CN107041361A - The store method and preservation reagent of a kind of tumor tissues - Google Patents
The store method and preservation reagent of a kind of tumor tissues Download PDFInfo
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- CN107041361A CN107041361A CN201710261155.4A CN201710261155A CN107041361A CN 107041361 A CN107041361 A CN 107041361A CN 201710261155 A CN201710261155 A CN 201710261155A CN 107041361 A CN107041361 A CN 107041361A
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- tumor tissues
- reagent
- enzymolysis
- tumor
- hyaluronidase
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The invention discloses a kind of store method of tumor tissues and preservation reagent.The inventive method comprises the following steps：1) tumor tissues to be saved are placed in tumor tissues enzymolysis reagent, 10 20min, tumor tissues after being digested is cultivated in 20 25 DEG C；Contain hyaluronidase and DNA enzymatic I in the tumor tissues enzymolysis reagent；Contain 40 150U hyaluronidases in every liter of tumor tissues enzymolysis reagent, containing higher than 40Kunitz U DNA enzymatics I；2) tumor tissues after the enzymolysis are placed in freezen protective liquid, 4 DEG C of 20min of pre-equilibration 10 obtain tumor tissues after pre-equilibration；3) tumor tissues after the pre-equilibration are subjected to sequencing freezing, after temperature is down to 150 DEG C, is transferred in liquid nitrogen container and is stored.The features such as tumor tissues store method of the present invention has form and the characterization of molecules that tumor tissues can be than more fully retaining patient's derived tissues after tumor formation rate height, transplanting.
Description
Technical field
The invention belongs to biomedical sector, it is related to a kind of store method of tumor tissues and preserves reagent.
Background technology
How the increase of influence and harmfulness with tumour to the mankind, protect, preserve and make full use of human tumor to lose
Resource is passed, China's treatment and prevention of tumour level is improved, has become the task of top priority.It is used as the work tumor tissues sample of tumor research material
Key player is played in tumor research progression is promoted, with important scientific research and clinical value, and tumour of living
Tissue preservation techniques turn into the basis for realizing these values.
The mode that tumor tissues living are preserved at present is generally directly freezed preservation, more than required cryoprotector using DMSO,
Serum and cell culture fluid composition are, it is necessary to matching while using, and the less stable of this tissue freezing solution so that its shelf-life compared with
Short, preservation condition is also more harsh.
It can not ensure the structural intergrity of tumor tissues, and pole after freezing and thawing using existing freezing and storing method
The tumour easy to lose hereditary capacity of itself, reduces the value of clinical studies of tissue specimen.
It is badly in need of a kind of more long-acting and more easily tumor tissues store method at present.
The content of the invention
It is an object of the invention to provide a kind of store method of tumor tissues and preservation reagent.
Tumor tissues store method provided by the present invention, specifically may include following steps：
(1) tumor tissues to be saved are placed in tumor tissues enzymolysis reagent, 10-20min is cultivated (such as in 20-25 DEG C
15min), tumor tissues after being digested；
Contain hyaluronidase and DNA enzymatic I in the tumor tissues enzymolysis reagent；Every liter of tumor tissues enzymolysis reagent
In the hyaluronidase containing 40-150U, contain the DNA enzymatic I higher than 40Kunitz U.
Wherein, the enzyme activity of the hyaluronidase defines the enzyme activity for the hyaluronidase that same sigma companies article No. is H3506
Definition.The enzyme activity of the DNA enzymatic I defines the enzyme activity definition for the DNA enzymatic I that same sigma companies article No. is DN25-1G.
In the present invention, the hyaluronidase is specially sigma Products, and article No. is H3506.The DNA enzymatic I has
Body is sigma Products, and article No. is DN25-1G.Accordingly, the hyaluronidase is in tumor tissues enzymolysis reagent
Concentration can be 100-150mg/mL；Concentration of the DNA enzymatic I in the tumor tissues digest reagent can be 100-120mg/
mL。
(2) tumor tissues after the enzymolysis are placed in freezen protective liquid, at 4 DEG C of pre-equilibration 10-20min (such as 15min),
Obtain tumor tissues after pre-equilibration；
(3) tumor tissues after the pre-equilibration are subjected to sequencing freezing, after temperature is down to -150 DEG C, are transferred to liquid nitrogen container
It is middle to be stored.
In view of most solid tumor tissues contain substantial amounts of collagenic connective tissue, make the spy for itself possessing compact structure
Property, thus the present invention digests the operation that reagent carries out incubated at room temperature to it by set tumor tissues, realizes tumor tissues
The effect that enzymolysis reagent is organized to be digested to collagen etc., makes the solid tumor tissue interstitial of densification loose, and then beneficial to follow-up
The smooth infiltration of freezen protective liquid in pre equilibrium process；And then shorten the follow-up equilibration time in freezen protective liquid.Pass through
After enzymolysis processing, during pre-equilibration, only can reach pre-equilibration effect with 10-20 minutes, (freezen protective liquid permeates
To tumor tissues center)；Compared with the pre-equilibration time of 30-90 minutes common in the art, pre-equilibration is greatly shortened
Time, it is to avoid due to pre-equilibration overlong time, and then cause its cryoprotector to the cytotoxic damages of tumor tissues.
In the process, the tumor tissues digest reagent by the hyaluronidase, the DNA enzymatic I and containing volume
Percentage composition constitutes for the DMEM nutrient solutions of 10% hyclone.
More specific, the hyaluronidase containing 48-120U in every liter of tumor tissues enzymolysis reagent contains
The DNA enzymatic I higher than 40Kunitz U, surplus is containing the DMEM nutrient solutions that volumn concentration is 10% hyclone.Its
In, the enzyme activity of the hyaluronidase defines the enzyme activity definition for the hyaluronidase that same sigma companies article No. is H3506.It is described
The enzyme activity of DNA enzymatic I defines the enzyme activity definition for the DNA enzymatic I that same sigma companies article No. is DN25-1G.In the present invention, it is described
The sour enzyme of bright matter is specially sigma Products, and article No. is H3506.The DNA enzymatic I is specially sigma Products, and article No. is
DN25-1G.Accordingly, concentration of the hyaluronidase in the tumor tissues digest reagent is specially 120mg/mL；Institute
State concentration specially 100mg/mL of the DNA enzymatic I in the tumor tissues digest reagent.
The freezen protective liquid is made up of DMSO with sucrose；In the freezen protective liquid, the concentration point of DMSO and sucrose
Wei not 1mol/L and 0.1mol/L.
In step (1), before the tumor tissues to be saved are placed in the tumor tissues enzymolysis reagent, may be used also
Comprise the following steps：Collect tumor tissues, be placed in containing volumn concentration for 10% hyclone DMEM nutrient solutions in, in 5-
It is transported in 10min with ice chest in Biohazard Safety Equipment, changes fresh containing the DMEM that volumn concentration is 10% hyclone
Nutrient solution, and tumor tissues are cut into (0.5mm-1.5mm) × (0.8mm-1.2mm) × (2.5mm-3.5mm) size, obtain
The tumor tissues to be saved.
In step (2), tumor tissues after the enzymolysis are placed in described in 2-4 pieces in the freezen protective liquid concretely
Tumor tissues are placed in freezen protective liquid described in 2ml after enzymolysis.
It is described " tumor tissues after the pre-equilibration to be subjected to sequencing freezing, treat that temperature is down to -150 DEG C in step (3)
Afterwards, it is transferred in liquid nitrogen container and is stored ", it specifically may include following steps：It will be equipped with the freezing of tumor tissues after the pre-equilibration
Pipe is put into programmed cooling instrument, is started cryogenic temperature and is set as 4 DEG C, and near -60 DEG C of 2.5 DEG C/min rate of temperature fall, then
- 80 DEG C are down to 1 DEG C/min speed, finally -150 DEG C are down to -30 DEG C/min rate of temperature fall；Whole cryovial is shifted
Stored into liquid nitrogen container.
The reagent provided by the present invention for being used to preserve tumor tissues, can be following (A) or (B)：
(A) tumor tissues enzymolysis reagent；
(B) reagent set that reagent and freezen protective liquid are constituted is digested by the tumor tissues；
Contain hyaluronidase and DNA enzymatic I in the tumor tissues enzymolysis reagent；Every liter of tumor tissues enzymolysis reagent
In contain 40-150U (such as 48-120U) the hyaluronidase, contain the DNA enzymatic I higher than 40Kunitz U.
Wherein, the enzyme activity of the hyaluronidase defines the enzyme activity for the hyaluronidase that same sigma companies article No. is H3506
Definition.The enzyme activity of the DNA enzymatic I defines the enzyme activity definition for the DNA enzymatic I that same sigma companies article No. is DN25-1G.
In the present invention, the hyaluronidase is specially sigma Products, and article No. is H3506.The DNA enzymatic I has
Body is sigma Products, and article No. is DN25-1G.Accordingly, the hyaluronidase is in tumor tissues enzymolysis reagent
Concentration can be 100-150mg/mL (such as 120mg/mL)；Concentration of the DNA enzymatic I in the tumor tissues digest reagent can
For 100-120mg/mL (such as 100mg/mL).
The freezen protective liquid is made up of DMSO with sucrose；In the freezen protective liquid, the concentration point of DMSO and sucrose
Wei not 1mol/L and 0.1mol/L.
Application of the described reagent in tumor tissues are preserved falls within protection scope of the present invention.
In the present invention, the tumor tissues can be body tumor tissue, concretely any human body solid malignant
Tissue.The malignant tumour such as stomach cancer, cancer of the esophagus, cancer of pancreas, nasopharyngeal carcinoma, carcinoma of urinary bladder, kidney, colon cancer, thyroid cancer, mammary gland
Cancer, cervical carcinoma, oophoroma, prostate cancer, carcinoma of urinary bladder, glioblastoma, osteosarcoma, liver cancer, lung cancer etc..
Tumor tissues store method provided by the present invention has that tumor formation rate is high, tumor tissues can be than more fully after transplanting
The features such as retaining form and the characterization of molecules of patient's derived tissues.
Brief description of the drawings
Fig. 1 freezes influence of the time to the tumor formation rate of colon cancer Transplanted tumor model for difference.
Fig. 2 be tumor tissues freeze, recover after hematoxylin eosin staining (HE dyeing) result.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
Hyaluronidase：Sigma Products, article No. is H3506 (400-1000units/mg solid).
DNA enzymatic I：Sigma Products, article No. is DN25-1G (>=400Kunitz units/mg protein).
SCID mice：Beijing Vital River Experimental Animals Technology Co., Ltd..
Embodiment 1, the preservation of body tumor tissue
First, tumor tissues preserve reagent
Tumor tissues digest reagent：It is 10% hyclone by hyaluronidase, DNA enzymatic I and containing volumn concentration
DMEM nutrient solutions are constituted.Wherein, concentration of the hyaluronidase in the tumor tissues digest reagent is 120mg/mL；Institute
It is 100mg/mL to state concentration of the DNA enzymatic I in the tumor tissues digest reagent.Equivalent to tumor tissues enzymolysis examination every liter described
The hyaluronidase containing 48-120U in agent, contains the DNA enzymatic I higher than 40Kunitz U.
Freezen protective liquid：It is made up of DMSO with sucrose；And in the freezen protective liquid, the concentration difference of DMSO and sucrose
For 1mol/L and 0.1mol/L.
2nd, tumor tissues store method of the present invention
Specimens Specimen origin is in 56 years old male's colon cancer with Patients with Liver Metastasis, and sample acquires trouble
The informed consent of person and the license of Ethics Committee.Patient is preoperative not to be received chemotherapy and other associated treatments.
1st, sample will be taken to be placed in the DMEM nutrient solutions containing 10% (volumn concentration) hyclone in art；Will dress
The DMEM nutrient solutions for having tumor tissues are transferred to ice chest, are transferred in 5-10min in Biohazard Safety Equipment, are replaced by fresh contain
In the DMEM nutrient solutions for having 10% (volumn concentration) hyclone；Tumor tissues are cut into 1mm × 1mm × 3mm sizes,
Obtain tumor tissues to be frozen.
2nd, tumor tissues to be frozen are cultivated into 15min under room temperature (20-25 DEG C) with tumor tissues enzymolysis reagent, obtained
Tumor tissues after enzymolysis.
3rd, tumor tissues are placed in freezen protective liquid described in 2ml after being digested described in 2-4 pieces, in 4 DEG C of pre-equilibration 15min,
Obtain tumor tissues after pre-equilibration.
4th, tumor tissues after the pre-equilibration are subjected to sequencing freezing, its temperature is down to after -150 DEG C, be transferred to liquid nitrogen
Tank is stored, and is specifically included：The cryovial that will be equipped with tumor tissues after pre-equilibration is put into programmed cooling instrument, starts freezing temperature
Degree is set as 4 DEG C, and near -60 DEG C of 2.5 DEG C/min rate of temperature fall, then is down to -80 DEG C with 1 DEG C/min speed, finally
- 150 DEG C are down to -30 DEG C/min rate of temperature fall；Whole cryovial is transferred in liquid nitrogen container and stored, with to be seeded.
3rd, conventional tumor tissues store method
1st, sample will be taken to be placed in the DMEM nutrient solutions containing 10% (volumn concentration) hyclone in art；Will dress
The DMEM nutrient solutions for having tumor tissues are transferred to ice chest, are transferred in 5-10min in Biohazard Safety Equipment, are replaced by fresh contain
In the DMEM nutrient solutions for having 10% (volumn concentration) hyclone；Tumor tissues are cut into 1mm × 1mm × 3mm sizes,
Obtain tumor tissues to be frozen.
2nd, tumor tissues are placed in conventional freezing and preserve liquid (formula：Hyclone 10%, DMSO 10%, surplus is DMEM
Culture medium, % represents volumn concentration) in, in 4 DEG C of pre-equilibration 60min, obtain tumor tissues after pre-equilibration.
3rd, tumor tissues after the pre-equilibration are subjected to sequencing freezing, its temperature is down to after -150 DEG C, be transferred to liquid nitrogen
Tank is stored, and is specifically included：The cryovial that will be equipped with tumor tissues after pre-equilibration is put into programmed cooling instrument, starts freezing temperature
Degree is set as 4 DEG C, and near -60 DEG C of 2.5 DEG C/min rate of temperature fall, then is down to -80 DEG C with 1 DEG C/min speed, finally
- 150 DEG C are down to -30 DEG C/min rate of temperature fall；Whole cryovial is transferred in liquid nitrogen container and stored, with to be seeded.
Embodiment 2, the foundation in body tumor tissues storehouse
For examination mouse：SCID mice (Severe combined immunodeficient mice), male or female, mouse age
4~5 weeks.
1st, the tumor tissues sample in the freezen protective of embodiment 1 is placed in 37 DEG C of water-baths and recovered, ice is transferred to after recovery
(containing the hyclone that volumn concentration is 10%) in the DMEM culture mediums of bath.
2nd, the tumor tissues after step 1 is recovered are migrated under the preceding oxter envelope of mouse.Specially：Animal is numb with isoflurane
After liquor-saturated, sterilization mouse back of the body skin of abdomen, sterile cotton balls is wiped, cut with blade near the preceding oxter in side of mouse one small
Mouthful, 18G piercing needle blunt separation skins are with subcutaneously, making into a sack made by leather, ready tumor tissue being placed in wherein, cut
Mouth is added dropwise one and drips dual anti-(100 × penicillin/streptomycin).
3rd, after after tumour growth to predefined size, mouse is put to death, tumour is aseptically exposed, obtained after image data
Excised tumor tissue, is divided into two groups, one of which carries out stored frozen by the Partial tumors tissue obtained with mouse.Will be another
Group tumor tissues repeat step 1 and 2 in the passage of body tissue, obtain in body tumor tissues storehouse.Or by the stored frozen
Tumor tissues recovery after repeat step 1 and 2 carry out tumor tissue cell passage, obtain in body tumor tissues storehouse.
Embodiment 3, difference freeze influence of the time to the tumor formation rate of colon cancer Transplanted tumor model
By embodiment 1 freeze 4-52 weeks (4,12,24,48,52 weeks) tumor tissues moved according to the method for embodiment 2
The foundation (each freeze time be respectively provided with least 5 mouse) of knurl model is planted, tumor tissues is moved to certainly difference is counted after 4 weeks and freeze
Influence of the time to the tumor formation rate of colon cancer Transplanted tumor model.
Experiment sets the fresh tumor tissues without in the embodiment 1 that freezes as control simultaneously.
As a result it is as shown in Figure 1.As seen from the figure：After cryopreservation methods recovery tumor tissues using the present invention, transplant in immune
Compared in deficient mice, 24 weeks after the transfer with transplanting the mouse of fresh tumor tissue, both transplanting success rates are
More than 90%, both is migrated to knurl success rate no significant difference.
The cryopreservation methods for the solid tumor tissue that the embodiment of the present invention 1 is provided, recover after can making tissue freezing, recovery
Rate reaches more than 90%, greatly improves the recovery survival rate of Primary Tumor tissue.The solid tumor tissue that the present invention is provided
Cryopreservation methods, tissue, the science of heredity that can maintain like the tumor tissues of the tumor tissues after recovery and fresh acquisition is biological special
Levy, studied with ensureing that it can replace fresh tumor tissue to carry out related cell, molecular biology experiment.
Embodiment 4, tumor tissues freeze, recover after hematoxylin eosin staining (HE dyeing) test
The tumor tissues that 24 weeks are frozen in embodiment 1 are placed in 37 DEG C of water-baths and recovered, it is solid with 4% paraformaldehyde after recovery
Fixed processing, then uses FFPE；Routine paraffin wax is cut into slices；HE dyeing is carried out after dewaxing, it is specific as follows：
1st, 2 × 10min of dimethylbenzene dewaxing；
2nd, absolute ethyl alcohol washes away 2 × 5min of dimethylbenzene；
3rd, 95%, 80% each 10min of ethanol, originally washing l min (should not allow anxious water directly to rush to the group on slide
Knit), distillation washing 1min；
4th, haematoxylin dyeing 4min, originally washes 2min；
5th, 1% hydrochloride alcohol differentiation 20s (being controlled under mirror), originally washes 2min；
6th, 1% weak aqua ammonia returns blue 30s (being controlled under mirror), originally washes 2 minutes, distillation washing 1min；
7th, eosin stains 90s；
8th, 80% ethanol dehydration 10s, 95% alcohol 10s, absolute ethyl alcohol 5min；
9th, absolute ethyl alcohol 10min；
10th, 2 × 10min of dimethylbenzene；
11st, neutral gum or canada balsam mounting.
In the micro- Microscopic observations of Olympus, and recorded.
Experiment sets the fresh tumor tissues without in the embodiment 1 that freezes as control simultaneously.
As shown in Fig. 2 A represents the HE coloration results of fresh tumor tissues, B represent the present invention freeze, recover after tumour
The HE coloration results of tissue.C represent conventional method freeze, recover after tumor tissues HE coloration results.As seen from the figure：The present invention
Method freezes, recover after tumor tissues compared with the tumor tissues after conventional cryopreservation, recovery, closer to fresh tumor tissue
Morphosis, point out the present invention freeze and resuscitation process be not apparent from influence tumor characteristic.
Claims (10)
1. a kind of tumor tissues store method, comprises the following steps：
(1) tumor tissues to be saved are placed in tumor tissues enzymolysis reagent, cultivate 10-20min in 20-25 DEG C, obtain enzyme
Tumor tissues after solution；
Contain hyaluronidase and DNA enzymatic I in the tumor tissues enzymolysis reagent；Contain in every liter of tumor tissues enzymolysis reagent
There is 40-150U hyaluronidase, contain the DNA enzymatic I higher than 40Kunitz U；
(2) tumor tissues after the enzymolysis are placed in freezen protective liquid, in 4 DEG C of pre-equilibration 10-20min, obtained after pre-equilibration
Tumor tissues；
(3) tumor tissues after the pre-equilibration are subjected to sequencing freezing, after temperature is down to -150 DEG C, is transferred in liquid nitrogen and carries out
Storage.
2. according to the method described in claim 1, it is characterised in that：The tumor tissues digest reagent by hyaluronidase, DNA
Enzyme I and containing volumn concentration for 10% hyclone DMEM nutrient solutions constitute；
Hyaluronidase containing 48-120U, the DNA containing higher than 40Kunitz U in every liter of tumor tissues enzymolysis reagent
Enzyme I, surplus is containing the DMEM nutrient solutions that volumn concentration is 10% hyclone.
3. method according to claim 1 or 2, it is characterised in that：The freezen protective liquid is made up of DMSO with sucrose；
In the freezen protective liquid, the concentration of DMSO and sucrose is respectively 1mol/L and 0.1mol/L.
4. according to any described method in claim 1-3, it is characterised in that：In step (1), swollen by described to be saved
Before tumor tissue is placed in the tumor tissues enzymolysis reagent, also comprise the following steps：Tumor tissues are collected, are placed in containing volume hundred
Divide content in the DMEM nutrient solutions of 10% hyclone, to be transported in Biohazard Safety Equipment, more renewed with ice chest in 5-10min
Fresh is the DMEM nutrient solutions of 10% hyclone containing volumn concentration, and tumor tissues are cut into (0.5mm-1.5mm)
× (0.8mm-1.2mm) × (2.5mm-3.5mm) size, obtains the tumor tissues to be saved.
5. method according to claim 4, it is characterised in that：In step (2), tumor tissues after the enzymolysis are placed in institute
State in freezen protective liquid is that tumor tissues are placed in freezen protective liquid described in 2ml after being digested described in 2-4 pieces.
6. according to any described method in claim 1-5, it is characterised in that：It is described " by the pre-equilibration in step (3)
Tumor tissues carry out sequencing freezing afterwards, after temperature is down to -150 DEG C, is transferred in liquid nitrogen container and is stored " include following step
Suddenly：The cryovial that will be equipped with tumor tissues after the pre-equilibration is put into programmed cooling instrument, is started cryogenic temperature and is set as 4 DEG C,
And near -60 DEG C of 2.5 DEG C/min rate of temperature fall, then with 1 DEG C/min speed -80 DEG C are down to, finally with -30 DEG C/min's
Rate of temperature fall is down to -150 DEG C；Whole cryovial is transferred in liquid nitrogen and stored.
7. the reagent for preserving tumor tissues, is following (A) or (B)：
(A) tumor tissues enzymolysis reagent；
(B) reagent set that reagent and freezen protective liquid are constituted is digested by the tumor tissues；
Contain hyaluronidase and DNA enzymatic I in the tumor tissues enzymolysis reagent；Contain in every liter of tumor tissues enzymolysis reagent
There is 40-150U hyaluronidase, contain the DNA enzymatic I higher than 40Kunitz U.
8. reagent according to claim 7, it is characterised in that：The tumor tissues digest reagent by hyaluronidase, DNA
Enzyme I and containing volumn concentration for 10% hyclone DMEM nutrient solutions constitute；
Hyaluronidase containing 48-120U, the DNA containing higher than 40Kunitz U in every liter of tumor tissues enzymolysis reagent
Enzyme I, surplus is containing the DMEM nutrient solutions that volumn concentration is 10% hyclone.
9. the reagent according to claim 7 or 8, it is characterised in that：The freezen protective liquid is made up of DMSO with sucrose；
In the freezen protective liquid, the concentration of DMSO and sucrose is respectively 1mol/L and 0.1mol/L.
10. application of any described reagent in tumor tissues are preserved in claim 7-9.
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| CN109315384A (en) * | 2018-11-12 | 2019-02-12 | 西南医科大学 | A kind of transfer of breast cancer tissue and cryopreservation methods |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107041362A (en) * | 2017-05-23 | 2017-08-15 | 黑龙江天晴干细胞股份有限公司 | A kind of tumor tissue's deep-frozen method |
| CN109315384A (en) * | 2018-11-12 | 2019-02-12 | 西南医科大学 | A kind of transfer of breast cancer tissue and cryopreservation methods |
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