CN110074093A - Source of people umbilical cord blood hematopoietic stem cell program cooling freezing method - Google Patents
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Abstract
The invention discloses a kind of source of people umbilical cord blood hematopoietic stem cell program cooling freezing methods, pre-process, and be cooled to 4 DEG C to source of people umbilical cord blood hematopoietic stem cell obtained after separation, obtain sample；Sample is transferred in program cooling box and is proceeded as follows: 1) 4 DEG C of waiting 3-4min；2) cooled down with the speed of 1 DEG C/min, until the temperature of sample is -5 DEG C；3) cooled down with the speed of 21 DEG C/min, until the temperature of program cooling box is -54 DEG C；4) cooled down with the speed of 17 DEG C/min, until the temperature of program cooling box is -17 DEG C；5) cooled down with the speed of 2 DEG C/min, until the temperature of sample is -40 DEG C；6) cooled down with the speed of 10 DEG C/min, until the temperature of sample is -80 DEG C, be transferred to liquid nitrogen.The invention has the advantages that on the one hand shortening frozen storage process by the way that rate of temperature fall and cooling control point is rationally arranged, program cooling freezing cost is reduced；On the other hand, so that cell is steadily spent transformation temperature, cell in frozen storage process is avoided to be destroyed, improve the Cell viability of cell after recovery.
Description
Technical field:
The present invention relates to a kind of programmed cell cooling freezing method, in particular to a kind of source of people umbilical cord blood hematopoietic stem cell journeys
Sequence cooling freezing method.
Background technique:
The endovascular blood in fetus close with placenta side in umbilical cord when source of people Cord blood is newborn's birth, wherein contain
Source of people cord blood stem cell is commonly used for a variety of diseases in the blood system of transplantation treatment and disease of immune system, including hematological system
Malignant tumour, marrow hematopoiesis function failure, congenital metabolic disease, congenital immune deficiency illness, autoimmune conditions,
Certain entity tumors, respiratory disease, cardiovascular disease, endocrine system disease, digestion class disease etc..Therefore, Cord blood has become
The important sources of candidate stem cell.
The quantity of source of people umbilical cord blood hematopoietic stem cell is to determine the direct acting factor of transplanting efficiency, due to source of people Cord blood
Candidate stem cell is without the image of Buddha other stem cells (such as mescenchymal stem cell) equally massive amplification, therefore source of people Cord blood in vitro
Candidate stem cell seems especially precious.Umbilical cord blood hematopoietic stem cell is usually to send to Cord Blood Bank and carry out after acquisition process
Freezen protective and wait be utilized again, the quantity after Cryopreservation can directly influence later phase clinical transplanting efficiency, therefore
Its cryopreservation resuscitation process requires higher than other types stem cell.
Biological sample successfully freezes refer to the chilled recovery of cell after, cell still maintains survival, and has certain life
Object activity.Biological sample successfully freezes the rate depending on freezing, the control of accurate temperature and reasonable (+50 DEG C of temperature gradient
~-80 DEG C).When temperature is down to -80 DEG C, most of biological sample is transferred in liquid nitrogen to be saved for a long time.In cell cryopreservation mistake
Cheng Zhong, liquid phase are solid phase, and cell metabolism stops, and loses moisture, cause cell inner salt, metabolite concentration to change, Jin Erzao
At osmotic unbalances, cell rupture to death will directly influence freeze-stored cell motility rate.Meanwhile freezing rate is too fast or too slow,
It is easy to cause intracellular water to lose or formed large ice crystals, is all unfavorable for cell survival.
Currently, for source of people umbilical cord blood hematopoietic stem cell freezen protective method be roughly divided into non-controlled slow freezing method and
Controlled slow freezing method.Non- controlled slow freezing method is that candidate stem cell is not passed through to Programmed freezing directly freezed to be stored in -196
In liquid nitrogen at DEG C, this freezing and storing method it is maximum the disadvantage is that, generated into the cell during sharp temperature drop big
The ice crystal of amount, ice crystal burst cell membrane and lead to cell death；Meanwhile with the extension of holding time, candidate stem cell recycling
Rate seriously reduces, and the holding time is no more than 2 years, and influence of the preservation effect vulnerable to low temperature refrigerator working condition, Wu Fabao
Demonstrate,proving the source of people umbilical cord blood hematopoietic stem cell frozen can be with successful resuscitation.Controlled slow freezing method is by using Programmed freezing instrument, choosing
Suitable frozen stock solution is selected, suitable probe is prepared, sets suitable temperature rate, can effectively ensure that a limited number of source of people navels
Motility rate with blood candidate stem cell；But total time-consuming is longer, about 10 minutes or so 1 hour, time-consuming just makes for refrigerating process
It is big at liquid nitrogen consumption, it is at high cost；Meanwhile the method cell recoveries, Cell viability be not high, is not able to satisfy source of people umbilical cord blood hematopoietic
The use demand of stem cell.
Summary of the invention:
The purpose of the present invention is to provide a kind of designs of cooling process rationally, frozen storage process time-consuming greatly shortens, and drops simultaneously
The rate of recovery of umbilical cord blood hematopoietic stem cell and Cell viability greatly improve after low stem cell program cooling freezing cost, recovery
Source of people umbilical cord blood hematopoietic stem cell program cooling freezing method.
The present invention is implemented by following technical solution: source of people umbilical cord blood hematopoietic stem cell program cooling freezing method, to separation
The source of people umbilical cord blood hematopoietic stem cell obtained afterwards is pre-processed, and is cooled to 4 DEG C, obtains sample；The sample is turned
It moves in program cooling box and proceeds as follows: 1) 4 DEG C of waiting 3-4min；2) cooled down with the speed of 1 DEG C/min, until the sample
The temperature of product is -5 DEG C；3) cooled down with the speed of 21 DEG C/min, until the temperature of described program cooling box is -54 DEG C；4) with 17 DEG C/
The speed of min cools down, until the temperature of described program cooling box is -17 DEG C；5) cooled down with the speed of 2 DEG C/min, until the sample
Temperature be -40 DEG C；6) cooled down with the speed of 10 DEG C/min, until the temperature of the sample is -80 DEG C, is transferred in liquid nitrogen, makes institute
It states liquid nitrogen and floods the sample completely.
Further, the pretreatment is specifically, be added cryoprotector, institute to the source of people umbilical cord blood hematopoietic stem cell
The addition volume ratio for stating source of people umbilical cord blood hematopoietic stem cell and the cryoprotector is 4-5:1, makes the source of people Cord blood
Hemocytoblast and the cryoprotector, which are sufficiently mixed to be placed in ice water, stands 10min to 4 DEG C, obtains the sample.
In the above process, cryoprotector is the Cryo-Sure DEX 40 of origen production, is commercial reagent consumptive material.
Advantages of the present invention: by the way that rate of temperature fall and cooling control point is rationally arranged, on the one hand, concern source of people Cord blood is made
The relationship of hemocytoblast transformation temperature and cooling control point, the control point that makes to cool down level off to source of people umbilical cord blood hematopoietic stem cell phase transformation
Point herein on basis, occurs temperature compensation effect by fast cooling and intracellular water crystallization exotherm, then makes cabinet fast
Speed heating, and source of people umbilical cord blood hematopoietic stem cell frozen storage process is allow steadily to spend transformation temperature, avoid source of people in frozen storage process
Umbilical cord blood hematopoietic stem cell is destroyed, and improves the Cell viability of source of people umbilical cord blood hematopoietic stem cell after recovery, and then can be extended
The source of people umbilical cord blood hematopoietic stem cell holding time；On the other hand, shorten frozen storage process, frozen storage process time-consuming control 55min with
It is interior, reduce the usage amount of liquid nitrogen during Programmed freezing, reduces stem cell program cooling freezing cost.
Detailed description of the invention:
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is the CD34+ cell rate fluidic cell figure before embodiment 3 freezes.
Fig. 2 is the CD34+ cell rate fluidic cell figure after embodiment 3 is recovered.
Fig. 3 is the program temperature lowering curve figure of embodiment 3.
Fig. 4 is that embodiment 3 freezes phase point temperature curve partial enlarged view.
Fig. 5 is the CD34+ cell rate fluidic cell figure before embodiment 4 freezes.
Fig. 6 is the CD34+ cell rate fluidic cell figure after 4 Cryopreservation of embodiment.
Fig. 7 is the CD34+ cell rate fluidic cell figure before embodiment 5 freezes.
Fig. 8 is the CD34+ cell rate fluidic cell figure after 5 Cryopreservation of embodiment.
Fig. 9 is the program temperature lowering curve figure that embodiment 5 freezes.
Figure 10 is that embodiment 5 freezes phase point temperature curve partial enlarged view.
Specific embodiment:
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
Embodiment 1:
Embodiment 1 is source of people umbilical cord blood hematopoietic stem cell program cooling freezing method of the invention, to obtained after separation
Source of people umbilical cord blood hematopoietic stem cell is pre-processed, and pretreatment is protected specifically, freezing is added to source of people umbilical cord blood hematopoietic stem cell
Agent is protected, cryoprotector is the Cryo-Sure DEX 40 of origen production, source of people umbilical cord blood hematopoietic stem cell and cryoprotection
The addition volume ratio of agent be 4:1, make source of people umbilical cord blood hematopoietic stem cell be sufficiently mixed with cryoprotector be placed in it is quiet in ice water
10min is set, 4 DEG C is cooled to, obtains sample.
Sample is transferred in program cooling box and is proceeded as follows:
1) 4 DEG C of waiting 3min；The purpose is to make program cooling box and probe temperature simultaneously close to 4 DEG C, to cool down in next step
It prepares；
2) cooled down with the speed of 1 DEG C/min, until the temperature of sample is -5 DEG C；The purpose is to gradually cool the temperature to source of people navel
Transformation temperature with blood candidate stem cell, make to cool down herein control point closer to source of people umbilical cord blood hematopoietic stem cell transformation temperature,
The process that cell crystallization exotherm can more accurately be controlled, keeps cell temperature lowering curve more steady, during subsequent fast cooling,
Sample temperature decline is no more than 5 degree, and cell can preferably be protected by reducing temperature change degree, improves the freezing survival rate of cell；
3) cooled down with the speed of 21 DEG C/min, until the temperature of program cooling box is -54 DEG C；The purpose is to be next step temperature
Compensation is prepared；In the process, sample temperature drops to -10 DEG C from -5 DEG C of previous step, and sample temperature decline is no more than 5
DEG C, cell activity is influenced small；
4) cooled down with the speed of 17 DEG C/min, until the temperature of program cooling box is -17 DEG C；Inside program cooling box
Source of people umbilical cord blood hematopoietic stem cell constantly discharges heat, and after reducing rate of temperature fall, program cooling box spin manifold temperature is risen by -54 DEG C
To -17 DEG C, Programmed freezing instrument can reduce the intracorporal nitrogen temperature of case, such source of people umbilical cord blood hematopoietic by a large amount of air drafts of fan
After stem cell heat release, the temperature of sample will not rapid decrease, temperature slowly declines always, in this way can be to avoid phase transformation
Point is destroyed to source of people umbilical cord blood hematopoietic stem cell bring, by the way that temperature is turned up rapidly, using temperature compensation effect, makes source of people navel
Band blood candidate stem cell steadily spends transformation temperature, improves the rate of recovery and Cell viability of umbilical cord blood hematopoietic stem cell after recovery,
And then extend the umbilical cord blood hematopoietic stem cell holding time；
5) cooled down with the speed of 2 DEG C/min, until the temperature of sample is -40 DEG C；It is dry thin not influencing source of people umbilical cord blood hematopoietic
On the basis of born of the same parents' motility rate, frozen storage process is shortened, reduces and freezes time-consuming；
6) cooled down with the speed of 10 DEG C/min, until the temperature of sample is -80 DEG C, be transferred to liquid nitrogen；The purpose is to fast coolings
To -80 DEG C, and transfers them in liquid nitrogen and save for a long time.
In the present embodiment, time-consuming is frozen to can be controlled within 55min, compared with existing process control cools down freezing,
It effectively shortens and freezes time-consuming, liquid nitrogen dosage can be reduced, reduced costs；Meanwhile in this embodiment, source of people umbilical cord is paid close attention to
The relationship of blood candidate stem cell transformation temperature and cooling control point, the control point that makes to cool down level off to source of people umbilical cord blood hematopoietic stem cell phase
Height herein on basis, occurs temperature compensation effect by fast cooling and intracellular water crystallization exotherm, then makes cabinet
It is rapidly heated and source of people umbilical cord blood hematopoietic stem cell frozen storage process is allow steadily to spend transformation temperature, avoid source of people in frozen storage process
Umbilical cord blood hematopoietic stem cell is destroyed, and improves the Cell viability of source of people umbilical cord blood hematopoietic stem cell after recovery, and then can be extended
The source of people umbilical cord blood hematopoietic stem cell holding time.
Embodiment 2:
Embodiment 2 is source of people umbilical cord blood hematopoietic stem cell program cooling freezing method of the invention, to obtained after separation
Source of people umbilical cord blood hematopoietic stem cell is pre-processed, and pretreatment is protected specifically, freezing is added to source of people umbilical cord blood hematopoietic stem cell
The addition volume ratio of shield agent, source of people umbilical cord blood hematopoietic stem cell and cryoprotector is 4.5:1, keeps source of people umbilical cord blood hematopoietic dry thin
Born of the same parents and cryoprotector, which are sufficiently mixed to be placed in ice water, stands 10min, is cooled to 4 DEG C, obtains sample.
Sample is transferred in program cooling box and is proceeded as follows:
1) 4 DEG C of waiting 3.5min；The purpose is to make program cooling box and probe temperature simultaneously close to 4 DEG C, to drop in next step
Temperature is prepared；
2) cooled down with the speed of 1 DEG C/min, until the temperature of sample is -5 DEG C；The purpose is to gradually cool the temperature to source of people navel
Transformation temperature with blood candidate stem cell, make to cool down herein control point closer to source of people umbilical cord blood hematopoietic stem cell transformation temperature,
The process that cell crystallization exotherm can more accurately be controlled, keeps cell temperature lowering curve more steady, during subsequent fast cooling,
Sample temperature decline is no more than 5 degree, and cell can preferably be protected by reducing temperature change degree, improves the freezing survival rate of cell；
3) cooled down with the speed of 21 DEG C/min, until the temperature of program cooling box is -54 DEG C；The purpose is to be next step temperature
Compensation is prepared；In the process, sample temperature drops to -10 DEG C from -5 DEG C of previous step, and sample temperature decline is no more than 5
DEG C, sample temperature variation is small, influences on cell activity small；
4) cooled down with the speed of 17 DEG C/min, until the temperature of program cooling box is -17 DEG C；Inside program cooling box
Source of people umbilical cord blood hematopoietic stem cell constantly discharges heat, and after reducing rate of temperature fall, program cooling box spin manifold temperature is risen by -54 DEG C
To -17 DEG C, Programmed freezing instrument can reduce the intracorporal nitrogen temperature of case, such source of people umbilical cord blood hematopoietic by a large amount of air drafts of fan
After stem cell heat release, the temperature of sample will not rapid decrease, temperature slowly declines always, in this way can be to avoid phase transformation
Point is destroyed to source of people umbilical cord blood hematopoietic stem cell bring, by the way that temperature is turned up rapidly, using temperature compensation effect, makes source of people navel
Band blood candidate stem cell steadily spends transformation temperature, improves the rate of recovery and Cell viability of umbilical cord blood hematopoietic stem cell after recovery,
And then extend the umbilical cord blood hematopoietic stem cell holding time；
5) cooled down with the speed of 2 DEG C/min, until the temperature of sample is -40 DEG C；It is dry thin not influencing source of people umbilical cord blood hematopoietic
On the basis of born of the same parents' motility rate, frozen storage process is shortened, reduces and freezes time-consuming；
6) cooled down with the speed of 10 DEG C/min, until the temperature of sample is -80 DEG C, be transferred to liquid nitrogen；The purpose is to fast coolings
To -80 DEG C, and transfers them in liquid nitrogen and save for a long time.
In the present embodiment, time-consuming is frozen to can be controlled within 55min, compared with existing process control cools down freezing,
It effectively shortens and freezes time-consuming, liquid nitrogen dosage can be reduced, reduced costs；Meanwhile in this embodiment, source of people umbilical cord is paid close attention to
The relationship of blood candidate stem cell transformation temperature and cooling control point, the control point that makes to cool down level off to source of people umbilical cord blood hematopoietic stem cell phase
Height herein on basis, occurs temperature compensation effect by fast cooling and intracellular water crystallization exotherm, then makes cabinet
It is rapidly heated and source of people umbilical cord blood hematopoietic stem cell frozen storage process is allow steadily to spend transformation temperature, avoid source of people in frozen storage process
Umbilical cord blood hematopoietic stem cell is destroyed, and improves the Cell viability of source of people umbilical cord blood hematopoietic stem cell after recovery, and then can be extended
The source of people umbilical cord blood hematopoietic stem cell holding time.
Embodiment 3:
Embodiment 3 is source of people umbilical cord blood hematopoietic stem cell program cooling freezing method of the invention, to obtained after separation
Source of people umbilical cord blood hematopoietic stem cell is pre-processed, and pretreatment is protected specifically, freezing is added to source of people umbilical cord blood hematopoietic stem cell
The addition volume ratio of shield agent, source of people umbilical cord blood hematopoietic stem cell and cryoprotector is 5:1, makes source of people umbilical cord blood hematopoietic stem cell
It is sufficiently mixed to be placed in ice water with cryoprotector and stands 10min, be cooled to 4 DEG C, obtain sample.
Sample is transferred in program cooling box and is proceeded as follows:
1) 4 DEG C of waiting 4min；The purpose is to make program cooling box and probe temperature simultaneously close to 4 DEG C, to cool down in next step
It prepares；
2) cooled down with the speed of 1 DEG C/min, until the temperature of sample is -5 DEG C；The purpose is to gradually cool the temperature to source of people navel
Transformation temperature with blood candidate stem cell, make to cool down herein control point closer to source of people umbilical cord blood hematopoietic stem cell transformation temperature,
The process that cell crystallization exotherm can more accurately be controlled, keeps cell temperature lowering curve more steady, during subsequent fast cooling,
Sample temperature decline is no more than 5 degree, and cell can preferably be protected by reducing temperature change degree, improves the freezing survival rate of cell；
3) cooled down with the speed of 21 DEG C/min, until the temperature of program cooling box is -54 DEG C；The purpose is to be next step temperature
Compensation is prepared；In the process, sample temperature drops to -10 DEG C from -5 DEG C of previous step, and sample temperature decline is no more than 5
DEG C, sample temperature variation is small, influences on cell activity small；
4) cooled down with the speed of 17 DEG C/min, until the temperature of program cooling box is -17 DEG C；Inside program cooling box
Source of people umbilical cord blood hematopoietic stem cell constantly discharges heat, and after reducing rate of temperature fall, program cooling box spin manifold temperature is risen by -54 DEG C
To -17 DEG C, Programmed freezing instrument can reduce the intracorporal nitrogen temperature of case, such source of people umbilical cord blood hematopoietic by a large amount of air drafts of fan
After stem cell heat release, the temperature of sample will not rapid decrease, temperature slowly declines always, in this way can be to avoid phase transformation
Point is destroyed to source of people umbilical cord blood hematopoietic stem cell bring, by the way that temperature is turned up rapidly, using temperature compensation effect, makes source of people navel
Band blood candidate stem cell steadily spends transformation temperature, improves the rate of recovery and Cell viability of umbilical cord blood hematopoietic stem cell after recovery,
And then extend the umbilical cord blood hematopoietic stem cell holding time；
5) cooled down with the speed of 2 DEG C/min, until the temperature of sample is -40 DEG C；It is dry thin not influencing source of people umbilical cord blood hematopoietic
On the basis of born of the same parents' motility rate, frozen storage process is shortened, reduces and freezes time-consuming；
6) cooled down with the speed of 10 DEG C/min, until the temperature of sample is -80 DEG C, be transferred to liquid nitrogen；The purpose is to fast coolings
To -80 DEG C, and transfers them in liquid nitrogen and save for a long time.
In the present embodiment, time-consuming is frozen to can be controlled within 55min, compared with existing process control cools down freezing,
It effectively shortens and freezes time-consuming, liquid nitrogen dosage can be reduced, reduced costs；Meanwhile in this embodiment, source of people umbilical cord is paid close attention to
The relationship of blood candidate stem cell transformation temperature and cooling control point, the control point that makes to cool down level off to source of people umbilical cord blood hematopoietic stem cell phase
Height herein on basis, occurs temperature compensation effect by fast cooling and intracellular water crystallization exotherm, then makes cabinet
It is rapidly heated and source of people umbilical cord blood hematopoietic stem cell frozen storage process is allow steadily to spend transformation temperature, avoid source of people in frozen storage process
Umbilical cord blood hematopoietic stem cell is destroyed, and improves the Cell viability of source of people umbilical cord blood hematopoietic stem cell after recovery, and then can be extended
The source of people umbilical cord blood hematopoietic stem cell holding time.
Embodiment 4:
Embodiment 4 is the non-program cooling freezing method of source of people umbilical cord blood hematopoietic stem cell of the prior art, is obtained to after separation
To source of people umbilical cord blood hematopoietic stem cell pre-processed, pretreatment specifically, to source of people umbilical cord blood hematopoietic stem cell be added it is cold
The addition volume ratio of jelly protective agent, source of people umbilical cord blood hematopoietic stem cell and cryoprotector is 5:1, keeps source of people umbilical cord blood hematopoietic dry
Cell and cryoprotector, which are sufficiently mixed to be placed in ice water, stands 10min, is cooled to 4 DEG C, obtains sample；Sample directly freezed
It is stored in the liquid nitrogen at -196 DEG C.
Embodiment 5:
Embodiment 5 is the existing program cooling freezing method of source of people umbilical cord blood hematopoietic stem cell of the prior art, after separation
Obtained source of people umbilical cord blood hematopoietic stem cell is pre-processed, and pretreatment to source of people umbilical cord blood hematopoietic stem cell specifically, be added
The addition volume ratio of cryoprotector, source of people umbilical cord blood hematopoietic stem cell and cryoprotector is 5:1, makes source of people umbilical cord blood hematopoietic
Stem cell and cryoprotector, which are sufficiently mixed to be placed in ice water, stands 10min, is cooled to 4 DEG C, obtains sample；Detailed process is such as
Under: 1. 4 DEG C waiting 3-4 minutes；2. 1 DEG C of cooling per minute, until sample temperature is -4 DEG C；3. 30 DEG C of cooling per minute, until
Spin manifold temperature is -60 DEG C；4. 17 DEG C of cooling per minute, until spin manifold temperature is -20 DEG C；5. 3 DEG C of cooling per minute, until sample
Temperature is -10 DEG C；6. 2 DEG C of cooling per minute, until sample temperature is -40 DEG C；7. 10 DEG C of cooling per minute, until sample temperature
It is -80 DEG C, is transferred to liquid nitrogen.
Embodiment 6:
Embodiment 1,2,3 is respectively the side that the present invention carries out source of people umbilical cord blood hematopoietic stem cell program cooling freezing
Method, embodiment 4 are method that is existing, carrying out non-program cooling freezing for source of people umbilical cord blood hematopoietic stem cell, and embodiment 5 is
Method existing, that program cooling freezing is carried out for source of people umbilical cord blood hematopoietic stem cell.Using above-described embodiment 1,2,3,4,5
Method carry out freezen protective processing to a batch of source of people umbilical cord blood hematopoietic stem cell, respectively number be test group 1,2,3,
4、5。
One, the comparison of liquid nitrogen usage amount, liquid nitrogen cost
It is frozen using the method for above-mentioned test group 1,2,3,4,5 to a batch of source of people umbilical cord blood hematopoietic stem cell
Processing, detects liquid nitrogen usage amount, the liquid nitrogen use cost during test group 1,2,3,4,5, liquid nitrogen price is according to city
Field 6 yuan/L of valence is calculated, and specific testing result is as shown in table 1.
1 liquid nitrogen usage amount of table and freezing Cost comparisons
| Project | Test group 1 | Test group 2 | Test group 3 | Test group 4 | Test group 5 |
| Liquid nitrogen usage amount (L/ times) | 29.4 | 29.7 | 30 | 0 | 40 |
| Liquid nitrogen cost (member/time) | 176.4 | 178.2 | 180 | 0 | 200 |
As shown in Table 1, with test group 4 (i.e. existing non-program cool down freezing method), (i.e. existing program cools down test group 5
Freezing method) it compares, the liquid nitrogen usage amount of test group 1,2,3 is low, to reduce liquid nitrogen use cost, being finally reached is reduced
Source of people umbilical cord blood hematopoietic stem cell Slow-rate freezing cost.
Two, the Other nucleated cells differential count statistical result comparison before the freezing of source of people umbilical cord blood hematopoietic stem cell and after Cryopreservation
It is frozen using the method for above-mentioned test group 1,2,3,4,5 to a batch of source of people umbilical cord blood hematopoietic stem cell
It is thin to have carried out core to the source of people umbilical cord blood hematopoietic stem cell before the freezing of test group 1,2,3,4,5 and after Cryopreservation respectively for processing
Born of the same parents' counting statistics, the Other nucleated cells differential count statistical result before freezing is as shown in table 2, the Other nucleated cells differential count statistics after Cryopreservation
The results are shown in Table 3.
Other nucleated cells differential count statistical result before 2 five groups of cell cryopreservations of table
Other nucleated cells differential count statistical result after 3 five groups of cell freezing recoveries of table
There is core to the source of people umbilical cord blood hematopoietic stem cell before the freezing of test group 1,2,3,4,5 and after Cryopreservation to use
Cell counting is counted, wherein Cell viability=(survivaling cell quantity/total number of cells amount) × 100%, to characterize tool
There is the cell quantity of bioactivity to account for the ratio of total number of cells, after the preceding Cell viability of freezing and Cryopreservation between Cell viability
Difference is smaller to show that refrigerating process is lower for the degree of injury of cell activity；Recovery rate of nucleated cell=(have after Cryopreservation
Number of nucleated cells before nucleus number/freezing) × 100%, to characterize refrigerating process for cyto-architectural extent of the destruction, there is core
Cell recoveries are higher to show that refrigerating process is lower for cyto-architectural extent of the destruction.
As shown in Table 2, before freezing processing, the Cell viability of each test group is identical.
As shown in Table 3, after Cryopreservation, the total number of nucleated cell of each test group declines, but from recovery rate of nucleated cell
As can be seen that (i.e. existing non-program cooling freezes for test group 1,2,3 (i.e. present procedure cooling freezing method) and test group 4
Method), test group 5 (i.e. existing program cool down freezing method) compare, (i.e. present procedure cools down freezing side test group 1,2,3
Method), using and protect be more advantageous to source of people umbilical cord blood hematopoietic stem cell Cryopreservation after low for cyto-architectural extent of the destruction
Deposit the extension of time.
It is compared by table 2 and table 3 it is found that the Cell viability of test group 1,2,3 (i.e. present procedure cooling freezing method) does not have
It changes, shows to carry out program cooling freezing processing to source of people umbilical cord blood hematopoietic stem cell using the method for the present invention, it can be effective
The cell activity of source of people umbilical cord blood hematopoietic stem cell is kept, and then the umbilical cord blood hematopoietic stem cell holding time can be extended；Test
Group 4 (i.e. existing non-program cool down freezing methods), test group 5 (i.e. existing program cool down freezing method) Cell viability have under
Drop shows dry to source of people umbilical cord blood hematopoietic thin using existing non-program cooling freezing method, existing program cooling freezing method
Born of the same parents carry out program cooling freezing processing, can destroy the cell activity of the cell activity of source of people umbilical cord blood hematopoietic stem cell, be unfavorable for
Use after source of people umbilical cord blood hematopoietic stem cell Cryopreservation.
Three, the flow-cytometry method statistical result comparison before the freezing of source of people umbilical cord blood hematopoietic stem cell and after Cryopreservation
It carries out freezing place to a batch of source of people umbilical cord blood hematopoietic stem cell using the method for above-mentioned test group 3,4,5
Reason uses flow cytometry to the source of people umbilical cord blood hematopoietic stem cell before the freezing of test group 3,4,5 and after Cryopreservation respectively
Method is counted, and the CD34+ cell rate fluidic cell figure before test group 3 freezes and after Cryopreservation is as shown in Figure 1 and Figure 2, test
CD34+ cell rate fluidic cell figure before 4 freezing of group and after Cryopreservation is as shown in Figure 5, Figure 6, before test group 5 freezes and freezes
CD34+ cell rate fluidic cell figure after recovery is as shown in Figure 7, Figure 8.To before the freezing of test group 3,4,5 and after Cryopreservation
Data are counted in CD34+ cell rate fluidic cell figure, and the results are shown in Table 4.
Flow-cytometry method statistical result before 4 three groups of cell freezings of table and after Cryopreservation
| Group | Test group 3 | Test group 4 | Test group 5 |
| CD34+ cell rate (%) before freezing | 0.32 | 0.28 | 0.58 |
| CD34+ cell rate (%) after Cryopreservation | 0.300 | 0.16 | 0.45 |
| CD34+ cell recoveries (%) | 93.7 | 57.1 | 77.6 |
Flow cytometry is used to the source of people umbilical cord blood hematopoietic stem cell before the freezing of test group 3,4,5 and after Cryopreservation
Method is counted, wherein the CD34+ in source of people umbilical cord blood hematopoietic stem cell before freezing before the freezing of CD34+ cell rate characterization is thin
The ratio of born of the same parents, the ratio of the CD34+ cell in source of people umbilical cord blood hematopoietic stem cell after freezing after the freezing of CD34+ cell rate characterization
Example, CD34+ cell recoveries characterize refrigerating process for cyto-architectural extent of the destruction, and CD34+ cell recoveries are higher to be shown
Refrigerating process is lower for cyto-architectural extent of the destruction.
As shown in Table 4, it can be seen that test group 3 (i.e. present procedure cooling freezing method) from CD34+ cell recoveries
It is right compared with test group 4 (i.e. existing non-program cooling freezing method), test group 5 (i.e. existing program cooling freezing method)
Use low in cyto-architectural extent of the destruction, after being more advantageous to source of people umbilical cord blood hematopoietic stem cell Cryopreservation, this with it is above-mentioned
Other nucleated cells differential count statistical result is consistent.
Embodiment 7:
Embodiment 3 is the method that the present invention carries out program cooling freezing for source of people umbilical cord blood hematopoietic stem cell, embodiment 5
For method that is existing, carrying out program cooling freezing for source of people umbilical cord blood hematopoietic stem cell.Using the side of above-described embodiment 3,5
Method carries out freezen protective processing to a batch of source of people umbilical cord blood hematopoietic stem cell, and number is test group 3,5 respectively, right respectively
The monitoring of the Slow-rate freezing temperature-fall period of test group 3,5.The program temperature lowering curve of test group 3 is as shown in figure 3, its partial enlarged view
As shown in Figure 4；The program temperature lowering curve of test group 5 is as shown in figure 9, its partial enlarged view is as shown in Figure 10；
By the comparison of Fig. 3 and Fig. 9 it is found that test group 3 (i.e. present procedure cooling freezing method) and test group 5 are (i.e. existing
Have program cooling freezing method) it compares, the phase point temperature variation of test group 3 is significantly less than test group 5, i.e. curvilinear indentations more
Small, this result can more obviously be found out in Fig. 4 and Figure 10；Thus, it is indicated, that (i.e. present procedure cools down test group 3
Freezing method) can slow down transformation temperature give source of people umbilical cord blood hematopoietic stem cell bring destroy, pass through fast cooling and intracellular water
Divide crystallization exotherm that temperature compensation effect occurs, so that cabinet is rapidly heated makes source of people umbilical cord blood hematopoietic stem cell frozen storage process can
Steadily to spend transformation temperature, source of people umbilical cord blood hematopoietic stem cell in frozen storage process is avoided to be destroyed, improves source of people umbilical cord after recovery
The Cell viability of blood candidate stem cell, and then the source of people umbilical cord blood hematopoietic stem cell holding time can be extended.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (2)
- The freezing method 1. source of people umbilical cord blood hematopoietic stem cell program cools down, which is characterized in that the source of people obtained after separation Umbilical cord blood hematopoietic stem cell is pre-processed, and is cooled to 4 DEG C, obtains sample；The sample is transferred in program cooling box And proceed as follows: 1) 4 DEG C of waiting 3-4min；2) cooled down with the speed of 1 DEG C/min, until the temperature of the sample is -5 DEG C； 3) cooled down with the speed of 21 DEG C/min, until the temperature of described program cooling box is -54 DEG C；4) cooled down with the speed of 17 DEG C/min, Temperature to described program cooling box is -17 DEG C；5) cooled down with the speed of 2 DEG C/min, until the temperature of the sample is -40 DEG C； 6) cooled down with the speed of 10 DEG C/min, until the temperature of the sample is -80 DEG C, is transferred in liquid nitrogen, floods the liquid nitrogen completely The sample.
- The freezing method 2. source of people umbilical cord blood hematopoietic stem cell program according to claim 1 cools down, which is characterized in that described Specifically, cryoprotector is added to the source of people umbilical cord blood hematopoietic stem cell, the source of people umbilical cord blood hematopoietic is dry thin for pretreatment The addition volume ratio of born of the same parents and the cryoprotector is 4-5:1, protects the source of people umbilical cord blood hematopoietic stem cell and the freezing Shield agent, which is sufficiently mixed to be placed in ice water, stands 10min to 4 DEG C, obtains the sample.
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| CN111642501A (en) * | 2020-06-11 | 2020-09-11 | 北京佳宸弘生物技术有限公司 | Optimized program-controlled cooling method for umbilical cord blood hematopoietic stem cells |
| CN112913833A (en) * | 2021-01-29 | 2021-06-08 | 华夏源细胞工程集团股份有限公司 | Programmed cooling method for human umbilical cord mesenchymal stem cell working cell bank |
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| WO2013096710A1 (en) * | 2011-12-21 | 2013-06-27 | Biocision, Llc | Method and devices for cryopreservation of biomaterials |
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