CN113142189A

CN113142189A - Kit for freezing and recovering tumor tissues and/or cells and treatment method thereof

Info

Publication number: CN113142189A
Application number: CN202110398739.2A
Authority: CN
Inventors: 孙晓娇; 刘星; 张红霞; 陆重益; 张文平
Original assignee: Jiangsu Antaikang Health Technology Co ltd
Current assignee: Jiangsu Antaikang Health Technology Co ltd
Priority date: 2020-04-12
Filing date: 2021-04-12
Publication date: 2021-07-23
Anticipated expiration: 2041-04-12
Also published as: CN111466369A; CN113142189B

Abstract

The invention discloses a tumor tissue/or cell cryopreservation and resuscitation kit and a treatment method thereof, and the tumor tissue/cell cryopreservation and resuscitation kit comprises a cryopreservation kit and a resuscitation kit, wherein the cryopreservation kit comprises a vitrification liquid, and the resuscitation kit comprises a resuscitation liquid, and the vitrification liquid and the resuscitation liquid contain completely clear, stable and controllable components, can be directly applied to cryopreservation and resuscitation of active tumor tissues/cells, and do not need to be diluted or prepared by self, so that the operation is simplified. The biological activity and morphological characteristics of the tumor tissues and/or cells are not obviously changed before and after vitrification cryopreservation by using the invention, which shows that the vitrification cryopreservation and resuscitation solution system better maintains the microenvironment and tumor heterogeneity characteristics of the tumor tissues and/or cells, and the cryopreserved tumor tissues and/or cells are transplanted in situ after resuscitation, so that the tumor formation rate is high, and the active tumor tissues and/or cells can be effectively cryopreserved.

Description

Kit for freezing and recovering tumor tissues and/or cells and treatment method thereof

Technical Field

The invention relates to the technical field of treatment of tumor tissues and/or cells, in particular to a kit for freezing and resuscitating a gravity tissue and a treatment method thereof.

Background

Vitrification cryopreservation is a novel low-temperature preservation technology, and has potential application value in the fields of scientific research basic research and clinic application of clinical live tumor tissues and/or cell samples. Conventional methods for preserving active tumor tissues and/or cells, such as slow programmed freezing with propylene glycol, direct liquid nitrogen injection (ultra-fast freezing) and dmso vitrification, can completely or partially inactivate the active tumor tissues and/or cells, resulting in a large amount of valuable clinically active tumor tissues and/or cells being wasted due to the inability to achieve long-term effective high-activity preservation.

The vitrification solution is developed by Rau and Fahy as early as 1985, and is mainly applied to cryopreservation of oocytes, spermatids and various stem cells. The method comprises the steps of fully reacting the high-concentration frozen stock solution, cooling in a rapid protective agent to convert the frozen stock solution into a glass-like semitransparent state, and storing at low temperature. In the process, no ice crystal is formed inside and outside the cell, so that the structural damage of the cell is avoided, and the tissue activity can be highly preserved.

Disclosure of Invention

The invention aims to provide a kit for freezing and resuscitating live tumor tissues and/or cells and a treatment method thereof.

In order to realize one of the above objects, the present invention provides a kit for cryopreservation and resuscitation of tumor tissue and/or cells, which is suitable for cryopreservation and resuscitation of primary breast cancer tumor tissue and/or cells of a human body, the kit for cryopreservation and resuscitation of tumor tissue and/or cells comprises a tumor tissue and/or cell cryopreservation kit and a tumor tissue and/or cell resuscitation kit,

the tumor tissue/cell cryopreservation kit comprises:

the glass transition liquid 1 comprises the following components: 65-95V/V% of Gibco DMEM medium, 5.5-20V/V% of dimethyl sulfoxide, 3.5-15V/V% of ethylene glycol, 0.5-4W/V% of bovine serum albumin, 1-5W/V% of cane sugar, 0.05-0.8W/V% of methyl cellulose (4000CP), 0.25-0.6W/V% of hydroxyethyl starch and 15-35W/V% of glucose; and

the glass liquid 2 comprises the following components: 65-95V/V% of Gibco DMEM medium, 5.5-20V/V% of dimethyl sulfoxide, 8-20V/V% of ethylene glycol, 0.5-4W/V% of bovine serum albumin, 10-20W/V% of cane sugar, 0.05-0.8W/V% of methyl cellulose (4000CP), 0.25-0.6W/V% of polyvinylpyrrolidone and 15-35W/V% of glucose;

the tumor tissue/cell resuscitation kit comprises:

the resuscitation fluid 1 comprises the following components: earl's balanced salt solution 65-85V/V%, 1x phosphate buffer solution 15-35V/V%, bovine serum albumin 1-3w/V%, sucrose 10-40w/V% and glucose 15-35 w/V%;

the resuscitation fluid 2 comprises the following components: earl's balanced salt solution 65-85V/V%, 1x phosphate buffer solution 15-35V/V%, bovine serum albumin 1-3w/V%, sucrose 10-20w/V% and glucose 15-35 w/V%; and

the resuscitation fluid 3 comprises the following components: earl's balanced salt solution 75-95V/V%, 1x phosphate buffer 5-25V/V%, bovine serum albumin 1-3w/V% and glucose 15-35 w/V%.

As an optional technical scheme, the components of the glass transition liquid 1 comprise: 80V/V% of Gibco DMEM medium, 10V/V% of dimethyl sulfoxide, 10V/V% of ethylene glycol, 2W/V% of bovine serum albumin, 1W/V% of sucrose, 0.05W/V% of methyl cellulose (4000CP), 0.25W/V% of hydroxyethyl starch and 25W/V% of glucose; and

the components of the glass transition liquid 2 comprise: DMEM medium 70V/V%, dimethyl sulfoxide 15V/V%, ethylene glycol 12V/V%, bovine serum albumin 3W/V%, sucrose 20W/V%, methyl cellulose (4000CP) 0.1W/V%, polyvinylpyrrolidone 0.25W/V% and glucose 30W/V%.

As an optional technical solution, the tumor tissue/cell section mould is further included, and the tumor tissue/cell section mould includes: the base, the base includes the upper surface, the upper surface be equipped with the depressed part and certainly the upper surface is perpendicular downwards, along transversely being equipped with many equidistance distributions, the degree of depth is less than the cutter guide way of depressed part minimum, wherein, the both sides of base still are equipped with the handle.

As an optional technical scheme, the concave part is an ellipsoid, the width of the ellipsoid is 30mm, and the length of the ellipsoid is 35 mm; the vertical distance from the lowest point of the concave part to the upper surface of the base is 25 mm.

As an optional technical scheme, the depth of the cutter guide groove is 5mm lower than the lowest point of the concave part; the distance between two adjacent cutter guide grooves is 1 mm; the number of the cutter guide grooves is 23.

As an optional technical solution, the resuscitation solution 1 comprises the following components: earl's balanced salt solution 65V/V%, 1x phosphate buffer 35V/V%, bovine serum albumin 1w/V%, sucrose 30w/V% and glucose 25 w/V%;

the components of the resuscitation fluid 2 comprise: earl's balanced salt solution 75V/V%, 1x phosphate buffer 25V/V%, bovine serum albumin 1w/V%, sucrose 15w/V% and glucose 25 w/V%; and

the components of the resuscitation fluid 3 comprise: earl's balanced salt solution 95V/V%, 1x phosphate buffer 5V/V%, bovine serum albumin 1w/V%, sucrose 10w/V% and glucose 15 w/V%.

The invention also provides a processing method for cryopreserving the tumor tissues and/or the cells, which is suitable for cryopreserving the primary breast cancer tumor tissues and/or the cells of a human body, the tumor tissues and/or the cells are cryopreserved by utilizing the tumor tissue and/or cell cryopreserving kit, and the processing method for cryopreserving the tumor tissues and/or the cells comprises the following steps:

s1, washing the tumor tissue/cell sample for 2 times by 1x phosphate buffer solution, and removing envelope, blood vessel and mucous necrotic tissue;

s2, slicing the tumor tissues and/or cells by using the tumor cutting die, and washing again by using 1x phosphate buffer solution;

s3, firstly immersing the sliced tumor tissues/cells into 5ml of the vitrification solution 1 for 10 min; then soaking the glass fiber in 5ml of the vitrification solution 2 for 10 min;

s4, taking out the tumor tissue and/or cells frozen by the vitrification in the S3, draining, rapidly transferring the tumor tissue and/or cells into a freezing tube, and storing in a sterile liquid nitrogen tank.

As an optional technical solution, in S1, the tumor tissue/cell sample is subjected to a blood vessel treatment process, a membrane covering process and a necrotic tissue treatment process, and the treated tumor tissue/cell sample needs to be completely immersed in a 1 × phosphate buffer solution, and the operation is gentle, so as to avoid damage to the tumor tissue/cell sample.

Alternatively, in S2, the tumor tissue/cells are sliced, and the slice of the tumor tissue/cells is 1 × 1 × 1 mm.

10. The method of claim 8, wherein the frozen tumor tissue and/or cells are processed by:

in the S4, the tumor tissues and/or cells after the S3 vitrification cryopreservation are drained and rapidly moved into a cryopreservation tube to be preserved in a liquid nitrogen tank.

The invention also provides a treatment method for recovering tumor tissues and/or cells, which is suitable for recovering primary breast cancer tumor tissues and/or cells of a human body, and is characterized in that the tumor tissues and/or cells are recovered by utilizing the tumor tissue and/or cell recovery kit, and the treatment method for recovering the tumor tissues and/or cells comprises the following steps:

s11, rapidly immersing the tumor tissue/cells taken out from the liquid nitrogen into 10-20ml of resuscitation solution 1 heated in water bath at 36-38 ℃ for 5-10 min;

s22, taking out the tumor tissues and/or cells treated by the resuscitation solution 1 and immersing the tumor tissues and/or cells in 5-10ml of resuscitation solution 2 for 5-10 min;

s33, taking out the tumor tissues and/or cells treated by the resuscitation solution 2 and immersing the tumor tissues and/or cells in 5-10ml of resuscitation solution 3 for 5-10 min;

s44, washing the tumor tissue/cells treated by the resuscitation solution 3 for 2 times by using 1x phosphate buffer solution, and immersing the tumor tissue/cells in sterile normal saline to obtain the tumor tissue/cells after resuscitation.

As an alternative technical scheme, in the S11, the tumor tissues and/or cells taken out of the liquid nitrogen are quickly immersed in 10ml of resuscitation solution 1 preheated by a water bath at 37 ℃ for 5 min.

As an optional technical scheme, in S22, the tumor tissue/cells treated by the resuscitation solution 1 is transferred into 5ml of room temperature resuscitation solution 2, and the immersion time is 5 min;

in the step S33, the tumor tissues and/or cells treated by the resuscitation solution 2 are immersed in 10ml of room-temperature resuscitation solution 3 for 10 min.

The invention also provides a processing method for freezing and recovering tumor tissues and/or cells, which is suitable for freezing and recovering primary breast cancer tumor tissues and/or cells of a human body, and the processing method for freezing and recovering the tumor tissues and/or cells comprises the following steps:

cryopreservation treatment, the cryopreservation treatment comprising:

s3, firstly immersing the sliced tumor tissues/cells into 5ml of the vitrification solution 1 for 10 min; then soaking the glass fiber in 5ml of the vitrification solution 2 for 10 min; and

s4, taking out the tumor tissues and/or cells subjected to vitrification cryopreservation in the S3, draining, quickly transferring the tumor tissues and/or cells into a cryopreservation tube, and storing in a sterile liquid nitrogen tank;

a resuscitation treatment comprising:

s33, taking out the tumor tissues and/or cells treated by the resuscitation solution 2 and immersing the tumor tissues and/or cells in 5-10ml of resuscitation solution 3 for 5-10 min; and

Compared with the prior art, the cryopreservation and resuscitation kit comprises the tissue vitrification solution and the resuscitation solution, and each component in a solution system contained in the kit is clear and stable. The tumor tissue/cell frozen and recovered by the freezing and recovering kit disclosed by the invention can maintain the heterogeneity of the tumor tissue/cell and the characteristics of the tumor microenvironment while ensuring the self activity of the tumor tissue/cell. And the tumor tissue/or cytological characteristics and biological characteristics are not changed on the establishment of a subcutaneous tumor implantation PDX model and a tumor in-situ transplantation tumor PDOX model. In addition, the tumor tissue/cell vitrification cryopreservation and resuscitation kit has simple and clear treatment method, and related instruments and equipment are commonly used, so that the operation, popularization and application are facilitated.

Drawings

FIG. 1 is a schematic diagram of PDX and PDOX modeling conditions of primary breast cancer tumor tissues and/or cells of a human body after being frozen and thawed and inoculated to an immunodeficiency mouse for one month.

FIG. 2 is a schematic diagram of morphological features of HE staining of PDX and PDOX mouse model tumor tissues and/or cells after one month of inoculation of immunodeficient mice after freezing and reviving of human primary breast cancer tumor tissues and/or cells.

FIG. 3A is a perspective view of a tumor tissue/cell section mold; fig. 3B is a top view of the tumor tissue/cell block module.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments shown in the drawings. These embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to these embodiments are included in the scope of the present invention.

The invention provides a kit suitable for freezing and resuscitating tumor tissues and/or cells, which comprises a freezing kit and a resuscitating kit, wherein the freezing kit is used for freezing and cryopreserving the tumor tissues and/or cells, and the resuscitating kit is used for resuscitating the tumor tissues and/or cells frozen and reserved by the freezing kit.

The invention provides a tumor tissue/cell cryopreservation kit, which comprises a vitrification liquid 1 and a vitrification liquid 2, wherein the vitrification liquid 1 comprises the following components: 65-95V/V% of Gibco DMEM medium, 5.5-20V/V% of dimethyl sulfoxide, 3.5-15V/V% of ethylene glycol, 0.5-4W/V% of bovine serum albumin, 1-5W/V% of cane sugar, 0.05-0.8W/V% of methyl cellulose (4000CP), 0.25-0.6W/V% of hydroxyethyl starch and 15-35W/V% of glucose; and a vitrification liquid 2, which comprises the following components: 65-95V/V% of Gibco DMEM medium, 5.5-20V/V% of dimethyl sulfoxide, 8-20V/V% of ethylene glycol, 0.5-4W/V% of bovine serum albumin, 10-20W/V% of cane sugar, 0.05-0.8W/V% of methyl cellulose (4000CP), 0.25-0.6W/V% of polyvinylpyrrolidone and 15-35W/V% of glucose.

The invention also provides a tumor tissue/cell resuscitation kit, which comprises: the resuscitation liquid comprises resuscitation liquid 1, resuscitation liquid 2 and resuscitation liquid 3, wherein the resuscitation liquid 1 comprises the following components: earl's balanced salt solution 65-85V/V%, 1x phosphate buffer solution 15-35V/V%, bovine serum albumin 1-3w/V%, sucrose 10-40w/V% and glucose 15-35 w/V%; the resuscitation fluid 2 comprises the following components: earl's balanced salt solution 65-85V/V%, 1x phosphate buffer solution 15-35V/V%, bovine serum albumin 1-3w/V%, sucrose 10-20w/V% and glucose 15-35 w/V%; and a resuscitation fluid 3, which comprises the following components: earl's balanced salt solution 75-95V/V%, 1x phosphate buffer 5-25V/V%, bovine serum albumin 1-3w/V% and glucose 15-35 w/V%.

Experiments prove that by adopting the kit for freezing and recovering the tumor tissues and/or cells provided by the invention, more than 99% of the tumor tissues and/or cells after freezing and recovering can be successfully recovered. The recovery liquid 1 and the recovery liquid 2 of the tumor tissue/cell recovery kit adopt Earl's balanced salt solution 65-85V/V%, and the recovery liquid 3 adopts Earl's balanced salt solution 75-95V/V%, the osmotic pressure range of the Earl's balanced salt solution is 273-297(mOSM), which is close to the osmotic pressure of the tumor tissue/cell to be recovered 280mmol/L, so that the recovery liquid is more in line with the optimal osmotic pressure for the survival of the tumor tissue/cell, and particularly has better effect on the recovery of the tumor microenvironment stem cells, immune cells or fibroblasts in the tumor tissue/cell. Namely, the frozen and recovered tumor tissues and/or cells are frozen and recovered by using the frozen and recovered tumor tissue and/or cells kit, the tumor tissue and/or cells heterogeneity and the tumor microenvironment characteristic can be maintained while the tumor tissue and/or cells self-activity is ensured, the tumor tissues and/or cells after subsequent recovery are transplanted into an immunodeficient mouse body, a PDX and PDOX mouse model can be successfully established, and the tumor tissues and/or cells are successfully activated in the mouse body.

The respective preparation processes of the above-mentioned tumor tissue/cell cryopreservation kit and tumor tissue/cell recovery kit, and the treatment method of tumor tissue/cell will be described below by specific examples.

EXAMPLE 1 preparation of tumor tissue/cell cryopreservation kit

Preparing a vitrification solution required by cryopreservation of tumor tissues/cells, wherein the ingredients of the specific formula are as follows:

the vitrification liquid 1 includes: 80V/V% of Gibco DMEM medium, 10V/V% of dimethyl sulfoxide (DMSO), 10V/V% of Ethylene Glycol (EG), 2W/V% of Bovine Serum Albumin (BSA), 1W/V% of sucrose, 0.05W/V% of methyl cellulose (4000CP), 0.25W/V% of hydroxyethyl starch and 25W/V% of glucose.

The vitrification liquid 2 includes: DMEM medium 70V/V%, dimethyl sulfoxide (DMSO) 15V/V%, Ethylene Glycol (EG) 12V/V%, Bovine Serum Albumin (BSA) 3W/V%, sucrose 20W/V%, methylcellulose (4000CP) 0.1W/V%, polyvinylpyrrolidone (PVP) 0.25W/V% and glucose 30W/V%.

Wherein, the Chinese and English names of each reagent and the purchasing manufacturer are listed in Table 1:

TABLE 1

EXAMPLE 2 cryopreservation of tumor tissue and/or cells

Filtering, subpackaging and freezing the prepared frozen reagent in a refrigerator at the temperature of-4 ℃ for storage; if the prepared frozen reagent needs to be stored for a long time, the frozen reagent needs to be frozen in a refrigerator at the temperature of minus 20 ℃.

The tumor tissue/cell is subjected to cryopreservation treatment, and the treatment method of the cryopreservation treatment comprises the following steps:

step S1, washing the tumor tissue/cell sample for 2 times by 1x phosphate buffer solution (English abbreviation: 1xPBS), and removing envelope, blood vessel and mucous necrotic tissue; wherein, the action is gentle in the operation process, and the damage of tumor tissues and/or cell masses is avoided. In addition, the tumor tissue and/or cell sample is treated by blood vessel, envelope and necrotic tissue, and the treated tumor tissue and/or cell sample is completely immersed in 1x phosphate buffer.

And step S2, slicing the tumor tissues and/or cells by using the tumor section mould, and washing the tumor tissues and/or cells again by using a 1x phosphate buffer solution. Wherein, after the tumor section mould section processing, tissue blocks with the size of 1X 1mm are obtained and washed again by 1X phosphate buffer solution.

The tumor tissue/cell slicing mold in step S2 is shown in fig. 3A and 3B, and the tumor tissue/cell processing mold includes a base 1, the base 1 includes an upper surface 2, the upper surface 2 is provided with a recess 3 and a cutter guide groove 4, the cutter guide groove 4 is vertically downward from the upper surface 2, and a plurality of minimum points with a depth lower than the recess 3 are transversely disposed at equal intervals.

In this embodiment, the cutter guide groove 4 in the base 1 is a 304 stainless steel cutter guide groove; the handles 5 on the two sides of the base 1 are ABS plastic injection molding handles which are embedded on the two sides of the base 1.

In a preferred embodiment, the recess 3 is an ellipsoid, the width of which is 30mm and the length of which is 35 mm; wherein, the vertical distance from the lowest point of the concave part 3 to the upper surface 2 of the base 1 is 25 mm.

In a preferred embodiment, the depth of the cutter guide groove 4 is less than 5mm below the lowest point of the recess 3, and the distance between any two adjacent cutter guide grooves is about 1 mm. Preferably, the number of the cutter guide grooves 4 is 23, but not limited thereto.

Step S3, firstly immersing the sliced tumor tissues/cells into 5ml of the vitrification solution 1 for 10 min; and then immersed in 5ml of the vitrification solution 2 for 10 min.

Wherein, 5ml of each of the vitrification solution 1 and the vitrification solution 2 prepared according to the content of each component in the above example 1 are respectively taken, and the tumor tissue/cell slice is respectively and sequentially immersed in 5ml of the vitrification solution 1 for 10 min; then the glass is immersed for 10min in 5ml of vitrification solution 2.

And S4, taking out the tumor tissues and/or cells subjected to vitrification cryopreservation in S3, draining, quickly transferring the tumor tissues and/or cells into a cryopreservation tube, and storing in a sterile liquid nitrogen tank.

In other embodiments of the present invention, the tumor tissue and/or cells may be rapidly transferred into the cryovial and stored in a liquid nitrogen tank in step S4.

EXAMPLE 3 preparation of tumor tissue/cell Resuscitation kit

The resuscitation solution required by the resuscitation of tumor tissues and/or cells is prepared, and the specific formula components are as follows:

the components of the resuscitation fluid 1 comprise: earl's balanced salt solution 65V/V%, 1x phosphate buffer 35V/V%, bovine serum albumin 1w/V%, sucrose 30w/V% and glucose 25 w/V%;

The volume concentration gradient of Earl's balanced salt solution used in the resuscitation solution 1, the resuscitation solution 2 and the resuscitation solution 3 is increased, and in the process of tumor tissue/cell resuscitation, the osmotic pressure of the final resuscitation solution 3 is isotonic with that of living tumor tissue/cells, so that the resuscitation of the tumor tissue/cells is facilitated, particularly, the osmotic pressure of the final resuscitation solution maintains the osmotic pressure of tumor microenvironment stem cells, immune cells or fibroblasts in the tumor tissue/cells, and the resuscitation success rate is remarkably improved.

Wherein, the Chinese and English names of each reagent and the 25w purchasing manufacturer are listed in Table 2:

TABLE 2

Serial number	Name of English	Name of Chinese	Manufacturer(s)
				1	Earl's balanced salt solution	Erer balanced salt solution	Gibco
2	1xPBS	1x phosphate buffer	Invitrogen
				3	BSA	Bovine serum albumin	Invitrogen
4	-	Sucrose	Shanghai worker
				5	-	Glucose	Shanghai worker

EXAMPLE 4 Resuscitation treatment of tumor tissues and/or cells

Step S11, rapidly immersing the tumor tissue/cells taken out from the liquid nitrogen into 10-20ml of resuscitation solution 1 heated in water bath at 36-38 ℃ for 5-10 min. Wherein, preferably, the tumor tissue/cells taken out from the liquid nitrogen are quickly immersed into 10ml of resuscitation solution 1 preheated in water bath at 37 ℃ for 5 min.

And step S22, taking out the tumor tissues and/or cells treated by the resuscitation solution 1 and immersing the tumor tissues and/or cells in 5-10ml of resuscitation solution 2 for 5-10 min. Wherein, preferably, the tumor tissue/cells treated by the resuscitation solution 1 is transferred into 5ml of room temperature resuscitation solution 2, and the immersion time is 5 min.

And step S33, taking out the tumor tissues and/or cells treated by the resuscitation solution 2 and immersing the tumor tissues and/or cells in 5-10ml of resuscitation solution 3 for 5-10 min. Wherein, preferably, the tumor tissue/cell treated by the resuscitation solution 2 is immersed in 10ml of room temperature resuscitation solution 3 for 10 min.

And step S44, washing the tumor tissue/cells treated by the resuscitation solution 3 for 2 times by using a 1x phosphate buffer solution, and immersing the tumor tissue/cells in sterile physiological saline to obtain the tumor tissue/cells after resuscitation.

Example 5 human Primary Breast cancer tumor tissue/cell cryopreservation experiment

Step S1, washing a human primary breast cancer tumor tissue/cell sample for 2 times by 1x phosphate buffer solution, and completely immersing the sample in the 1x phosphate buffer solution to remove envelope, blood vessels and mucous necrotic tissues; wherein, the action is gentle in the operation process, and the damage of tumor tissues and/or cell masses is avoided.

Step S2, slicing the human primary breast cancer tumor tissue/cell by using a tumor cutting die to obtain tissue slices with the size of 1x 1mm, and cleaning again by using 1x phosphate buffer solution.

Step S3, immersing the sliced primary breast cancer tumor tissue/cells of the human body into 5ml of pre-prepared vitrification solution 1 (the components and the dosage are shown in example 1) for 10 min; then, the glass is immersed in 5ml of a previously prepared glass solution 2 (the components and the amounts thereof are shown in example 1) for 10 min.

Example 6 Resuscitation experiment of human Primary Breast cancer tumor tissues and/or cells

Step S11, rapidly immersing the human primary breast cancer tumor tissue/cells taken out from the liquid nitrogen into 10ml resuscitation solution 1 heated in water bath at 36-38 ℃ for 5 min.

And step S22, transferring the human primary breast cancer tumor tissues/cells treated by the resuscitation solution 1 into 5ml of room-temperature resuscitation solution 2, and immersing for 5 min.

And step S33, immersing the human primary breast cancer tumor tissues/cells treated by the resuscitation solution 2 into 10ml of room-temperature resuscitation solution 3 for 10 min.

And step S44, washing the primary breast cancer tumor tissue/cells of the human body treated by the resuscitation solution 3 for 2 times by using 1x phosphate buffer solution, and immersing the cells in sterile normal saline to obtain the resuscitated tumor tissue/cells.

And step S55, subcutaneously planting or in-situ transplanting the revived human primary breast cancer tumor tissues and/or cells into an immunodeficient mouse body to establish a new generation of PDX and PDOX models for subsequent medical treatment or scientific research.

As shown in figure 1, after the primary breast cancer tumor tissues and/or cells of a human body are recovered by the cryopreservation and recovery method of the invention and are planted subcutaneously or transplanted in situ in an immunodeficiency mouse for one month, the tumor-bearing of the experimental group adopting the cryopreservation method of the invention still exists and grows progressively, and the model building of PDX and PDOX is successful.

In fig. 1, the upper left picture establishes a PDX model for fresh human primary breast cancer tumor tissue/or cell transplantation, and the upper right picture establishes a PDOX model for fresh human primary breast cancer tumor tissue/cell transplantation; the lower left picture is a PDX model established after the primary breast cancer tumor tissue and/or cells of the human body are subjected to cryopreservation and resuscitation in the invention are transplanted; the lower right picture is the PDOX model established after the primary breast cancer tumor tissue and/or cells of the human body are subjected to cryopreservation and resuscitation in the invention are transplanted.

The primary breast cancer tumor tissue/cell of the human body recovered by freezing and storing by the kit and the processing method can be successfully transplanted on an immunodeficiency mouse, and an effective PDX and PDOX mouse model is established. After verification, after primary breast cancer tumor tissues and/or cells of a human body are cryopreserved and recovered and are planted under the skin or transplanted in situ in an immunodeficiency mouse for one month, the tumor tissues and/or cells are dissected to grow into new blood vessels, and the tissues are gradually proliferated and enlarged. The results show that: after primary breast cancer tumor tissues and/or cells of a human body recovered by cryopreservation are transplanted on an immunodeficiency mouse, a PDX and PDOX mouse model is successfully established, the tumor tissues and/or cells are activated in the mouse, and the tumor formation rate is more than 99%.

In addition, the results of the tumor formation rate of human primary breast cancer tumor tissue/cells transplanted into mice after resuscitation of experimental and another control group are listed in table 3

	Experimental group	Another control group
			Tumor formation rate	＞99％	83％

The cryopreservation and resuscitation kit is adopted for cryopreservation and resuscitation of human primary breast cancer tumor tissues and/or cells of an experimental group.

The cryopreservation and resuscitation kit for cryopreservation and resuscitation of human primary breast cancer tumor tissues and/or cells in another control group comprises: the tumor tissue cryopreservation kit and the tumor tissue resuscitation kit are characterized in that DMEM culture medium (high-glucose modified Du's eagle's culture medium) is adopted as a basic culture medium in vitrification liquid and resuscitation liquid in the cryopreservation kit and the resuscitation kit.

According to the cryopreservation and resuscitation kit, Earl's balanced salt solution with increased concentration gradient is adopted in the resuscitation solution 1, the resuscitation solution 2 and the resuscitation solution 3, in the resuscitation process, the provided osmotic pressure environment is more adaptive to the osmotic pressure environment for survival of human primary breast cancer tumor tissues/cells, and the better effect is achieved in the aspect of maintaining resuscitation of tumor microenvironment stem cells, immune cells or fibroblasts in the human primary breast cancer tumor tissues/cells, so that the tumor formation rate of the recovered human primary breast cancer tumor tissues/cells transplanted into a mouse body for establishing a PDOX model can be close to 100%.

Example 7: human body primary breast cancer tumor tissue/cell cryopreservation and resuscitation hematoxylin-eosin staining (HE staining) experiment

1. Test method

(1) In an ultra-clean workbench, conventionally preparing human primary breast cancer tumor tissues and/or cell sediments to be cryopreserved; during the preparation process, attention is paid to prevent part of primary breast cancer tumor tissues and/or cells of a human body from dying due to a force clamp or a sharp instrument;

(2) after the primary breast tumor tissues and/or cells of the cryopreserved human body are resuspended by using the sample preservation solution, subpackaging the cryopreserved tissues and/or cells into a cryopreservation tube with the capacity of 2ml, adding 1ml of tissue cryopreservation solution into the cryopreservation tube with the capacity of 2ml, putting the cryopreservation solution into a programmed cooling box, cooling to-80 ℃, and transferring the cryopreservation solution into a liquid nitrogen tank for preservation after 24 hours;

(3) after being frozen and recovered by the freezing and recovery kit provided by the invention, primary breast cancer tumor tissues/cells of a human body are fixed by 4% paraformaldehyde and then are embedded by an OCT (optical coherence tomography) embedding medium; the tissue slice is frozen conventionally, the slice thickness is generally 14 μm, the slice is preserved at-20 ℃ in a short period, and the slice can be preserved at-80 ℃ for a long period;

(4) dyeing: 3-10 min of hematoxylin dip-dyeing, washing with tap water for a moment, differentiating for 3-5 sec by using 1% hydrochloric acid alcohol, washing with tap water for several minutes to several hours, washing with a lithium carbonate saturated aqueous solution for 1-2 min, washing with tap water for 15min, and dip-dyeing with a 0.5% eosin solution for 1-2 min; and (3) dehydrating: 1-2 min of first 95% ethanol, 1-2 min of second 95% ethanol and 1-2 min of 100% ethanol; and (3) transparency: 2min of a xylene-carbolic acid mixed solution, 1-2 min of xylene for the first time, 1-2 min of xylene for the second time and 1-2 min of xylene for the third time; sealing: sealing neutral gum into a sheet; observed under an Olympus microscope and photographed.

2. Results

As shown in fig. 2, a represents the HE staining result of the fresh human primary breast cancer tumor tissue/cell, and B represents the HE staining result of the cryopreserved and recovered human primary breast cancer tumor tissue/cell, which shows that more than 99% of the cryopreserved and recovered human primary breast cancer tumor tissue/cell is recovered, and the cryopreserved and recovered human primary breast cancer tumor tissue/cell and the fresh human primary breast cancer tumor tissue/cell have similar morphological structures, suggesting that the tumor characteristics are not significantly affected by the cryopreservation and recovery processes. The results indicate that the cryopreservation and resuscitation process did not significantly affect tumor characteristics.

Mainly considering that the recovery liquid 1 and the recovery liquid 2 adopt Earl's balanced salt solution 65-85V/V%, and the recovery liquid 3 adopts Earl's balanced salt solution 75-95V/V%, in the recovery process, the provided osmotic pressure environment is more adaptive to the osmotic pressure environment for survival of human primary breast cancer tumor tissues/cells, and the recovery liquid has better effect on promoting recovery of tumor microenvironment stem cells, immune cells or fibroblasts in the human primary breast cancer tumor tissues/cells. In addition, the recovery of the tumor microenvironment stem cells, immune cells or fibroblasts in the human primary breast cancer tumor tissues/cells is maintained, and the morphological structure of the recovered tumor of the human primary breast cancer tumor tissues/cells is similar to the morphological structure of fresh human primary breast cancer tumor tissues/cells.

In addition, the invention also provides a tumor tissue/or cell cryopreservation and recovery kit, which comprises the tumor tissue/cell cryopreservation kit and the tumor tissue/cell recovery kit.

In addition, the present invention provides a method for freezing and resuscitating tumor tissue and/or cells, comprising the steps of:

cryopreservation treatment, the cryopreservation treatment comprising:

a resuscitation treatment comprising:

In conclusion, the cryopreservation and resuscitation kit comprises the tissue vitrification liquid and the resuscitation liquid, and the components of the vitrification liquid and the resuscitation liquid are clear and stable. The cryopreserved and resuscitated human primary breast cancer tumor tissues and/or cells by using the cryopreserved and resuscitated kit disclosed by the invention can maintain the heterogeneity of the tumor tissues and/or cells and the characteristics of a tumor microenvironment while ensuring the self-activity of the human primary breast cancer tumor tissues and/or cells. And on the establishment of a subcutaneous tumor implantation PDX model and a tumor in-situ transplantation tumor PDOX model, the tumor tissue and/or the cytological characteristics and the biological characteristics of the primary breast cancer of the human body are not changed.

In addition, the tumor tissue/cell vitrification cryopreservation and resuscitation kit provided by the invention has simple and clear treatment method, and related instruments and equipment are commonly used, so that the operation, popularization and application are facilitated.

It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.

The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims

1. A kit for freezing and recovering tumor tissues and/or cells is suitable for freezing and recovering primary breast cancer tissues and/or cells of a human body and is characterized in that the kit for freezing and recovering the tumor tissues and/or cells comprises a tumor tissue and/or cell freezing kit and a tumor tissue and/or cell recovering kit,

the tumor tissue/cell cryopreservation kit comprises:

the tumor tissue/cell resuscitation kit comprises:

2. The kit for cryopreservation and resuscitation of tumor tissue and/or cells according to claim 1,

the components of the glass transition liquid 1 comprise: 80V/V% of Gibco DMEM medium, 10V/V% of dimethyl sulfoxide, 10V/V% of ethylene glycol, 2W/V% of bovine serum albumin, 1W/V% of sucrose, 0.05W/V% of methyl cellulose (4000CP), 0.25W/V% of hydroxyethyl starch and 25W/V% of glucose; and

3. The tumor tissue/cell cryopreservation and resuscitation kit of claim 1, further comprising a tumor tissue/cell slice mold comprising: the base, the base includes the upper surface, the upper surface be equipped with the depressed part and certainly the upper surface is perpendicular downwards, along transversely being equipped with many equidistance distributions, the degree of depth is less than the cutter guide way of depressed part minimum, wherein, the both sides of base still are equipped with the handle.

4. The tumor tissue/cell cryopreservation and resuscitation kit of claim 3, wherein the recess is an ellipsoid having a width of 30mm and a length of 35 mm; the vertical distance from the lowest point of the concave part to the upper surface of the base is 25 mm.

5. The tumor tissue/cell cryopreservation and resuscitation kit of claim 3, wherein the depth of the cutter guiding groove is 5mm below the lowest point of the depression; the distance between two adjacent cutter guide grooves is 1 mm; the number of the cutter guide grooves is 23.

6. The kit for cryopreservation and resuscitation of tumor tissue and/or cells according to claim 1,

7. A method for processing frozen tumor tissue/cell, which is suitable for frozen storage processing of human primary breast cancer tumor tissue/cell, and is characterized in that the tumor tissue/cell is frozen and stored by using the tumor tissue/cell frozen and stored kit according to any one of claims 1-5, and the method for processing frozen tumor tissue/cell comprises the following steps:

8. The method of claim 7, wherein the frozen tumor tissue and/or cells are processed by:

in the step S1, the tumor tissue/cell sample is treated by blood vessels, envelopes and necrotic tissues, and the treated tumor tissue/cell sample needs to be completely immersed in 1 × phosphate buffer solution, and the operation is gentle, so as to avoid damage to the tumor tissue/cell sample.

9. The method of claim 8, wherein the frozen tumor tissue and/or cells are processed by:

in S2, the tumor tissue and/or cells are sliced to a size of 1X 1 mm.

11. A treatment method for recovering tumor tissues and/or cells, which is suitable for recovering primary breast cancer tumor tissues and/or cells of a human body, and is characterized in that the tumor tissues and/or cells are recovered by using the tumor tissue and/or cell recovery kit as claimed in claim 1 or 7, and the treatment method for recovering the tumor tissues and/or cells comprises the following steps:

12. The method for treating tumor tissue and/or cell resuscitation as claimed in claim 11,

in the S11, tumor tissues and/or cells taken out of liquid nitrogen are quickly immersed in 10ml of resuscitation solution 1 preheated in a water bath at 37 ℃ for 5 min.

13. The method for treating tumor tissue and/or cell resuscitation as claimed in claim 11,

in the step S22, transferring the tumor tissues and/or cells treated by the resuscitation solution 1 into 5ml of room-temperature resuscitation solution 2, and immersing for 5 min;

14. A method for freezing and resuscitating tumor tissue/cells, which is suitable for freezing and resuscitating primary breast cancer tumor tissue/cells in human body, and comprises the steps of:

cryopreservation treatment, the cryopreservation treatment comprising:

a resuscitation treatment comprising: