Medium for preserving biological materials

Details Medium for preserving biological materials Medium for preserving biological materials

1998-12-14

2001-06-19

Ware, Deborah K. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Culture medium, per se

C435S001300, C435S002000, C435S235100, C435S325000, C435S374000, C424S093100, C424S093600, C424S093700

Reexamination Certificate

active

06248588

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a medium allowing the preservation and cryopreservation of biological materials such as animal cells and viral particles, which is directly injectable or reinjectable into an organism. It relates more particularly to a medium for the preservation of biological material comprising a saline solution, modified fluid gelatin and human serum albumin.
In cell therapy or gene therapy as well as in blood transfusion or bone marrow transplantation, one of the principal problems encountered is that of the preservation of biological material. It is indeed important to be able to preserve biological material, under good conditions of viability, for a sufficiently long period of time compatible with industrial scale production and storage and also to make it possible to carry out certain tests. The most commonly used method of preservation consists in freezing the said material. However, during the freezing of cells, for example, they undergo a very high stress due in particular to a phenomenon of exosmosis and to the formation of ice inside the cell. These phenomena cause numerous cell lyses both during freezing and during thawing. The capacity to divide or to differentiate of even the non-lysed cells may be irreversibly impaired. It is very important to be able to obtain a high viability level after thawing for cells which have to be injected into a living organism (blood transfusion, bone marrow transplantation or cell therapy for example), it is indeed futile to reinject dead or damaged cells. Likewise, when cells have to be recultured, it is equally important that the viability level is high. Up until now, a cryoprotectant which prevented cell lysis was added to the medium. The protecting agent most widely used and which gives the best results is a non-injectable preservative, dimethyl sulphoxide or DMSO. DMSO becomes inserted into the cell membranes and makes it possible to stabilize them. It thus prevents the destruction of the cells. A system such as this makes it possible, at the time of thawing, to have a large number of live and viable cells which are capable of dividing. However, this method poses a major problem when the said cells have to be introduced or reintroduced into an organism. Indeed, DMSO is a product which is toxic to the cells at room temperature. DMSO permeabilizes the membranes, causing the death of the cell. It should therefore be removed before being able to reimplant the cells in the case of an autograft of modified cells or to implant them in the case of a heterograft, for example, of bone marrow cells or alternatively to carry out transfusion in the case of blood cells. The elimination of the DMSO is achieved, after thawing, generally by diluting the sample in ten volumes of DMSO-free medium, followed by centrifugation and elimination of the supernatant. This operation is repeated several times until practically all the DMSO is eliminated. This treatment involves a loss of time and, by multiplying the manipulations, increases the risks of contamination by external pathogenic agents and of losses of the said biological material.
In the presence of DMSO, the percentage of viable cells after thawing is greater than seventy per cent under optimum freezing/thawing conditions as defined later. Without DMSO, this percentage is less than twenty per cent. Up until now, freezing in the presence of DMSO was the most effective and therefore the most widely used method.
BRIEF SUMMARY OF THE INVENTION
The applicant investigated a new type of medium which makes it possible to avoid manipulations subsequent to the thawing while maintaining a high percentage of viable cells. For that, the applicant developed a freezing/preservation medium which makes it possible to obtain a high viability on thawing without using DMSO or another cytotoxic cryopreservative. The advantage of such a medium stems from the fact that the solution is injectable immediately after the thawing without any manipulation being necessary. It then becomes possible to carry out the thawing directly in the operating theatre, thereby reducing the time between the thawing and use, which also makes it possible to remain constantly in a sterile medium and therefore to reduce to a minimum the risks of external contaminations.
A first subject of the invention relates to a medium for the preservation and/or freezing of biological material comprising a saline solution, modified fluid gelatin and human serum albumin (HSA).
As indicated above, this medium lacks any toxic agent and may be administered directly to an organism. It may be used for preserving, optionally in frozen form, various biological materials such as viruses, cells, platelets and the like.
DETAILED DESCRIPTION OF THE INVENTION
The first element entering into the composition of the medium according to the invention is the saline solution. The saline solution is more particularly a solution which is isotonic with the plasma. The salts entering into the composition of this solution may vary. Advantageously, it comprises chlorides, such as sodium chloride, potassium chloride, calcium chloride and/or magnesium chloride, and lactates, such as, for example, sodium lactate. More particularly, the isotonic saline solution generally comprises sodium chloride, potassium chloride, magnesium chloride and sodium lactate. According to another variant, magnesium chloride is replaced by calcium chloride. In this case, the salt concentrations of the saline solution are equivalent or practically equivalent to those of a “Ringer-lactate” solution. Such a solution is usually used in perfusion to compensate for a dehydration or a loss of physiological saline for example.
According to a specific embodiment of the invention, the saline solution is essentially composed of NaCl, MgCl
2
, KCl and lactate whose respective final concentrations in the medium are given in Table 1 below.
TABLE 1
Minimum
Maximum
concentration
concentration
Specific
Salt
(g/l)
(g/l)
example
NaCl
2.0
9
5.7
MgCl
2
0.05
0.2
0.093
KCl
0.05
0.5
0.247
Lactate
0.5
4
2.25
Gelatin is the second constituent entering into the composition of the medium according to the invention. It is a protein composed of various amino acids linked by adjacent amino and carbonyl groups, so as to give the conventional peptide bond. The molecular weight of gelatin is characteristic and high (the average molecular weight values vary from about 10,000 to 100,000) and it is substantially heterogeneous for a given gelatin type or quality. Gelatin is composed of rod or asymmetric type molecules resulting from the hydrolysis of the long chains of the polypeptide residues in the white connective tissue. Experience has shown that the primary hydrolysis of collagen occurs at intervals at reactive sites of these chains to produce a non-degraded related ideal gelatin molecule. This continues to a varying degree in a secondary hydrolysis at random intervals on the less reactive bonds of the ideal gelatin molecule. This explains how the degradation reaction is responsible for the random heterogenous molecular pattern of a specific gelatin sample. Likewise, each protein constituting the gelatin has a defined isoelectric point at which the ionization and, consequently, the physical and chemical reactivity is minimal. These properties are especially the solubility, the viscosity and the colloidal osmotic pressure. The asymmetry of the gelatin molecule therefore gives, with the heterogenous molecular pattern, intrinsic properties of gel formation and viscosity to the gelatin solutions prepared for the medium according to the invention.
The gelatin itself (sterilized and freed from pyrogenic and antigenic substances) has already been used as product for replacing blood plasma, but it has raised a number of problems, in particular for its preservation, because it gels at room temperature. This has led to other compounds derived from gelatin, generally designated by the term modified fluid gelatin, which make it possible to overcome these disadvantages in particular.
Among the modified

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