CN106212443B - 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
- Publication number
- CN106212443B CN106212443B CN201610665376.3A CN201610665376A CN106212443B CN 106212443 B CN106212443 B CN 106212443B CN 201610665376 A CN201610665376 A CN 201610665376A CN 106212443 B CN106212443 B CN 106212443B
- Authority
- CN
- China
- Prior art keywords
- cell
- injection
- clinical grade
- protective solutions
- serum albumin
- 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.)
- Active
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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
Abstract
The present invention relates to a kind of clinical grade Cell protective solutions and its preparation method and application, the clinical grade Cell protective solutions include human serum albumin injection, dextran and amino acid injection and isotonic sodium chloride injection；Its preparation method is to be uniformly mixed human serum albumin injection, dextran and amino acid injection and isotonic sodium chloride injection；It is saved applied to cell；Compared with prior art, suitable human serum albumin injection and dextran and amino acid injection is added in the invention in isotonic sodium chloride injection, it can be while being supplied to cytotrophy, adjust acid-base imbalance caused by cell metabolism, alleviate cell because damaging caused by acid poisoning, greatly prolong cell survival rate, keep cell viability, so that it is longer and being capable of direct clinical use to save cell stage, using more safe ready, preparation method is simple, convenient, fast.
Description
Technical field
The invention belongs to biomedicine fields more particularly to a kind of Cell protective solutions and its preparation method and application.
Background technique
For cell as an independent life entity, it has the plastic of very strong vitality, Proliferation, Differentiation ability and function
Sexuality.The mechanism of cytotherapeutic treatment disease is broadly divided into two major classes: first is that the direct effect of cell, directly specific with its
Bioactivity repairing damage tissue and organ；Or play specificity/non-specific lethal effect；Second is that cell is indirect
Effect, secretes the relevant factor or bioactive molecule such as to adjust the proliferation and functional activity of patients own cells.
With the development of the society, science and technology flourishes, there has also been the higher phases to quality of life and life expectancy by the mankind
It hopes.Possess the dream that a health, happiness, happy life cycle are everyone.But such as persistent ailments such as tumours, traditional is controlled
Treatment means have come into plateau, and brought side effect also allows many patient sufferings can't bear.
Cell therapy is the disease treatment new technology risen in recent years, refers to and utilizes certain cells with specific function
Characteristic has these cells and increases after being obtained using biological engineering method and/or being handled by amplification in vitro, specific culture etc.
Strong immune, kill pathogen and tumour cell promote the therapeutic efficiencies such as tissue and organ regeneration and physical recovery, to reach treatment
The purpose of disease.
Cell therapy is some difficulties with its good curative effect, the unique advantages such as Small side effects, more individuation, personalization
The treatment of the property controlled disease provides a kind of selection, sometimes even last selection.In history rank very long at present and in the future
Section, cell therapy will all take on important role in clinical treatment, and 21st century will be that cell therapy plays a significant role
Epoch.
Cell therapy mostly uses greatly physiological saline as common infusion medium at present, by the good cell of cultured and amplified in vitro
It is injected into sodium chloride injection, is added or is added without suitable human serum albumin, be placed in 0~10 DEG C and save, transport extremely
Destination；Although this store method is simple, clinically using conveniently, safely, of short duration preservation can only be used as, when preservation
Between the decline of too long cell viability quickly, be unable to satisfy strange land and use at a distance；Also have at present and increases appropriate model in physiological saline
The ingredients such as the potassium chloride, magnesium chloride, the sodium acetate that enclose are for saving the good cell of cultured and amplified in vitro, 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, be unable to satisfy strange land and use at a distance, and cannot
It is directly used in clinical use.
Summary of the invention
Technical problem to be solved by the invention is to provide it is a kind of prepare it is simple and convenient, using it is convenient, save cell stage
Clinical grade Cell protective solutions and its preparation method and application that are longer and being capable of direct clinical use.
The technical scheme to solve the above technical problems is that
A kind of clinical grade Cell protective solutions, it includes human serum albumin injection, the injection of D-40 amino acid
Liquid and isotonic sodium chloride injection.
Compared with prior art, the beneficial effects of the present invention are:
Dextran and amino acid injection is added to human serum albumin injection and waits by the invention
It seeps in sodium chloride injection, is configured to Cell protective solutions and is saved for cell, can be adjusted thin while being supplied to cytotrophy
Born of the same parents be metabolized caused by acid-base imbalance, alleviate cell because being damaged caused by acid poisoning, and with human serum albumin injection and isotonic chlorine
Change sodium injection and play advantageous synergistic effect, greatly prolong cell survival rate, keep cell viability, saves cell stage than single
It is solely longer using human serum albumin injection and isotonic sodium chloride injection, unexpected effect is played in terms of cell preservation
Fruit, holding time greatly prolong, and at least can reach 72 hours, better meet cell strange land and use at a distance, and clinical grade
The component of Cell protective solutions is injection, by its save cell can direct clinical use, using more safe ready,
It is convenient and saves trouble.
Based on the above technical solution, the present invention can also be improved as follows.
As a kind of preferred embodiment of the invention, in terms of volumetric concentration, its each group distribution ratio are as follows: 2.5%~
7.5% human serum albumin injection, 3%~8% dextran and amino acid injection, remaining is isotonic sodium chloride
Injection.
Beneficial effect using above-mentioned preferred embodiment is:
Clinical grade Cell protective solutions in the present invention save cell using this formula rate, not only prepare it is more convenient, more
Add and be suitable for promoting, and can guarantee while being supplied to cytotrophy, adjust acid-base imbalance caused by cell metabolism, alleviates
Cell greatly prolongs cell survival rate, keeps cell viability, preservation cell stage is longer, thin because damaging caused by acid poisoning
Born of the same parents save aspect and play unexpected effect, and the holding time can reach 96 hours, and it is remote to better meet cell strange land
It uses, and the component proportion of clinical grade Cell protective solutions is within this range, preserves the clinical grade cytoprotection of target cell
Liquid can be directly injected into human body, and direct clinical use using more safe ready, is convenient and saves trouble.
As another preferred embodiment of the invention, the volume of human serum albumin in the human serum albumin injection
Concentration is 20%, and dextran and amino acid injection described in every 1000ml includes water and following components: 4.1g contains 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.
Beneficial effect using above-mentioned preferred embodiment is:
Human serum albumin injection in the present invention is patent medicine preparation, and dextran and amino acid injection is compound
Preparation patent medicine passes through many years clinical verification, and use is safer, can be straight by the cell that clinical grade Cell protective solutions save
Connect clinical use, application is more convenient, and the human serum albumin of this concentration can preferably with D-40 amino
Acid injection plays synergistic effect, preferably gives cells with nutrient, adjusts acid-base imbalance caused by cell metabolism, alleviates cell
Because the effect damaged caused by acid poisoning is more preferable, to greatly prolong cell survival rate, cell viability is preferably kept, saves cell
Time is longer, at least can reach 96 hours, better meets cell strange land and uses at a distance.
As another preferred embodiment of the invention, in terms of volumetric concentration, its each group distribution ratio are as follows: 5% people
Blood albumin injection, 5% dextran and amino acid injection, remaining be isotonic sodium chloride injection.
Beneficial effect using above-mentioned preferred embodiment is:
Clinical grade Cell protective solutions of the invention can preferably give cells with nutrient using this formula rate, adjust thin
Born of the same parents be metabolized caused by acid-base imbalance, alleviate cell because caused by acid poisoning damage effect it is more preferable, to greatly prolong cell survival
Rate preferably keeps cell viability, and preservation cell stage is longer, can reach 96 hours or more, it is remote to better meet cell strange land
Distance uses, and the component proportion of clinical grade Cell protective solutions is in this way, the clinical grade cell for preserving target cell is protected
Shield liquid can be directly injected into human body, and direct clinical use using more safe ready, is convenient and saves trouble.
A kind of preparation method of clinical grade Cell protective solutions as described above, its steps are as follows: inject human serum albumin
Liquid, dextran and amino acid injection and isotonic sodium chloride injection are uniformly mixed to get thin to the clinical grade
Born of the same parents protect liquid.
Compared with prior art, the beneficial effects of the present invention are:
The method of the present invention is easy to operate, easy to implement quick, creative by dextran and amino acid injection
It is added in human serum albumin injection and isotonic sodium chloride injection, is configured to Cell protective solutions and is saved for cell, it can be
While being supplied to cytotrophy, acid-base imbalance caused by cell metabolism is adjusted, alleviates cell because damaging caused by acid poisoning, and
Advantageous synergistic effect is played with human serum albumin injection and isotonic sodium chloride injection, greatly prolongs cell survival rate, is protected
Cell viability is held, preservation cell stage is longer than human serum albumin injection and isotonic sodium chloride injection is used alone, thin
Born of the same parents save aspect and play unexpected effect, and the holding time can reach 96 hours, and it is remote to better meet cell strange land
Use, and the component of clinical grade Cell protective solutions is injection, the cell saved by it can direct clinical use, answer
With more safe ready, be convenient and save trouble.
A kind of application of clinical grade Cell protective solutions as described above, it is saved applied to cell.
Compared with prior art, the beneficial effects of the present invention are:
Clinical grade Cell protective solutions of the invention are saved applied to cell, can be adjusted while being supplied to cytotrophy
Acid-base imbalance caused by cell metabolism alleviates cell because damaging caused by acid poisoning, and with human serum albumin injection and isotonic
Sodium chloride injection plays advantageous synergistic effect, greatly prolongs cell survival rate, keeps cell viability, saves cell stage ratio
Human serum albumin injection is used alone and isotonic sodium chloride injection is longer, is played in terms of cell preservation unexpected
Effect, holding time at least can reach 96 hours, better meet cell strange land and use at a distance, and clinical grade cytoprotection
The component of liquid is injection, by its save cell can direct clinical use, using more safe ready, province of saving worry
Thing.
As a kind of preferred embodiment of the invention, the cell that it is applied to clinical medicine fields is saved.
Beneficial effect using above-mentioned preferred embodiment is:
The cell that clinical grade Cell protective solutions of the invention may be directly applied to clinical medicine fields saves, clinical grade cell
Protect liquid component be injection, by its save cell can direct clinical use, using more safe ready, save worry
It saves trouble.
As another preferred embodiment of the invention, it is 2~8 DEG C that it, which is applied to storage temperature when cell saves,.
Beneficial effect using above-mentioned preferred embodiment is:
Clinical grade Cell protective solutions of the invention save cell at this temperature, can preferably extend cell survival rate, protect
Cell viability is held, preservation cell stage is longer, better meets cell strange land and uses at a distance.
As another preferred embodiment of the invention, it is applied to the preservation of immunocyte CIK cell, and when preservation exempts from
The concentration of epidemic disease cell CIK cell is 0.8~1.5 × 107A/ml.
Beneficial effect using above-mentioned preferred embodiment is:
Clinical grade Cell protective solutions of the invention can be applied to the preservation of various cells, as the guarantor of immunocyte CIK cell
When depositing, cell concentration is controlled 0.8~1.5 × 107In the range of a/ml, preservation effect is more preferable, can preferably extend cell
Survival rate keeps cell viability, and preservation cell stage is longer, at least can reach 96 hours, it is thin to better meet immunocyte CIK
Born of the same parents strange land uses at a distance.
As another preferred embodiment of the invention, it is applied to the preservation of umbilical cord mesenchymal stem cells, when preservation
The concentration of umbilical cord mesenchymal stem cells is 3~7 × 105A/ml.
Beneficial effect using above-mentioned preferred embodiment is:
Clinical grade Cell protective solutions of the invention can be applied to the preservation of various cells, when preservation umbilical cord mesenchymal stem cells
When, cell concentration is controlled 3~7 × 105In the range of a/ml, preservation effect is more preferable, can preferably extend cell survival
Rate keeps cell viability, and preservation cell stage is longer, at least can reach 96 hours, better meets umbilical cord mesenchymal stem cells
Strange land uses at a distance.
The present invention is further elaborated below.
A kind of clinical grade Cell protective solutions, it includes human serum albumin injection, the injection of D-40 amino acid
Liquid and isotonic sodium chloride injection, in terms of volumetric concentration, its each group distribution ratio are as follows: 2.5%~7.5% human serum albumin note
The dextran and amino acid injection of liquid, 3%~8% is penetrated, remaining is isotonic sodium chloride injection；
Wherein, the dextran and amino acid injection is compound preparation patent medicine, every 1000ml low molecule dextrorotation
Sugared acid anhydride amino acid injection includes water and following components: 4.1g contains 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, auxiliary material are sodium hydrogensulfite；
The human serum albumin injection is patent medicine preparation, and the volumetric concentration of human serum albumin is 20%.
A kind of preparation method of clinical grade Cell protective solutions as described above, its steps are as follows: inject human serum albumin
Liquid, dextran and amino acid injection and isotonic sodium chloride injection are uniformly mixed to get thin to the clinical grade
Born of the same parents protect liquid.
A kind of application of clinical grade Cell protective solutions as described above, it saves applied to cell, may be directly applied to face
The cell of bed field of medicaments saves, and it is preferably 2~8 DEG C that it, which is applied to storage temperature when cell saves, can be applied to immunocyte
The preservation of CIK cell, the concentration of immunocyte CIK cell is preferably 0.8~1.5 × 10 when preservation7A/ml, can also be applied to
The preservation of umbilical cord mesenchymal stem cells, the concentration of umbilical cord mesenchymal stem cells is preferably 3~7 × 10 when preservation5A/ml.
Dextran and amino acid injection is typically just used as the injection of trophism blood volume expander to use, and has
Strong antigen can improve plasma colloid osmotic pressure after intravenous, absorb blood vessel free surface moisture and increase blood volume, increase and maintain blood
Pressure；The effect of its expanding blood volume is weaker than macrodex and of short duration, but the effect for improving microcirculation is stronger than macrodex；It can
Make aggregated red blood cell and platelet disaggregation, reduce viscosity of blood, improves microcirculation, prevent thrombosis；In addition, it
Also there is osmotic diuresis effect, internal essential amino acid can also be supplemented, protein synthesis is made to dramatically increase and improve nutrition
Situation has and promotes human body protein Metabolism of Normal, corrects negative nitrogen balance, supplements protein, accelerates the effect of wound healing.
Dextran and amino acid injection is added to human serum albumin injection and waits by the invention
It seeps in sodium chloride injection, is configured to Cell protective solutions and is saved for cell, cell survival rate can be greatly prolonged, keep cell living
Power, preservation cell stage is longer than human serum albumin injection and isotonic sodium chloride injection is used alone, in cell preservation side
Unexpected effect is played in face, develops the new application of dextran and amino acid injection, is anticipated to field of medicaments
Justice is great.
In the present invention isotonic sodium chloride injection (i.e. mass concentration be 0.9% sodium chloride injection) mainly as molten
Agent medium uses, for dissolving mixing dextran and amino acid injection and human serum albumin injection, thus more preferably
Performance dextran and amino acid injection and human serum albumin injection synergistic effect, extend cell survival rate,
Cell viability is kept, the cell holding time is extended.
Specific embodiment
The principles and features of the present invention are described below, and the given examples are served only to explain 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 includes human serum albumin injection, the injection of D-40 amino acid
Liquid and isotonic sodium chloride injection, each group distribution ratio are as follows: the D-40 of the human serum albumin injection of 7.5ml, 3ml
The isotonic sodium chloride injection of amino acid injection and 89.5ml；
Wherein, the dextran and amino acid injection is preferably compound preparation patent medicine, every 1000ml low molecule
Dextran amino acid injection include 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, auxiliary material is sodium hydrogensulfite；
The human serum albumin injection is preferably patent medicine preparation, and the volumetric concentration of human serum albumin is 20%.
A kind of preparation method of clinical grade Cell protective solutions as described above, its steps are as follows: by the white egg of people's blood of 7.5ml
The isotonic sodium chloride of white injection (50ml, 20% specification), the dextran and amino acid injection of 3ml and 89.5ml is infused
Liquid is penetrated to be uniformly mixed to get the clinical grade Cell protective solutions are arrived.
A kind of application of clinical grade Cell protective solutions as described above, it saves applied to cell, may be directly applied to face
The cell of bed field of medicaments saves.
Embodiment 2
A kind of clinical grade Cell protective solutions, it includes human serum albumin injection, the injection of D-40 amino acid
Liquid and isotonic sodium chloride injection, each group distribution ratio are as follows: the D-40 ammonia of the human serum albumin injection of 5ml, 5ml
The isotonic sodium chloride injection of base acid injection and 90ml；
Wherein, the dextran and amino acid injection is preferably compound preparation patent medicine, every 1000ml low molecule
Dextran amino acid injection include 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, auxiliary material is sodium hydrogensulfite；
The human serum albumin injection is preferably patent medicine preparation, and the volumetric concentration of human serum albumin is 20%.
A kind of preparation method of clinical grade Cell protective solutions as described above, its steps are as follows: by the human serum albumin of 5ml
The isotonic sodium chloride injection of injection (50ml, 20% specification), the dextran and amino acid injection of 5ml and 90ml
It is uniformly mixed to get the clinical grade Cell protective solutions are arrived.
A kind of application of clinical grade Cell protective solutions as described above, it saves applied to cell, may be directly applied to face
The cell of bed field of medicaments saves.
Embodiment 3
A kind of clinical grade Cell protective solutions, it includes human serum albumin injection, the injection of D-40 amino acid
Liquid and isotonic sodium chloride injection, each group distribution ratio are as follows: the D-40 of the human serum albumin injection of 2.5ml, 8ml
The isotonic sodium chloride injection of amino acid injection and 89.5ml；
Wherein, the dextran and amino acid injection is preferably compound preparation patent medicine, every 1000ml low molecule
Dextran amino acid injection include 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, auxiliary material is sodium hydrogensulfite；
The human serum albumin injection is preferably patent medicine preparation, and the volumetric concentration of human serum albumin is 20%.
A kind of preparation method of clinical grade Cell protective solutions as described above, its steps are as follows: by the white egg of people's blood of 2.5ml
The isotonic sodium chloride of white injection (50ml, 20% specification), the dextran and amino acid injection of 8ml and 89.5ml is infused
Liquid is penetrated to be uniformly mixed to get the clinical grade Cell protective solutions are arrived.
A kind of application of clinical grade Cell protective solutions as described above, it saves applied to cell, may be directly applied to face
The cell of bed field of medicaments saves.
Embodiment 4
Clinical grade Cell protective solutions of the Example 1 into embodiment 3 are as experimental group 1, experimental group 2 and experimental group respectively
3 Cell protective solutions carry out the experiment of the preservation of immunocyte CIK cell, while using current industry inner cell protection liquid
Common prescription as a control group 1 Cell protective solutions, carry out control experiment.
1, the preparation of 1 Cell protective solutions of control group
5ml human serum albumin injection (50ml, 20% specification) and the isotonic sodium chloride injection of 95ml are mixed well.
2, separation of lymphocytes and culture
1) coating of culture bottle: preparing a 50ml centrifuge tube, and the DPBS buffer of 20ml is added with pipette, is added
The RetroNectin T100B fibronectin (TAKARA) of 250ul after mixing gently, adds people's CD3 antibody
(TAKARA) 100ul, piping and druming mix.Coating buffer is evenly distributed in the culture bottle of 2 T75, is put into 4 DEG C of refrigerator coatings overnight
For use.
2) blood isolation and culture: 30-50mL peripheral blood or bleeding of the umbilicus are taken, is operated by taking 30ml as an example below.First by blood
With DPBS, 1:1 is diluted by volume, is then separated with lymph separating liquid, concrete operations are as follows: take two 50ml from
Heart pipe, it is each that 15mL human lymphocyte separating liquid (Tianjin TBD company) is added, after mixing 60mL blood dilution liquid, edge from
Heart tube wall is slowly added to lymphocyte separation medium upper layer, is careful not to destroy the liquid level boundary of the two.Every pipe 30mL, 2000rpm
It is centrifuged 20min, delays and rises slow drop；After centrifugation, drawing cyclic annular tunica albuginea layer is lymphocyte confluent monolayer cells into new centrifuge tube,
Guarantee is collected into whole lymphocytes.Wash cell twice with DPBS, Flow cytometry, according to count results by 1 ×
106A/mL density is inoculated in culture bottle；GT-551H3 culture medium is added into culture bottle, concentration is 100000U/mL's
IFN-γ (Peprotech), the IL-2 that next day adds GT-551H3 culture medium (Japanese TaKaRa), concentration is 3000ng/mL
(interleukin 2, the double aigret medicine companies in Beijing), Fiber differentiation CIK cell carried out counting fluid infusion processing every 2~3 days.
3) finished product is collected: dispelling and extract the CIK cell by 14 days amplification in vitro Fiber differentiations, centrifugation removal culture medium
Afterwards twice with brine cell.It is counted by flow cytometer and detects cell surface marker object CD3, CD56
Expression quantity.Testing result, double positive cells ratio are greater than 20%.
4) it is sampled in four times using 4 centrifuge tubes immune in 4 centrifuge tubes to get adjusting to immunocyte CIK cell
Cell CIK cell quantity is respectively 1 × 109It is a, 0.8 × 109It is a, 1 × 109It is a, 1.5 × 109It is a, respectively correspond 1 He of control group
Experimental group 1,2,3, marks, spare.
3, finished product cell saves
By above-mentioned spare 4 parts of immunocyte CIK cells centrifugation, discard supernatant, respectively with control group 1 and experimental group 1,2,
3 Cell protective solutions are made into cell suspending liquid again, are subsequently placed in refrigerator, carry out 4 DEG C, 2 DEG C, 4 DEG C, 8 in refrigerator respectively
DEG C it is stored refrigerated, and in 0h, for 24 hours, after 48h, 72h, 96h to group of cells carry out survival rate detection, testing result such as 1 institute of table
Show:
The viability examination result for the CIK cell that 1 control group 1 of table and experimental group 1~3 are cultivated
| Control 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 with the passage of holding time.When CIK cell concentration is 0.8~1.5
×107When a/ml, 1 Cell viability of control group is lower than 80% after 48h, and experimental group motility rate after saving 96h is still greater than
80% (at present in industry clinical use CIK cell standard are as follows: cell concentration 1 × 107A/ml, specification 100ml, survival rate are big
In 80%).
It can be seen that the clinical grade Cell protective solutions in the present invention are suitable for the preservation of CIK cell, cell can be supplied to
While nutrition, acid-base imbalance caused by cell metabolism is adjusted, alleviates cell because damaging caused by acid poisoning, to extend cell
Survival rate keeps cell viability, and saving cell stage at least can reach 96 hours, and meet cell strange land well makes at a distance
With.
Embodiment 5
Clinical grade Cell protective solutions of the Example 1 into embodiment 3 are as experimental group 4, experimental group 5 and experimental group respectively
6 Cell protective solutions carry out the experiment for saving umbilical cord mesenchymal stem cells, while using the normal of current industry inner cell protection liquid
With the Cell protective solutions for being formulated as a control group 2, control experiment is carried out.
1, the preparation of 2 Cell protective solutions of control group
5ml human serum albumin injection (50ml, 20% specification) and the isotonic sodium chloride injection of 95ml are mixed well.
2, the separation, culture and phenotypic evaluation of umbilical cord mesenchymal stem cells
1) umbilical cord is transferred to sterile petri dish, and 10mlPBS buffer (Solarbio company) soaking and washing to umbilical cord is added to send out
It is white, no bloodstain.
2) umbilical cord tissue is cut into segment, removes blood vessel and exocuticle, obtains magnificent Tong Shi glue.
3) magnificent Tong Shi glue is shredded as far as possible, shreds into 1mm3Fritter.
4) tissue block shredded is uniformly laid on culture dish bottom.
5) 1ml serum free medium is added and soaks tissue block, overturning culture dish waits for its patch jail, sets 37 DEG C, 5%CO2It is full
With culture in humidified incubator (Thermo company, the U.S.).
6) it overturns every other day, supplementing culture medium to 10ml.
7) full dose changes DMEM culture medium after uterus tissue pieces 1 week, discards tissue block and non-adherent cell.
8) every 3 days half amounts change culture medium after, observe cell growth state daily, when observation cell to 80% merges, use
Above-mentioned digestive juice vitellophag, by 1: 5 passage.
9) third generation cell dissociation is centrifuged, is sampled after PBS washing.
10) flow cytomery cell quantity, survival rate and cell surface marker object.
Umbilical cord mesenchymal stem cells flow cytometry cell phenotype analysis the result shows that: mescenchymal stem cell CD73, CD90,
CD105 expression quantity is greater than 95%, and CD45, CD34 and HLA-DR expression quantity are less than 2%.
11) it is sampled in four times using 4 centrifuge tubes to get to umbilical cord mesenchymal stem cells, adjusts the navel in 4 centrifuge tubes
Band mescenchymal stem cell quantity is respectively 5 × 107It is a, 3 × 107It is a, 5 × 107It is a, 7 × 107It is a, respectively correspond 2 He of control group
Experimental group 4,5,6, marks, spare.
3, finished product cell saves
By above-mentioned spare 4 parts of umbilical cord mesenchymal stem cells centrifugation, discard supernatant, respectively with control group 2 and experimental group 4,
5,6 Cell protective solutions are made into cell suspending liquid again, are subsequently placed in refrigerator, respectively in refrigerator carry out 4 DEG C, 2 DEG C, 4 DEG C,
8 DEG C stored refrigerated, and in 0h, for 24 hours, after 48h, 72h, 96h to group of cells carry out survival rate detection, testing result such as table 2
It is shown:
4~6 umbilical cord mesenchymal stem cells viability examination result of 2 control group 2 of table and experimental group
As shown in Table 2, umbilical cord mesenchymal stem cells survival rate is successively decreased with the passage of holding time.When cell concentration is 3
~7 × 105When a/ml, 2 Cell viability of control group is already below 80% after 72h, and motility rate is still after saving 96h for experimental group
So be greater than 85% (at present in industry clinical use umbilical cord mesenchymal stem cells standard are as follows: cell quantity be 1 × 105A/kg body
Weight, i.e. amount commonly are 3~7 × 10580%) a/ml, specification 100ml, survival rate are greater than.
It, can be it can be seen that clinical grade Cell protective solutions in the present invention are suitable for the preservation of umbilical cord mesenchymal stem cells
While being supplied to cytotrophy, adjust cell metabolism caused by acid-base imbalance, alleviate cell because being damaged caused by acid poisoning, from
And extend cell survival rate, cell viability is kept, saving cell at least can reach 96 hours, meet cell strange land long distance well
From using.
Integrated embodiment 4 and embodiment 5 are it is found that the clinical grade Cell protective solutions in the present invention can greatly prolong cell survival
Rate keeps cell viability, and is suitable for the preservation of variety classes cell, and the holding time at least can reach 96 hours, meanwhile, and because
Its each component is all clinical preparation, and ingredient is simple, and it is convenient to obtain, and clinical use is less prone to incompatibility, highly-safe, is fitted
Cooperation is the Cell protective solutions of clinical use, can meet longer-distance dispatching and use.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of clinical grade Cell protective solutions, which is characterized in that it includes human serum albumin injection, D-40 ammonia
Base acid injection and isotonic sodium chloride injection, its each group distribution ratio are as follows: 2.5%~7.5% human serum albumin injection,
3%~8% dextran and amino acid injection, remaining is isotonic sodium chloride injection.
2. clinical grade Cell protective solutions according to claim 1, which is characterized in that people in the human serum albumin injection
The volumetric concentration of blood albumin is 20%, and dextran and amino acid injection described in every 1000ml is comprising water and with the 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.
3. clinical grade Cell protective solutions according to claim 1 or 2, which is characterized in that in terms of volumetric concentration, its each group
Distribution ratio are as follows: 5% human serum albumin injection, 5% dextran and amino acid injection, remaining be isotonic chlorination
Sodium injection.
4. a kind of preparation method of clinical grade Cell protective solutions as claimed any one in claims 1 to 3, which is characterized in that
Its steps are as follows: mix human serum albumin injection, dextran and amino acid injection and isotonic sodium chloride injection
Close uniformly to get arrive the clinical grade Cell protective solutions.
5. a kind of application of clinical grade Cell protective solutions as claimed any one in claims 1 to 3, which is characterized in that it is answered
It is saved for cell.
6. the application of clinical grade Cell protective solutions according to claim 5, which is characterized in that it leads applied to clinical medicine
The cell in domain saves.
7. the application of clinical grade Cell protective solutions according to claim 5, which is characterized in that when it is applied to cell preservation
Storage temperature is 2~8 DEG C.
8. the application of clinical grade Cell protective solutions according to claim 5, which is characterized in that it is applied to immunocyte
The preservation of CIK cell, the concentration of immunocyte CIK cell is 0.8~1.5 × 10 when preservation7A/ml.
9. the application of clinical grade Cell protective solutions according to claim 5, which is characterized in that it is applied to umbilical cord mesenchyma
The preservation of stem cell, the concentration of umbilical cord mesenchymal stem cells is 3~7 × 10 when preservation5A/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610665376.3A CN106212443B (en) | 2016-08-12 | 2016-08-12 | Clinical grade Cell protective solutions and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610665376.3A CN106212443B (en) | 2016-08-12 | 2016-08-12 | Clinical grade Cell protective solutions and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106212443A CN106212443A (en) | 2016-12-14 |
| CN106212443B true CN106212443B (en) | 2019-08-06 |
Family
ID=57547292
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610665376.3A Active CN106212443B (en) | 2016-08-12 | 2016-08-12 | Clinical grade Cell protective solutions and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106212443B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107090434B (en) * | 2017-04-28 | 2020-10-13 | 蓝莲(杭州)生物科技有限公司 | Blood anticoagulation and preservation method for improving CIK cell culture effect |
| 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 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186338A (en) * | 2008-08-20 | 2011-09-14 | 人类起源公司 | Improved cell composition and methods of making the same |
| CN102920734A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis |
| CN103404509A (en) * | 2013-08-07 | 2013-11-27 | 彭乐 | Cell preserving fluid as well as preparation method and application of cell preserving fluid |
| CN105132370A (en) * | 2015-09-28 | 2015-12-09 | 丛秀丽 | Clinic-level adipose-derived stem cell preparation and storage methods |
| CN105340876A (en) * | 2014-08-23 | 2016-02-24 | 瑞安市普罗生物科技有限公司 | Cryopreservation method and medium for CIK (Cytokine Induced Killer) cells |
-
2016
- 2016-08-12 CN CN201610665376.3A patent/CN106212443B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102186338A (en) * | 2008-08-20 | 2011-09-14 | 人类起源公司 | Improved cell composition and methods of making the same |
| CN102920734A (en) * | 2012-11-14 | 2013-02-13 | 青岛奥克生物开发有限公司 | Mesenchymal stem cell injection and preparation method thereof as well as application in preparation of medicine for treating ulcerative colitis |
| CN103404509A (en) * | 2013-08-07 | 2013-11-27 | 彭乐 | Cell preserving fluid as well as preparation method and application of cell preserving fluid |
| CN105340876A (en) * | 2014-08-23 | 2016-02-24 | 瑞安市普罗生物科技有限公司 | Cryopreservation method and medium for CIK (Cytokine Induced Killer) cells |
| CN105132370A (en) * | 2015-09-28 | 2015-12-09 | 丛秀丽 | Clinic-level adipose-derived stem cell preparation and storage methods |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106212443A (en) | 2016-12-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106212443B (en) | Clinical grade Cell protective solutions and its preparation method and application | |
| CN104542578B (en) | Cell preservation solution and preparation method and applications thereof | |
| King et al. | Sphingosine-1-phosphate prevents egress of hematopoietic stem cells from liver to reduce fibrosis | |
| CN106701681B (en) | A kind of external evoked amplification of immunocyte, the method for freezing and recovering | |
| CN107148967B (en) | Antigen-specific T lymphocyte cryopreservation solution and preparation method and application thereof | |
| CN105076116A (en) | Cell cryopreservation liquid, application thereof and cryopreservation method of megakaryocyte progenitor cells | |
| CN102349500B (en) | Mesenchymal stem cell self-preserving liquid | |
| AU2013284340B2 (en) | Isolation of stem cells from adipose tissue by ultrasonic cavitation, and methods of use | |
| CN103210903B (en) | Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof | |
| CN111248191A (en) | Normal-temperature cell preservation solution and cell preparation for injection | |
| CN104920340A (en) | Immune cell preserving fluid and application thereof | |
| CN103966164B (en) | A kind of hemizygote CAPRI cell preparation method | |
| JP2022088551A (en) | Uses of expanded populations of hematopoietic stem/progenitor cells | |
| CN106665559B (en) | A kind of immunocyte frozen stock solution and its application | |
| TW200902718A (en) | Procurement, isolation, and cryopreservation of endometrial/menstrual cells | |
| TW201629212A (en) | Method of preparing injection solution | |
| CN104222069A (en) | Erythroid progenitor cell cryopreservation liquid and application thereof | |
| CN108184818A (en) | A kind of Human plactnta mesenchyma stem cell suspension protective agent | |
| JP2023060299A (en) | Mesenchymal stem cells obtained from wharton's jelly for treatment of sepsis | |
| CN115039763A (en) | Immune cell cryopreservation liquid | |
| CN108350419A (en) | stem cell transplantation method and stem cell transplantation composition | |
| CN106754704A (en) | The method of the external evoked amplification of immunocyte | |
| CN107502592A (en) | A kind of immunocyte serum free medium and preparation method thereof | |
| CN106474157B (en) | Liver stem cell injection and preparation method thereof | |
| CN106754688A (en) | A kind of efficient method for resuscitation for freezing PMNC |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |