CN117683716A

CN117683716A - Method for in vitro amplification of tumor-infiltrating lymphocytes

Info

Publication number: CN117683716A
Application number: CN202311653770.1A
Authority: CN
Inventors: 赵彪希; 张潮; 孙开菊; 孙逗祥; 曹永�; 顾晔星; 隋礼丽
Original assignee: Binoji Shanghai Biotechnology Co ltd
Current assignee: Binoji Shanghai Biotechnology Co ltd
Priority date: 2022-12-08
Filing date: 2023-12-05
Publication date: 2024-03-12

Abstract

The application discloses a method for in vitro amplification of tumor-infiltrating lymphocytes, which comprises the following steps: releasing tumor-infiltrating lymphocytes from tumor tissues, and performing amplification culture on the released tumor-infiltrating lymphocytes; wherein the step of using UW fluid storage to transport tumor-infiltrating lymphocytes from the tumor tissue is further included prior to releasing the tumor tissue. The step of using UW liquid for storage and transportation of the tumor tissue is added before the tumor-infiltrating lymphocytes are released from the tumor tissue, so that the amplification efficiency of the tumor-infiltrating lymphocytes can be remarkably improved, and more tumor-infiltrating lymphocytes can be obtained.

Description

Method for in vitro amplification of tumor-infiltrating lymphocytes

Technical Field

The application belongs to the technical field of biology, and particularly relates to a method for in-vitro amplification of tumor-infiltrating lymphocytes.

Background

Tumor infiltrating lymphocytes (Tumor Infiltrating Lymphocytes, TIL) are cells that infiltrate a large number of T cells within the tumor tissue, and the presence of some of these T cells against tumor-specific antigens is an immune cell that can penetrate into the tumor tissue to kill the tumor. TIL therapy is a method of isolating T cells from tumor tissue, stimulating and expanding them in vitro, and infusing them back into the patient, thereby expanding the immune response and treating primary or secondary tumors.

The number of cells infused with TIL therapy is far greater than CAR-T therapy, and the difficulty of expansion is much more difficult than CAR-T therapy. Second, TIL has naturally homing to tumor tissue once, and cellular properties are more prone to depletion, so how to obtain a large number of active TILs in a short period of time has been a research difficulty in the art.

Disclosure of Invention

The purpose of the application is to provide a method for in vitro amplification of tumor-infiltrating lymphocytes, so that the tumor-infiltrating lymphocytes maintain high amplification efficiency and cell quality.

In a first aspect, the present application provides a method for in vitro expansion of tumor-infiltrating lymphocytes, comprising the steps of:

releasing tumor-infiltrating lymphocytes from tumor tissues, and performing amplification culture on the released tumor-infiltrating lymphocytes;

wherein the step of transporting the tumor tissue using UW fluid is further included prior to releasing tumor-infiltrating lymphocytes from the tumor tissue.

In a second aspect the present application provides tumour infiltrating lymphocytes prepared by the method of the first aspect of the present application.

In a third aspect, the present application provides the use of UW fluid in the in vitro expansion of tumor-infiltrating lymphocytes.

The beneficial effects of this application: the inventors found in the study that by adding a step of transporting tumor tissue with UW fluid before releasing the tumor-infiltrating lymphocytes from the tumor tissue and then performing cell expansion, the expansion efficiency of the tumor-infiltrating lymphocytes is significantly improved, and more tumor-infiltrating lymphocytes are easily obtained.

Drawings

FIG. 1 is a photograph of a tumor tissue patch of example 1.

FIG. 2 is a schematic diagram of TIL confluence gradient classification.

FIG. 3 shows the degree of TIL confluence at 12 days of culture after release of the TIL in the UW liquid storage group in example 1.

FIG. 4 shows the degree of TIL confluence at 12 days of culture following release of TIL in the RPMI-1640 storage group in example 1.

Detailed Description

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will briefly introduce the drawings that are required to be used in the embodiments or the description of the prior art, it is obvious that the drawings in the following description are only one embodiment of the present application, and other embodiments may be obtained according to these drawings to those skilled in the art.

Definition of the definition

As used herein, the terms "a" and "an" and "the" and similar referents refer to the singular and the plural, unless the context clearly dictates otherwise.

As used herein, the terms "about," "substantially" and "similar to" refer to an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on the manner in which the value is measured or determined, or on the limitations of the measurement system.

"tumor infiltrating lymphocytes" or "TILs" herein refer to a population of cells initially obtained as leukocytes, which have left the blood stream of a subject and migrated to a tumor. TILs include, but are not limited to, cd8+ cytotoxic T cells (lymphocytes), th1 and Th17 cd4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. "Primary TIL" is a cell (pre-REP cell) obtained or released directly from a patient tumor tissue sample as described herein, and "secondary TIL" is any population of TILs that has been expanded or proliferated as discussed herein, including but not limited to a cell (REP cell) obtained by rapid expansion culture.

TIL can generally be defined biochemically using cell surface markers, or functionally by its ability to infiltrate tumors and affect treatment. TIL can generally be classified by expressing more than one of the following biomarkers: CD3, CD4, CD8, tcrαβ, CD27, CD28, CD56, CD39, CD69, CCR7, CD45Ra, CD95, PD-1 and CD25. Alternatively, or in addition, the TIL may be functionally defined by its ability to infiltrate a solid tumor after re-infusion into the patient. TIL may also be characterized by potency-e.g., if, for example, the release of Interferon (IFN) is greater than about 50pg/mL, greater than about 100pg/mL, greater than about 150pg/mL, or greater than about 200pg/mL, then TIL may be considered effective.

In a first aspect the present application provides a method of amplifying TIL in vitro comprising the steps of:

releasing TIL from tumor tissue (herein referred to as releasing stage), and amplifying and culturing the released TIL;

wherein the step of transporting the tumor tissue using UW fluid is further included prior to releasing the TIL from the tumor tissue.

UW fluid (the University of Wisconsin solution) was developed by Folkert Belzer, a university of Weisconsin surgeon, and James Southard, a basic scientist, in 1980 and introduced into clinical use in the United states in 1987. The UW solution mimics the intracellular environment similar electrolyte composition (high potassium, low sodium) using a phosphate buffer system; mainly contains raffinose and lactobionic acid as osmotic membrane, and can prevent cell edema; the solution is mainly used for human liver transplantation preservation at present by adding additives for maintaining cell activity, such as hydroxyethyl starch, allopurinol, antioxidant glutathione, adenosine and the like. Prior art culturing TIL cells generally includes the steps of releasing TIL from tumor tissue and amplifying the released TIL, and the inventors found in studies that increasing the step of transporting the tumor tissue with UW fluid prior to releasing TIL from tumor tissue, and contacting tumor tissue with UW fluid for a period of time, significantly increases the efficiency of amplification of TIL in tumor tissue.

The tumor tissue is transported by using UW liquid, and the tumor tissue is contacted with the UW liquid for a period of time, which can be the cleaning of the tumor tissue by using the UW liquid or the storage of the tumor tissue by using the UW liquid before the step of releasing TIL; in some embodiments, the transporting the tumor tissue with UW fluid may be at any stage prior to the release of TIL, e.g., the transporting the tumor tissue with UW fluid directly after the tumor tissue is removed, until the step of releasing TIL begins; alternatively, the tumor tissue may be placed in RPMI-1640 and washed with UW fluid prior to the TIL release step, or preferably stored, etc., without limitation.

The inventors found that the TIL expansion efficiency and cell activity were higher when tumor tissue was stored with UW fluid than when it was not stored at the same time in vitro. In addition, the efficiency of amplification and cellular activity of TIL gradually decrease with increasing tumor time, for example, after 72 hours of tumor ex vivo, the efficiency of amplification and cellular activity of TIL decreases significantly. In some embodiments, the tumor tissue may be transported using UW fluid for a period of time ranging from 5 minutes to 72 hours.

In some embodiments, the tumor tissue may be transported using UW fluid for a period of 2 hours to 60 hours, 6 hours to 48 hours, 12 hours to 36 hours, 16 hours to 36 hours, etc.; more specifically, it may be 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 24 hours, 30 hours, 32 hours, 36 hours, 40 hours, 45 hours, 48 hours, 50 hours, 55 hours, 60 hours, 64 hours, 72 hours, etc., or a time range between any two time points.

In some embodiments, to obtain TIL with higher amplification efficiency and activity, it is preferred that the tumor tissue is transported using UW fluid for a period of 2-48 hours, 6-48 hours, 12-48 hours, 20-48 hours, etc.

In some embodiments, the TIL in the tumor tissue is still capable of maintaining high amplification efficiency and activity for more than 24 hours in the UW fluid.

In some embodiments, the temperature of the UW liquid is 0-25 ℃, preferably 4-10 ℃. The inventors have found that lower temperatures are beneficial for maintaining cell activity and expansion efficiency.

In some embodiments, the TIL may be released from the tumor tissue using different methods, or extracted, such as tissue pellet extraction, enzymatic digestion (e.g., enzymatic digestion single cell suspension), milling (e.g., mechanical tissue slice milling), and the like. Wherein, the different extraction methods all require using sterile scissors to cut tumor tissue into 1-3mm ³ Small blocks. The inventors have found that shearing tumor tissue into small pieces facilitates faster release or extraction of TIL from tumor tissue.

In some embodiments, the release of tumor-infiltrating lymphocytes can be achieved by tissue-pellet extraction, which comprises placing each pellet in, for example, a 24-well plate for culturing, which can be 1-20 days, and the inventors found that too short a culturing time is unfavorable for the sufficient release of TIL cells, too long a time, and the TIL cells cannot be effectively expanded after the release is completed, resulting in cell waste; in some preferred embodiments, the time to release tumor infiltrating lymphocytes using tissue clot extraction is between 5 and 8 days.

In some embodiments, the TIL may be released by tissue pellet extraction using a first medium, such as X-vivo15, AIM-V medium, RPMI-1640 medium, optmizer ^TM Medium or Optmizer ^TM Pro medium. In some embodiments, the first medium may be replaced with any medium that is functionally equivalent. For example, including but not limited to DMEM, FUJIFILM Irvin MHM-C, MEM, DMEM/F12.

In some embodiments, the first medium may be a T cell medium selected from the group consisting of X-vivo15, AIM-V medium,RPMI-1640 medium and Optmizer ^TM Medium or Optmizer ^TM One or more of Pro medium; in some embodiments, the T cell culture medium is RPMI-1640 medium; in some embodiments, the T cell culture medium is an OpTmizer ^TM A culture medium; in some embodiments, the T cell culture medium is an OpTmizer ^TM Pro medium.

In some embodiments, the first medium further comprises one, several or all of 100 to 6000IU/ml IL-2,1 to 10% serum replacement and 1 to 5mM glutamine.

In some embodiments, the first medium may further comprise at least one of IL7, IL15 and/or IL21, 10 to 500. Mu.M optional amino acids, 1 to 50mM sodium pyruvate, and 1 to 10% AB serum in an amount of 1 to 100 ng/ml.

In some embodiments, 10 to 500U/ml of the green streptomycin and/or 10 to 500U/ml of the gentamicin may be further included in the first medium.

In some embodiments, the serum replacement comprises at least one of amino acids, proteins, inorganic salts, ethanolamine, sodium pyruvate, ascorbyl phosphate, glutathione, insulin.

In some embodiments, the first medium may further comprise at least one of 2.5% hepes, 1% l-glutamine, 1% non-essential amino acid, 1% sodium pyruvate.

In this application, the inventors have found that TIL is more rapidly and effectively released after storage via UW fluid.

In some embodiments, the release of TIL may be achieved using enzymatic digestion, which may be at least one of collagenase, hyaluronidase, and DNase, e.g., a combination of collagenase and hyaluronidase may be used for tumor mass digestion.

In some embodiments, the tumor tissue pieces may be ground into a cell suspension using mechanical grinding.

The above are all conventional TIL extraction methods in the art, and the present application is not limited herein.

In some embodiments, the amplifying culturing of the released TIL comprises: slowly amplifying and culturing in a second culture medium; and optionally performing a rapid expansion culture in a third medium.

In some embodiments, the second medium is selected from the group consisting of Optmizer ^TM Culture medium, optmizer ^TM Pro medium, RPMI-1640 medium or X-vivo15 medium. In some embodiments, the second medium may be replaced with any medium that is functionally equivalent. For example, including but not limited to DMEM, FUJIFILM Irvin MHM-C, MEM, DMEM/F12.

In some embodiments, the second medium further comprises one, several or all of 100 to 6000IU/ml IL-2,1 to 10% serum replacement and 1 to 5mM glutamine.

In some embodiments, the second medium may further comprise at least one of IL7, IL15 and/or IL21, 10-500. Mu.M optional amino acids, 1-50 mM sodium pyruvate and 1-10% AB serum at 1-100 ng/ml.

In some embodiments, the second medium may further comprise 10 to 500U/ml of penicillin and/or 10 to 500U/ml of gentamicin.

In some embodiments, the time of slow amplification culture is 2-10 days, preferably 5-8 days.

In some embodiments, for example when the TIL is released using tissue-pellet extraction, the slow expansion culture stage and the release stage may employ exactly the same medium, i.e., the first medium and the second medium may be the same; in other embodiments, the slow expansion culture stage and the release stage may employ different media, i.e., the first and second media may be different.

In some embodiments, the slow expansion culture phase and the release phase may be distinguished by changing the medium (to the same or a different medium); in other embodiments, the slow expansion culture phase and the release phase may also be distinguished without changing the medium, simply by observation of culture time and/or cell status.

In some embodiments, the slow expansion culture phase and the release phase may not require distinction between the two phases by manipulation, e.g., culturing the tumor tissue in a first medium after corresponding treatment, e.g., for 5-10 days, to observe the release of TIL from the tumor tissue, which is the release phase; after, for example, 5-10 days, amplification of the released TIL can be observed, i.e.into the slow amplification incubation phase; wherein the release phase and the slow expansion culture phase are continuous, wherein the first medium and the second medium are the same.

In some embodiments, the total time of the slow amplification incubation period and the release period is 10-28 days.

In some embodiments, for example when the TIL is released using enzymatic digestion or milling, the resulting digested or milled tumor tissue may be directly inoculated into a second medium for slow expansion culture after completion of enzymatic digestion or milling.

In some embodiments, the third medium is selected from AIM-V medium, optmizer ^TM Medium or Optmizer ^TM Pro medium; in some embodiments, the third medium may be replaced with any medium that is functionally equivalent. For example, including but not limited to DMEM, FUJIFILM Irvin MHM-C, MEM, DMEM/F12.

In some embodiments, the third medium further comprises one, several or all of 100 to 6000IU/ml IL-2,1 to 10% serum replacement, 1 to 5mM glutamine, and 10 to 60ng/ml OKT 3.

In some embodiments, the third medium may further comprise at least one of IL7, IL15 and/or IL21, 10 to 500. Mu.M optional amino acids, 1 to 50mM sodium pyruvate, and 1 to 10% AB serum at 1 to 100 ng/ml.

In some embodiments, the third medium may further comprise 10 to 500U/ml of penicillin and/or 10 to 500U/ml of gentamicin.

In some embodiments, the rapid expansion culture further comprises adding antigen presenting cells to the culture system; peripheral Blood Mononuclear Cells (PBMC) are preferred; more preferably irradiated peripheral blood mononuclear cells.

In some embodiments, slow expansion cultured TIL is mixed with irradiated PBMCs and cultured in medium.

In some embodiments, the time of the rapid amplification culture is 2-20 days, preferably 2-14 days.

In some embodiments, the rapid amplification culture may increase the amount of TIL by at least 50-fold, at least 100-fold, at least 200-fold, at least 1000-fold, at least 2000-fold, or at least 5000-fold.

A second aspect of the present application provides a TIL prepared according to the method of the first aspect of the present application. In some embodiments, the proportion of cd3+ T cells in the TIL is increased.

In a third aspect the present application provides the use of UW fluid in the in vitro amplification of TIL.

In some embodiments, the UW fluid is used to soak, wash, preserve, or transport tumor tissue that includes the TIL therein.

In the following examples, various aspects of the present application will be described, which are for illustrative purposes only and do not limit the scope of the present application in any way. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.

Examples

Example 1 Effect of UW fluid and RPMI-1640 storage on ovarian cancer TIL amplification

1. Tissue transport: immersing ovarian tumor tissue in UW liquid; the tumor tissue with the same size is immersed in RPMI-1640, stored at the low temperature of 4 ℃ and transported to a laboratory.

Til release and slow amplification culture (pre-REP): removing necrosis and fibrous tissue in the soaked tumor tissue by using a tumor small block extraction method and using sterilized scissors; then the tumor mass is sheared into 1-3mm ³ Small pieces, as shown in fig. 1. Transferring tumor small pieces into culture plate, adding culture medium, and culturing in 5% carbon dioxide incubator at 37deg.C. The culture medium comprises the following components: t cell culture medium +SR +L-alanyl-L-glutamine +IL-2 (expressed as T cell culture medium in this example).

At 6 days of culture, observing the amplification capability of TIL (divided into four gradients of extremely low, medium and high according to the amplification confluence) by a microscope, wherein a tumor cell confluence gradient classification schematic diagram is shown in FIG. 2; the TIL amplification capacity results for different transport fluid storages are shown in table 1.

TABLE 1 results of the TIL amplification capacities of different transport fluid storage groups

As can be seen from the results in Table 1, the amplification capacity of TIL reached a medium-high level after UW fluid storage, whereas the amplification capacity of TIL was at a low level in the RPMI-1640 storage group, indicating that UW fluid storage was able to increase the amplification capacity of TIL. In addition, in the pre-REP stage, a T cell culture medium is adopted, so that the amplification capability of TIL is improved.

When the culture is carried out for 12 days, in the TIL of the UW liquid storage group, the confluence of 4-hole cells in the T cell culture medium group reaches more than 80 percent (shown in figure 3); whereas the TIL of the RPMI-1640 storage group, the confluency of all the well cells was not 80%, and the results are shown in FIG. 4; further illustrating that UW fluid storage can increase the amplification capacity of TIL.

3. Rapid amplification culture (REP):

pre-REP cells and irradiated PBMC are uniformly mixed and inoculated into a culture plate, a T cell culture medium +SR +L-alanyl-L-glutamine +IL-2+OKT3 is added, and the mixture is placed into an incubator for culture. The cells were collected and counted after culturing for two weeks with liquid changes every 2-3 days, and the results indicate that TIL was able to amplify more than 1000-fold in REP phase.

EXAMPLE 2 UW liquid storage increases the amount of ovarian cancer TIL pre-REP cells

The tumor tissue pellet obtained in example 1 was transferred to a culture plate containing a medium, and subjected to pre-REP culture at 37℃in a 5% carbon dioxide incubator. The culture medium comprises the following components: optmizer ^TM Pro culturebase+SR+L-alanyl-L-Glutamine+IL-2 (in this example, opTmizer) ^TM Pro medium); RPMI-1640 medium+10% AB serum+L-alanyl-L-glutamine+IL-2 (in this example, RPMI-1640 medium); x-vivo15 Medium+SR+L-alanyl-L-glutamine+IL-2 (denoted by X-vivo15 Medium in this example).

TIL was collected on day 14 for cell count analysis. The results are shown in Table 2.

TABLE 2 TIL number after 14 days of culture for different storage groups

Culture medium	UW liquid storage group	RPMI-1640 storage group
			RPMI-1640 medium	5.29E6	2E5
X-vivo15 Medium	12.6E6	14.4E5
			OpTmizer ^TM Pro medium	7.39E6	6.3E5

As can be seen from the results in Table 2, the number of TIL cells in the UW liquid storage group was significantly increased by about 10-fold as compared with the RPMI-1640 storage group by culturing TIL in the same medium.

Example 3 effects of UW fluid and RPMI-1640 storage on renal cancer TIL amplification

Immersing kidney tumor tissue in UW solution; the kidney tumor tissue is immersed in RPMI-1640, stored at low temperature and transported to a laboratory.

Tumor tissue pieces were obtained in the same manner as in example 1, and T cell medium+sr+alanyl-L-glutamine+il-2 (represented by T cell medium in this example), cultured in a 5% carbon dioxide incubator at 37 ℃, and the cell confluency was calculated as the number of pores at 80% and 100% on days 11 to 20, respectively, and the results are shown in table 3, which demonstrate that TIL in kidney tumor tissue stored in UW fluid has a stronger proliferation capacity.

TABLE 3 culture results for different transport fluid storage groups

The porosity is expressed as: on day 20, the cell confluency was a proportion of the number of wells of 100%; porosity = cell confluence 100% of the number of pores per 24×100%.

On day 20, the viability and phenotype of the cells in the above-described hole sites with a porosity of greater than 80% were examined by flow cytometry fluorescence sorting (FACS), and the results are shown in table 4 below.

TABLE 4 cell viability and phenotypic outcome for different transport fluid storage groups

Group of	Cell viability	White blood cells (CD45+)	CD3+
				uW group (n=7)	85.58％	64.82％	86.8％
1640 set (n=4)	90.6％	67.5％	18.04％

As can be seen from Table 4, the proportion of CD3+ T cells in the pre-REP cells obtained after UW fluid storage was significantly increased compared to the RPMI-1640 storage group. The results show that the obtained TIL has higher purity by using kidney tumor tissue after UW liquid storage.

EXAMPLE 4 application of the method of the present application in TIL culture

The method for in vitro amplification of tumor-infiltrating lymphocytes disclosed by the application can be applied to amplification culture of multiple ovarian cancer and renal cancer TIL in our subsequent research.

In this example, 3 cases of ovarian tumor tissue and 3 cases of kidney tumor tissue were selected, respectively, immersed in UW solution, and transported to laboratory after low-temperature preservation.

Tumor tissue pieces were obtained in the same manner as in example 1, and pre-REP and REP stage cultures were performed using T cell medium +SR +L-alanyl-L-glutamine +IL-2, and the harvested TILs were counted and subjected to flow test. The experimental results are shown in table 5 below.

TABLE 5 TIL culture results of ovarian tumor tissue and renal tumor tissue after UW fluid storage

As can be seen from table 5, TIL was amplified using the method of the present application for ovarian tumor tissue and kidney tumor tissue: considerable cell quantity can be obtained in the pre-REP stage, and the cell expansion times can reach more than 1500 times after the REP stage culture, and the CD3+T proportion in the living lymphocyte can be obviously improved. The results show that the method for amplifying tumor-infiltrating lymphocytes in vitro disclosed by the application can successfully obtain ovarian cancer TIL and renal cancer TIL with high amplification factors and high purity.

The foregoing description of the preferred embodiments of the present invention is not intended to limit the invention to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and scope of the invention.

Claims

1. A method of expanding tumor-infiltrating lymphocytes in vitro comprising the steps of:

2. The method of claim 1, wherein the tumor tissue is placed in the UW fluid for a period of time ranging from 5 minutes to 72 hours.

3. The method of claim 1, wherein the UW liquid has a temperature of 0-25 ℃.

4. The method of claim 1, wherein the method of releasing tumor-infiltrating lymphocytes from tumor tissue comprises at least one of tissue-pellet extraction, enzymatic digestion, or milling.

5. The method of claim 4, wherein the tissue-patch extraction method comprises: the tumor tissue pieces are cultured in a first medium for 1-20 days.

6. The method of claim 5, wherein the first medium is selected from the group consisting of X-vivo15, AIM-V medium, RPMI-1640 medium, optmizer ^TM Medium or Optmizer ^TM Pro medium.

7. The method of claim 4, wherein the enzymatic digestion comprises digestion of small pieces of the tumor tissue with collagenase, hyaluronidase, and optionally dnase.

8. The method of claim 1, wherein said expanding the released tumor-infiltrating lymphocytes comprises: slowly amplifying and culturing in a second culture medium; and optionally performing a rapid expansion culture in a third medium.

9. The method of claim 8, wherein the second medium is selected from the group consisting of: optmizer ^TM Culture medium, optmizer ^TM Pro medium, RPMI-1640 medium or X-vivo15 medium.

10. The method of claim 8, wherein the slow expansion culture is performed for a period of 2-10 days.

11. The method of claim 8, wherein the third medium is selected from AIM-V medium, optmizer ^TM Medium or Optmizer ^TM Pro medium.

12. The method of claim 8, wherein the rapid amplification culture is performed for a period of 2-20 days;

preferably, the rapid amplification culture is carried out for a period of 2 to 14 days.

13. Tumor infiltrating lymphocytes prepared according to the method of any one of claims 1-12.

Use of uw fluid in the in vitro expansion of tumor-infiltrating lymphocytes.