An Optimized Feeder-Free Method for Isolation and Expansion of Functional ILC2s Isolated From Human Peripheral Blood.

Innate lymphoid cells (ILCs) are tissue-resident innate counterparts of T cells. Group 2 ILCs (ILC2s) produce cytokines such as IL-5 and IL-13 in response to alarmins, including IL-25 and IL-33 and play a key role in type 2 immunity. Lately, ILC2s have become a major focus across several research areas, including asthma, allergic lung inflammation, atopic dermatitis, and liver fibrosis. However, ILC2s are rare in human peripheral blood, making their isolation and expansion for functional studies challenging. Existing methods commonly rely on flow-cytometry-based cell sorting, which requires large sample volumes, expensive reagents, and considerable hands-on time. Moreover, in vitro expansion protocols often depend on feeder cell lines and activating alarmins. Here, we present a simple and efficient approach for isolating and expanding human ILC2s from peripheral blood, without the need for flow-cytometry-based cell sorting, feeder cells, or external alarmins. This method yields pure and viable ILC2s from both large and small blood volumes, enabling the use of limited or rare patient samples. Expanded ILC2s responded robustly to alarmin stimulation by proliferating and producing signature cytokines and retained functionality after cryopreservation and reculture. Together, this method provides a user-friendly and cost-effective alternative to current human ILC2 isolation and expansion protocols.