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Cryopreserved ECP‐treated lymphocytes maintain apoptotic response and anti‐proliferative effect
Author(s) -
Radwanski Katherine,
Heber Cheryl,
Min Kyungyoon
Publication year - 2015
Publication title -
journal of clinical apheresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.697
H-Index - 46
eISSN - 1098-1101
pISSN - 0733-2459
DOI - 10.1002/jca.21354
Subject(s) - cryopreservation , andrology , apoptosis , medicine , phosphatidylserine , apheresis , annexin , peripheral blood mononuclear cell , immunology , flow cytometry , biology , in vitro , embryo , biochemistry , microbiology and biotechnology , platelet , phospholipid , membrane
Background : The ability to cryopreserve a portion of the cells treated during extracorporeal photopheresis (ECP) would improve therapy logistics, particularly for pediatric patients, by allowing multiple therapeutic doses to be collected from a single apheresis session. However, the effect of cryopreservation on ECP‐treated cells is unknown (e.g., ECP‐induced lymphocyte apoptosis and inhibition of proliferation). Study Design and Methods : Mononuclear cell (MNC) apheresis products collected from healthy subjects were ECP‐treated using offline methods. Fresh samples of ECP‐treated and control cells were placed immediately in culture. The remainder of the cells were frozen in cryovials ( n = 8) or cryobags ( n = 8) at −80°C. After 1 week of −80°C storage, ECP‐treated and control cells were thawed rapidly and samples were placed in culture. Lymphocyte apoptosis was assessed by phosphatidylserine exposure using Annexin V/7‐AAD labeling. Lymphocyte proliferation after 3 days culture was measured using the carboxyfluorescein succinimidyl ester labeling technique. Results : On Day 0, apoptosis levels were <5% in fresh ECP‐treated and control cells and approximately 20% on thawing of cryopreserved ECP‐treated and control cells. Apoptosis levels were comparable between the two cryopreserved groups immediately on thawing, indicating that ECP‐treated cells were no more sensitive to the cryopreservation process than control cells. During 72‐h culture, apoptosis levels increased to >80% in fresh and cryopreserved ECP‐treated cells but remained near constant in both control groups. Inhibition of lymphocyte proliferation was >95% in all ECP‐treated cells with no significant difference between fresh and cryopreserved cells ( P = 0.12). Conclusion : Cryopreservation did not impair the apoptotic response or anti‐proliferative effect of ECP‐treated lymphocytes, thereby demonstrating early feasibility of this approach. J. Clin. Apheresis 30:154–161, 2015. © 2014 Wiley Periodicals, Inc.