CN106719604A - Pet peripheral blood directly freezes reagent and method - Google Patents
Pet peripheral blood directly freezes reagent and method Download PDFInfo
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- CN106719604A CN106719604A CN201710064441.1A CN201710064441A CN106719604A CN 106719604 A CN106719604 A CN 106719604A CN 201710064441 A CN201710064441 A CN 201710064441A CN 106719604 A CN106719604 A CN 106719604A
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- peripheral blood
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- frozen
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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
-
- 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
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- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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Abstract
The present invention provides a kind of pet peripheral blood and freezes reagent, including：10~20W/V% of sodium cellulose glycolate, dimethyl sulfoxide (DMSO) 15~35V/V%, MEM 65~85V/V% of culture medium, preferably also include 0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose, 3~7W/V% of inosine, 0.5~2.5W/V% of sodium acid carbonate.Pet peripheral blood of the invention freezes reagent and can directly freeze pet peripheral blood, without carrying out centrifugal concentrating to blood, the beneficiating ingredient in blood is retained to greatest extent.
Description
Technical field
The present invention relates to pet peripheral blood Cryopreservation Technology field, specifically, it is related to a kind of direct for pet peripheral blood
The reagent and method for freezing.
Background technology
Pet (referring mainly to as the dog and cat of companion animals) can accompany the mankind, alleviate the pressure in daily life, pleased
Happy mood, promotes human health, as a member in family.With China's expanding economy, the exacerbation of aging degree, to doting on
The demand of thing is increasing.While pet quantity increases, the industry such as pet food, pet supplies, pet training has obtained fluffy
Vigorous development.Pet health domain variability is paid close attention to by enough, also not yet forms specification.Compared to human health market, dote on
Thing health market has very big space to wait to excavate.Love pet, pays close attention to pet health.
In pet peripheral blood in addition to accounting for most of haemocyte, also contain abundant monocyte, cell factor.Most
Closely study and show, by replacing the blood of young animals and geriatric animals, young animals can be allowed to show aging state, and it is old
Year, animal can recover one's youthful vigour, and these secrets may be relevant with the cell factor in young blood.Peripheral blood not only convenient material drawing,
And it is feature-rich, wherein T lymphocytes can be transformed by Chimeric antigen receptor, be used to treat lymthoma etc. after amplification in vitro
Malignant tumour；NK cells can help body to resist aging, for beauty；MSC cells can effectively alleviate body inflammatory reaction,
For treatment of arthritis etc..But these beneficial effects each depend on the peripheral blood of a health, once pet aging or trouble
The vigor of these cells can be all remarkably decreased after being ill, and immunological rejection can be produced again using non-autologous PBC.Cause
This, for it, to freeze a peripheral blood very necessary when pet young healthy.
At present on the market not yet with the presence of the product directly frozen for pet peripheral blood.With reference to mankind's health field, outward
All blood is typically frozen by two ways, and one kind is to add after centrifugal concentrating to freeze reagent and freeze, and another kind is in extracting blood
Different cells frozen respectively.Both modes operate all sufficiently complex, and can lose including such as cell factor
Some beneficiating ingredients.
The content of the invention
The invention technical problem to be solved
In view of above-mentioned technical task, it was unexpectedly observed that by dimethyl sulfoxide (DMSO), hydroxymethyl cellulose after inventor studies repeatedly
Sodium, MEM culture mediums are configured to freeze reagent, and the concentration of dimethyl sulfoxide (DMSO), sodium cellulose glycolate is adjusted to far above biography
When system freezes the concentration in reagent, it is possible to achieve the effect for directly freezing, i.e. using the concentration when freezing reagent, without right
Peripheral blood carries out the operations such as concentration centrifugation, and this is frozen into reagent with peripheral blood with substantially 1:1 ratio mixing after directly freeze in-
80 DEG C or so, total cell vigor is almost identical with new blood after recovery, can reach and very excellent freeze effect.
Scheme for solving above-mentioned technical problem
(1) one kind freezes reagent, including：10~20W/V% of sodium cellulose glycolate, 15~35V/V% of dimethyl sulfoxide (DMSO),
65~85V/V% of MEM culture mediums.
(2) one kind freezes reagent, including：10~15W/V% of sodium cellulose glycolate, 15~30V/V% of dimethyl sulfoxide (DMSO),
65~85V/V% of MEM culture mediums.
(3) reagent is frozen as described in (1) or (2), is also included：
0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose, 3~7W/V% of inosine, sodium acid carbonate
0.5~2.5W/V%.
(4) purposes of the reagent in pet peripheral blood freezes is frozen as described in (1) or (2).
(5) purposes of the reagent in pet peripheral blood freezes is frozen as described in (3).
(6) a kind of direct cryopreservation methods of peripheral blood, comprise the following steps：
A. the reagent that freezes any one of (1)~(3) is injected separately into cell cryopreservation tube；
B. pet peripheral blood is gathered；
C.30 in minute, the peripheral blood that will be gathered in step b is added in the cryopreservation tube of step a, is mixed；
D. one day night will be frozen at cryopreservation tube -80 ± 5 DEG C；
E. cryopreservation tube is moved into liquid nitrogen longer-term storage.
Invention effect
Pet peripheral blood of the invention freezes reagent and can directly freeze pet peripheral blood, compared with conventional method, without
Centrifugal concentrating is carried out to blood, the beneficiating ingredient in blood is remained to greatest extent.During use, only reagent need to will be frozen
With blood with substantially 1：1 ratio mixing, directly can freeze, when freezing reagent without using tradition after mixing at -80 DEG C or so
Programmed cooling step.In addition, pet peripheral blood of the invention freezes cryopreservation methods and coordinates this to freeze reagent and uses, side
Just, fast, effect stability, the peripheral blood for freezing is after recovery, and Cell viability is high, can facilitate pet in aging state and disease
The immunization therapy of beauty, treatment of arthritis, anti-aging, tumour is carried out during state using cell component in own health peripheral blood
Deng.
Brief description of the drawings
Fig. 1 is the monocyte 2h differential velocity adherent 100X photograph via bright fields of control group.
Fig. 2 is the 9m monocyte 2h differential velocity adherent 100X photograph via bright fields of the embodiment of the present invention 4.
Specific embodiment
The first aspect of the present invention is that pet peripheral blood freezes reagent.
Pet peripheral blood of the invention is frozen in reagent, and main component is sodium cellulose glycolate, dimethyl sulfoxide (DMSO)
And MEM culture mediums (DMSO).
Of the invention to freeze reagent, the concentration of sodium cellulose glycolate is preferably 10~20W/V%, if methylol is fine
The concentration of the plain sodium of dimension is less than 10W/V%, and if peripheral blood directly freezes in the reagent, the cell survival rate after recovery is relatively low,
The requirement for freezing cannot be met.If the concentration of sodium cellulose glycolate is higher than 20W/V%, peripheral blood directly freezes in the reagent
If depositing, the sodium cellulose glycolate of high concentration can cause cell dehydration in peripheral blood, directly result in cell death.Methylol is fine
The concentration of the plain sodium of dimension is more preferably 10~15W/V%.
Of the invention to freeze reagent, the concentration of dimethyl sulfoxide (DMSO) is preferably 15~35V/V%, if dimethyl sulfoxide (DMSO)
Concentration is less than 15V/V%, and if peripheral blood directly freezes in the reagent, the cell survival rate after recovery is relatively low, it is impossible to meet
The requirement for freezing.If the concentration of dimethyl sulfoxide (DMSO) is higher than 35V/V%, highly concentrated if peripheral blood directly freezes in the reagent
The dimethyl sulfoxide (DMSO) of degree can cause serious cell membrane toxicity, influence cryopreservation resuscitation efficiency.The concentration of dimethyl sulfoxide (DMSO) is more preferably
It is 15~30V/V%, such as more preferably 20V/V%.
It is of the invention to freeze reagent preferably also comprising synergistic component 0.1~0.5W/V% of polyvinylpyrrolidone, glucose 1
~10W/V%, 3~7W/V% of inosine, 0.5~2.5W/V% of sodium acid carbonate.By adding these compositions, invention effect can be made
More preferably.
Preferably, the reagent that freezes of the invention contains sodium cellulose glycolate 10W/V%, dimethyl sulfoxide (DMSO) 20V/V%,
MEM culture mediums 80V/V%；And polyvinylpyrrolidone 0.3W/V%, glucose 5W/V%, inosine 4W/V%, sodium acid carbonate
1.5W/V%.
The second aspect of the present invention is to coordinate this directly to freeze the cryopreservation methods of the peripheral blood that reagent is used, including following step
Suddenly：
A. peripheral blood of the invention is frozen into reagent and is injected separately into cell cryopreservation tube；
B. pet peripheral blood is gathered；
C.30 in minute, the peripheral blood that will be gathered in step b is added in the cryopreservation tube of step a, is mixed；
D. one day night will be frozen at cryopreservation tube -80 ± 5 DEG C；
E. cryopreservation tube is moved into liquid nitrogen longer-term storage.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition or according to institute of manufacturer
The condition of suggestion.Unless otherwise indicated, otherwise percentage and number is calculated by weight.For example, W/V% contains in representing every 100ml
Some weight, unit is g.V/V% represents the volume contained in every 100ml, and unit is ml.
Unless otherwise defined, all specialties used in text and scientific words and meaning familiar to one skilled in the art institute
Justice is identical.Additionally, during any method similar to described content or impartial and material all can be applied to the present invention.Described in text
Preferable implementation only presented a demonstration with material and be used.
1. pet peripheral blood freezes the preparation of reagent
Respectively according to the amount shown in table 1, measure dimethyl sulfoxide (DMSO) (abbreviation DMSO, purchased from SIGMA), MEM culture mediums and (be purchased from
GIBCO 250ml indigo plant mouths bottle (purchased from another name for Sichuan Province ox)) is put into, jog is mixed.Then hydroxymethyl cellulose is weighed according to the amount shown in table 1
Sodium (being purchased from SIGMA), polyvinylpyrrolidone (abbreviation PVP, purchased from SIGMA), glucose (being purchased from SIGMA), inosine (are purchased from
SIGMA during), sodium acid carbonate (being purchased from SIGMA) adds above-mentioned blue mouth bottle, mixing is rocked.With 0.22 μm after solid all dissolves
Filter (being purchased from MILLIPORE) filtration sterilization, filtering is prepared real to by autoclaved 100ml indigo plant mouths bottle (being purchased from another name for Sichuan Province ox)
Example and comparative example are applied, 4 DEG C of refrigerators is stored in standby.
Table 1：Freeze the preparation of reagent
2. cryopreservation methods of pet peripheral blood
Freeze pet peripheral blood in accordance with the following steps successively：
A. to be injected separately into each 10ml thin for each embodiment and the reagent that freezes of comparative example for about 5ml being prepared as shown in table 1
In born of the same parents' cryopreservation tube (being purchased from SIGMA)；
B. purple skull vacuum test tube (being purchased from Kang Jie) the collection pet periphery containing ethylenediamine tetra-acetic acid anti-coagulants is used
Blood about 5ml, gentle inversion is mixed, and is placed on ice；
C.30min it is interior, the peripheral blood gathered in step b is separately added into the cryopreservation tube of step a, gentle inversion is mixed；
D. -80 DEG C of refrigerators (purchased from PANASONIC) are immediately placed in freeze in short term；
E. liquid nitrogen (being purchased from MVE) longer-term storage is moved into after -80 DEG C of refrigerators freeze 24h；
When f. needing recovery, it is put into 37 DEG C of water-baths rapidly after taking out cryopreservation tube, rocks defrosting, is carried out at once after defrosting follow-up
Operation.
3. effect test
Total cell viability examination after 3.1 pet peripheral blood cryopreservation resuscitations
After taking out the cryopreservation tube for freezing reagent and peripheral blood containing embodiment/comparative example respectively, 37 DEG C of water are put into rapidly
In bath cabinet (purchased from power occasion science and technology), defrosting is rocked, different cryopreservation resuscitation time points is set, recovery is labeled as 3m after freezing 3 months,
Recovery is labeled as 6m after freezing 6 months, and recovery is labeled as 9m after freezing 9 months.
Separately take the fresh peripheral bloods of 5ml to be well mixed with the frozen stock solution of 5ml embodiments 4, without freezing, labeled as blank pair
According to group.
UseAutomated cell calculating instrument (is purchased from LIFE TECH), cell count is carried out to each group and is deposited
Motility rate is calculated.Each group in triplicate, is averaged as a result respectively.
Table 2：Total cell viability examination (n=3) after pet peripheral blood cryopreservation resuscitation
From experimental result, using embodiments of the invention 1~6 freeze reagent directly freeze peripheral blood 3 months, 6
Total cell vigor compares new blood after recovering within individual month, 9 months does not have significant changes, illustrates that the reagent that directly freezes of the invention exists
Convenient, fast realization can well protect cell in peripheral blood while freezing.
In comparative example 1,2, the concentration of sodium cellulose glycolate is less than 10W/V%, and the cell survival rate after recovery is relatively low, nothing
Method meets the requirement for freezing.
Comparative example 3 freezes reagent, and the concentration of dimethyl sulfoxide (DMSO) is less than 15V/V%, the cell survival rate after recovery compared with
It is low, it is impossible to meet the requirement for freezing.
Comparative example 4 freezes reagent, and the concentration of sodium cellulose glycolate is higher than 20W/V%, the cell after recovery is universal
Shrinkage, survival rate is relatively low, it is impossible to meet the requirement for freezing.
Comparative example 5 freezes reagent, and the concentration of dimethyl sulfoxide (DMSO) is higher than 35V/V%, the cell fragment after recovery is more,
Survival rate is relatively low, it is impossible to meet the requirement for freezing.
Monocyte differential attachment method culture is compared after 3.2 pet peripheral blood cryopreservation resuscitations
After falling into a trap and count up to and finish at above-mentioned 3.1, the peripheral blood that the 9m of blank control group and embodiment 4 recovers is moved into 15ml
In sterile centrifugation tube.900 turns of 3min centrifugations of centrifuge (being purchased from Hunan instrument), supernatant discarded contains 10% hyclone using 10ml
The DMEM high glucose mediums (being purchased from GIBCO) of (being purchased from GIBCO) are resuspended.Plant 100mm Tissue Culture Dish (being purchased from CORNING)
In, 5%CO2, 37 DEG C of incubator cultures.Liquid is changed after 2h, most of non-attached cell, attached cell is discarded for monocyte.It is real
Test result as depicted in figs. 1 and 2, Fig. 1 is blank control group monocyte 2h differential velocity adherent 100X photograph via bright fields, and Fig. 2 is in reality
Apply after example 4 to freeze and freeze 24h in reagent and move into liquid nitrogen 9m, monocyte 2h differential velocity adherent 100X photograph via bright fields.By Fig. 1 and Tu
2 is visible, and both illustrate that the reagent that directly freezes of the invention can be very well while convenient, fast realization freezes without significant difference
Protection peripheral blood in cell.
Below the preferred embodiment to the invention is illustrated, but the invention be not limited to it is described
Embodiment, those of ordinary skill in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (6)
1. one kind freezes reagent, including：
10~20W/V% of sodium cellulose glycolate, 65~85V/V% of dimethyl sulfoxide (DMSO) 15~35V/V%, MEM culture medium.
2. one kind freezes reagent, including：
10~15W/V% of sodium cellulose glycolate, 65~85V/V% of dimethyl sulfoxide (DMSO) 15~30V/V%, MEM culture medium.
3. reagent is frozen as claimed in claim 1 or 2, also included：
0.1~0.5W/V% of polyvinylpyrrolidone, 1~10W/V% of glucose, 3~7W/V% of inosine, sodium acid carbonate 0.5~
2.5W/V%.
4. purposes of the reagent in pet peripheral blood freezes is frozen as claimed in claim 1 or 2.
5. purposes of the reagent in pet peripheral blood freezes is frozen as claimed in claim 3.
6. a kind of direct cryopreservation methods of peripheral blood, comprise the following steps：
A. the reagent that freezes any one of claims 1 to 3 is injected separately into cell cryopreservation tube；
B. pet peripheral blood is gathered；
C.30 in minute, the peripheral blood that will be gathered in step b is added in the cryopreservation tube of step a, is mixed；
D. one day night will be frozen at cryopreservation tube -80 ± 5 DEG C；
E. cryopreservation tube is moved into liquid nitrogen longer-term storage.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710064441.1A CN106719604A (en) | 2017-02-04 | 2017-02-04 | Pet peripheral blood directly freezes reagent and method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710064441.1A CN106719604A (en) | 2017-02-04 | 2017-02-04 | Pet peripheral blood directly freezes reagent and method |
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| CN201710064441.1A Pending CN106719604A (en) | 2017-02-04 | 2017-02-04 | Pet peripheral blood directly freezes reagent and method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112544611A (en) * | 2020-12-16 | 2021-03-26 | 生物岛实验室 | Cell cryopreservation agent and cell cryopreservation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112544611A (en) * | 2020-12-16 | 2021-03-26 | 生物岛实验室 | Cell cryopreservation agent and cell cryopreservation method |
| CN112544611B (en) * | 2020-12-16 | 2021-10-29 | 生物岛实验室 | Cell cryopreservation agent and cell cryopreservation method |
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Application publication date: 20170531 |