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Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions

Abstract

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4–positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm2, where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

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Figure 1: Representative features of hPS cells culture systems based on passaging in cell aggregates and single-cell suspensions.
Figure 2: Transfer of hPS cells from feeder layer to LN-521 substratum at Step 15.
Figure 3: Bright-field images of living hPS cells plated on LN-521 at different densities at different time points (Step 6 or 9).
Figure 4: Flowchart of rapid passaging of hPS cell in single-cell suspensions at Step 6.
Figure 5: Routine characterization of hPS cells cultured on LN-521.

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Acknowledgements

This work was supported in part by grants from the Swedish Research Council (to K.T. and O.H.), the Knut and Alice Wallenberg Foundation (to K.T.), the AFA Insurance Foundation (to K.T. and O.H.), Söderberg's Foundation (to K.T.), the European Commission (to O.H.) and the National Medical Research Council, Singapore (to K.T.).

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S.R. and L.A. conducted in vitro experiments with the pluripotent cells. S.R., L.A. and O.H. contributed to the planning and design of experiments and to the writing of the manuscript. K.T. planned and designed the project and contributed to the writing of the manuscript.

Corresponding author

Correspondence to Karl Tryggvason.

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Competing interests

K.T. and S.R. are shareholders in BioLamina, AB.

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Rodin, S., Antonsson, L., Hovatta, O. et al. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions. Nat Protoc 9, 2354–2368 (2014). https://doi.org/10.1038/nprot.2014.159

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