title: Cryopreservation of cellular products in a closed-bag system with an incorporated dimethyl sulfoxide-resistant sterile filter outside of cleanroom facilities.
abstract: BACKGROUND
  Manipulations, for example, cryopreservation, of cellular therapeutics carried out in an open system must be performed in a class A environment with surrounding class B environment. To avoid cleanroom facilities, a new closed-bag system with an incorporated dimethyl sulfoxide-resistant sterile filter for cryopreservation of cellular products was evaluated at two different centers.
  
  
  METHODS
  A total of 44 different products (22 buffy coats [BCs] and 22 leukapheresis [LK] products) were split and cryopreserved in parallel in cleanroom facilities (Method I) and with the closed system on the bench of a "normal" laboratory (Method II). Viability analyzed by 7-aminoactinomycin D staining and flow cytometric analysis and sterility of the products were analyzed.
  
  
  RESULTS
  Independent of the cellular source (BC or LK), the median viability of CD45+ cells decreased significantly (p < 0.01) during cryopreservation: namely, in BCs, -15.8 percent with both methods, and in LK products, -5.4 percent with Method I and -4.8 percent with Method II, respectively. CD3+ as well as CD14+ cells exhibited a similar pattern and were also found significantly (p < 0.01) diminished after thawing independent of the handling system. For CD19+ cells, the small decrease of viability was only for the BC group significant (p = 0.027) when the cells had been processed with Method I. No bacterial contamination was detected neither in fresh products nor in products after cryopreservation.
  
  
  CONCLUSIONS
  The closed system for cryopreservation of cellular products appears to be equivalent to cleanroom-based methods regarding cellular integrity and sterility when appropriate quality of sterile filters is assured.
authors.affiliation: Division of Stem Cell Transplantation and Immunotherapy, Second Department of Medicine, Schleswig-Holstein University Hospital, Campus Kiel, Kiel, Germany. a.humpe@med2.uni-kiel.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Humpe
authors.forename: Pal
authors.initials: P
authors.lastname: Jansen
authors.forename: Bernd Karsten
authors.initials: BK
authors.lastname: Tischer
authors.forename: Sabine
authors.initials: S
authors.lastname: Schubert
authors.forename: Christian
authors.initials: C
authors.lastname: Beck
authors.forename: Ilse-Dorothea
authors.initials: ID
authors.lastname: Adamzik
authors.forename: Jens-Holger
authors.initials: JH
authors.lastname: Maas
authors.forename: Alexander
authors.initials: A
authors.lastname: Strate
authors.forename: Martin
authors.initials: M
authors.lastname: Gramatzki
authors.forename: Joachim
authors.initials: J
authors.lastname: Riggert
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D002470:Cell Survival; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods; D004121:Dimethyl Sulfoxide; D006801:Humans; D007937:Leukapheresis
chemical_list: D015703:Antigens, CD; D004121:Dimethyl Sulfoxide