Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems
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{"title"=>"Defined essential 8′ medium and vitronectin efficiently support scalable xeno-free expansion of human induced pluripotent stem cells in stirred microcarrier culture systems", "type"=>"journal", "authors"=>[{"first_name"=>"Sara M.", "last_name"=>"Badenes", "scopus_author_id"=>"36058384500"}, {"first_name"=>"Tiago G.", "last_name"=>"Fernandes", "scopus_author_id"=>"22934439300"}, {"first_name"=>"Cláudia S.M.", "last_name"=>"Cordeiro", "scopus_author_id"=>"57188695717"}, {"first_name"=>"Shayne", "last_name"=>"Boucher", "scopus_author_id"=>"7005146153"}, {"first_name"=>"David", "last_name"=>"Kuninger", "scopus_author_id"=>"6507946116"}, {"first_name"=>"Mohan C.", "last_name"=>"Vemuri", "scopus_author_id"=>"6604057187"}, {"first_name"=>"Maria Margarida", "last_name"=>"Diogo", "scopus_author_id"=>"6701492791"}, {"first_name"=>"Joaquim M.S.", "last_name"=>"Cabral", "scopus_author_id"=>"7201350203"}], "year"=>2016, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"609296768", "pmid"=>"26999816", "doi"=>"10.1371/journal.pone.0151264", "issn"=>"19326203", "scopus"=>"2-s2.0-84962476807", "sgr"=>"84962476807"}, "id"=>"17645e1c-0c32-3e0f-b631-cfd14f0dc107", "abstract"=>"Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.", "link"=>"http://www.mendeley.com/research/defined-essential-8-medium-vitronectin-efficiently-support-scalable-xenofree-expansion-human-induced", "reader_count"=>63, "reader_count_by_academic_status"=>{"Unspecified"=>3, "Professor > Associate Professor"=>2, "Researcher"=>13, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>18, "Student > Postgraduate"=>1, "Student > Master"=>11, "Other"=>5, "Student > Bachelor"=>6, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>3, "Professor > Associate Professor"=>2, "Researcher"=>13, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>18, "Student > Postgraduate"=>1, "Student > Master"=>11, "Other"=>5, "Student > Bachelor"=>6, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>12, "Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>10, "Materials Science"=>1, "Agricultural and Biological Sciences"=>27, "Medicine and Dentistry"=>3, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Veterinary Science and Veterinary Medicine"=>1, "Chemical Engineering"=>2, "Chemistry"=>2}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>12}, "Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Chemistry"=>{"Chemistry"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>27}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>10}, "Unspecified"=>{"Unspecified"=>4}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Chemical Engineering"=>{"Chemical Engineering"=>2}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"Switzerland"=>1, "Portugal"=>1, "Germany"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/4867702"], "description"=>"<p>(A) Three different inoculation strategies: a. accutase, b. EDTA and c. EDTA+ROCKi were compared. (B) Seeding yield after 5 days of culture of hiPS cells on Geltrex coated polystyrene microcarriers (GM) and plate (GP), vitronectin coated polystyrene microcarriers (VtnM) and plate (VtnP), using the three different inoculation strategies: accutase (green), EDTA (purple) and EDTA+ROCKi (blue). Cells were inoculated at a 5x10<sup>4</sup> cells/cm<sup>2</sup> density. Error bars represent SEM (standard error of the mean). Three replicate wells were performed for each condition. (C) Immunostaining of hiPS cells cultured on microcarriers with E8 medium at day 5. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm.</p>", "links"=>[], "tags"=>["hiPS cells", "spinner flask cultures", "Human Induced Pluripotent Stem Cells", "hiPS cell functionality", "embryoid body formation", "microcarrier surface area", "Stirred Microcarrier Culture Systems Human", "cell density b", "support hiPS cell expansion", "50 mL spinner flask"], "article_id"=>3128818, "categories"=>["Biophysics", "Biochemistry", "Space Science", "Cell Biology", "Physiology", "Biotechnology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology"], "users"=>["Sara M. Badenes", "Tiago G. Fernandes", "Cláudia S. M. Cordeiro", "Shayne Boucher", "David Kuninger", "Mohan C. Vemuri", "Maria Margarida Diogo", "Joaquim M. S. Cabral"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0151264.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/hiPS_cell_expansion_on_microcarriers_in_E8_medium_under_static_conditions_/3128818", "title"=>"hiPS cell expansion on microcarriers in E8 medium, under static conditions.", "pos_in_sequence"=>3, "defined_type"=>1, "published_date"=>"2016-03-21 05:31:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/4867768"], "description"=>"<p>EDTA clump inoculation was performed using 55,000 cells/cm<sup>2</sup> and a continuous agitation at 44 rpm (optimal values obtained solving the quadratic model). (A) Total cell numbers during the 10 days expansion. Maximum cell yield obtained solving the quadratic model is represented by the black line. Error bars represent SEM (standard error of the mean) of duplicate samples. (B) Immunostaining of hiPS cells cultured on microcarriers in the spinner flask with E8 medium at day 10. Cells were stained for pluripotency markers NANOG (left panel) and OCT4 (right panel), and nuclei counterstained with DAPI. Scale bars– 100 μm. (C) Immunostaining of hiPS cells harvested from microcarriers after 10 days expansion in spinner flask and re-plated on GP. Cells were stained after 3 days for intracellular pluripotency markers NANOG, OCT4 and SOX2, and nuclei counterstained with DAPI; and for surface markers SSEA4 and TRA-1-60. Scale bars– 100 μm. (D) Flow cytometry analysis of hiPS cells harvested from microcarriers after 10 days expansion in spinner flask. Cells were stained for OCT4 and NANOG. (E) mRNA was isolated from hiPSC at day 0 and at the end of the spinner flask culture (day 12) on microcarriers, and the undifferentiated hiPSC marker transcripts (<i>OCT4</i> and <i>NANOG</i>) were analysed by RT-PCR. (F) Quantitative RT-PCR analysis of spontaneous differentiated EB of hiPSC cultured in spinner flask. The relative expression of each gene was measured against the same gene prior to differentiation. (G) Immunostaining showing the formation of cells expressing SOX17 (endoderm), TUJ1 (ectoderm) and α-SMA (mesoderm) after EB formation and spontaneous differentiation assay with hiPSC cultured in spinner flask. Nuclei were counterstained with DAPI. Scale bar: 50 μm.</p>", "links"=>[], "tags"=>["hiPS cells", "spinner flask cultures", "Human Induced Pluripotent Stem Cells", "hiPS cell functionality", "embryoid body formation", "microcarrier surface area", "Stirred Microcarrier Culture Systems Human", "cell density b", "support hiPS cell expansion", "50 mL spinner flask"], "article_id"=>3128884, "categories"=>["Biophysics", "Biochemistry", "Space Science", "Cell Biology", "Physiology", "Biotechnology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology"], "users"=>["Sara M. Badenes", "Tiago G. Fernandes", "Cláudia S. M. Cordeiro", "Shayne Boucher", "David Kuninger", "Mohan C. Vemuri", "Maria Margarida Diogo", "Joaquim M. S. Cabral"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0151264.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/hiPS_cell_expansion_in_E8_medium_in_a_50_mL_spinner_flask_/3128884", "title"=>"hiPS cell expansion in E8 medium, in a 50 mL spinner flask.", "pos_in_sequence"=>5, "defined_type"=>1, "published_date"=>"2016-03-21 05:31:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/4867552", "https://ndownloader.figshare.com/files/4867564", "https://ndownloader.figshare.com/files/4867576", "https://ndownloader.figshare.com/files/4867591", "https://ndownloader.figshare.com/files/4867597", "https://ndownloader.figshare.com/files/4867609"], "description"=>"<div><p>Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm<sup>2</sup> of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x10<sup>6</sup> cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.</p></div>", "links"=>[], "tags"=>["hiPS cells", "spinner flask cultures", "Human Induced Pluripotent Stem Cells", "hiPS cell functionality", "embryoid body formation", "microcarrier surface area", "Stirred Microcarrier Culture Systems Human", "cell density b", "support hiPS cell expansion", "50 mL spinner flask"], "article_id"=>3128713, "categories"=>["Biophysics", "Biochemistry", "Space Science", "Cell Biology", "Physiology", "Biotechnology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology"], "users"=>["Sara M. Badenes", "Tiago G. Fernandes", "Cláudia S. M. Cordeiro", "Shayne Boucher", "David Kuninger", "Mohan C. Vemuri", "Maria Margarida Diogo", "Joaquim M. S. Cabral"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0151264.s001", "https://dx.doi.org/10.1371/journal.pone.0151264.s002", "https://dx.doi.org/10.1371/journal.pone.0151264.s003", "https://dx.doi.org/10.1371/journal.pone.0151264.s004", "https://dx.doi.org/10.1371/journal.pone.0151264.s005", "https://dx.doi.org/10.1371/journal.pone.0151264.s006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Defined_Essential_8_Medium_and_Vitronectin_Efficiently_Support_Scalable_Xeno_Free_Expansion_of_Human_Induced_Pluripotent_Stem_Cells_in_Stirred_Microcarrier_Culture_Systems/3128713", "title"=>"Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems", "pos_in_sequence"=>1, "defined_type"=>4, "published_date"=>"2016-03-21 05:31:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/4867729"], "description"=>"<p>(A) The procedure consists in 4 steps: a. EDTA+ROCKi inoculation strategy into 20 g/L VtnM; b. Attachment is performed in half of the total volume in static conditions for 24 h (in the presence of ROCKi), following by intermittent stirring overnight to allow cell- microcarrier interaction; c. Expansion is performed with a specific continuous agitation with 80% E8 medium change everyday; and d. At the end of the culture, cell pluripotency and differentiation potential are evaluated. Quadratic model relating initial cell density and agitation speed with cell yield during spinner flask culture of human iPS cells: 3D representation (B) and 2D heat map (C) are shown. For initial cell density: 30 000 cells/cm<sup>2</sup> (−1 level) ≤ Initial Cell Density ≤ 70 000 cells/cm<sup>2</sup> (1 level); for agitation speed: 30 rpm (−1 level) ≤ Agitation Speed ≤ 70 rpm (1 level).</p>", "links"=>[], "tags"=>["hiPS cells", "spinner flask cultures", "Human Induced Pluripotent Stem Cells", "hiPS cell functionality", "embryoid body formation", "microcarrier surface area", "Stirred Microcarrier Culture Systems Human", "cell density b", "support hiPS cell expansion", "50 mL spinner flask"], "article_id"=>3128842, "categories"=>["Biophysics", "Biochemistry", "Space Science", "Cell Biology", "Physiology", "Biotechnology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology"], "users"=>["Sara M. Badenes", "Tiago G. Fernandes", "Cláudia S. M. Cordeiro", "Shayne Boucher", "David Kuninger", "Mohan C. Vemuri", "Maria Margarida Diogo", "Joaquim M. S. Cabral"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0151264.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Scaling_up_hiPS_cell_expansion_in_E8_medium_to_a_50_mL_spinner_flask_under_dynamic_conditions_/3128842", "title"=>"Scaling-up hiPS cell expansion in E8 medium to a 50 mL spinner flask, under dynamic conditions.", "pos_in_sequence"=>4, "defined_type"=>1, "published_date"=>"2016-03-21 05:31:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/4867798"], "description"=>"<p>Cardiomyocyte (CM) differentiation was performed using Life Technologies Cardiomyocyte Differentiation Kit (A) of hiPS cells harvested from microcarriers and re-plated on GP. Immunostaining depicting CM for the cTNT marker. Nuclei were counterstained with DAPI. Scale bar– 100 μm; and (B) of hiPS cells on VtnM in static conditions in a low attachment plate. Immunostaining for the cTNT marker depicting CM on microcarriers (left panel) and plated CM on GP after differentiation protocol (right panel). Nuclei were counterstained with DAPI. Scale bars– 50 μm. Neural induction by Dual-SMAD inhibition (C) of hiPS cells harvested from microcarriers and re-plated on GP. Immunostaining depicting neural progenitor (NP) cells for the neuroectoderm markers PAX6 and NESTIN. Nuclei were counterstained with DAPI. Scale-bar: 100 μm; and (D) of hiPS cells on VtnB in static conditions in a low attachment plate. Immunostaining for the neuroectoderm markers PAX6 and NESTIN depicting NP cells on microcarriers (left panel) and plated NP cells on GP after differentiation protocol (right panel). Nuclei were counterstained with DAPI. Scale bars– 50 μm. (E) Quantitative RT-PCR analysis of differentiated hiPSC cultured in spinner flask, subject to cardiomyocytes or neural differentiation. The relative expression of each gene was measured against the same gene prior to differentiation.</p>", "links"=>[], "tags"=>["hiPS cells", "spinner flask cultures", "Human Induced Pluripotent Stem Cells", "hiPS cell functionality", "embryoid body formation", "microcarrier surface area", "Stirred Microcarrier Culture Systems Human", "cell density b", "support hiPS cell expansion", "50 mL spinner flask"], "article_id"=>3128908, "categories"=>["Biophysics", "Biochemistry", "Space Science", "Cell Biology", "Physiology", "Biotechnology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology"], "users"=>["Sara M. Badenes", "Tiago G. Fernandes", "Cláudia S. M. Cordeiro", "Shayne Boucher", "David Kuninger", "Mohan C. Vemuri", "Maria Margarida Diogo", "Joaquim M. S. Cabral"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0151264.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/hiPS_cells_cultured_in_spinner_flasks_with_E8_medium_and_VtnM_retain_their_differentiation_potential_/3128908", "title"=>"hiPS cells cultured in spinner flasks with E8 medium and VtnM retain their differentiation potential.", "pos_in_sequence"=>6, "defined_type"=>1, "published_date"=>"2016-03-21 05:31:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/4867669"], "description"=>"<p>(A) Colonies cultured with E8 medium in Geltrex (left panel), and in vitronectin (right panel). Scale bars—100μm. (B) Cumulative fold increase in total cell numbers over four passages with E8 medium in Geltrex (green), in vitronectin (purple), in Synthemax<sup>®</sup> (blue) or in CELLStart™ (red). (C) Average fold increase per passage. The fold increase was 5.2 for cells growing in Geltrex, 4.3 for vitronectin, 4.5 for Synthemax<sup>®</sup> and 1.9 for CELLStart™. Error bars represent SEM (standard error of the mean). Four passages and two independent experiments were performed. (D) Immunostaining of colonies cultured with E8 medium in vitronectin. Colonies were stained for pluripotency markers OCT4, SOX2, and NANOG, and nuclei counterstained with DAPI. Scale bars—100μm. (E) Immunostaining of colonies cultured with E8 medium in vitronectin. Colonies were stained for pluripotency surface markers TRA-1-60, TRA-1-81, and SSEA4. Scale bars—100μm. (F) and (G) Flow cytometry analysis of pluripotency surface markers (TRA-1-60 and SSEA4) (F) and transcription factors (OCT4, NANOG, SOX2) (G) in cells expanded with E8 medium and vitronectin.</p>", "links"=>[], "tags"=>["hiPS cells", "spinner flask cultures", "Human Induced Pluripotent Stem Cells", "hiPS cell functionality", "embryoid body formation", "microcarrier surface area", "Stirred Microcarrier Culture Systems Human", "cell density b", "support hiPS cell expansion", "50 mL spinner flask"], "article_id"=>3128794, "categories"=>["Biophysics", "Biochemistry", "Space Science", "Cell Biology", "Physiology", "Biotechnology", "Ecology", "Biological Sciences not elsewhere classified", "Developmental Biology"], "users"=>["Sara M. Badenes", "Tiago G. Fernandes", "Cláudia S. M. Cordeiro", "Shayne Boucher", "David Kuninger", "Mohan C. Vemuri", "Maria Margarida Diogo", "Joaquim M. S. Cabral"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0151264.g001", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/hiPS_cell_maintenance_in_E8_medium_and_vitronectin_/3128794", "title"=>"hiPS cell maintenance in E8 medium and vitronectin.", "pos_in_sequence"=>2, "defined_type"=>1, "published_date"=>"2016-03-21 05:31:10"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2016-01-01T00:00:00Z", "end_date"=>"2016-12-31T00:00:00Z", "subject_areas"=>[]}
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