Serum replacement with albumin‐associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture

Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2‐ to 10‐fold increase) without any influence on pluripotency. In addition, albumin‐associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009–1016, 2016

[1]  Steven Jupe,et al.  A family of fatty acid binding receptors. , 2005, DNA and cell biology.

[2]  K. Jakobs,et al.  Lysophospholipid receptors: signalling, pharmacology and regulation by lysophospholipid metabolism. , 2007, Biochimica et biophysica acta.

[3]  J. Itskovitz‐Eldor,et al.  Insulin production by human embryonic stem cells. , 2001, Diabetes.

[4]  Anneli Ritala,et al.  Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells. , 2014, Plant biotechnology journal.

[5]  G. Lyons,et al.  The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells. , 2010, Biomaterials.

[6]  P. Burridge,et al.  Improved Human Embryonic Stem Cell Embryoid Body Homogeneity and Cardiomyocyte Differentiation from a Novel V‐96 Plate Aggregation System Highlights Interline Variability , 2007, Stem cells.

[7]  Y. Kishi,et al.  Autotaxin Stabilizes Blood Vessels and Is Required for Embryonic Vasculature by Producing Lysophosphatidic Acid* , 2006, Journal of Biological Chemistry.

[8]  K. Woodhouse,et al.  Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories , 2008, Stem cells.

[9]  B. Staels,et al.  Sorting out the roles of PPAR alpha in energy metabolism and vascular homeostasis. , 2006, The Journal of clinical investigation.

[10]  N. Lambert,et al.  Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells. , 2000, Biochemical and biophysical research communications.

[11]  P. Casey,et al.  Gα12 and Gα13 Negatively Regulate the Adhesive Functions of Cadherin* , 2002, The Journal of Biological Chemistry.

[12]  O. Blaschuk,et al.  Cadherins as novel targets for anti-cancer therapy. , 2009, European journal of pharmacology.

[13]  Philippe Lefebvre,et al.  Sorting out the roles of PPARα in energy metabolism and vascular homeostasis , 2006 .

[14]  Robert Zweigerdt,et al.  Suspension culture of human pluripotent stem cells in controlled, stirred bioreactors. , 2012, Tissue engineering. Part C, Methods.

[15]  L. Pike The challenge of lipid rafts This work was supported by National Institutes of Health grants RO1 GM064491 and R01 GM082824 to LJP. Published, JLR Papers in Press, October 27, 2008. , 2009, Journal of Lipid Research.

[16]  S. Cowley,et al.  Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach , 2014, Journal of visualized experiments : JoVE.

[17]  S. Reuveny,et al.  Agitation can induce differentiation of human pluripotent stem cells in microcarrier cultures. , 2011, Tissue engineering. Part C, Methods.

[18]  J. I. Izpisúa Belmonte,et al.  Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal , 2008, PloS one.

[19]  J. Büchs,et al.  Scaled‐up manufacturing of recombinant antibodies produced by plant cells in a 200‐L orbitally‐shaken disposable bioreactor , 2015, Biotechnology and bioengineering.

[20]  M. Schuldiner,et al.  Selective Ablation of Human Embryonic Stem Cells Expressing a “Suicide” Gene , 2003, Stem cells.

[21]  J. Chun,et al.  Lysophospholipid receptors: signaling and biology. , 2004, Annual review of biochemistry.

[22]  Jongil Ju,et al.  Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies. , 2014, Biomaterials.

[23]  Masayuki Yamato,et al.  Creation of human cardiac cell sheets using pluripotent stem cells. , 2012, Biochemical and biophysical research communications.

[24]  Robert Zweigerdt,et al.  Large scale production of stem cells and their derivatives. , 2009, Advances in biochemical engineering/biotechnology.

[25]  S. Bennett,et al.  Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells , 2012, Cell adhesion & migration.

[26]  Peter W. Zandstra,et al.  Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics. , 2012, Biotechnology and bioengineering.

[27]  Sean P. Palecek,et al.  3-D microwell culture of human embryonic stem cells. , 2006, Biomaterials.

[28]  Michael S Kallos,et al.  Mass Transfer Limitations in Embryoid Bodies during Human Embryonic Stem Cell Differentiation , 2012, Cells Tissues Organs.

[29]  Robert Zweigerdt,et al.  Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control. , 2005, Biotechnology and bioengineering.

[30]  A. Russell,et al.  Abrogation of E‐Cadherin‐Mediated Cell–Cell Contact in Mouse Embryonic Stem Cells Results in Reversible LIF‐Independent Self‐Renewal , 2009, Stem cells.

[31]  H. Baharvand,et al.  Bioprocess development for mass production of size-controlled human pluripotent stem cell aggregates in stirred suspension bioreactor. , 2012, Tissue engineering. Part C, Methods.

[32]  Smadar Cohen,et al.  Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation , 2004, Biotechnology and bioengineering.

[33]  N. Nakatsuji,et al.  A 3D Sphere Culture System Containing Functional Polymers for Large-Scale Human Pluripotent Stem Cell Production , 2014, Stem cell reports.

[34]  S. Yamanaka,et al.  Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells , 2010, The Journal of experimental medicine.

[35]  R. Colman,et al.  Platelet hypersensitivity induced by cholesterol incorporation. , 1975, The Journal of clinical investigation.

[36]  Andrew J Racher,et al.  Antibody production. , 2006, Advanced drug delivery reviews.

[37]  R. A. Cooper Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells. , 1978, Journal of supramolecular structure.

[38]  Y. Sakai,et al.  Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production , 2014, Biotechnology progress.