CN108849859A - A kind of frozen stock solution and preparation method thereof of leukaemia cell's sample - Google Patents
A kind of frozen stock solution and preparation method thereof of leukaemia cell's sample Download PDFInfo
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- CN108849859A CN108849859A CN201810852423.4A CN201810852423A CN108849859A CN 108849859 A CN108849859 A CN 108849859A CN 201810852423 A CN201810852423 A CN 201810852423A CN 108849859 A CN108849859 A CN 108849859A
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention discloses a kind of frozen stock solutions and preparation method thereof of leukaemia cell's sample, belong to cell cryopreservation technical field.Frozen stock solution of the invention is by solution A, autologous plasma and DMSO by volume 1:1:0.04 composition, wherein the solvent of solution A is ten thousand river in Shangdong Province solution, comprising vitamin A, vitamin C, tea polyphenols, trehalose, vitamin A in solution A, vitamin C, tea polyphenols, trehalose final concentration be respectively 10mg/mL, 10mg/mL, 0.5mg/mL, 20mg/mL.Solution A, autologous plasma and DMSO are proportionally uniformly mixed, frozen stock solution of the invention can be obtained.Frozen stock solution preparation of the present invention is simple, at low cost, can not be changed with long-term preservation leukaemia cell, cell activity, and brings back to life cell survival rate up to 95% or more.
Description
Technical field
The present invention relates to cell cryopreservation technical fields, and in particular to a kind of frozen stock solution of leukaemia cell's sample and its preparation
Method.
Background technique
Leukaemia is to endanger one of important tumour of the mankind, and in the research of leukemia diagnosis and treatment, clinic is frozen
Leukaemia cell's sample to the research of the diagnosis typing of leukaemia, state of illness monitoring and guiding treatment, the building in clinical sample library
It is significant, it is also very big to its demand, therefore carrying out eased effective method to freeze leukaemia sample is ability
The urgent demand in domain, a kind of method for establishing prolonged cold preservation leukaemia cell have the research of leukemia diagnosis and treatment
It is of great importance.
If simple freezes, cell will receive the damage of two aspects in frozen storage process：First, in the process frozen
In, extracellular moisture freezes first, so that the electrolyte concentration in the solution not frozen increases, causes cell death, belongs to infiltration
Permeability damage；Second, since low temperature causes cell formation ice crystal intracellular that can destroy intracellular structure, belongs to mechanical damage.Therefore
In conjunction with the above two o'clock reason, it is necessary to which the speed for solving freezing and freezes the formula of agent, and the speed of thawing also directly affects when recovery
The activity of cell.
In traditional leukaemia cell's frozen storage process, tire ox blood slurry, the growth for cell is added in frozen stock solution big city
It plays an important role, but also becomes the obstacle of clinical application simultaneously.Tire ox blood starches the possibility that may have bacterium and virus infection, special
It is not that prion can lead to rabid ox disease, additionally, it is possible to cause local immunity reaction or inflammatory reaction.With commercialization without blood plasma
Culture medium substitutes animal blood plasma culture cell, and there are still some problems in practical applications, if culture medium development cost is high, does not have
There is versatility, is only limitted to specific cell culture etc..Be added specific cell factor in the medium, cost then costly,
Its extraction and preparation technique is complicated, and low yield, cost is considerable, also has the shortcomings that potential toxicity, cause gene mutation, prevent its from
Meet the needs of clinical application.
Summary of the invention
It is an object of the invention to overcome shortcoming and deficiency of the existing technology, a kind of leukaemia cell's sample is provided
Frozen stock solution, using the frozen stock solution can pole increase substantially the survival rate of leukaemia cell.The object of the invention is also to provide
The preparation method of the frozen stock solution.
The purpose of the invention is achieved by the following technical solution：
A kind of frozen stock solution of leukaemia cell's sample is made of solution A, autologous plasma and dimethyl sulfoxide (DMSO).Institute
The solvent for stating solution A is ten thousand river in Shangdong Province solution, includes vitamin A, vitamin C, tea polyphenols, trehalose.
It is further preferred that the volume ratio of solution A, autologous plasma and DMSO is 1:1:0.04.
It is further preferred that the final concentration of vitamin A in the solution A, vitamin C, tea polyphenols, trehalose is respectively
10mg/mL、10mg/mL、0.5mg/mL、20mg/mL。
The preparation method of the frozen stock solution of above-mentioned leukaemia cell's sample, includes the following steps：
(1) vitamin A, vitamin C, tea polyphenols, trehalose are added into ten thousand rivers in Shangdong Province and prepares solution A.
(2) acquisition will freeze the blood of the patient of leukaemia cell's sample, separate autologous plasma.
(3) solution A, autologous plasma and DMSO are proportionally uniformly mixed, obtain the frozen stock solution of leukaemia cell's sample.
The frozen stock solution of above-mentioned leukaemia cell's sample is freezing the application in leukaemia cell.
In frozen stock solution of the present invention, trehalose, vitamin E can resist the damage of ice crystal, and vitamin C and tea polyphenols can be with
Cellular oxidation is resisted, keeps the activity of cell, the albumin in ten thousand rivers in Shangdong Province and autologous plasma can preferably protect stem cell.This is several
Kind substance is combined manufactured frozen stock solution and the survival after freeze-stored cell recovery is greatly improved for freezing leukaemia cell, pole
Rate has extraordinary application prospect.
The invention has the advantages that and beneficial effect：
(1) frozen stock solution of the present invention is prepared simple, at low cost；Leukaemia cell is frozen using it, it is easy to operate, have good
Application prospect.
(2) leukaemia is frozen using frozen stock solution of the present invention, brings back to life cell survival rate up to 95% or more, relatively using conventional thin
The recovery survival rate of born of the same parents' frozen stock solution increases significantly, and there is no cell depletion.
(3) frozen stock solution of the present invention can not be changed with long-term preservation leukaemia cell, cell activity, ensure that cell is raw
Object activity.
Detailed description of the invention
Fig. 1 is the aspect graph for freezing Preleukemia cell.
Fig. 2 is the aspect graph of the leukaemia cell to recover after being frozen 6 months using frozen stock solution of the present invention.
Fig. 3 is the aspect graph of the leukaemia cell to recover after being frozen 6 months using control frozen stock solution.
Fig. 4 is the cell survival rate result figure frozen after the recovery of leukaemia cell's different time using frozen stock solution.
Fig. 5 is the cell activity result figure frozen after leukaemia cell's recovery using frozen stock solution.
Specific embodiment
Following embodiment should not be construed as limiting the invention for further illustrating the present invention.If not referring in particular to
Conventional means bright, that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1
One, the preparation of frozen stock solution
(1) vitamin A 100mg, vitamin C 100mg, tea polyphenols 5mg, trehalose 200mg are added into ten thousand river in Shangdong Province 10mL,
Make vitamin A, vitamin C, tea polyphenols, trehalose final concentration be respectively 10mg/mL, 10mg/mL, 0.5mg/mL, 20mg/
ML is filtered with 0.2 μm of filter, obtains solution A.
(2) acquisition will freeze the blood of the patient of leukaemia cell's sample, be added into the purple pipe containing EDTA,
3000r/min is centrifuged 15min, and blood plasma is sucked into new centrifuge tube, the syringe filter mistake for the use of aperture being then 0.2 μm
Filter obtains autologous plasma, and 4 DEG C save for use.
(3) by solution A, autologous plasma and DMSO by volume 1:1:0.04 ratio is uniformly mixed, and is obtained of the invention
Frozen stock solution.Meanwhile by calf serum and DMSO by volume 9:1 ratio is uniformly mixed, as control frozen stock solution.
Two, cell cryopreservation
Cultivate leukaemia cell, cell density about 5 × 107/ mL photographs to record its form, and 1000g is centrifuged 5min, abandons
Fall supernatant, be divided into two groups of implementation group, control group, is separately added into frozen stock solution of the present invention (implementation group) or control frozen stock solution (control group)
1mL is mixed, is placed in sterile cryopreservation tube.Cryopreservation tube is put in refrigerator first 4 DEG C 2 hours, then -20 DEG C 2 hours, then -80 DEG C of mistakes
It is transferred in liquid nitrogen container and freezes after night.
Three, cell recovery
By freeze respectively 3 days, 1 week, January, June cryopreservation tube take out, be quickly placed into 37 DEG C of water-baths, concussion is until cell is outstanding
Liquid melts completely, photographs to record its form, is diluted with 5 times of DMEN liquid, and 1000g is centrifuged 5min, is centrifuged off supernatant, trypan blue
Dyeing is repeated 3 times calculating cell survival rate and is averaged.
Fig. 1-3 is multiple after freezing Preleukemia cell, being frozen 6 months using frozen stock solution of the present invention or control frozen stock solution respectively
The aspect graph of the leukaemia cell of Soviet Union, after making discovery from observation before freezing and freezing 6 months using the present invention or control frozen stock solution
Cellular morphology indifference calculates its cell density about 5 × 107/mL。
Fig. 4 is that the cell survival rate knot after the recovery of leukaemia cell's different time is frozen using the present invention or control frozen stock solution
Fruit figure, its survival rate is 95% or more after freezing recovery in leukaemia cell 6 months using frozen stock solution of the present invention as the result is shown, hence it is evident that
Higher than control frozen stock solution.
Four, CCK8 measures Cell viability
The cryopreservation tube for freezing June is taken out, 37 DEG C of water-baths are quickly placed into, concussion is melted completely up to cell suspension, is centrifuged
The cell suspension uniformly obtained is resuspended with DMEN culture medium afterwards.Inoculating cell suspension (5000 cells/wells) in 96 orifice plates, to
The CCK8 of 10 μ L is added in each hole, by culture plate in incubator (37 DEG C, 5%CO2Condition) middle culture 4 hours, it is surveyed with microplate reader
Determine absorbance at 450nm, and Cell viability is calculated by absorbance.
Fig. 5 is to freeze the cell activity result figure after leukaemia cell's recovery using the present invention or control frozen stock solution, as a result
Display is significantly higher than control frozen stock solution using the cell activity that frozen stock solution of the present invention freezes after leukaemia cell's recovery.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. a kind of frozen stock solution of leukaemia cell's sample, it is characterised in that：It is made of solution A, autologous plasma and DMSO；It is described molten
The solvent of liquid A is ten thousand rivers in Shangdong Province, includes vitamin A, vitamin C, tea polyphenols, trehalose.
2. frozen stock solution according to claim 1, it is characterised in that：The volume ratio of solution A, autologous plasma and DMSO is 1:1:
0.04。
3. frozen stock solution according to claim 1, it is characterised in that：Vitamin A in the solution A, vitamin C, tea polyphenols,
The final concentration of trehalose is respectively 10mg/mL, 10mg/mL, 0.5mg/mL, 20mg/mL.
4. the preparation method of the described in any item frozen stock solutions of claim 1-3, it is characterised in that：Include the following steps：
(1) vitamin A, vitamin C, tea polyphenols, trehalose are added into ten thousand rivers in Shangdong Province and prepares solution A；
(2) acquisition will freeze the blood of the patient of leukaemia cell's sample, separate autologous plasma；
(3) solution A, autologous plasma and DMSO are proportionally uniformly mixed, obtain the frozen stock solution of leukaemia cell's sample.
5. the described in any item frozen stock solutions of claim 1-3 are freezing the application in leukaemia cell.
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| CN201810852423.4A CN108849859B (en) | 2018-07-27 | 2018-07-27 | Cryopreservation solution for leukemia cell sample and preparation method thereof |
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| CN201810852423.4A CN108849859B (en) | 2018-07-27 | 2018-07-27 | Cryopreservation solution for leukemia cell sample and preparation method thereof |
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Citations (3)
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|---|---|---|---|---|
| US20060251683A1 (en) * | 2002-12-20 | 2006-11-09 | David Frey | Directly injectable formulations which provide enhanced cryoprotection of cell products |
| CN103070161A (en) * | 2013-01-09 | 2013-05-01 | 广州爱菲科生物科技有限公司 | Cryopreservation liquid and cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC) |
| CA3013187A1 (en) * | 2016-02-01 | 2017-08-10 | Green Cross Lab Cell Corporation | Medium composition for cryopreservation of cell and use thereof |
-
2018
- 2018-07-27 CN CN201810852423.4A patent/CN108849859B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060251683A1 (en) * | 2002-12-20 | 2006-11-09 | David Frey | Directly injectable formulations which provide enhanced cryoprotection of cell products |
| US20160345574A1 (en) * | 2002-12-20 | 2016-12-01 | Aduro Gvax Inc. | Directly injectable formulations which provide enhanced cryoprotection of cell products |
| CN103070161A (en) * | 2013-01-09 | 2013-05-01 | 广州爱菲科生物科技有限公司 | Cryopreservation liquid and cryopreservation method for adipose tissue-derived mesenchymal stem cells ( ADSC) |
| CA3013187A1 (en) * | 2016-02-01 | 2017-08-10 | Green Cross Lab Cell Corporation | Medium composition for cryopreservation of cell and use thereof |
Non-Patent Citations (1)
| Title |
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| 张俊萍，等: "不同冷冻保护剂对外周血干细胞保存的研究", 《癌症》 * |
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