This protocol outlines the techniques used to routinely passage hPSCs in the SKI Stem Cell Research Facility at Sloan-Kettering. We prefer to culture our cells on mouse embryo fibroblasts (MEFs) but occasionally grow the cells feeder free for particular applications, such as nucleofection, viral transduction or karyotyping.
Individualization leads to severe cell death in human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). This phenomenon leads to difficulties in handling cells in applications such as passaging, cryopreservation and transfection, and the treatment with ROCK inhibitors has been found to effectively improve cell survival.
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are known to be vulnerable to apoptosis upon various technical manipulation, such as single cell dissociation, freezing and thawing, etc., which hinder their use for clonal isolation in gene transfer, differentiation and FACS cell sorting.
This protocol was developed in the Salk STEM Cell Core to enable researchers to consistently and reproducibly produce reprogrammed iPS cells, the initial idea came via word of mouth reports of its effectiveness to increase the efficiency of viral transduction. It has most commonly been used on retrovirus and lentivirus factors. Initial evaluation of this method showed 5–10× increase in transduction efficiency.
There are a number of methods to expand human pluripotent stem cells (hPSCs) without feeders. I prefer to culture my hPSCs with mouse embryonic fibroblasts (MEFs) but there are occasions when feeder free growth is required (e.g., nucleofection, viral transduction or karyotyping to name a few).
This protocol is used for general maintenance and passaging of hES and iPS cells in a feeder-independent culture system such as mTeSR1/Matrigel. It assumes that the cells are grown in a 6-well plate format.
This protocol is used for general maintenance and passaging of hES and iPS cells grown on MEFs (Feeder-Dependent). It assumes that the cells are grown in a 6-well plate format.
Cryopreservation is a critical step to preserve the integrity of human pluripotent stem cells, however, the recovery after cryopreservation is inefficient with traditional enzymatic methods, such as dispase and collagenase. Due to the technical difficulties of cryopreservation, regular passaging methods are often different from harvest methods used in cryopreservation.
Stem cell research is a rapidly expanding field with the potential to develop therapeutic agents to treat diseases as well as study disease development from early stages. The culture of human pluripotent stem cells shares many of the same protocols as standard mammalian cell culture. However, the successful culture and maintenance of human...