Efficient automatable and aseptic vitrification of human pluripotent stem cells using bio-safe and chemically defined media

Abstract

Human pluripotent stem (hPS) cells, mainly represented by human embryonic stem (hES) and human induced pluripotent stem (hiPS) cells, are considered as a virtually unlimited material for biomedical research and cell-based therapies. However, their use in the clinics requires efficient and bio-safe handling. Cryopreservation is an obligate key step of storage and transportation, during which the cells undergo extreme physical and chemical conditions prone to alter their viability as well as their biological properties. Human PS cells cryopreservation routinely consists in conventional slow-freezing resulting in poor survival rates mainly due to cell damages due to excessive dehydration and water crystallization. In order to prevent this, several groups have adapted vitrification protocols to hPS cells. Vitrification is a cryopreservation method based on the conversion of a liquid into a glass-like state by an infinite enhancement of its viscosity and without formation of ice crystals. It has been shown that vitrification protocols are more effective than slow freezing (1–3). However, they cannot provide any insurance of biological safety since cells are stored in containers that are predisposed to leakage when plunged into liquid nitrogen (i.e. non aseptic containers). This study presents our newly developed hPS cells cryopreservation method.