CN106701681B - A kind of external evoked amplification of immunocyte, the method for freezing and recovering - Google Patents
A kind of external evoked amplification of immunocyte, the method for freezing and recovering Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
Abstract
The invention discloses the external evoked amplification of immunocyte, the method for freezing and recovering.This method includes：Culture vessel, the culture vessel after being coated with are coated with using CD16 antibody；The first induced amplification culture is carried out in the culture vessel that mononuclearcell is placed in after being coated with using immune cell activation culture medium, obtains the immunocyte of preliminary induced amplification；The immunocyte of preliminary induced amplification is subjected to the second induced amplification culture, the immunocyte broken up using immune cell expansion culture medium；The immunocyte of differentiation is subjected to the 3rd induced amplification culture using immunocyte scale amplification culture medium, obtains the immunocyte of large-scale function activation；Immunocyte is frozen using immunocyte frozen stock solution, the immunocyte frozen；The immunocyte water-bath frozen is melted, and mixed with the Cryopreservation liquid of immunocyte, obtains recovery cell mixture；Recovery cell mixture is subjected to centrifugal treating, the immunocyte recovered.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to the external evoked amplification of immunocyte, freeze and recover
Method.
Background technology
In recent years, oneself warp of biological therapy turns into the fifth-largest Therapeutic mode after operation, chemicotherapy and endocrine therapy,
And gradually it is taken seriously.Adoptive cellular immunotherapy method (ACI) is one of cell biological treatment method, and it is directed to tumour trouble
Immunocyte of person's infusion with antitumor activity, direct killing tumour cell or excitating organism immune response are thin to kill tumour
Born of the same parents, reach the purpose for the treatment of tumour.At present clinically using adoptive immunity cell include DC-CIK cells, til cell,
LAK cells and NK cells, wherein, CIK cell, LAK cells and A-NK cells are all the anticancers based on NK
System.
NK (natural killer cells) is also referred to as NK cells, is mainly derived from marrow CD34+'s
Lymphocyte, marrow, peripheral blood and spleen are distributed in, account for the 10%-20% of PBLC.NK cells are exempted from tumour
Epidemic disease, the non-own cell of removing etc. play a significant role：It is the main composition of innate immune defence, positioned at body defenses system
The first line of defence, the killing activities of NK cells limits without MHC, can be i.e. recognizable without antigen presensitization independent of antibody
And the cell of tumour and virus infection is killed, by perforin-particle enzymatic pathway and Fas-FasL paths directly to tumour cell
Play CDCC killing tumor cell；Simultaneously its and can morbidity Early insulin secretion cytokine profiles and chemotactic factor (CF) such as
TNF-α, IFN-γ and IL-1 etc., these cell factors participate in anticancer and regulation Acquired immune response, therefore NK cells are also to connect
Connect the bridge of the innate immunity and acquired immunity.Although the security and curative effect of the anticancer effect of NK cells are affirmed, by
Only account for the 10%-20% of PBLC in it, thus how to obtain high-purity, NK cell products are that NK is controlled in high quality
The key for the treatment of.Found that by external stimulation culture relatively large-scale NK cells preparation can be carried out in recent years, oneself know IL-2,
The cell factors such as IL-15, IL-18 and IL-7 play a significant role on the amplification in vitro to NK cells, their amplification in vitro NK
The multiple of cell is at several times to decades of times.IL-2 is the cell factor of important induced NK cell proliferation, and it can be activated
NK cells, the generation for promoting NK cells propagation and cell factor.IL-15 and IL-7 effect is similar to IL-2, while they are also
Can be by the coreceptor γ chain combinations with NK cell surface expressions, it is NK cells to promote candidate stem cell directed differentiation, and right
Development, differentiation and maintenance long-term in vitro survival of NK cells etc. play a significant role.IL-15 and IL-2 synergy
Both is cooperated with the amplification in vitro of NK cells, be the most traditional combination of cytokines of current NK cell expansion ex vivos.According to
Report IL-18 not only can the THi cells secretion of induced activation produce substantial amounts of IFN-γ, more can be by promoting Fas-FasL paths
Opening, strengthen the cytotoxicity of NK cells, and be in dose dependent.But have NK cell therapy products in the market to deposit
, but freeze numerous and diverse cultivating system that rear resuscitation effect is poor, uses, amplification times and ineffective, the part cultivating system that kills knurl
The problems such as security risk be present.
Thus, the culture of NK, freeze and have much room for improvement with method for resuscitation.
The content of the invention
It is contemplated that at least solves one of technical problem present in prior art.Therefore, one object of the present invention
It is to propose a kind of external evoked amplification of immunocyte, the method for freezing and recovering, this method induced amplification immunocyte, lures
Lead that efficiency high, amplification rate are fast, safe low with cost, and the large-scale function activation that obtains of induced amplification is immune
Cell long-period is recovered the cell characteristics for still keeping good after preserving.
According to an aspect of the present invention, the invention provides a kind of external evoked amplification of immunocyte, freeze and answer
The method of Soviet Union.According to an embodiment of the invention, this method includes：
Culture vessel is coated with using CD16 antibody, so as to the culture vessel after being coated with；
In culture vessel mononuclearcell being placed in using immune cell activation culture medium after the coating carry out first
Induced amplification culture, to obtain the immunocyte of preliminary induced amplification；
The immunocyte of the preliminary induced amplification is subjected to the second induced amplification training using immune cell expansion culture medium
Support, so as to the immunocyte broken up；
The immunocyte of the differentiation is subjected to the 3rd induced amplification culture using immunocyte scale amplification culture medium,
To obtain the immunocyte of large-scale function activation；
The immunocyte of the large-scale function activation is frozen using immunocyte frozen stock solution, so that what is frozen exempts from
Epidemic disease cell；
The immunocyte frozen is placed in 37-42 DEG C of water-bath and melted, and is mixed with the Cryopreservation liquid of immunocyte
Close, to obtain recovery cell mixture；And
The recovery cell mixture is subjected to centrifugal treating, so as to the immunocyte recovered,
Wherein,
The immune cell activation culture medium be with the addition of the first blood plasma, proleulzin and Sapylin serum-free lymph it is thin
Born of the same parents' culture medium；
The immune cell expansion culture medium is the serum-free lymphocytes culture medium that with the addition of proleulzin and Sapylin；
The immunocyte scale amplification culture medium is the serum-free lymphocytes culture medium that with the addition of proleulzin；
The immunocyte frozen stock solution includes 2-10 volumes % DMSO, 2-15 volume % HSA, 20-88 volume %'s
Second blood plasma and surplus freezing media；
Conyza japonica system composite family Conyza plant Conyza japonica (Thunb.) Less herb, face generation human relations etc. was once
Through reporting, isolated compound Conyza japonica saponin(e R, Structural Identification are 3-O- β-D-glucopyranosyl from Conyza japonica
medicagenic acid 28-O-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-[β-D-
Apiofuranosyl- (1 → 3)]-α-L-rhamnopyranosyl- (1 → 2)-α-L-arabinopyranosyl ester,
Its structural formula is：
Inventor have further surprisingly found that micro Conyza japonica saponin(e R, blood plasma and HSA (human serum albumins, Human
Serum Albumin) frozen stock solution for preparing immunocyte is combined with DMSO, DMSO usage amount, this frozen stock solution can be reduced
There is preferable protective effect to freeze-stored cell, good cell viability, cell recoveries height are still kept after freeze-stored cell recovery.
According to an aspect of the present invention, the invention provides a kind of immunocyte frozen stock solution, it includes 0.2-0.8 bodies
Product % DMSO；2-15 volumes % HSA；0.001g/ml Conyza japonica saponin(e R, 20-88 volume % blood plasma；And surplus
Culture medium.Inventor has surprisingly found that the frozen stock solution can still keep good effective for freezing immunocyte after cell recovery
Good cell viability, and cell recoveries are high.
The Cryopreservation liquid includes albumin and dextran.
Exempted from a large scale it is surprisingly found by the inventors that carrying out induced amplification culture to mononuclearcell using this method
Epidemic disease cell, with induced efficiency is high, amplification rate is fast, safe and low cost and other advantages.Also, the big rule that this method freezes
The immunocyte of mould, effective holding time length, cell still keeps good cell viability after recovery, and cell recoveries are high, from
And meet the needs of a large amount of immunocytes of clinical treatment.
In addition, the external evoked amplification of immunocyte according to the above embodiment of the present invention, the method that freezes and recover are also
There can be technical characteristic additional as follows：
According to an embodiment of the invention, in the immune cell activation culture medium and the immune cell expansion culture medium,
The concentration of the Sapylin is 0.007-0.013KE/ml, it is preferable that is 0.01KE/ml.
According to an embodiment of the invention, the immune cell activation culture medium, the immune cell expansion culture medium and institute
State in immunocyte scale amplification culture medium, the concentration of the proleulzin is 700-1300IU/ml, it is preferable that is
1000IU/ml。
According to an embodiment of the invention, the immune cell activation culture medium, the immune cell expansion culture medium and institute
State in immunocyte scale amplification culture medium, the serum-free lymphocytes culture medium is OpTmi zerTM CTSTMWithout blood
Clear culture medium or SuperCulture TM L500 human lymphocyte serum free mediums.
According to an embodiment of the invention, in the immune cell activation culture medium, the concentration of first blood plasma is 7-13
Volume %, it is preferable that be 10 volume %.
According to an embodiment of the invention, first blood plasma and second blood plasma are autologous plasma.
According to an embodiment of the invention, in the immunocyte frozen stock solution, the concentration of the DMSO is 10 volume %；It is described
HSA concentration is 5 volume %；The concentration of second blood plasma is 40 volume %；The freezing media be 1640 culture mediums or
Stemspan culture mediums.
According to an embodiment of the invention, in the Cryopreservation liquid, the albumin is human albumin, optionally, described
Human albumin derives from commercially available human serum albumins or autologous plasma.
According to an embodiment of the invention, the content of the albumin is 1-5%, it is preferable that is 2.5%.
According to an embodiment of the invention, the dextran is Dextran 40.
According to an embodiment of the invention, the content of the dextran is 2-8 weight %, it is preferable that is 5 weight %.
According to an embodiment of the invention, the immunocyte is NK and BMDC.
According to an embodiment of the invention, when the tentatively CD56 of the immunocyte of the induced amplification expression is more than 70%
When, carry out the second induced amplification culture.
According to an embodiment of the invention, when the differentiation immunocyte CD56 expression more than 90% when, carry out institute
State the 3rd induced amplification culture.
According to an embodiment of the invention, in the first induced amplification incubation, passage in every 1 day is once.
According to an embodiment of the invention, in the second induced amplification incubation, passage in every 2 days is once.
According to an embodiment of the invention, in the 3rd induced amplification incubation, passage in every 2 days is once.
According to an embodiment of the invention, every 106-109The immunocyte of the individual large-scale function activation uses 1ml institutes
State immunocyte frozen stock solution.
According to an embodiment of the invention, the volume that the immunocyte frozen mixes after melting with the Cryopreservation liquid
Than for 1：0.5-5, it is preferable that be 1：1.
The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description
Obtain substantially, or recognized by the practice of the present invention.
The present invention is advantageous in that：
A kind of method that the present invention proposes external evoked amplification of immunocyte, freezes and recover, this method induced amplification
Immunocyte, induced efficiency is high, amplification rate is fast, safe low with cost, and the large-scale work(that induced amplification obtains
Can activation immunocyte preserve for a long time after recover the cell characteristics for still keeping good.
Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination accompanying drawings below to embodiment
Substantially and it is readily appreciated that, wherein：
Fig. 1 shows the PMNC number and cell of culture different time according to an embodiment of the invention
The result schematic diagram of activity；
Fig. 2 shows the lymph in the PMNC according to an embodiment of the invention before and after cultivating 14 days
The ratiometric result schematic diagram of cell；
Fig. 3 is shown according to the PMNC number of the culture different time of another of the invention embodiment and thin
The result schematic diagram of cytoactive；
Fig. 4 shows the lymph in the PMNC according to an embodiment of the invention before and after cultivating 12 days
The result schematic diagram of cell proportion；
Fig. 5 shows the result schematic diagram of nude mice tumorigenesis experiment according to an embodiment of the invention.
Embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from beginning to end
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
The embodiment of figure description is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that instruction or hint phase
To importance or the implicit quantity for indicating indicated technical characteristic.Thus, define " first ", the feature of " second " can be with
Express or implicitly include one or more this feature.Further, in the description of the invention, unless otherwise saying
Bright, " multiple " are meant that two or more.
According to an aspect of the present invention, the invention provides a kind of external evoked amplification of immunocyte, freeze and answer
The method of Soviet Union.According to an embodiment of the invention, this method includes：Culture vessel is coated with using CD16 antibody, after being coated with
Culture vessel；Mononuclearcell is placed in the culture vessel after being coated with using immune cell activation culture medium and carries out the first induction
Amplification cultivation, obtain the immunocyte of preliminary induced amplification；Utilize immune cell expansion culture medium exempting from preliminary induced amplification
Epidemic disease cell carries out the second induced amplification culture, the immunocyte broken up；Will using immunocyte scale amplification culture medium
The immunocyte of differentiation carries out the 3rd induced amplification culture, obtains the immunocyte of large-scale function activation；It is thin using being immunized
Born of the same parents' frozen stock solution freezes the immunocyte of large-scale function activation, the immunocyte frozen；The immunocyte frozen is put
Melt in 37-42 DEG C of water-bath, and mixed with the Cryopreservation liquid of immunocyte, obtain recovery cell mixture；And will be multiple
Cell mixture of reviving carries out centrifugal treating, the immunocyte recovered.
Exempted from a large scale it is surprisingly found by the inventors that carrying out induced amplification culture to mononuclearcell using this method
Epidemic disease cell, with induced efficiency is high, amplification rate is fast, safe and low cost and other advantages.Also, the big rule that this method freezes
The immunocyte of mould, effective holding time length, cell still keeps good cell viability after recovery, and cell recoveries are high, from
And meet the needs of a large amount of immunocytes of clinical treatment.
According to an embodiment of the invention, immune cell activation culture medium is that with the addition of the first blood plasma, proleulzin and sand culture
The serum-free lymphocytes culture medium of woods；Immune cell expansion culture medium is the serum-free leaching that with the addition of proleulzin and Sapylin
Bar cell culture medium；Immunocyte scale amplification culture medium is the serum-free lymphocytes culture medium that with the addition of proleulzin.
Induced amplification culture is carried out to mononuclearcell using the culture medium group and obtains large-scale immunocyte, there is induced efficiency
Height, amplification rate are fast, safe and low cost and other advantages, so as to meet the needs of a large amount of immunocytes of clinical treatment.
According to an embodiment of the invention, immunocyte frozen stock solution includes：2-10 volumes % DMSO, 2-15 volume %'s
HSA, 20-88 volume % the second blood plasma and surplus freezing media.Thus, it with the addition of the second blood in the immunocyte frozen stock solution
Slurry, and the ratio of each component is suitable, and the length of effective holding time of immunocyte, cell still keeps good cell after recovery
Vigor, cell recoveries are high.
, wherein it is desired to explanation, when the percentage by volume sum of described DMSO, HSA and blood plasma is less than 1, is immunized thin
Born of the same parents' frozen stock solution further includes the culture medium of residual volume.For example, in immunocyte frozen stock solution, when DMSO concentration is 10 bodies
Product %, HSA concentration are 2% volume, and when the concentration of blood plasma is 88% volume, the immunocyte frozen stock solution does not include culture medium；
When the concentration that DMSO concentration is 5 volumes %, HSA is 2% volume, and the concentration of blood plasma is 88% volume, the immunocyte freezes
Liquid storage includes the culture medium of 5% volume；When the concentration that DMSO concentration is 10 volumes %, HSA is 5% volume, the concentration of blood plasma
For 40% volume when, the immunocyte frozen stock solution includes the culture medium of 45% volume.
According to an embodiment of the invention, Cryopreservation liquid includes albumin and dextran.Thus, using including albumin
The immunocyte that Cryopreservation liquid dilution with dextran is melted, at normal temperatures, is not only reduced in cells frozen storing liquid
Injuries of the DMSO to immunocyte, and albumin in Cryopreservation liquid and dextran are to the immunocyte osmotic pressure of thawing
Played regulatory role with nutrition.
According to an embodiment of the invention, in immune cell activation culture medium and the immune cell expansion culture medium, sand culture
The concentration of woods is 0.007-0.013KE/ml.Thus, the induction of immunocyte and growth rate are fast, and amplification efficiency is high, obtain
Immunocyte purity it is high, there is more preferable immunologic function, is controlled available for clinics such as anti-infective, antitumor and raising immunity
Treat.
According to a preferred embodiment of the invention, in immune cell activation culture medium and the immune cell expansion culture medium,
The concentration of Sapylin is 0.01KE/ml.Thus, faster, amplification efficiency is high, acquisition for the induction of immunocyte and growth rate
Immunocyte purity is high, has more preferable immunologic function, available for clinical treatments such as anti-infective, antitumor and raising immunity.
According to an embodiment of the invention, immune cell activation culture medium, immune cell expansion culture medium and immunocyte rule
In modelling amplification culture medium, the concentration of proleulzin is 700-1300IU/ml.Thus, the induction of immunocyte and propagation speed
Degree is fast, and amplification efficiency is high, and the immunocyte purity of acquisition is high, has more preferable immunologic function, available for anti-infective, antitumor and
Improve the clinical treatments such as immunity.
According to a preferred embodiment of the invention, immune cell activation culture medium, immune cell expansion culture medium and immune thin
In born of the same parents' scale amplification culture medium, the concentration of proleulzin is 1000IU/ml.Thus, the induction of immunocyte and propagation speed
Faster, amplification efficiency is high for degree, and the immunocyte purity of acquisition is high, has more preferable immunologic function, available for anti-infective, antitumor
With improve the clinical treatment such as immunity.
According to an embodiment of the invention, immune cell activation culture medium, immune cell expansion culture medium and immunocyte rule
In modelling amplification culture medium, serum-free lymphocytes culture medium is：OpTmizerTM CTSTMSerum free medium or
SuperCulture TML500 human lymphocyte serum free mediums.Thus, the external efficient amplification training of immunocyte is advantageous to
Support, and keep higher functional activity.
According to an embodiment of the invention, in immune cell activation culture medium, the concentration of the first blood plasma is 7-13 volumes %.By
This, is advantageous to the external efficient amplification culture of immunocyte, and keep higher functional activity.
According to a preferred embodiment of the invention, in immune cell activation culture medium, the concentration of the first blood plasma is 10 volume %.
Thus, the induction of immunocyte and growth rate and efficiency significantly improve, and the immunocyte purity of acquisition is high, has and preferably exempts from
Epidemic disease function.
According to an embodiment of the invention, the first blood plasma and the second blood plasma are autologous plasma.Thus, immunocyte is to blood plasma
Rejection effect it is small, the induction of immunocyte and growth rate and efficiency significantly improve, and the immunocyte purity of acquisition is high, has
More preferable immunologic function.
, wherein it is desired to explanation, used term " autologous plasma " herein refers to and treated induced amplification and jelly
The cell deposited has the blood plasma of same source.In other words, i.e. the blood plasma is with treating that it is same that the mononuclearcell of induced amplification is derived from
Body.Thus, it is small to autologous immunocyte rejection effect, be advantageous to the protection of freeze-stored cell.
According to an embodiment of the invention, in immunocyte frozen stock solution, DMSO concentration is 10 volume %；HSA concentration is 5
Volume %；The concentration of second blood plasma is 40 volume %；Freezing media is 1640 culture mediums or Stemspan culture mediums.Thus,
Osmotic pressure regulating effect is good in freeze-thaw treatment, the protective effect to cell protrudes, and is still kept well after cell recovery
Cell viability, so as to effectively protect cell, cell survival rate is improved, and rationally control the cost of frozen stock solution.According to this
The embodiment of invention, in Cryopreservation liquid, albumin is human albumin.Albumin in blood has transport, maintains osmotic pressure
And trophism.External albumin has the function that to protect cell.And human albumin and mononuclearcell are blood constituent,
It is more beneficial for protecting cell, makes the vigor of cell more preferable, it is CIK cell to promote mononuclearcell induction, and obtains CIK cell
For clinic, the rejection effect of patient is small.
According to an embodiment of the invention, the source of human albumin is not particularly limited, as long as human albumin can be provided i.e.
Can, you can so that from commercially available human serum albumins, human plasma can also be derived from.People of people's mononuclearcell to humanized
The rejection effect of seralbumin or blood plasma is small, is advantageous to the recovery of freeze-stored cell.
According to an embodiment of the invention, the content of albumin is not particularly limited, and is lured as long as mononuclearcell can be improved
Lead the induced efficiency for CIK cell, amplification times or activity.According to some embodiments of the present invention, the content of albumin
For 1-5% or 1-5.Thus, the multiplication rate of cell for recovering to obtain is fast, and induction differentiation efficiency is high, and cytoactive is good.According to this
The preferred embodiment of invention, the content of albumin is 2.5.Thus, the obtained multiplication rate of cell is recovered faster, induction differentiation
Efficiency high, cytoactive is more preferably.
Wherein it should be noted that it can also be percentage by weight that the content of albumin, which can be percent by volume, root
It is adjusted according to the state of albumin, if albumin is solid-state, then the content of albumin is 1-5 weight %, if white egg
White is liquid, then the content of albumin is 1-5 volumes %.
According to an embodiment of the invention, dextran is Dextran 40.Thus, be advantageous to improve the recovery effect of cell
Fruit.
According to an embodiment of the invention, the content of dextran is 2-8 weight %.Thus, the obtained increasing of cell is recovered
It is fast to grow speed, induction differentiation efficiency is high, and cytoactive is good.According to a preferred embodiment of the invention, the content of the dextran
For 5 weight %.Thus, the obtained multiplication rate of cell is recovered faster, induction differentiation efficiency is high, and cytoactive is more preferably.According to
Another aspect of the present invention, the invention provides a kind of Cryopreservation reagent of the killing cell for cytokine profiles induction
Box.According to an embodiment of the invention, the kit includes the freezing for the killing cell for being previously described for cytokine profiles induction
Resuscitation fluid.Thus, obtained mononuclearcell is recovered using the Cryopreservation kit, in follow-up Induction Process, induction
It is fast to form the multiplication rate of the killing cell of cytokine profiles induction, induces differentiation efficiency high, cytoactive is good.
According to an embodiment of the invention, the immunocyte is NK and BMDC.Thus, induction is expanded
Increasing Efficiency is high.
According to an embodiment of the invention, when the activation immunocyte CD56 expression more than 70% when, carry out the
Two induced amplification cultures.Thus, the second induction can be carried out when the induction of most of mononuclearcell is differentiated to form immunocyte
Amplification cultivation so that immunocyte purity is further lifted, and cell quantity is improved significantly, while is immunized in amplification procedure thin
Born of the same parents' function further enhances.
According to an embodiment of the invention, when the differentiation immunocyte CD56 expression more than 90% when, carry out institute
State the 3rd scale induced amplification culture.Thus, when the induction of most mononuclearcells is differentiated to form immunocyte
Carry out the 3rd induced amplification culture so that immunocyte obtains further scale amplification, while keeps immunologic function, and being possible
Clinical immunotherapy sufficient cell is provided.
According to an embodiment of the invention, during immune cell activation culture medium, passage in every 1 day is once.Thus, it is immunized thin
During born of the same parents activate culture medium, immunocyte is substantially activated, while is effectively expanded, and other cell proportions substantially drop
It is low.
According to an embodiment of the invention, in the second induced amplification incubation, passage in every 2 days is once.Thus, the second induction
During amplification cultivation, immunocyte purity is further enhanced, while other cell proportions further reduce, immunocyte
Function is strengthened.
According to an embodiment of the invention, in the 3rd induced amplification incubation, passage in every 2 days is once.Thus, the 3rd induction
During amplification cultivation, immunocyte realizes that scale expands under high-purity, obtains the immunocyte of the function activation of abundance
For possible clinical immunotherapy.
According to an embodiment of the invention, every 106-109The immunocyte of individual large-scale function activation is immune thin using 1ml
Born of the same parents' frozen stock solution.Thereby, it is possible to effectively realize freezing for immunocyte, and the cell concentration frozen is high, suitable for mass immunization
Cell freezes, and freezes that cost is low, and effect is good.According to the present invention specific example, every 107-108Individual immunocyte uses 1ml institutes
State immunocyte frozen stock solution.Thus, the cell concentration that freezes is high, and suitable for freezing for mass immunization cell, it is low to freeze cost, and
And cell cryopreservation effect is good.
According to an embodiment of the invention, the immunocyte that freezes with the Cryopreservation liquid is 1 by volume after melting：
0.5-5 is mixed.Thereby it is ensured that the DMSO concentration after the dilution of Cryopreservation liquid is relatively low, the damage to immunocyte is small.According to
The preferred embodiments of the present invention, the immunocyte that freezes melt after with the Cryopreservation liquid be 1 by volume：1 is mixed.
Thus, DMSO concentration is low after ensureing to dilute, and on the premise of the damage to immunocyte is small, the dosage of Cryopreservation liquid is small,
The cost of Cryopreservation is low.
Below with reference to specific embodiment, the present invention will be described, it is necessary to which explanation, these embodiments are only explanation
Property, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or bar in embodiment
Part, carried out according to the technology described by document in the art or condition or according to product description.Agents useful for same or instrument
The unreceipted production firm person of device, being can be by the conventional products of acquisition purchased in market, such as can purchase from Sigma companies.
Embodiment 1：
Using the induced amplification method of the immunocyte of the embodiment of the present invention, it is by the mononuclearcell induced amplification of separation
Immunocyte, and detect the activity of immunocyte.
First, experimental method
1. prepare the anti-human coated T75 bottles of CD16
1.1 add the anti-human CD16 monoclonal antibodies of 2.5 μ g/mL dissolved through medical saline in sterile culture flask
5mL, gently rocking blake bottle makes antibody confluent cultures face, and 4 DEG C of lucifuges are stayed overnight.
1.2 use preceding recovery antibody coating buffer, with 5mL brines blake bottle once, then the T cell expansion with 5mL
Increase culture medium (OpTmizerTM CTSTMT-cell expansion SFM) it washed once.
2. gathering peripheral blood, peripheral blood blood plasma and mononuclearcell are separated
2.1 with the sterile blood sampling bag collection human peripheral about 100ml for adding anti-coagulants, reserved 1ml peripheral bloods do fast inspection and
Bracket for blood grouping, sterile packaged, sterile 4 DEG C of preservations transport, accurate recording gather information to blood taking bag immediately.Will after fast inspection screening is qualified
Peripheral blood is sent into GMP laboratories, if examining unqualified, blood sample waste treatment soon.
2.2 take out blood bag in GMP laboratories, alcohol disinfecting blood taking bag, observe after no blood coagulation and haemolysis in super-clean bench
Blood bag is opened, is transferred blood into 50ml sterile centrifugation tubes (≤40ml/ pipes), 2500rpm centrifugations 15min.
2.3 transfer upper plasmas, from sterile centrifugation tube, 3500rpm centrifugation 15min, collect supernatant blood plasma to newly to another
Sterile centrifugation tube in, with membrana oralis seal centrifugation the mouth of pipe, haemocyte be used for separate mononuclearcell.
Blood plasma is put into water-bath 30-50min in 56 DEG C of water-baths by 2.4 inactivates complement, and 3500rpm centrifugations 15min, which is removed, to be mended
Body, 10ml every are dispensed into 15ml sterile centrifugation tubes, -20 DEG C freeze it is standby；And leave and take 7.5ml blood plasma and examined slowly：Virus
Five, mycoplasma, endotoxin and microorganism, wherein virus is defined by third party's detection.
2.5 the haemocyte precipitation in step 2 is resuspended with the isometric physiological saline of blood plasma after be transferred to the sterile glass of 250ml
In glass bottle, the HES of the amount of total blood volume 1/3 is added, gently rocking saline bottle makes to be well mixed, and standing makes red blood cell
Sedimentation.
2.6 after red blood cell layer sedimentation layering, by upper strata milky suspension gentle aspiration to sterile centrifugation tube, 1800rpm
5min, supernatant discarding are centrifuged, precipitation is resuspended with 10ml physiological saline.
2.7 take sterile 15ml centrifuge tubes 2, each to add 5ml normal temperature human peripheral lymphocyte separating liquids, respectively on upper strata
Gently add each 5ml cell suspensions.Room temperature 2000rpm rises slow drop centrifugation 25min slowly.
2.8 gently take out centrifuge tube, and careful interface centre cloud and mist layer leucocyte of drawing is added into new 15ml centrifuge tubes
Brine 2 times.
Cell is resuspended in 2.9 1ml physiological saline, takes 5 μ l cells to be added in 245 μ l physiological saline and dilutes 50 times, counts,
And with Trypan Blue meter cell survival rate.The band blood stains thing terminated is tested by proportioning bromogeramine soaked overnight
It can abandon.
2.10 reserved 4.5x106 cells carry out the ratio of streaming antibody labeling detection NK cells；Remaining single core is thin
Born of the same parents' cell is inoculated into the blake bottle added with 20ml culture mediums, and Medium Proportion is：18ml OpTmizerTM CTSTM T-cell
Expansion SFM culture medium+2ml autologous plasmas (Autologous plasma)+final concentration 1000IU/ml Pepro Tech
IL-2+ final concentration 0.01KE/ml Sapylins (OK432), 37 DEG C, 5%CO2 sterile cultures, it is designated as the 0th day.
2.11 daily observation cells, and appropriate passage amplification is carried out, Medium Proportion is that Medium Proportion is：
OpTmizerTM CTSTMThe autologous plasma (Autologous plasma) of T-cell expansion SFM culture mediums+10%+end
Concentration 1000IU/ml Pepro Tech IL-2.When autologous plasma has run out, blood plasma, i.e. OpTmizer are no longer addedTM
CTSTMT-cell expansion SFM culture mediums+final concentration 1000IU/ml PeproTech IL-2.
2.12 when cultivating system has reached 2L, changes cultivating system, i.e. SuperCulture TM L500 people lymph
Cell non-serum culture medium+domestic the IL-2 of final concentration 1000IU/ml, observes cell, and suitably passed on daily.Treat 14 days
When, cell recovery is carried out, 10ml culture mediums supernatant is left and taken and carries out Elisa secretion factor detections.
2.13 the cell of recovery is resuspended with 200ml physiological saline, and adds 10ml human serum albumins, is mixed；And from
In take 10ml cell suspensions to be checked, the injection of remaining cell is fed back to be prepared to feed back in bag.Hanged from extraction 10ml cells in bag are fed back
Liquid is detected：Mycoplasma, endotoxin, microorganism and five, virus.
3. flow cytomery cell medium size lymphocyte pedigree
By the 10ml cell suspensions of taking-up, 1800rpm centrifugal recovery cells, streaming antibody labeling is carried out.Homotype pair is set
According to, single standard specimen product and Dyeing pipe, often pipe sample cell number about 5 × 105, then add corresponding antibody staining.4 DEG C, 30min is put
Put, with brine, then above machine testing analyzes the NK cell proportions in lymphocyte populations.
4. by remaining 10ml cell suspensions supernatant, dispensed, to detect five, virus, endotoxin, mycoplasma, micro- life
Thing.
5. nude mice tumorigenesis experiment SPF levels female BAl BIc/c nude mices, is purchased from Institute of Experimental Animals, Chinese Academy of Medical Sciences, 4-
6 week old, body weight 16-20g, raised in lamina air flow frame in mouse cage with cover, drinking water, standard feed and other and animal contact
The sterilized processing of product.Take the NK of the 21st day of positive control Ragi cells and K562 cells and Differentiation Induction in vitro to be detected
Cell is subcutaneous by 3 × 107/0.2ml inoculation nude mice flanks, is marked with picric acid, observes into knurl situation within 2 months by a definite date.
6. the culture medium supernatant for receiving cell is subjected to Elisa secretion factor detections, i.e. IFN-γ, TNF-α and Perforin
Detection.
2nd, experimental result
The experimental result of 1 culture 14 days
Amplifiable 150 times of cell after 1.1 cultures 14 days
6 × 107 PMNCs taken after the separation of PBLC separating liquid are inoculated into 20ml
In cultivating system, cell enters logarithm growth stage quickly.After cultivating 14d, cultivating system is expanded to 4L, cell number amplification to 9
× 109, for amplification times at up to 150 times, viable count cultivates the PMNC of different time more than 90%
Number and cytoactive are the results detailed in Fig. 1.
The NK cell proportions in PMNC after 1.2 amplifications are significantly raised
PMNC after amplification in 14 days, NK cell proportions (CD3-CD56+) in lymphocyte by
18.15% rises to 95.27%, and at the same time T lymphocytes (CD3+) ratio drops to 4.61% by 72.15%, complementary T
Cell (Th, CD3+CD4+) and cytotoxic T cell (Tc, CD3+CD8a+) have reduction, bone-marrow-derived lymphocyte (CD3-CD19+) base
This disappearance, cell uniformity significantly improve；Calculate and understand with reference to the cell count in Fig. 1, NK cells amplification 780 after cultivating 14 days
Times, the result of the percentage of lymphocyte in PMNC before and after cultivating 14 days is shown in Fig. 2, wherein, NK cells：CD3-
CD56+；T cell：CD3+；Helper T lymphocyte (Th)：CD3+CD4+；Cytotoxic T cell (Tc cells)：CD3+CD8a+；B is thin
Born of the same parents：CD3-CD9+.
The cell products that 1.3 culture amplifications obtain detect the body-sensing dye that do not find the cause of disease
We by the cell after culture and cell suspension commission detection platform have detected hepatitis B surface antigen, biscuit antigen,
Human immune defect virus antibody, Treponema pallidum specific antibody, cytomegalovirus and mycoplasma, bacterium and endogenous toxic material
Element, testing result are negative, and it is safe to illustrate the batch products, is not polluted in incubation, the results detailed in Table
1。
NK cells clinical safety after table 1 is cultivated 14 days detects
2nd, the experimental result of 12 days is cultivated
Amplifiable 75 times of cell after 2.1 cultures 12 days
9 × 107 PMNCs after the separation of PBLC separating liquid are taken to be inoculated into 20ml cultures
In system, cell enters logarithm growth stage quickly.After cultivating 12d, cultivating system is expanded to 4L, and cell number amplification to 6.76 ×
109, amplification times are up to 75 times, and for viable count more than 90%, experimental result refers to Fig. 3.
The NK cell proportions in PMNC after 2.2 amplifications are significantly raised
PMNC is after amplification in 12 days, and the NK cell proportions (CD3-CD56+) in lymphocyte were from the 7th day
32.87% rise to the 12nd day 92.19%, at the same time T lymphocytes (CD3+) ratio drop to 3.24%, it is complementary
T cell (Th, CD3+CD4+) and cytotoxic T cell (Tc, CD3+CD8a+) have reduction, bone-marrow-derived lymphocyte (CD3-CD19+)
Basic to disappear, cell uniformity significantly improves, and the percentage of lymphocyte in PMNC before and after cultivating 12 days is detailed
See Fig. 4, wherein, NK cells：CD3-CD56+；T cell：CD3+；Helper T lymphocyte (Th)：CD3+CD4+；Cytotoxic T cell
(Tc cells)：CD3+CD8a+；B cell：CD3-CD9+.
The cell products that 2.3 culture amplifications obtain detect the body-sensing dye that do not find the cause of disease
We by the cell after culture and cell suspension commission detection platform have detected hepatitis B surface antigen, biscuit antigen,
Human immune defect virus antibody, Treponema pallidum specific antibody, cytomegalovirus and mycoplasma, bacterium and endogenous toxic material
Element, testing result are negative, and it is safe to illustrate the batch products, is not polluted in incubation, the results detailed in Table
2。
NK cells clinical safety after table 2 is cultivated 12 days detects
The culture medium supernatant of table 3. carries out Elisa detections
| Experiment 1 | Experiment 2 | |
| IFN-γ | 1.48x105pg/ml | 1.435x105pg/ml |
| TNF-α | 61.1pg/ml | 48.75pg/ml |
| Perforin | 19ng/ml | 17.48ng/ml |
The cell cultivated has IFN-γ, TNF-α, Perforin secretion.
3. the NK cells after culture amplification will not form tumour in vivo
Nude mice tumorigenesis experimental result as shown in figure 5, wherein, A saline control groups (into knurl number of mice/group in number of mice,
0/5), B Raji cell controls group (3/5), C K562 cell controls groups (4/5), D NK groups of cells (0/5), i.e., seen at 2 months
The NK groups of cells mouse for examining phase subendothelial injection 0.2ml physiological saline groups and 3 × 107/0.2ml cultures 21 days are showed no tumour
Formed, inject has 3/5 and 4/5 mouse to form visible swell respectively just as the Raji cells of volume and the mouse of K562 cells
Knurl.Even if by 21 days, NK cells were still safely and effectively, will not lead oncogenic formation for result explanation culture.
Embodiment 2
In the present embodiment, 6 kinds of frozen stock solutions without blood plasma and 6 kinds of frozen stock solutions containing blood plasma are respectively adopted, to embodiment 1
The NK cells of external evoked amplification are frozen, and are compared and are frozen effect, to observe the influence that blood plasma preserves to NK cell cryopreservations,
And DMSO concentration is reduced to reduce the possibility of cytotoxicity.It is specific as follows：
1st, NK cells are frozen using the frozen stock solution without blood plasma
1.1st, NK cells freeze and method for resuscitation：
The 10th day NK cell obtained of external evoked amplification of embodiment 1 is respectively adopted into four kinds of frozen stock solutions containing HSA to carry out
Freeze, wherein the composition of four kinds of frozen stock solutions is as follows：
Frozen stock solution 1：5 volume %DMSO+10 volumes %HSA；
Frozen stock solution 2：5 volume %DMSO+15 volumes %HSA；
Frozen stock solution 3：10 volume %DMSO+10% volumes HSA；
Frozen stock solution 4：10 volume %DMSO+15 volumes %HS
Frozen stock solution 5：0.2 volume % DMSO+15 volumes % HSA, Conyza japonica saponin(e R is added after the completion of preparation to its end
Concentration is 0.001g/ml；
Frozen stock solution 6：0.4 volume % DMSO+10 volumes % HSA, Conyza japonica saponin(e R is added after the completion of preparation to its end
Concentration is 0.001g/ml；A,
Wherein, residual volume is 1640 or Stemspan culture mediums in frozen stock solution 1-6.
NK cells are frozen according to following steps：After freezen protective liquid and cell are mixed, speed moves into cryopreservation tube, and is put into jelly
Deposit in box, overnight, next day is transferred in liquid nitrogen for -70 DEG C of program coolings.Wherein, every 107 NK cells use 1ml frozen stock solutions.
Freezen protective NK cell 30d, are then recovered.
Detection freezes cell recoveries after the survival rate of front and rear cell, and recovery.Specifically, before freezing and freeze simultaneously
Cell survival rate computational methods after recovery are：【Viable count/(viable count+dead cell number)】× 100%.Cell after recovery
The computational methods of the rate of recovery are：While freezing (viable count after recovery/viable count) × 100%.
1.2nd, recovery NK cytoscopies result：
As a result show, viability rate is below before freezing.Specifically, the cell survival that frozen stock solution 3 and 4 preserves
Rate is substantially better than frozen stock solution 1 and 2, and has no difference between frozen stock solution 3 and 4, shows 10 volume % DMSO and has to NK cells
Preferable protective effect, and protective effects of the HSA of various concentrations for NK cells is without marked difference；The cell of frozen stock solution 3 and 4
The rate of recovery is also superior to frozen stock solution 1 and 2.The cell recoveries of frozen stock solution 5 and 6 also superior to frozen stock solution 1 and 2, especially frozen stock solution 5,
Cell recoveries are up to 99.3%.
The cell recoveries of each group frozen stock solution are：
Frozen stock solution 1：62.3%；
Frozen stock solution 2：61.2%；
Frozen stock solution 3：73.5%；
Frozen stock solution 4：75.3%,
Frozen stock solution 5：99.3%；
Frozen stock solution 6：77.2%；
2nd, NK cells are frozen using the frozen stock solution containing blood plasma
2.1st, separation obtains human umbilical cord blood mononuclear cell, and mononuclearcell is inoculated into the blake bottle added with 20ml culture mediums
In, Medium Proportion is：18ml OpTmizerTM CTSTMT-cell expansion SFM culture medium+2ml autologous plasmas
(Autologous plasma)+final concentration 1000IU/ml Pepro Tech IL-2+ final concentration 0.01KE/ml Sapylins
(OK432), 37 DEG C, 5%CO2 sterile cultures, it is designated as the 0th day.Daily observation cell, and carry out appropriate passage amplification, culture medium
Match and be for Medium Proportion：OpTmizerTM CTSTMThe autologous plasma of T-cell expansion SFM culture mediums+10%
(Autologous plasma)+final concentration 1000IU/ml Pepro Tech IL-2, induced amplification obtain NK cells.
2.2, NK cells freeze and method for resuscitation：
Four kinds of freezen protective liquid containing blood plasma are respectively adopted, the NK cells that above-mentioned external evoked amplification obtains for 10 days are carried out
Freezen protective, wherein the composition of four containing blood plasma kinds of frozen stock solutions is as follows：
Frozen stock solution 9：5 volume %DMSO+40 volume % blood plasma；
Frozen stock solution 10：10 volume %DMSO+40 volume % blood plasma；
Frozen stock solution 11：5 volume %DMSO+5 volume %HSA+40 volume % blood plasma；
Frozen stock solution 12：10 volume %DMSO+5 volume %HSA+40 volume % blood plasma,
Frozen stock solution 13：0.2 volume % DMSO+15 volumes % HSA+40 volume % blood plasma, added after the completion of preparation white
Wine grass saponin(e R to its final concentration of 0.001g/ml；
Frozen stock solution 14：0.4 volume % DMSO+10 volumes % HSA+40 volume % blood plasma, added after the completion of preparation white
Wine grass saponin(e R to its final concentration of 0.001g/ml；
Wherein, in frozen stock solution 9-14, blood plasma is autologous plasma, and residual volume is 1640 or Stemspan cultures
Base.
Wherein, NK cells are frozen according to following steps：After freezen protective liquid and cell are mixed, speed moves into cryopreservation tube, and
It is put into freezing storing box, overnight, next day is transferred in liquid nitrogen for -70 DEG C of program coolings.Wherein, every 107 NK cells are frozen using 1ml
Liquid.
Freezen protective NK cell 30d, are then recovered, wherein, resuscitation fluid contains 2.5% blood plasma and 5%Dextran40.
Detection freezes cell recoveries after survival rate, the cell surface marker expression of front and rear cell, and recovery.Tool
Body, the cell survival rate computational methods before freezing and after freezing and recovering are：【Viable count/(viable count+dead cell
Number)】× 100%.The computational methods of cell recoveries are after recovery：While freezing (viable count after recovery/viable count) ×
100%.Utilize flow cytomery NK cells cell surface marks CD3-CD56+ expression.
2.3rd, recovery NK cytoscopies result：
As a result show, six kinds preserve immunocyte survival rates and cell surface marker expression that liquid preserve and before freezing
Compared to without marked difference, cell recoveries are higher than the frozen stock solution that blood plasma is free of in " step 1 ".Also, freezen protective liquid 5 and 6,9
With certain multiplication capacity is respectively provided with after 10 cell recoveries frozen, the cell that 13 and 14 frozen stock solutions freeze has vigorous breeding
Ability.Thus, show that blood plasma and Conyza japonica saponin(e R have good protective effect to cell.
The cell recoveries of each group frozen stock solution are：
Frozen stock solution 7：71.5%；
Frozen stock solution 8：72.3%；
Frozen stock solution 9：83.3%；
Frozen stock solution 10：83.2%,
Frozen stock solution 11：99.7%；
Frozen stock solution 12：92.3%；
To sum up, inventor is matched using DMSO, HAS, Conyza japonica saponin(e R and blood plasma various concentrations, preserves the single core of bleeding of the umbilicus
The immunocyte of cell induction, observe frozen stock solution effect, the results showed that, there is 10 volume %DMSO preferable cytoprotection to make
With；And protective effects of the HSA of various concentrations to immunocyte is without marked difference.And use the autologous plasma of the embodiment of the present invention
External evoked immunocyte is preserved with HSA and DMSO combinations or the frozen stock solution for being further combined Conyza japonica saponin(e R, after recovery
Cell recoveries are high, and cytoactive is good, have certain multiplication capacity, and the cell recoveries that remaining each group freezing liquid preserves are inclined
It is low.
Embodiment 3
The NK cells that the frozen stock solution 9 of embodiment 2 freezes are recovered, specific method is as follows
1st, resuscitation process
(1) the NK cells of the freezen protective of 2 frozen stock solution of embodiment 7 are taken out in liquid nitrogen, are melted using 37~42 DEG C of water-bath speed.
(2) use isometric X-VIVO15 culture mediums (being designated as P groups) respectively, and it is isometric contain 2.5%HSA, 5%
Dextran40 resuscitation fluid (being designated as F1 groups) two ways processing cell.
(3) centrifuge, take cell respectively, counted using cell viability analyzer, survey cell survival rate.
2nd, cultivated after recovering
The cell of P groups and F1 groups is used into immunocyte scale amplification culture medium, i.e. SuperCulture TM respectively
The L500 human lymphocytes serum free medium+domestic IL-2 of final concentration 1000IU/ml, in vitro culture is carried out, next day carries out cell
Technology and activity analysis.
3rd, calculate
During due to recovery cell, initiator cell number is inconsistent, need to quote correction factor, calculates CD3/CD56 positive cells
Number.Computational methods are as follows：
(1) average value of two groups of initiator cell numbers is calculated；
(2) average value number divided by each group initiator cell number are used, obtains the correction factor K of each group；
(3) each group final cell number is multiplied by corresponding correction factor K and CD3/CD56 positive percentage respectively, is
CD3/CD56 positive cell numbers.
4. result
(1) recovering experiment result
The cell number and cell viability result for the NK cells that " step 1 " recovery obtains are as shown in table 4.
Influence of the resuscitation fluid of table 4 to cell number and cell viability
| Packet | Cell number | Cell viability |
| P | (0.90±0.08)×107 | (80.30 ± 0.01) % |
| F1 | (0.95±0.04)×107 | (90.25 ± 0.02) % |
From table 4, the cell of recovery is handled using culture medium P or resuscitation fluid F1, both cell numbers are without marked difference (p>
0.05), but cell viability has significant difference (p<0.05).As a result show, compared with control group P resuscitation fluids, F1 resuscitation fluids
It can be good at the vigor for the NK cells that lifting recovery obtains.
(2) recovery cultivation results
In " step 2 ", P groups and F1 groups are as shown in table 5 in the result of next day NK cell culture.
The resuscitation fluid of table 5 is to cultivating 24h cell number and the influence of cell viability
| Packet | Cell number | Cell viability |
| P | (0.89±0.08)×107 | (90.20 ± 0.01) % |
| F1 | (0.96±0.04)×107 | (93.10 ± 0.02) % |
From table 5, the cell recovered is handled using culture medium P or resuscitation fluid F1, after carrying out 24h in vitro cultures, both
Cell number and cell viability are without marked difference (p>0.05).
5. conclusion
The above results show that NK cells are recovered after Large scale in vitro culture is expanded and frozen using P groups and F1 groups
Liquid handles the cell of freezen protective, although both quantity are higher than P groups without significant difference, the cell viability of F1 groups processing, and then
Prove the Cryopreservation liquid phase ratio with P group ordinary culture mediums, the Cryopreservation liquid of F1 groups (containing 2.5%HSA, 5%Dextran40)
Preferably cell can be promoted to rejuvenate during cell recovery.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or the spy for combining the embodiment or example description
Point is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term not
Necessarily refer to identical embodiment or example.Moreover, specific features, structure, material or the feature of description can be any
One or more embodiments or example in combine in an appropriate manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not
In the case of departing from the principle and objective of the present invention a variety of change, modification, replacement and modification can be carried out to these embodiments, this
The scope of invention is limited by claim and its equivalent.
Claims (9)
1. a kind of external evoked amplification of immunocyte, the method for freezing and recovering, it is characterised in that including：
Culture vessel is coated with using CD16 antibody, so as to the culture vessel after being coated with；
Mononuclearcell is placed in using immune cell activation culture medium in the culture vessel after the coating and carries out the first induction
Amplification cultivation, to obtain the immunocyte of preliminary induced amplification；
The immunocyte of the preliminary induced amplification is subjected to the second induced amplification culture using immune cell expansion culture medium, with
Just the immunocyte broken up；
The immunocyte of the differentiation is subjected to the 3rd induced amplification culture using immunocyte scale amplification culture medium, so as to
Obtain the immunocyte of large-scale function activation；
The immunocyte of the large-scale function activation is frozen using immunocyte frozen stock solution, it is immune thin so as to what is frozen
Born of the same parents；
The immunocyte frozen is placed in 37-42 DEG C of water-bath and melted, and is mixed with the Cryopreservation liquid of immunocyte, with
Just recovery cell mixture is obtained；And
The recovery cell mixture is subjected to centrifugal treating, so as to the immunocyte recovered,
Wherein,
The immune cell activation culture medium is the serum-free lymphocyte training that with the addition of the first blood plasma, proleulzin and Sapylin
Support base；
The immune cell expansion culture medium is the serum-free lymphocytes culture medium that with the addition of proleulzin and Sapylin；
The immunocyte scale amplification culture medium is the serum-free lymphocytes culture medium that with the addition of proleulzin；
The immunocyte frozen stock solution includes the second of 2-10 volumes % DMSO, 2-15 volume % HSA, 20-88 volume %
Blood plasma and surplus freezing media；
The Cryopreservation liquid includes albumin and dextran；
The concentration of the Sapylin is 0.007-0.013KE/ml,
The immune cell activation culture medium, the immune cell expansion culture medium and the immunocyte scale amplification cultivation
In base, the concentration of the proleulzin is 700-1300IU/ml,
The immune cell activation culture medium, the immune cell expansion culture medium and the immunocyte scale amplification cultivation
In base, the serum-free lymphocytes culture medium is OpTmizerTMCTSTMSerum free medium or SuperCultureTM
L500 human lymphocyte serum free mediums,
In the immune cell activation culture medium, the concentration of first blood plasma is 7-13 volumes %；
The freezing media is 1640 culture mediums or Stemspan culture mediums.
2. according to the method for claim 1, it is characterised in that first blood plasma and second blood plasma are autologous blood
Slurry.
3. according to the method for claim 1, it is characterised in that in the immunocyte frozen stock solution,
The concentration of the DMSO is 10 volume %；
The concentration of the HSA is 5 volume %；
The concentration of second blood plasma is 40 volume %.
4. according to the method for claim 1, it is characterised in that in the Cryopreservation liquid, the white egg of the albumin behaviour
In vain, optionally, the human albumin derives from commercially available human serum albumins or autologous plasma,
Optionally, the content of the albumin is 1-5%,
Optionally, the dextran is Dextran 40,
Optionally, the content of the dextran is 2-8 weight %.
5. according to the method for claim 1, it is characterised in that the immunocyte is that NK and dendron shape are thin
Born of the same parents.
6. according to the method for claim 1, it is characterised in that when the tentatively CD56's of the immunocyte of induced amplification
When expression is more than 70%, the second induced amplification culture is carried out,
Optionally, when the differentiation immunocyte CD56 expression more than 90% when, carry out the 3rd induced amplification training
Support.
7. according to the method for claim 1, it is characterised in that in the first induced amplification incubation, pass within every 1 day
Once,
Optionally, in the second induced amplification incubation, pass on once within every 2 days,
Optionally, in the 3rd induced amplification incubation, passage in every 2 days is once.
8. according to the method for claim 1, it is characterised in that every 106-109The individual large-scale function activation is immunized
Cell uses immunocyte frozen stock solution described in 1ml.
9. according to the method for claim 1, it is characterised in that the immunocyte frozen is multiple with the freezing after melting
The volume ratio that liquid of reviving mixes is 1：0.5-5.
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| CN108251366A (en) * | 2016-12-28 | 2018-07-06 | 华南生物医药研究院 | Cryopreservation liquid and its application for the killing cell of cytokine profiles induction |
| CN107347871A (en) * | 2017-06-07 | 2017-11-17 | 江苏善之源健康科技有限公司 | A kind of immunocyte, which is fed back, preserves liquid |
| CN107904175B (en) * | 2017-12-29 | 2018-09-25 | 广州沙艾生物科技有限公司 | The incubator of induced amplification for immunocyte |
| CN108192868B (en) * | 2017-12-29 | 2018-12-07 | 广州沙艾生物科技有限公司 | The induced amplification method of immunocyte |
| CN108060130B (en) * | 2017-12-29 | 2018-12-07 | 广州沙艾生物科技有限公司 | Control the method that incubator is suitable for the induced amplification of immunocyte |
| CN111019892A (en) * | 2019-12-16 | 2020-04-17 | 杭州恩格生物医疗科技有限公司 | Immune cell in-vitro induction amplification method |
| CN111387175A (en) * | 2020-05-06 | 2020-07-10 | 河北生命原点生物科技有限公司 | Cryopreservation solution and cryopreservation method for lymphoid cells |
| CN111454903B (en) * | 2020-05-06 | 2023-10-20 | 青岛瑞思德生物科技有限公司 | Immune cell in vitro culture, induction, activation and cryopreservation method and cell bank establishment thereof |
| CN111676193A (en) * | 2020-06-19 | 2020-09-18 | 珠海贝索细胞科学技术有限公司 | Recovery liquid and recovery method for PBMC of cryopreserved human |
| CN115843784A (en) * | 2022-12-30 | 2023-03-28 | 广东康盾高新技术产业集团股份公司 | CAR-NK cell preservation solution and preparation method and preservation method thereof |
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| CN102994449A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of NK cells |
| CN104222069B (en) * | 2014-08-27 | 2016-06-29 | 中国人民解放军军事医学科学院野战输血研究所 | CFU-E frozen stock solution and application thereof |
| CN104651310B (en) * | 2015-02-28 | 2017-10-10 | 浙江卫未生物医药科技有限公司 | A kind of method of external evoked amplification NK cells |
| CN105039253B (en) * | 2015-08-05 | 2019-02-19 | 广州赛莱拉干细胞科技股份有限公司 | Serum free medium suitable for immunocyte large-scale culture |
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| CN105754941B (en) * | 2016-04-18 | 2020-06-02 | 广州复大医疗有限公司 | In-vitro induced amplification culture method for peripheral blood NK cells |
| CN105754942A (en) * | 2016-05-18 | 2016-07-13 | 天津普瑞赛尔生物科技有限公司 | Method for amplifying NK cells in vitro and NK cells obtained by same |
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