Tumor tissue cryopreservation kit, tumor tissue resuscitation kit, and method of cryopreserving and/or resuscitating tumor tissue

Abstract

A kit, including: a first solution, a second solution, and a metal mesh. The first solution includes: dulbecco's modified eagle medium (DMEM), 65-95 V/V %; dimethyl sulfoxide (DMSO), 5.5-20 V/V %; ethylene glycol (EG), 3.5-15 V/V %; bovine serum albumin (BSA), 0.5-4 W/V %; sucrose 1-5 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05-0.8 W/V %; hetastarch 0.25-0.6 W/V %; and glucose 15-35 W/V %. The second solution includes: DMEM, 65-95 V/V %; DMSO, 5.5-20 V/V %; EG, 8-20 V/V %; BSA, 0.5-4 W/V %; sucrose, 10-20 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05-0.8 W/V %; polyvinyl pyrrolidone (PVP), 0.25-0.6 W/V %; and glucose 15-35 W/V %. The metal mesh has a thickness of 0.15-0.2 mm and includes a plurality of square holes. The side length of the square holes is 2.0-3.0 mm, and the spacing between adjacent holes is 0.5-2.0 mm.

Claims

1. A kit, comprising: a first solution, comprising: dulbecco's modified eagle medium (DMEM), 65-95 V/V %; dimethyl sulfoxide (DMSO), 5.5-20 V/V %; ethylene glycol (EG), 3.5-15 V/V %; bovine serum albumin (BSA), 0.5-4 W/V %; sucrose 1-5 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05-0.8 W/V %; hetastarch 0.25-0.6 W/V %; glucose 15-35 W/V %; a second solution, comprising: DMEM, 65-95 V/V %; DMSO, 5.5-20 V/V %; EG, 8-20 V/V %; BSA, 0.5-4 W/V %; sucrose, 10-20 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05-0.8 W/V %; polyvinyl pyrrolidone (PVP), 0.25-0.6 W/V %; glucose 15-35 W/V %; and a metal mesh, with a thickness of 0.15-0.2 mm, and comprising a plurality of square holes, a side length of the square holes being 2.0-3.0 mm, and a spacing between adjacent holes being 0.5-2.0 mm.

2. The kit of claim 1, wherein: the first solution comprises: dulbecco's modified eagle medium (DMEM), 80 V/V %; dimethyl sulfoxide (DMSO), 10 V/V %; ethylene glycol (EG), 10 V/V %; bovine serum albumin (BSA), 3 W/V %; sucrose 1 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05 W/V %; hetastarch 0.25 W/V %; glucose 25 W/V %; and the second solution comprises: DMEM, 70 V/V %; DMSO, 18 V/V %; EG, 12 V/V %; BSA, 3 W/V %; sucrose, 20 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05 W/V %; PVP, 0.25 W/V %; glucose 30 W/V %.

3. The kit of claim 1, further comprising a slicer; wherein the slicer comprises a pedestal; the pedestal comprises an upper surface, a recessed portion disposed on the upper surface, and a plurality of guiding grooves which are equidistantly distributed and vertically downward from the upper surface of the pedestal; and a depth of the plurality of guiding grooves is greater than a depth of the recessed portion.

4. The kit of claim 3, wherein the depth of the plurality of guiding grooves is greater than the depth of the recessed portion by 1.8-2.3 mm, and a spacing between two adjacent guiding grooves is 1 mm.

5. The kit of claim 3, wherein the recessed portion is an ellipsoid with a width of 16 mm and a length of 25 mm; and a vertical distance from a lowest point of the recessed poriton to the upper surface of the pedestal is 8.5 mm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1B are diagrams illustrating the modeling of PDXs after one month of implantation in immunodeficient mice with human primary cholangiocarcinoma tumor tissue cryopreserved according to one embodiment of the disclosure and the conventional cryopreservation method (Example 6);

(2) FIG. 2 is a diagram illustrating the anatomy of PDXs tumor derived from the cryopreserved-rewarmed human primary cholangiocarcinoma tumor tissue after one month of implantation in immunodeficient mice (Example 6);

(3) FIG. 3 is a diagram illustrating the comparative percent bar chart of PDXs tumor generation rate derived from fresh and cryopreserved-rewarmed human primary cholangiocarcinoma tumor tissue (Example 6);

(4) FIGS. 4A-4B are diagrams illustrating the comparison of the results of H&E staining of fresh or cryopreserved-rewarmed tumor tissue; FIG. 4A represents the H&E staining result of fresh tumor tissue, FIG. 4B represents the H&E staining result of cryopreserved-rewarmed tumor tissue (Example 7);

(5) FIGS. 5A-5C are diagrams illustrating a tumor tissue slicer of a tumor tissue cryopreservation kit as described in the disclosure, FIG. 5A is a perspective view, FIG. 5B is a top view, and FIG. 5C is a cross-sectional view in the A-A direction; and

(6) FIG. 6 is a diagram illustrating a front view of the metal mesh of a tumor tissue cryopreservation kit as described in the disclosure.

DETAILED DESCRIPTION

(7) To further illustrate, examples detailing a tumor tissue cryopreservation kit and a tumor tissue resuscitation kit are described below. It should be noted that the following examples are intended to describe and not to limit the description.

Example 1: Preparation of Tumor Tissue Cryopreservation Kit

(8) The formulation components of the solutions for tumor tissue cryopreservation kit are as follows:

(9) A first solution (V1), comprising: dulbecco's modified eagle medium (DMEM), 65-95 V/V %; dimethyl sulfoxide (DMSO), 5.5-20 V/V %; ethylene glycol (EG), 3.5-15 V/V %; bovine serum albumin (BSA), 0.5-4 W/V %; sucrose 1-5 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05-0.8 W/V %; hetastarch 0.25-0.6 W/V %; glucose 15-35 W/V %. Specifically, in this example, the first solution comprises: DMEM 80 V/V %, DMSO 10 V/V %, EG 10 V/V %, BSA 3 W/V %, sucrose 1 W/V %, methylcellulose 4000 cP 0.05 W/V %, hetastarch 0.25 W/V %, glucose 25 W/V %; and

(10) A second solution (V2), comprising: DMEM, 65-95 V/V %; DMSO, 5.5-20 V/V %; EG, 8-20 V/V %; BSA, 0.5-4 W/V %; sucrose, 10-20 W/V %; methylcellulose with a viscosity of 4000 centipoise (cP), 0.05-0.8 W/V %; polyvinyl pyrrolidone (PVP), 0.25-0.6 W/V %; glucose 15-35 W/V %. Specifically, in this example, the first solution comprises: DMEM 70 V/V %, DMSO 18 V/V %, EG 12 V/V %, BSA 3 W/V %, sucrose 20 W/V %, methylcellulose 4000 cP 0.05 W/V %, PVP 0.25 W/V %, glucose 30 W/V %.

(11) The English name and the manufacturer of each reagent are as follows:

(12) DMEM: dulbecco's modified eagle medium (high glucose); Sigma-Aldrich.

(13) DMSO: dimethyl sulfoxide; Sigma-Aldrich.

(14) BSA: Bovine Serum Albumin; Sigma-Aldrich.

(15) EG: Ethylene Glycol; Sigma-Aldrich.

(16) Sucrose: Sigma-Aldrich.

(17) methylcellulose 4000 cP: Sigma-Aldrich.

(18) hetastarch: Sigma-Aldrich.

(19) glucose: Sigma-Aldrich.

(20) PVP: Polyvinyl Pyrrolidone; Sigma-Aldrich.

Example 2: Method of Cryopreserving Tumor Tissue

(21) The prepared cryopreservation kit is filtered, packaged, and stored temporarily in a refrigerator at −4° C., and stored in a refrigerator at −20° C. for long-term preservation.

(22) The method of cryopreserving tumor tissue are as follows:

(23) a. The tumor tissue is washed three times with physiological saline. The blood vessel, the capsule, and the necrotic tissue are trimmed and peeled off, and the tumor tissue is sliced with the slicer into a size of 1×10×10 mm, and washed again with physiological saline.

(24) The slicer, as shown in FIGS. 5A-5C, comprises: a pedestal 1 comprising an upper surface and a recessed portion 2 formed on the upper surface; twelve guiding grooves 3, which are equidistantly distributed and have a depth 2 mm deeper than the lowest point of the recessed portion 2, are vertically downward from the upper surface of the pedestal 1. The spacing between the two adjacent guiding grooves 3 is 1 mm. The recessed portion 2 is an ellipsoid with a width of 16 mm and a length of 25 mm; and a vertical distance from the lowest point of the recessed portion 2 to the upper surface of the pedestal 1 is 8.5 mm.

(25) b. The first solution (V1) and the second solution (V2) are prepared, and then filtered to eliminate bacteria;

(26) c. the tumor tissue slices are immersed in 10 mL of the V1 at room temperature for 25 min;

(27) d. the tumor tissue slices are immersed in 10 mL of the V2 at room temperature for 15 min, or until they precipitate to the bottom of the tube; and

(28) e. the tumor tissue slices are placed in a minimum volume of solution onto a metal mesh (as shown in FIG. 6), and immersed directly into liquid nitrogen for at least 5 minutes; and

(29) f. the metal mesh with tumor tissue slices is inserted into a marked cryotube, and stored in a liquid nitrogen tank for long-term preservation.

Example 3: Preparation of Tumor Tissue Resuscitation Kit

(30) The formulation components of the solutions for tumor tissue resuscitation kit are as follows:

(31) Solution 1 (T1), comprising: dulbecco's modified eagle medium (DMEM), 65-85 V/V %; phosphate buffer saline (1×), 15-35 V/V %; bovine serum albumin (BSA), 1-3 W/V %; sucrose 10-40 W/V %; glucose, 15-35 W/V %; specifically, in this example, the Solution 1 (T1) comprises: DMEM 65 V/V %, 1×PBS 35 V/V %, BSA 2 W/V %, sucrose 40 W/V %, glucose 25 W/V %;

(32) Solution 2 (T2), comprising: dulbecco's modified eagle medium (DMEM), 65-85 V/V %; phosphate buffer saline (1×), 15-35 V/V %; bovine serum albumin (BSA), 1-3 W/V %; sucrose 10-20 W/V %; glucose, 15-35 W/V %; specifically, in this example, the Solution 2 (T2) comprises: DMEM 75 V/V %, 1×PBS 25 V/V %, BSA 2 W/V %, sucrose 20 W/V %, glucose 25 W/V %; and

(33) Solution 3 (T3), comprising: dulbecco's modified eagle medium (DMEM), 75-95 V/V %; phosphate buffer saline (1×), 5-25 V/V %; bovine serum albumin (BSA), 1-3 W/V %; glucose, 15-35 W/V %. Specifically, in this example, the Solution 3 (T3) comprises: DMEM 95 V/V %, 1×PBS 5 V/V %, BSA 2 W/V %, glucose 15 W/V %.

(34) The English name and the manufacturer of each reagent are as follows:

(35) DMEM: dulbecco's modified eagle medium (high glucose); Sigma-Aldrich.

(36) 1×PBS: 0.01M Phosphate Buffered Saline; Sigma-Aldrich.

(37) BSA: Bovine Serum Albumin; Sigma-Aldrich.

(38) Sucrose: Sigma-Aldrich.

(39) glucose: Sigma-Aldrich.

Example 4: Method of Resuscitating Tumor Tissue

(40) The method for resuscitating tumor tissue are as follows:

(41) a. The tumor tissue slices are taken out of the liquid nitrogen tank and immersed into 10 mL of the T1 at 37° C. for 3 min;

(42) b. the tumor tissue slices are immersed into 10 mL of the T2 at room temperature for 5 min;

(43) c. the tumor tissue slices are immersed into 10 mL of the T3 at room temperature for 10 min; and

(44) d. the tumor tissue slices are immersed into 10 mL of fresh T3 at room temperature for 10 min; the resuscitated tumor tissue is obtained and can be used in subsequent studies.

Example 5: Cryopreservation of Human Primary Cholangiocarcinoma Tumor Tissue

(45) a. The human primary cholangiocarcinoma tumor tissue is washed three times with physiological saline. The blood vessel, the capsule, and the necrotic tissue are trimmed and peeled off, and the tumor tissue is sliced with the slicer into a size of 1×10×10 mm, and washed again with physiological saline;

(46) b. the first solution (V1), the second solution (V2), solution 1 (T1), solution 2 (T2) and solution 3 (T3) are prepared, and then filtered to eliminate bacteria;

(47) c. the tumor tissue slices are immersed in 10 mL of the V1 at room temperature for 25 min;

(48) d. the tumor tissue slices are immersed in 10 mL of the V2 at room temperature for 15 min, or until they precipitate to the bottom of the tube;

(49) e. the tumor tissue slices are placed in a minimum volume of solution onto a metal mesh, and immersed directly into liquid nitrogen for at least 5 minutes; and

(50) f. the metal mesh with tumor tissue slices is inserted into a marked cryotube, and stored in a liquid nitrogen tank for long-term preservation.

Example 6: Resuscitation of Human Primary Cholangiocarcinoma Tumor Tissue

(51) a. The tumor tissue slices are taken out of the liquid nitrogen tank and immersed into 10 mL of the T1 at 37° C. for 3 min;

(52) b. the tumor tissue slices are immersed into 10 mL of the T2 at room temperature for 5 min;

(53) c. the tumor tissue slices are immersed into 10 mL of the T3 at room temperature for 10 min;

(54) d. the tumor tissue slices are immersed into 10 mL of fresh T3 at room temperature for 10 min; and the resuscitated tumor tissue is obtained;

(55) e. the resuscitated tumor tissue can be transplanted to immunodeficient mice to establish a new generation of PDX model for subsequent medical or scientific research after being washed with physiological saline.

(56) As shown in FIGS. 1A-1B, the human primary cholangiocarcinoma tumor tissue is cryopreserved and resuscitated by the method as described in the disclosure or the conventional method, and then transplanted to immunodeficient mice for one month. It can be seen that the tumor of the experimental group using the method as described in the disclosure still exists, the progressive growth is distinct, so the PDX modeling is successful; while the tumor of the control group using the conventional method disappears, leaving only a slight scar tissue, which means PDX modeling failed. Results: The human primary cholangiocarcinoma tumor tissue, cryopreserved and resuscitated by the kit and the method as described in the disclosure, can be successfully transplanted to immunodeficient mice to establish an effective PDX mouse model. However, using the conventional method to cryopreserve and resuscitate tumor tissue, the PDX model could not be established in immunodeficient mice.

(57) As shown in FIG. 2, the anatomy of human primary cholangiocarcinoma tumor tissue, cryopreserved, resuscitated and transplanted to immunodeficient mice for one month, shows that the tumor tissue forms new vessels, and progressively proliferated and enlarged.

(58) Results: The human primary cholangiocarcinoma tumor tissue, cryopreserved and resuscitated by the kit and the method as described in the disclosure, can be transplanted to immunodeficient mice to establish a PDX mouse model successfully, and the tumor tissue is activated in the mouse.

(59) As shown in FIG. 3, 27 cases of human primary cholangiocarcinoma tumor tissue are cryopreserved and resuscitated by the kits as described in the disclosure, and then transplanted to immunodeficient mice. Compared with the fresh tissue, the formation rate of transplanted tumor of the two are both about 85%. Results: Compared with the fresh ones, the human primary cholangiocarcinoma tumor tissue, cryopreserved and resuscitated by the kit and the method as described in the disclosure, showed no significant difference in the success rate of transplanting tumor formation.

Example 7: Hematoxylin-Eosin Staining (H&E Staining) Experiment of Human Primary Cholangiocarcinoma Tumor Tissue after Cryopreservation and Resuscitation

(60) Experimental Method

(61) Tumor tissue is cryopreserved, resuscitated, fixed with 4% paraformaldehyde, embedded in paraffin, sliced using conventional paraffin, dewaxed (first xylene 3-5 min, second xylene 3-5 min, 100% ethanol 1-2 min, first 95% ethanol 1-2 min, second 95% ethanol 1-2 min, 80% ethanol 1 min, 70% ethanol 1 min), rinsed with tap water, rinsed with distilled water, dyed using hematoxylin for 3-10 min, rinsed with tap water, differentiated using 1% hydrochloric acid and alcohol for 3-5 sec, rinsed with tap water, saturated lithium carbonate solution for 1-2 min, tap water for 15 min, soaked in 0.5% eosin solution for 1-2 min, dehydrated (first 95% ethanol 1-2 min, second 95% ethanol 1-2 min, 100% ethanol 1-2 min), hyalinized (xylene-carbolic acid mixture 2 min, first xylene 1-2 min, second xylene 1-2 min, third xylene 1-2 min), and sealed using neutral balsam seal. Observe the resulting product under microscope, and take photos.

(62) Experimental Result

(63) FIG. 4A represents the H&E staining result of fresh tumor tissue, and FIG. 4B represents the H&E staining result of the cryopreserved-rewarmed tumor tissue, showing that the cryopreserved-rewarmed tumor tissue has a similar morphological structure to the fresh one, suggesting that the cryopreservation and resuscitation processes do not affect tumor characteristics significantly. The results indicate that the cryopreservation and resuscitation processes do not significantly affect tumor characteristics.

(64) It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.