Feeder-dependent culture proven more reliable than FBS

Gibco KnockOut Serum Replacement (SR) – Multi-Species is a serum-free supplement that supports the growth of pluripotent stem cells (PSCs) cultured on fibroblast feeder cells. Designed to directly replace serum in existing protocols, this research-use product uses the same KnockOut Serum Replacement (KSR) formulation that has been trusted by many for almost 20 years in over 2,000 publications.

View protocol    View app note    Transition from FBS

KnockOut SR – Multi-Species offers:

  • A more defined formulation for feeder-dependent culture of mouse, human, and other mammalian PSCs
  • More compact colony morphology compared to serum (Figure 1, Table 1)
  • Better maintenance of undifferentiated PSCs (Figure 2, Table 1)
  • Stable price and supply

Phase contrast images of human and mouse ESC colonies cultured in FBS-based medium or KSR-MS-based medium. Figure 1. PSC morphology in cultures grown using KnockOut SR – Multi-Species vs. FBS. H9 human embryonic stem cells (ESCs) were cultured on mouse embryonic fibroblasts (MEFs) with 20% fetal bovine serum (FBS) or 20% KnockOut SR – Multi-Species. 129S6 mouse ESCs were cultured on MEFs using 15% FBS or 15% KnockOut SR – Multi-Species. The human ESC colonies were thicker and more compact when cultured with KnockOut SR – Multi-Species than with FBS. The mouse ESC colonies cultured with KnockOut SR – Multi-Species were dome-shaped and more compact than with FBS.

Four-panel image series showing mouse and human ESCs cultured in the presence of FBS or KSR - Multi-Species and stained for PSC markers OCT4 and SOX2. Figure 2. PSC marker expression in cultures grown with KnockOut SR – Multi-Species vs. FBS. H9 human ESCs and 129S6 mouse ESCs were grown on MEFs under the same conditions as described in Figure 1. Human ESCs grown using KnockOut SR – Multi-Species exhibit uniformly high Oct4 (red) and Sox2 (green) expression, while those grown using FBS are heterogeneous and show more faint Sox2 staining. Mouse ESCs grown using KnockOut SR – Multi-Species also show higher and more homogeneous expression of Oct4 than those cultured in FBS. DAPI (blue) was used as a nuclear stain. Both cultures showed tighter, more desirable colony morphology when grown with KnockOut SR – Multi-Species.

Transitioning human PSCs from FBS to KnockOut SR – Multi-Species

Human PSCs already in culture can be passaged into medium containing KnockOut SR – Multi-Species using standard passaging protocols.

Illustration showing the basic steps to transition human PSCs from FBS to KnockOut SR - Multi-Species.

Transitioning mouse PSCs from FBS to KnockOut SR – Multi-Species

Mouse PSCs can thawed or passaged directly into medium containing KnockOut SR – Multi-Species using standard thawing or passaging protocols.

Illustration showing the basic steps to transition mouse PSCs from FBS to KnockOut SR - Multi-Species.

Table 1. Comparison of growth characteristics of human and mouse cells grown in serum and in KSR – Multi-Species.

  Species Serum KSR – Multi-Species References
Colony morphology Human Less compact More compact H. Koivisto, 2004; M. Amit, 2000
Colony morphology Mouse Flatter & less compact Dome-shaped & more compact R. Blelloch, 2007; T. Davies, 2012
Growth rate Human Slower Faster H. Koivisto, 2004; J. Inzunza, 2005; H. Skottman, 2006
Unwanted differentiation Human and mouse High Low J. Inzunza, 2005; H. Koivisto, 2004; H. Skottman, 2006; P. Hong-mei, 2006; C. Denning, 2006; G. Guo, 2016
Efficiency of ESC derivation Human and mouse Low Higher P. Hong-mei, 2006; T. Davies, 2012J. Cheng, 2004; V. Bryja, 2006
Clonal efficiency Human and mouse Low Higher M. Amit, 2000; J. Cheng, 2004
Reprogramming Mouse Slower Faster, selectively supports PSCs R. Blelloch, 2007; X. Zhao, 2010; H. Hirai, 2012

Complete media system

Supplement Basal medium Recommended reagent
KnockOut Serum Replacement – Multi-Species KnockOut DMEM or KnockOut DMEM/F-12 bFGF or LIF