Enzyme-free passage of human pluripotent stem cells by controlling divalent cations.
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 Mg(2+) and Ca(2+) 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 Mg(2+) and remained as large colonies in high concentrations of Ca(2+). Using enzyme-free solutions containing Ca(2+) without Mg(2+), 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.
1] Top Runner Incubation Center for Academia-Industry Fusion, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan [2] Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 Japan.
Kiyoshi
K
Ohnuma
Laboratory of Stem Cell Cultures, Department of Disease Bioresources Research, National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Ayaka
A
Fujiki
Laboratory of Stem Cell Cultures, Department of Disease Bioresources Research, National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Kana
K
Yanagihara
Top Runner Incubation Center for Academia-Industry Fusion, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
Saoko
S
Tachikawa
Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 Japan.
Yohei
Y
Hayashi
Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, 5 Ibaraki 305-8562, Japan.
Yuzuru
Y
Ito
Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, 5 Ibaraki 305-8562, Japan.
Yasuko
Y
Onuma
Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 Japan.
Techuan
T
Chan
Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 Japan.
Tatsuo
T
Michiue
Laboratory of Stem Cell Cultures, Department of Disease Bioresources Research, National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Miho K
MK
Furue
1] Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 Japan [2] Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, 5 Ibaraki 305-8562, Japan.
Makoto
M
Asashima
D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D010450:Endopeptidases / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D007621:Karyotyping; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism
D002413:Cations, Divalent; D005353:Fibronectins; D010450:Endopeptidases; C023139:dispase; D008274:Magnesium; D002118:Calcium