Sensitivity of human pluripotent stem cells to insulin precipitation induced by peristaltic pump-based medium circulation: considerations on process development

Controlled large-scale production of human pluripotent stem cells (hPSCs) is indispensable for their envisioned clinical translation. Aiming at advanced process development in suspension culture, the sensitivity of hPSC media to continuous peristaltic pump-based circulation, a well-established technology extensively used in hydraulically-driven bioreactors, was investigated. Unexpectedly, conditioning of low protein media (i.e. E8 and TeSR-E8) in a peristaltic pump circuit induced severe viability loss of hPSCs cultured as aggregates in suspension. Optical, biochemical, and cytological analyses of the media revealed that the applied circulation mode resulted in the reduction of the growth hormone insulin by precipitation of micro-sized particles. Notably, in contrast to insulin depletion, individual withdrawal of other medium protein components (i.e. bFGF, TGFβ1 or transferrin) provoked minor reduction of hPSC viability, if any. Supplementation of the surfactant glycerol or the use of the insulin analogue Aspart did not overcome the issue of insulin precipitation. In contrast, the presence of bovine or human serum albumin (BSA or HSA, respectively) stabilized insulin rescuing its content, possibly by acting as molecular chaperone-like protein, ultimately supporting hPSC maintenance. This study highlights the potential and the requirement of media optimization for automated hPSC processing and has broad implications on media development and bioreactor-based technologies.

[1]  C. R. Thomas,et al.  Effects of shear on proteins in solution , 2011, Biotechnology Letters.

[2]  J. Eckel,et al.  Insulin analogues: action profiles beyond glycaemic control. , 2008, Archives of Physiology and Biochemistry.

[3]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[4]  Amandine Calvet,et al.  Monitoring cell culture media degradation using surface enhanced Raman scattering (SERS) spectroscopy. , 2014, Analytica chimica acta.

[5]  C. Dobson,et al.  Chemical modification of insulin in amyloid fibrils , 2003, Protein science : a publication of the Protein Society.

[6]  J. Clemente,et al.  Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. , 2010, Journal of biotechnology.

[7]  Feng Zhao,et al.  Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development. , 2005, Biotechnology and bioengineering.

[8]  A. Hawe,et al.  Formulation Development for Hydrophobic Therapeutic Proteins , 2007, Pharmaceutical development and technology.

[9]  Melissa A. Kinney,et al.  Hydrodynamic modulation of pluripotent stem cells , 2012, Stem Cell Research & Therapy.

[10]  Ivan Martin,et al.  Three dimensional multi‐cellular muscle‐like tissue engineering in perfusion‐based bioreactors , 2016, Biotechnology and bioengineering.

[11]  A. Klibanov,et al.  Mechanism of insulin aggregation and stabilization in agitated aqueous solutions , 1992, Biotechnology and bioengineering.

[12]  R. McKay,et al.  Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. , 2014, Cell stem cell.

[13]  J. Mirouze,et al.  Stable Insulin for Implantable Delivery Systems: In Vitro Studies With Different Containers and Solvents , 1987, Diabetes Care.

[14]  R. Passier,et al.  Insulin Redirects Differentiation from Cardiogenic Mesoderm and Endoderm to Neuroectoderm in Differentiating Human Embryonic Stem Cells , 2008, Stem cells.

[15]  Fred Jacobson,et al.  Protein aggregation and bioprocessing , 2006, The AAPS Journal.

[16]  Sean C. Bendall,et al.  IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro , 2007, Nature.

[17]  Ying Wang,et al.  Efficient and Scalable Expansion of Human Pluripotent Stem Cells Under Clinically Compliant Settings: A View in 2013 , 2013, Annals of Biomedical Engineering.

[18]  S. Heyner.  Growth factors in preimplantation development: role of insulin and insulin-like growth factors. , 1997, Early pregnancy : biology and medicine : the official journal of the Society for the Investigation of Early Pregnancy.

[19]  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.

[20]  R. Zweigerdt,et al.  Bioreactors for Expansion of Pluripotent Stem Cells and Their Differentiation to Cardiac Cells , 2016 .

[21]  Y. Schneider,et al.  Stem cell bioprocess engineering towards cGMP production and clinical applications , 2014, Cytotechnology.

[22]  L. Langkjaer,et al.  Precipitation of insulin products used for continuous subcutaneous insulin infusion. , 2005, Diabetes technology & therapeutics.

[23]  J. Rossant,et al.  Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells , 2010, In Vitro Cellular & Developmental Biology - Animal.

[24]  Robert Zweigerdt,et al.  Scalable expansion of human pluripotent stem cells in suspension culture , 2011, Nature Protocols.

[25]  Wim Jiskoot,et al.  The Molecular Chaperone α-Crystallin as an Excipient in an Insulin Formulation , 2010, Pharmaceutical Research.

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

[27]  Jun Wu,et al.  Cellular Metabolism and Induced Pluripotency , 2016, Cell.

[28]  Robert Zweigerdt,et al.  Impact of Feeding Strategies on the Scalable Expansion of Human Pluripotent Stem Cells in Single‐Use Stirred Tank Bioreactors , 2016, Stem cells translational medicine.

[29]  Roland Hetzer,et al.  Tissue-engineering bioreactors: a new combined cell-seeding and perfusion system for vascular tissue engineering. , 2002, Tissue engineering.

[30]  Hans R. Schöler,et al.  Small Molecule-Assisted, Line-Independent Maintenance of Human Pluripotent Stem Cells in Defined Conditions , 2012, PloS one.

[31]  Jennifer M. Bolin,et al.  Chemically defined conditions for human iPS cell derivation and culture , 2011, Nature Methods.

[32]  J. Chow,et al.  Physical Stability of Insulin Formulations , 1983, Diabetes.

[33]  V. Uversky,et al.  Effect of environmental factors on the kinetics of insulin fibril formation: elucidation of the molecular mechanism. , 2001, Biochemistry.

[34]  John E. Hambor,et al.  Bioreactor Design and Bioprocess Controls for Industrialized Cell Processing Bioengineering Strategies and Platform Technologies , 2012 .

[35]  J. Brange,et al.  Insulin structure and stability. , 1993, Pharmaceutical biotechnology.

[36]  Robert Zweigerdt,et al.  Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture , 2014, Stem cell reports.

[37]  R. Passier,et al.  NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes , 2011, Nature Methods.

[38]  Yuquan Wei,et al.  Development of a Xeno-Free Substrate for Human Embryonic Stem Cell Growth , 2015, Stem cells international.

[39]  L. Schad,et al.  Characterization of a chip-based bioreactor for three-dimensional cell cultivation via Magnetic Resonance Imaging. , 2013, Zeitschrift für Medizinische Physik.

[40]  D. F. Waugh,et al.  Studies of the Nucleation and Growth Reactions of Selected Types of Insulin Fibrils , 1953 .

[41]  M-Y Chang,et al.  Insulin concentration is critical in culturing human neural stem cells and neurons , 2013, Cell Death and Disease.

[42]  J. Tamada,et al.  Kinetics of insulin aggregation in aqueous solutions upon agitation in the presence of hydrophobic surfaces. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[43]  J. Thomson,et al.  Derivation of human embryonic stem cells in defined conditions , 2006, Nature Biotechnology.

[44]  C. Mummery,et al.  Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. , 2008, Differentiation; research in biological diversity.

[45]  Catarina Brito,et al.  Process engineering of human pluripotent stem cells for clinical application. , 2012, Trends in biotechnology.

[46]  Artur Lichtenberg,et al.  In vitro re-endothelialization of detergent decellularized heart valves under simulated physiological dynamic conditions. , 2006, Biomaterials.

[47]  Umberto Morbiducci,et al.  A Versatile Bioreactor for Dynamic Suspension Cell Culture. Application to the Culture of Cancer Cell Spheroids , 2016, PloS one.

[48]  M. Yap,et al.  FGF‐2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3‐K/GSK3β signaling , 2010, Journal of cellular physiology.

[49]  D. Eibl,et al.  Modification and qualification of a stirred single-use bioreactor for the improved expansion of human mesenchymal stem cells at benchtop scale , 2014 .

[50]  A. Kirschning,et al.  A practical synthesis of Rho-Kinase inhibitor Y-27632 and fluoro derivatives and their evaluation in human pluripotent stem cells. , 2011, Organic & biomolecular chemistry.

[51]  Maria Margarida Diogo,et al.  Stem cell cultivation in bioreactors. , 2011, Biotechnology advances.

[52]  Robert Zweigerdt,et al.  Cardiac differentiation of human pluripotent stem cells in scalable suspension culture , 2015, Nature Protocols.

[53]  Bradford Stanley,et al.  Control of Protein Particle Formation During Ultrafiltration/Diafiltration Through Interfacial Protection , 2014, Journal of pharmaceutical sciences.

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

[55]  R. David,et al.  Directing cardiomyogenic differentiation of human pluripotent stem cells by plasmid-based transient overexpression of cardiac transcription factors. , 2013, Stem cells and development.

[56]  Thomas Scheper,et al.  Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium. , 2010, Stem cell research.

[57]  Diana Nada Caterina Massai,et al.  Progress and challenges in large-scale expansion of human pluripotent stem cells , 2017 .

[58]  R. Pedersen,et al.  Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells , 2005, Journal of Cell Science.

[59]  Diego Gallo,et al.  Bioreactors as engineering support to treat cardiac muscle and vascular disease. , 2013, Journal of healthcare engineering.

[60]  Marc Peschanski,et al.  Improvement of culture conditions of human embryoid bodies using a controlled perfused and dialyzed bioreactor system. , 2008, Tissue engineering. Part C, Methods.

[61]  V. T. G. Chuang,et al.  Recombinant human serum albumin. , 2007, Drugs of today.

[62]  Yonatan Y Lipsitz,et al.  CD24 tracks divergent pluripotent states in mouse and human cells , 2015, Nature Communications.

[63]  P. Blackshear,et al.  Glycerol Prevents Insulin Precipitation and Interruption of Flow in an Implantable Insulin Infusion Pump , 1983, Diabetes Care.

[64]  R Fumero.  Optimization of blood pumping systems. , 1980, Journal of biomechanics.

[65]  U. Mura,et al.  Chaperone-like features of bovine serum albumin: a comparison with α-crystallin , 2005, Cellular and Molecular Life Sciences CMLS.

[66]  A. Del Corso,et al.  Chaperone-like features of bovine serum albumin: a comparison with alpha-crystallin. , 2005, Cellular and molecular life sciences : CMLS.

[67]  Geoffrey L. Francis,et al.  Albumin and mammalian cell culture: implications for biotechnology applications , 2010, Cytotechnology.

[68]  Robert Zweigerdt,et al.  Up-scaling single cell-inoculated suspension culture of human embryonic stem cells. , 2010, Stem cell research.

[69]  J. Lebkowski,et al.  Basic Fibroblast Growth Factor Supports Undifferentiated Human Embryonic Stem Cell Growth Without Conditioned Medium , 2005, Stem cells.

[70]  K. Drejer.  The bioactivity of insulin analogues from in vitro receptor binding to in vivo glucose uptake. , 1992, Diabetes/metabolism reviews.

[71]  Chih-Ming Ho,et al.  An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells. , 2011, Nature communications.

[72]  W Wang,et al.  Instability, stabilization, and formulation of liquid protein pharmaceuticals. , 1999, International journal of pharmaceutics.

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

[74]  QingYun Mai,et al.  Comparison of a xeno-free and serum-free culture system for human embryonic stem cells with conventional culture systems , 2016, Stem Cell Research & Therapy.

[75]  J. Thomson,et al.  Basic Fibroblast Growth Factor Support of Human Embryonic Stem Cell Self‐Renewal , 2006, Stem cells.

[76]  R. Weissmann,et al.  Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells* , 2014, Molecular & Cellular Proteomics.

[77]  A. Nunez,et al.  Serum Albumin Prevents Protein Aggregation and Amyloid Formation and Retains Chaperone-like Activity in the Presence of Physiological Ligands , 2012, The Journal of Biological Chemistry.

[78]  Shinya Yamanaka,et al.  Elite and stochastic models for induced pluripotent stem cell generation , 2009, Nature.

[79]  Roberto Fumero,et al.  OPTIMISTISATION OF BLOOD PUMPING SYSTEMS , 1980 .

[80]  Angelique M. Nelson,et al.  Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB 2 receptor signaling , 2007 .

[81]  Robert Zweigerdt,et al.  Large-scale production of human pluripotent stem cell derived cardiomyocytes. , 2016, Advanced drug delivery reviews.