CN110024775A - The clinical cytology preparation formula and purposes for saving liquid - Google Patents
The clinical cytology preparation formula and purposes for saving liquid Download PDFInfo
- Publication number
- CN110024775A CN110024775A CN201910388402.6A CN201910388402A CN110024775A CN 110024775 A CN110024775 A CN 110024775A CN 201910388402 A CN201910388402 A CN 201910388402A CN 110024775 A CN110024775 A CN 110024775A
- Authority
- CN
- China
- Prior art keywords
- cell
- liquid
- clinical cytology
- preparation
- clinical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of clinical cytology preparations with liquid is saved, and it includes the components of following concentration:
Description
Technical field
The invention belongs to cell cryopreservation fields, more particularly to a kind of clinical cytology preparation preservation liquid and purposes.
Background technique
Cells frozen storing liquid protects cell in frozen storage process when saving cell, through cryoprotector, traditional
Cell-protecting is only intracellular protective agent, the damage by ice crystal is easy when cell is frozen, Cell viability is low, can not be effective
Protect cell.In addition, cells frozen storing liquid in the prior art usually contains animal derived substance, complicated component, in clinical application
On there are potential risks, also increase the probability of pollution, while most of frozen stock solutions all contain DMSO (dimethyl is sub- at present
Sulfone), there are certain toxicity for clinical application.
DMSO freezen protective is related to the xicity related risk of DMSO, especially in the application of cell adoptive therapy.For example,
The infusion for the Cell Cryopreservation that Zambelli et al. had evaluated transplanting in 1998 is xicity related, and determines and exist in graft
DMSO amount it is related with toxic grade.Davis et al. has found that nearly all patient for receiving freezen protective is in nineteen ninety
Self cellular transplant shows as expiratory dyspnea (83%), and heart rate reduces (98%) and transient hypertension (96%), these
All due to infusion DMSO.In 1991, he had found infusion correlated response, mainly nausea and shiver with cold, the bone with freezen protective
Implantation of marrow is related.In addition, occurring the nausea of higher level after the bone-marrow transplantation of freezen protective in pediatric patients, vomit, the rhythm of the heart
Not normal and low blood pressure (Okamoto, 1993).
Traditional cell preparation needs to be fed back in 12h after completing preparation, drops to more than 12h Cell viability
80% or so, therapeutic effect is influenced, while cell preparation can not carry out the indexs such as the sterile, mycoplasma of cell immediately and finally determine.
There is a need in the art for the clinical cytology preparation preservation liquid for solving above-mentioned technical problem.
Summary of the invention
For the above-mentioned prior art, the present invention provides a kind of clinical cytology preparations with liquid is saved, and can be used for cell long-period
Preservation, can be applied to the foundation of cell bank, scientific research cell strain saves, clinical mescenchymal stem cell, monocyte, immunocyte
And the long-term preservation of involved all mammalian cells, while DMSO ingredient and animal sources are free of in frozen stock solution
Ingredient, it is safer, meet clinical needs.
Since most of lymphocyte therapies need the cell of regular injections multi-dose, the toxic effect of DMSO is can
With accumulation, therefore invention removes DMSO ingredient, it ensure that cells frozen storing liquid is safer in clinical application.Using facing
Bed cell preparation with save liquid, freeze-stored cell preparation can be shifted to an earlier date, and be stored for a long time, freeze before sampling carry out cell without
The Indexs measures such as bacterium, mycoplasma, reuse after qualification, not only ensure that cell clinical quality in this way, but also pass through clinical grade
The cell preparation that serum-free frozen stock solution obtains has passed through security test, and motility rate is greater than 90% after recovery, can be directly using note
Enter human body.
Clinical cytology preparation of the invention is as shown in table 1 with preservation liquid and the comparison of conventional cell frozen stock solution application method.
Table 1: clinical cytology preparation of the invention saves liquid compared with the application method of conventional cell frozen stock solution
Clinical cytology preparation of the invention is saved liquid and can be saved with 2-8 DEG C, without packing；2-8 degrees Celsius of preservation, without anti-
Multiple freeze thawing link, that is, take and use；Cold process of depositing is to deliver directly -80 DEG C of refrigerator overnights, and next day enters liquid nitrogen, operates more simplified.
Using clinical cytology preparation of the invention, can in the case where keeping cell phenotype Long-term Cryopreservation, such as 1
Year, 2 years or longer time freeze.
Compared with traditional frozen stock solution, the motility rate of freeze-stored cell can be improved using clinical cytology preparation of the invention, it is such as multiple
48 hours microphotos determine after Cell viability measurement and recovery after Soviet Union.
Using clinical cytology preparation of the invention, cell is directly recovered, cell preparation is made, during which without cleaning,
90% or more motility rate is able to maintain in 12h.
On the one hand, the present invention provides a kind of clinical cytology preparations with liquid is saved, and may include the group of following concentration
Point:
The concentration unit of each component is mg/L.
In one embodiment, the concentration of potassium dihydrogen phosphate can be 100-500, preferably 200-400, preferably 210-
300, preferably 150,200,210,220 or 250mg/L.
In one embodiment, the concentration of disodium hydrogen phosphate can be 300-900, preferably 400-800, preferably 600-
750, preferably 500,650,700,726,730 or 850mg/L.
In one embodiment, the concentration of sodium chloride can for 3000-10000, preferably 5000-9500, preferably 4000,
6000,7000,8000,8500,8800,8900,9000,9100,9200,9300 or 9400mg/L.
In one embodiment, the concentration of glucose can be 3000-15000；It is preferred that 4000-10000, preferably
4500-8000, preferably 3000,4100,4500,4600,5000,6000,7000mg/L.
In one embodiment, the concentration of extracellular cryoprotector can be 1000-6000, preferably 2000-5000,
It is preferred that 2500-3000, preferably 2400,2500,2600,2700 or 4000mg/L.
In one embodiment, the concentration of intracellular cryoprotector can be 1000-6000, preferably 2000-5500,
It is preferred that 3000-5000, preferably 5000mg/L.
In one embodiment, clinical cytology preparation is with saving the pH value of liquid to 7-10,7-8,7-7.5,7.1-7.4,
It is preferred that 7.2-7.3.
In one embodiment, disodium hydrogen phosphate is seven hypophosphite monohydrate disodium hydrogens.
In one embodiment, extracellular cryoprotector is selected from: hydroxyethyl starch, polyvinylpyrrolidone (PVP),
Sucrose, polyethylene glycol, glucan and arabogalactan.
In one embodiment, intracellular cryoprotector is selected from: glycerol, propylene glycol and ethylene glycol.
In one embodiment, clinical cytology preparation is free of DMSO, protein and/or serum with liquid is saved.
In one embodiment, the concentration of extracellular cryoprotector is 2000-5000mg/L and/or the cell
The concentration of interior cryoprotector is 2000-5000mg/L.
In one embodiment, the concentration of extracellular cryoprotector is 2000mg/L and the intracellular cryoprotection
The concentration of agent is 5000mg/L.In one embodiment, the concentration of extracellular cryoprotector is 5000mg/L and described thin
The concentration of cryoprotector intracellular is 2000mg/L.In one embodiment, the concentration of extracellular cryoprotector is
The concentration of 5000mg/L and the intracellular cryoprotector is 5000mg/L.In one embodiment, extracellular freezing is protected
Shield agent is polyvinylpyrrolidone and intracellular cryoprotector is propylene glycol.
In one embodiment, clinical cytology preparation, which saves liquid, can be free of one or more amino acid.Amino acid
Can be l-amino acid, such as 20 kinds of natural L-amino acids, for example, glycine, alanine, valine, leucine, isoleucine,
Phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, Soviet Union's ammonia
Acid, aspartic acid, glutamic acid, lysine, arginine and histidine.
On the other hand, the present invention provides the clinical cytology preparation preparation methods for saving liquid comprising mixing is clinical
The cell preparation various components for saving liquid.In one embodiment, the method includes adjusting pH value to 7-10,7-8,7-
7.5,7.1-7.4, preferably 7.2-7.3.
On the other hand, the application the present invention provides clinical cytology preparation with preservation liquid in freeze-stored cell.
In one embodiment, it freezes as Long-term Cryopreservation, such as 1 year, 2 years, 3 years or the cold of longer time are deposited.
In one embodiment, cell is selected from: mescenchymal stem cell, mononuclearcell, T cell and NK cell.
The present invention is achieved by the following technical solutions:
A kind of clinical grade clinical cytology preparation preservation liquid, is to be grouped as by the group of following concentration, if each concentration unit
It is mg/L without special instruction:
The extracellular cryoprotector is chosen as hydroxyethyl starch, polyvinylpyrrolidone (PVP), sucrose, poly- second two
Alcohol, glucan, arabogalactan.
The intracellular cryoprotector is glycerol, propylene glycol, ethylene glycol.
Clinical grade clinical cytology preparation of the invention save liquid the preparation method comprises the following steps: taking above-mentioned component in addition to water, root
It according to its respectively dissolution characteristics classification dissolution, then mixes, water, which is added, makes each component final concentration as described above, adjusting pH value to 7.1
~7.4 to get, industrial filter element filtering, while nitrogen protection is dispensed and (unstability ingredient avoided to be oxidized).
Clinical cytology preparation of the invention contains duplicate protection composition with liquid is saved, from it is intracellular with extracellularly protect simultaneously
The damage of ice crystal, substantially increases the motility rate of cell when protecting cell from freezing, and adapts to extensive cell line and saves, while this hair
It is complete serum-free frozen stock solution that bright clinical cytology preparation, which saves in liquid, avoid the heterologous foreign that is introduced in clinical application at
Part, it is safer.
Clinical cytology preparation of the invention improves the motility rate of cell cryopreservation, cell cryopreservation adaptability is wider with liquid is saved
It is general, freeze motility rate and averagely reach 90% or more, while the product can be reserved for and cool down when 2~8 DEG C, freeze-stored cell without program, it can
It is placed directly within -80 DEG C to freeze, Liquid nitrogen storage is placed directly within after 12h, operationally simplify and largely freeze program, while product
More stable, without having to worry about multigelation to product influence.
The present invention also provides the following contents:
1. a kind of clinical cytology preparation preservation liquid, it is characterised in that: it is to be grouped as by the group of following concentration, each concentration
Unit is mg/L unless otherwise noted:
2. according to the preservation liquid of clinical cytology preparation described in item 1, it is characterised in that: the extracellular cryoprotector choosing
From hydroxyethyl starch.
3. according to the preservation liquid of clinical cytology preparation described in aforementioned item, it is characterised in that: be by the component of following concentration
Composition, each concentration unit is mg/L unless otherwise noted:
4. according to the preservation liquid of clinical cytology preparation described in aforementioned item, it is characterised in that: be by the component of following concentration
Composition, each concentration unit is mg/L unless otherwise noted:
5. according to the preservation liquid of clinical cytology preparation described in aforementioned item, it is characterised in that: be by the component of following concentration
Composition, each concentration unit is mg/L unless otherwise noted:
6. according to the preparation method for saving liquid of clinical cytology preparation described in aforementioned item, it is characterised in that: take in addition to water
Component, according to its respectively dissolution characteristics classification dissolution, then mix, be added water make each component final concentration as described in aforementioned item,
Adjust pH value to 7.1~7.4 to get.
7. the application according to clinical cytology preparation described in aforementioned item with preservation liquid in freeze-stored cell.
Detailed description of the invention
Fig. 1: the photo for freezing peripheral blood mononuclear cells using the clinical cytology preparation of optimization with preservation liquid and recovering
(200 times of magnifying powers).
Fig. 2: the photo (200 times of magnifying powers) that peripheral blood mononuclear cells is frozen using conventional cell frozen stock solution and is recovered.
Fig. 3: the photo (200 after being frozen NK cell with preservation liquid using the clinical cytology preparation of optimization and recovered 48 hours
Times magnifying power).
Fig. 4: the photo (200 times of magnifying powers) after freezing NK cell using conventional cell frozen stock solution and recover 48 hours.
Fig. 5: using the clinical cytology preparation photo for saving liquid freezing and storing umbilical mesenchymal stem cells and recovering of optimization
(200 times of magnifying powers).
Fig. 6: using conventional cell frozen stock solution freezing and storing umbilical mesenchymal stem cells and the photo (200 times of magnifying powers) of recovery.
Fig. 7: using the clinical cytology preparation preservation liquid cryopreserved T cells of optimization and (200 times of photo after recovery 48 hours
Magnifying power).
Fig. 8: the photo (200 times of magnifying powers) with conventional cell frozen stock solution cryopreserved T cells and after recovering 48 hours.
The fluidic cell phenotypic map of various phenotypic markers before Fig. 9 A-9G:MSC freezes.
Figure 10 A-10G:MSC saves the fluidic cell phenotypic map of 1 year various phenotypic marker after freezing.
Specific embodiment
It is provided below defined below in order to understanding the present invention.
Term " extracellular cryoprotector " refers to the impermeability protective agent for being unable to penetrating cell film.In the present invention, carefully
Extracellular cryoprotector can be hydroxyethyl starch, polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, glucan and/or
Arabogalactan.
Term " intracellular cryoprotector " refers to can penetrating cell permeability of the membrane protective agent.In the present invention, into the cell
Cryoprotector can be glycerol, propylene glycol, ethylene glycol.
Term " clinical cytology preparation preservation liquid " is referred to as cells frozen storing liquid, refers to liquid used in cell cryopreservation
Body.
In the present invention, clinical cytology preparation can not add amino acid with liquid is saved.Amino acid can be l-amino acid,
Such as one of 20 kinds of natural L-amino acids, such as glycine, alanine, valine, leucine, isoleucine, phenylalanine, dried meat
Propylhomoserin, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid,
Glutamic acid, lysine, arginine and/or histidine.In one embodiment, clinical cytology preparation preservation liquid can be different
Shi Tianjia or amino containing 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 kind or more
Acid.
In the present invention, frozen storage process is by cell, such as MSC (mescenchymal stem cell), (primary point of mononuclearcell
From), T cell or NK cell clinical cytology preparation is added with saving (about 1E7 cell of total number of cells, clinical cytology preparation in liquid
With preservation liquid 1mL), it is placed directly within -80 DEG C and freezes, Liquid nitrogen storage is placed directly within after 12h.Resuscitation process is by cell recovery, fastly
Recovery, adds phosphate buffer solution.Specifically, water-bath is opened, is adjusted to 37 DEG C, it is thin by what is frozen after temperature to be placed is stablized
Born of the same parents, which are placed in 37 DEG C of water-baths, dissolves, time 2min, after the cell suspension wait freeze melts, is rapidly added phosphate buffer solution
In, protection liquid is taken out in centrifugation (1000rpm, 5min).Then by cell precipitate injection physiological saline+human serum albumins
Clinic preparation is made in suspension.
In tradition freezes and recovers, with conventional cell frozen stock solution (traditional frozen stock solution formula are as follows: 70% DMEM+10%
The fetal calf serum of DMSO+20%) it is frozen.Specifically, with 1E7 cell of total number of cells, conventional cell frozen stock solution 1mL into
Row, 4 DEG C of 30min of program cooling；-20℃1h；- 80 DEG C overnight, and next day, which puts into, saves 12h in liquid nitrogen.It is then turned on water-bath, is adjusted
To 37 DEG C, after temperature to be placed is stablized, the cell frozen is placed in 37 DEG C of water-baths and is dissolved, time 2min, to be frozen is thin
It after born of the same parents' suspension melts, is rapidly added in appropriate phosphate buffer solution, protection liquid is taken out in centrifugation (1000rpm, 5min).Melt
Afterwards, it needs to be cleaned repeatedly with PBS three times, 1000rpm, 5min, abandons supernatant.Then by cell precipitate injection stage physiological saline
Clinic preparation is made in the suspension of+human serum albumins.
Embodiment
Below with reference to embodiment, the present invention is further illustrated.
Instrument involved in following embodiments, reagent, material etc. are unless otherwise noted existing in the prior art
Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Experimental method involved in following embodiments, inspection
Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..
Embodiment 1: it prepares clinical cytology preparation and saves liquid
With following ingredient and concentration prepare clinical cytology preparation save liquid, each concentration unit unless otherwise noted,
For mg/L:
The extracellular cryoprotector is hydroxyethyl starch, is purchased from sigma, similarly hereinafter.
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjusting pH value to 7.2 to get, industrial filter element filtering, while nitrogen protection is divided
It fills (unstability ingredient is avoided to be oxidized).
Embodiment 2: it prepares clinical cytology preparation and saves liquid
With following ingredient and concentration prepare clinical cytology preparation save liquid, each concentration unit unless otherwise noted,
For mg/L:
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjusting pH value to 7.2 to get, industrial filter element filtering, while nitrogen protection is divided
It fills (unstability ingredient is avoided to be oxidized).
Embodiment 3: it prepares clinical cytology preparation and saves liquid
With following ingredient and concentration prepare clinical cytology preparation save liquid, each concentration unit unless otherwise noted,
For mg/L:
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjusting pH value to 7.2 to get, industrial filter element filtering, while nitrogen protection is divided
It fills (unstability ingredient is avoided to be oxidized).
Embodiment 4: it prepares clinical cytology preparation and saves liquid
With following ingredient and concentration prepare clinical cytology preparation save liquid, each concentration unit unless otherwise noted,
For mg/L:
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjusting pH value to 7.2 to get, industrial filter element filtering, while nitrogen protection is divided
It fills (unstability ingredient is avoided to be oxidized).
Embodiment 5: it prepares clinical cytology preparation and saves liquid
Clinical cytology preparation is prepared with following ingredient and concentration and saves liquid, is mg/L:
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjusting pH value to 7.2 to get, industrial filter element filtering, while nitrogen protection is divided
It fills (unstability ingredient is avoided to be oxidized).
Embodiment 6: it prepares clinical cytology preparation and saves liquid
With following ingredient and concentration prepare clinical cytology preparation save liquid, each concentration unit unless otherwise noted,
For mg/L:
The preparation method comprises the following steps: taking above-mentioned component in addition to water, according to its respectively dissolution characteristics classification dissolution, then mixes, add
Enter water and make each component final concentration as described above, adjusting pH value to 7.2 to get, industrial filter element filtering, while nitrogen protection is divided
It fills (unstability ingredient is avoided to be oxidized).
Embodiment 7: the clinical cytology preparation of the invention Performance for saving liquid
1. motility rate measures
The clinical cytology preparation prepared using embodiment 1,2,3,4,5,6 freezes mode with liquid, freeze-stored cell is saved are as follows:
By cell shown in table 2, i.e. the training of MSC (mescenchymal stem cell), mononuclearcell (primary separation), T cell or NK cell
Above-mentioned clinical cytology preparation (1E7 cell of total number of cells in preservation liquid is added in sediment after nutrient solution centrifugation；Clinical cytology system
Agent preservation liquid 1mL), it is placed directly within -80 DEG C and freezes, Liquid nitrogen storage is placed directly within after 12h.After saving 1 week, by cell recovery,
Fast recovery, adds phosphate buffer solution.Specifically, water-bath is opened, 37 DEG C are adjusted to, after temperature to be placed is stablized, by what is frozen
Cell, which is placed in 37 DEG C of water-baths, to be dissolved, time 2min, and after the cell suspension wait freeze melts, it is molten to be rapidly added phosphate-buffered
In liquid, centrifugation (1000rpm, 5min) discards protection liquid.In parallel, with conventional cell frozen stock solution (traditional frozen stock solution formula are as follows:
The fetal calf serum of 70% DMEM (friendly health biological product, article No.: CM0101)+10%DMSO+20%) it is that control is tested.
Specifically, with 1E7 cell of total number of cells, conventional cell frozen stock solution 1mL is carried out, 4 DEG C of 30min of program cooling；-20℃1h；-80
DEG C overnight, next day put into liquid nitrogen in save 1 week；It is then turned on water-bath, 37 DEG C is adjusted to, after temperature to be placed is stablized, will freeze
Cell be placed in 37 DEG C of water-baths and dissolve, time 2min is rapidly added 10mL phosphate after the cell suspension wait freeze melts
In buffer solution, protection liquid is taken out in centrifugation (1000rpm, 5min).It after thawing, needs to be cleaned with PBS, 1000rpm, 5min, abandon
Supernatant.Appropriate phosphate buffer solution is added to sediment later, suspension is made, it is spare.
Then above-mentioned respective frozen stock solution is calculated treated Cell viability.Motility rate calculates as follows: the cell after recovery is hanged
Liquid draws 100ul, and the trypan blue solution of 100ul0.04% is added, and 10ul is drawn after mixing well and is added in blood counting chamber, quilt
It is dead cell that trypan blue, which dyes blue, and undyed is living cells, by calculating the cell of survival and the ratio of total cell quantity
Value, to calculate cell recovery motility rate).Comparison is as shown in table 2.
It is compared by the motility rate of the preservation liquid to different formulations, determines two kinds of cryoprotector combinations in embodiment 4
Effect is best.Two kinds of cryoprotectors are PVP (polyvinylpyrrolidone) and propylene glycol, and are formulated for embodiment 4
Optimization is formulated as follows:
In terms of mg/L:
The formula of embodiment 4 (or preservation liquid of referred to as optimization) after optimization is also shown in the comparison of conventional cell frozen stock solution method
Table 2.
Table 2: the different clinical cytology preparations of the invention cell recovery motility rate ratio for saving liquid and conventional cell frozen stock solution
Compared with
MSC (mescenchymal stem cell), mononuclearcell (primary separation), T cell and NK cell are internal separation and training
It supports.In short, MSC cell: planting the inoculation of block method by umbilical cord tissue, climbed out of by the magnificent Tong Shi glue of umbilical cord, by thin to what is climbed out of
Born of the same parents freeze after passing on.Mononuclearcell: peripheral blood is extracted, it is direct that mononuclearcell is isolated by lymphocyte separation medium
It freezes.T cell: extract peripheral blood, by lymphocyte separation medium isolate mononuclearcell directly add the induced t cell factor into
Row culture, inducing T cell directly freeze.NK cell: peripheral blood is extracted, mononuclearcell is isolated by lymphocyte separation medium
Directly plus NK cell inducible factors are cultivated, and the NK cell of induction directly freezes.It the preparation process of these cells and cultivated
Cheng Junwei conventional manufacturing process.These cells can also directly be bought by commercial sources acquisition and according to the explanation of supplier into
Row culture.
2. the phenotypic analysis for passing through flow cytometry
If the above-mentioned frozen stock solution with optimization saves MSC cell, after freezing 1 year, by before being frozen to MSC and after recovery
Phenotype detected.Prove in the frozen stock solution of optimization it is long-term it is cold deposit after on MSC phenotype without influence, cell function can be maintained not
Become, guarantees cell better quality.
Specifically, the present embodiment before freeze-stored cell and freezes carry out table after 1 year using the preservation liquid of above-mentioned optimization
Type detection, by the antibody of label (for CD73 CD105 CD45 CD34 CD90 CD44 antibody；Supplier is
Peprotech, article No. are as follows: CD45:07111-60-100, CD44:06511-50-100, CD105:17111-60-100；CD73:
05811-80-100；Supplier: BioGEMS, CD90:03011-50-100；Supplier BD, CD34:555822) it is incubated for and
Dyeing.In short, cell 1000rpm, the 5min eccentric cleaning that will be detected, each sample detection total number of cells are 1E5, dissolution
In 200ul PBS solution, antibody 5ul is added, while setting negative control (antibody is not added) room temperature and being protected from light incubation 15min, is incubated for
After, 1000rpm, 5min centrifugation remove supernatant, and be resuspended with PBS, and direct flow cytometer detection detects 10000 thin
Born of the same parents, slow running, the fluorescent dye channel according to corresponding to antibody are configured, and testing result is shown in Fig. 9 A-9G and Figure 10 A-
10G).Using instrument BD c6 stream type cell analyzer (setting corresponding fluorescence channel according to fluorescent dye to detect), according to supply
The explanation that quotient provides is measured.As a result as shown in table 3.
Table 3: the result obtained according to Flow Cytometry Assay: phenotype summarizes before MSC freezes and after freezing 1 year
Note: umbilical cord mesenchymal stem cells negative phenotype is CD34-, CD45-；Positive phenotypes are CD44+, CD73+, CD105+
And CD90+.Umbilical cord mesenchymal stem cells judge index is negative phenotype < 2%, positive phenotypes > 95%.
Table 3 is demonstrated before freeze-stored cell and cell function is not affected after saving 1 year in the frozen stock solution of optimization,
It still conforms to require, such as be determined by Phenotypic examination.
3. influence of the cell preparation holding time (12 hours and 24 hours) to Cell viability in liquid is saved
1 is frozen to MSC cell (the MSC cell in umbilical cord source) by the preservation liquid and traditional serum-free formula of above-mentioned optimization
Zhou Hou, 4 DEG C of preservations 12 are small after clinical preparation then is made with injection physiological saline+human serum albumins suspension after recovery
When and measure motility rate after 24 hours, freeze it is as described above with resuscitation process, after the dissolution of cell recovery that wherein this experiment freezes
It is directly prepared into cell preparation under conditions of not cleaned and carries out dependence test；Traditional serum-free formula, the cell frozen is at 37 DEG C
It after water-bath dissolution, is cleaned repeatedly with PBS three times, 1000rpm, 5min, abandons supernatant, be made into preparation progress phase after collecting cell precipitation
Close test.(motility rate calculates: the cell suspension after recovery being drawn 100ul, the trypan blue solution of 100ul0.04% is added, sufficiently
It draws 10ul after mixing to be added in blood counting chamber, dying blue by trypan blue is dead cell, and undyed is living cells, is led to
The ratio of the cell and total cell quantity that calculate survival is crossed, to calculate cell recovery motility rate).Experiment is duplicate to be carried out, and is made even
Mean value.
Table 4: the Cell viability of optimization saved in liquid
Table 5: the Cell viability in traditional serum-free formula
| Cell activity records the time | Holding time | 1 motility rate of pipe | 2 motility rate of pipe | Average motility rate |
| October in 2018 27 22:00 | 12h | 94% | 95.3% | 94.65% |
| October in 2018 28 10:00 | 24h | 89% | 91% | 90% |
As shown in table 4 and table 5, cell preparation is made in the MSC that the preservation liquid of optimizing application freezes, directly recovery, interior for 24 hours
It is able to maintain 90% or more motility rate.Using clinical cytology preparation of the invention with liquid is saved, gradient cooling, cell can not had to
Motility rate can reach 90% or more, hence it is evident that be better than conventional cell frozen stock solution.It is not cleaned after the cell recovery dissolution that this experiment freezes
Under conditions of be directly prepared into cell preparation progress dependence test.Traditional serum-free formula due to containing the ingredients such as DMSO and
It needs to clean, the cell frozen is cleaned three times, 1000rpm, 5min repeatedly after 37 DEG C of water-baths are dissolved with PBS, abandons supernatant, is received
It is spare that preparation is made into after collection cell precipitation.
4. micro-imaging
The preservation liquid and conventional cell frozen stock solution of optimization of the invention for shooting handle the microphoto after various cells.It is aobvious
Micro mirror is six factory's 37XB type of glazing, is shot by the slr camera equipment on microscope.
It is cold to deposit with resuscitation process as described in " motility rate measurement " above, 48 hours shooting culture solutions are only cultivated after recovery
Photo (cultivating 48h after referring to inoculation medium for 48 hours after recovery, motility rate calculates consistent with above-mentioned motility rate method) (1, MSC training
It supports: culture medium: MSC serum free medium brand: friendly health biology article No. NC0103 and NC0103.S condition of culture: 37 DEG C, 5% 2
Carbonoxide incubator.2, T cell culture: culture medium: T cell serum free medium brand: friendly health biology article No.: NC0105 culture
Condition: 37 DEG C, 5% carbon dioxide incubator.3, NK cell culture: culture medium: NK cell non-serum culture medium brand: friendly Kang Sheng
Object article No.: NC0102 and AN0102 condition of culture: 37 DEG C, 5% carbon dioxide incubator.4, PBMC is cultivated: the training of T cell serum-free
Feeding base brand: friendly health biology article No.: NC0105 condition of culture: 37 DEG C, 5% carbon dioxide incubator.Motility rate measurement is directly to take
Culture solution dyeing measurement motility rate, as described in " motility rate measurement " above.
Microphoto is shot to the various cells after recovery.
The recovery comparison diagram of MSC cell is as shown in Figure 1 and Figure 2, and Fig. 1 is that the preservation liquid of use optimization freezes MSC cell and answers
The microphoto of Soviet Union, Fig. 2 are the microphoto (200 times of magnifying powers) for freezing MSC cell using conventional cell frozen stock solution and recovering,
By comparison as it can be seen that the motility rate (96%) of the preservation liquid of optimization is substantially better than conventional cell frozen stock solution (85%).Pass through after recovery 48
The photo of hour is it can be concluded that the motility rate after cell cryopreservation is higher.
The recovery comparison diagram of peripheral blood mononuclear cells is as shown in Figure 3, Figure 4.Fig. 3 is outside being frozen using the preservation liquid of optimization
All blood mononuclear cells and the microphoto recovered, Fig. 4 are to freeze peripheral blood mononuclear cells simultaneously using conventional cell frozen stock solution
The microphoto of recovery.By comparison as it can be seen that the motility rate (95%) of the preservation liquid of optimization was substantially better than biography by 48 hours after recovery
It unites cells frozen storing liquid (90%).
T cell recovery comparison diagram is as shown in Figure 5, Figure 6, and Fig. 5 is small using the preservation liquid cryopreserved T cells and recovery 48 that optimize
When after microphoto, Fig. 6 be using conventional cell frozen stock solution cryopreserved T cells and recover 48 hours after microphoto.By right
Than as it can be seen that by 48 hours after recovery photos it can be concluded that the motility rate after cell cryopreservation is higher.
The recovery comparison diagram of NK cell (natural killer cells) is as shown in Figure 7, Figure 8, and Fig. 7 is to be frozen using the preservation liquid of optimization
The photo depositing NK (natural killer cells) and recovering, Fig. 8 are to freeze NK (natural killer cells) simultaneously using conventional cell frozen stock solution
The photo of recovery, by comparison as it can be seen that the motility rate (95%) in the preservation liquid of optimization is substantially better than conventional cell frozen stock solution (85%),
By 48 hours after recovery photos it can be concluded that the motility rate after cell cryopreservation is higher.
Above-mentioned, although specific embodiments of the present invention have been described in conjunction with the embodiments, not protects to the present invention
The limitation of range, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art
The various modifications or changes that can be made are not needed to make the creative labor still within protection scope of the present invention.
Claims (10)
1. a kind of clinical cytology preparation is with liquid is saved, it includes the components of following concentration:
The concentration unit of each component is mg/L；
Preferably pH value is to 7-10,7-8,7-7.5 or 7.1-7.4, preferably 7.2-7.3；
The preferably described disodium hydrogen phosphate is seven hypophosphite monohydrate disodium hydrogens.
2. clinical cytology preparation according to claim 1 is with liquid is saved, wherein the extracellular cryoprotector is selected from: hydroxyl
Hydroxyethyl starch, polyvinylpyrrolidone (PVP), sucrose, polyethylene glycol, glucan and arabogalactan.
3. clinical cytology preparation according to claim 1 or 2 is with liquid is saved, wherein the intracellular cryoprotector choosing
From: glycerol, propylene glycol and ethylene glycol.
4. clinical cytology preparation as claimed in one of claims 1-3 is with liquid is saved, wherein the clinical cytology preparation is protected
Liquid storage is free of DMSO, protein and/or serum.
5. clinical cytology preparation as claimed in one of claims 1-4, with liquid is saved, wherein clinical cytology preparation, which is used, saves liquid
PH be 7.2.
6. clinical cytology preparation as claimed in one of claims 1-5 is with liquid is saved, wherein the extracellular cryoprotector
It is polyvinylpyrrolidone and the intracellular cryoprotector is propylene glycol.
7. clinical cytology preparation as claimed in one of claims 1-6 is with liquid is saved, wherein the clinical cytology preparation is protected
Liquid storage is free of one or more amino acid.
8. the preparation method for saving liquid of clinical cytology preparation described in any one of -7 according to claim 1 comprising mixing
The clinical cytology preparation various components for saving liquid, preferably adjusting pH value to 7-10,7-8,7-7.5 or 7.1-7.4, preferably
7.2-7.3 preferably 7.2.
9. application of the clinical cytology preparation described in any one of -7 with preservation liquid in freeze-stored cell according to claim 1, excellent
Selection of land wherein freezes as Long-term Cryopreservation, such as 1 year, 2 years, 3 years or longer time freeze.
10. application according to claim 9, wherein the cell is selected from: mescenchymal stem cell, mononuclearcell, T cell
With NK cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910388402.6A CN110024775A (en) | 2019-05-10 | 2019-05-10 | The clinical cytology preparation formula and purposes for saving liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910388402.6A CN110024775A (en) | 2019-05-10 | 2019-05-10 | The clinical cytology preparation formula and purposes for saving liquid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110024775A true CN110024775A (en) | 2019-07-19 |
Family
ID=67241668
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910388402.6A Pending CN110024775A (en) | 2019-05-10 | 2019-05-10 | The clinical cytology preparation formula and purposes for saving liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110024775A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112042637A (en) * | 2020-09-17 | 2020-12-08 | 深圳普瑞金生物药业有限公司 | Cell cryopreservation agent and cell cryopreservation method |
| CN113424820A (en) * | 2021-08-04 | 2021-09-24 | 北京全式金生物技术有限公司 | Serum-free, protein-free and DMSO-free cell cryopreservation liquid and application thereof |
| CN114041456A (en) * | 2021-12-09 | 2022-02-15 | 安徽中盛溯源生物科技有限公司 | Clinical-grade NK cell cryopreservation liquid and using method thereof |
| CN114199653A (en) * | 2021-10-28 | 2022-03-18 | 济南德亨医学科技有限公司 | Microbial immunofluorescence staining solution for vagina |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090123905A1 (en) * | 2005-10-17 | 2009-05-14 | Tomoo Eto | Method of Preserving Early Embryos of Experimental Animals by Vitrification |
| CN105660606A (en) * | 2016-03-10 | 2016-06-15 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryopreservation fluid |
| CN107047542A (en) * | 2017-06-23 | 2017-08-18 | 友康恒业生物科技（北京）有限公司 | A kind of serum-free cell frozen stock solution |
| CN107439536A (en) * | 2017-08-17 | 2017-12-08 | 重庆斯德姆生物技术有限公司 | A kind of cells frozen storing liquid of shelf-stable |
| CN107996558A (en) * | 2017-12-21 | 2018-05-08 | 湖南丰晖生物科技有限公司 | Cells frozen storing liquid and its application |
| CN108902129A (en) * | 2018-06-26 | 2018-11-30 | 吉林省太阳鸟再生医学工程有限责任公司 | Cell cryopreservation composition and its application |
| CN109479870A (en) * | 2017-09-11 | 2019-03-19 | 中国科学院动物研究所 | A kind of method for glass frozen preservation of biomaterial |
| CN109673623A (en) * | 2019-02-21 | 2019-04-26 | 白晓红 | A kind of glass freezing reagent and vitrifying defrosting reagent and its application and application method |
-
2019
- 2019-05-10 CN CN201910388402.6A patent/CN110024775A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090123905A1 (en) * | 2005-10-17 | 2009-05-14 | Tomoo Eto | Method of Preserving Early Embryos of Experimental Animals by Vitrification |
| CN105660606A (en) * | 2016-03-10 | 2016-06-15 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryopreservation fluid |
| CN107047542A (en) * | 2017-06-23 | 2017-08-18 | 友康恒业生物科技（北京）有限公司 | A kind of serum-free cell frozen stock solution |
| CN107439536A (en) * | 2017-08-17 | 2017-12-08 | 重庆斯德姆生物技术有限公司 | A kind of cells frozen storing liquid of shelf-stable |
| CN109479870A (en) * | 2017-09-11 | 2019-03-19 | 中国科学院动物研究所 | A kind of method for glass frozen preservation of biomaterial |
| CN107996558A (en) * | 2017-12-21 | 2018-05-08 | 湖南丰晖生物科技有限公司 | Cells frozen storing liquid and its application |
| CN108902129A (en) * | 2018-06-26 | 2018-11-30 | 吉林省太阳鸟再生医学工程有限责任公司 | Cell cryopreservation composition and its application |
| CN109673623A (en) * | 2019-02-21 | 2019-04-26 | 白晓红 | A kind of glass freezing reagent and vitrifying defrosting reagent and its application and application method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112042637A (en) * | 2020-09-17 | 2020-12-08 | 深圳普瑞金生物药业有限公司 | Cell cryopreservation agent and cell cryopreservation method |
| CN113424820A (en) * | 2021-08-04 | 2021-09-24 | 北京全式金生物技术有限公司 | Serum-free, protein-free and DMSO-free cell cryopreservation liquid and application thereof |
| CN114199653A (en) * | 2021-10-28 | 2022-03-18 | 济南德亨医学科技有限公司 | Microbial immunofluorescence staining solution for vagina |
| CN114041456A (en) * | 2021-12-09 | 2022-02-15 | 安徽中盛溯源生物科技有限公司 | Clinical-grade NK cell cryopreservation liquid and using method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110024775A (en) | The clinical cytology preparation formula and purposes for saving liquid | |
| US20220272965A1 (en) | Cell freezing medium for clinical use | |
| McCarey et al. | Improved corneal storage | |
| CN102511471B (en) | Mesenchymal stem cell frozen stock solution and preparation method thereof | |
| CN104770363B (en) | A kind of frozen stock solution of mescenchymal stem cell and cryopreservation methods | |
| CN109090100A (en) | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method | |
| CN108040460A (en) | The method of freezen protective tumor infiltrating lymphocyte | |
| CN113424820B (en) | Serum-free, protein-free and DMSO-free cell cryopreservation liquid and application thereof | |
| CN111838139A (en) | Protein-free cell cryopreservation liquid and application thereof | |
| US20200170242A1 (en) | Cell cryopreservation formulation and cell recovery method | |
| CN114946830A (en) | Extracellular vesicle storage liquid based on response surface optimization and preparation method thereof | |
| CN109329272A (en) | A kind of spermatozoa cryopreservation liquid and its preparation method and application | |
| CN105394029A (en) | Cell cryopreservation liquid used for treatment of leukemia | |
| CN105454220A (en) | Placenta preservation method, placenta preservation solution and preparation method thereof | |
| WO1990009432A1 (en) | Fluorophore-labeled cryopreserved cells for use as targets in cytotoxicity assays | |
| Salzman | Animal Cell Cultures: Tissue culture is a powerful tool in the study of nutrition, physiology, virology, and genetics. | |
| CN107446023A (en) | It is a kind of can antagonism HuR protein rna binding activity polypeptide HIP 13 and its application | |
| WO2020227843A1 (en) | Formulation and use of preservation solution for clinical cell preparations | |
| CN110195037A (en) | A kind of human umblilical vein endothelial primary separation and culture method | |
| CN106190979A (en) | Method for culturing hematopoietic stem/progenitor cells in vitro and compositions thereof | |
| CN110192552B (en) | Application of glycylglycine conditioned medium in mesenchymal stem cell cryopreservation and preparation of commercial cryopreservation protective agent | |
| CN113632784A (en) | Stem cell cryopreservation protective agent and application method thereof | |
| Abrahams et al. | Assessment of viability of frozen bone marrow cells using a cell‐culture method | |
| Seto et al. | Cultivation of tissues and leukocytes from amphibians | |
| CN115802889A (en) | Low temperature storage of biological samples |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190719 |