WO2005053396A1 - Means for the transport and storage of cells or living tissues - Google Patents

Abstract

The invention relates to the use of polymeric hydrogels in the form of grains, essentially comprising biocompatible hydrophilic polysaccharides, for the transport and storage of cells in culture, or of living tissues.

Description

 "Means for the transport and conservation of living cells or tissues"

The subject of the invention is means, in particular kits, devices and methods, for transporting and preserving living cells or tissues. The marketing of cells in the form of ready-to-use culture plates requires a transport phase between the producer or the distributor and the end user. The most frequently used and least expensive technical solution consists in covering the plates filled with culture medium with an adhesive plastic film. The air bubbles trapped under the film must be removed manually using a syringe, in each well: they can indeed be in contact with the cells during transport and are likely to cause cell damage . It is then necessary to cover the plate with a second adhesive plastic film to plug the holes made by the syringe. This process of preparing the plates for the transport has the drawback of being long and therefore not very productive in industrial terms. In addition, the use of adhesive films to seal the culture plates encounters various types of problems, such as the leakage of potentially toxic products, the tightness of the system cannot be guaranteed. Another problem concerns the possible contamination between the different wells of the plate during the removal of the adhesive film. Inter-well contamination becomes almost inevitable when using 96-well plates or beyond. In addition, cells placed in these conditions hardly survive more than three days, which limits the possibility of exporting these plates over long distances and in particular on other continents. These various drawbacks are found with other closure systems for transport, such as stoppers in plastic or rubber. In addition, their cost price is high, which makes them hardly usable in this industrial sector. The transport and storage of organs such as, for example, the corneas, arteries, skin, • tumors, are carried out in a liquid medium, in bottles closed hermetically by caps. However, the organs thus placed in solution are subjected to shocks during transport. These shocks cause the dilation of soft organs such as the tonsils or fatty tissues, which makes them difficult to exploit by the person who receives them. Furthermore, since the fragments of organs cannot be oriented in a precise manner, it is necessary to use as many vials as there are fragments to be analyzed. It is in fact impossible to differentiate several fragments once placed in the same bottle (which may be the case, for example, when different fragments of organs are removed from a patient in the operating environment and must be identified in order to be analyzed). In addition, the fragments of non-immobilized organs cannot be subjected directly to certain analysis techniques such as magnetic resonance imaging (MRI) due to their movement in liquid medium. The objective of the invention consists in particular in developing transport means making it possible to make the transport medium solid. In the case of cell transport, the invention makes it possible to free the producer from the use of adhesive plastic films or other closure systems, which thus avoids contact of air bubbles with the cells, leakage of liquid. and inter-well contamination. In the case of the transport of tissues (organs or fragments of organs), the solidification of the transport medium makes it possible to immobilize the tissues. The invention thus makes it possible to reduce the shocks suffered by the tissues during transport and to maintain their integrity while awaiting their usage. The invention also allows, in this case, the implementation of medical analysis techniques requiring immobilization of tissues (such as MRI analyzes). To this end, the work of the inventors has focused on hydrogel grains in the form of a calibrated powder meeting specific characteristics. The _. hydrogels are hydrophilic systems made up of an insoluble polymer matrix swollen in an aqueous medium. They are among other things defined by their swelling rate (TG) which is established as follows:

TG = 100x [(wet gel weight) - (dry gel weight)] / (dry gel weight). It is assumed that the TG corresponds to the maximum volume of a given aqueous solution that the hydrogel can absorb. This volume is called the swelling volume. In the present invention, the hydrogel grains are prepared in the form of a dehydrated calibrated powder or in the form of pellets (compressed powder). The addition of a physiological solution (a cell culture medium or an organ transport medium for example) causes them to swell. Below 90% of the TG, the gel grains are agglomerated in a compact form. Beyond the swollen grains begin to detach from each other. There is an excess liquid phase in which the dissociated grains. are submerged. The gels are preferably used between 80 and 90% of their TG. Below 80% the gel grains are liable to continue their swelling by taking up water from the cells or tissues with which they are in contact, resulting in less biocompatibility. Unexpectedly, such hydrogel grains allow prolonged survival of cells and tissues, thereby ensuring an extension of the time for their transport. In the case of tissues, they make it possible to immobilize them by embedding them in the mass of the grains between the swollen hydrogel grains. The invention therefore relates to the use, for the transport and preservation of cells or living tissues, of polymers capable of forming hydrogels. compatible with cell survival. It also relates to kits comprising these hydrogels in the form of a calibrated powder or of a tablet (compacted powder) ready to be rehydrated with the medium. Another object of the invention relates to devices for transporting and preserving cells or living tissues produced using such hydrogels. The invention also relates to a method for making such devices. In accordance with the invention, the transport and storage of cells in culture and living tissues are carried out using polymer hydrogels in the form of grains, characterized in that they are essentially constituted by hydrophilic polysaccharides, insolubilized by crosslinking, before contact with cells, compatible with cell survival, including swelling with an aqueous medium _. is made in si tu, without prior heating. It will be noted that the hydrogel grains are completely distinct from the cells and living tissues from which they facilitate transport and storage. Advantageously, the disintegration of these hydrogel grains can be carried out at room temperature by simple addition of liquid medium. Preferably, said hydrogels are used between 80 and 90% of their swelling rate. The polysaccharide hydrogels used according to the invention are more especially chosen from those which can be handled in different compacted forms, such as pellets or in powder form, which allow rapid distribution of precise quantities of polymers in cell culture plates of different formats (from 6 wells to 96 wells and beyond) or in containers for the transport of living tissue, especially organs of different sizes. The invention relates specifically to the use of pullulan hydrogel grains for the transport and storage of cells in culture or living tissue. Other suitable polysaccharide hydrogels include the grains of dextran hydrogels, or of scleroglucan, curdlane, and mixtures thereof. The invention relates in particular to the use of mixed pullulans _. with at least one of said polysaccharides. The polysaccharides used in accordance with the invention are. optionally chemically modified by cationic, neutral, hydrophobic or anionic functional groups (in particular by carboxylate, sulphate or phosphate groups). Preferably, said grains of polysaccharide hydrogels are crosslinked by bridging the hydroxyl bonds of the polysaccharide (s). The crosslinking agent and the level of crosslinking are chosen so as to have a material which meets the above characteristics, in particular as regards compatibility with cell survival. A suitable crosslinking agent is, for example, TMPS (sodium trimetaphosphate). 'The crosslinking rates are in particular of the order of 3 to 20%, in particular from 5 to 10%. In a variant, the grains of polysaccharide hydrogels are co-crosslinked with natural polysaccharides such as glycosaminoglycans (in particular heparin). Advantageously, said grains of polysaccharide hydrogels can be sterilized according to the techniques classic for cell culture and tissue transport, and easily separable to allow recovery of cells or living tissue and functional at the end of ^'transport. . The invention ^'also relates to kits for the transportation and • la conservation of cells or living tissue, characterized in that they comprise at least one polysaccharide hydrogel in the form of grain, as defined above. Advantageously, such kits comprise: • a container or ^a plurality of containers for cells or tissues, • at least one hydrogel of polysaccharide in compacted form or powder, • a medium for the transport of cells or tissues. In the case of transport cells, the kits further comprise ^{advantageously} an agent for solubilizing the hydrogel grains after the transport and / or a solution for the separation of grains. As the dissociation agent, use is preferably made of the transport medium added to the excess stopper. A specific enzyme of the hydrogel to ^"solubilize, particularly in the form of an enzyme solution as may also be used. The invention also relates to devices for transporting and preserving cells or living tissues obtained by using said hydrogel grains. . These devices are characterized in that they comprise a receptacle or a plurality of receptacles containing the cells or the tissues, and a number of hydrogels as defined above, in the form of grains, the swelling of which if in a medium , such as the transport medium, covers cells or tissues. For the transport of living tissue, it should be noted that the tissue, in particular the organ or the fragment of an organ, may be more or less embedded in the hydrogel grains. A simple addition of a solution, in particular of the transport medium, to the hydrogel ensures the separation of the agglomerated grains and makes it possible to recover the organ or the tissue. In the case of cell transport, a conventional container is used, consisting of a multiwell plate, the stopper consisting of hydrogel grains covering the cells and thus preventing any leakage. In accordance with the invention, the covering of cells or tissues by ^" polysaccharide hydrogels is carried out by adding the grains of polysaccha.ride hydrogels in compacted form (tablet) or powder, to the container containing the cells or tissues and a transport medium in an amount suitable for obtaining the desired swelling of the hydrogel grains in si tu, without heating. The cells to be transported are more especially in monolayers. A preferred embodiment of the invention for covering the cells, includes ^' : the seeding of cells in a container to a confluence of 90 to 100%, the addition of transport medium in an amount sufficient for the swelling of the polysaccharide to ensure the recovery of the cells by a stopper constituted of agglomerated hydrogel grains, the deposition of the polysaccharide in compacted form or powder, subjected to a prior sterilization treatment, in a quanti appropriate tee to obtain the recovery sought. Transport can advantageously be carried out at room temperature, generally around 15 to 30 ° C or at a lower temperature which can be of the order of 4 ° C. At the time of use, an additional step is used which consists in adding a solution constituted for example by the medium of transport to dissociate the stopper. Alternatively, an agent for dissolving the gel can be added. Digestion is generally carried out in ten minutes at 37 ° C. The disintegration of the plugs by excess of medium or the solubilization of the hydrogel grains by enzymatic digestion, using specific enzymes of the polysaccharide, constitute standard means applicable whatever the cell type transported. The invention is applicable to the transport of living tissues, in particular organs or fragments of organs such as corneas, arteries, skin, tumors, umbilical cords, tonsils, hair bulbs, fatty tissues. ^' It also applies to the transport of human, animal or plant cells. ..On include such use for transportation of SIRC cells, ^• the ^• dermal fibroblasts, keratinocytes in the epidermis, endothelial cells and hepatocytes. The devices produced can in particular be used for customer experiments (for example toxicity tests on pig corneas, cosmetic tests on skin fragments, or eye irritation tests on cells such as the Predisafe ™ tests ), 'transplants on a patient (for example corneal, artery, skin grafts ...), analyzes of tissues taken in the operating environment (for example histological analysis of tumors in a laboratory distant from the hospital). The invention thus provides suitable means for the supply or the production of ready-to-use cell or tissue transport devices. Other characteristics and advantages of the invention are given in the examples which follow by way of illustration and with reference to FIGS. 1 to 5, which represent: - Figure 1: a photo of SIRC cells after transport according to the invention. - Figure 2: the% cell mortality after testing a cytotoxic product on the cells transported according to the invention or with a film. ^" Figure 3: a photo of the pig's cornea transported according to the invention. ^' - Figure ^' 4: a photo of rabbit aortas included in a hydrogel according to the invention, and - Figures 5A and 5B: analyzes IRM corresponding to Figure 4. / TRANSPORTATION aND STORAGE CELLS I polysaccharides gels Preparation Pullulan [Polysciences (Polysciences, arrington, PA, _USA;. Lot # 21115). or Hayashibara (Hayashibara, Japan; Lot #

PI20)]. In the following descriptions, the term "pullulan" alone refers to the pullulan Polysciences, the pullulan

Hayashibara being mentioned explicitly. Dextran [Sigma D-5251; Lot # 81K1082] Scleroglucan [Carbomer Inc; Cat # 4, -00043-; Lot # 5- 8787] Carboxymethyl dextran CMD (Laboratory ERIT-M 0204) Carboxymethyl pullulan CMP (Laboratory ERIT-M 0204) Heparin [Sanofi; Lot # H-702; anticoagulant activity: 159 IU / mg; unfractionated (15000-18000 g / mol)] '- Method 2 g of polysaccharide are deposited in the tube, then 4.3 mL of 1M NaOH is added. The assembly ^is' placed in a water bath at 50 ° C and with stirring (10 min).

The TMPS (Sigma) is then added according to the desired level of crosslinking in the reaction mixture. The medium is kept under stirring. The gel takes a few minutes. • Example 1: Synthesis of GP4 (5% of TMPS) 10 g of pullulan + 21.5 ml of 1M sodium hydroxide homogenization for 10 minutes addition of 5% (by weight) of TMPS: 0.5 g - gel setting: 2 30 minutes aspect of the gel:. rigid and very elastic. • Example 2: Synthesis of GPH2 (5% heparin, 10% TMPS) 9.5 g of pullulan + 0.5 g of heparin + 21.5 mL of 1M sodium hydroxide - homogenization for 10 minutes - addition of 10 % ^' (by weight) of TMPS: 1 g ^' setting of the gel: 1 minute 50 aspect of the gel: very rigid - Recovery of the gel

The gel is in the form of a more or less rigid block; we pass it in. force through a sieve by adding double-distilled water to facilitate its passage. This step makes it possible to optimize the gel washes which will follow. The ground gel is recovered before washing. -> Washing of the gels The washes are carried out in a neutral PBS buffer (phosphate buffered saline) at pH 7.4. The recovered gel is first ground, then carried out: 3 washes with 1.5 M PBS; 2 washes with 0.15 M PBS; 2 to 3 washes with PBS 0.015 M. In the end, a pH close to 7.4 must be reached, each washing is carried out for 20 minutes. -> Drying of the gels The gel beads obtained are dried in ^' 0.015M ethanol / PBS mixtures at the following concentrations: 2 ethanol / 0.015 M PBS baths at ^' 70% ethanol 2 ethanol / 0.015 M PBS baths at 50% d ethanol 1 bath 100% ^'ethanol. The beads are then dried in a vacuum oven at 40 ° C to evaporate the alcohol, then sieved.

II. Formatting, characterization and digestion of gels Gels are used in the form of powders or in the form of lozenges (compressed grains). • Swelling rate of synthesized gels → Simple pullulan or dextran gels The following table shows the swelling rates of simple gels measured in PBS (0.15 M)

- → Mixed gels The following table shows the swelling rates of mixed gels measured in 0.15 M PBS

The gels are then swollen between 80% and 90% of their swelling rate for good mechanical strength in 24 and 96 well plates. III. Polysaccharide cap formation protocol • MATERIALS AND REAGENTS

24 or 96 well plates, fibroblasts

Predisafe 'plates (Bioprédic) 24 or 96' wells

24 or 96 well plates, rat hepatocytes

24 or 96 well plates, keratinocytes

Bacillus acidopullulyticus pullulanase enzyme solution ('SIGMA): Product number: P2986;

Activity: 44.1 units per mL

• TEST SYSTEMS => The gels are put in the form of tablets:

Grain size: 100 to 500 μm. 24-well plates: 13 mm diameter pellet, 60 mg (specifications: below). 96-well plates: 5 mm diameter pellet, 8 mg (specifications below).

Weight of Pressure Diameter Duration of powder (mg) tablets (mm) exerted (tonnes pressure (min.) 8 5 3 5 20 5 - 3 • 4 ^'40 5 2 3 40 13 2 4 60 ^' 13 3 4

=> The different types of cells used are: Hepatocytes of rats, SIRC (rabbit corneal fibroblasts), Keratinocytes of human epidermis, human endothelial cells, human hepatocytes. = The "plugs ^" of polysaccharides are degraded enzymatically ^' : Pullulanase solution Final concentration: units / mL.

• METHOD => Seeding of cells Defrosting of cells and seeding ^' on D0 Observation of cells and determination of their confluence => Placement of plugs on confluent cells Sterilization of the pellets: - under UV for a minimum duration of 15 minutes per side _. aspiration of the culture medium introduction of a volume of the transport medium corresponding to 80% of the TG of the hydrogel, deposit of the pellets in the wells The following table groups together the volumes of mil. medium of transport to be added to the wells to obtain swelling of pellets of different weights to 80% of the TG of the GPH2 and GP4 hydrogels.

=> Conservation Transport à température ambiante ^' o à 4°C sur 1 à 4 jours: cellules et^' « bouchons » de polysaccharides Présence d'un témoin : Film adhésif (technique actuelle) = Digestion enzymatique des bouchons Utilisation d'une solution de pullulanase à 88 unités/mL diluée dans le milieu de transport. . digestion pendant 10 minutes à 37 °C. On obtient alors un milieu liquide dont 1 à 2 rinçages au PBS permettent' d'éliminer toutes traces de débris de gel. Distribution des volumes enzymatiques : 1/3 du volume du gel gonflé Le tableau suivant regroupe les volumes de solution enzymatique dans les puits pour obtenir le délitement des pastilles préparées à partir des hydrogels GPH2 et.GP4.

=> Storage Transport at room temperature ^' o at 4 ° C over 1 to 4 days: cells and ^' plugs' of polysaccharides Presence of a control: Adhesive film (current technique) = Enzymatic digestion of plugs Use of a 88-unit / mL pullulanase solution diluted in the transport medium. . digestion for 10 minutes at 37 ° C. A liquid medium is then obtained, of which 1 to 2 rinses with PBS make it possible to remove all traces of gel debris. Distribution of the enzymatic volumes: 1/3 of the volume of the swollen gel The following table groups together the volumes of enzymatic solution in the wells to obtain the disintegration of the pellets prepared from the hydrogels GPH2 and .GP4.

Depending on the use ^desired at this stage, the cells are placed in a suitable medium. We carry out for example: a replacement of the culture medium to take photos of the morphological state of the cells, a HES, MGG staining for a histological analysis, - neutral red tests to determine the viability of the cells, tests functional for measuring the cytotoxicity and ocular irritancy of a cosmetic product. IV. Results obtained according to cell type

• Rat hepatocytes The hepatocytes are seeded on 24-well plates. The cells are brought into contact with plugs of pullulan-dextran (mixed gel), pullulan and heparin pullulan and left on the bench at room temperature for 24 hours. After enzymatic degradation of the gels, the cells are observed under a microscope. The observations of cell density and quality of the monolayers are summarized in the table below:

• SIRC cells (rabbit corneal fibroblasts) The results obtained are illustrated in FIG. 1. Immediately after digestion, cells spread from D1 to D2 are observed, some cell losses on D3 and ^' D4'. The May Grunwald Giemsa coloration shows a normal appearance of the nuclei.

Neutral red test: Percentage of cell viability Reference: 100% = Jl film; average of 3 experiences

Test fonctionnel : test d'une lotion colorante modérément toxique : On rapporte sur la figure 2 le % de mortalité des cellules après addition du ^"colorant. Cellules après 1 jour de transport avec les bouchons GPH2, GP4 et le film. L' examen de cette figure montre que les cellules sont fonctionnelles ; les bouchons- de polysaccharide GP4 et GPH2 sont biocompatibles avec les cellules SIRC et la réponse des cellules dans ce test de cytotoxicité est normale. • Kératinocytes d' épidémie humain . —» Transport en plaque 24 puits Le bouchonnage se fait avec les gels de polysaccharides GP4 et GPH2 sous forme de pastilles de 13 mm (poids : 60 mg, granulométrie : .100 à 500 μm) . Les cellules sont conservées 3 jours à température ambiante et à 4°C. La digestion se fait selon le mode opératoire présenté dans la méthode. Observation après digestion : cellules avec une morphologie intacte pour les deux températures de conservation. • Cellules endothéliales humaines -» Transport en plaques 24 puits Le bouchonnage est effectué avec les gels GD4, GD5 et GDH1._. Les cellules sont conservées 3 jours à température ambiante. Observation après digestion : cellules avec morphologie intacte. • Hépatocytes humains Les hépatocytes humains ensemencés à confluence sont mis en contact des grains d' hydrogels puis conservés pendant 3 jours à température ambiante sur la paillasse (conditions du transport habituel) . Après dégradation des gels par une solution de pullulanase à 88 u/ml, la qualité des cellules est observée sous microscope, après digestion et 24 heures après la remise en culture dans un incubateur avec un milieu approprié. Les résultats sont résumés dans le tableau' ci- après :

Functional test: test of a moderately toxic coloring lotion: The% of cell mortality is reported in FIG. 2 after addition of the ^" dye. Cells after 1 day of transport with the GPH2, GP4 caps and the film. this figure shows that the cells are functional, the polysaccharide plugs GP4 and GPH2 are biocompatible with SIRC cells and the response of the cells in this cytotoxicity test is normal • Human epidemic keratinocytes - »24-well plate transport The capping is done with GP4 and GPH2 polysaccharide gels in the form of 13 mm pellets (weight: 60 mg, particle size: .100 to 500 μm). The cells are stored for 3 days at room temperature and at 4 ° C. digestion is carried out according to the operating mode presented in the method Observation after digestion: cells with an intact morphology for the two storage temperatures • Human endothelial cells - »24-well plate transport The capping is carried out with GD4, GD5 and GDH1 gels. _. The cells are kept for 3 days at room temperature. Observation after digestion: cells with intact morphology. • Human hepatocytes Human hepatocytes seeded at confluence are brought into contact with hydrogel grains and then stored for 3 days at room temperature on the bench (conditions of usual transport). After degradation of the gels by a pullulanase solution at 88 u / ml, the quality of the cells is observed under a microscope, after digestion and 24 hours after re-culture in an incubator with an appropriate medium. The results are summarized in the table below:

B / TRANSPORT AND CONSERVATION OF ORGANS

I. General protocol Materials and reagents 12-well plates Pig corneas Rat aortas. Human skin PF4% (parafor aldehyde 4%) D-sucrose 4% ^• GP4 (pullulan gel crosslinked with 5% TMPS) GPH2 (pullulan gel crosslinked with 10% TMPS and Co-crosslinked with 5% heparin ) GPyl (Hayashibara pullulan gel crosslinked with 10% TMPS) GPy4 (Hayashibara pullulan gel crosslinked with 5% TMPS) GpyH4 (Hayashibara pullulan gel crosslinked with 5% TMPS and co-crosslinked with 5% heparin ) Medium for transporting corneas Aorta transport medium Test system => The gels are put in the form of pellets with a grain size of 100 to 500 μm in 12-well plates (pellet of 13 mm in diameter and weight 60 mg) or in the form of powder, for the other containers. => The hydrogels of polysaccharides are eliminated by an excess of medium

METHODE => Sampling tissue to carry Sample, and tissue dissection according to appropriate protocols Decontamination in a bath of NaCl 0.9% - Stable and antibiotics in the transport medium = Placing ^'grains hydrogels at D0 Sterilization of the Tablets: under UV for a minimum duration of 15 minutes, per side. Optimal hydrogel swelling volumes (at 85% swelling rate) The hydrogel swelling time ^• varies between 10 and 20 minutes. ^Examples: GPH2 gels and GP4

=> Storage Transport at room temperature organ and grains of polysaccharide hydrogels. Presence of a witness: Adhesive film (parallel test) => Organ recovery By excess of transport medium Distribution of twice the medium introduced for swelling.

II. RESULTS OBTAINED ACCORDING TO THE TYPE OF ORGAN • PIG CORNEES Preparation of the pellets The swelling is carried out in two stages so as to embed the organ to be transported inside the hydrogel .: Weight of the tablet 100 mg (1 tablet of 60 mg + 1 tablet of 40 mg) - Granularity: 100 to 500 μm Shape of the tablets: 13 mm in diameter. Contacting corneas / gels of polysaccharides was swelled the first wafer 60 mg in 1.1 ml of transport medium from the outlet ^by weight of the hydrogel, placing the cornea on the hydrogel, are then added 0.7 ml of transport medium and the second tablet is inflated by 40 mg on top. Transport in 12-well plates The hydrogel is put in place in 20 minutes. The cells are stored for 3 days at room temperature and at 4 ° C. Contacting corneas and grains of hydrogels The results obtained are illustrated by the photo in FIG. 3. Histological analyzes Good preservation of the corneas in general until D3. Results of functional tests at MTT Experiments carried out on the left periphery, in the center and on the right periphery of the cornea. The 100% reference is MTT on the cornea on D0

GPy4 : gel de pullulane Hayashibara réticulé à -5% de TMPS GPyH4 : gel de pullulane Hayashibara co-réticulé à 5% d'héparine et réticulé à 5% de TMPS • AORTES DE RAT - Transport en plaque 24 puits Les grains d' hydrogel utilisés sont des grains de gel de pullulane Hayashibara à 10% de TMPS (GPyl) et gonflé à 50, 80 ou 90% de son taux de gonflement. - Le gel est mis en contact avec l'artère sous forme de poudre dé granulosité 38 à 100 μm. ^"Introduction des artères dans le gel Le gonflement se fait en deux temps, dans une plaque de 24 puits, on introduit 2 ml de milieu (sérum physiologique) . La moitié de la quantité de poudre nécessaire est introduite dans le puits et gonfle pendant 10 minutes, ensuite on y dépose l'artère. La moitié de poudre restante est versée par dessus. La conservation se fait à 4°C durant 5 jours.

GPy4: Hayashibara pullulan gel cross-linked with -5% TMPS GPyH4: Hayashibara pullulan gel co-cross-linked with 5% heparin and cross-linked with 5% TMPS • RAT AORTES - 24-well plate transport Hydrogel grains used are Hayashibara pullulan gel grains at 10% TMPS (GPyl) and swollen at 50, 80 or 90% of its swelling rate. - The gel is brought into contact with the artery in the form of a powder with a grain size of 38 to 100 μm. ^" Introduction of the arteries into the gel The swelling takes place in two stages, in a 24-well plate, 2 ml of medium (physiological saline) are introduced. Half the quantity of powder required is introduced into the well and swells for 10 minutes, then the artery is placed there. Half of the remaining powder is poured on top. Storage is done at 4 ° C for 5 days.

• Artery recovery The gel-covered artery is recovered using a spatula. Histological analyzes The control: aorta preserved in the conservation medium has an intact endothelium The aorta preserved in the gel at a swelling rate of less than 80% has a damaged endothelium (round cells) The aorta preserved in the gel at 90 % of its rate of swelling has an intact endothelium ^(cells' well conserved). • • RABBIT AORTES (FOR MRI ANALYSIS) The hydrogel grains used are pullulan gel grains with 10% TMPS (GPyl) and swells to 90% of their swelling rate. -. ^'The protocol is identical to the previous protocol for the addition of the gel fragments organs. These fragments come from rabbits which have been marked with metal nanoparticles of iron oxide which mark the inflammatory zones (plaques at ^' heromatous). Detection by Magnetic Resonance Imaging (1.5 Tesla) is performed on a clinical device. These are thoracic aortas (non-inflammatory AT), abdominal aortas (AA) after 3 hours (H3), 12 hours (H12) and 5 days (D5). Witness: non-incubated control. Figure 4 gives a photo of the aortas included in the hydrogel. The MRI analyzes are given in FIGS. 5A and 5B. Transport at room temperature over 5 days. • HUMAN SKIN EXPLANTS Transport. at room temperature over 2 days and 7 days - Transport and recovery technique for the organ 'Swelling of the polysaccharide gel to 3/4 of its maximum swelling. Deposition of the explant: epidermis face against frost and dermis face facing outwards The finished gel to swell (up to 90% of the rate of inflation) and imprisons 1 ^'• The skin explant explant is ready for use after flushing of the skin against the PBS - Results of tests MTT The measurements are made on D0, D2 and D7. Reference: J0 = 100%

GPy4 : gel de pullulane Hayashibara réticulé à 5% de TMPS GPyH4 : gel de pullulane Hayashibara coréticulé à 5% d'héparine et réticulé à 5% de TMPS GPH^'4 : gel de pullulane Polysciences réticulé à 5% de STMP.

GPy4: Hayashibara pullulan gel crosslinked with 5% TMPS GPyH4: Hayashibara pullulan gel crosslinked with 5% heparin and crosslinked with 5% TMPS GPH ^' 4: Polysciences pullulan gel crosslinked with 5% STMP.

Claims

RESELL I CAT I ON S 1 / The use for the transport and conservation of cells in culture ^'or grains of living tissues of polymer hydrogels essentially consist of hydrophilic polysaccharide insolubilized by crosslinking, compatible with cell survival, including -the swelling with an aqueous medium is made in si tu, without prior heating. 2 / Use according to claim 1, characterized in that the swelling of the hydrogel grains is 8.0 to 90% of their swelling rate. 3 / ^' Use according to claim 1 or 2, characterized in that the polysaccharide used to prepare said hydrogel grains is pullulan. 4 / Use according to claim ^' 1 or 2, -characterized in that the polysaccharides used to prepare said hydrogel grains are chosen from dextrans, scleroglucan, curdlane, and their mixtures and are optionally mixed with pullulan. 5 / Use according to any one of the claims 1 to 4, characterized in that _. that said polysaccharides are modified by functional, cationic, neutral, hydrophobic or anionic groups, in particular carboxylate, sulphate or phosphate groups. 6 / Use according to any one of claims 1 to 5, characterized in that said grains of polysaccharide hydrogels are crosslinked by bridging the hydroxyl groups of the polysaccharide (s), or are co-crosslinked with natural polysaccharides such as glycosaminoglycans, in particular heparin. 7 / Use according to any one of claims 1 to 6, characterized in that said grains of polysaccharide hydrogels are present before swelling in the form of pellets or powder. 8 / Use according to any one of claims 1 to 7, characterized in that the tissues transported are organs ^' or fragments of organs of human, animal or plant origin. 9 / The use according to any one of claims 1 to 7, characterized in ^'that' the transported cells are human cells, animal or plant. 10 / Kits ^' for the. transport and storage of isolated cells or living tissues, characterized in that they comprise at least one hydrogel of polysaccharides, in the form of. grains, as used according to any one of claims 1 to 9. 11 / kits according to claim 10, characterized in that they comprise: • a container or a plurality of containers for cells or tissues, • at least one polysaccharide hydrogel in compacted or powder form and, preferably, • a medium for the transport of cells or tissues. 12 / Kits according to claim 11, characterized in that they comprise. further an agent for disintegrating the hydrogel after the ^"transport and / or solution. 13 / Kit according to claim 12, characterized in that the dissociating agent is a specific enzyme. the hydrogel dissociate. 14 / devices for the transport and storage of cells or living tissue, characterized in that ⁷ they comprise a container or a plurality of containers _•. containing cells or tissue, covered by a hydrogel in the form of grains , as used according to one of claims 1 to 9, obtained by swelling in if you in a medium such as the transport medium. 15 / Devices according to claim 14, characterized in that the tissues transported are organs or fragments of organs of human, animal or plant origin and the cells transported are human, animal or plant cells. 16 / Method for ^' the development of devices according to claim 14 or 15, characterized in that it comprises the addition of grains of hydrogels of polysaccharides as used according to any one of claims 1 to 9, under compacted or powdered form in the container containing the cells or tissues and a transport medium in an appropriate quantity - to obtain the desired swelling of the hydrogel grains. 17 / A method according to claim 16 for the transport and preservation of cells in culture, characterized in that it comprises: the seeding of cells, • - the addition of. transport medium in an amount sufficient for the swelling of the polysaccharide to ensure the tight closure of the container or the covering of the cells, the deposition of the polysaccharide under. compacted or powdered form. subjected to a prior sterilization treatment, in an appropriate quantity to obtain the desired recovery. 18 / A method according to claim 16 or 17, characterized in that at the time of use, an agent for dissolution is added _. of the gel, in particular a specific enzyme ^• the hydrogel, or a solution.