CN107094752A - A kind of tumor tissues storage in vitro liquid and preparation method thereof - Google Patents

Abstract

The invention discloses a tumor tissue preservation solution in vitro and a preparation method thereof. The method comprises: adding antibiotics to a basal culture medium to obtain liquid A; dissolving taurine in a natural culture medium, and then adding polyethylene glycol octyl The phenyl ether is used to obtain liquid B; the liquid B is added to the liquid A and mixed uniformly to obtain the tumor tissue preservation liquid in vitro. The in vitro preservation solution prepared by the above method can maintain the biological activity of the tumor tissue when it is preserved in vitro, prolong the preservation time of the tumor tissue in vitro, and improve the success rate of tumor transplantation.

Description

A kind of tumor tissue preservation solution in vitro and its preparation method

technical field

The invention relates to the field of biotechnology, in particular to a tumor tissue preservation solution in vitro and a preparation method thereof.

Background technique

Human-derived tumor xenograft (Patient-Derived Xenograft, PDX) model is a model in which tumors from cancer patients are directly transplanted into mice to grow tumors. Because it is highly consistent with the real clinical practice and can accurately reflect the pathological characteristics and drug response of its source tumor patients, it has become the preferred model for the development of anticancer drugs by major pharmaceutical companies. The construction of PDX models requires fresh human tumor tissue samples. Therefore, in order to ensure the success rate of transplantation, during the tumor tissue transplantation experiment, it is necessary to use a preservation solution to maintain the biological activity of the tumor tissue during the transplantation process, so as to improve the success rate of transplantation. .

At present, common tumor tissue preservation solutions in vitro are phosphate buffer saline (Phosphate Buffer Saline, PBS) and cell culture medium. Among them, the main components of the PBS buffer include disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and potassium chloride, etc., which have a salt balance and adjustable pH buffering effect; the main components of the cell culture medium include basal culture Base, bovine serum, antibiotics, etc., can be used to maintain the growth and proliferation of cells.

The inventors of the present application found in the long-term research and development process that the PBS buffer solution is simple in composition, but its function is limited, and it cannot well maintain the biological activity of tumor tissue in vitro; the cell culture medium is mainly used to maintain the growth and proliferation of cells, not as a tumor The best choice for tissue preservation solution. When the above two are used as tumor tissue preservation solutions in vitro, the activity of retaining tumor cells is limited, and the longer the storage time in vitro, the worse the activity of tumor cells, resulting in a very low success rate of tumor tissue transplantation.

Contents of the invention

The technical problem mainly solved by the present invention is to provide a tumor tissue preservation solution in vitro and its preparation method, which can maintain the biological activity of the tumor tissue in vitro, prolong the storage time of the tumor tissue in vitro, and thereby improve the success rate of tumor transplantation.

In order to solve the above-mentioned technical problems, a technical solution adopted by the present invention is: provide a tumor tissue in vitro preservation solution, the preservation solution includes basal medium, antibiotics, natural medium, taurine and polyethylene glycol octylbenzene base ether.

In order to solve the above technical problems, another technical solution adopted by the present invention is to provide a method for preparing a tumor tissue in vitro preservation solution, the method comprising: adding antibiotics to the basal medium to obtain liquid A; dissolving in the natural medium taurine, and then add polyethylene glycol octylphenyl ether to obtain liquid B; add liquid B to liquid A and mix well to obtain a tumor tissue preservation solution in vitro.

The beneficial effects of the present invention are: different from the situation of the prior art, the tumor tissue in vitro preservation solution provided by the present invention includes basal medium, antibiotics, natural medium, taurine and polyethylene glycol octylphenyl ether. Among them, basal medium and antibiotics provide basic growth conditions for tumor tissue, while inhibiting the growth of fungi and bacteria, and can maintain the morphological structure and biological activity of tumor tissue; natural medium, taurine and polyethylene glycol octylbenzene The base ether provides abundant nutrients for the survival of tumor cells, and at the same time promotes the absorption of nutrients by tumor cells, and protects, promotes growth and regulates tumor tissues. The combined effect of the above two enables the tumor tissue to maintain its morphological structure and biological activity in the preservation solution, prolongs the storage time of the tumor tissue in vitro, and thus improves the success rate of tumor transplantation.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained based on these drawings without creative effort. in:

Fig. 1 is a schematic flow diagram of an embodiment of a method for preparing a tumor tissue in vitro preservation solution of the present invention;

Fig. 2 is a pathological diagram of the PDX original patient tumor established by using the tumor tissue in vitro preservation solution of the present invention and the tumor tissue after three passages.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The first embodiment of the tumor tissue in vitro preservation solution of the present invention includes basal medium, antibiotics, natural medium, taurine, and polyethylene glycol octylphenyl ether.

Basal medium usually refers to basic synthetic medium, the main components of which are amino acids, vitamins, carbohydrates, inorganic salts and auxiliary substances (such as nucleic acid degradation products, redox agents, etc.), providing a basic growth environment for tumor tissues in vitro. The basal medium in the embodiment of the present invention can specifically be 199 cell culture medium and its improved varieties, basic Eagle medium (Basal Medium Eagle, BME), minimal amount of Eagle medium (Minimal Essential Medium, MEM), Dulbecco's Modified Eagle Medium (DMEM) and its improved varieties, Iscove's Modified Eagle Medium (IMEM), Roswell Park Memorial Institute-1640 medium, (Roswell Park Memorial Institute, RPMI), At least one of DMEM/F12 cell culture medium, etc.

Antibiotics mainly play an antibacterial and bactericidal role, providing a sterile environment for tumor tissues in vitro, thereby preventing microbial infections such as bacteria and fungi, and ensuring the biological activity of tumor tissues.

The natural medium is rich in nutrients necessary for cell growth, and can provide nutrients that are absent or in a small amount in the basic medium, and the cell culture effect is good. The natural medium in the present invention can be at least one of biological liquid (such as serum), tissue infusion (such as embryo infusion) and coagulant (such as plasma).

Taurine is an essential amino acid for the human body. It participates in a series of physiological processes in the body. It has multiple functions such as regulating cell calcium homeostasis, scavenging oxygen free radicals and stabilizing cell membranes. It can protect, promote growth and regulate tumor tissue in vitro. effect.

Polyethylene glycol octylphenyl ether can increase the permeability of tumor cell membrane in vitro, which is beneficial to the absorption of nutrients by tumor cells.

In the above-mentioned tumor tissue preservation solution in vitro, the basal medium and antibiotics provide basic growth conditions for the tumor tissue, inhibit the growth of fungi and bacteria at the same time, and can maintain the morphological structure and biological activity of the tumor tissue; the natural medium, taurine and Polyethylene glycol octylphenyl ether provides rich nutrients for the survival of tumor cells, and at the same time promotes the absorption of nutrients by tumor cells, and plays a role in protecting, promoting growth and regulating tumor tissues. The combined effect of the above two enables the tumor tissue to maintain its morphological structure and biological activity in the preservation solution, prolongs the storage time of the tumor tissue in vitro, and thus improves the success rate of tumor transplantation.

The second embodiment of the tumor tissue in vitro preservation solution of the present invention includes DMEM cell culture medium, amphotericin B, penicillin-streptomycin, fetal bovine serum, taurine and polyethylene glycol octylphenyl ether. It should be noted that, for detailed descriptions of content related to this implementation manner and the above-mentioned first implementation manner, refer to the above-mentioned implementation manner, and details are not repeated here.

Among them, DMEM cell culture medium is a basic medium containing various amino acids and glucose, which is improved and developed on the basis of MEM medium, and the content of various components is increased. This cell culture medium is suitable for tumor cells that grow faster and are difficult to attach, and can provide a basic growth environment for tumor cells in vitro, especially the high-glucose cell culture medium.

Amphotericin B is a polyene antifungal antibiotic that inhibits fungal growth mainly by affecting cell membrane permeability.

Penicillin-streptomycin is an antibiotic that is often used in cell culture. Penicillin mainly inhibits Gram-positive bacteria, and streptomycin mainly inhibits Gram-negative bacteria. The complementary use of the two can inhibit most common bacteria, thereby providing In vitro tumor tissue provides a sterile environment.

Fetal bovine serum is the most used natural medium in cell culture. It is often used in the in vitro culture of animal cells. It is rich in nutrients necessary for cell growth and has extremely important functions. It can provide Nutrients.

Compared with the preservation solution in the first embodiment, the above storage solution uses DMEM cell culture medium as the base medium, amphotericin B and penicillin-streptomycin as antibiotics, and uses fetal calf serum as the natural medium at the same time, which can protect the tumor The in vitro preservation of tissues provides a better environment, so that the morphology and biological activity of tumor tissues can be more efficiently maintained, and at the same time, the in vitro preservation time of tumor tissues and the success rate of transplantation can be further extended.

Optionally, the volume percentage of DMEM cell culture medium in the preservation solution is 87.1% to 91.5%, specifically 88%, 89%, 90%, 91%, and the content of amphotericin B and penicillin-streptomycin Volume percentage content is 0.87%～0.91%, specifically can be 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, fetal bovine serum is 8.7%～9.1%, specifically can be 8.7%, 8.8%, 8.9% %, 9.0%, 9.1%. The content of polyethylene glycol octyl phenyl ether is 0.019%-0.021%, and the concentration of taurine in the preservation solution is 0.019-0.021g/ml.

Optionally, the concentration of amphotericin B is 10 mg/ml, the concentration of penicillin is 10 kU/ml, and the concentration of streptomycin is 10 mg/ml.

In one application scenario, the components of tumor tissue in vitro preservation solution are as follows: 500ml DMEM, 5ml amphotericin B, 5ml penicillin-streptomycin, 50ml fetal bovine serum, 0.1g taurine, 0.1ml polyethylene glycol octane phenyl ether. Wherein, the concentration of amphotericin B is 10 mg/ml, the concentration of penicillin is 10 kU/ml, and the concentration of streptomycin is 10 mg/ml. Of course, the content of each component in the preservation solution may also be other content within the range required in this embodiment, which is not limited here.

Please refer to Figure 1, Figure 1 is a schematic flow chart of an embodiment of the method for preparing a tumor tissue in vitro preservation solution of the present invention, the method includes:

Step S101: adding antibiotics to the basal medium to obtain liquid A;

Step S102: dissolving taurine in natural culture medium, and then adding polyethylene glycol octylphenyl ether to obtain liquid B;

Step S103: Add liquid B into liquid A and mix well to obtain a tumor tissue preservation solution in vitro.

It should be noted that the various components used in the preparation of the tumor tissue in vitro preservation solution in this embodiment are the same as those in the first embodiment and the second embodiment of the tumor tissue in vitro preservation solution of the present invention. Refer to the foregoing implementation manners, and details are not repeated here.

In one application scenario, the preparation method of tumor tissue in vitro preservation solution is as follows: add 5ml amphotericin B to 500ml DMEM cell culture medium to obtain solution A; dissolve 0.1g taurine in 50ml fetal bovine serum, and then add 0.1 ml polyethylene glycol octylphenyl ether to obtain solution B, and then add solution B to solution A and mix well to obtain a tumor tissue preservation solution in vitro.

The tumor tissue in vitro preservation solution prepared by the above method contains DMEM cell culture medium, antibiotics, fetal bovine serum, taurine and polyethylene glycol octylphenyl ether. Among them, in the above-mentioned in vitro preservation solution of tumor tissue, the DMEM cell culture medium, amphotericin B, and penicillin-streptomycin in liquid A provide basic growth conditions for tumor tissue, and can inhibit the growth of fungi and basically all common bacteria , to maintain the morphological structure and biological activity of tumor tissue; fetal bovine serum, taurine and polyethylene glycol octylphenyl ether provide rich nutrients for the survival of tumor cells, and at the same time promote the absorption of nutrients by tumor cells , to protect, promote growth and regulate tumor tissue. In turn, the tumor tissue can maintain its morphological structure and biological activity in the preservation solution, prolong the storage time of the tumor tissue in vitro, and improve the success rate of tumor transplantation.

The present invention will be described in detail below by way of examples.

The tumor tissue in vitro preservation solutions 1, 2, and 3 of the present invention used in the following examples are all prepared from the first example of the preparation method of the tumor tissue in vitro preservation solution of the present invention. The formulas of the three preservation solutions for tumor tissue in vitro preservation are as follows shown in the table.

Table 1 3 kinds of tumor tissue in vitro preservation solution formulations of the present invention

Example 1

Divide the same volume of PBS buffer solution, cell culture solution, and tumor tissue in vitro preservation solutions 1, 2, and 3 of the present invention into 5 groups as preservation solutions for tumor tissue preservation. Wherein, the preserved tumor tissues are equal in weight. The above five groups of tumor tissue transplantation experiments were completed within 2 hours, and each group was subcutaneously transplanted into five 6-week-old Non Obese Diabetes-Server Combined Immune-deficiency (NOD-SCID) mice. One month later, the tumor formation status of the tumor tissue was observed, and the success rate of colon cancer transplantation in the three groups was compared, as shown in Table 2. Compared with the other two groups, P<0.05.

It should be pointed out that, except for the differences indicated above, other conditions are the same.

Table 2 The success rate of colon cancer transplantation using each preservation solution

PBS buffer cell culture medium Preservation solution 1 Preservation solution 2 Preservation solution 3 Total number of transplants 5 5 5 5 5 total number of tumors 1 2 4 5 5 Transplant success rate 20% 40% 80% 100% 100%

It can be seen from Table 2 that the transplantation of the tumor is completed within 2 hours, that is to say, the storage time of the tumor in the preservation solution is within 2 hours. The result at this time is that the success rate of transplantation using PBS buffer solution is 20%, the success rate of transplantation using cell culture medium is slightly higher, which is 40%, and the success rate of transplantation using tumor tissue in vitro preservation solution 1, 2, and 3 of the present invention is 20%. Respectively up to 80%, 100%, 100%. Therefore, the success rate of the three tumor tissue preservation solutions in vitro with different components in the present invention is significantly higher than that of the first two preservation solutions.

Example 2

Divide the same volume of PBS buffer solution, cell culture solution, and tumor tissue in vitro preservation solutions 1, 2, and 3 of the present invention into 5 groups as preservation solutions for tumor tissue preservation. Wherein, the preserved tumor tissues are equal in weight. The above five groups of tumor tissue transplantation experiments were completed within 6 hours, and each group was subcutaneously transplanted into five 6-week-old NOD-SCID mice, and the tumor formation status of the tumor tissue was observed after one month, and the success rate of colon cancer transplantation in the three groups was compared. For comparison, as shown in Table 3. Compared with the other two groups, P<0.05.

It should be pointed out that, except for the differences indicated above, other conditions are the same.

Table 3 The success rate of colon cancer transplantation using each preservation solution

PBS buffer cell culture medium Preservation solution 1 Preservation solution 2 Preservation solution 3 Total number of transplants 5 5 5 5 5 total number of tumors 0 1 3 3 3 Transplant success rate 0% 20% 60% 60% 60%

It can be known from Table 3 that the tumor transplantation time is completed within 6 hours, that is to say, the storage time of the tumor in the preservation solution is within 6 hours. The result at this time is that the success rate of transplantation using PBS buffer solution is 0%, the success rate of transplantation using cell culture medium is slightly higher, which is 20%, and the success rate of transplantation using tumor tissue in vitro preservation solution 1, 2, and 3 of the present invention is 0%. Both are as high as 60%, which is also significantly higher than the success rate of the first two preservation solutions.

It can be clearly seen from Example 1 and Example 2 that the in vitro preservation solution for tumor tissue prepared by the present invention can significantly prolong the in vitro preservation time of tumor tissue, and significantly improve the success rate of in vitro tumor transplantation, even if the transplantation time is extended to 6 hours, The transplant success rate is still as high as 60%.

Example 3

Please refer to Fig. 2, Fig. 2 is the pathological picture of the PDX original patient tumor established by using the tumor tissue in vitro preservation solution 1, 2, 3 of the present invention and the tumor tissue after three passages (the original picture is in color). Among them, Figure a is the pathological map of the tumor of the original patient, and Figures b, c, and d are the pathological pictures of the tumors of the third-generation NOD-SCID mice transplanted with preservation solutions 1, 2, and 3, respectively.

It can be seen from Fig. 2 that the pathological characteristics of the tumors of the mice transplanted with tumor tissue in vitro preservation solutions 1, 2, and 3 of the present invention after three passages are still consistent with those of the original patient's tumor. This shows that the tumor tissue in vitro preservation solutions 1, 2, and 3 prepared by the present invention did not cause damage to tumor tissue, or the destructive effect was very weak, and had a good effect on maintaining the original morphological structure and biological activity of tumor tissue in vitro.

The above is only the embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process conversion made by using the description of the present invention and the contents of the accompanying drawings, or directly or indirectly used in other related technologies fields, all of which are equally included in the scope of patent protection of the present invention.

Claims (10)

1. A tumor tissue preservation solution in vitro, characterized in that, The preservation solution includes basal medium, antibiotics, natural medium, taurine and polyethylene glycol octylphenyl ether. 2. preservation solution according to claim 1, is characterized in that, The basic medium is Duchenne's DMEM cell culture medium, and the volume percentage of the DMEM cell culture medium in the preservation solution is 87.1%-91.5%. 3. preservation solution according to claim 1, is characterized in that, The antibiotics are amphotericin B and penicillin-streptomycin. 4. preservation solution according to claim 3, is characterized in that, The volume percentages of the amphotericin B and the penicillin-streptomycin in the preservation solution are both 0.87%-0.91%. 5. preservation solution according to claim 3, is characterized in that, The concentration of the amphotericin B is 10 mg/ml. 6. preservation solution according to claim 3, is characterized in that, The concentration of the penicillin is 10 kU/ml, and the concentration of the streptomycin is 10 mg/ml. 7. preservation solution according to claim 1, is characterized in that, The natural culture medium is fetal bovine serum, and the volume percentage of the fetal bovine serum in the preservation solution is 8.7%-9.1%. 8. preservation solution according to claim 1, is characterized in that, The concentration of the taurine in the preservation solution is 0.019-0.021 g/ml. 9. preservation solution according to claim 1, is characterized in that, The volume percentage of the polyethylene glycol octylphenyl ether in the preservation solution is 0.019%-0.021%. 10. A method for preparing a tumor tissue in vitro preservation solution, characterized in that the method comprises: Add antibiotics to the basal medium to obtain liquid A; Dissolving taurine in natural medium, then adding polyethylene glycol octylphenyl ether to obtain liquid B; Add the B solution to the A solution and mix well to obtain the tumor tissue preservation solution in vitro.