TILs (tumor-infiltrating lymphocytes) extraction and culture strategy

1. Experimental preparation

TILs Extraction Culture Kit (abs90045)

Additional reagents and consumables are required

Reagent Arrival Handling:

1) TIL cell expansion medium A and TIL cell activation medium B can be stored at 4 °C for 3 months, and stored at 4 °C after receiving the goods, it is recommended to use them within 1 month, and it is not recommended to store them at -20 °C for a long time, so as to avoid repeated freezing and thawing more than 2 times;

2) Tissue preservation solution G and tissue digestion solution C contain nutrients to maintain cell activity, in order to maintain the activity of reagent nutrients, it is not recommended to store at -20 °C for a long time, and avoid repeated freezing and thawing more than 2 times.

2. Steps and methods:

1 Tissue pretreatment

1.1 Experimental Materials

Buffer F (pre-chilled at 4°C), tissue preservation solution G, sampling tubes, tissue transport box, and ice packs
need to be prepared in advance

1.2 Acquisition and transportation of the organization

● Tissue sampling and transportation is the first and most neglected link of TIL extraction, if the tissue is not properly stored in the early stage, it will lead to problems such as poor cell viability, pollution, and few normal cells, which will reduce the success rate of TIL extraction;

● The tissue should be washed 3-5 times with tissue preservation solution G and buffer F within 30min of leaving the body, the blood on the surface of the tissue should be rinsed, put into the tissue preservation solution G, and the sampling tube should be stored and transported at 4°C cryotemperature (within 72h).

2 TIL cells extracted

2.1 Experimental Materials

Buffer F (4°C), tissue digest C (37°C), separation solution D, separation solution E, TIL expansion medium A, TIL activation medium B (room temperature or 37°C), forceps (10 cm), pointed ophthalmic scissors/scalpel blades, disposable 60 mm Petri dishes, 1.5 mL/15 mL/50 mL centrifuge tubes, 100 μm cell strainers, 3 mL sterile Pasteur pipettes/pipettes, 24-well cell culture plates, and metal ice boxes.

2.2 Organizational Processing

After the collection of materials, it is recommended to store and transport at 2-8°C, and quickly transfer to a clean laboratory for TIL extraction experimental procedures, tissue photography and registration of details.

2.2.1 Tissue cleaning

After the sample tube is sterilized, the tissue is taken out in the ultra-clean table, placed in a Petri dish, buffer F is added, and the cleaning operation is repeated three times or more times with a 3mL pasteurized pipette or 1000μL pipette pipette.

2.2.2 Dissociation and digestion of tissues

Tissue impurities are removed with ophthalmic scissors or scalpel blades, forceps are transferred into a 1.5 mL EP tube, and the tissue is further mechanically dissociated into a volume of approximately 1-3 mm with ophthalmic scissors³The tissue block was transferred to a 15mL EP tube, and 8mL of tissue digest was added to the 37°C shaking for 60min. During the digestion process, the tissue digestion was observed under the microscope every 20 minutes, and a small amount of digestive juice was taken to observe under the microscope, and more single cells were observed for the next step.

2.2.3 Filtering of organizations

Add 3 volumes of buffer F to terminate the digestion, and the digested tissue is filtered through a cell sieve with a 100 μm pore size, and the filtrate is collected.

2.3 TIL cell isolation

In order, 2mL of TIL separation solution D and 4mL of separation solution E were added to a 15mL centrifuge tube, and then 6mL of tissue filtrate was gently added along the wall of the centrifuge tube, and the cells on the interface of the D separation solution were collected at 2000 rpm to collect the cells on the interface of the D separation solution, and the suspension rich in TIL cells was collected in this layer, and centrifuged at 1500 rpm for 10min. The interfacial layer of the separator E is mainly tumor cells. TIL cells were washed 2 times with buffer F to remove the cell separation solution and obtain a TIL cell pellet. If cell activation and expansion are not performed at this time, the appropriate amount of TIL cells can be selected for programmed cryopreservation.

3 TIL cell activation expansion culture (24-well plate as an example)

3.1 Experimental Materials

Buffer F (4°C), TIL Expansion Medium A, TIL Activation Medium B (room temperature or 37°C), 15mL/centrifuge tube, 3mL sterile Pasteur pipette/pipette, and 24-well cell culture plates need to be prepared in advance.

3.2 TIL cell activation

● Count the isolated TIL cells;

● TIL cells were resuspended in TIL Cell Activation Medium B (concentration 200000cell/mL), and 1.5mL of TIL Cell Activation Medium B was added to each well for continuous activation for 48 hours.

3.3 TIL cell expansion

● After activation, slowly aspirate TIL cell activation medium B along the side wall of the cell plate (note: do not aspirate the cells);

● Add 1.5mL of TIL cell expansion medium A to each well for continuous expansion culture;

● On the 4th day of continuous culture (at this time, obvious clumping of T cells can be observed), slowly absorb TIL cell expansion medium A along the side wall of the cell plate, add fresh TIL cell expansion medium A, and continue to culture for 5 days (at this time, a large number of T cells can be observed to be obviously clumping and suspended in the medium);

● Cultured cells can also be identified by flow cytometry.

4 TIL cell passaging (24-well plate as an example)

The T cell clusters suspended in the medium were collected in a 15 mL centrifuge tube, centrifuged at 1000 rpm for 5 min, and the cells were counted (concentration was 200000 cells/mL). TIL Cell Expansion Medium A re-seeded 24-well cell culture plates. Depending on the inoculation density, amplification can be completed in 48-72 hours.

5 TIL cells were cryopreserved

5.1 Experimental Materials

Passaging buffer F (4 °C), TIL cryopreservation solution H (4 °C), 15 mL centrifuge tubes, cell cryopreservation tubes, programmed cooling cassette, pipette gun

5.2 Cryopreservation steps

The T cell mass suspended in the medium was collected in a 15mL centrifuge tube, centrifuged at 1000 rpm for 5min, the cells were counted, TIL cryopreservation solution H (concentration was 5000000cell/mL), and transferred to the cell cryovial for program cooling.

6 TIL cell recovery (24-well plate as an example)

There are two types of recovery of TIL cells, one is that after TIL cells are extracted, they are cryopreserved immediately, and subsequent recovery needs to be activated; One is that TIL cells are activated and expanded and then cryopreserved, and subsequent thawing does not require activation.

6.1 TIL cells are cryopreserved immediately after extraction, and recovery needs to be activated by following the following steps:

6.1.1 Experimental Materials

Buffer F, TIL Cell Expansion Medium A, TI Cell L Activation Medium B, 24-well cell culture plates, 15 mL centrifuge tubes, water bath, 3 mL sterile Pasteur pipette/pipette.

6.1.2 Resuscitation steps

● Take a 15mL centrifuge tube and add 8mL of buffer F for later use;

● Remove the cryopreserved cells from the low temperature environment and quickly thaw them in a 37°C water bath, and gently shake the cryopreservation tube to ensure that the cryopreservation solution is completely thawed in a short time during the thawing process of the water bath. Quickly transfer the thawed T cells to a 15 mL centrifuge tube, gently pipette 6-8 times with a pipette, and centrifuge at 1000 rpm for 5 min to discard the supernatant. The T cell pellet was counted, the appropriate TIL cell activation medium B was added and mixed (concentration 500000cell/mL), and 1.5mL of the mixture was added to each well and observed and cultured in a 24-well cell culture plate for 48 h.

● After activation, slowly aspirate TIL cell activation medium B along the side wall of the cell plate (note: do not aspirate the cells);

● Add 1.5mL of TIL cell expansion medium A to each well for continuous expansion culture;

● On the 4th day of continuous culture (at this time, obvious clumping of T cells can be observed), slowly absorb TIL cell expansion medium A along the side wall of the cell plate, add fresh TIL cell expansion medium A, and continue to culture for 5 days (at this time, a large number of T cells can be observed to be obviously clumping and suspended in the medium);

● Cultured cells can also be identified by flow cytometry.

6.2 TIL cells are cryopreserved after activation and expansion, and subsequent thawing does not require activation, the steps are as follows:

6.2.1 Experimental Materials

Buffer F, TIL Cell Expansion Medium A, 24-well cell culture plates, 15 mL centrifuge tubes, water bath, 3 mL sterile Pasteur pipettes/pipettes

6.2.2 TIL cell recovery culture

● Take a 15mL centrifuge tube and add 8mL of buffer F for later use;

● Remove the cryopreserved cells from the low temperature environment and quickly thaw them in a 37°C water bath, and gently shake the cryopreservation tube to ensure that the cryopreservation solution is completely thawed in a short time during the thawing process of the water bath. Quickly transfer the thawed T cells to a 15 mL centrifuge tube, gently pipette 6-8 times with a pipette, and centrifuge at 1000 rpm for 5 min to discard the supernatant. The T cell pellet was counted, the appropriate TIL cell expansion medium A (concentration 500,000 cells/mL) was added and mixed, and 1.5mL of the mixture was added to each well to observe the culture in a 24-well cell culture plate.

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