Human iPS Cell Dissociation Reagents

Solutions for Pluripotent Stem Cell Passaging

Human induced pluripotent stem cells have the capacity to give rise to differentiated progeny arising from of all 3 germ layers and have been historically grown on MEF feeder cells or ECM substrates such as vitronectin in serum-free media. Common cell dissociation enzymes, such as trypsin, cause unwanted differentiation and cell death of iPSCs. A variety of gentler iPS cell dissociation reagents have been utilized to successfully detach and passage human iPS cells from their substrates while maintaining high cell viabilities and pluripotent phenotypes.

Dispase II: Proven to be a rapid, yet gentle, enzyme for passaging human iPS cells as aggregates in both feeder and feeder-free conditions. The enzyme does not damage cell membranes as much as other enzymatic dissociation reagents. Dispase has been used alongside PluriSTEM™ Human ES/iPS Cell Media to maintain undifferentiated human iPSCs.

Accutase®/Accumax®: A cell detachment solution of proteolytic and collagenolytic enzymes that can be used for the routine detachment of stem cells from standard tissue culture plasticware. When used with rho-associated kinase inhibitor (ROCKi), single cell passaging of human iPSCs with Accutase® reagent can be successfully achieved.

EZ-LiFT™ Stem Cell Passaging Reagent: A proprietary enzyme-free and chemically defined stem cell dissociation reagent that selectively passages only undifferentiated pluripotent stem cells. The reagent eliminates the need for manual colony selection or cell scraping and maintains high cell viability without the need for ROCKi. EZ-LiFT™ reagent can also rescue highly differentiated iPS cell cultures.

Protocol Guide

Table 1. Comparison of various human iPS cell dissociation reagents.
 

  Dispase II Accutase®/Accumax™ EZ-LiFT™ Reagent
Mechanism Enzymatic Enzymatic Non-Enzymatic
Composition Bacteria Derived,
Undefined
Animal Derived,
Undefined
Animal-Free,
Chemically Defined
Passage Method Aggregates Single Cell Small Aggregates
Recommended Split Ratio 1:6* 1:30* 1:30*
Pluripotent Cell Enrichment No No Yes
Cell Survival ++ ++ +++
Plating Consistency Low High High
Lot to Lot Variability Med Low Low
ROCKi Requirement No Yes No
Membrane Protein Damage High Med Low
Cryopreservation (Vials/Well) 1 vial 2 vials 5 vials
Transfection Ready No Yes Yes

* Suggested split ratio. Optimal split ratio must be determined by end user.  
 

Human iPS cell morphology utilizing various stem cell dissociation reagents

Figure 1. Human iPS cell morphology utilizing various stem cell dissociation reagents. A) iPS cell colonies exposed to Dispase II results in the colony edge detachment from the plate, producing a curled appearance (arrows). Colonies are then manually passaged to a new ECM coated plates using cell scraping B) Exposure of iPS cells to Accutase® reagent with ROCKi results in a single cell suspension of cells C) EZ-LiFT™ reagent produces small cell aggregates that can be subsequently passaged at high cell viabilities.

 

EZ-LiFT™ reagent selectively dissociates and passages only undifferentiated human iPS cells

Figure 2. EZ-LiFT™ reagent selectively dissociates and passages only undifferentiated human iPS cells. A) An overgrown and neglected human iPS cell culture with highly differentiated phenotypes (arrows) that should not be passaged). B) Exposure of cells to EZ-LiFT™ reagent for 10 minutes selectively detaches only undifferentiated iPS cells (arrows) and produces small cell aggregates that can be further passaged. C) Homogeneous undifferentiated iPS cell colonies emerge 5-7 days after passaging with EZ-LiFT™ reagent.

 

EZ-LiFT™ reagent can rescue highly differentiated human ES/iPSC cultures

Figure 3. EZ-LiFT™ reagent can rescue highly differentiated human ES/iPSC cultures. A) Human iPS cells derived from PBMCs were allowed to spontaneously differentiate (~60% differentiation) before passaging with EZ-LiFT™ reagent. B) 6 to 8 days after the first passage, the culture contains mostly undifferentiated pluripotent colonies with less than 5% differentiated cells observed. C) By the 2nd passage with EZ-LiFT™ reagent, culture is ~100% undifferentiated and contains only pluripotent stem cells.

Ordering Table

Description Part Number
PluriSTEM™ Dispase-II Solution, 100 mL SCM133
Accutase® Cell Detachment Solution (Sigma) A6964
Accutase® Cell Detachment Solution (EMD Millipore) SCR005
Accutase®XL Lyophilized Accutase, 500 mL SCR104
ESGRO™ Complete Accutase, 100 mL SF006
Accumax® Cell Detachment Solution (Sigma) A7089
Accumax® Cell Detachment Solution (EMD Millipore) SCR006
EZ-LiFT™ Stem Cell Passaging Reagent, 100 mL SCM139-100ML
EZ-LiFT™ Stem Cell Passaging Reagent, 500 mL SCM139-500ML