Cryopreservation of cellular products in a closed-bag system with an incorporated dimethyl sulfoxide-resistant sterile filter outside of cleanroom facilities.
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.
Humpe
Andreas
A
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
Jansen
Pal
P
Tischer
Bernd Karsten
BK
Schubert
Sabine
S
Beck
Christian
C
Adamzik
Ilse-Dorothea
ID
Maas
Jens-Holger
JH
Strate
Alexander
A
Gramatzki
Martin
M
Riggert
Joachim
J
D015703:Antigens, CD / Q000032:analysis; D002470:Cell Survival; D015925:Cryopreservation / Q000295:instrumentation* / Q000379:methods; D004121:Dimethyl Sulfoxide; D006801:Humans; D007937:Leukapheresis
D015703:Antigens, CD; D004121:Dimethyl Sulfoxide