CN111849892A - In-vitro amplification method and application of glioma-derived Tumor Infiltrating Lymphocytes (TILs) - Google Patents
In-vitro amplification method and application of glioma-derived Tumor Infiltrating Lymphocytes (TILs) Download PDFInfo
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Abstract
The embodiment of the invention discloses an in-vitro amplification method and application of glioma-derived Tumor Infiltrating Lymphocytes (TILs), belonging to the technical field of cell culture. The method comprises the following steps: pre-culturing TIL cells; and culturing and expanding TIL cells. According to the invention, under the condition of not separating glioma infiltrating lymphocytes, TIL cells are directly induced and cultured and then are massively amplified, the operation procedure is simple, the time consumption is short, the amplification quantity and the cell activity of the TIL cells can be obviously improved, and the effect of killing glioma tumor cells is good. In the steps of TIL cell culture and amplification, the low-concentration cytokine composite nutrient medium is adopted, so that the requirement of TIL cell culture amplification is met, and the potential risk of cytokine storm in the subsequent treatment process is reduced.
Description
Technical Field
The embodiment of the invention relates to the technical field of cell culture, in particular to an in-vitro amplification method and application of glioma-derived Tumor Infiltrating Lymphocytes (TILs).
Background
Glioma is the most common primary intracranial tumor originated from brain neuroepithelium, is difficult to cure radically and is easy to relapse. The main treatment methods at present are surgical treatment, radiotherapy, chemotherapy and the like. The 5-year survival of high grade gliomas, especially glioblastoma, is less than 10%.
Tumor Infiltrating Lymphocytes (TILs) are lymphocytes isolated from tumor tissue. The existing TIL cell culture technology is to separate tumor infiltrating lymphocytes from tumor tissues by using a mechanical treatment and/or enzyme digestion method, and add high-dose interleukin-2 (IL-2, 6000U/mL) to carry out in-vitro culture. Studies have shown that IL-2-activated TILs have higher tumoricidal activity and better targeting than LAK from autologous Peripheral Blood Mononuclear Cells (PBMCs).
TIL cells have been used for more than 20 years as a specific immune cell for tumor therapy and have been studied in a variety of tumors. The currently used TIL in vitro amplification technology is to separate TIL cells from tumor tissues and then perform TIL induction culture, and the TIL cells cultured in vitro have been proved to have the anti-tumor effect. However, the existing method for expanding TIL cells needs to separate TIL cells first, which has the defects of slow expansion speed, small number of obtained lymphocytes and the like, and in order to obtain more effector cells quickly, the dosage of cytokines (such as IL-2) is higher, and the patients are easy to generate factor storm in the treatment process, so that the method is not beneficial to treatment and even generates life risk to the patients.
The related report of glioma TIL is not found in the prior art, so that the development of a novel method for efficiently amplifying glioma TIL in vitro is of great significance.
Disclosure of Invention
Therefore, the embodiment of the invention provides an in vitro amplification method of glioma-derived Tumor Infiltrating Lymphocytes (TILs) and application thereof, so as to solve the problems of complicated operation procedures, long time consumption, low activity and high IL-2 usage amount of the existing TIL cell amplification method.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:
according to a first aspect of embodiments of the present invention, there is provided a method for in vitro expansion of glioma-derived Tumor Infiltrating Lymphocytes (TILs), the method comprising the steps of:
(1) pre-culture of TIL cells
The glioma tissue is washed by PBS and cut into 1-3mm3The tissue blocks with different sizes are cultured in pre-culture liquid at 37 deg.C and 5% CO2Culturing in an incubator to fully release PBMC in the tissue mass capillaries;
(2) culture and expansion of TIL cells
The glioma tissues obtained in the step (1) are placed in a TIL cell culture medium I at 37 ℃ and 5% CO2Culturing in an incubator, changing the culture solution once every 2-3 days, adding the TIL cell culture medium II after culturing for 6-8 days, continuing culturing for 6-8 days, centrifuging for 5-8min at the speed of 1000rpm/min of 800-.
Further, the composition of the pre-culture solution is as follows: on the basis of RMPI 1640 medium, 1% P/S (penicillin/streptomycin) was added.
Further, the composition of the TIL cell culture medium i is as follows: on the basis of GMP DC medium, 5% human A/B serum, 1% human platelet lysate, 1% P/S, 2mM L-glutamine, 1 XMEM-Eagle, 1mM Skodium pyrolate, 10mM HEPES, 1 XSeta-mercaptoethanol, 500U/mL IL-2, 50U/mL IL-15, 50U/mL IL-21 were added.
Further, the composition of the TIL cell culture medium ii was as follows: based on GMP DC medium, 10% human A/B serum, 1% human platelet lysate, 1% P/S, 2mM L-glutamine, 1 XMEM-Eagle, 1mM Skovate, 10mM HEPES, 1 XSeta-mercaptoethanol, 1000U/mL IL-2, 100U/mL IL-15, 100U/mL IL-21 were added.
Further, in step (1), glioma tissues were seeded in 100mm cell culture dishes.
Further, in the step (1), the culture time is 30 min.
Further, in step (2), glioma tissues were seeded in 24-well plates, 2 pieces/well.
According to a second aspect of embodiments of the present invention, there is provided the use of Tumour Infiltrating Lymphocytes (TILs) obtained by the in vitro expansion method described above in the treatment of glioma.
The embodiment of the invention has the following advantages:
1. according to the invention, under the condition of not separating glioma infiltrating lymphocytes, TIL cells are directly induced and cultured and then are massively amplified, the operation procedure is simple, the time consumption is short, the amplification quantity and the cell activity of the TIL cells can be obviously improved, and the effect of killing glioma tumor cells is good.
2. In the steps of TIL cell culture and amplification, the low-concentration cytokine composite nutrient medium is adopted, so that the requirement of TIL cell culture amplification is met, and the potential risk of cytokine storm in the subsequent treatment process is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
FIG. 1 is a culture diagram of glioma primary tumor cells of the present invention;
FIG. 2 is a diagrammatic view of a TIL cell culture according to the invention;
FIG. 3 shows the flow cytometry results of TILs of the present invention (including the detection of CD45, CD3, CD4, CD8 content);
FIG. 4 shows the flow cytometry results of TILs of the present invention (including the detection of CD19, CD127, CD4, CD8 levels);
FIG. 5 is a schematic diagram of the process of killing glioma tumor tissue cells by TIL cells of the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Culture of glioma tumor tissue cells
Taking part of glioma tumor tissue, grinding, digesting, removing red blood cells, centrifuging at 800rpm/min for 5min to collect cells, discarding supernatant, and culturing with culture solution at 37 deg.C and 5% CO2And culturing the cells in the incubator. Wherein the culture solution comprises the following components: on the basis of DMEM/F12 medium, 10% FBS, 1% P/S (penicillin/streptomycin), 2mM L-glutamine, 1 XMEM-Eagle, 1mM Sodium pyruvate, 10mM HEPES were added. After 1-3 generations of culture, the cells are digested, centrifuged at 800rpm/min for 5min to collect the cells, resuspended and washed by adding culture medium, and then stored or subjected to the next experiment. The culture results of glioma primary tumor cells are shown in figure 1.
Example 2
In vitro amplification method of glioma Tumor Infiltrating Lymphocytes (TILs)
(1) Pre-culture of TIL cells
The collection tube containing the RMPI 1640 culture medium containing 1% P/S is placed on ice, and glioma tissues collected in an operating room are quickly placed in the collection tube, so that the activity of the tissues can be preserved in the transportation process.
Taking out glioma tissue, washing with 1 × PBS for 3 times, and cutting into 1-3mm3The tissue piece of (4), the tissue piece was seeded on a 100mm cell culture dish containing 1% P/S RMPI 1640 medium and placed in 5% CO2And culturing in an incubator at 37 ℃ for 30min to fully release the PBMC in the capillary vessel of the tissue block.
(2) Culture and expansion of TIL cells
Inoculating the glioma tissue obtained in the step (1) on a 24-well plate containing a TIL cell culture medium I, wherein 2 glioma tissues are inoculated in each well, and the glioma tissues are placed at 37 ℃ and 5% CO2Culturing in an incubator, changing the culture solution once every 2 days, after culturing for 8 days, adding a TIL cell culture medium II, continuing culturing for 8 days, centrifuging at 1000rpm/min for 5min, discarding the supernatant, and suspending and washing the cells by using the culture medium.
Wherein, the composition of the TIL cell culture medium I is as follows: on the basis of GMP DC medium, 5% human A/Bserum, 1% human platelet lysate, 1% P/S, 2mM L-glutamine, 1 XMEM-Eagle, 1mM Skodiuvate, 10mM HEPES, 1 XBeta-mercaptoethanol, 500U/mL IL-2, 50U/mL IL-15, 50U/mL IL-21 were added.
The composition of TIL cell culture medium ii was as follows: based on GMP DC medium, 10% humanA/Bserum, 1% human platelet lysate, 1% P/S, 2mM L-glutamine, 1 XMEM-Eagle, 1mM Smodiumpyrolate, 10mM HEPES, 1 XSeta-mercaptoethanol, 1000U/mLIL-2, 100U/mL IL-15, 100U/mL IL-21 were added.
The results of TIL cell culture are shown in fig. 2.
Example 3
TIL cell composition analysis
Flow cytometry analysis of CD3 in TIL cells released from glioma tissue blocks obtained by the amplification method of example 2+-T，CD4+-T，CD8+-T，CD19+-T, and CD127+T, see FIG. 3, FIG. 4 and Table 1.
TABLE 1TIL cell component content and expansion (including CD4 and CD8)
The results showed that CD8 was present in expanded culture of TIL cells in vitro+The proportion of T cells is at a steady high level, and CD8+The proportion of T cells increases continuously during the slow expansion phase and at a steady high level during the fast expansion phase.
Example 4
In vitro killing activity of TIL cells against glioma tumor tissue cells
TIL cells (referred to as effector cells) cultured in example 2 were collected and mixed with glioma tumor tissue cells (referred to as target cells) cultured in example 1, and the ratio of effector cells to target cells was set at 1:1, 2:1, 5:1, and 10: 1. The killing effect of the TIL cells obtained in example 2 on glioma tumor tissue cells was examined by taking pictures at different time periods (after mixed culture, the shooting test was carried out on days 2,4 and 6, respectively), and the results are shown in fig. 5, which shows that the expanded TIL cells cultured in vitro can kill almost all glioma tumor cells, and the results show that the expanded TIL cells have high killing ability.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for the in vitro expansion of Tumor Infiltrating Lymphocytes (TILs) of glioma origin, comprising the steps of:
(1) pre-culture of TIL cells
The glioma tissue is washed by PBS and cut into 1-3mm3The tissue blocks with different sizes are cultured in pre-culture liquid at 37 deg.C and 5% CO2Culturing in an incubator to fully release PBMC in the tissue mass capillaries;
(2) culture and expansion of TIL cells
The glioma tissues obtained in the step (1) are placed in a TIL cell culture medium I at 37 ℃ and 5% CO2Culturing in an incubator, changing the culture solution once every 2-3 days, after culturing for 6-8 days, adding a TIL cell culture medium II, continuing culturing for 6-8 days, centrifuging at 800-.
2. The in vitro amplification method of claim 1, wherein the composition of the pre-culture fluid is as follows: on the basis of RMPI 1640 medium, 1% P/S was added.
3. The in vitro amplification method of claim 1, wherein said TIL cell culture medium i has the following composition: on the basis of GMP DC medium, 5% humanA/B serum, 1% human platelet lysate, 1% P/S, 2mML-glutamine, 1 XMEM-Eagle, 1mM Sodium pyrolate, 10mM HEPES, 1 XBeta-mercaptoethanol, 500U/mL IL-2, 50U/mL IL-15, 50U/mLIL-21 were added.
4. The in vitro amplification method of claim 1, wherein said TIL cell culture medium ii has the following composition: based on GMP DC medium, 10% humanA/B serum, 1% human platelet lysate, 1% P/S, 2mML-glutamine, 1 XMEM-Eagle, 1mM Sodium pyrolate, 10mM HEPES, 1 XBeta-mercaptoethanol, 1000U/mL IL-2, 100U/mL IL-15, 100U/mLIL-21 were added.
5. The in vitro amplification method of claim 1, wherein in step (1), the glioma tissue is seeded in a 100mm cell culture dish.
6. The in vitro amplification method of claim 1, wherein the culture time in step (1) is 30 min.
7. The in vitro amplification method according to claim 1, wherein in step (2), the glioma tissue is seeded in 24-well plates at 2 blocks/well.
8. Use of Tumor Infiltrating Lymphocytes (TILs) obtained by the in vitro expansion method of claim 1 in glioma treatment.
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| CN113215099A (en) * | 2021-04-28 | 2021-08-06 | 广东康盾创新产业集团股份公司 | TIL cell amplification culture medium and use method thereof |
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