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~T cell activation이 되질 않아 궁금한 점이 있어서 여기에 남깁니다.
현재 miltenyi biotec ？T Cell Activation/Expansion Kit, human를 이용하여
T cell clone을 activation 시킬려고 하는데 잘 안됩니다.
위 제품에 protocol에는 PBMC에 Beads를 처리하여 사용하는데 현재는 T Cell clone에만
처리를 하고 있습니다.
1. T cell clone에만 CD3 / CD28 beads를 처리하여서 잘 안되는 걸까요?? feeder cell이나 PBMC를 조금이나마 같이 처리를 해줘야 하나요??
2. PBMC와 Feeder cell을 이용하여 T Cell clone을 expansion 시키는 방법도 같이 하고 있는데 PBMC에 radiation 처리 할 수 없어서 mitomycin C를 이용 합니다. 15ml tube에 PBMC와 mitomycin C 25ug/ml 농도로 처리후 4시간 37도시에서 incubation 후 centrifuge를 돌리면 PBMC가 서로 엉겨붙어서 pellet 자체가 resuspend를 많이 해도 떨어지지 않아 손실이 굉장히 많습니다. 혹시 MMC를 다르게 처리 해야 하나요?
T cell activation에 대해 잘 아시면 메일을 알려주시면 따로 메일 보내겠습니다
- T cell
- expansion
각 분야 한인연구자와 현업 전문가분들의 답변을 기다립니다.
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답변
신소연님의 답변
2016-01-20- 1
cell 분리관련해서는 magnetic bead 로 분리하시면 원심분리후 엉겨붙는 현상 때문에 어려운 일은 없지 않을까싶구요. (PBMNC가 잘 엉겨붙지는 않는데 fibroblast contam.은 아닌지요.)
expansion관련해서는 아래 논문이 도움되시면 좋겠네요.
Methods Mol Biol. 2016;1371:29-41. doi: 10.1007/978-1-4939-3139-2_3.Expansion of Regulatory T Cells In Vitro and In Vivo by IL-33.
Matta BM1, Turnquist HR2,3.
Author informationAbstract
Thymic-derived, regulatory T cells (Treg) represent a subset of CD4(+) T cells that are required for normal immune homeostasis and suppression of unwanted responses against self-antigens (Ags) that prevent autoimmunity. Their role as immune regulators and potent ability to suppress T cell responses has been the focus of intense investigations aimed at utilizing these cells therapeutically, particularly in the settings of autoimmunity and transplantation. Many methods for expanding Treg have been described; however, efforts to generate large numbers of Treg for use in vivo often compromise their suppressor function or rely on the induction of Treg rather than their expansion. Our recent studies have focused on the barrier tissue-derived cytokine IL-33, a recently described IL-1 family member. IL-33 has emerged as a multifunctional protein, with reported roles in driving potent Type 1 and Type 2 immunity, as well as facilitating profound Treg expansion in vitro and in vivo. IL-33-expanded Treg express the IL-33 receptor (R) ST2, and express classical markers associated with Treg phenotype and suppressor function. They suppress both CD4(+) and CD8(+) T cell proliferation and effector functions in vitro, and Treg expressing ST2 have been identified as important regulators of detrimental immune responses in vivo. In the present chapter, we detail methods for expanding significant numbers of Treg using IL-33 both in vitro and in vivo that may potentially be used to promote/maintain organ transplant tolerance or suppress autoimmunity.