Enzyme-Free Passaging and Generation of Single Cells from Human ES and iPS Cells with Gentle Cell Dissociation Reagent

Traditional methods for passaging human embryonic stem (ES) and induced pluripotent stem (iPS) cell cultures often involve the use of mechanical dissection of undifferentiated colonies under the microscope, or partial enzymatic dissociation of colonies into clumps using collagenase or dispase. The mechanical dissection method is laborious, while extra care must be taken when working with enzymes to ensure that they are inactivated or diluted sufficiently, to prevent reduced attachment and poor survival of clumps in subsequent passages.

With the Gentle Cell Dissociation Reagent (GCDR), these difficulties above are avoided. The GCDR is an enzyme-free, animal component-free, and chemically defined solution which does not require washing/centrifugation steps after clump dissociation, and allows for great expansion (12-40 fold) of human ES and iPS cell cultures. Here are some tips to keep in mind while using GCDR:

  • Incubation time in GCDR: For cultures maintained in mTESR™1, TeSR™-E8™, or TeSR™2 on Matrigel™, incubate in GCDR for 6-8 minutes at room temperature. For cultures maintained in mTESR™1, TeSR™-E8™, or TeSR™2 on Vitronectin XF™, incubate in GCDR for 6 - 12 minutes at room temperature. For cultures maintained in mTeSR™ Plus on Corning® Matrigel®, incubate in GCDR for 8-10 minutes at room temperature. For cultures maintained in mTeSR™ Plus on Vitronectin XF™, incubate in GCDR for 8-12 minutes at room temperature. For other matrices or media, incubation times may vary and likely require optimization. Please note the incubation times may also vary when using different cell lines or other non-enzymatic cell passaging reagents.
  • Once the aggregates are broken up into suitable clumps, there is no need to wash out the GCDR by centrifugation. Instead, replate clumps immediately onto plates coated with appropriate matrices.
  • Test a series of split ratios for the first few passages, in order to determine the optimal passaging density.
For more information, including a detailed protocol passaging human ES and iPS cell cultures using GCDR and the characteristics of these cultures, please refer to the GCDR Technical Bulletin.

In addition to its utility in clump passaging of ES and iPS cell cultures, GCDR can also be used for the generation of a single cell suspension in an enzyme-free fashion. The difference between these two applications lies in the incubation temperature and time used for dissociation. For generating single cells for downstream differentiation experiments, we recommend incubating in GCDR for 8-10 minutes at 37°C (instead of the recommended 6-8 minutes at room temperature). We also recommend including ROCK Inhibitor (Y-27632) when preparing single cell suspensions of ESCs/iPSCs. Single cells generated using GCDR are functional by their successful differentiation into definitive endoderm (DE) using the STEMdiff™ Definitive Endoderm Kit.

If you have questions on how to use GCDR in your system, please contact Technical Support at techsupport@stemcell.com.

You may also be interested in ReLeSR™, another enzyme-free reagent for dissociation and passaging of human pluripotent stem cells (hPSCs) as aggregates. Please refer to this Technical Tip for more details.