Methods of growing tumor infiltrating lymphocytes in gas-permeable containers
a technology of infiltrating lymphocytes and gas-permeable containers, which is applied in the direction of cell culture active agents, drug compositions, antibody medical ingredients, etc., can solve the problems of technical, regulatory, logistical and logistic challenges that are introduced
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example 1
[0059]This example demonstrates that TIL cultured in gas-permeable containers is better than, or at least comparable to, that in a 24-well plate.
[0060]The growth of TIL from tumors using gas permeable flasks with a 40 mL capacity and 10 cm2 gas permeable silicone bottom (G-Rex10, Wilson Wolf Manufacturing Corporation, New Brighton, Minn., USA (providing about 10 cm2 of surface area for growth of the TIL)) or 24-well plates (Corning Corning, N.Y.) was compared. A total of 14 melanoma samples were tested, including 9 freshly prepared tumor digests (Table 1A) and 5 thawed samples from previously frozen tumor digests (Table 1B). TIL from frozen tumor from patient 2653 were not able to be cultured in either the G-Rex10 or 24-well plates. Except for one fresh sample (#3522), the ratio of harvested TIL to initially seeded cells at day 17 to 29 was similar to or better in the G-Rex10 flasks than in the 24-well plate (Table 1A and 1B).
TABLE 1AComparison of initial TIL culture in G-Rex10flask...
example 2
[0063]This example demonstrates that the culture of TIL from tumor fragments in gas permeable flasks produces a greater number of TIL as compared to culture in 24-well plates after 7 to 13 days.
[0064]The growth of TIL from tumor fragments in G-Rex10 flasks or 24-well plates was next compared. For each tumor sample, fragments approximately 1 to 8 mm3 in size were seeded into 24-well plates at 1 piece per well and into G-Rex10 flasks at 5, 10, 20, or 30 pieces per flask. The growth of TIL from 2 lymph nodes and 1 liver metastasis was assessed (Table 2). TIL could be grown from tumor fragments in both gas-permeable flasks and 24-well plates, but after 7 to 13 days greater quantities of TIL were obtained from the G-Rex10 flasks than the wells (Table 2).
TABLE 2Initial TIL culture using tumor fragments in G-Rex10 flasks and 24-well plates.Number of cellsPatient# of TIL PhenotypeNumber TILTIL(% TIL expressing each antigen)and TumorCultureTumor TIL#perViabilityCD3+CD3+SourceVesselFragments*...
example 3
[0066]This example demonstrates the kinetics of TIL growth in gas-permeable flasks.
[0067]In order to assess the kinetics of TIL growth in gas-permeable cultureware, TIL from one patient were cultured in G-Rex100 flasks seeded at a density of 5×106 and 10×106 cells per flask. The cells were counted daily after day 6. On Day 6 the number of cells in the G-Rex100 flask seeded at 5×106 cells was 255×106 cells and at 10×106 cells was 300×106 cells. The quantity of TIL in G-Rex100 flasks seeded at each cell density increased steadily until day 9, but there was little increase in cell counts between days 9 and 10. After 10 days 906×106 cells were harvested from flasks seeded with 5×106 TIL and 1,050×106 cells from flasks seeded with 10×106 TIL. Although TIL expanded well for 9 days, in order to keep the G-Rex100 flask expansion process similar to REP in T-flasks and gas-permeable bags where TIL are transferred from T-flasks to bags on day 7, further studies focused on TIL expansion in the ...
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