CN108378021A - A kind of stored refrigerated system of lymphocyte - Google Patents
A kind of stored refrigerated system of lymphocyte Download PDFInfo
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- CN108378021A CN108378021A CN201810223238.9A CN201810223238A CN108378021A CN 108378021 A CN108378021 A CN 108378021A CN 201810223238 A CN201810223238 A CN 201810223238A CN 108378021 A CN108378021 A CN 108378021A
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- 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
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
- A01N1/0273—Transport containers
Abstract
The invention discloses a kind of stored refrigerated system of lymphocyte, this preservation system is made of preservation liquid, gas permeability storage bag.It includes following each component that liquid, which is preserved, with volume percent：The isotonic electrolyte solution with blood plasma, 78%~93%；Human serum albumin injection 5%~20%；Glucose injection 1%~4%.Wherein, electrolyte solution is any one of sodium chloride injection, compound sodium chloride injection, Multiple electrolytes injection, Lactated Ringer'S Solution or their mixed solution；Or it is physiological saline, the mixed solution of potassium chloride injection, calcium gluconate injection.The present invention also provides application of the above-mentioned preservation liquid in Preservation of lymphocytes.Refrigeration protection liquid used in the present invention uses injection to configure, and ingredient is simple, can be directly used for feeding back.Storage bag is made with the high material of oxygen transit dose, and the cell survival time is extended, and vigor reduces slowly, suitable for storage and transport under the conditions of 4 15 DEG C, facilitates the clinical application of cell preparation.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of stored refrigerated system of lymphocyte.
Background technology
After cell leaves incubator and culture solution, the vigor that is easy to happen reduces and dead phenomenon.Living cells class drug is (such as
T, the products such as NK, DC, stem cell) it is different from traditional drug, this kind of drug needs to consider formulation ingredients to cell on preparation
The influence of metabolic activity.Cell needs constantly to carry out metabolism maintenance activity, therefore in cell preparation other than cell itself, also
There is the condition for meeting cell activity：Suitable osmotic pressure, the pH value of stabilization, basic nutriment, sufficient oxygen content
Deng, just can guarantee cell preparation before feedback by detection, transport, preserve etc. processing procedures remain to keep activity.
In order to maintain vigor of cell during transport and preservation, generally there are 2 kinds of store methods at present：It freezes and cold
It hides.
Freezing is that cell is stored in frozen solution, cell storage temperature is dropped to using the method for program cooling-
80 DEG C as low-temperature receiver using liquid nitrogen or dry ice in transport hereinafter, after arriving at, by cell recovery, wash away cryoprotection
After liquid, cell, which is resuspended in the isotonic solution such as physiological saline, (also to be had for feeding back and does not remove frozen solution direct feedback
Method).This store method cell holding time is longer, but there are the shortcomings that：1) step is complicated, more than stored refrigerated method
Go out many operating procedures；2) of high cost；3) cell viability reduces larger after recovering；4) time lengthening is fed back, is not suitable for needing
The patient fed back immediately.
And traditional stored refrigerated method the problem is that：1) it is not prepared as raw material using injection, it cannot
For feeding back；2) cell preservation effect was bad when being formulated simple, and formula cannot be satisfied the requirement of injection when complicated；3) existing
Store method does not all account for the influence of preparation packaging bag gas permeability, for cell for a kind of this drug of work, packaging bag
Gas permeability is to influence one of the key factor of the quality of the pharmaceutical preparations.
Invention content
Deficiency in for the above-mentioned prior art, the present invention provides a kind of stored refrigerated systems of lymphocyte, including leaching
Bar cell-preservation liquid, gas permeability storage bag.Wherein preserving liquid uses injection to configure, and ingredient is simple, can be directly used for back
It is defeated；Coordinate dedicated gas permeability shipping container simultaneously, the cell survival time is long, and vigor reduces slowly, can be protected in 4-15 DEG C of condition
It deposits and transports.The stored refrigerated liquid of the present invention is applied to Preservation of lymphocytes, can maintain cell activity (80% or more survival rate) 48
Hour or more, transportation cost is relatively low, and need not do recovery and cleaning operation before feeding back again, and storage life inner cell vigor is higher than jelly
It deposits.
A kind of stored refrigerated system of lymphocyte is provided to achieve the above object, and present invention employs following technical schemes：
A kind of stored refrigerated system of lymphocyte, this preservation system include Preservation of lymphocytes liquid, gas permeability storage bag.
Preferably, the gas permeability storage bag material is EVA or FEP or silicon rubber.
Further, oxygen transit dose is more than 500cm3/m2·24h·0.1MPa。
Further, oxygen transit dose is more than or equal to 2500cm3/m2·24h·0.1MPa。
Preservation of lymphocytes liquid in the stored refrigerated system of lymphocyte, includes following each group with volume percent
Point：
The electrolyte solution 78%~93% isotonic with blood plasma；
Human serum albumin injection 5%~20%；
Glucose injection 1%~4%.
Preferably, include following each component with volume percent：
The electrolyte solution 88% isotonic with blood plasma；
Human serum albumin injection 10%；
Glucose injection 2%.
Further, the electrolyte solution is sodium chloride injection, compound sodium chloride injection, compound electrolyte injection
Any one of liquid, Lactated Ringer'S Solution appoint several mixed liquors.
Further, the electrolyte solution is the mixing of physiological saline, potassium chloride injection, calcium gluconate injection
Solution.
The proportioning of above-mentioned each injection is respectively：
Contain in per 1L injections：
Compound sodium chloride injection：Sodium chloride 8.50g, potassium chloride 0.3g, calcium chloride 0.33g, auxiliary material are water for injection；
Multiple electrolytes injection：Sodium chloride (NaCl) 5.26g；Sodium gluconate (C6H11NaO7) 5.02g；Sodium acetate
(C2H3NaO2·3H2O)3.68g；Potassium chloride (KCl) 0.37g；Magnesium chloride (MgCl2·6H2O) 0.30g, auxiliary material are injection
Water；
Lactated Ringer'S Solution：Sodium lactate (C3H5O3Na) 3.10g, sodium chloride (NaCl) 6.00g, potassium chloride (KCl) 0.30g,
Calcium chloride (CaCl2·2H20) 0.20g, auxiliary material are water for injection；
The human serum albumin injection of specification 10g, 50ml：200g total proteins, wherein no less than 96% human serum albumin,
Sodium Caprylate is no more than 266mg, and auxiliary material is water for injection；
10% glucose injection：Glucose 100g, auxiliary material are water for injection.
Above-mentioned injection is clinical special medicament, is dosage form and concentration as defined in corresponding injection.
The present invention also provides application of the stored refrigerated system of above-mentioned lymphocyte in Preservation of lymphocytes.
Preferably, the temperature of the Preservation of lymphocytes is 4 DEG C~15 DEG C.
Further, the temperature of the Preservation of lymphocytes is 4 DEG C~10 DEG C.
Preferably, the time of the Preservation of lymphocytes is 0h~48h.
Preferably, the density of the lymphocyte is no more than 10,000,000/ml.
The beneficial effects of the present invention are：
1) refrigeration protection liquid used in the present invention uses injection to configure, and ingredient is simple, can be directly used for back
It is defeated；Coordinate gas permeability storage bag provided by the invention as shipping container simultaneously, the cell survival time can be made to be extended, vigor
It reduces slow；Through experiment application the present invention refrigeration protect liquid cooperation gas permeability shipping container carry out Preservation of lymphocytes when, suitable for
Storage and transport under the conditions of 4-15 DEG C, compared with the prior art middle most using cryogenic freezing cold chain transportation, this is greatly facilitated
The clinical application of cell preparation.
2) such cell preservation method of the present invention can maintain cell activity 48 hours under the premise of 80% or more survival rate
More than, the suitable holding time is in 48 hours；And transportation cost is relatively low, need not do recovery and cleaning behaviour before feedback again
Make, short time inner cell vigor is higher than the method frozen.
Description of the drawings
Fig. 1 is the cell preservation effect comparison diagram that the present invention preserves that liquid uses different electrolyte solutions.
Fig. 2 is different concentration of glucose in present invention preservation liquid to the influence diagram of cell survival rate.
Fig. 3 is the influence diagram that the present invention preserves different human serum albumin concentration versus cell survival rates in liquid.
Influence diagram of the difference cell density to cell survival rate when Fig. 4 is present invention preservation liquid application.
Influence diagram of the difference storage temperature to cell survival rate when Fig. 5 is present invention preservation liquid application.
Fig. 6 is influence diagram of the oxygen transit dose to cell survival rate.
Fig. 7 is freezen protective and the cell survival rate comparison diagram under stored refrigerated mode.
Fig. 8 is freezen protective and the IFN-γ release experiment comparison diagram under stored refrigerated mode.
Specific implementation mode
The technical solution in the present invention is clearly and completely described below in conjunction with the embodiment of the present invention.Following reality
It applies example to be only used for clearly illustrating technical scheme of the present invention, and not intended to limit the protection scope of the present invention.
A kind of stored refrigerated system of lymphocyte
This preservation system includes Preservation of lymphocytes liquid, gas permeability storage bag.
The gas permeability storage bag material is EVA or FEP or silicon rubber；Its oxygen transit dose is more than 500cm3/m2·
24h·0.1MPa.In order to obtain better preservation effect, the selection of oxygen transit dose is more than or equal to 2500cm3/m2·24h·
0.1MPa.The EVA refers to ethylene-vinyl acetate copolymer material；The FEP refers to fluorinated ethylene propylene copolymer (perfluor
Ethylene propylene copolymer) material.
A kind of Preservation of lymphocytes liquid
Since as pharmaceutical preparation, final preparation ingredient will feed back to human body together with cell, therefore, formulation ingredients by
To stringent limitation, to meet the strict demand as drug ingredient.It can be expired as cell preparation ingredient using existing injection
The requirement of sufficient safety, however, there remains the reaction considered between different injection ingredients, with prevent from generating precipitation, toxicity,
Allergy.Therefore the ingredient of cell preparation should reach the requirement for meeting cell activity under the premise of as simple as possible.
Preservation liquid provided by the invention is configured using injection, and ingredient is simple, meets the requirement of cell activity, can be direct
For feeding back.Specifically, including following each component with volume percent：
The electrolyte solution 78%~93% isotonic with blood plasma；
Human serum albumin injection 5%~20%；
Glucose injection 1%~4%.
Proportioning as one preferred includes following each component with volume percent：
The electrolyte solution 88% isotonic with blood plasma；
Human serum albumin injection 10%；
Glucose injection 2%.
The above-mentioned stored refrigerated liquid of lymphocyte includes the electrolyte solution and albumin isotonic with blood plasma, glucose；Wherein
Albumin be the highest protein of content in blood plasma, have following functions：Maintain the constant of colloid osmotic pressure；Albumin belongs to non-
The transport protein of specificity can reversibly be combined to form ease of solubility with the small organic molecule of many slightly solubilities and inorganic ions
Compound plays the role of removing toxic substances and transport；Albumin can guarantee the exchange between intracellular fluid, extracellular fluid；Albumin is to thin
The stability of after birth has protective effect；Albumin is a kind of important nutriment；When albumin encounters heavy metal ion, meeting
Automatically it is combined with heavy metal ion, plays the role of removing toxic substances.Glucose therein is the important energy matter of cell, for generating
ATP and partially synthetic metabolism.
Wherein, the electrolyte solution be sodium chloride injection, compound sodium chloride injection, Multiple electrolytes injection,
Any one of Lactated Ringer'S Solution.
Above-mentioned each injection is clinical special medicament, and dosage form and concentration meet corresponding injection regulation, specifically
Proportioning is respectively：
Contain in per 1L injections：
Compound sodium chloride injection：Sodium chloride 8.50g, potassium chloride 0.3g, calcium chloride 0.33g, auxiliary material are water for injection；
Multiple electrolytes injection：Sodium chloride (NaCl) 5.26g；Sodium gluconate (C6H11NaO7) 5.02g；Sodium acetate
(C2H3NaO2·3H2O)3.68g；Potassium chloride (KCl) 0.37g；Magnesium chloride (MgCl2·6H2O) 0.30g, auxiliary material are injection
Water；
Lactated Ringer'S Solution：Sodium lactate (C3H5O3Na) 3.10g, sodium chloride (NaCl) 6.00g, potassium chloride (KCl) 0.30g,
Calcium chloride (CaCl2·2H20) 0.20g, auxiliary material are water for injection；
The human serum albumin injection of specification 10g, 50ml：200g total proteins, wherein no less than 96% human serum albumin,
Sodium Caprylate is no more than 266mg, and auxiliary material is water for injection；
10% glucose injection：Glucose 100g, auxiliary material are water for injection.
The mixing of physiological saline+potassium chloride injection+calcium gluconate injection also can be selected in electrolyte solution therein
Solution.
Application of the above-mentioned stored refrigerated system of lymphocyte in Preservation of lymphocytes
As preferred embodiment, the temperature of the Preservation of lymphocytes is 4 DEG C~15 DEG C.
As preferred embodiment, the density of the lymphocyte is no more than 10,000,000/ml.
【Embodiment 1】The difference of different electrolyte solution cell preservation effects
According to the form below 1 prepares the stored refrigerated liquid of lymphocyte with percent by volume：
The proportioning of the stored refrigerated liquid of 1. lymphocyte of table
Wherein, the concrete component of each injection is as shown in table 2：
The component of each injection in the stored refrigerated liquid of 2. lymphocyte of table
The T cell for activating amplification cultivation to the 10th day by OKT3 and IL-2 is taken, is made into respectively with above-mentioned preservation liquid a concentration of
1x107Cell suspension, be added made of eva film in storage bag, be put into 4 DEG C of refrigerator and preserve.Respectively the 0th, 17,
It samples within 23,40,48 hours, platform phenol indigo plant dyeing counting cell survival rate.The results are shown in Figure 1.
The result shows that group 2, group 3, organize 4 preservations formula of liquid be substantially better than organize 1 preservation formula of liquid, namely with compound chlorine
Change sodium injection, the preservation liquid that Multiple electrolytes injection or Lactated Ringer'S Solution add glucose and human serum albumin to be made into is better than
Sodium chloride injection adds the preservation liquid that glucose and human serum albumin are made into, but organizes 42, group 3, group no significant differences.This is
Because group 2, group 3 organize the inorganic ions that 4 these electrolyte solutions provide basis for cell preservation, the current potential of cell membrane is maintained
With other electrophysiological functions；Maintain osmotic pressure；The nutriments such as cotransport glucose help excretion metabolism product etc., to
The normal energetic supersession of cell is maintained, ensures survival rate of cell during preservation.And simple sodium chloride injection is used for
When cell preserves, due to lacking K+、Ca2+、Mg2+Plasma can only maintain the activity of cell the short time.
It should be noted that the present embodiment is with the proportioning (ratio range outside table 1：The isotonic electrolyte solution with blood plasma
78%~93%；Human serum albumin injection 5%~20%；Glucose injection 1%~4%), it can obtain similarly
As a result.
【Embodiment 2】Influence of the different concentration of glucose to cell preservation effect
According to the form below 3 prepares the stored refrigerated liquid of lymphocyte with percent by volume：
The proportioning of the stored refrigerated liquid of 3. lymphocyte of table
The T cell that amplification cultivation to the 10th day is activated by OKT3 and IL-2 is taken, such as upper table compound sodium chloride injection+Portugal
The preservation liquid that grape sugar injection+human serum albumin injection is made into is made into a concentration of 1x107Cell suspension, be added eva film
In manufactured storage bag, it is put into 4 DEG C of refrigerators and preserves.It was sampled respectively at the 0th, 17,23,40,48 hour, platform phenol indigo plant dyeing counting is thin
Born of the same parents' survival rate.The results are shown in Figure 2.
The result shows that with the promotion of glucose relative concentration, cell survival rate is higher；Glucose has rush to cell survival
Into effect.
It should be noted that the present embodiment is with the proportioning (ratio range outside table 3：The isotonic electrolyte solution with blood plasma
78%~93%；Human serum albumin injection 5%~20%；Glucose injection 1%~4%), it can obtain similarly
As a result.
【Embodiment 3】Influence of the different human serum albumins to cell preservation effect
According to the form below 4 prepares the stored refrigerated liquid of lymphocyte with percent by volume：
The proportioning of the stored refrigerated liquid of 4. lymphocyte of table
The T cell that amplification cultivation to the 10th day is activated by OKT3 and IL-2 is taken, such as upper table compound sodium chloride injection+Portugal
The preservation liquid that grape sugar injection+human serum albumin injection is made into is made into a concentration of 1x107Cell suspension, be added eva film
In manufactured storage bag, it is put into 4 DEG C of refrigerators and preserves.It was sampled respectively at the 0th, 12,24,36,48 hour, platform phenol indigo plant dyeing counting is thin
Born of the same parents' survival rate.The results are shown in Figure 3.
The result shows that cell survival rate is directly proportional to human serum albumin concentration.
It should be noted that the present embodiment is with the proportioning (ratio range other than table 4：The isotonic electrolyte solution with blood plasma
78%~93%；Human serum albumin injection 5%~20%；Glucose injection 1%~4%), it can obtain similar knot
Fruit.
【Embodiment 4】Influence of the different cell densities to cell preservation effect
The T cell for activating amplification cultivation to the 10th day by OKT3 and IL-2 is taken, is noted with compound sodium chloride injection+glucose
It is respectively 5,000,000/ml, 10,000,000/ml, 20,000,000/ml to penetrate the preservation liquid that liquid+human serum albumin injection is made into and be made into concentration,
The cell suspension of 50000000/ml；It is added in storage bag made of eva film, is put into 4 DEG C of refrigerators and preserves.Respectively the 0th, 17,
It samples within 23,40,48 hours, platform phenol indigo plant dyeing counting cell survival rate.
The results are shown in Figure 4, and curve corresponds to 5,000,000/ml, 10,000,000/ml, 20,000,000/ml respectively from top to bottom in figure,
The cell suspension of 50000000/ml；The result shows that cell preserves density in 5,000,000/ml, 10,000,000/ml survival rates are higher, if super
10,000,000/ml is crossed, cell survival rate will be greatly reduced.
【Embodiment 5】Influence of the different storage temperatures to cell survival rate
The T cell for activating amplification cultivation to the 10th day by OKT3 and IL-2 is taken, is noted with compound sodium chloride injection+glucose
It penetrates the preservation liquid that liquid+human serum albumin injection is made into and is made into a concentration of 1x107Cell suspension, be added eva film made of
In storage bag, be individually placed to be put into 1 DEG C, 4 DEG C, 10 DEG C, preserve in 25 DEG C of thermal environment.Respectively the 0th, 17,23,40,48
Hour sampling, platform phenol indigo plant dyeing counting cell survival rate.
The results are shown in Figure 5, shows：In 4-15 DEG C of storage temperature range, cell survival rate is higher.4-10 DEG C is
Best storage temperature.
【Embodiment 6】Different oxygen transit doses preserve influence of the container to cell preservation effect
Existing clinical cytology feed back frequently be that Normal Saline bag (bottle) feeds back cell.Normal Saline bag
(bottle) common material is polyethylene or polypropylene material, it is desirable that low-permeable.And the physiological metabolism of cell has to oxygen.Therefore
The present invention tests the cell preservation effect that different oxygen transit doses preserve container.Specific experiment process is as follows：Take by OKT3 and
IL-2 activates the T cell of amplification cultivation to the 10th day, is noted with compound sodium chloride injection+glucose injection+human serum albumin
It penetrates the preservation liquid that liquid is made into and is made into a concentration of 1x107Cell suspension, be individually placed to non-PVC multi-layer co-extruded film (southwestern medicine company,
100ml sodium chloride injection bags, Chinese medicines quasi-word H50021772, oxygen transit dose：50cm3/m20.1MPa for 24 hours), polypropylene
Film (day sage's pharmacy, 100ml sodium chloride injection bags, Chinese medicines quasi-word H20033147, oxygen transit dose：500cm3/m2·
0.1MPa for 24 hours), eva film (Dupon, 3165SB, oxygen transit dose：2500cm3/m20.1MPa for 24 hours), FEP films
(OriGen, PL70, oxygen transit dose：3500cm3/m20.1MPa for 24 hours), silicon rubber film (wilsonwolf, G-Rex
10M, oxygen transit dose：230000cm3/m20.1MPa for 24 hours) etc. made of in closed container, be put into 4 DEG C of refrigerators and preserve.Point
It was not sampled at the 0th, 17,23,40,48 hour, platform phenol indigo plant dyeing counting cell survival rate.The results are shown in Figure 6.
The result shows that cell survival rate is directly proportional to oxygen gas permeability amount, the higher kyto- of oxygen gas permeability amount preserves effect
Fruit is better.Due to lacking many nutriments in cell-preservation liquid, to improve the holding time, need to protect by the way of refrigeration
It deposits.When stored refrigerated, cell metabolism slows down, but can't stop, and still needs the supply of oxygen.The present invention is sent out through experiment
Existing, ventilative conditions on cell preservation has a significant impact, and good ventilative condition is more preferable than air-locked conditioned cell preservation effect,
The cell holding time is longer.
【Embodiment 7】Freezen protective and stored refrigerated comparison
It takes peripheral blood a, PBMC is detached using Ficoll, with Pan T cell Isolation Kit human
(Miltenyi, 130-096-535) detaches T cell.With X-VIVO15 (LONZA, 04-418Q) culture mediums by cell concentration tune
Whole is 1x106/ ml, addition 300IU/ml IL-2,2% heat inactivation autologous plasma, 25ul/ml t cell activations magnetic bead (GIBCO,
11132D), 37 DEG C, 5% carbon dioxide culture are put into.CD19CAR slow virus is added after 48 hours.4th day cleaning cell, with containing
Cell is resuspended in 300IU/ml IL-2, the fresh culture of 2% heat inactivation autologous plasma.It is close according to cell growth condition and cell
Degree carries out fluid infusion and amplification cultivation.It takes culture to the 10th day CAR-T cell, is divided into 2 groups：Group 1 uses compound sodium chloride injection
The preservation liquid that+glucose injection+human serum albumin injection is made into is made into a concentration of 1x107Cell suspension, be packed into EVA it is thin
In film strips, it is put into 4 DEG C of refrigerators and preserves；Group 2 prepares frozen stock solution with according to following table：
CAR-T cells are made into 1x10 with above-mentioned frozen stock solution7Cell suspension, be packed into cryopreservation tube in, be put into program cooling
Box (NALGENE, 5100-0001), -80 DEG C freeze.Respectively in the 0th, 12,24,36,48 hour sampling bench phenol indigo plant dyeing counting
Cell survival rate.The results are shown in Figure 7.
In addition at the 0th, 12,24,36,48 hour, IFN-γ release experiment was done in sampling.Take target cell (raji of CD19+)
With effector cell (the CAR-T cells of CD19CAR+), 2 × 10 are made into the culture mediums of RPMI1640 containing 5%FBS5/ ml cells are outstanding
Liquid is grouped and is loaded according to following table：
3 multiple holes of every group of setting, 37 DEG C, 5%CO2Under the conditions of co-culture 4h, receive supernatant 100 μ l, ELISA and detect IFN-
γ secretory volumes.
IFN-γ burst size (pg/ml) after effector cell is induced by target cell=by (the effect target cell after correction is incubated altogether
The corresponding target cell control group after corresponding effector cell's control group mean OD value-correction after group mean OD value-correction is flat
Equal OD values) it is brought into concentration (pg/mL) * samples dilution times of calculated IFN-γ in the obtained formula according to standard curve
Number.As a result see Fig. 8.
The result shows that method using the present invention, freezen protective is substantially better than in 24 hours inner cell preservation effects, performance
For cell survival rate higher, there is higher IFN-γ secretion amount when being incubated with target cell.
Therefore the stored refrigerated liquid of lymphocyte provided by the invention coordinates dedicated gas permeability when carrying out Preservation of lymphocytes
Storage bag makes the cell survival time be extended, and vigor reduces slow, storage and transport under the conditions of 4-15 DEG C, to convenient thin
The clinical application of born of the same parents' preparation.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvement and deformations can also be made, these improvement and deformations
Also it should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of stored refrigerated system of lymphocyte, this preservation system includes Preservation of lymphocytes liquid, gas permeability storage bag.
2. the stored refrigerated system of lymphocyte according to claim 1, which is characterized in that the gas permeability storage bag material
For EVA or FEP or silicon rubber.
3. preservation system according to claim 1, storage bag oxygen transit dose is more than 500cm3/m2·24h·
0.1MPa。
4. preservation system according to claim 3, storage bag oxygen transit dose is more than or equal to 2500cm3/m2·24h·
0.1MPa。
5. a kind of Preservation of lymphocytes liquid, which is characterized in that with volume percent include following each component：
The electrolyte solution 78%~93% isotonic with blood plasma；
Human serum albumin injection 5%~20%；
Glucose injection 1%~4%.
6. Preservation of lymphocytes liquid according to claim 5, which is characterized in that with volume percent include following each group
Point：
The electrolyte solution 88% isotonic with blood plasma；
Human serum albumin injection 10%；
Glucose injection 2%.
7. Preservation of lymphocytes liquid according to claim 5 or 6, it is characterised in that：The electrolyte solution is sodium chloride
Any one of injection, compound sodium chloride injection, Multiple electrolytes injection, Lactated Ringer'S Solution appoint several mixing
Liquid；Or the mixed solution that the electrolyte solution is physiological saline, potassium chloride injection, calcium gluconate injection.
8. application of the stored refrigerated system of claim 1-4 any one of them lymphocytes in Preservation of lymphocytes.
9. application according to claim 8, which is characterized in that the temperature of the Preservation of lymphocytes is 4 DEG C~15 DEG C.
10. application according to claim 8, which is characterized in that the density of the lymphocyte is no more than 10,000,000/ml.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109845725A (en) * | 2019-01-25 | 2019-06-07 | 北京益华生物科技有限公司 | Cell transport saves liquid and its application |
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| CN114762497A (en) * | 2021-01-11 | 2022-07-19 | 京东方再生医学科技有限公司 | Cell cryopreservation solution and cell cryopreservation method |
| CN114762497B (en) * | 2021-01-11 | 2023-08-11 | 京东方再生医学科技有限公司 | Cell cryopreservation liquid and cell cryopreservation method |
| CN113100227A (en) * | 2021-03-31 | 2021-07-13 | 北京益华生物科技有限公司 | NK cell transfusion liquid capable of being directly input into human body and preparation method and application thereof |
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