Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment

J Immunother. 2012 Apr;35(3):283-92. doi: 10.1097/CJI.0b013e31824e801f.


Adoptive cell therapy of metastatic melanoma with autologous tumor infiltrating lymphocytes (TIL) is clinically effective, but TIL production can be challenging. Here we describe a simplified method for initial TIL culture and rapid expansion in gas-permeable flasks. TIL were initially cultured from tumor digests and fragments in 40 mL capacity flasks with a 10 cm² gas-permeable silicone bottom, G-Rex10. A TIL rapid expansion protocol (REP) was developed using 500 mL capacity flasks with a 100 cm² gas-permeable silicone bottom, G-Rex100. TIL growth was successfully initiated in G-Rex10 flasks from tumor digests from 13 of 14 patients and from tumor fragments in all 11 tumor samples tested. TIL could then be expanded to 8-10×10⁹ cells in a 2-step REP that began by seeding 5×10⁶ TIL into a G-Rex100 flask, followed by expansion at day 7 into 3 G-Rex100 flasks. To obtain the 30-60×10⁹ cells used for patient treatment, we seeded 6 G-Rex100 flasks with 5×10⁶ cells and expanded into 18 G-Rex100 flasks. Large-scale TIL REP in gas-permeable flasks requires approximately 9-10 L of media, about 3-4 times less than other methods. In conclusion, TIL initiation and REP in gas-permeable G-Rex flasks require fewer total vessels, less media, less incubator space, and less labor than initiation and REP in 24-well plates, tissue culture flasks, and bags. TIL culture in G-Rex flasks will facilitate the production of TIL at the numbers required for patient treatment at most cell processing laboratories.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Humans
  • Immunotherapy, Adoptive*
  • Lymphocytes, Tumor-Infiltrating / cytology*
  • Primary Cell Culture / instrumentation*
  • Primary Cell Culture / methods*
  • Primary Cell Culture / standards