CN113207871A - Storage liquid for in-vitro preservation of T memory stem cells and application thereof - Google Patents
Storage liquid for in-vitro preservation of T memory stem cells and application thereof Download PDFInfo
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Abstract
The invention discloses a storage solution for in vitro preservation of T memory stem cells and application thereof. One technical scheme to be protected by the invention is a composition for in vitro preservation of T memory stem cells. The active ingredients of the composition consist of TWS119, IL-7 and IL-15. In the composition, the proportion of TWS119, IL-7 and IL-15 can be from TWS 1191.5 to 3nmol, from IL-73 to 10ng and from IL-153 to 10 ng. Experiments prove that the storage liquid containing the composition can store T in vitro for a short time compared with the common storage liquidSCMThe cell can keep the survival rate, proliferation and differentiation of the cell at a higher level, and can be applied to clinical and basic research.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a storage solution for in-vitro storage of T memory stem cells and application thereof.
Background
T memory stem cell (T memory cell, T)SCM) Is a T cell subgroup discovered in the early differentiation stage in recent years, has the characteristics of stem cells and memory T cells, the stem cells are characterized by self-renewal and differentiation potential, and the memory T cells are characterized by T cells when antigen is stimulated againSCMCells rapidly proliferate and differentiate into effector T cells, mediate cytokine production and cytotoxic effects, and clear antigens in a short time. T isSCMHas good implantation in vivo, and can differentiate into various memory and effector T cells. T isSCMHas extremely high stem cell characteristics, namely strong self-renewal capacity, anti-apoptosis capacity and directed proliferation and differentiation potential. Thus, TSCMShows the potential of high-efficiency and durable tumor resistance and infectious diseases resistance, is an ideal seed cell for adoptive therapy, and has stronger application prospect. .
TSCMOnly 2% -4% of human peripheral blood mononuclear cells, and sufficient amount of T is preparedSCMAnd preserving its activity for a certain period of time in vitro is a prerequisite for its use. Because of the T prepared in vitroSCMIf the cells cannot be used in time, the cells are usually required to be stored in vitro for a period of time, and the stem cells are easy to lose the characteristics of the cells in vitro, namely the cells are differentiated into late-stage cells and lose the application value of the cells, so that the method for searching the cells in vitro to keep the characteristics unchanged is extremely important. At present, a sufficient amount of T can be prepared by a combined method of antigen stimulation, differentiation inhibitor and cytokine amplificationSCMAnd meets the requirements of subsequent research and clinical application. T isSCMThe in vitro preparation period is long, and the T cells are not easy to be frozen and stored, so that the in vitro storage T is neededSCMTo maintain its characteristics.
Disclosure of Invention
The technical problem to be solved by the invention is how to preserve T memory stem cells or how to preserve T memory stem cells in vitro for a short time or how to keep the characteristics of cell morphology, phenotype, proliferation, differentiation and the like of the T memory stem cells stored for a short time unchanged.
In order to solve the above technical problems, the present invention first provides a composition for in vitro preservation of T memory stem cells. The active ingredients of the composition consist of TWS119, IL-7 and IL-15. In the composition, the proportion of TWS119, IL-7 and IL-15 can be from TWS 1191.5 to 3nmol, from IL-73 to 10ng and from IL-153 to 10 ng.
In the composition, the proportions of the TWS119, the IL-7 and the IL-15 can be 1191.5-3 nmol, 73-8 ng and 153-8 ng.
In the composition, the ratio of TWS119, IL-7 and IL-15 can be TWS 1192 nmol, IL-75 ng and IL-155 ng.
Human serum and RPMI-1640 medium may also be included in the compositions described above.
In the composition, the proportions of TWS119, IL-7, IL-15, human serum and RPMI-1640 culture solution can be TWS 1191.5-3 nmol, IL-73-8 ng, IL-153-8 ng, 80-120 muL of human serum and 880-920 muL of RPMI-1640 culture solution.
In the composition, the ratio of TWS119, IL-7, IL-15, human serum and RPMI-1640 culture solution can be TWS 1192 nmol, IL-75 ng, IL-155 ng, human serum 100 μ L, and RPMI-1640 culture solution 900 μ L.
The individual components of the compositions described above may be packaged separately or stored in admixture.
In the composition described above, the serum may be human serum. The IL-7 can be recombinant human IL-7; the IL-15 may be a recombinant human IL-15.
In order to solve the above technical problems, the present invention also provides a reagent or a kit comprising the composition described above. The reagent or the kit is used for storing the T memory stem cells.
In order to solve the technical problems, the invention also provides a method for preserving the T memory stem cells in vitro. The method comprises storing the T memory stem cells with a stock solution comprising the composition described above.
The T memory stem cells described above can be obtained by culturing in RPMI-1640 medium containing 10% human serum, 5. mu. mol/L TWS119, 20ng/mL IL-7 and 20ng/mL IL-15.
The time for storing the T memory stem cells using the stock solution described above may be 30 days.
The conditions for storing the T memory stem cells using the stock solution described above may be 37 ℃ and 5% CO2Storing and culturing under the condition.
In order to solve the technical problem, the invention also provides any one of the following applications:
use of P1, a composition as described above and/or a reagent or kit as described above and/or a method as described above for the storage of a T memory stem cell product;
use of P2, a composition as described above, and/or a reagent or kit as described above, and/or a method as described above, in the manufacture of a product for maintaining the biological activity of T memory stem cells in vitro;
use of P3, a composition as described above and/or a reagent or kit as described above and/or a method as described above for the manufacture of a product for maintaining a phenotype of T memory stem cells in vitro;
use of P4, a composition as described above and/or a reagent or kit as described above and/or a method as described above for the manufacture of a product for improving the proliferative status of T memory stem cells in vitro;
use of P5, a composition as described above and/or a reagent or kit as described above and/or a method as described above for the preparation of a product for maintaining the killing ability of effector cells differentiated from T memory stem cells in vitro.
The T memory stem cells stored by the method described above also belong to the scope of the present invention.
The T memory stem cells described above may be human peripheral blood-derived T memory stem cells.
In the examples of the present invention, the self-prepared cell stock solution of the present invention and the known common stem cell stock solution were used to store human peripheral blood-derived T for a short period of timeSCMA cell. Wherein the self-made cell storage solution is as follows: 2 μ M/L TWS119+5ng/mL IL-7+5ng/mL IL-15+ 10% human serum + RPMI-1640; common cell culture cell stocks are: 10% human serum + RPMI-1640. After 30 days of storage, the cells were observed for morphological changes and cell death rate was counted, and the cells were flow-thinnedThe cytometric instrument detects the cell proliferation capacity and cell phenotype; detecting TSCMKilling ability of differentiated effector cells, as freshly prepared TSCMCells were used as positive controls. The result shows that TSCMThe cell morphology has no obvious difference among all groups; self-made preservation solution preservation cultured TSCMThe cell survival rate is higher than that of the common cell culture solution group, and the proliferation capacity, the phenotype and the killing capacity of effector cells differentiated from the cell culture solution group are not obviously different from those of fresh cells. Therefore, the self-made T of the inventionSCMThe cell stock solution can be used for storing T in vitro for a short timeSCMThe cell can keep the survival rate, proliferation and differentiation of the cell at a higher level, and can be applied to clinical and basic research.
Drawings
Fig. 1 shows a comparison of cell death rates (n-5, mean ± SD) for each group.
FIG. 2 shows the morphological observation (. times.10) of the cells of each group. A is freshly cultured TSCMA cell; b is a cell preserved by the self-made preservation solution; c is a cell preserved by a common culture solution.
FIG. 3 is TSCMAnd (4) detecting the phenotype of the cells. T isSCMThe circle gate strategy of (1). Lymphocytes were first trapped by forward scattered light and side scattered light, labeled with CD45RA + CCR7+ and CD95SCM. A is a flow chart representing the example of FIG. 1, with the ordinate having the first column for side scattered light (SSC), the second column for CCR7 (representing chemokine receptor), and the third column for CD95 (representing inhibitor of apoptosis); the abscissa shows forward scattered light (FSC), CD45RA, and FSC from left to right. B is TSCMData analysis of phenotype positive cell ratios (n-5, mean ± SD).
FIG. 4 is a comparison of the proliferation potency of three groups of cells. A is a flow chart representing the example of FIG. 1, the ordinate is the number of cells, and the abscissa is CFSE (representing the number of generations of cell proliferation); b is a data analysis chart of the ratio of cells in which proliferation occurs (n ═ 5, mean ± SD).
FIG. 5 is a comparison of THP-1 killing ability of effector cells differentiated from three groups of cells. A represents the flow chart 1 example, the ordinate is PI, and the abscissa is forward scattered light (FSC); b is the ratio of killed THP-1 cells, (n ═ 5, mean ± SD).
FIG. 6 is a graph of TWS119 and rapamycin versus T at various concentrationsSCMThe influence of cell frequency and absolute value.
FIG. 7 shows the results of different concentrations of cytokines (IL-7 and IL-15) for TSCMThe effect of cell preservation.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The experiments in the embodiment of the invention are all provided with three repetitions, and data are processed and analyzed by SPSS21.0 statistical software.
The sources of the reagents in the examples of the invention are as follows:
sources of relevant antibodies:
example one, T memory Stem cells (T)SCM) Preparation, storage and characterization of
T memory stem cells (T)SCM) Preparation and storage of
1.1. Preparation of peripheral blood-derived TSCM
On day 0, 20mL of peripheral blood was collected from healthy subjects (the experimental contents were all reported to volunteers and signed with informed consent, afterExperiments were performed after approval by the ethics committee of the new country medical college). Heparin is added into peripheral blood for anticoagulation to obtain anticoagulation, and human peripheral blood mononuclear cells are separated by using a density gradient centrifugation method. Adding PBS with the same volume into the anticoagulated blood, and uniformly mixing to obtain diluted blood; taking another test tube, adding a lymphocyte separation solution with the same volume as the diluted blood: sucking diluted blood, slowly adding lymphocyte separation liquid along the tube wall, centrifuging at 2500rpm at room temperature for 25 min, taking out the centrifuged liquid, sucking white layer mononuclear lymphocyte, washing with PBS for three times, adding 10% human serum + RPMI-1640 (liquid obtained by adding human serum to 10% of human serum volume content into RPMI-1640 cell culture medium), and placing in cell culture box at 37 deg.C with 5% CO2Incubated under conditions overnight.
On day 1, removing adherent cells, collecting suspended cells, namely human peripheral blood lymphocytes, counting, adding microspheres coated with CD3/CD28 antibodies, wherein the ratio of the microspheres to the cells is 1: 5, adding inhibitor TWS119 to TWS119 content of 5 μmol/L, placing in cell culture box at 37 deg.C and 5% CO2Culturing under the condition.
On day 6, recombinant human IL-7 (hereinafter referred to as IL-7) and recombinant human IL-15 (hereinafter referred to as IL-15) were added to the cells until the contents of both were 20ng/mL, and the cells were further placed in a cell culture incubator at 37 ℃ with 5% CO2Culturing under the condition, half-amount changing the cell every other day, half-amount changing the TWS119, and full-amount changing the cytokine IL-7 and IL-15 to obtain the amplified TSCMA cell.
The specific operation steps are as follows:
TWS119 half-amount liquid change: at a position close to the wall of the culture dish, half of the culture solution is aspirated, then the same half volume of fresh 10% human serum + RPMI-1640 culture solution is added, and then TWS119 is added to make the working concentration reach 2.5 mu mol/L (usually half of the initial addition);
total exchange of cytokine IL-7 and IL-15: IL-7 and IL-15 were added in the same amount as the initial amount (since cytokine effect disappeared in an environment at 37 ℃ for 3-4 days), and the final concentrations were 20 ng/mL.
1.2 TSCMStorage of cells
On day 14, the expanded T cells were preserved with home-made preservation solution or ordinary cell culture solution, respectivelySCMA cell.
The self-made preservation solution in the invention is storage TSCMComposition of cells prepared TSCMAnd (4) storing the cells. Wherein the invention is used for storing TSCMThe cellular composition consists of the GSK-3 beta inhibitors TWS119, IL-7 and IL-15. The proportions of TWS119, IL-7 and IL-15 are TWS 1192 nmol, IL-75 ng and IL-155 ng.
T of the inventionSCMThe cell stock solution consists of GSK-3 beta inhibitors TWS119, IL-7, IL-15 and human serum and RPMI-1640 culture solution. T of the inventionSCMIn the cell stock solution, the proportions of the GSK-3 beta inhibitors TWS119, IL-7 and IL-15, human serum and RPMI-1640 culture solution are TWS 1192 nmol, IL-75 ng, IL-155 ng, 1mL (10%) of human serum and 9mL (90%) of RPMI-1640 culture solution. The components of the composition may be packaged separately or may be stored in admixture.
Adding 1mL of human serum into 9mL of RPMI-1640 culture solution to obtain 10% human serum-containing RPMI-1640 culture solution, respectively adding 10 uL of 2mmol/L TWS119 into 10mL of 10% human serum-containing RPMI-1640 culture solution to make the working concentration reach 2nmol/L, adding 10 uL of 5mg/mL IL-7 to make the working concentration reach 5ng/mL, adding 10 uL of 5ng/mL IL-15 to make the working concentration reach 5ng/mL, so as to obtain the invented homemade TSCM10mL of cell stock solution. Per ml of homemade T of the inventionSCMThe cell stock solution contains: human serum 100 μ L, RPMI-1640 culture medium 900 μ L, IL-75 ng, IL-155 ng, TWS 1192 nmol.
The common preservation solution is 10% human serum + RPMI-1640: human serum was added to RPMI-1640 cell culture medium to a volume content of 10% of human serum.
The storage step is as follows: in a cell culture incubator (37 ℃, 5% CO)2) The culture medium TWS119 half amount liquid change and the cytokine IL-7 and IL-15 full amount liquid change are carried out at intervals of 3-4 days, and the culture medium TWS119 half amount liquid change and the cytokine IL-7 and IL-15 full amount liquid change are taken out for detection after 30 days.
2. Detecting TSCMCharacterization of cells
Respectively take out of the product prepared in step 1T preserved by self-made preservation solution and prepared for 30 days in vitroSCMCell and general cell culture solution preserved TSCMCell, assay TSCMChange in cell death rate, proliferation ability and phenotype and killing ability of effector cells differentiated from the same, and freshly prepared cultured T cells are takenSCMCells were completed as positive controls.
2.1 TSCMObservation of cell death rate: freshly prepared and cultured cells and cells preserved in the home-made cell preservation solution and the ordinary culture solution after 30 days were taken, respectively stained with trypan blue, counted under a microscope, and the cell death rates of the respective groups were compared. Results are shown in FIG. 1, fresh preparation of TSCMThe death rate of the cells (shown in FIG. 1 as fresh cell group) was lowest, and the self-made preservation solution group TSCMCell mortality was lower than in the normal cell culture broth group.
2.2 TSCMAnd (3) observing cell morphology: after removing dead cells by centrifugation, the cells were observed under an inverted microscope and cultured with fresh TSCMCells were control. As a result, as shown in FIG. 2, a self-prepared preservation solution set T was preparedSCMCells (B in FIG. 2), general Medium group TSCMCells (C in FIG. 2) and fresh culture group TSCMThe cells (A in FIG. 2) were not morphologically distinct and were in suspension, round, and shiny.
2.3 TSCMDetection of cell phenotype: three groups of cells are respectively taken, mouse anti-human fluorescent antibodies CD45RA, CCR7 and CD95 are stained, incubated for 30 minutes in a dark place, washed by PBS and then put on a computer for detection. High-expression T of both self-made cell preservation solution and fresh culture group cellsSCMCell phenotype: can be combined with CCR7, CD45RA and CD95 fluorescent antibodies to generate fluorescence (figure 3), and only (52.63 +/-3.24)% of common culture solution group cells express TSCMCell phenotype (as in B of figure 3). FIG. 3A shows homemade T of the present inventionSCMThe cell stock solution can well maintain TSCMThe cell phenotype is not changed, and the common culture solution enables TSCMThe change of cell phenotype is not suitable for the next research and application.
2.4 TSCMDetection of cell proliferation: three groups of cells were labeled with CFSE, and coated CD3/CD2 was added8, stimulating the microspheres of the antibody, wherein the ratio of the microspheres to the cells is 1: 10. after 6 days, each group was subjected to flow-based detection of cell proliferation. CFSE (CFDA-SE) is a cell stain that can fluorescently label living cells, and after entering cells, CFSE can be coupled to cell proteins by binding with intracellular amino groups. During cell division and proliferation, CFSE labeled fluorescence can be equally distributed to two daughter cells, and thus its fluorescence intensity is half that of the parent cell, which can be analyzed by flow cytometry at 490nm excitation light. The results show (FIG. 4), self-made preservation solution set TSCMCell proliferation potency and fresh cell group TSCMThe cell proliferation capacities are not different and are all higher than that of the common culture solution group TSCMCell proliferation capacity (B in FIG. 4).
2.5 TSCMKilling ability of effector cells of cell differentiation: t isSCMCells have stem cell characteristics that can differentiate into other subpopulations of memory and effector T cells upon antigenic stimulation. Three groups of cells were placed in 24-well plates, and THP-1 cells (GmbH, Cat: CL-0233) labeled with Celltrace Violet were added at a ratio of THP-1 to T cells of 1: three groups of cells were observed for killing of THP-1. After 12 hours, three groups of cells were stained with PI (propidium iodide) and their differentiated effector cells were examined for killing by flow cytometry (fig. 5). The self-made preservation solution group is composed ofSCMThe THP-1 cell killing ability of effector cells differentiated from the cells was indistinguishable from that of the fresh cell group and was higher than that of the normal culture medium group (B in FIG. 5).
T provided by the inventionSCMCan store T in vitro for a short period of timeSCMThe cells keep high survival rate, proliferation and differentiation, provide experimental basis for clinical application and have wide application prospect.
EXAMPLE II screening of other differentiation inhibitors
1. Other differentiation inhibitors on TSCMEffect of cell storage
Preparation of rapamycin-containing stock solution: adding 1mL of human serum into 9mL of RPMI-1640 culture solution to obtain 10% human serum-containing RPMI-1640 culture solution, respectively adding different amounts of rapamycin into 10mL of 10% human serum-containing RPMI-1640 culture solution to obtain different concentrations (100ng/mL, 200ng/mL, 500ng/mL and 1000ng/mL) of rapamycin-containing stock solutions, respectively adding 10 μ L of 5mg/mL IL-7 into the stock solutions to make the working concentration reach 5ng/mL, and adding 10 μ L of 5mg/mL IL-15 to make the working concentration reach 5 ng/mL.
Also according to the method of step 1.2 of example I, the self-made T containing TWS119 of different concentrations (1. mu. mol/L, 2. mu. mol/L, 4. mu. mol/L, 6. mu. mol/L)SCMAnd (4) storing the cells.
Rapamycin-containing stock solution and TWS 119-containing T prepared by the inventionSCMComparison of the storage effect of the cell stock solution:
TSCMpreparation and storage of cells in the same manner as in example one, T was assayed 30 days after storageSCMCellular characteristics, results are shown in FIG. 6, low concentrations of rapamycin induced TSCMThe cell frequency is reduced, and the high concentration of rapamycin can enable TSCMIncreased cell frequency (B in fig. 6); morphological observation of T in stock solutions containing rapamycin at various concentrationsSCMCell death is high, TSCMThe absolute value of the number of cells is lower than that of TWS119 containing 2 mu mol/LSCMCell stock (C, D in FIG. 6).
Concentration screening of TWS119
By analyzing different TWS119 concentrations in stock solutions versus TSCMCell storage Effect (including T)SCMCell frequency and number morphology) found as TWS119 concentration increased, TSCMThe frequency of cells increased (a in fig. 6), but the frequency of cell death observed under the mirror also increased. The results of viable cell count showed that TWS119 of 2. mu. mol/L can induce T in a short period of timeSCMThe cells were maintained at a good level at the same time (C in FIG. 6).
3. Screening for cytokine concentration
The present invention has explored the possibility of varying concentrations of cytokines (IL-7 and IL-15) for T, with other factors being constantSCMThe results of the cellular effects show that T is increased with increasing cytokine concentrationSCMFrequency of cellsIncrease, but T after cytokine concentration increased to 5ng/mLSCMThe cell frequency is in a steady state, and the cytokine concentration of 5ng/mL can ensure TSCMThe cells maintained a higher frequency (FIG. 7), and finally a cytokine concentration of 5ng/mL (IL-7 and IL-15) was selected to maintain TSCMViability of cells stored in vitro.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A composition for in vitro preservation of T memory stem cells, the active ingredients of the composition consisting of TWS119, IL-7, IL-15; in the composition, the proportion of TWS119, IL-7 and IL-15 is TWS 1191.5-3 nmol, IL-73-10 ng and IL-153-10 ng.
2. The composition of claim 1, wherein: in the composition, the proportion of TWS119, IL-7 and IL-15 is TWS 1191.5-3 nmol, IL-73-8 ng and IL-153-8 ng.
3. The composition of claim 2, wherein: in the composition, the proportion of TWS119, IL-7 and IL-15 is TWS 1192 nmol, IL-75 ng and IL-155 ng.
4. The composition according to any one of claims 1-3, characterized in that: the composition also comprises human serum and RPMI-1640 culture solution.
5. The composition according to any one of claims 1 to 4, wherein: in the composition, the serum is human serum; the IL-7 is recombinant human IL-7; the IL-15 is recombinant human IL-15.
6. A reagent or kit comprising the composition of any one of claims 1-5 for storing T memory stem cells.
7. A method for in vitro preservation of T memory stem cells comprising storing T memory stem cells with a storage solution comprising the composition of any one of claims 1-5.
8. The method of claim 7, wherein: the time for storing the T memory stem cells using the stock solution was 30 days.
9. Any one of the following applications:
use of P1, a composition according to any one of claims 1 to 5 and/or a reagent or kit according to claim 6 and/or a method according to any one of claims 7 to 8 for the storage of a T memory stem cell product;
use of P2, a composition according to any one of claims 1 to 5 and/or a reagent or kit according to claim 6 and/or a method according to any one of claims 7 to 8 in the manufacture of a product for maintaining the biological activity of T memory stem cells in vitro;
use of P3, a composition according to any one of claims 1 to 5 and/or a reagent or kit according to claim 6 and/or a method according to any one of claims 7 to 8 in the manufacture of a product for maintaining the phenotype of T memory stem cells in vitro;
use of P4, a composition according to any one of claims 1 to 5 and/or a reagent or kit according to claim 6 and/or a method according to any one of claims 7 to 8 for the manufacture of a product for improving the proliferative status of T memory stem cells in vitro;
use of P5, a composition according to any one of claims 1 to 5 and/or a reagent or kit according to claim 6 and/or a method according to any one of claims 7 to 8 for the preparation of a product for maintaining the killing ability of effector cells differentiated from T memory stem cells in vitro.
10. T memory stem cells stored by the method of any one of claims 7-8.
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