Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K5/00—Heat-transfer, heat-exchange or heat-storage materials, e.g. refrigerants; Materials for the production of heat or cold by chemical reactions other than by combustion
  • C09K5/02—Materials undergoing a change of physical state when used
  • C09K5/06—Materials undergoing a change of physical state when used the change of state being from liquid to solid or vice versa
  • C09K5/063—Materials absorbing or liberating heat during crystallisation; Heat storage materials
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/10—Petri dish
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M45/00—Means for pre-treatment of biological substances
  • C12M45/09—Means for pre-treatment of biological substances by enzymatic treatment
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M45/00—Means for pre-treatment of biological substances
  • C12M45/20—Heating; Cooling
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
  • C12N5/0626—Melanocytes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/18—Means for temperature control
  • B01L2300/1855—Means for temperature control using phase changes in a medium
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/18—Means for temperature control
  • B01L2300/1877—Means for temperature control using chemical reactions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L9/00—Supporting devices; Holding devices
  • B01L9/52—Supports specially adapted for flat sample carriers, e.g. for plates, slides, chips
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2523/00—Culture process characterised by temperature
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
  • Y02E60/14—Thermal energy storage

Definitions

• the present invention relates to the field of molecular and cellular biology kits, as well as that of kits for implementing a chemical reaction requiring a moderate heat input. It particularly relates to kits comprising a simple and inexpensive autonomous heating means for treating biopsies.
• the treatment of the samples essentially amounts to a more or less extensive dissociation of the cells, followed, if necessary, by the separation of different cell types to select the appropriate cells for the intended application (melanocytes for achromatic dermatoses). for example) and filtration to remove aggregates.
• Cell dissociation is most often performed by incubating the sample in a trypsin solution.
• the optimum temperature for trypsin activity is 37 ° C. At this temperature, and depending on the enzyme concentration and the degree of dissociation desired, the incubation times described in the literature are between 50 minutes and 3 hours (Guerra et al., 2003, Uiekar, 2003, van Geel and al., 2004).
• Cîinical Cell Culture offers a kit (ReCell®) containing the necessary consumables to perform all the steps, from the collection to the reimplantation of the cells, as well as an electronic heating unit equipped with batteries.
• ReCell® a kit containing the necessary consumables to perform all the steps, from the collection to the reimplantation of the cells, as well as an electronic heating unit equipped with batteries.
• This device has two major disadvantages, which are its very high price and its ecological impact.
• the present invention provides an advantageous alternative to the solutions of the prior art, in particular the ReCell® kit, since it relies on the use of heaters for heating and maintaining at a suitable temperature an enzymatic solution for the time necessary for the action of the enzyme.
• the term “heater” here refers to small objects capable of emitting heat, also called “hot water bottles” or “stand-alone heat packs”, and whose operation is based on exothermic physical or chemical processes. They are commonly used to warm hands or feet exposed to the cold. In what follows, the terms “heater” and “heat pack” are used interchangeably.
• reusable heaters consisting of a pouch containing a saturated aqueous solution of sodium acetate supercooled.
• a metal wafer By twisting a metal wafer inside the liquid, solidified acetate crystals are generated, which trigger crystallization, and the solution becomes solid. This phase transition is at the melting temperature (54 e for a 20% solution, for example), there is heating of the pouch then cooling to room temperature once solidification is complete.
• the pouch When the pouch is cooled, it is possible to return the sodium acetate (become solid) in the liquid state, placing the pouch in very hot water.
• chemical heaters whose principle is activated by oxidation in contact with the air. They are effective longer (8 to 60 hours compared to about 1 hour for supercooled sodium acetate heaters) but are only used once.
• a solution placed in a container typically a Petri dish, placed on a heater, reaches in a few minutes an optimal temperature for the activity of many enzymes such as trypsin, and maintains for at least 15 to 20 minutes.
• the invention therefore relates, in the first place, to a method for carrying out a biological, biochemical or chemical reaction requiring an incubation at a temperature of between 30 and 40 ° C., characterized in that it comprises an activation step. a heater which operates on an exothermic physical or chemical process, and a step of contacting said heater with a container containing the reagents involved in said reaction.
• This method is particularly advantageous in the context of a biological, medical or diagnostic application.
• the step of contacting the heater with the container is performed by placing the heater and at least the inner portion of the container in a cavity of a support provided for this purpose.
• a support makes it possible, on the one hand, to wedge the container securely on the heater, thus limiting the risk of overturning.
• the use of an insulating support, having a low thermal conductivity limits the heat loss of the heater and promote the transfer of heat from the heater to the solution to be heated.
• the depth of the cavity makes it possible to place the heater and only the lower part In this way, the container is well seated on the heater, does not slip, and remains easy to grasp by the operator.
• the use of a heater and, where appropriate, an insulating support makes it possible to maintain the temperature of a biological, biochemical or chemical solution between 30 and 40 ° C. for the necessary duration. (from a few minutes to a few tens of minutes, for example 5, 10, 15, 18. 20 minutes or more).
• This use of the heater a simple object, inexpensive and generally intended for outdoor recreation (skiing, mountaineering etc.), - to implement a biological process (molecular or cellular biology) or chemical replacement of sophisticated laboratory equipment is as advantageous as it is surprising, since it allows considerable savings without altering the quality of the results obtained.
• biological reaction any reaction involving elements from a living being, such as for example living cells, (animal cells derived from a biopsy, plant cells, yeasts or bacteria), viruses, organelles, enzymes, metabolism products, etc.
• a “biochemical reaction” involves substances involved in chemical reactions of living matter (enzymes, sugars, lipids, etc.).
• the container contains an enzymatic solution and the contact between the heater and the container is maintained for at least 10 minutes.
• the heater is used in the context of a cell biology process
• the enzyme solution contains cells from a biopsy, for example a tissue sample to be dissociated.
• the enzyme solution contains, for example, trypsin.
• the heater preferably consists of a sealed plastic bag containing a saturated aqueous sodium acetate solution.
• the invention can also be implemented with a chemical heater, allowing a longer incubation.
• the used pouch-type heating device containing sodium acetate is a disc whose thickness is between 3 and 7 mm, preferably 4 to 6 mm. the diameter is between 7 and 11 cm, preferably 8 to 10 cm, and the solution containing the reagents involved in the reaction has a volume of between 3 and 10 ml and is contained in a petri dish with a diameter of between 7 and 10 cm. and 1 1 cm, preferably between S e 10 cm, or a compartment of said box.
• petri dish is meant here a shallow transparent cylindrical box, glass or plastic, provided with a lid.
• the petri dish is compartmentalized, for example by a small wall along a diameter.
• the cavity intended to receive the heater is also circular, of diameter slightly greater than that of the heater (between 7 and 1 1, 5 cm) and has a depth between 5 and 20 night),
• the present invention also provides a method of dissociating cells from a freshly harvested tissue sample comprising the steps of:
• tissue sample placed in said solution and incubating for at least 10 minutes, preferably 15 to 20 minutes;
• the heater has a disc shape and the container is a petri dish, as mentioned above.
• the diameters of these two elements are preferably identical or almost identical; if a support is used, the cavity of the support for receiving at least the heater is also circular, of diameter slightly greater than the diameter of the heater and the container.
• step (iv) may, if appropriate, be preceded or followed by a step of inhibiting trypsin by adding an inhibitor.
• an inhibitor for example, no trypsin inhibitor is added, rinsing tissue sample sheet sufficient to stop the action of trypsin. This allows in particular to limit the number of substances that will be applied to the patient with the cells.
• the present invention also relates to a method for preparing a cell suspension suitable for dermal application to a patient, comprising the steps of:
• step (iii) filtering the solution obtained in step (ii) on a cell sieve.
• the above method comprises an additional step of adding hyaluronic acid to the cell suspension obtained in step (Iii). This step makes it possible to obtain one. Viscous mixture that is easily applied into the wound bed. In addition, hyaluronic acid promotes viability. cells.
• a particular application of the above methods is the treatment of vitiligo; in this application, the cells recovered in step (ii) comprise at least melanoeytes.
• the present invention also relates to a kit for biological application (cell biology kit and / or kit of molecular biology), characterized in that it comprises a heater whose operation is based on an exothermic physical or chemical process.
• the heater consists of a sealed plastic bag containing a saturated aqueous sodium acetate solution in a supercooled state.
• the kit also contains a support which comprises a cavity for receiving the heater.
• This support comprises, for example, a plate of thermal insulating material with a thickness of between 5 and 35 mm, in which the cavity intended to receive the heater has a depth of between 5 and 25 mm.
• the support may also be constituted by a thinner plate (for example 1 m thick), folded or. assembled to form a hollow support.
• the material used for the support has a thermal eonducti ity less than or equal to 0.4 W.nf '. "1 to 20 ° C. More preferably, thermal eonduct reinstate is less than 0.35 W, Ni '! .K" ⁇ or less than 0.08 Wm *.
• the insulating nature of the support is secondary, since the premises in which the kit will be used are generally at least 18 ° C, and the incubation time of the cell sample with the irypsine is not very long. on the other hand, some applications may require extreme thermal conditions and / or require a time i longer incubation at a temperature between 30 and 40 ° C. In this case, it is important to use an insulating support, which can, if necessary be completed by a lid to keep the heat in the container longer.
• kits may also comprise a container the base of which has the same geometry 'than .the heater and, where appropriate of the cavity of the support, to enable efficient heat exchange.
• the heater may be a disc whose thickness is between 3 and? mm, preferably 4 to 6 ram and the diameter is between 7 and 11 cm, preferably 8 to. 10 cm; in this case, the container is preferably a petri dish of diameter substantially equal to that of the heater and, if a support is present, the cavity for receiving the heater is also circular, a diameter slightly greater than that of the heater.
• a kit according to the present invention may also contain an enzyme, for example trypsin.
• an enzyme for example trypsin.
• a kit is for example designed for the treatment of a biopsy, and includes in particular the means necessary for the dissociation of the cells of said biopsy.
• this kit comprises a compartmentalized Petri dish whose diameter is identical or very similar to that of the heater, a cell sieve, and trypsin (in freeze-dried form or in solution).
• Figure 1 Evolution of the temperature of a buffer solution in a Petri dish of 9 cm diameter, placed on a heater to sodium acetate, having a disk shape with a diameter substantially equal to that of the petri dish, over a period of 40 minutes, in a room at 20 ° C
• FIG. 2 photos of the device comprising a support and a heat pack (heater). 1: heat pack; 2: support; 3: compartmentalized box placed on the heat pack.
• Figure 6 Evolution of the temperature as a function of time (comparison of means without support and with supports V2 or V3).
• a wound bed is seeded with autologous cells.
• the cell suspensio obtained after graft disintegration consists of a mixed population, mainly basal cells of keratinocytes. but also Lan.gerh.ans cells, melanocytes and fibroblasts.
• Example 1 A heater as described in. Example 1, and a disposable kit-composed of elements and ancillary instruments; enzymatic and application solutions, sterile instruments, including a two-compartment petri dish.
• the kit On receipt, the kit is stored at + 4 ° C until use. The kit is placed at room temperature 10 min before use.
• the area to be sampled is defined surgical felt, disinfected with chlorhexidine 0.5 '% alcoholic solution, and anesthetized with Xylocaine 2%.
• a thin skin flap with a minimum surface area of 4 cm 2 and 0.2-0.3 mm thickness is removed with a dermatome.
• Biopsy 1 0.4% 1.2 95.0% 3.4%,> 0.1%
• Biopsy 2 0.4% 98.0% 3.3%> 0J%
• the inhibition of trypsin was not achieved by addition of fetal serum-type inhibitor, but by rinsing with PBS, in order to decrease the number of products of biological origin used in the manufacture of the product. The cell viability after this mode of inhibition of trypsin has been validated,
• the determination of cell viability was carried out according to a conventional cell culture technique: the trypan blue exclusion test. Volume to volume dilution of trypan blue and cell suspension was performed in a hemolysis tube. After a contact time of 1 to 2 minutes, the mixture was deposited with a micropipette between slide and coverslip on a counting cell. Dead cells, stained blue. and. live, non-stained cells were counted using an optical microscope-phase contrast.
• the number of cells isolated varied from one individual to another depending on the size of the biopsies treated, but the cell / cm 2 yield was relatively constant.
• the cell density of the final suspension therefore depends greatly on the size of the biopsy treated.
• the percentage of cloning efficiency makes it possible to evaluate the level of cells capable of adhering and forming colonies.
• a known quantity of dermal epithelial cells was seeded into 3 T25 cm 2 flasks. To the 1 th day of culture, when the clones were large enough but not contiguous, the flasks were stained with a solution of crystal violet (10% formaldehyde / 0.5% crystal violet, qs distilled water). Cloning efficiency was calculated in. performing the ratio of the average total number of colonies per T25em flask 2 x 100 to the number of cells seeded per T25 cm 2 .
• the behavior of the cells after re-culture and the cloning efficiency were homogeneous from one biopsy to another and showed that the cell suspension contained cells capable of proliferating after trypsination.
• the cell membrane was permeabilized in a solution of PBS-BSA (Bovine Serum Albumin) 1% - 0.5% triton.
• PBS-BSA Bovine Serum Albumin
• the primary antibody specific for normal melanocytes KI / beieb antibody
• the secondary antibody Alexa Fluor 488 anti-mouse donkey antibody
• 1% BSA The labeled cells were taken up in PBS and passed to FACS-SCAN.
• the ratios of metabolites / keratinocytes in the epidermal suspensions obtained were comparable to those calculated by Guerra et al (between 1: 30 and 1: 200) (Guerra et al, 2003).
• isolated epidermal cells were not cultured and the entire process was performed in the daytime (biopsy, finished product manufacturing, and non-cultured autologous epidermal cell transplant).
• Example 3 Use of an insulating support to optimize the rise and the maintenance of the temperature of the medium by the heater
• Heat packs (heaters) PVC, CE marking Support plates consisting of a polypropylene sheet (PP) 1 to 2 mm thick, folded to obtain a support with a total thickness of 15 mm, having a circular cavity 90 mm in diameter ( Figure 2).
• thermobouton is in contact with the medium, making it possible to measure directly within the solution the temperature variations, after ac ivation of the heat pack.
• the experiment is carried out by placing or not the heat pack and the petri dish in the circular cavity of the support. ( Figure 2). Three different media were tested. They differ in the more or less central position of the circular cavity intended to accommodate the heater and the petri dish.
• the temperature data collected by the temperature probe plunged into the medium are shown schematically in Figure 3.
• the average temperature on the 6 tests was 35.5 ° C. with an average standard deviation of 3.9 * 0.
• the temperatures reached are higher when the heaters are placed in a support (1.1 ° C difference with V2 and 0.8 ° C with V3);
• the medium contained in the compartmentalized Pelri box is maintained at a temperature greater than 35 ° for at least 18 minutes;
• the supports therefore make it possible to optimize the rise in temperature and / or its maintenance.
• VitiC'ell® kit support during an enzymatic digestion of fine skin biopsy in order to achieve a dermo-epidermal separation, according to the epidermal suspensions production process described in Example 2, makes it possible to on average get a higher temperature and faster.
• the support therefore has a double interest: worktable and optimization of the temperature rise of the heat packs.

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