Method of processing allograft skin for transplantation, and cryopreserved allograft skin produced thereby

Abstract

The present invention relates to a method of processing allograft skin for transplantation and a cryopreserved allograft skin produced thereby. More specifically, the present invention relates to a method in which a cryoprotectant is prepared by adding sucrose to basic constituents comprising dimethyl sulfoxide, an animal cell culture medium and fetal bovine serum, and then the resulting solution is used to subject skin tissue for transplantation to a freezing process.

Claims

1. A method of processing allograft of viable skin for transplantation comprising: i) mixing a cryoprotectant solution to include dimethyl sulfoxide, sucrose, an animal cell culture medium and fetal bovine serum to obtain a cryoprotectant solution in which the mixing ratio of dimethyl sulfoxide, the animal cell culture medium and fetal bovine serum is 1:3-5:4-6 based on weight, and the sucrose has a final concentration of 25% to 40% by weight of the cryoprotectant solution; ii) penetrating the cryoprotectant solution into a separated allograft of viable living skin for 6 to 24 hours; and iii) freezing the cryoprotectant-penetrated skin in a controlled rate freezer at a freezing rate of from −0.1 to −7° C. per minute.

2. The method of processing allograft skin for transplantation according to claim 1, wherein the final concentration of sucrose in the cryoprotectant solution is 30% by weight.

3. The method of processing allograft skin for transplantation according to claim 1, wherein the animal cell culture medium is selected from the group consisting of MEM, DMEM, RPMI 1640 and IMDM.

4. The method of processing allograft skin for transplantation according to claim 1, wherein the cryoprotectant is penetrated into the separated skin in a 4° C. low temperature bath for 6-24 hours.

5. The method of processing allograft skin for transplantation according to claim 1, wherein the cryoprotectant-penetrated skin is frozen in a controlled rate freezer with a freezing rate of −1° C. per minute.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph representing the cell death rate of glycerol-preserved allograft (GPA) and cryopreserved allograft (CPA) measured by TUNEL assay.

(2) FIG. 2 is an optical microscope photograph of glycerol-preserved allograft after H&E staining (200 times magnification), and

(3) FIG. 3 is an optical microscope photograph of cryopreserved allograft after H&E staining (200 times magnification).

(4) FIG. 4 is a fluorescence microscope photograph of glycerol-preserved allograft after TUNEL staining (100 times magnification), and

(5) FIG. 5 is a fluorescence microscope photograph of cryopreserved allograft after TUNEL staining (100 times magnification).

(6) FIG. 6 is a graph representing the change of the cell death rate according to the sucrose concentration measured by TUNEL assay.

DESCRIPTION OF EMBODIMENTS

(7) The present invention is explained in more detail with the following examples. However, it must be understood that the protection scope of the present invention is not limited to the examples.

(8) Because human skin tissue harvested from a donor (cadaver) is prohibited from being used in an experiment, pig skin—which is the closest to human skin—is used for preparing ten (10) of both cryopreserved skins and glycerol-preserved skins according to the following methods of Example and Comparative Example.

EXAMPLE

(9) Cryopreserved skin was prepared with pig skin according to the following steps.

(10) (1) Pig skin was washed with saline solution.

(11) (2) The pig skin was cut at the size of 5×10 cm.sup.2.

(12) (3) Dimethyl sulfoxide (Sigma, USA), DMEM (Gibco, USA) and fetal bovine serum (Gibco, USA) were mixed in the weight ratio of 1:4:5.

(13) (4) Sucrose was added to the solution of step (3) as the final concentration of 30 wt % and dissolved to obtain a cryoprotectant.

(14) (5) The pig skin of step (2) was immersed in the cryoprotectant of step (4).

(15) (6) A low temperature bath (P-039, CoreTech, Korea) was set at 4° C.

(16) (7) The pig skin of step (5) was put in the 4° C. low temperature bath, and then the cryoprotectant was penetrated into the pig skin for 12 hours.

(17) (8) The penetration-completed pig skin and 50 ml of the cryoprotectant were put in a polyamide bag (CryoBag™, Origen, USA).

(18) (9) A controlled rate freezer was prepared.

(19) (10) The polyamide bag of step (8) was put in the controlled rate freezer and frozen to −150° C. with the rate of −1° C. per minute.

(20) (11) After freezing, the polyamide bag was kept frozen in a dry shipper until analysis experiments.

Comparative Example

(21) A glycerol-preserved skin was prepared with pig skin by sequentially treating it with 50% glycerol and 85% glycerol according to the following steps.

(22) (1) Pig skin was washed with saline solution.

(23) (2) The pig skin was cut at the size of 5×10 cm2.

(24) (3) The pig skin was immersed in 50% glycerol diluted with distilled water for 72 hours.

(25) (4) The pig skin of step (3) was immersed in 85% glycerol for 72 hours.

Experimental Example 1

(26) The pig skins prepared according to the above Example and Comparative Example were analyzed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. TUNEL assay was carried out by using an In Situ Cell Death Detection Kit, Fluorescein (Roche, Germany) according to the manufacturer's instructions. The results of the cell death rate are represented in FIG. 1. (The results are a mean of 10 samples.)

(27) Pictures of the skins of the Example and Comparative Example were taken with a microscope (Olympus BX51), and the results are shown in FIGS. 2, 3, 4 and 5.

(28) As can be seen from FIG. 1, the cell death rate in the Example is 4-5 times lower than that of the Comparative Example.

(29) In addition, from the microscope photographs of FIGS. 2, 3, 4 and 5, it can be seen that cell death in the skin tissue in the Example is clearly smaller than that in the Comparative Example.

(30) That is, the method according to the present invention shows higher cell viability compared with the conventional glycerol preservation method. As a result, the cryopreserved allograft skin according to the present invention can increase the success rate of acellular dermis grafting and curtail the treatment duration by helping to regenerate granulation tissue at the transplanted region.

Experimental Example 2

(31) To investigate the change of the cell death rate according to the difference of sucrose concentration, the cryopreserved allograft skin was prepared by the same method as in the Example except that the sucrose concentration in the cryoprotectant was 5, 10, 15, 20, 25, 30, 35 and 40 wt %, and it was then analyzed by TUNEL assay. TUNEL assay was carried out by using an In Situ Cell Death Detection Kit, Fluorescein (Roche, Germany) according to the manufacturer's instructions, and 4′,6-diamidino-2-phenylindole (DAPI) was used as a fluorescent marker. The results of cell death rate are shown in the following Table 1, and represented in FIG. 6 as a graph.

(32) TABLE-US-00001 TABLE 1 Sucrose Conc. n 5 wt % 10 wt % 15 wt % 20 wt % 25 wt % 30 wt % 35 wt % 40 wt % 1 45 49 31 35 23 20 25 36 2 36 55 22 21 28 23 10 33 3 64 57 47 22 64 39 23 41 4 39 56 57 23 12 11 28 26 5 41 41 66 68 20 5 21 20 6 65 26 25 43 19 20 30 13 AVE 48 47 41 35 28 20 23 28 SD 13 12 18 18 19 12 7 10 SE 5.251455 4.91709 7.378648 7.441625 7.574812 4.744588 2.891559 4.284987 (Unit of cell death rate: %)

(33) As can be seen from Table 1 and FIG. 6, when sucrose is comprised as 25 to 40 wt % in the final concentration, the cell death rate is quite low.