Warmer for promoting a biological reaction

Abstract

The present invention relates to the use of warmers, or autonomous heat packs, for heating and maintaining a solution at a suitable temperature, for the period of time required to accomplish a chemical, biochemical or biological reaction, in particular in molecular biology or cell biology applications. Biology kits containing warmers are also part of this invention.

Claims

1. A kit comprising a warmer including a hermetic plastic pouch containing a sodium acetate-saturated aqueous solution, the operation of which is based on an exothermic physical or chemical process; and a container for culturing a patient's biopsy tissue sample; wherein the warmer maintains the temperature of the liquid in the container at 30°−40° C. for at least 10 minutes.

2. The kit of claim 1, further comprising a support which has a cavity configured to receive the warmer.

3. The kit of claim 2, wherein the support cavity is further configured to receive at least the lower part of the container.

4. The kit of claim 1, further comprising a solution comprising trypsin.

5. The kit of claim 2, further comprising a solution comprising trypsin.

6. The kit of claim 3, further comprising a solution comprising trypsin.

7. The kit of claim 1, wherein the container has a base having the same geometry as that of the warmer.

8. The kit of claim 2, wherein the cavity of the support is circular, with a diameter from 7 to 11.5 cm, and a depth from 5 to 20 mm.

9. The kit of claim 3, wherein the cavity of the support is circular, with a diameter from 7 to 11.5 cm, and a depth from 5 to 20 mm.

10. The kit of claim 1, further comprising an enzyme in lyophilized form or in solution.

11. The kit of claim 2, further comprising an enzyme in lyophilized form or in solution.

12. The kit of claim 3, further comprising an enzyme in lyophilized form or in solution.

Description

FIGURE LEGENDS

(1) FIG. 1: Change in the temperature of a buffer solution in a Petri dish 9 cm in diameter, placed on a sodium acetate warmer, having the shape of a disk with a diameter approximately equal to that of the Petri dish, over a period of 40 minutes, in a room at 20° C.

(2) FIG. 2: Photos of the device comprising a support and a heat pack (warmer). 1: heat pack; 2: support; 3: compartmentalized dish placed on the heat pack.

(3) FIG. 3: Change in the temperature as a function of time, without support.

(4) FIG. 4: Change in the temperature as a function of time, with the support V2.

(5) FIG. 5: Change in the temperature as a function of time, with the support V3.

(6) FIG. 6: Change in the temperature as a function of time (comparison of the means without support and with supports V2 or V3).

EXAMPLES

Example 1: Temperature of the Medium in a Petri Dish Placed on a Warmer

(7) The temperature of a (buffer) solution in a Petri dish 9 cm in diameter, placed on a sodium acetate warmer, having the shape of a disk with a diameter approximately equal to that of the Petri dish and a thickness between 4 and 6 mm, was measured.

(8) The results (FIG. 1) show quite a rapid increase in temperature, since the temperature reaches 30° C. in a few minutes. The temperature then remains between 30 and 40° C. for a little more than 20 minutes.

(9) These results showed good reproducibility.

Example 2: Application to Dermo-Epidermal Separation, in the Context of the Treatment of Vitiligo by Autologous Graft of Melanocytes

(10) Using a thin skin biopsy (0.2 mm-0.3 mm), the bed of a wound is seeded with autologous cells.

(11) The cell suspension obtained after disaggregation of the graft consists of a mixed population, mainly basal keratinocyte cells, but also Langerhans cells, melanocytes and fibroblasts.

(12) Materials Used

(13) A warmer as described in example 1 and a disposable kit composed of ancillary elements and instruments: enzymatic and application solutions, sterile instruments, including a two-compartment Petri dish.

(14) Protocol

(15) A sterile surgical field is prepared.

(16) Upon reception, the kit is stored at +4° C. until use. The kit is placed at ambient temperature 10 min before use.

(17) Preparation and Heating of the Enzymatic Solution Using a 5 ml syringe and a needle, take 5 ml of PBS solution and inject them into the bottle of lyophilized trypsin. Homogenize thoroughly before taking up the regenerated solution and in one compartment of the two-compartment Petri dish. Activate the heat pack instantaneously by crumpling the metal plate; the crystal solidifies instantaneously. Place the two-compartment Petri dish on the activated heat pack and wait 5 min.

(18) Taking a Skin Sample

(19) The zone from which a sample is to be taken is delimited with a surgical felt tip pen, disinfected with 0.5% chlorhexidine in alcoholic solution, and anesthetized with 2% xylocaine. A thin strip of skin which has a minimum surface area of 4 cm.sup.2 and a thickness of 0.2-0.3 mm is taken with a dermatome.

(20) Carrying Out the Dermo-Epidermal Separation (DES) Using sterile forceps (not provided), transfer the biopsy into the Petri dish compartment containing the trypsin in solution at 0.4%. Incubate for 15 minutes. Rinsing of the biopsy: With the same 5 ml syringe and a needle, take 5 ml of PBS solution and place in the second compartment of the Petri dish. At the end of the incubation, using sterile forceps, transfer the biopsy into the second compartment, for rapid rinsing with PBS. Using sterile forceps, place the biopsy inside the lid of the Petri dish, taking care to keep the direction of the dermo-epidermal junction facing upward. Using the forceps, separate the two fragments.

(21) Preparation of the Cell Suspension Using the second 5 ml syringe and a needle, take 1.5 ml of PBS and place them on the biopsy fragments, scrape the cells from the junction surfaces with a scalpel (not provided) and coarsely cut up the epidermal part so as to produce a mixture of cells. Set aside and immobilize the dermal part. Tilt the Petri dish so as to suction up the entire volume using the 5 ml syringe, draw the cells up into the syringe and suction several times so as to create a cell suspension.

(22) Cell Filtration Transfer the cell suspension into the sieve that will have been placed on the pot with the red cap.

(23) Preparation of the Cell Suspension in Hyaluronic Acid Remove the sieve. Place the hyaluronic acid contained in the syringe (i.e. 1.5 ml) in the pot with the red cap containing the filtered cell suspension. Using the syringe, mix the hyaluronic acid and the cell suspension so as to obtain a viscous but homogeneous suspension. The suspension thus prepared is ready to be deposited by syringe on the wound bed.

(24) Results

(25) The protocol above was carried out several times, on three different skin samples, using two trypsin concentrations (0.4% and 0.8%).

(26) The results obtained are summarized in table 1 below.

(27) TABLE-US-00001 TABLE 1 Number of Presence of Trypsin cells Cell Cloning melanocytes Identi- concen- isolated × viability efficiency by flow fication tration 10.sup.6/cm.sup.2 (%) test cytometry Biopsy 1 0.4% 1.2 95.0% 3.4% >0.1% 0.8% 1.2 97.0% 3.4% >0.1% Biopsy 2 0.4% 3.4 98.0% 3.3% >0.1% 0.8% 2.3 97.0% 3.8% >0.1% Biopsy 3 0.4% 4.2 97.4% 4.6% >0.1% 0.8% 3.9 99.4% 4.5% >0.1%

(28) Trypsin Concentration

(29) The process for obtaining the active substance was adapted from the technique described by van Geel et al. (van Geel et al., 2004).

(30) The concentration of the trypsin solution was optimized (0.4% or 0.8% trypsin instead of 0.25% trypsin/0.08% EDTA). Unlike the technique described by van Geel et al., the inhibition of the trypsin was not carried out by adding inhibitor of fetal calf serum type, but by rinsing with PBS, in order to reduce the number of products of biological origin used for the production of the product. The cell viability after this mode of trypsin inhibition was validated.

(31) Cell Viability

(32) The cell viability was determined according to a conventional cell culture technique: the trypan blue exclusion test. A volume-for-volume dilution of trypan blue and of the cell suspension was carried out 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. The dead cells, stained blue, and the live cells, unstained, were counted using a phase contrast optical microscope.

(33) The cell viability was very satisfactory after isolation of the epidermal cells (greater than 90%).

(34) The behavior of the cells after being put back in culture and the cloning efficiency were uniform from one biopsy to the other and showed that the cell suspension contained cells capable of proliferating after trypsinization.

(35) Number of Cells Isolated/Cm of Biopsy

(36) Calculation carried out on the basis of the results of the test described above.

(37) The number of cells isolated was variable from one individual to another depending on the size of the biopsies treated, but the cell yield/cm.sup.2 was relatively constant. The cell density of the final suspension therefore depends greatly on the size of the biopsy treated.

(38) Cloning Efficiency Test (CFE)

(39) The cloning efficiency percentage makes it possible to evaluate the amount of cells capable of adhering and of forming colonies. A known amount of epidermal cells was seeded into 3 T25 cm.sup.2 flasks. Around the 14th day of culture, when the clones were sufficiently large but not joining up, the flasks were stained with a solution of crystal violet (10% formaldehyde/0.5% crystal violet, qs distilled water). The cloning efficiency was calculated by producing the ratio of the mean of the total number of colonies per T25 cm.sup.2 flask×100 to the number of cells seeded per T25 cm.sup.2.

(40) The behavior of the cells after being placed back in culture and the cloning efficiency were uniform from one biopsy to the other and showed that the cell suspension contained cells capable of proliferating after trypsinization.

(41) Melanocyte Content in the Cell Suspension by Flow Cytometry

(42) The cell membrane was permeabilized in a solution of PBS-1% BSA (bovine serum albumin)—0.5% triton. The primary antibody specific for normal melanocytes (NKI/beteb antibody) was incubated, and then rinsed with PBS-1% BSA. The secondary antibody (Alexa Fluor 488 donkey antimouse antibody) was incubated and then rinsed with PBS-1% BSA. The labeled cells were taken up in PBS and passed through a FACS-SCAN.

(43) The melanocyte/keratinocyte ratios in the epidermal suspensions obtained were comparable to those calculated by Guerra et al. (between 1:30 and 1:200) (Guerra et al., 2003).

(44) In the process described here, the isolated epidermal cells were not placed in culture and the entire process was carried out in one day (biopsy, production of the final product and grafting of noncultured autologous epidermal cells).

Example 3: Use of an Insulating Support for Optimizing the Increasing and the Maintaining of the Temperature of the Medium Using the Warmer

(45) 3.1. Materials and Methods

(46) Materials and Reagents PVC heat packs (warmers), EC labeling. Support plates consisting of a polypropylene (PP) sheet 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 (FIG. 2). Compartmentalized Petri dish (ref. Dutscher 020012). Temperature probe (thermobouton) programmed to operate with the ThermoTrack V4 and/or A BT 529 software.

(47) Methods

(48) Six milliliters of PBS stored at +4° C., equilibrated at ambient temperature for 20 minutes, are placed in one of the compartments of a Petri dish 90 mm in diameter, in which a thermobouton probe (device for recording temperature variations) is placed in order to take temperature recordings every minute. The Petri dish is then covered with its lid.

(49) The thermobouton probe is in contact with the medium, making it possible to measure temperature variations directly within the solution, after activation of the heat pack. The experiment is carried out by placing or not placing the heat pack and the Petri dish in the circular cavity of the support (FIG. 2). Three different supports were tested. They differ in terms of the more or less central position of the circular cavity intended to receive the warmer and the Petri dish.

(50) Each condition was tested 5 to 6 times, with different temperature probes. The temperature measurements were carried out for 20 min. The mean of the temperatures was calculated over all the values recorded during this period of time.

(51) A statistical analysis of the temperatures measured in the medium with or without support was carried out by means of a student's t test.

(52) 3.2. Results

(53) Change in the Temperature of the Medium with a Heat Pack without Support

(54) The temperature data collected by the temperature probe immersed in the medium are represented diagrammatically in FIG. 3. The mean temperature over the 6 tests was 35.5° C., with a mean standard deviation of 3.9° C.

(55) These results demonstrate the reproducibility of the temperatures obtained with the various warmers, without support.

(56) Change in the Temperature of the Medium with a Heat Pack in a Support

(57) Two supports (prototypes V2 and V3) were tested. They differ in terms of the position of the circular cavity intended to receive the warmer. Indeed, in the support V3, this cavity was centralized so as to allow the heat inside the support to be uniform, in order to optimize the increasing and the maintaining of the temperature in the compartmentalized Petri dish placed on the warmer.

(58) The temperature data collected for 20 minutes by the temperature probe immersed in the medium are represented diagrammatically in the graphs presented in FIG. 4 (support V2), in FIG. 5 (support V3) and in FIG. 6 (comparison of the two supports and of the temperatures obtained without support), and summarized in the table below.

(59) TABLE-US-00002 TABLE 2 Period of Time to time during Maximum reach the which temper- temper- maximum Mean temper- ature main- ature temper- ature of tained >35° C.* reached ature the 6 tests Test 18 min 39.5° C. 9 min 35.5° C. ± 3.9° C. without support Test 18 min 40.5° C. 6 min 36.7° C. ± 3.6° C. with support V2 Test 18 min .sup. 40° C. 9 min 36.3° C. ± 1° C..sup.  with support V3 *period of time obtained over a total time of the experiment of 21 minutes

(60) These results demonstrate: the reproducibility between the warmers; the temperatures reached are higher when the warmers are placed in a support (1.1° C. of difference with V2 and 0.8° C. with V3); with or without support, the medium contained in the compartmentalized Petri dish is maintained at a temperature above 35° C. for at least 18 minutes; without support, the maximum temperature of 39.5° C. is reached in 9 min, whereas, with the support V2, it reaches 40.5° C. in 6 min.

(61) In order to determine whether the differences observed are statistically significant, a statistical analysis by means of a student's t test was carried out using the values obtained with the support V3. This test indicates a probability of 0.03<0.05, demonstrating that the difference in the temperatures obtained without support and with the support V3 is significant (table 3). The support V3 therefore makes it possible to obtain a temperature which is on average higher than without support, more rapidly and more reproducibly (s.d. ±1° C.)

(62) TABLE-US-00003 TABLE 3 Mean without support Mean with support 35.4 35.9 35.9 35.7 35.7 36.9 35.3 36.3 35.3 36.8 35.6 NA Mean 35.5  36.32 t 0.03

(63) The supports therefore make it possible to optimize the increase in temperature and/or the maintaining thereof.

(64) The use of the support of the VitiCell® kit during an enzymatic digestion of a thin skin biopsy in order to produce a dermo-epidermal separation thereof, according to the process for producing epidermal suspensions described in example 2, makes it possible to obtain on average a higher temperature and more rapidly. The support therefore has a double advantage: work plate and optimization of the temperature increase of the heat packs.

REFERENCES

(65) Gauthier, Y. and Surleve-Bazeille, J. E. (1992) Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol, 26, 191-194. Guerra, L., Primavera, G., Raskovic, D., Pellegrini, G., Golisano, O., Bondanza, S., Paterna, P., Sonego, G., Gobello, T., Atzori, F., Piazza, P., Luci, A. and De Luca, M. (2003) Erbium: YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. Arch Dermatol, 139, 1303-1310. Mulekar, S. V. (2003) Melanocyte-keratinocyte cell transplantation for stable vitiligo. Int J Dermatol, 42, 132-136. van Geel, N., Ongenae, K., De Mil, M., Haeghen, Y. V., Vervaet, C. and Naeyaert, J. M. (2004) Double-blind placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting vitiligo. Arch Dermatol, 140, 1203-1208.