CN108148805A - A kind of people Tscm cells and its preparation method and application - Google Patents
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Abstract
The invention belongs to biological immunology fields, and in particular to a kind of people Tscm cells and its preparation method and application, the preparation method includes the following steps：(1) human cord blood is acquired, human cord blood mononuclearcell is obtained using density-gradient centrifugation method；(2) CD8+CD45RA+CCR7+ T cells subgroups are sorted using flow cell sorter；(3) the T cells subgroup of sorting is added in the culture medium containing stimulant CD3/CD28 beads, cell factor IL 2 and melbine (metformin) and cultivated；(4) it carries out changing liquid every 2d and adds cell factor IL 2, the CD8+T cells with Tscm phenotypes can be obtained when being expanded to 14d.The method of the present invention, which can obtain largely, has memory characteristic while the low T cell of differentiation state is used for the treatment of tumour.
Description
Technical field
The invention belongs to biological immunology fields, and in particular to a kind of people Tscm cells and its preparation method and application.
Background technology
With deepening continuously for biomedical sector research, Immunology technology is maked rapid progress, and immunization therapy becomes
The mankind defeat the new hope of tumour.Important composition portion of the adoptive immunotherapy of immunocompetent cell infusion as immunization therapy
Point, refer to the immune effector cell of Activation In Vitro being transfused to patient, to kill the tumour cell of patient's body.But the therapy exists
It breeds and huge challenge is also faced with while hope：The immunocyte that adoptive cellular immunotherapy is transfused to patient is in eventually
Last differentiation state, the time-to-live is short in vivo, and antitumous effect is limited.How more rejuvenations are obtained, the anti-of lasting stability swells
Oncocyte is the hot spot of current immunization therapy research.
CD8+ memory T cells play a significant role in antitumor reaction, are divided into different subgroups i.e. stem-like cell T cell
(Tscm), center memory t cell (Tcm) and effect memory t cell (Tem).Wherein Tscm subgroups because its have it is good from
My updating ability and multipotency begetting power and ability with stronger killing tumor cell and be concerned.
At present, the method for the Tscm cells of culture amplification both at home and abroad can not meet clinical practice demand, and one side blood comes
Source comes from patient or patients' relatives peripheral blood mostly so that the acquisition of initial cell is significantly limited；On the other hand, Tscm is thin
Born of the same parents cultivate in vitro should not stablize maintenance, easily further differentiation so that culture gained Tscm cell numbers are less.Therefore, such as
What solves the acquisition of initial cell and the maintenance of Tscm cells is the maximum bottleneck of current Tscm in vitro cultures.
Melbine (metformin) is a kind of safe and efficient biguanides, as insulin sensitizer,
Metformin can by reducing hepatic gluconeogenesis, increase surrounding tissue to the sensibility of insulin, intestinal cell is inhibited to absorb
Glucose plays blood sugar reducing function, effectively reduces blood insulin level.Meanwhile it is found in cellular energy metabolism research, diformazan
Biguanides is regulation of energy signaling molecule Adenylate cyclase (AMP-activated protein kinase, AMPK)
Agonist, activation AMPK negative regulations downstream mammal rapamycin target spot (mammalian target ofrapamycin,
MTOR) access.
Invention content
Invention broadly provides a kind of people Tscm cells and its preparation method and application, can obtain a large amount of with memory
Characteristic while the low T cell of differentiation state are used for the treatment of tumour.Its technical solution is as follows：
A kind of preparation method of people Tscm cells, includes the following steps：
(1) human cord blood is acquired, human cord blood mononuclearcell is obtained using density-gradient centrifugation method；
(2) CD8+CD45RA+CCR7+ T cells subgroups are sorted using flow cell sorter；
(3) the T cells subgroup of sorting added in containing stimulant CD3/CD28beads, cell factor IL-2 and
It is cultivated in the culture medium of metformin；
(4) it carries out changing liquid every 2d and adds cell factor IL-2, can be obtained with Tscm phenotypes when being expanded to 14d
CD8+T cells.
Preferably, it is aseptically to acquire people the specific method of human cord blood mononuclearcell to be obtained in step (1)
Cord blood first centrifuges under the conditions of 1500rpm, 10min, by 56 DEG C of inactivation 25min in serum transfers to sterile centrifuge tube, then
4000rpm, 20min centrifuge serum, and with the haemocyte of buffer solution dilution precipitation, haemocyte dilution is slowly then added to leaching
It in bar separating liquid, is centrifuged under the conditions of 2500rpm, 25min, finally draws tunica albuginea layer, and with buffer solution for cleaning, 1500rpm,
10min is centrifuged, that is, obtains human cord blood mononuclearcell.
Preferably, the buffer solution is PBS buffer solution, when diluting the haemocyte of precipitation with buffer solution, using 1/2-1/4 blood
The PBS buffer solution dilution of cell concentration, the volume of the lymph separating liquid are the 1/2-1/4 of haemocyte diluent liquid accumulated amount.
Preferably, it is with the flow cell sorter sorting specific method of T cells subgroup in step (2), according to every 2
×106A mononuclearcell, which is protected from light, adds in 1-3 μ L antibody, and the antibody is CD3-FITC, CD8-APC-cy-7, CD45RA-APC
And CCR7-Percp, carry out airflow classification CD8+CD45RA+CCR7+ T cells.
Preferably, the volume of step (3) moderate stimulation agent CD3/CD28beads is the 1/3 of T cells volume, carefully
A concentration of 50-150 μM of a concentration of 500-1000IU/mL of intracellular cytokine IL-2, metformin.
Preferably, culture medium described in step (3) is Takara GT-551 culture mediums, and condition of culture is 37 DEG C, 5%CO2
Environment.
Preferably, the autoserum, penicillin and streptomysin of 5% volume are further included described in step (3) in culture medium,
T cell density is (1-3) × 10 in culture medium6/mL。
Preferably, CD8+T cells expandeds reach 180-220 times when being expanded to 14d.
A kind of people Tscm cells, are obtained by the above method.
People's Tscm cells can be applied in treatment tumor disease preparation.
People's Tscm cells are prepared using the above method, are had the following advantages：
Compared with traditional peripheral blood obtains primary immune cell, using Cord blood with unique excellent in the method for the present invention
Gesture：Cord Blood-Derived enriches, and has unique biology, immunological characteristic and contains a large amount of T cells, is more easy to induce dry
Celliform T cell perfectly solves initial cell and obtains problem；
In testing in vitro, we are for the first time the study found that metformin can activate the AMPK signal paths of T cell to tie up
The number of Tscm cells is held, and inhibits the further differentiation of Tscm cells, substantially increases the amplification of Tscm cell injuring models
Efficiency, also remain the efficiently anti-swollen of Tscm cells maintaining this group of cell self-renewal abilities and diving property of multipotency while
Knurl ability.
T cells are obtained from Cord blood, add in CD3/CD28beads and IL-2 induced activations, and using metfromin
The ratio of Tscm cells is maintained, there is the memory characteristic low T cell of differentiation state simultaneously so as to obtain largely, it is anti-swollen so as to obtain
The T cell of high, the internal time-to-live length of tumor activity is used for clinical adoptive cellular immunotherapy.
This method can expand the external mass propgation of Tscm cells, can effectively push the further of cellular immunotherapy
Development solves clinical culture technique bottleneck at present, extends adoptive immunity cell in the patient's body time-to-live, it is persistently high to enhance its
The anti-tumor capacity of effect further carries out larger scale clinical treatment for cellular immunotherapy and takes a firm foundation.
Description of the drawings
Fig. 1 is human umbilical cord blood mononuclear cell Tn cell proportion figures；
Fig. 2 is adult peripheral mononuclear cell Tn cell proportion figures；
Fig. 3 is human umbilical cord blood mononuclear cell and peripheral blood mononuclear cells Tn Cell counts figures；
Fig. 4 is Tscm cell proportion figures prepared by comparative example 1；
Fig. 5 is Tscm cell proportion figures prepared by embodiment 1；
Fig. 6 is the statistical chart for the Tscm cell proportions that comparative example 1 is prepared with embodiment 1；
Fig. 7 is the expression figure of the lethal marker CD107a of method in flow cytometer detection comparative example 1；
Fig. 8 is the expression figure of the lethal marker CD107a of method in flow cytometer detection embodiment 1；
Fig. 9 is the statistical chart for the Tscm Cell killing ratios that comparative example 1 is prepared with embodiment 1.
Specific embodiment
Embodiment 1
Cord blood 50mL is acquired under aseptic condition, human cord blood mononuclearcell is obtained using density-gradient centrifugation method
CBMCs is as follows：First 1500rpm, 10min are centrifuged, and raising speed and the acceleration to slow down are 9m/s2, serum is turned
It moves in sterile 50mL centrifuge tubes, 56 DEG C of inactivation 25min, then 4000rpm, 20min centrifugation serum, raising speed adds with what is slowed down
Speed is 9m/s2.Using PBS buffer solution according to haemocyte amount 1:The haemocyte of 3 dilution proportion precipitation is then thin by blood
Born of the same parents' dilution is slowly added in lymph separating liquid, and the volume of the lymph separating liquid is the 1/ of haemocyte diluent liquid accumulated amount
3.Last 2500rpm, 25min are centrifuged, and raising speed and the acceleration to slow down are 5m/s2, it is careful to draw tunica albuginea layer, delayed with PBS
Fliud flushing cleans twice, 1500rpm, 10min centrifugation, and raising speed and the acceleration to slow down are 9m/s2, you can obtain Cord blood list
A nucleus.
The CBMCs of above-mentioned separation is counted, according to every 2 × 106A cells from light adds in 2 μ L streaming antibody, the streaming
Antibody is CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp, carries out airflow classification CD8+CD45RA+
CCR7+T cells.
The T cells subgroup of sorting is placed in the stimulant CD3/CD28beads containing 1/3T cell subsets volumes
In the GT551 culture mediums of 1000IU/mL IL-2, while 5% autoserum, penicillin and streptomysin are added in, adjustment cell is close
Spend is 2 × 106/mL.At 37 DEG C, 5%CO2It is cultivated in incubator, adds in 100 μM of metformin afterwards for 24 hours, it is thin every 2d observations
Intracellular growth state, half amount change liquid and add the cell factor IL-2 of full dose.Culture can obtain 90% to 7d has Tscm tables
The CD8+T cells of type, its amplification times reaches 220 times when being expanded to 14d.
In reagent used above, it is public that the lymphocyte separation medium is selected from Tianjin Hao oceans biological products science and technology Limited Liability
Department, GT-551 culture mediums are selected from Takara companies, and IL-2 is produced for Beijing Shuanglu Pharmaceutical Co., Ltd., metformin purchases
From sigma companies, CD3/CD28beads is produced for Life technology companies, and the streaming antibody is purchased from Biologend
Company.
Embodiment 2
Cord blood 50mL is acquired under aseptic condition, human cord blood mononuclearcell is obtained using density-gradient centrifugation method
CBMCs is as follows：First 1500rpm, 10min are centrifuged, and raising speed and the acceleration to slow down are 9m/s2, serum is turned
It moves in sterile 50mL centrifuge tubes, 56 DEG C of inactivation 25min, then 4000rpm, 20min centrifugation serum, raising speed adds with what is slowed down
Speed is 9m/s2.Using PBS buffer solution according to haemocyte amount 1:The haemocyte of 2 dilution proportion precipitation is then thin by blood
Born of the same parents' dilution is slowly added in lymph separating liquid, and the volume of the lymph separating liquid is the 1/ of haemocyte diluent liquid accumulated amount
4.Last 2500rpm, 25min are centrifuged, and raising speed and the acceleration to slow down are 5m/s2, it is careful to draw tunica albuginea layer, delayed with PBS
Fliud flushing cleans twice, 1500rpm, 10min centrifugation, and raising speed and the acceleration to slow down are 9m/s2, you can obtain Cord blood list
A nucleus.
The CBMCs of above-mentioned separation is counted, according to every 2 × 106A cells from light adds in 1 μ L antibody, and the antibody is
It is thin to carry out airflow classification CD8+CD45RA+CCR7+T by CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp
Born of the same parents.
The T cells subgroup of sorting is placed in the stimulant CD3/CD28beads containing 1/2T cell subsets volumes
In the GT551 culture mediums of 900IU/mL IL-2, while 5% autoserum, penicillin and streptomysin are added in, adjustment cell is close
Spend is 3 × 106/mL.At 37 DEG C, 5%CO2It is cultivated in incubator, adds in 50 μM of metformin afterwards for 24 hours, cell is observed every 2d
Growth conditions, half amount change liquid and add the cell factor IL-2 of full dose.Culture can obtain 80% to 7d has Tscm phenotypes
CD8+T cells, its amplification times reaches 180 times when being expanded to 14d.
In reagent used above, it is public that the lymphocyte separation medium is selected from Tianjin Hao oceans biological products science and technology Limited Liability
Department, GT-551 culture mediums are selected from Takara companies, and IL-2 is produced for Beijing Shuanglu Pharmaceutical Co., Ltd., metformin purchases
From sigma companies, CD3/CD28beads is produced for Life technology companies, and the streaming antibody is purchased from Biologend
Company.
Embodiment 3
Cord blood 50mL is acquired under aseptic condition, human cord blood mononuclearcell is obtained using density-gradient centrifugation method
CBMCs is as follows：First 1500rpm, 10min are centrifuged, and raising speed and the acceleration to slow down are 9m/s2, serum is turned
It moves in sterile 50mL centrifuge tubes, 56 DEG C of inactivation 25min, then 4000rpm, 20min centrifugation serum, raising speed adds with what is slowed down
Speed is 9m/s2.Using PBS buffer solution according to haemocyte amount 1:The haemocyte of 4 dilution proportion precipitation is then thin by blood
Born of the same parents' dilution is slowly added in lymph separating liquid, and the volume of the lymph separating liquid is the 1/ of haemocyte diluent liquid accumulated amount
2.Last 2500rpm, 25min are centrifuged, and raising speed and the acceleration to slow down are 5m/s2, it is careful to draw tunica albuginea layer, delayed with PBS
Fliud flushing cleans twice, 1500rpm, 10min centrifugation, and raising speed and the acceleration to slow down are 9m/s2, you can obtain Cord blood list
A nucleus.
The CBMCs of above-mentioned separation is counted, according to every 2 × 106A cells from light adds in 3 μ L antibody, and the antibody is
It is thin to carry out airflow classification CD8+CD45RA+CCR7+T by CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp
Born of the same parents.
The T cells subgroup of sorting is placed in the stimulant CD3/CD28beads containing 1/4T cell subsets volumes
In the GT551 culture mediums of 500IU/mL IL-2, while 5% autoserum, penicillin and streptomysin are added in, adjustment cell is close
Spend is 1 × 106/mL.At 37 DEG C, 5%CO2It is cultivated in incubator, adds in 150 μM of metformin afterwards for 24 hours, it is thin every 2d observations
Intracellular growth state, half amount change liquid and add the cell factor IL-2 of full dose.Culture can obtain 85% to 7d has Tscm tables
The CD8+T cells of type, its amplification times reaches 200 times when being expanded to 14d.
In reagent used above, it is public that the lymphocyte separation medium is selected from Tianjin Hao oceans biological products science and technology Limited Liability
Department, GT-551 culture mediums are selected from Takara companies, and IL-2 is produced for Beijing Shuanglu Pharmaceutical Co., Ltd., metformin purchases
From sigma companies, CD3/CD28 beads are produced for Life technology companies, and the streaming antibody is purchased from Biologend
Company.
Comparative example 1
Difference lies in be not added with metformin, other condition of culture are consistent to the present embodiment with embodiment 1.
Detection and result
The phenotype, activity and metformin of Tscm cells are detected the maintenance of Tscm and the enhancing of function, with
Sample in embodiment 1 is research object.Concrete operation step is as follows：
1. the human umbilical cord blood mononuclear cell obtained in Example 1, and the Tn cell proportions compared with adult peripheral blood, as a result
See Fig. 1-3.Wherein Fig. 1 is with flow cytometer detection human umbilical cord blood mononuclear cell Tn cell proportions, and Fig. 2 is with flow cytometer detection adult human peripheral
Blood mononuclear cell Tn cell proportions, Fig. 3 are human umbilical cord blood mononuclear cell and peripheral blood mononuclear cells Tn Cell counts figures.By
Fig. 1-3 is it is found that human umbilical cord blood mononuclear cell Tn cells are low compared to peripheral blood mononuclear cells Tn cell contents, so need to be to navel
Band mononuclearcell carries out induced activation, has the memory characteristic low T cell of differentiation state simultaneously so as to obtain largely.
2.Tscm cellular morphologies and Activity determination：Micro- Microscopic observation cell state is cultivated to 3d cells agglomerate, quickly
Growth, is cultivated to the apoptosis situation of 7d Flow cytometry cells.Collect 1 × 106A cell is resuspended in 200 μ L
In Annexin-V binding buffer, it is protected from light and adds in 2 μ L Annexin-V-APC antibody, 4 DEG C are protected from light incubation 15min, on
PI is added in before machine.
3.Tscm cell phenotypes and Function detection：Culture is collected to the cell 1 × 10 of 7d and 14d6It is a, it is resuspended in
In PBS of the 200 μ L containing 1%FBS (fetal calf serum), it is protected from light and adds in streaming antibody：CD8-APC-cy-7、CD45RA-APC、CCR7-
Percp and CD95-PE-cy-7,4 DEG C are protected from light incubation 15min, the phenotype of flow cytometer detection Tscm.It is analyzed and cultivated using Diva softwares
The cell phenotype result of acquisition shows that Tscm ratios reach 95%, the Tscm cells high 30% obtained compared with peripheral blood Fiber differentiation with
On.
4. the Tscm cell proportions that Tscm cells prepared by flow cytometer detection detection embodiment 1 are prepared with comparative example 1, as a result such as
Shown in Fig. 4-6, wherein Fig. 4 is Tscm cell proportions prepared by comparative example 1, and Fig. 5 is Tscm cell proportions prepared by embodiment 1,
Fig. 6 is the statistical chart for the Tscm cell proportions that comparative example 1 is prepared with embodiment 1.By result it is found that using 1 method system of embodiment
Standby Tscm cell proportions are high, conducive to the induction and activation of Tscm cells.
5. the Tscm cells that Tscm cells prepared by embodiment 1 are prepared with comparative example 1 respectively with lung cancer tumor cell line
A549 and cancer of the esophagus tumor cell line TE7 are incubated altogether, the expression of the cell killing marker CD107a of detection Tscm, as a result as schemed
Shown in 7-9, wherein Fig. 7 is the expression of the lethal marker CD107a of method in flow cytometer detection comparative example 1, and Fig. 8 is examined for streaming
The expression of the lethal marker CD107a of method in embodiment 1 is surveyed, Fig. 9 is the Tscm cells that comparative example 1 is prepared with embodiment 1
The statistical chart of killing ratio, Fig. 7-9 show that Tscm cell killings function prepared by the present invention induces Tscm compared with method in embodiment 1
Cell killing function is 5-10 times high, has stronger antitumor action.
For Tscm cells prepared by embodiment 2-3, the testing result phase of its result and embodiment 1 after above-mentioned detection is done
Seemingly, this will not be repeated here.
It will be apparent to those skilled in the art that technical solution that can be as described above and design, make other various
Corresponding change and deformation, and all these changes and deformation should all belong to the protection domain of the claims in the present invention
Within.
Claims (10)
1. a kind of preparation method of people Tscm cells, it is characterised in that：Include the following steps：
(1) human cord blood is acquired, human cord blood mononuclearcell is obtained using density-gradient centrifugation method；
(2) CD8+CD45RA+CCR7+ T cells subgroups are sorted using flow cell sorter；
(3) the T cells subgroup of sorting added in containing stimulant CD3/CD28beads, cell factor IL-2 and
It is cultivated in the culture medium of metformin；
(4) it carries out changing liquid every 2d and adds cell factor IL-2, the CD8+ with Tscm phenotypes can be obtained when being expanded to 14d
T cell.
2. the preparation method of people Tscm cells according to claim 1, it is characterised in that：People's umbilical cord is obtained in step (1)
The specific method of blood mononuclear cell is aseptically to acquire human cord blood, first 1500rpm, 10min centrifugation, by serum
55-57 DEG C of inactivation 20-30min in sterile centrifuge tube, then 4000rpm, 20min centrifugation serum are transferred to, is diluted with buffer solution
Then the haemocyte of precipitation is slowly added to haemocyte dilution in lymph separating liquid, 2500rpm, under the conditions of 25min from
The heart finally draws tunica albuginea layer, and with buffer solution for cleaning, 1500rpm, 10min centrifugation, i.e. acquisition human cord blood mononuclearcell.
3. the preparation method of people Tscm cells according to claim 2, it is characterised in that：The buffer solution is buffered for PBS
Liquid when diluting the haemocyte of precipitation with buffer solution, is diluted using the PBS buffer solution of 1/2-1/4 haemocyte amounts, the lymph separation
The volume of liquid is the 1/2-1/4 of haemocyte diluent liquid accumulated amount.
4. the preparation method of people Tscm cells according to claim 1, it is characterised in that：Step uses fluidic cell in (2)
The sorter sorting specific method of T cells subgroup is, according to every 2 × 106A mononuclearcell is protected from light addition 1-3 μ L and resists
Body, the antibody are CD3-FITC, CD8-APC-cy-7, CD45RA-APC and CCR7-Percp, carry out airflow classification CD8+
CD45RA+CCR7+ T cells.
5. the preparation method of people Tscm cells according to claim 1, it is characterised in that：Step (3) moderate stimulation agent CD3/
The volume of CD28beads is the 1/2-1/4, a concentration of 500-1000IU/ of cell factor IL-2 of T cells volume
A concentration of 50-150 μM of mL, metformin.
6. the preparation method of people Tscm cells according to claim 1, it is characterised in that：Culture medium described in step (3)
For Takara GT-551 culture mediums, condition of culture is 37 DEG C, 5%CO2Environment.
7. the preparation method of people Tscm cells according to claim 1, it is characterised in that：Culture medium described in step (3)
In further include the autoserum, penicillin and streptomysin of 5% volume, T cell density is (1-3) × 10 in culture medium6/mL。
8. the preparation method of people Tscm cells according to claim 1, it is characterised in that：CD8+T cells when being expanded to 14d
Amplification times reach 180-220 times.
9. a kind of people Tscm cells, it is characterised in that：It is obtained by claim 1-8 any one of them method.
10. a kind of application of the people Tscm cells in tumor disease preparation is treated described in claim 9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109580716A (en) * | 2018-11-23 | 2019-04-05 | 浙江大学 | The recognition methods of recirculating fluidized bed raising section gas-solid flow pattern based on electrostatic detection |
| CN109943527A (en) * | 2019-04-19 | 2019-06-28 | 四川药智联恒科技有限公司 | Promote Cord blood T cell amplification in vitro and maintains high proportion TSCMThe method of subgroup |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109580716A (en) * | 2018-11-23 | 2019-04-05 | 浙江大学 | The recognition methods of recirculating fluidized bed raising section gas-solid flow pattern based on electrostatic detection |
| CN109580716B (en) * | 2018-11-23 | 2020-10-30 | 浙江大学 | Method for identifying gas-solid flow pattern of lifting section of circulating fluidized bed based on electrostatic detection |
| CN109943527A (en) * | 2019-04-19 | 2019-06-28 | 四川药智联恒科技有限公司 | Promote Cord blood T cell amplification in vitro and maintains high proportion TSCMThe method of subgroup |
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