Min Lu1, Naomi Guyette1, Nick Asbrock1, Boris Greber2, Vi Chu1
1MilliporeSigma, Cellular Assays Biological Reagents & Kits, Temecula, CA, USA., 2Max Planck Institute for Molecular Biomedicine, Münster, Germany
Introduction to Stem Cell Culture in PluriSTEM™ Media
Methods for Stem Cell Culture in PluriSTEM™ Media
Preparation of Coated Plates for Stem Cell Culture
Preparation of Dispase II for Stem Cell Culture
Transition of Human ES/iPS Cells to PluriSTEM™ Media
Single Cell Passaging of Human ES/iPSCs in PluriSTEM™ Media
Results for Culture of Stem Cells in PluriSTEM™ Media
Materials for Culture of Stem Cells in PluriSTEM™ Media
References
Current serum-free media formulations used to culture human pluripotent stem cells require daily media replenishment and at least one media exchange on the weekend. The costs in media, reagents, and human resources become prohibitively expensive as increasing numbers of core labs and consortia are focused on generating disease models using human iPS cells. PluriSTEM™ Human ES/iPS Cell Media are small molecule-based media that enable weekend-free culture of human pluripotent stem cells and allow for media exchanges every other day without compromising the morphology or long-term functionality of pluripotent stem cells. Pluripotent cells maintained in this moderate feeding regimen exhibited robust cell health with minimal spontaneous differentiation, expressed high levels of pluripotency markers (Nanog, Oct-4, Sox-2, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), retained differentiation potential, and possessed a normal karyotype.
Figure 1. How does PluriSTEM™ media work?Human pluripotent stem cells grown in PluriSTEM™ media retain pluripotency and demonstrate robust growth rates via two mechanisms: 1) bFGF, TGFβ1 and Activin A promote stem cell self-renewal and 2) small molecule inhibitors suppress unwanted spontaneous extraembryonic and mesodermal differentiation pathways.
Expansion of pluripotent human ES and iPS cells with PluriSTEM™ media requires cultureware that are coated with ECM Gel Matrix (CC131) or xeno-free substrates such as Vitronectin (CC130) or ECMatrix™-511 Laminin Substrates. Below are general guidelines for the coating of 6-well plates and culture flasks with ECM Gel Matrix:
Pluripotent human ES and iPS cells maintained in PluriSTEM™ media may be enzymatically passaged using Dispase II protease. PluriSTEM™ Dispase II Solution (SCM133) is a ready-to-use 1 mg/mL solution validated to work alongside PluriSTEM™ Human ES/iPS Media for the culture and passage of human embryonic and induced pluripotent stem cells.
(Applicable for both Feeder-Based and Feeder-Free Media Systems)
Figure 2. Time course of pluripotent stem cell expansion in PluriSTEM™ media.Human iPS cells expanded over a 6-day period in feeder-free conditions are ready to be passaged at day 6 when cells are 80% confluent.
Figure 3. Pluripotent stem cells cultured in PluriSTEM™ media retain pluripotency markers.H9 human ES cells were maintained using an every-other-day/no weekend feeding schedule in PluriSTEM™ media for over 20 passages. Cells retained characteristic pluripotent morphology (i.e. homogeneous round colonies with defined borders) and high expression levels of alkaline phosphatase plus pluripotency markers Oct-4, Nanog, Sox-2, TRA-1-60, TRA-1-81, SSEA-4. Cells demonstrated an absence of staining for SSEA-1.
Figure 4. Karyotyping of pluripotent stem cells cultured in PluriSTEM™ media. H9 human ES cells maintained using an every-other-day/no weekend feeding schedule in PluriSTEM™ media for 9 and 20 passages have normal female karyotypes.
Figure 5. Human iPS cells cultured in PluriSTEM™ media vs. mTeSR1® media.Similar pluripotent morphologies were observed after 3 passages in both mTesr1® and PluriSTEM™ media.
Figure 6. In vivo differentiation of pluripotent stem cells cultured in PluriSTEM™ media.Human pluripotent stem cells cultured in PluriSTEM™ media for >20 passages differentiate in vivo to expected germ layers. H&E stained teratoma sections courtesy of Dr. Boris Greber.
Figure 7. In vitro differentiation of pluripotent stem cells cultured in PluriSTEM™ media.Human pluripotent stem cells cultured in PluriSTEM™ media for >20 passages In vitro differentiate to all 3 germ layers (mesoderm, endoderm and ectoderm) using an embryoid body differentiation protocol.
Figure 8. PluriSTEM™ media supports single cell passaging of pluripotent stem cells In vitro.H9 hESCs cultured in PluriSTEM™ media for 22 passages were dissociated into single cells using Accumax™ reagent. One hour before dissociation, 10 μM ROCK Inhibitor was added. 100,000 dissociated cells were seeded into each well of a coated 6-well-plate. Unlike cells cultured in mTesr1® media, single cells are highly viable in PluriSTEM™ media and continue to form pluripotent colonies after three rounds of single cell passaging using Accumax™ reagent.
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