FR2862980A1 - MEANS FOR TRANSPORTING AND PRESERVING LIVING CELLS OR FABRICS - Google Patents

Abstract

The invention relates to the use of polymer hydrogels in the form of grains essentially constituted by biocompatible hydrophilic polysaccharides for the transport and preservation of cells in culture or of living tissues.

Description

Means for transporting and preserving living cells or tissues

  The subject of the invention is means, in particular kits, devices and methods, for the transport and preservation of 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 inexpensive 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 manually flushed with a syringe into each well: they may indeed be in contact with the cells during transport and may cause damage to the cells. . It is then necessary to cover the plate with a second adhesive plastic film to seal the holes made by the syringe. This plate preparation process for transportation has the disadvantage of being long and therefore unproductive in industrial terms. In addition, the use of adhesive films to seal the culture plates encounters various types of problems, such as leakage of potentially toxic products, the sealing of the system can not 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, the 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 disadvantages are found with other sealing systems for transportation, such as plastic or rubber plugs. In addition, their cost price is high, which makes them not very usable in this industrial sector.

  The transport and preservation of organs such as, for example, corneas, arteries, skin and tumors are carried out in liquid medium, in vials sealed with stoppers. But the organs thus placed in solution undergo shocks during transport. These shocks cause the laceration of soft organs such as tonsils or fatty tissue, which makes them difficult to exploit by the person who receives them.

  Furthermore, since the organ fragments can not be oriented precisely, it is necessary to use as many vials as fragments to be analyzed. It is indeed impossible to differentiate several fragments once placed in the same vial (which may be the case for example when different organ fragments are taken from a patient in the operating environment and must be identified for analysis). In addition, non-immobilized organ fragments can not be directly subjected to certain analytical techniques such as magnetic resonance imaging (MRI) because of their movement in a liquid medium.

  The object of the invention is in particular to develop means of transport 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, thus avoiding the contact of air bubbles with the cells, the 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 waiting for their use. The invention also makes it possible, in this case, to use medical analysis techniques that require the immobilization of tissues (such as, for example, MRI analyzes).

  For this purpose the work of the inventors focused on hydrogels in the form of calibrated powder meeting specific characteristics. Hydrogels are hydrophilic systems consisting of an insoluble polymer matrix which is swollen in an aqueous medium. Among other things, they are 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 TG is the maximum volume of a given aqueous solution that the hydrogel can absorb. This volume is called swelling volume.

  In the present invention, the hydrogels 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 their swelling. Below 90% of the TG, the gel grains are agglomerated in a compact form. Beyond the swollen grains begin to detach from each other. An excess liquid phase appears in which the dissociated grains are immersed.

  The gels are preferably used between 80 and 90% of their TG. Below 80% the gel grains are likely to continue their swelling by taking water cells or tissues with which they are in contact, resulting in less biocompatibility. Unexpectedly, such hydrogels provide prolonged survival of cells and tissues, thereby ensuring longer time for transport. In the case of tissues, they make it possible to immobilize them by embedding them in the mass of grains between the inflated hydrogel grains.

  The invention therefore relates to the use, for the transport and preservation of living cells or tissues, of polymers capable of forming hydrogels compatible with cell survival.

  It also relates kits comprising these hydrogels in the form of a calibrated powder or lozenge (compacted powder) ready to be rehydrated with the medium.

  Another object of the invention relates to devices for transporting and preserving living cells or tissues made using such hydrogels.

  The invention also relates to a method for producing such devices.

  According to the invention, the transport and preservation of cells in culture and of living tissues are carried out using polymeric hydrogels in the form of grains, characterized in that they consist essentially of hydrophilic polysaccharides, insolubilized by crosslinking, compatible with cell survival, whose swelling with an aqueous medium is achieved in situ without prior heating.

  Advantageously, the dissociation of these hydrogel grains can be carried out at ambient 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 that can be handled in various compacted forms, such as pellets or in powder form, which allow rapid distribution of precise amounts of polymers in cell culture plates of different sizes ( from 6 wells to 96 wells and beyond) or in containers for transporting living tissues, including organs of different sizes.

  The invention is especially directed to the use of pullulan hydrogels for transporting and preserving cells in culture or living tissues.

  Other suitable polysaccharide hydrogels include dextran, or scleroglucan, curdlane hydrogels, and mixtures thereof.

  The invention aims in particular the use of pullulanes mixed with at least one of said polysaccharides.

  The polysaccharides used in accordance with the invention are optionally chemically modified with cationic, neutral, hydrophobic or anionic functional groups (in particular with carboxylate, sulphate or phosphate groups).

  Preferably, said polysaccharide hydrogels are crosslinked by bridging the hydroxyl linkages of the polysaccharide (s).

  The crosslinking agent and the degree of crosslinking are chosen so as to have a material meeting the above characteristics, in particular as regards compatibility with cell survival. A suitable crosslinking agent is constituted for example by TMPS (sodium trimetaphosphate). In one embodiment, the polysaccharide hydrogels are co-crosslinked with natural polysaccharides such as glycosaminoglycans (in particular heparin).

  Advantageously, said polysaccharide hydrogels are sterilizable according to conventional techniques in cell culture and tissue transport, and easily dissociable to allow the recovery of living cells and tissues and functional after transport.

  The invention also provides kits for the transport and preservation of living cells or tissues, characterized in that they comprise at least one polysaccharide hydrogel, as defined above.

  Advantageously, such kits comprise: E a container or a plurality of containers for the cells or tissues, E at least one polysaccharide hydrogel in compacted form or powder, É a medium for transporting cells or tissues.

  In the case of cell transport, the kits also advantageously comprise an agent for solubilizing the hydrogel grains at the end of the transport and / or a solution allowing dissociation of the grains.

  As the dissociation agent, the transport medium added to the excess plug is preferably used. An enzyme specific for the hydrogel to be solubilized, in particular in the form of an enzymatic solution, may also be used.

  The invention is also directed to devices for transporting and preserving living cells or tissues obtained using said hydrogels.

  These devices are characterized in that they comprise a container or a plurality of receptacles enclosing the cells or tissues, and one or more hydrogels as defined above, including the swelling in situ by a medium, such as the medium of transport, ensures the recovery of cells or tissues.

  For the transport of living tissue, it will be appreciated that the tissue, particularly the organ or organ fragment, may be more or less embedded in the hydrogel grains. A simple addition of a solution, in particular of the transport medium, on 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 consisting of a multiwell plate is used, the cap consisting of hydrogel grains covering the cells and thus avoiding any leakage.

  According to the invention, the cell or tissue covering by the polysaccharide hydrogels is carried out by adding the polysaccharide hydrogels in compacted form (pellet) or powder, in the container containing the cells or tissues and a transport medium in an amount appropriate to achieve the desired swelling of the hydrogel grains in situ, without heating.

  A preferred embodiment of the invention for the recovery of cells comprises: - seeding 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 ensures the recovery of the cells by a plug consisting of agglomerated hydrogel grains, - the deposition of polysaccharide in compacted form or powder, subjected to sterilization pretreatment, in an appropriate amount to obtain the desired recovery.

  The transport may advantageously be carried out at room temperature, generally from about 15 to 30 ° C. or at a lower temperature, which may be of the order of 4 C.

  At the moment of use, an additional step of adding a solution constituted for example by the transport medium is used to dissociate the plug. Alternatively, an agent for dissolution of the gel may be added. The digestion is generally carried out in about ten minutes at 37 ° C. The disintegration of the caps by excess of medium or the solubilization of the hydrogel grains by enzymatic digestion, using polysaccharide-specific enzymes, constitute standard means applicable. whatever cell type is 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, hairballs, fatty tissues.

  It also applies to the transport of human, animal or plant cells. It may be mentioned, for example, its use for the transport of SIRC cells, dermal fibroblasts, epidermal keratinocytes, endothelial cells and hepatocytes.

  The devices made are particularly useful for experiments at the customer (for example porcine cornea toxicity tests, cosmetological tests on skin fragments, or ocular irritation tests on cells such as PredisafeTM tests), transplants on a patient (for example corneal, arterial, skin grafts ...), tissue analyzes taken in the operating environment (for example histological analysis of tumors in a laboratory remote from the hospital).

  The invention thus provides appropriate means for providing or producing 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: FIG. 1: a photo of SIRC cells after a transport according to the invention .

  - Figure 2: the% of cell mortality after testing a cytotoxic product on the cells transported according to the invention or with a film.

  - Figure 3: a photo of porcine cornea transported according to the invention.

  FIG. 4: a photo of rabbit aortas included in a hydrogel according to the invention, and FIGS. 5A and 5B: MRI analyzes corresponding to FIG. 4.

  A / TRANSPORT AND PRESERVATION OF CELLS 1. Preparation of polysaccharide gels Pullulane [Polysciences (Warrington, PA, USA, Lot # 21115) or Hayashibara (Hayashibara, Japan; Lot # PI20)]. In the rest of the descriptions, the term "pullulan" alone refers to Polysciences pullulan, Hayashibara pullulan being mentioned explicitly.

  Dextran [Sigma D-5251; Lot # 81K1082] Scleroglucan [Carbomer Inc .; Cat # 4,00043; Lot # 5- 8787] Carboxymethyl Dextran CMD (ERIT-M Laboratory 0204) Carboxymethyl Pullulan CMP (ERIT-M Laboratory 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 are added. The whole is placed in a water bath at 25 50 C and stirring (10 min).

  The TMPS (Sigma) is then added according to the desired degree of crosslinking in the reaction mixture. The medium is kept under stirring. The gel takes in 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 taken of the gel: 2 minutes 30 aspect of the gel: rigid and very elastic.

  EXAMPLE 2 Synthesis of GPH2 (5% heparin, 10% TMPS) 9.5 g pullulan + 0.5 g heparin + 21.5 ml 1M sodium hydroxide - homogenization for 10 minutes 10% addition (by weight) of TMPS: 1 g taken 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; it is passed through a sieve by adding doubly distilled water to facilitate its passage. This step optimizes the gel washes that will follow. The ground gel is recovered before washing.

  Washings of the gels The washes are carried out in a neutral buffer PBS (phosphate buffer saline) at pH 7.4. The recovered gel is first milled, followed by 3 washes with 1.5 M PBS; 2 washes with PBS 0.15 M; 2 to 3 washings with PBS 0.015 M. Finally, a pH of 7.4 should be reached, each wash is carried out for 20 minutes.

  Drying of the Gels The obtained gel beads are dried in 0.015M ethanol / PBS mixtures at the following concentrations: 2 ethanol baths / 0.015 M PBS at 70% ethanol 2 ethanol baths / 0.015 M PBS at 50% strength ethanol 1 bath with 100% ethanol.

  The beads are then dried in a vacuum oven at 40 C to evaporate the alcohol and then sieved.

  II. Formatting, characterization and digestion of gels Gels are used in the form of powders or in the form of pellets (compressed grains).

  Taux Swelling rate of synthesized gels Single pullulan or dextran gels The following table groups together the swelling rates of single gels measured in PBS (0.15 M). Gels Composition Rate of Crosslinking (TR) (TG) 5 % GP1 Pullulan 10% 1800% (Polysciences) GP2 Pullulan 3% 3600% (Polysciences) GP3 Pullulan 1% 4000% (Polysciences) GP4 Pullulan 5% 2000% (Polysciences) GPyl Pullulan 10% 3600% (Hayashibara) GPy4 Pullulan 5% 2400% (Hayashibara) GD1 Dextran 10% 2800% 000g / mol GD2 Dextran 5% 3600% 000g / mol GD3 Dextran 10% 3400% 500,000g / mol GD4 Dextran 3% 4400% 500,000g / mol GD5 Dextran 5% 4000% 500 000g / mol Mixed Gels The following table groups the swelling rates of mixed gels measured in PBS 0.15M Gels Composition Rate Reticulation Rate swelling (TR) (TG) 5% GPS1 Pullulan: 85% 10% 2000% Scleroglucan : 15% GPS2 Pullulan: 85% 3% 3000% Scleroglucan: 15% GPS3 Pullulan: 85% 1% 2200% Scleroglucan: 15% GPS4 Pullulan: 95% 10% 3000% Scleroglucan: 5% GPS5 Pullulan: 99% 10% 3300% Scleroglucan: 1% GPS6 Pullulan: 70% 10% 2000% Scleroglucan: 30% GPS7 Pullulan: 99% 5% 2000% Scleroglucan: 1% GPCMP1 Pullulan: 90% 1% 2000% CMP: 10% GPCMP3 Pullulan: 90% 10% 1600% CMP: 10% GPCMP4 Pullulan: 90% 25% 1300% CMP: 10% GPCMP5 Pullulan: 90% 50% 1000% CMP: 10% GPH1 Pullulan: 90% 10% 2800% Heparin: 10% GPH2 Pullulan: 95% 10% 2200% Heparin: 5% GPH3 Pullulan: 99% 10% 4000% Heparin: 1% GPH4 Pullulan: 95% 5% 3000% Heparin: 5 % GpyH4 Pullulan 5% 2800% (Hayashibara): 95% Heparin 5% GPSH1 Pullulan 85% 10% 4000% Scleroglucan: 10% Heparin: 5% GPD1 Pullulan 75% 1% 3000% Dextran (500,000g / mol): 25% GPD2 Pullulan: 75% 10% 3000% Dextran (500,000g / mol): 25% GPD3 Pullulan: 50% 1% 2000% Dextran (500,000g / mol): 50% GDH1 Dextran 10% 2800% (500,000g / mol) : 95% Heparin: 5% The gels are then inflated between 80% and 90% of their swelling rate for a good mechanical resistance in 24 th plates t 96 wells.

  III. Polysaccharide plug formation protocol MATERIALS AND REAGENTS Plates 24 or 96 wells, fibroblasts Plates Predisafe (Bioprédic) 24 or 96 wells Plates 24 or 96 wells, rat hepatocytes Plates 24 or 96 wells, keratinocytes Enzyme solution of pullulanase of Bacillus acidopullulyticus (SIGMA): Product Number: P2986; Activity: 441 units per mL É TEST SYSTEMS: The gels are put in the form of pellets Granulometry: 100 to 500} gym.

  24-well plates: 13 mm diameter pellet, 60 mg (specifications: below).

  96 well plates: 5 mm diameter lozenge, 8 mg (specifications below).

  Pressure Diameter Weight Powder Length (mg) pellets (mm) exerted (tons pressure (min) 8 5 3 5 5 3 4 5 2 3 13 2 4 13 3 4 = The different types of cells used are: Hepatocytes rats, SIRC (rabbit cornea fibroblasts), human epidermal keratinocytes, human endothelial cells, human hepatocytes.

  The polysaccharide plugs are enzymatically degraded: - Pullulanase solution: Final concentration: 88 units / mL.

  METHOD = Cell inoculation Thawing cells and inoculation at OJ Observation of the cells and determination of their confluence = Placement of the caps on the confluent cells Sterilization of the pellets: under UV for a minimum duration of 15 minutes per suction face of the medium of culture 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 transport medium to be added in the wells to obtain the swelling of pellets of different weight at 80% TG of the hydrogels GPH2 and GP4.

  Volume of Pellets Volume Volume (mg) Transport Medium Medium (mL) Transport (mL) GPH2 GP4 8 0.150 0.200 0.370 0.500 0.750 0.800 0.800 1,000 = Storage Transport at room temperature or 4 C over 1 to 4 days: cells and caps of polysaccharides Presence of a control: Adhesive film (current technique) = Enzymatic digestion of caps Using a solution of pullulanase 88 units / mL diluted in the transport medium.

  digestion for 10 minutes at 37 C. A liquid medium is then obtained, from which 1 to 2 rinses with PBS make it possible to remove any traces of gel debris. 20

  Distribution of enzymatic volumes: 1/3 of the volume of the swollen gel The following table groups the volumes of enzymatic solution in the wells to obtain the disintegration of the pellets prepared from the hydrogels GPH2 and GP4.

  Weight of Volume of Pellet Volume (mg) enzymatic enzyme solution (mL) (mL) GPH2 GP4 8 0.059 0.053 0.146 0.133 0.294 0.267 0.440 0.400 Depending on the desired use at this stage, the cells are placed in a suitable medium.

  For example: - a delivery of the culture medium to make photos of the morphological state of the cells, - an HES staining, MGG to have a histological analysis, - neutral red test to determine the viability of the cells, functional tests for measuring the cytotoxicity and ocular irritancy of a cosmetic product. IV. Results obtained according to the cell type Hepatocytes are seeded on 24-well plates. The cells are put in contact with the pullulan-dextran plugs (mixed gel), pullulan and heparinous 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: 2 hours after delivery in 20 hours after return to incubator incubator Gels Loss of Quality of Loss of Quality of cells cells cells cells Control film No +++ No +++ GPD2 Yes ++ Yes +++ GPH2 No + No +++ GP1 Little ++ No + E SIRC (rabbit cornea fibroblasts) cells The results obtained are shown in Figure 1. Just after digestion, cells spread from J1 to J2, some cell losses at J3 and J4 are observed. The May Grunwald Giemsa stain shows a normal appearance of the nuclei.

  Neutral red test: Percentage of cell viability Reference: 100% = film at day 1; average of 3 experiments J1 J2 J3 GP4 105 10 100 7 91 7 GPH2 95 3 91 4 83 6 Film 100 11 98 8 88 5 Functional test: test of a moderately toxic dye lotion: The mortality rate is shown in FIG. cells after addition of the dye.

  Cells after 1 day of transport with the caps GPH2, GP4 and the film.

  Examination of this figure shows that the cells are functional; GP4 and GPH2 polysaccharide plugs are biocompatible with SIRC cells and the cell response in this cytotoxicity assay is normal.

  E Keratinocytes of human epidermis - + Transport in 24-well plate The capping is done with polysaccharide gels GP4 and GPH2 in the form of pellets of 13 mm (weight: 60 mg, granulometry: 100 to 500 μm). The cells are stored for 3 days at ambient temperature and at 4 C.

  The digestion is done according to the procedure presented in the method.

  Observation after digestion: cells with intact morphology for both storage temperatures.

  É Human endothelial cells -> 24-well plate transport Capping is performed with GD4, GD5 and GDH1 gels. The cells are stored for 3 days at room temperature.

  Observation after digestion: cells with intact morphology.

  Human Hepatocytes Human hepatocytes inoculated at confluence are contacted with hydrogels and then stored for 3 days at room temperature on the bench (usual transport conditions). 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 a suitable medium. The results are summarized in the following table: J3 Observation after Observation 24 h digestion after re-culture Control Film + ++ GPy4 ++ ++ GPH4 ++ ++ GpyH4 ++ + B / TRANSPORT AND CONSERVATION OF ORGANS 1. General protocol 5 Materials and reagents Plates 12 wells Pork horns Rat aortas PF4% human skin (4% paraformaldehyde) D-sucrose 4% GP4 (5% crosslinked pullulan gel of TMPS) GPH2 (crosslinked pullulan gel) 10% TMPS and Co-crosslinked with 5% heparin) GPyl (Hayashibara pullulan gel crosslinked at 10% TMPS) GPy4 (Hayashibara crosslinked pullulan gel at 5% TMPS) GpyH4 (crosslinked Hayashibara pullulan gel) at 5% of TMPS and co-crosslinked with 5% heparin) Medium of transport of corneas Transport medium of the aortas Test system = The gels are formed into pellets of granulosity 100 to 500 μm in 12 well plates ( pellet 13 mm in diameter and weight 60 mg) or in powder form for s other containers.

  Polysaccharide hydrogels are removed by an excess of medium

METHOD

  Tissue collection and dissection according to 5 adapted protocols Decontamination in a bath of 0.9% NaCl and antibiotics Storage in the transport medium = Installation of hydrogels at OJ Sterilization of the pellets: under UV for a minimum of 15 minutes per side.

  Optimum swelling volumes of the hydrogels (at 85% swelling rate) The swelling time of the hydrogels varies between 10 and 15 minutes.

  Examples: GPH2 and GP4 gels Weight of Medium Volume Volume of Medium Transport Pellets (mg) (mL) (mL) GPH2 GP4 1.8 1.6 2.7 2.6 3.6 3.3 = Storage Transport at room temperature: organ and hydrogels of polysaccharides.

  Presence of a control: Adhesive film (parallel test) = Recovery of the organ By excess of transport medium Distribution of twice the medium introduced for the swelling.

  II. RESULTS OBTAINED FROM THE ORGAN TYPE PORK HUMPS Preparation of the pellets The swelling is done in two stages so as to embed the organ to be transported inside the hydrogel: Weight of pellet 100 mg (1 pellet of 60 mg + 1 lozenge of 40 mg) Granulity: 100 to 500} .gym - Form of the pellets: 13 mm in diameter.

  Contacting corneas / polysaccharide gels The first pellet of 60 mg is swollen in 1.1 ml of transport medium, as soon as the weight of the hydrogel is taken up, the cornea is placed on the hydrogel and then 0.7 ml of transport medium and the second pellet is swollen 40 mg above.

  Transport in 12-well plates The installation of the hydrogel is done in 20 minutes. The cells are stored for 3 days at ambient temperature and at 20 ° C.

  Contacting corneas and hydrogels The results obtained are illustrated by the photo of FIG.

  Histological analyzes Good general preservation of corneas until D3. Results of functional tests at MTT Experiments performed at the left periphery, at the center and at the right periphery of the cornea.

  The 100% reference is the MTT on the cornea at 0% viability. Measurement points GPy4 film Cell GPyH4 Conditioning Left periphery 38 6 96 12 112 20 J3 Center 57 13 112 13 82 12 Right periphery 73 11 128 21 108 14 J6 Left periphery 47 15 115 26 47 9 Center 41 13 82 12 52 8 Periphery right 32 9 106 9 108 9 GPy4: Hayashibara pullulan gel crosslinked at 5% of TMPS GPyH4: Hayashibara pullulan gel co-crosslinked with 5% heparin and crosslinked 5% TMPS RAT AORTES 24-well plate transport The hydrogel used is Hayashibara pullulan gel with 10% TMPS (GPyl) and inflated to 50, 80 or 90% of its swelling rate.

  The gel is contacted with the artery as powder of granulosity 38 to 100 μm.

  Introduction of the arteries in the gel The swelling is done in two stages, in a 24-well plate, 2 ml of medium (physiological saline) is introduced. Half of the amount of powder required is introduced into the well and inflated for 10 minutes, after which the artery is deposited. Half of the remaining powder is poured over. Preservation is at 4 C for 5 days.

  Recovery of the artery The artery covered with gel is recovered using a spatula. Histological analyzes The control: aorta preserved in the preservation 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% Its swelling rate has an intact endothelium (well-preserved cells).

  RABBIN AORES (FOR MRI ASSAYS) The hydrogel used is a pullulan gel with 10% TMPS (GPyl) and swollen at 90% of its swelling rate.

  The protocol is identical to the previous protocol for the addition of the gel to the organ fragments.

  These fragments come from rabbits that have been labeled with metallic iron oxide nanoparticles that mark the inflammatory zones (atheromatous plaques). Detection by Magnetic Resonance Imaging (1.5 Tesla) is performed on a clinical device.

  These are thoracic aorta (non-inflammatory AT), abdominal aorta (AA) after 3 hours (H3), 12 hours (H12), and 5 days (D5). Tem: control not incubated.

  Figure 4 gives a picture of the aortas included in the hydrogel.

  MRI analyzes are given in Figures 5A and 5B. Transport at room temperature over 5 days.

  HUMAN SKIN EFFECTS Transport at room temperature over 2 days and 7 days - Transport technique and organ recovery Swelling of polysaccharide gel 3/4 of maximum swelling.

  Deposit of the explant: face epidermis against frost and face 30 dermis directed outwards The gel finished swelling (up to 90% of its swelling rate) and traps the epidermis of the explant The explant is ready for use after rinsing of the epidermal surface with PBS - MTT test results Measurements are made on D0, D2 and D7. Reference: JO = 100%% viability GPy4 GPH4 GPyH4 Packaging J2 93 6 38 6 96 12 J7 104 6 113 5 92 7 GPy4: Hayashibara pullulan gel cross-linked to 5% of TMPS GPyH4: Hayashibara pullulan gel co-cured at 5% d heparin and 5% crosslinked with TMPS GPH4: Polysciences pullulan gel crosslinked at 5% STMP.

Claims (18)

CLAIMS S   1 / Use for transporting and preserving cells in culture or living tissues of polymer hydrogel grains essentially consisting of hydrophilic polysaccharides, insolubilized by crosslinking, compatible with cell survival, whose swelling with an aqueous medium is carried out in situ, without heating.   2 / The use according to claim 1, characterized in that the swelling of the hydrogels is 80 to 90% of their swelling rate.   3 / The use according to claim 1 or 2, characterized in that the polysaccharide used to prepare said hydrogels is pullulan.   4 / The use according to claim 1 or 2, characterized in that the polysaccharides used to prepare said hydrogels are selected from dextrans, scleroglucan, curdlane, and mixtures thereof and are optionally mixed with pullulan.   5 / The use according to any one of claims 1 to 4, characterized in that said polysaccharides are modified by functional groups, cationic, neutral, hydrophobic or anionic, in particular carboxylate groups, sulfates or phosphates.   6 / The use according to any one of claims 1 to 5, characterized in that said polysaccharide hydrogels are crosslinked by bridging the hydroxyl groups of the polysaccharide or are co-crosslinked with natural polysaccharides such as glycosaminoglycans, in particular heparin.   7 / The use according to any one of claims 1 to 6, characterized in that said polysaccharide hydrogels are before swelling in the form of pellets or powder.   8 / Use according to any one of claims 1 to 7, characterized in that the transported tissues are organs or fragments of organs of human, animal or vegetable origin.   9 / Use according to any one of claims 1 to 7, characterized in that the cells transported are human, animal or plant cells.   10 / Kits for the transport and preservation of isolated cells or living tissues, characterized in that they comprise at least one polysaccharide hydrogel, as used according to any one of claims 1 to 9.   11 / kits according to claim 10, characterized in that they comprise: E a container or a plurality of containers for the cells or tissues, É at least one polysaccharide hydrogel in compacted form or powder and, preferably, É a medium for transporting cells or tissues.   12 / kits according to claim 11, characterized in that they further comprise an agent for the dissociation of the hydrogel after transport and / or a solution.   13 / kits according to claim 12, characterized in that the dissociating agent is an enzyme specific to the hydrogel to be dissociated.   14 / Devices for the transport and preservation of living cells or tissues, characterized in that they comprise a container or a plurality of receptacles enclosing the cells or tissues, covered by a hydrogel as used according to one of the Claims 1 to 9, obtained by swelling in situ in a medium such as the transport medium.   15 / Devices according to claim 14, characterized in that the transported tissues 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 the polysaccharide hydrogels as used according to any one of claims 1 to 9, in compacted form or in powder in the container containing the cells or tissues and a transport medium in an amount appropriate to achieve the desired swelling of the hydrogel.   17 / 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 ensures the tight closure of the container or the covering of the cells, - the deposition of the polysaccharide in compacted form or powder, subjected to a sterilization pretreatment, in an appropriate amount to obtain the desired recovery.   18 / Method according to claim 16 or 17, characterized in that at the time of use, an agent is added for the dissolution of the gel, in particular a specific enzyme of the hydrogel, or a solution.