Safe and effective cryopreservation methods for long-term storage of human-amniotic-fluid-derived stem cells.

Objectives: Stem cells (SCs) can be isolated from amniotic fluid (AF) for a variety of perinatal applications. In view of this, we compared different cryopreservation protocols for these AFSCs.

Methods: We screened seven freezing and thawing protocols using two well-established human AFSC lines: freezing protocol 1 (FP1), 10% dimethyl sulfoxide (DMSO); FP2, 2.5% DMSO, caspase inhibitor, and catalase; FP3, 5% glycerol, caspase inhibitor, and catalase; FP4, sperm freezing medium; FP5, slow-freezing solution; FP6, ethylene glycol, sucrose, and Ficoll 70; and FP7, vitrification solution. Outcome measures were post-thawing cell viability, recovery, doubling time and mesenchymal SC markers. The three best performing protocols were subsequently tested on cells isolated from clinical consecutive freshly harvested AF samples from two fetal medicine units.

Results: Protocols 1, 5, and 6 performed significantly better on well-characterized cell lines. They performed equally well on cell pellets from freshly harvested AF (n = 28).

Conclusions: We identified three suitable cryopreservation protocols because of high cell recovery and unchanged SC characteristics. Given one of these, the slow-freezing solution, is compatible with current good manufacturing practice legislation, it may be ultimately clinically used.