Splitting hPSCs with Dispase

Mark J. Tomishima

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.

Splitting hESC/hiPSC lines with EDTA in feeder-free conditions

Guokai Chen

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.

Splitting hESC/hiPSC lines on MEF using Accutase

Hyesoo Kim

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.

General spinfection

W. Travis Berggren, Margaret Lutz, and Veronica Modesto

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.

Conditioning pluripotent stem cell media with mouse embryonic fibroblasts (MEF-CM)

Mark J. Tomishima

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).

Cryopreservation of human pluripotent stem cells in defined conditions

Guokai Chen

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.

Basic pluripotent stem cell culture protocol

Maria Borowski, Maria Giovino-Dohert, Lan Ji, Meng-Jiao Shi, Kelly P. Smith, Joseph Laning

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...