Investigation of the influence of cell density of human fibroblasts cryopreserved inside collagen sponges at various cooling rates.
The influence of cell density of cells cryopreserved inside a collagen matrix at various cooling rates was investigated. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 10(5) to 10(7) cells/cm(3). Four artificial tissues were first stacked in a test chamber, frozen at a cooling rate of 0.3 to 50 degrees C/min in a solution of Dulbecco's Modified Eagle Medium, 20% fetal bovine serum and 10% dimethylsulfoxide, kept frozen below -185 degrees C for 2 hours, and then finally thawed. Membrane integrity of fibroblasts using a trypan blue exclusion assay was evaluated as an index for post-thaw cellular viability. Results show that with increasing cell density, the post-thaw membrane integrity decreased. Therefore, in the cryopreservation of biological tissue, it seems high cell density is one factor which causes a decline in viability.
Matsumura
Yumi
Y
Graduate School of Medical Sciences. Kitasato University, 1-15-1 Kitazato Sagamihara-shi, Kanagawa, Japan.
Ujihira
Masanobu
M
Nogawa
Satoshi
S
Kimura
Keita
K
Ichikawa
Hinako
H
Mabuchi
Kiyoshi
K
D001187:Artificial Organs; D002452:Cell Count; D002470:Cell Survival; D003094:Collagen; D015925:Cryopreservation / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology*; D006801:Humans; D014021:Tissue Preservation / Q000379:methods*
D003094:Collagen