Enzyme-free Passage of Human Pluripotent Stem Cells by Controlling Divalent Cations

Abstract

Enzymes used for passaging human pluripotent stem cells (hPSCs) digest cell surface proteins, resulting in cell damage. Moreover, cell dissociation using divalent cation-free solutions causes apoptosis. Here we report that Mg2+ and Ca2+ control cell-fibronectin and cell-cell binding of hPSCs, respectively, under feeder- and serum-free culture conditions without enzyme. The hPSCs were detached from fibronectin-, vitronectin- or laminin-coated dishes in low concentrations of Mg2+ and remained as large colonies in high concentrations of Ca2+. Using enzyme-free solutions containing Ca2+ without Mg2+, we successfully passaged hPSCs as large cell clumps that showed less damage than cells passaged using a divalent cation-free solution or dispase. Under the same conditions, the undifferentiated and early-differentiated cells could also be harvested as a cell sheet without being split off. Our enzyme-free passage of hPSCs under a serum- and feeder-free culture condition reduces cell damage and facilitates easier and safer cultures of hPSCs.

Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach

“…However, because of the longer handling time compared with explant cutting and the possible consequences of the tissue enzymatic dissociation on cell fate, enzymatic dissociation of tissues appears not to be adapted to medicinal manufacturing (19). In addition, tissues and cells exposed to collagenase, an enzyme frequently used to recover HDPCs from the pulp tissue, are considered to be ''more than minimally manipulated'' by the Food and Drug Administration.…”

Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells

“…Finally, we did not evaluate the dissociation methods for their effects on the long-term survivability and maturation of our cells, as this was beyond the scope of our study. Enzymatic dissociation is known to have many effects on survivability and maturation, including such parameters as ability to re-attach post-dissociation, increased apoptosis, increased need for small molecules such as Rho-associated protein kinase (ROCK) inhibitor to prevent apoptosis-induced cell loss, loss of cell-surface antigens, and effects on karyotype stability [1, 2, 6, 7, 20]. Therefore, we caution investigators to further examine the long-term effects of each dissociation method before selecting the one that best suits their needs.…”

“…An obstacle to some of these applications is the fact that they often grow in large clusters that are difficult to dissociate without substantial cell loss, likely from loss of cell-cell contact or disruption of adherent cell processes in this cell population [1, 2]. To avoid this cell loss, multiple practices have been developed to work around having to dissociate these clusters [3-5].…”

“…Mechanical dissociation methods include the use of filters, chopping techniques, microfluidic devices, and various trituration strategies using a variety of pipettes [2-5]. Enzymatic dissociation methods include the application of proteolytic enzymes such as trypsin, TrypLE, dispase, and Accutase, with or without also manipulating ion concentrations [1, 4, 7-10]. We sought to determine which method optimally balanced neural cell cluster dissociation with cell survival by directly comparing a wide range of mechanical, enzymatic, and combination dissociation methods.…”

Imaging-cytometry revealed spatial heterogeneities of marker expression in undifferentiated human pluripotent stem cells

“…Flow-cytometric analysis was performed as described previously (Ohnuma et al 2014). Briefly, all cells were dissociated into single cell suspension using 0.02% ( w / v ) ethylenediaminetetraacetic acid tetrasodium salt dihydrate in PBS and fixed in 4% formaldehyde.…”