is systematically performed 24 to 48 hours before the transplant when the organ is kept in culture.

Preferably, the medium according to the invention comprises Natamycin at a concentration greater than 5 mg / l, preferably greater than 10 mg / l, more preferably greater than 14 mg / l.

Preferably, the preservation medium according to the invention comprises Natamycin at a concentration of less than 50 mg / l, preferably less than 40 mg / l, more preferably less than 30 mg / l, and even more preferably less than 20 mg / L.

Preferably, the preservation medium according to the invention comprises Natamycin at a concentration of between 10 and 20 mg / l, preferably around 15 mg / l.

Preferably, the medium according to the present invention is a liquid medium. In a particular embodiment, the medium according to the present invention is not a Mc Carey-Kaufman medium.

Preferably, the medium of the present invention comprises fetal calf serum or the like, and most preferably irradiated fetal calf serum or the like.

Equivalents, having functional properties identical or similar to those of fetal calf serum, can easily be identified by those skilled in the art in view of his general knowledge.

The invention also relates to the use of a medium as defined above for the preservation, organoculture, cell culture, transport or deturgescence of organs, biological tissues or living cells and in particular of living human corneas. By "living organs, cells or living tissues" is meant human or animal components comprising fibroblasts, endothelial cells and / or living epithelial cells. Preferably, the term "organs, cells or living biological tissues", a biological tissue, preferably a living cornea, and even more preferably a living human cornea.

Filamentous fungi include zygomycetes and hyphomycetes selected from the group consisting of Fusarium spp, Aspergillus spp and Scedosporium and species of the genus Acremonium.

The medium of the invention may be described as an "adjunct therapeutic product" in

France or "medical device" as described in European Directive 93/42 EEC.

The medium according to the invention may contain viscoelastic substances (EVS) intended to protect the endothelial cells and the surrounding tissues, such as, for example, hyaluronic acid.

The media currently on the market intended for the deturgence of corneas contain dextran or poloxamer 188, whose function is to refine the thickness of the cornea and can therefore be used in the media according to the invention intended for detemescence. of corneas.

Methylcellulose is another EVS that may contain the medium according to the invention. Methylcellulose is of plant origin and is obtained from cellulose fibers from cotton flocks or wood pulp. These cellulose fibers are treated with caustic soda solution for etherification with methylene chloride. The degree of substitution corresponding to the number of methoxylated substituents per glucosidic unit is between 1.64 and 1.92. In particular, the methylcellulose marketed by the company SEPPIC under the trade name Metolose® SM 4000 with a Brookfield 4000 centipoise viscosity (2% water at +20 ^° C.) and a molecular weight of 20 ^° C., can be used to prepare the medium of the invention. 86000 Daltons. The medium according to the invention will preferably contain from 210 to 5000 mg / l, preferably from 1900 to 2500 mg / l, preferably 2205 mg / l of methylcellulose.

The EVS used allow hydration of the cells by water retention and have a certain adhesiveness or attachment to the cells and tissues that surround them, thus ensuring the protection of the latter against chemical attack or the toxic effects of air.

The medium according to the invention also contains other components commonly used in the field of the preservation of living cells.

In particular, the medium according to the invention contains a chemical nutrient aqueous base, conventionally used in organ preservation media or cell culture. Reference may in particular be made to the article "Biofutur Technoscope", No. 133, April 1994, pages 3-16, which states that a nutrient base contains:

- Amino acids, whose role in cellular metabolism is to provide - nitrogen and carbon. Some cells, in addition to the 13 essential amino acids, have specific needs (serine, for example, for lymphoid cells);

- sugars, glucose is the most commonly used, although it can be replaced by galactose when it is necessary to limit the accumulation of lactic acid;

- vitamins, mainly of group B, of which 8 are considered indispensable;

ions, provided in the form of balanced salt solutions, which play an important role in maintaining the membrane potential and the osmotic pressure and which are also the co-factors of numerous enzymatic reactions;

trace metals appear to play an increasingly important role, especially when the culture is carried out in a defined medium. The most important are selenium, cadmium and lithium. This type of bases can be prepared from these different constituent elements. Some chemical nutrients also exist commercially, either in liquid form or in solid form: the latter must then be reconstituted in water. In particular, IMDM (Iscove's Modified Dulbecco's Medium ref: Iscove, NN and Melchers (1978) J. of Exp Med 147: 923-933), MEM ALPHA from STAlNERS, CP, can be used. et al., Nature New Biol. 230.52 (1971), the Click RPMI middle of Click et al, CeIl. Immunol. 3, 264 (1972), CMRL1066 medium of PARKER, RC, et al. Special Publications, NY Academy of Sciences, 5, 303, (1957), Leibovitz L15 medium of A. LEIBOVITZ, Am. J. Hyg. 78, 173 (1963) and HJ. MORTON, In vitro, 6, 89 (1970), M199 medium of MORGAN, JF et al., Proc. Exp.Biol.Med. 73, 1 (1950), DMEM / HAM F12 medium, D. BARNES and G. SATO, Anal. Biochem. 102, 255 (1980), aqueous feeder bases that contain various substances necessary for the maintenance of cells and tissues, including various trace elements, amino acids, vitamins, electrolytes, a pH stabilizing buffer, a pH indicator (for example, phenol red), glucose or galactose (L 15). Advantageously, the medium according to the invention contains, therefore, amino acids, trace elements, vitamins, electrolytes, pH stabilizing buffer, mainly from the nutrient base used. If the base used does not contain these elements in sufficient quantity, these will be completed.

The medium according to the invention preferably contains, and independently, from 1 to 50 mg / l of chondroitin sulfate, from 0.1 to 25 mg / l of heparan sulfate, from 500 to

2000 mg / l of alginic acid, from 1000 to 10,000 mg / l of hydroxyethyl starch.

In the case where the medium is intended to be used on human corneal grafts, it will preferably contain components present in the aqueous humor such as sodium lactate, sodium acetate, sodium citrate iron ascorbate II, iron gluconate II, sodium pyruvate and calcium chloride.

In a particularly advantageous manner, the medium according to the invention contains independently or in combination:

^• from 0.01 to 350 mg / 1 of vitamins, preferably selected from tocopherol acetate, retinol acetate, hydroquinone, ascorbic acid, Bl-HCL thiamine, riboflavin B2, D-Ca pantothenate B5, pyridoxal HCl B6, biotin B8, folic acid B9, cyancobalamin B 12, nicotinamide B3 PP, chromium orotate B13 ^• from 0.01 to 650 mg / 1 trace elements, preferably selected from among these:

- CaCl ₂ , 2H ₂ O

- KCl

- CaH ₂ PO ₄ , 2H ₂ O

- NaH ₂ PO ₄₅ H ₂ O

- NaHCO ₃

- MgCO ₃ , 7H ₂ O

MgSO ₄ , 7H ₂ O

- FeSO ₄ , 7H ₂ O

CuSO ₄ , 5H ₂ O

- MnCO ₃ , 4H ₂ O

MnCl ₂ , 4H ₂ O

Na ₂ SiO ₃ , 9H ₂ O

- H ₂ SeO ₃

- NH ₄ VO ₃

- (NH ₄ ) OMO ₇ O ₂₄ , 4H ₂ O

- SnCl ₂ , 2H ₂ O

- ZnSO ₄ , 7H ₂ O

- Zinc oxide

- MC1 ₂ .6H ₂ O

^• from 0.005 to 150 mg / 1 of nucleosides, preferably selected from adenosine, cytidine, deoxyadenosine, deoxycytidine, deoxyguanosine, guanosine, uridine, and thymidine,

^• from 800 to 4000 mg / l of amino acids,

from 500 to 9000 mg / l of oste, and preferably glucose and / or galactose, ^• other elements, in an amount of from 0.001 to 75,000 mg / 1 in total, preferably selected from sodium acetate (3H2O), sodium citrate, sodium lactate, sodium pyruvate, iron gluconate II, sodium selenite, poloxamer 188, oleic acid, linoleic acid, linolenic acid, palmitic acid and Tween 80.

The medium according to the invention may be in liquid or semi-solid form. It has a viscosity important enough to promote the protection of cells. Advantageously, the Brookfield viscosity of the preservation medium according to the invention is between 1 and 15 centipoise (cps), preferably between 2.5 and 10 cps at + 20 ° C.

The medium according to the invention may be used for the preservation, organoculture, cell culture, freezing, transport and / or deturgescence of organs, biological tissues or living cells, in particular the corneas, and also more particularly living human corneas. The medium according to the invention can be used at temperatures between -196 ° C. and + 37 ° C. in particular, preferably between +4 and + 34 ° C., and particularly preferably between +28 and + 34 ° C.

Depending on the intended application, the medium according to the invention may undergo certain adaptations.

For example, in the case where the medium according to the invention is intended to be used for preoperative deturgence, it will advantageously contain poloxamer 188 or dextran, at a rate of 20000 to 75000 mg / l, preferably from 40000 to 60000 mg / ml. 1, and even more preferably about 50000 mg / l.

In another aspect, the present invention relates to a method of preserving organs, biological tissues or living cells, comprising the step of placing the organ, biological tissue or living cells in the composition such that previously defined.

In general, when the tissue concerned is a human cornea, preservation is carried out at physiological temperature, namely +31 ^° C. in a volume of medium which is preferably between 30 and 200 ml. The technical operations are carried out taking care to respect an aseptic in order to avoid contamination of the sample. According to yet another aspect, the present invention relates to a method for preparing a medium according to the present invention, comprising a step of adding liquid Natamycin to said cell culture medium.

Alternatively, said method for preparing a medium according to the present invention comprises a step of adding solid Natamycin. Preferably, in this case, said Natamycin becomes liquid in contact with said medium.

The following examples illustrate the invention, but are not limiting in nature.

Example 1 Comparison of the Fungistatic Nature of Natamycin and Amphotericin B

The A. fumigatus 1, A fumigatus 2, A.niger and C.albicans strains were duplicated on Sabouraud agar plates, then placed at +20 ^° C. or + 31 ° C.

Realization of a range of Natamycine:

In a flask containing 40 mg of Delvocid® (50% of Natamycin and 50% of lactose), 20 ml of DMSO are added in order to obtain a stock solution at 2 g / l. From this, a range of Delvocid® is performed (2 mg / L, 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L and 100 mg / L) in a Cornea Prep medium HE

Macfarland is used as a standard (water + calcium carbonate) of concentration 1.10 ⁹ germs / mL.

10 .mu.l of the mother solution at 10 ^"3 to 10 ⁹ are mixed mold / ml for 10 ⁴ molds in lOμL.

Sterile water served as a negative control.

The various Cornéa Prep II media containing the Delvocid range were distributed in wells of a 96-well plate, these wells were inoculated with ⁴ molds and the plate was incubated for one week at 31 ^° C. Sabouraud agar plates. A daily reading was performed, a disorder in the wells indicates a growth of the germ thus an absence of fungistatic action.

Thus, the results show the concentration of fungistatic Natamicin. This is compared with Amphotericin B at 2.5 mg / L.

The results are shown in the table below:

The results show that there is no growth of mold at +31 ^° C. for a concentration of Delvocid® at 20 mg / L, ie 10 mg / l of Natamycin. This phenomenon has also been observed for Amphotericin at 2.5 mg / L. At a lower concentration, Amphotericin B is more fungistatic than Natamycin.

Example 2: Demonstration of the fungicidal nature of Natamycin.

To 100 mL of peptone water was added the contents of the following wells of Example 1 vial 1: content of the CA well without Delvocid®

bottle 2: content of the AN well without Delvocid®

bottle 3: content of the AFl well without Delvocid®

vial 4: content of the AF2 well without Delvocid® vial 5: content of the AC + 40 mg / L well of Delvocid® vial 6: content of the AN + 40 mg / L well of Delvocid® vial 7: content of the AFl + 40 mg / L well of Delvocid® vial 8: contents of the wells AF2 + 40 mg / L of Delvocid® vial 9: contents of the AC + 2.5 mg / L well of Amphotericin B

vial 10: AN well content + 2.5 mg / L Amphotericin B vial 11: AFl + well content 2.5 mg / L Amphotericin B vial 12: AF2 + 2.5 mg / L content Amphotericin B

This test makes it possible to highlight the fungicidal or non-fungicidal nature of the antifungal.

If it is only fungistatic (prevents growth), then sprouting is observed. If no regrowth is observed, it can be concluded that the sprouts were killed during the first test of Example 1 and that the antifungal is well fungicidal.

The results are shown in the following Table 2:

The vial analysis shows that the medium contains yeasts for vial 1 and filamentous fungi for vials 2, 3 and 4. Vials 5 to 8 are clear and colorless.

The results clearly show that Delvocid® is a fungicide at 40 mg / L (so that Natamycin is a fungicide at 20 mg / L) on both yeast (CA) and fungi. filamentous. Vial 9 remains clear indicating a fungicidal action on the yeast (CA) of amphotericin B, however, vials 10 to 12 show a disorder and indicating a fungistatic action of amphotericin B on these filamentous fungi.

Example 3: Cytotoxicity test of Natamycin and Amphotericin B.

The cytotoxicity of Natamycin and Amphotericin B was tested on the following cell lines:

Adherent cells

- Vero: African green monkey kidney cells

Chinese hamster ovary (CHO): Chinese hamster ovary cell

BHK

HEP II: tumor cells of human laryngeal carcinoma

3T3

1. A stock solution of Delvocid (Delvocid® Instant) at 2 g / L is obtained by mixing 40 mg of Delvocid® and 20mL of DMSO in a sterile 5OmL vial. The mixture is then vortexed until a clear solution is obtained.

Cornea Prep II at 80 mg / L Delvocid® is prepared by taking 2.4 mL of the stock solution and added to the Cornea medium.

Cornea Prep II 40 mg / L Delvocid®: is prepared by taking 1.2 ml of the stock solution and added to the Cornea medium.

Cell culture with Vero, CHO, BHK, HEP II, and 3T3 cells is prepared.

2. A stock solution of Amphotericin at 1 g / L is obtained by mixing 20 mg of Amphotericin and 2 ml of sterile water in a sterile 15 ml vial. The mixture is then vortexed to obtain a yellowish milky solution.

Cornea Prep II medium containing 2.5 mg / L of Amphotericin is prepared by taking 150 μL of the stock solution at 1 g / L, which is added to the Cornea Prep IL medium. A cell culture with Vero cells, Jurkat, CHO, BHK, HEP II, and 3T3 is prepared.

A 6-well plate is used for each cell type.

The tables below show the results obtained:

++: Complies at least 80%

+/-: less than 80% compliant cells

- : Improper

The results show that Delvocid® 40 mg / L has an almost negligible effect on cell lines.

Example 4: Example of media formulation according to the present invention.

The table below shows examples of formulation of the medium according to the present invention.

Claims

1. Gold medium gano culture including Natamycin as antifungal.

2. Medium according to claim 1, free of any other antifungal compound.

The medium of claim 1 or 2, further comprising at least one compound as an antibiotic.

4. Medium according to any one of the preceding claims, comprising Natamycin at a concentration greater than 5 mg / l, preferably greater than 10 mg / l, more preferably greater than 14 mg / l.

5. A medium according to any one of the preceding claims, comprising Natamycin at a concentration of less than 50 mg / L, preferably less than 40 mg / L, more preferably less than 30 mg / L, and even more preferred less than 20 mg / L.

The medium according to any one of the preceding claims, comprising Natamycin at a concentration of between 10 and 20 mg / L, preferably about 15 mg / L.

7. Medium according to any one of the preceding claims, characterized in that Natamycin is in liquid form.

8. Use of a medium according to any preceding claim for the preservation of organs, biological tissues or living cells.

9. Use of a medium according to any one of the preceding claims for the preservation of living human corneas.

10. A method of preserving organs, biological tissues or living cells, comprising the step of placing the organ, biological tissue or living cells in the medium according to any one of claims 1 to 7.

11. A process for preparing a medium according to any one of claims 1 to 7, comprising a step of adding liquid Natamycin to said medium.

12. Process for preparing a culture medium according to any one of claims 1 to 7, comprising a step of adding solid Natamycin, said Natamycin becoming liquid in contact with said medium.