title: Cryopreserved ECP-treated lymphocytes maintain apoptotic response and anti-proliferative effect.
abstract: 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).
  
  
  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).
  
  
  CONCLUSIONS
  Cryopreservation did not impair the apoptotic response or anti-proliferative effect of ECP-treated lymphocytes, thereby demonstrating early feasibility of this approach.
authors.affiliation: Research and Development, Fresenius Kabi USA, Lake Zurich, Illinois.
authors.forename: Katherine
authors.initials: K
authors.lastname: Radwanski
authors.affiliation: Research and Development, Fresenius Kabi USA, Lake Zurich, Illinois.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Heber
authors.affiliation: Research and Development, Fresenius Kabi USA, Lake Zurich, Illinois.
authors.forename: Kyungyoon
authors.initials: K
authors.lastname: Min
mesh_terms: D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis; D001781:Blood Component Removal / Q000379:methods*; D049109:Cell Proliferation; D002469:Cell Separation; D015925:Cryopreservation; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008214:Lymphocytes / Q000166:cytology*; D010718:Phosphatidylserines / Q000737:chemistry; D017893:Photopheresis / Q000379:methods*; D013388:Succinimides / Q000737:chemistry
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D017304:Annexin A5; D005452:Fluoresceins; D010718:Phosphatidylserines; D013388:Succinimides
keywords: cryopreservation; extracorporeal photochemotherapy; photopheresis