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Tumor tissue preservation method and preservation reagent

A technology of tumor tissue and preservation method, applied in the field of tumor tissue preservation method and preservation reagent, which can solve the problems of short shelf life, easy loss of tumor genetic characteristics, poor stability of cryopreservation solution, etc., and achieve the effect of high tumor formation rate

Inactive Publication Date: 2017-08-15
北京奥康华医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the way of preservation of living tumor tissue is generally direct cryopreservation, and the required cryoprotectant is mostly composed of DMSO, serum and cell culture medium, which needs to be prepared immediately after use, and the stability of this tissue cryopreservation solution is poor, making its The shelf life is short and the storage conditions are relatively harsh
[0004] The existing cryopreservation method cannot guarantee the structural integrity of tumor tissue after freezing and thawing, and it is easy to lose the genetic characteristics of the tumor itself, which reduces the clinical research value of tissue samples

Method used

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  • Tumor tissue preservation method and preservation reagent

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1, preservation of human tumor tissue

[0039] 1. Tumor tissue preservation reagents

[0040] Tumor tissue enzymatic hydrolysis reagent: composed of hyaluronidase, DNase I and DMEM culture solution containing 10% fetal bovine serum by volume. Wherein, the concentration of the hyaluronidase in the tumor tissue enzymatic hydrolysis reagent is 120 mg / mL; the DNase I concentration in the tumor tissue enzymatic hydrolysis reagent is 100 mg / mL. It is equivalent to containing 48-120 U of the hyaluronidase per liter of the tumor tissue enzymatic hydrolysis reagent, and containing more than 40 Kunitz U of the DNase I.

[0041] Cryopreservation solution: composed of DMSO and sucrose; and in the cryopreservation solution, the concentrations of DMSO and sucrose are 1 mol / L and 0.1 mol / L respectively.

[0042] 2. Tumor tissue preservation method of the present invention

[0043] The patient's tumor tissue samples were obtained from a 56-year-old male patient with colon...

Embodiment 2

[0052] Example 2, establishment of in vivo tumor tissue bank

[0053] Mice to be tested: SCID mice (Severe combined immunodeficient mice), regardless of sex, aged 4 to 5 weeks.

[0054] 1. The tumor tissue specimens in cryopreservation in Example 1 were resuscitated in a 37° C. water bath, and then transferred to an ice-bathed DMEM medium (containing 10% fetal bovine serum by volume).

[0055] 2. Transplant the tumor tissue resuscitated in step 1 under the anterior axillary capsule of the mouse. Specifically: after the animal is anesthetized with isoflurane, disinfect the skin on the back and abdomen of the mouse, wipe it dry with a sterile cotton ball, cut a small incision near the anterior armpit on one side of the mouse with a blade, and bluntly separate the skin from the subcutaneous tissue with an 18G puncture needle. , make it into a leather bag, place the prepared tumor tissue block in it, and add a drop of double antibody (100×penicillin / streptomycin) to the incision....

Embodiment 3

[0057] Embodiment 3, the impact of different cryopreservation time on the tumor formation rate of transplanted tumor model of colon cancer

[0058] The tumor tissues of Example 1 were cryopreserved for 4-52 weeks (4, 12, 24, 48, 52 weeks) to establish a xenograft model according to the method of Example 2 (at least 5 mice were set for each cryopreservation time) ), the effects of different freezing periods on the tumor formation rate of colon cancer xenograft models were counted 4 weeks after transplantation into the tumor tissue.

[0059] At the same time, the fresh tumor tissue in Example 1 without freezing was set as a control.

[0060] The result is as figure 1 shown. It can be seen from the figure: after adopting the cryopreservation method of the present invention to resuscitate the tumor tissue, transplant it to the immunodeficiency mouse, and compare it with the mouse transplanted with fresh tumor tissue within 24 weeks after transplantation, the transplantation succ...

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Abstract

The invention discloses a tumor tissue preservation method and a preservation reagent. The method comprises the following steps: 1) placing the to-be-preserved tumor tissue in a tumor tissue enzymatic hydrolysis reagent, culturing the to-be-preserved tumor tissue for 10-20 min at the temperature of 20-25 DEG C to obtain the tumor tissue at the temperature of 20-25 DEG C; wherein the tumor tissue enzymatic hydrolysis reagent contains hyaluronidase and DNA enzyme I; each liter of the tumor tissue enzymatic hydrolysis reagent contains 40-150 U hyaluronidase and contains DNA enzyme I with content higher than 40 Kunitz U; 2) placing the tumor tissue after enzymatic hydrolysis in refrigeration preservation liquid, pre-balancing the material for 10-20 min at the temperature of 4 DEG C to obtain the pre-balanced tumor tissue; and 3) performing programmed refrigeration on the pre-balanced tumor tissue, after the temperature is reduced to -150 DEG C, and transferring the material into a liquid nitrogen tank for storage. The tumor tissue preservation method has the characteristics of high tumor formation rate, and after transplantation, the tumor tissue can completely preserve the form and molecule characteristics of the patient-sourced tissue.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a preservation method and a preservation reagent of tumor tissue. Background technique [0002] With the increasing impact and harmfulness of tumors on humans, how to protect, preserve and make full use of human tumor genetic resources and improve the level of cancer prevention and treatment in my country has become a top priority. Live tumor tissue specimens as tumor research materials play an important role in promoting the progress of tumor research, and have important scientific research and clinical application values, and live tumor tissue preservation technology becomes the basis for realizing these values. [0003] At present, the way of preservation of living tumor tissue is generally direct cryopreservation, and the required cryoprotectant is mostly composed of DMSO, serum and cell culture medium, which needs to be prepared immediately after use, and the stability of this tissu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 卢戌梁孟儒王燕飞刘雪松刘静维王跃黄彩庭李京坡吴璇
Owner 北京奥康华医学检验所有限公司
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