CN106212443A - Clinical grade Cell protective solutions and its preparation method and application - Google Patents
Clinical grade Cell protective solutions and its preparation method and application Download PDFInfo
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- CN106212443A CN106212443A CN201610665376.3A CN201610665376A CN106212443A CN 106212443 A CN106212443 A CN 106212443A CN 201610665376 A CN201610665376 A CN 201610665376A CN 106212443 A CN106212443 A CN 106212443A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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Abstract
The present invention relates to a kind of clinical grade Cell protective solutions and its preparation method and application, described clinical grade Cell protective solutions comprises human serum albumin injection, dextran and amino acid injection and isotonic sodium chloride injection；Its preparation method is by human serum albumin injection, dextran and amino acid injection and isotonic sodium chloride injection mix homogeneously；It is applied to cell and preserves；Compared with prior art, the invention in isotonic sodium chloride injection, add appropriate human serum albumin injection and dextran and amino acid injection, can be while being supplied to cytotrophy, the acid base imbalance that regulation cellular metabolism causes, alleviate the damage that cell causes because of acidosis, it is greatly prolonged cell survival rate, keep cell viability, make to preserve cell stage longer and can direct Clinical practice, applying more safe ready, its preparation method is simple, convenient, fast.
Description
Technical field
The invention belongs to biomedicine field, particularly relate to a kind of Cell protective solutions and its preparation method and application.
Background technology
Cell is as an independent life entity, and it has the plastic of the strongest vitality, proliferation and differentiation ability and function
Sexuality.The mechanism of cytotherapeutic treatment disease is broadly divided into two big classes: one is the direct effect of cell, directly uses it specific
The tissue of biological activity repairing damage and organ；Or play specificity/non-specific lethal effect；Two is the indirect of cell
Effect, the factor as relevant in secretion or bioactive molecule regulate propagation and the functional activity of patients own cells.
Along with the progress of society, scientific and technological is flourish, and the mankind there has also been the higher phase to quality of life and life expectancy
Hope.Have a health, happiness, happy life cycle are everyone dream.But such as persistent ailments such as tumors, traditional controls
Treatment means have come into plateau, and the side effect brought also allows a lot of patient sufferings can't bear.
Cell therapy is the disease treatment new technique risen in recent years, refers to utilize some to have the cell of specific function
Characteristic, after using biological engineering method to obtain and/or being processed by amplification in vitro, specific culture etc., makes these cells have increasing
Strong immunity, kill the therapeutic efficiency such as pathogen and tumor cell, promotion tissue and organ regeneration and physical recovery, thus reach treatment
The purpose of disease.
Cell therapy is with its good curative effect, and side effect is little, the advantage that more individuation, personalization etc. are unique, difficult for some
The treatment of the property controlled disease, it is provided that a kind of selection, the most last selection.On the longest history rank
Section, cell therapy all will take on important role in clinical treatment, and 21st century will be that cell therapy plays a significant role
Epoch.
Cell therapy uses normal saline as conventional infusion medium, the cell good by cultured and amplified in vitro mostly at present
Be expelled in sodium chloride injection, add or be added without appropriate human albumin, be placed in 0～10 DEG C preserve, transport to
Destination；Although this store method is simple, application safety, convenience clinically, but only as of short duration preservation, during preservation
Between long cell viability decline quickly, it is impossible to meet strange land and use at a distance；Currently also have in normal saline, increase appropriate model
The compositions such as the potassium chloride that encloses, magnesium chloride, sodium acetate are for preserving the cell that cultured and amplified in vitro is good, although this method can be thin
The holding time of born of the same parents, but more than 48 hours after cell viability decline quickly, it is impossible to meet strange land and use at a distance, and can not
It is directly used in Clinical practice.
Summary of the invention
The technical problem to be solved is to provide one and prepares simple and convenient, application convenience, preservation cell stage
Longer and can the clinical grade Cell protective solutions and its preparation method and application of direct Clinical practice.
The technical scheme is that
A kind of clinical grade Cell protective solutions, it comprises human serum albumin injection, the injection of low molecular dextran aminoacid
Liquid and isotonic sodium chloride injection.
Compared with prior art, the invention has the beneficial effects as follows:
The invention by dextran and amino acid injection add to human serum albumin injection and etc.
Ooze in sodium chloride injection, be configured to Cell protective solutions and preserve for cell, can regulate thin while being supplied to cytotrophy
The acid base imbalance that born of the same parents' metabolism causes, alleviates the damage that causes because of acidosis of cell, and with human serum albumin injection and isotonic chlorine
Change sodium injection and play favourable synergism, be greatly prolonged cell survival rate, keep cell viability, preserve cell stage ratio single
Solely use human serum albumin injection and isotonic sodium chloride injection longer, in terms of cell preservation, give play to beyond thought effect
Really, the holding time is greatly prolonged, and at least can reach 72 hours, better meets cell strange land and uses at a distance, and clinical grade
The component of Cell protective solutions is injection, through its cell preserved can direct Clinical practice, apply more safe ready,
Be convenient and save trouble.
On the basis of technique scheme, the present invention can also do following improvement.
As a kind of preferred implementation of the present invention, in terms of volumetric concentration, its each component proportion is: 2.5%～
The dextran and amino acid injection of human serum albumin injection, 3%～8% of 7.5%, remaining is isotonic sodium chloride
Injection.
Above-mentioned preferred version is used to provide the benefit that:
Clinical grade Cell protective solutions in the present invention uses this formula proportion to preserve cell, not only prepares convenient, more
Add and be suitable to promote, and ensure that while being supplied to cytotrophy, the acid base imbalance that regulation cellular metabolism causes, alleviate
The damage that cell causes because of acidosis, is greatly prolonged cell survival rate, keeps cell viability, preserves cell stage longer, carefully
Born of the same parents' preservation aspect has given play to beyond thought effect, and the holding time can reach 96 hours, better meets cell strange land remote
Use, and the component proportion of clinical grade Cell protective solutions is in this range, preserves the clinical grade cytoprotective of target cell
Liquid can be directly injected into human body, direct Clinical practice, apply more safe ready, be convenient and save trouble.
As the another kind of preferred implementation of the present invention, the volume of human albumin in described human serum albumin injection
Concentration is 20%, and dextran and amino acid injection described in every 1000ml comprises water and following components: 4.1g containing bright ammonia
Acid, 1.8g isoleucine, 2.9g phenylalanine, 1.8g threonine, 2.0g valine, 0.6g tryptophan, 2.4g methionine,
3.4g glycine, 5.0g lysine, 2.2g arginine, 1.0g histidine, 60.0g Dextran 40.
Above-mentioned preferred version is used to provide the benefit that:
Human serum albumin injection in the present invention is patent medicine preparation, and dextran and amino acid injection is compound recipe
Preparation patent medicine, all through clinical verification for many years, uses safer, and the cell preserved through clinical grade Cell protective solutions can be straight
Connect Clinical practice, apply convenient, and the human albumin of this concentration can preferably and low molecular dextran amino
Acid injection plays synergism, preferably gives cells with nutrient, regulates acid base imbalance, alleviation cell that cellular metabolism causes
Because acidosis causes the effect of damage more preferably, thus it is greatly prolonged cell survival rate, preferably keeps cell viability, preserve cell
Time is longer, at least can reach 96 hours, better meets cell strange land and uses at a distance.
As the another kind of preferred implementation of the present invention, in terms of volumetric concentration, its each component proportion is: the people of 5%
Blood albumin injection, the dextran and amino acid injection of 5%, remaining is isotonic sodium chloride injection.
Above-mentioned preferred version is used to provide the benefit that:
The clinical grade Cell protective solutions of the present invention uses this formula proportion can regulate thin preferably to cells with nutrient
Acid base imbalance that born of the same parents' metabolism causes, alleviate cell and cause the effect of damage more preferable because of acidosis, thus be greatly prolonged cell survival
Rate, preferably keeps cell viability, preserves cell stage longer, can reach more than 96 hours, better meet cell strange land remote
Distance uses, and the component proportion of clinical grade Cell protective solutions is so arranged, and the clinical grade cell preserving target cell is protected
Protect liquid and can be directly injected into human body, direct Clinical practice, apply more safe ready, be convenient and save trouble.
The preparation method of a kind of clinical grade Cell protective solutions as above, its step is: human albumin injected
Liquid, dextran and amino acid injection and isotonic sodium chloride injection mix homogeneously, i.e. obtain described clinical grade thin
Born of the same parents protect liquid.
Compared with prior art, the invention has the beneficial effects as follows:
The inventive method is simple to operate, implements convenient and swift, creative by dextran and amino acid injection
Add in human serum albumin injection and isotonic sodium chloride injection, be configured to Cell protective solutions and preserve for cell, can be
While being supplied to cytotrophy, the acid base imbalance that regulation cellular metabolism causes, alleviate the damage that cell causes because of acidosis, and
Play favourable synergism with human serum albumin injection and isotonic sodium chloride injection, be greatly prolonged cell survival rate, protect
Holding cell viability, preservation cell stage ratio is used alone human serum albumin injection and isotonic sodium chloride injection is longer, carefully
Born of the same parents' preservation aspect has given play to beyond thought effect, and the holding time can reach 96 hours, better meets cell strange land remote
Use, and the component of clinical grade Cell protective solutions be injection, through its cell preserved can direct Clinical practice, answer
With more safe ready, be convenient and save trouble.
The application of a kind of clinical grade Cell protective solutions as above, it is applied to cell and preserves.
Compared with prior art, the invention has the beneficial effects as follows:
The clinical grade Cell protective solutions of the present invention is applied to cell and preserves, and can regulate while being supplied to cytotrophy
The acid base imbalance that cellular metabolism causes, alleviates the damage that causes because of acidosis of cell, and with human serum albumin injection and isotonic
Sodium chloride injection plays favourable synergism, is greatly prolonged cell survival rate, keeps cell viability, preserves cell stage ratio
It is used alone human serum albumin injection and isotonic sodium chloride injection is longer, given play to beyond thought in terms of cell preservation
Effect, the holding time at least can reach 96 hours, better meets cell strange land and use at a distance, and clinical grade cytoprotective
The component of liquid is injection, through its cell preserved can direct Clinical practice, apply more safe ready, province of saving worry
Thing.
As a kind of preferred implementation of the present invention, it is applied to the cell of clinical medicine fields and preserves.
Above-mentioned preferred version is used to provide the benefit that:
The clinical grade Cell protective solutions of the present invention may be directly applied to the cell of clinical medicine fields and preserves, clinical grade cell
The component of protection liquid is injection, through its cell preserved can direct Clinical practice, apply more safe ready, save worry
Save trouble.
As the another kind of preferred implementation of the present invention, it is applied to storage temperature when cell preserves is 2～8 DEG C.
Above-mentioned preferred version is used to provide the benefit that:
The clinical grade Cell protective solutions of the present invention preserves cell at this temperature, can preferably extend cell survival rate, protects
Hold cell viability, preserve cell stage longer, better meet cell strange land and use at a distance.
As the another kind of preferred implementation of the present invention, it is applied to the preservation of immunocyte CIK cell, exempts from during preservation
The concentration of epidemic disease cell CIK cell is 0.8～1.5 × 107Individual/ml.
Above-mentioned preferred version is used to provide the benefit that:
The clinical grade Cell protective solutions of the present invention can be applicable to the preservation of various cell, as the guarantor of immunocyte CIK cell
When depositing, cell concentration is controlled 0.8～1.5 × 107In the range of individual/ml, preservation effect is more preferable, can preferably extend cell
Survival rate, keeps cell viability, preserves cell stage longer, at least can reach 96 hours, better meet immunocyte CIK thin
Born of the same parents strange land uses at a distance.
As the another kind of preferred implementation of the present invention, it is applied to the preservation of umbilical cord mesenchymal stem cells, during preservation
The concentration of umbilical cord mesenchymal stem cells is 3～7 × 105Individual/ml.
Above-mentioned preferred version is used to provide the benefit that:
The clinical grade Cell protective solutions of the present invention can be applicable to the preservation of various cell, when preserving umbilical cord mesenchymal stem cells
Time, cell concentration is controlled 3～7 × 105In the range of individual/ml, preservation effect is more preferable, can preferably extend cell survival
Rate, keeps cell viability, preserves cell stage longer, at least can reach 96 hours, better meet umbilical cord mesenchymal stem cells
Strange land uses at a distance.
Below the present invention is further elaborated.
A kind of clinical grade Cell protective solutions, it comprises human serum albumin injection, the injection of low molecular dextran aminoacid
Liquid and isotonic sodium chloride injection, in terms of volumetric concentration, its each component proportion is: human albumin's note of 2.5%～7.5%
Penetrating the dextran and amino acid injection of liquid, 3%～8%, remaining is isotonic sodium chloride injection；
Wherein, described dextran and amino acid injection is compound preparation patent medicine, every 1000ml low molecule dextrorotation
Sugar acid anhydride amino acid injection comprises water and following components: 4.1g containing leucine, 1.8g isoleucine, 2.9g phenylalanine, 1.8g
Threonine, 2.0g valine, 0.6g tryptophan, 2.4g methionine, 3.4g glycine, 5.0g lysine, 2.2g arginine,
1.0g histidine, 60.0g Dextran 40, adjuvant is sodium sulfite；
Described human serum albumin injection is patent medicine preparation, and the volumetric concentration of its human albumin is 20%.
The preparation method of a kind of clinical grade Cell protective solutions as above, its step is: human albumin injected
Liquid, dextran and amino acid injection and isotonic sodium chloride injection mix homogeneously, i.e. obtain described clinical grade thin
Born of the same parents protect liquid.
The application of a kind of clinical grade Cell protective solutions as above, it is applied to cell and preserves, may be directly applied to face
The cell of bed field of medicaments preserves, and it is applied to storage temperature when cell preserves and is preferably 2～8 DEG C, can be applicable to immunocyte
The preservation of CIK cell, during preservation, the concentration of immunocyte CIK cell is preferably 0.8～1.5 × 107Individual/ml, it is possible to be applied to
The preservation of umbilical cord mesenchymal stem cells, during preservation, the concentration of umbilical cord mesenchymal stem cells is preferably 3～7 × 105Individual/ml.
Dextran and amino acid injection typically just uses as the injection of trophism blood volume expander, has
Strong antigen, can improve plasma colloid osmotic pressure after intravenous, absorb blood vessel free surface moisture and increase blood volume, raise and maintain blood
Pressure；Its expanding blood volume effect is more weak and of short duration than macrodex, but it is stronger than macrodex to improve microcirculatory effect；It can
Make the most aggregated erythrocyte and platelet disaggregation, reduce viscosity of blood, improve microcirculation, prevent thrombosis；Additionally, it
Also there is osmotic diuresis effect, it is also possible to essential amino acids in added body, make protein synthesis dramatically increase and improve nutrition
Situation, has promotion human body protein Metabolism of Normal, corrects negative nitrogen balance, supplement protein, accelerate the effect of wound healing.
The invention by dextran and amino acid injection add to human serum albumin injection and etc.
Ooze in sodium chloride injection, be configured to Cell protective solutions and preserve for cell, cell survival rate can be greatly prolonged, keep cell to live
Power, preservation cell stage ratio is used alone human serum albumin injection and isotonic sodium chloride injection is longer, in cell preservation side
Beyond thought effect has been given play in face, develops the new application of dextran and amino acid injection, anticipates field of medicaments
Justice is great.
Isotonic sodium chloride injection (i.e. mass concentration is the sodium chloride injection of 0.9%) in the present invention is mainly as molten
Agent medium uses, and is used for dissolving mixing dextran and amino acid injection and human serum albumin injection, thus more preferably
Play dextran and amino acid injection and the synergism of human serum albumin injection, extend cell survival rate,
Keep cell viability, extend the cell holding time.
Detailed description of the invention
Principle and feature to the present invention are described below, and example is served only for explaining the present invention, is not intended to limit
Determine the scope of the present invention.
Embodiment 1
A kind of clinical grade Cell protective solutions, it comprises human serum albumin injection, the injection of low molecular dextran aminoacid
Liquid and isotonic sodium chloride injection, each component proportion is: the human serum albumin injection of 7.5ml, the low molecular dextran of 3ml
The isotonic sodium chloride injection of amino acid injection and 89.5ml；
Wherein, described dextran and amino acid injection is preferably compound preparation patent medicine, every 1000ml low molecule
Dextran amino acid injection comprise water and following components: 4.1g containing leucine, 1.8g isoleucine, 2.9g phenylalanine,
1.8g threonine, 2.0g valine, 0.6g tryptophan, 2.4g methionine, 3.4g glycine, 5.0g lysine, 2.2g essence ammonia
Acid, 1.0g histidine, 60.0g Dextran 40, adjuvant is sodium sulfite；
Described human serum albumin injection is preferably patent medicine preparation, and the volumetric concentration of its human albumin is 20%.
The preparation method of a kind of clinical grade Cell protective solutions as above, its step is: by white for the human blood of 7.5ml egg
The isotonic sodium chloride note of white injection (50ml, 20% specification), the dextran and amino acid injection of 3ml and 89.5ml
Penetrate liquid mix homogeneously, i.e. obtain described clinical grade Cell protective solutions.
The application of a kind of clinical grade Cell protective solutions as above, it is applied to cell and preserves, may be directly applied to face
The cell of bed field of medicaments preserves.
Embodiment 2
A kind of clinical grade Cell protective solutions, it comprises human serum albumin injection, the injection of low molecular dextran aminoacid
Liquid and isotonic sodium chloride injection, each component proportion is: the human serum albumin injection of 5ml, the low molecular dextran ammonia of 5ml
The isotonic sodium chloride injection of base acid injection and 90ml；
Wherein, described dextran and amino acid injection is preferably compound preparation patent medicine, every 1000ml low molecule
Dextran amino acid injection comprise water and following components: 4.1g containing leucine, 1.8g isoleucine, 2.9g phenylalanine,
1.8g threonine, 2.0g valine, 0.6g tryptophan, 2.4g methionine, 3.4g glycine, 5.0g lysine, 2.2g essence ammonia
Acid, 1.0g histidine, 60.0g Dextran 40, adjuvant is sodium sulfite；
Described human serum albumin injection is preferably patent medicine preparation, and the volumetric concentration of its human albumin is 20%.
The preparation method of a kind of clinical grade Cell protective solutions as above, its step is: by the human albumin of 5ml
Injection (50ml, 20% specification), the dextran and amino acid injection of 5ml and the isotonic sodium chloride injection of 90ml
Mix homogeneously, i.e. obtains described clinical grade Cell protective solutions.
The application of a kind of clinical grade Cell protective solutions as above, it is applied to cell and preserves, may be directly applied to face
The cell of bed field of medicaments preserves.
Embodiment 3
A kind of clinical grade Cell protective solutions, it comprises human serum albumin injection, the injection of low molecular dextran aminoacid
Liquid and isotonic sodium chloride injection, each component proportion is: the human serum albumin injection of 2.5ml, the low molecular dextran of 8ml
The isotonic sodium chloride injection of amino acid injection and 89.5ml；
Wherein, described dextran and amino acid injection is preferably compound preparation patent medicine, every 1000ml low molecule
Dextran amino acid injection comprise water and following components: 4.1g containing leucine, 1.8g isoleucine, 2.9g phenylalanine,
1.8g threonine, 2.0g valine, 0.6g tryptophan, 2.4g methionine, 3.4g glycine, 5.0g lysine, 2.2g essence ammonia
Acid, 1.0g histidine, 60.0g Dextran 40, adjuvant is sodium sulfite；
Described human serum albumin injection is preferably patent medicine preparation, and the volumetric concentration of its human albumin is 20%.
The preparation method of a kind of clinical grade Cell protective solutions as above, its step is: by white for the human blood of 2.5ml egg
The isotonic sodium chloride note of white injection (50ml, 20% specification), the dextran and amino acid injection of 8ml and 89.5ml
Penetrate liquid mix homogeneously, i.e. obtain described clinical grade Cell protective solutions.
The application of a kind of clinical grade Cell protective solutions as above, it is applied to cell and preserves, may be directly applied to face
The cell of bed field of medicaments preserves.
Embodiment 4
Clinical grade Cell protective solutions in Example 1 to embodiment 3 is as experimental group 1, experimental group 2 and experimental group respectively
The Cell protective solutions of 3, carries out the experiment of the preservation of immunocyte CIK cell, uses current industry inner cell protection liquid simultaneously
Common prescription as a control group 1 Cell protective solutions, carry out control experiment.
1, the preparation of matched group 1 Cell protective solutions
5ml human serum albumin injection (50ml, 20% specification) and the isotonic sodium chloride injection of 95ml are fully mixed.
2, separation of lymphocytes and cultivation
1) being coated of culture bottle: prepare a 50ml centrifuge tube, adds the DPBS buffer of 20ml with pipet, adds
RetroNectin T100B fibronectin (TAKARA) of 250ul, gently after mixing, adds people's CD3 antibody
(TAKARA) 100ul, piping and druming mixing.It is evenly distributed to being coated liquid in the culture bottle of 2 T75, puts into 4 DEG C of refrigerators and be coated overnight
Stand-by.
2) blood separation and cultivation: take 30-50mL peripheral blood or umbilical blood, below operate as a example by 30ml.First by blood
Being diluted with DPBS 1:1 by volume, then separate with lymph separation liquid, concrete operations are as follows: take two 50ml from
Heart pipe, each adds 15mL human lymphocyte separation liquid (Tianjin TBD company), 60mL blood dilution liquid mix homogeneously after, edge from
Heart tube wall is slowly added to lymphocyte separation medium upper strata, is careful not to the liquid level boundary both destroying.Often pipe 30mL, 2000rpm
Centrifugal 20min, slow liter delays fall；Centrifugal terminate after, draw ring-type tunica albuginea layer and be lymphocyte confluent monolayer cells in new centrifuge tube,
Ensure to collect whole lymphocytes.With DPBS washed cell twice, Flow cytometry, according to count results by 1 ×
106Individual/mL density is inoculated in culture bottle；Add GT-551H3 culture medium in culture bottle, concentration is 100000U/mL's
IFN-γ (Peprotech), adds GT-551H3 culture medium (Japan TaKaRa) next day, concentration is the IL-2 of 3000ng/mL
(interleukin II, the double aigret Pharmaceutical in Beijing), inducing culture CIK cell, carried out counting fluid infusion every 2～3 days and process.
3) finished product is collected: dispel and extract the CIK cell through 14 days amplification in vitro inducing culture, centrifugal segregation culture medium
Use brine cell twice afterwards.Carry out counting and detect cell surface marker thing CD3, CD56 by flow cytometer
Expression.Testing result, double positive cell ratios are more than 20%.
4) use 4 centrifuge tubes to sample in four times, i.e. obtain immunocyte CIK cell, adjust the immunity in 4 centrifuge tubes
Cell CIK cell quantity is respectively 1 × 109Individual, 0.8 × 109Individual, 1 × 109Individual, 1.5 × 109Individual, the most corresponding matched group 1 He
Experimental group 1,2,3, carries out labelling, standby.
3, finished product cell preserves
Above-mentioned 4 parts of standby immunocyte CIK cell are centrifuged, supernatant discarded, respectively with matched group 1 and experimental group 1,2,
The Cell protective solutions of 3 is made into cell suspending liquid again, is subsequently placed in refrigerator, carry out in refrigerator respectively 4 DEG C, 2 DEG C, 4 DEG C, 8
DEG C stored refrigerated, and after 0h, 24h, 48h, 72h, 96h, each group of cell is carried out survival rate detection, testing result such as table 1 institute
Show:
The viability examination result of the CIK cell that table 1 matched group 1 and experimental group 1～3 are cultivated
| Matched group 1 | Experimental group 1 | Experimental group 2 | Experimental group 3 | |
| 0h | 92.6% | 92.6% | 92.6% | 92.6% |
| 24h | 84.4% | 90.6% | 89.9% | 88.3% |
| 48h | 82.4% | 84.8% | 85.4% | 84.5% |
| 72h | 72.6% | 85.8% | 84.7% | 82.7% |
| 96h | 65.9% | 81.1% | 82% | 80.6% |
As shown in Table 1, CIK cell survival rate is successively decreased along with the passage of holding time.When CIK cell concentration is 0.8～1.5
×107Individual/ml time, matched group 1 Cell viability after 48h less than 80%, and experimental group after preserving 96h motility rate still above
80% (in current industry, the standard of Clinical practice CIK cell is: cell concentration 1 × 107Individual/ml, specification 100ml, survival rate is big
In 80%).
As can be seen here, the clinical grade Cell protective solutions in the present invention is applicable to the preservation of CIK cell, can be supplied to cell
While nutrition, the acid base imbalance that regulation cellular metabolism causes, alleviate the damage that cell causes because of acidosis, thus extend cell
Survival rate, keeps cell viability, preserves cell stage and at least can reach 96 hours, well meets cell strange land and make at a distance
With.
Embodiment 5
Clinical grade Cell protective solutions in Example 1 to embodiment 3 is as experimental group 4, experimental group 5 and experimental group respectively
The Cell protective solutions of 6, carries out preserving the experiment of umbilical cord mesenchymal stem cells, uses the normal of current industry inner cell protection liquid simultaneously
With formula as a control group 2 Cell protective solutions, carry out control experiment.
1, the preparation of matched group 2 Cell protective solutions
5ml human serum albumin injection (50ml, 20% specification) and the isotonic sodium chloride injection of 95ml are fully mixed.
2, the separation of umbilical cord mesenchymal stem cells, cultivation and phenotypic evaluation
1) umbilical cord is transferred to sterile petri dish, adds 10mlPBS buffer (Solarbio company) soaking and washing and sends out to umbilical cord
In vain, without bloodstain.
2) umbilical cord tissue is cut into segment, peels off blood vessel and exocuticle, obtain China's Tong Shi glue.
3) China's Tong Shi glue is shredded as far as possible, shred into 1mm3Fritter.
4) piece of tissue shredded uniformly is laid on bottom culture dish.
5) adding 1ml serum-free medium moistening piece of tissue, its patch jail treated by upset culture dish, puts 37 DEG C, 5%CO2Full
Cultivate with in humidified incubator (Thermo company, the U.S.).
6) overturn every other day, supplementing culture medium to 10ml.
7) uterus tissue pieces after 1 week full dose change DMEM culture medium, discard piece of tissue and non-adherent cell.
8) the most every 3 days half amounts change culture medium, and every day, observation of cell growth conditions, when observation of cell to 80% merges, used
Above-mentioned Digestive system peptic cell, passes on by 1: 5.
9) third generation cell dissociation is centrifuged, samples after PBS washing.
10) flow cytomery cell quantity, survival rate and cell surface marker thing.
Umbilical cord mesenchymal stem cells flow cytometry cell phenotype analysis result shows: mescenchymal stem cell CD73, CD90,
CD105 expression is more than 95%, and CD45, CD34 and HLA-DR expression is less than 2%.
11) use 4 centrifuge tubes to sample in four times, i.e. obtain umbilical cord mesenchymal stem cells, adjust the umbilicus in 4 centrifuge tubes
Band mescenchymal stem cell quantity is respectively 5 × 107Individual, 3 × 107Individual, 5 × 107Individual, 7 × 107Individual, the most corresponding matched group 2 He
Experimental group 4,5,6, carries out labelling, standby.
3, finished product cell preserves
Above-mentioned 4 parts of standby umbilical cord mesenchymal stem cells are centrifuged, supernatant discarded, respectively with matched group 2 and experimental group 4,
5, the Cell protective solutions of 6 is made into cell suspending liquid again, is subsequently placed in refrigerator, carry out in refrigerator respectively 4 DEG C, 2 DEG C, 4 DEG C,
The stored refrigerated of 8 DEG C, and after 0h, 24h, 48h, 72h, 96h, each group of cell is carried out survival rate detection, testing result such as table 2
Shown in:
Table 2 matched group 2 and experimental group 4～6 umbilical cord mesenchymal stem cells viability examination result
As shown in Table 2, umbilical cord mesenchymal stem cells survival rate is successively decreased along with the passage of holding time.When cell concentration is 3
～7 × 105Individual/ml time, matched group 2 Cell viability is already below 80% after 72h, and experimental group is preserving after 96h motility rate still
So more than 85%, (in current industry, the standard of Clinical practice umbilical cord mesenchymal stem cells is: cell quantity is 1 × 105Individual/kg body
Weight, i.e. amount commonly is 3～7 × 105Individual/ml, specification 100ml, survival rate is more than 80%).
As can be seen here, the clinical grade Cell protective solutions in the present invention is applicable to the preservation of umbilical cord mesenchymal stem cells, can be
While being supplied to cytotrophy, the acid base imbalance that regulation cellular metabolism causes, alleviate the damage that cell causes because of acidosis, from
And extend cell survival rate, and keep cell viability, preserving cell at least can reach 96 hours, well meets cell strange land long distance
From use.
Integrated embodiment 4 and embodiment 5 understand, and the clinical grade Cell protective solutions in the present invention can be greatly prolonged cell survival
Rate, keeps cell viability, and is applicable to the preservation of variety classes cell, and the holding time at least can reach 96 hours, meanwhile, again because of
Its each component is all clinic preparation, and composition is simple, and it is convenient to obtain, and Clinical practice is difficult to incompatibility occur, and safety is high, suitable
Cooperation is the Cell protective solutions of Clinical practice, can meet longer-distance dispensing and use.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of the spirit or essential attributes of the present invention, it is possible to realize the present invention in other specific forms.Therefore, no matter
From the point of view of which point, all should regard embodiment as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all by fall in the implication of equivalency and scope of claim
Change is included in the present invention.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and
Within principle, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.
Claims (10)
1. a clinical grade Cell protective solutions, it is characterised in that it comprises human serum albumin injection, low molecular dextran ammonia
Base acid injection and isotonic sodium chloride injection.
Clinical grade Cell protective solutions the most according to claim 1, it is characterised in that in terms of volumetric concentration, its each component
Proportioning is: the dextran and amino acid injection of human serum albumin injection, 3%～8% of 2.5%～7.5%, its
Remaining is isotonic sodium chloride injection.
Clinical grade Cell protective solutions the most according to claim 2, it is characterised in that people in described human serum albumin injection
The volumetric concentration of blood albumin is 20%, and dextran and amino acid injection described in every 1000ml comprises water and following group
Point: 4.1g containing leucine, 1.8g isoleucine, 2.9g phenylalanine, 1.8g threonine, 2.0g valine, 0.6g tryptophan,
2.4g methionine, 3.4g glycine, 5.0g lysine, 2.2g arginine, 1.0g histidine, 60.0g Dextran 40.
4. according to the clinical grade Cell protective solutions described in Claims 2 or 3, it is characterised in that in terms of volumetric concentration, its each group
Distribution ratio is: the human serum albumin injection of 5%, the dextran and amino acid injection of 5%, and remaining is isotonic chlorination
Sodium injection.
5. the preparation method of a clinical grade Cell protective solutions as claimed any one in claims 1 to 3, it is characterised in that
Its step is: human serum albumin injection, dextran and amino acid injection and isotonic sodium chloride injection are mixed
Close uniformly, i.e. obtain described clinical grade Cell protective solutions.
6. the application of a clinical grade Cell protective solutions as claimed any one in claims 1 to 3, it is characterised in that it should
Preserve for cell.
The application of clinical grade Cell protective solutions the most according to claim 6, it is characterised in that it is applied to clinical medicine neck
The cell in territory preserves.
The application of clinical grade Cell protective solutions the most according to claim 6, it is characterised in that when it is applied to cell preservation
Storage temperature is 2～8 DEG C.
The application of clinical grade Cell protective solutions the most according to claim 6, it is characterised in that it is applied to immunocyte
The preservation of CIK cell, during preservation, the concentration of immunocyte CIK cell is 0.8～1.5 × 107Individual/ml.
The application of clinical grade Cell protective solutions the most according to claim 6, it is characterised in that it is applied between umbilical cord fill
The preservation of matter stem cell, during preservation, the concentration of umbilical cord mesenchymal stem cells is 3～7 × 105Individual/ml.
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| CN108378021A (en) * | 2018-03-19 | 2018-08-10 | 英普乐孚生物技术(上海)有限公司 | A kind of stored refrigerated system of lymphocyte |
| CN108899106A (en) * | 2018-07-10 | 2018-11-27 | 长沙健金电子技术有限公司 | It is a kind of for protecting the devices and methods therefor of cell |
| CN110352952A (en) * | 2019-07-22 | 2019-10-22 | 苏州四正柏生物科技有限公司 | A kind of composition saved for cell, aqueous compositions and its application |
| CN110622956A (en) * | 2019-09-27 | 2019-12-31 | 广州南医大生物工程有限公司 | Umbilical cord mesenchymal stem cell preservation solution |
| CN112772637A (en) * | 2021-01-28 | 2021-05-11 | 朱灏 | DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution |
| CN114600868A (en) * | 2022-02-22 | 2022-06-10 | 张帅军 | Cell preservation solution and application thereof in preservation of CIK cells |
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| CN112772637A (en) * | 2021-01-28 | 2021-05-11 | 朱灏 | DMSO-free human umbilical cord mesenchymal stem cell injection frozen stock solution |
| CN114600868A (en) * | 2022-02-22 | 2022-06-10 | 张帅军 | Cell preservation solution and application thereof in preservation of CIK cells |
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| CN106212443B (en) | 2019-08-06 |
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