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Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.

Correia C, Serra M, Espinha N, Sousa M, Brito C, Burkert K, Zheng Y, Hescheler J, Carrondo MJ, Sarić T, Alves PM - Stem Cell Rev (2014)

Bottom Line: The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated.Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures.This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal.

ABSTRACT
Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

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Experimental scheme for differentiation and purification of iPSC-derived CMs in stirred tank and WAVE bioreactors. In stirred tank bioreactors, an aggregation step was first performed in an Erlenmeyer for 48 h. After this time, aggregates were transferred to bioreactors to yield 150 aggregates/mL and cultured in the presence of ascorbic acid for additional 7 days. At day 9, puromycin was added to the medium to eliminate non-CMs. After 7 days of antibiotic-based CM selection, pure CM aggregates (cardiospheres) were harvested, dissociated and CMs were cultured in 2D plates for characterization studies (immunofluorescence microscopy and electrophysiological studies). In WAVE bioreactor cultures, cells were inoculated as single cells directly into the WAVE bioreactor and at day 2 the culture volume was adjusted to obtain 150 aggregates/mL. Aggregates were cultured in the presence of ascorbic acid for additional 7 days. At day 9, CM lineage selection was initiated and lasted 2 days. At day 11, aggregates were harvested, dissociated and plated in 2D plates for further characterization. Culture conditions evaluated in each bioreactor system are depicted in the bottom of the schematic workflow
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Fig1: Experimental scheme for differentiation and purification of iPSC-derived CMs in stirred tank and WAVE bioreactors. In stirred tank bioreactors, an aggregation step was first performed in an Erlenmeyer for 48 h. After this time, aggregates were transferred to bioreactors to yield 150 aggregates/mL and cultured in the presence of ascorbic acid for additional 7 days. At day 9, puromycin was added to the medium to eliminate non-CMs. After 7 days of antibiotic-based CM selection, pure CM aggregates (cardiospheres) were harvested, dissociated and CMs were cultured in 2D plates for characterization studies (immunofluorescence microscopy and electrophysiological studies). In WAVE bioreactor cultures, cells were inoculated as single cells directly into the WAVE bioreactor and at day 2 the culture volume was adjusted to obtain 150 aggregates/mL. Aggregates were cultured in the presence of ascorbic acid for additional 7 days. At day 9, CM lineage selection was initiated and lasted 2 days. At day 11, aggregates were harvested, dissociated and plated in 2D plates for further characterization. Culture conditions evaluated in each bioreactor system are depicted in the bottom of the schematic workflow

Mentions: To promote cell aggregation 0.7 × 105 cell/mL were inoculated into plastic Erlenmeyer flasks (Corning, USA) containing 100 mL of differentiation medium (Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX, supplemented with 20 % (v/v) FBS, 1 × NEAA, 1 % (v/v) Pen/Strep, 50 μM β-mercaptoethanol (all from Invitrogen, UK) and 100 μM ascorbic acid (Wako, Germany), and incubated at 37 ºC in a 5 % CO2 humidified atmosphere on an orbital shaker at 80–90 rpm. After two days aggregates were transferred into stirred tank bioreactors (DasGip cellferm-pro bioreactor system, Germany) and cultured at a concentration of 150 aggregates/mL in 200 mL of differentiation medium. Medium was partially changed at days 9 (50 % v/v), 12 (70 % v/v) and 14 (50 % v/v) by selection medium (differentiation media without ascorbic acid supplemented with puromycin at a final concentration of 8 μg/mL (InvivoGen, USA)) to eliminate non-CMs and promote CM selection. Antibiotic treatment resulted in the generation of pure aggregates of CMs (designated hereafter as cardiospheres). The experimental set up is illustrated in Fig. 1. All cultures were performed in computer-controlled stirred tank bioreactors equipped with a trapezoid shaped paddle impeller with arms and operated under defined conditions (CO2: 5 %; temperature: 37 °C; DO: 20 % O2 tension (atmospheric normoxia) or 4 % O2 tension (atmospheric hypoxia); surface aeration rate: 0.1 vvm (gas volume flow per unit of liquid volume per minute); agitation rate: 90 rpm for complete continuous stirred-tank reactor (CSTR) behavior; agitation profile: continuous or intermittent (ON: 30 s, OFF: 0 s) with or without direction change; cyclic mechanical frequency (defined by the number of stirring interruptions per unit of time): 0.033Hz). Data acquisition and process control were performed using DasGip Control Software 4.0. Three independent bioreactor runs were performed for every experimental setting.Fig. 1


Combining hypoxia and bioreactor hydrodynamics boosts induced pluripotent stem cell differentiation towards cardiomyocytes.

Correia C, Serra M, Espinha N, Sousa M, Brito C, Burkert K, Zheng Y, Hescheler J, Carrondo MJ, Sarić T, Alves PM - Stem Cell Rev (2014)

Experimental scheme for differentiation and purification of iPSC-derived CMs in stirred tank and WAVE bioreactors. In stirred tank bioreactors, an aggregation step was first performed in an Erlenmeyer for 48 h. After this time, aggregates were transferred to bioreactors to yield 150 aggregates/mL and cultured in the presence of ascorbic acid for additional 7 days. At day 9, puromycin was added to the medium to eliminate non-CMs. After 7 days of antibiotic-based CM selection, pure CM aggregates (cardiospheres) were harvested, dissociated and CMs were cultured in 2D plates for characterization studies (immunofluorescence microscopy and electrophysiological studies). In WAVE bioreactor cultures, cells were inoculated as single cells directly into the WAVE bioreactor and at day 2 the culture volume was adjusted to obtain 150 aggregates/mL. Aggregates were cultured in the presence of ascorbic acid for additional 7 days. At day 9, CM lineage selection was initiated and lasted 2 days. At day 11, aggregates were harvested, dissociated and plated in 2D plates for further characterization. Culture conditions evaluated in each bioreactor system are depicted in the bottom of the schematic workflow
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4225049&req=5

Fig1: Experimental scheme for differentiation and purification of iPSC-derived CMs in stirred tank and WAVE bioreactors. In stirred tank bioreactors, an aggregation step was first performed in an Erlenmeyer for 48 h. After this time, aggregates were transferred to bioreactors to yield 150 aggregates/mL and cultured in the presence of ascorbic acid for additional 7 days. At day 9, puromycin was added to the medium to eliminate non-CMs. After 7 days of antibiotic-based CM selection, pure CM aggregates (cardiospheres) were harvested, dissociated and CMs were cultured in 2D plates for characterization studies (immunofluorescence microscopy and electrophysiological studies). In WAVE bioreactor cultures, cells were inoculated as single cells directly into the WAVE bioreactor and at day 2 the culture volume was adjusted to obtain 150 aggregates/mL. Aggregates were cultured in the presence of ascorbic acid for additional 7 days. At day 9, CM lineage selection was initiated and lasted 2 days. At day 11, aggregates were harvested, dissociated and plated in 2D plates for further characterization. Culture conditions evaluated in each bioreactor system are depicted in the bottom of the schematic workflow
Mentions: To promote cell aggregation 0.7 × 105 cell/mL were inoculated into plastic Erlenmeyer flasks (Corning, USA) containing 100 mL of differentiation medium (Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX, supplemented with 20 % (v/v) FBS, 1 × NEAA, 1 % (v/v) Pen/Strep, 50 μM β-mercaptoethanol (all from Invitrogen, UK) and 100 μM ascorbic acid (Wako, Germany), and incubated at 37 ºC in a 5 % CO2 humidified atmosphere on an orbital shaker at 80–90 rpm. After two days aggregates were transferred into stirred tank bioreactors (DasGip cellferm-pro bioreactor system, Germany) and cultured at a concentration of 150 aggregates/mL in 200 mL of differentiation medium. Medium was partially changed at days 9 (50 % v/v), 12 (70 % v/v) and 14 (50 % v/v) by selection medium (differentiation media without ascorbic acid supplemented with puromycin at a final concentration of 8 μg/mL (InvivoGen, USA)) to eliminate non-CMs and promote CM selection. Antibiotic treatment resulted in the generation of pure aggregates of CMs (designated hereafter as cardiospheres). The experimental set up is illustrated in Fig. 1. All cultures were performed in computer-controlled stirred tank bioreactors equipped with a trapezoid shaped paddle impeller with arms and operated under defined conditions (CO2: 5 %; temperature: 37 °C; DO: 20 % O2 tension (atmospheric normoxia) or 4 % O2 tension (atmospheric hypoxia); surface aeration rate: 0.1 vvm (gas volume flow per unit of liquid volume per minute); agitation rate: 90 rpm for complete continuous stirred-tank reactor (CSTR) behavior; agitation profile: continuous or intermittent (ON: 30 s, OFF: 0 s) with or without direction change; cyclic mechanical frequency (defined by the number of stirring interruptions per unit of time): 0.033Hz). Data acquisition and process control were performed using DasGip Control Software 4.0. Three independent bioreactor runs were performed for every experimental setting.Fig. 1

Bottom Line: The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated.Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures.This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, Oeiras, 2780-157, Portugal.

ABSTRACT
Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) hold great promise for patient-specific disease modeling, drug screening and cell therapy. However, existing protocols for CM differentiation of iPSCs besides being highly dependent on the application of expensive growth factors show low reproducibility and scalability. The aim of this work was to develop a robust and scalable strategy for mass production of iPSC-derived CMs by designing a bioreactor protocol that ensures a hypoxic and mechanical environment. Murine iPSCs were cultivated as aggregates in either stirred tank or WAVE bioreactors. The effect of dissolved oxygen and mechanical forces, promoted by different hydrodynamic environments, on CM differentiation was evaluated. Combining a hypoxia culture (4 % O2 tension) with an intermittent agitation profile in stirred tank bioreactors resulted in an improvement of about 1000-fold in CM yields when compared to normoxic (20 % O2 tension) and continuously agitated cultures. Additionally, we showed for the first time that wave-induced agitation enables the differentiation of iPSCs towards CMs at faster kinetics and with higher yields (60 CMs/input iPSC). In an 11-day differentiation protocol, clinically relevant numbers of CMs (2.3 × 10(9) CMs/1 L) were produced, and CMs exhibited typical cardiac sarcomeric structures, calcium transients, electrophysiological profiles and drug responsiveness. This work describes significant advances towards scalable cardiomyocyte differentiation of murine iPSC, paving the way for the implementation of this strategy for mass production of their human counterparts and their use for cardiac repair and cardiovascular research.

Show MeSH
Related in: MedlinePlus