Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only. Page 1 of 40 Stem Cells and Development
© Mary Ann Liebert, Inc.
DOI: 10.1089/scd.2017.0090
1
Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture
Guoliang Meng, Shiying Liu, Anna Poon, Derrick Rancourt Department of Biochemistry and Molecular Biology
University of Calgary, Calgary, AB, Canada, T2N 4N1
Running title: Suspension Culture of hiPSCs
Corresponding author: Derrick E. Rancourt, Department of Biochemistry & Molecular
biology,
Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, T2N
4N1.
[email protected]
2 Human induced pluripotent stem cells (hiPSCs) hold great hopes for application in regenerative medicine due to their inherent capacity to self‐renew and differentiate into Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 2 of 40
cells from the three embryonic germ layers. For clinical applications, a large quantity of hiPSCs produced in standardized and scalable culture processes is required. Several groups including ours have developed methodologies for scaled‐up hiPSC production in stirred bioreactors in chemically defined medium. Here, we optimized the critical steps and factors that affect hiPSC expansion and yield in stirred suspension cultures including inoculation conditions, seeding density, aggregate size, agitation rate, and cell passaging method. After multiple passages in stirred suspension bioreactors, hiPSCs remained pluripotent, karyotypically normal, and capable of differentiating into all three germ layers. Key words: stirred suspension bioreactor, human induced pluripotent stem cells (hiPSCs), cell aggregate, agitation rate, seeding density
3 Introduction Human induced pluripotent stem cells (hiPSCs) hold great promise for regenerative Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 3 of 40
medicine as the pluripotent nature of these cells allow them to differentiate into virtually any cell type in the body [1‐3]. Therapeutic applications of hiPSCs and their derivatives require large quantities of cells that would be difficult to achieve using traditional two‐ dimensional culture. Currently, hiPSCs are derived and expanded in static feeder‐ dependent or feeder‐free culture systems which have several disadvantages such as limited cell yields and process control. In recent years, several groups have reported the different culture systems for the scale‐up production of hiPSCs. Oh et al. first developed a method for expanding human embryonic stem cells in suspension culture on microcarriers, and this approach was later applied to the expansion of hiPSCs [4‐10]. Microcarriers provide a high surface‐to‐volume ratio for hiPSC attachment and expansion [5]. Stirring motion in the microcarrier‐based system further minimizes the heterogeneity of the culture environment and distribution of oxygen. There are currently different types of microcarriers with variable cell attachment properties as well as bead‐to‐bead variability in cell confluency [11]. To further minimize variability in the culture system, other groups have explored microcarrier‐free systems with different vessel types [12‐24]. Table 1 provides a summary of suspension‐based bioprocessing for hESC and hiPSC expansion including the current study. The culture vessels mainly fall into 5 categories: spinner flask with a magnetic stir bar (NDS), spinner flask with a glass stirrer pendulum (Integra Biosciences), spinner flask with a suspended impeller (Corning), Erlenmeyer flask on an orbital shaker (Corning), and bioreactor systems (DASGIP, BioLevitator™). Specific operational protocols have been developed for these culture vessels with different hiPSC expansion rates. Key parameters that affect cell yield are cell inoculation condition, cell inoculation density, stirring speed, and culture period. In this study, we systematically assessed and optimized these parameters for our microcarrier‐free bioreactor system (Fig. 1) with chemically‐defined medium, mTeSR1. A strong emphasis was placed on the inoculation condition as this is a critical initial step for microcarrier‐free systems where hiPSCs are grown and expanded as aggregates.
4 Currently, two methods, single cell inoculation and aggregate inoculation, are used for the initiation of microcarrier‐free suspension culture. For single cell inoculation, Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 4 of 40
dissociated single cells are transferred directly into stirred culture vessel and then aggregates form from the union of individual cells from the stirring/agitation. The aggregate inoculation method employs pre‐formed aggregates from static culture, which are then transferred to the culture vessel for stirred suspension culture. Variable cell yields have been reported using the two inoculation methods with some even lower than that from the static culture. Here, we compared the two methods and developed a highly efficient method for aggregate formation in static suspension prior to cell inoculation. By using this method, majority of the single cells participated in aggregate formation. We further examined the combinatorial effects of expansion period, aggregation size, agitation rate, and pH on the number of viable hiPSCs in stirred suspension culture. Our optimized protocol consistently yielded a 12‐fold expansion of viable hiPSCs per culture run. The hiPSCs maintained their pluripotent state, differentiation potential, and normal karyotype after multiple expansions and passages in suspension culture. Our efforts contribute the translation of scalable, chemically‐defined suspension culture systems towards good manufacturing practice (GMP) conditions for clinical applications. Materials and Methods In this study, the suspension culture system we employed includes 125mL spinner flasks with a magnetic stir bar (NDS) (Fig. 1). Every experiment related to bioreactor culture was performed in triplicate. Static culture and maintenance of hiPSCs We cultured hiPSC lines, 4YA and 4YF, that were derived from infant fibroblasts (BJ cell lines) and reprogrammed using retrovirus 4 factors (OSKM) with EOS reporter. Both hiPSC lines have normal karyotypes (46, XY) and the cells were obtained from Dr. James Ellis’ laboratory at the University of Toronto (Canada). Cells were cultured and maintained in chemical defined, feeder free Matrigel/mTeSR1 system under standard culture conditions
5 (37°C, 5% CO2, 95% relative humidity). We coated 35mm and 60mm culture dishes with hESC‐qualified BD Matrigel (BD Biosciences) prior to cell culture for at least 2 hours at Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 5 of 40
room temperature. Cells were grown on these Matrigel‐coated dishes containing complete mTeSR1 medium (StemCell Technologies). Cells were passaged every 4 days as small cell clumps via enzymatic and mechanical treatment. Briefly, cultures were washed once with DMEM/F12 (Invitrogen) and then treated with Dispase (1 mg/mL, StemCell Technologies). After incubating the culture dishes for 7‐9 minutes at 37°C, the hiPSC colonies started to curl‐up around the edges and Dispase was removed followed by two washes with DMEM/F12. Colonies were scraped from the bottom of the culture dishes and broken into smaller cell clumps by trituration using a 1mL pipette tip. Cell clumps were then replated onto Matrigel‐coated dishes in mTeSR1 medium at a 1:6 split ratio. hiPSC inoculation in the bioreactor Inoculation conditions were previously reported to have a significant effect on cell survival and growth characteristics during the initial stages of suspension culture in bioreactors. In this study, we explored three types of hiPSC inoculum: (1) single cells, (2) cell clumps, and (3) sphere‐like hiPSC aggregates. hiPSC aggregate formation We developed three different methods of generating hiPSC aggregates for suspension culture in bioreactors: Method 1: Aggregates were formed on Poly‐D‐Lysine (PDL)‐coated dishes in mTeSR1 medium supplemented with 1‐3μM Y‐27632. For the preparation of PDL‐coated dishes, 35mm or 60mm culture dishes were treated with PDL (1‐3μg/mL, Millipore) for 2‐3 hours at room temperature followed by three washes with DMEM/F12. Cells maintained in the Matrigel/mTeSR1 culture system were enzymatically dissociated into single cells using Accutase (1mg/mL, StemCell Technologies) supplemented with 5μM Y‐27632. Single cells were transferred to the PDL‐coated dishes in mTeSR1 medium supplemented with 1‐3μM Y‐27632. Small hiPSC aggregates were formed overnight and these aggregates were
6 loosely attached to the PDL‐coated surface. Detachment of the cell aggregates from the culture dishes was achieved by introducing a stream of medium across the culture surface Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 6 of 40
with a 1mL pipette. Cell aggregates were then transferred into bioreactors with a working volume of 100 mL. Culture medium in the bioreactors was supplemented with 10μM Y‐ 27632. Method 2: Dissociated single cells were grown in static suspension culture condition where cells are cultured in non‐coated 35mm or 60mm petri dishes containing complete mTeSR1 medium supplemented with 10μM Y‐27632. After 3‐4 hours of static suspension cultures, hiPSC aggregates were observed, and these aggregates were pipetted up and down several times to generate smaller aggregates (25‐50μm) before inoculating them in 100 mL bioreactors. Method 3: Dissociated single cells were cultured in 15ml centrifuge tube containing complete mTeSR1 medium supplemented with 10μM Y‐27632. After 3‐4 hours, hiPSC aggregates were observed, and these aggregates were inoculated in 100 mL bioreactors. Several steps were taken to achieve the optimized size of aggregates. First, the single cell density we employed for aggregate formation in static condition was around 106 cells/cm2 in mTeSR1 medium supplemented 10µM Y‐27632. Second, we incubate the suspension cultures in a standard cell culture incubator for 3‐4 hours, depending on different cell lines. Third, after incubation, we gently pipetted the aggregates about 10 times and break them into smaller ones. Then randomly measure 30‐60 aggregates under microscope using measuring software. Aggregate‐forming efficiency To determine the effectiveness of the three methods in forming hiPSC aggregates, we estimated the percentage of viable hiPSCs that participated in aggregate formation by using this following equation: (Starting Cell Number –Number of Dead Cells after Seeding)/Starting Cell Number × 100%. Dead cells were stained positive for Trypan Blue and counted with a hemocytometer. For Method 1, dead cells were observed floating in the culture medium and some were also attached to the culture surface. Both viable aggregates and dead cells attached to the
7 culture surface were detached by introducing streams of culture media across the surface with a pipette. Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 7 of 40
Cell suspension was collected and then filtered through a 40μm cell strainer. The flow‐ through contained dead cells that were calculated after centrifugation and re‐suspension. For Method 2 and 3, the dead cells were observed floating in the culture medium. Cell suspension was collected and then filtered through 40μm cell strainer. The flow‐through contained dead cells that were calculated after centrifugation and re‐suspension. Removal of dead cells prior to inoculation Cell suspension containing hiPSC aggregates and dead cells were collected into a 15mL centrifuge tube and centrifuged at 300rpm for 3 minutes. The supernatant that contained the dead cells were aspirated, leaving the hiPSC aggregates at the bottom of the tube for downstream inoculation in bioreactors. Suspension culture in stirred bioreactors For single cell inoculation, single hiPSCs were seeded in stirred suspension bioreactors at low (1×105cells/mL), medium (2.5×105cells/mL), and high (5×105cells/mL) cell densities, which were calculated from the starting cell number minus the number of dead cells. For other inoculation conditions, either cell clumps or cell aggregates were seeded in the bioreactors at low (~ 2×104cells/mL), medium (~ 4×104cells/mL), and high (~ 8×104cells/mL) densities, which were estimated from the starting cell number minus the number of single cells remaining in the culture dish after cell aggregation. First, we estimated the percentage of viable hiPSCs that participated in aggregate formation by using this following equation: (Starting Cell Number –Number of Dead Cells after Seeding)/Starting Cell Number × 100%. Dead cells were stained positive for Trypan Blue and counted with a hemocytometer. Second, we estimated live cell density in the form of cell aggregates by using this following equation:
8 (Starting Cell Number × Percentage of Viable Cells (%)/Cell Suspension Volume (mL) Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 8 of 40
Suspension cultures were passaged every five days via enzymatic dissociation with Accutase and the hiPSC inoculation processes were repeated new cultures initiated through the same single cells and aggregation process, respectively as described above for each passage. Agitation speeds were set at 80, 100, and 120rpm for low, medium, and high, respectively. Determination of hiPSC number and viability from bioreactor suspension cultures Bioreactors were removed from the incubator and briefly swirled to even out the distribution of aggregates before withdrawing 2.0 mL of cell suspension for downstream cell count and viability assessment. Each collected sample was placed in a 35mm culture dish for observation and photomicrographs. Then, the cell aggregates in the dish were treated with 0.25% Trypsin‐EDTA (Invitrogen) for 5‐7 minutes at 37°C for dissociation into single cells. Subsequently, two 100μL samples were used for cell counting with a hemocytometer and cell viability was determined using the Trypan blue exclusion assay. The fold expansion at each passage was determined by the cell number at the time of harvest divided by the starting cell number. pH value measurements It has been well documented that pH in the culture environment affects cell metabolism and growth. In general, an alkalescent environment supports cell growth whereas acidic environment induces apoptosis. For each culture period, we monitored the pH of the culture media and used it as an indicator of medium replacement. Beginning from the third day (low and medium densities) or the second day (high density) after cell inoculation, a 5‐mL sample was taken from each bioreactor each day. When collecting medium sample, transferred the bioreactor into the cell culture hood, let it stand still for several minutes, gently aspirated a 5‐mL sample from the supernatant medium and then transferred it into a 50 mL tube. A digital pH meter with a probe (VWR SympHony B10P pH Meter) was used for the pH measurement of the culture media.
9 In vitro and in vivo hiPSC differentiation For in vitro differentiation, aggregates grown in suspension culture were dissociated into Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 9 of 40
single cells and re‐plated on 35mm agar‐coated dishes in a differentiation medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 10μM Y‐27632,1 mM L‐GlutaMax, 0.1 mM β ‐mercaptoethanol, and 1mM nonessential amino acids (all reagents were from Life Technologies). After 8‐10 days, the cells aggregated and formed cystic embryoid bodies (EBs), which were then collected and replated in the same medium without Y‐27632 onto gelatin‐treated four‐well plates. After 6 days, the EBs were fixed and stained for immunocytochemical analysis of early differentiation markers of the three germ layers. For in vivo differentiation, hiPSC aggregates were harvested and injected into the rear leg muscles of 6‐ to 8‐week‐old SCID‐beige mice (1‐2×106 cells per injection). Teratomas consisting of all three germ layers were removed from injection sites after 10~12 weeks, fixed overnight in 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and examined histologically after staining with eosin and haematoxylin. Immunocytochemistry Cells were fixed in 4% PFA for 15 minutes at room temperature, washed three times with 1X phosphate buffered saline (PBS), permeabilized with 0.1% Triton‐x 100 (Sigma Aldrich) for 15 minutes at room temperature, and then washed three more times with PBS before blocking with 10% normal serum or 3% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature to minimize non‐specific binding of antibodies. Fixed hiPSCs were incubated with primary antibodies (Millipore) against pluripotency markers: Nanog, Oct4, SSEA‐4, TRA‐1‐60, and TRA‐ 1‐81 at 1:100 dilution, 4°C overnight. Differentiated EBs were incubated with primary antibodies (Sigma Aldrich) against β‐tubulin III (ectoderm marker), smooth muscle actin (mesoderm marker), and α‐fetoprotein (endoderm marker) at 1:400 dilution, 4°C overnight. The next day, cells were incubated with the appropriate secondary antibodies, Alexa Fluor 546 goat anti‐mouse and Alexa Fluor 594 donkey anti‐rabbit (1:200; Invitrogen) for at least two hours at room temperature and then washed 3 times with 1XPBS before imaging with confocal microscope.
Page 10 of 40
10 Flow cytometry The expression of pluripotency markers, Oct4, Nanog, and SSEA‐4 of hiPSCs grown in Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
stirred bioreactors was analyzed by fluorescence‐activated cell sorting (FACS). Cell aggregates were dissociated into single cells via trypsinization, fixed with 4% PFA, washed three times with PBS, permeabilized with 0.75% Saponin (Sigma Aldrich). After two washes with PBS, cells were then resuspended in PBS containing 3% BSA for 30 minutes at 37°C. Re‐suspended cells were incubated with the following antibodies (Millipore) for at least 1 hour at 37°C: anti‐Oct4 (Alexa Fluor 488 conjugate; 1:50), anti‐Nanog (FITC conjugate; 1:50), anti‐SSEA‐4 (PE conjugate, 1:50 dilution). Mouse IgG Alexa Fluor 488, mouse IgG1 FITC, mouse IgG3PE were used as isotope controls for the primary antibodies. Flow cytometric analysis was performed using BD FACSAria III. Karyotype analysis Karyotype analysis of hiPSCs was carried out using G‐banding method. Briefly, hiPSCs were incubated with culture medium containing 0.1 mg/mL of colcemid (Sigma Aldrich) at 37°C for an hour, trypsinized into single cells, resuspended, and incubated in 68 mM KCl for 20 minutes at room temperature, and then fixed with fixative (3:1 methanol: glacial acetic acid), and dropped to make the spread of chromosomes on the slides. The slides were dried at 37°C on slide warmer overnight, baked at 80°C for 90 min, treated with 0.05% trypsin (Life Technologies) for 45 seconds to 1 minutes, and then stained with the Giemsa and Leishman’s solution (Sigma Aldrich). At least 10 metaphase spreads were analyzed for each of hiPSC lines grown in stirred bioreactors. Statistical analysis Statistical analysis on data was carried out using unpaired Student’s t‐test for the comparison of two groups with a minimal significance of p
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only. Page 1 of 40 Stem Cells and Development
© Mary Ann Liebert, Inc.
DOI: 10.1089/scd.2017.0090
1
Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture
Guoliang Meng, Shiying Liu, Anna Poon, Derrick Rancourt Department of Biochemistry and Molecular Biology
University of Calgary, Calgary, AB, Canada, T2N 4N1
Running title: Suspension Culture of hiPSCs
Corresponding author: Derrick E. Rancourt, Department of Biochemistry & Molecular
biology,
Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta, T2N
4N1.
[email protected]
2 Human induced pluripotent stem cells (hiPSCs) hold great hopes for application in regenerative medicine due to their inherent capacity to self‐renew and differentiate into Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 2 of 40
cells from the three embryonic germ layers. For clinical applications, a large quantity of hiPSCs produced in standardized and scalable culture processes is required. Several groups including ours have developed methodologies for scaled‐up hiPSC production in stirred bioreactors in chemically defined medium. Here, we optimized the critical steps and factors that affect hiPSC expansion and yield in stirred suspension cultures including inoculation conditions, seeding density, aggregate size, agitation rate, and cell passaging method. After multiple passages in stirred suspension bioreactors, hiPSCs remained pluripotent, karyotypically normal, and capable of differentiating into all three germ layers. Key words: stirred suspension bioreactor, human induced pluripotent stem cells (hiPSCs), cell aggregate, agitation rate, seeding density
3 Introduction Human induced pluripotent stem cells (hiPSCs) hold great promise for regenerative Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 3 of 40
medicine as the pluripotent nature of these cells allow them to differentiate into virtually any cell type in the body [1‐3]. Therapeutic applications of hiPSCs and their derivatives require large quantities of cells that would be difficult to achieve using traditional two‐ dimensional culture. Currently, hiPSCs are derived and expanded in static feeder‐ dependent or feeder‐free culture systems which have several disadvantages such as limited cell yields and process control. In recent years, several groups have reported the different culture systems for the scale‐up production of hiPSCs. Oh et al. first developed a method for expanding human embryonic stem cells in suspension culture on microcarriers, and this approach was later applied to the expansion of hiPSCs [4‐10]. Microcarriers provide a high surface‐to‐volume ratio for hiPSC attachment and expansion [5]. Stirring motion in the microcarrier‐based system further minimizes the heterogeneity of the culture environment and distribution of oxygen. There are currently different types of microcarriers with variable cell attachment properties as well as bead‐to‐bead variability in cell confluency [11]. To further minimize variability in the culture system, other groups have explored microcarrier‐free systems with different vessel types [12‐24]. Table 1 provides a summary of suspension‐based bioprocessing for hESC and hiPSC expansion including the current study. The culture vessels mainly fall into 5 categories: spinner flask with a magnetic stir bar (NDS), spinner flask with a glass stirrer pendulum (Integra Biosciences), spinner flask with a suspended impeller (Corning), Erlenmeyer flask on an orbital shaker (Corning), and bioreactor systems (DASGIP, BioLevitator™). Specific operational protocols have been developed for these culture vessels with different hiPSC expansion rates. Key parameters that affect cell yield are cell inoculation condition, cell inoculation density, stirring speed, and culture period. In this study, we systematically assessed and optimized these parameters for our microcarrier‐free bioreactor system (Fig. 1) with chemically‐defined medium, mTeSR1. A strong emphasis was placed on the inoculation condition as this is a critical initial step for microcarrier‐free systems where hiPSCs are grown and expanded as aggregates.
4 Currently, two methods, single cell inoculation and aggregate inoculation, are used for the initiation of microcarrier‐free suspension culture. For single cell inoculation, Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
Page 4 of 40
dissociated single cells are transferred directly into stirred culture vessel and then aggregates form from the union of individual cells from the stirring/agitation. The aggregate inoculation method employs pre‐formed aggregates from static culture, which are then transferred to the culture vessel for stirred suspension culture. Variable cell yields have been reported using the two inoculation methods with some even lower than that from the static culture. Here, we compared the two methods and developed a highly efficient method for aggregate formation in static suspension prior to cell inoculation. By using this method, majority of the single cells participated in aggregate formation. We further examined the combinatorial effects of expansion period, aggregation size, agitation rate, and pH on the number of viable hiPSCs in stirred suspension culture. Our optimized protocol consistently yielded a 12‐fold expansion of viable hiPSCs per culture run. The hiPSCs maintained their pluripotent state, differentiation potential, and normal karyotype after multiple expansions and passages in suspension culture. Our efforts contribute the translation of scalable, chemically‐defined suspension culture systems towards good manufacturing practice (GMP) conditions for clinical applications. Materials and Methods In this study, the suspension culture system we employed includes 125mL spinner flasks with a magnetic stir bar (NDS) (Fig. 1). Every experiment related to bioreactor culture was performed in triplicate. Static culture and maintenance of hiPSCs We cultured hiPSC lines, 4YA and 4YF, that were derived from infant fibroblasts (BJ cell lines) and reprogrammed using retrovirus 4 factors (OSKM) with EOS reporter. Both hiPSC lines have normal karyotypes (46, XY) and the cells were obtained from Dr. James Ellis’ laboratory at the University of Toronto (Canada). Cells were cultured and maintained in chemical defined, feeder free Matrigel/mTeSR1 system under standard culture conditions
5 (37°C, 5% CO2, 95% relative humidity). We coated 35mm and 60mm culture dishes with hESC‐qualified BD Matrigel (BD Biosciences) prior to cell culture for at least 2 hours at Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
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room temperature. Cells were grown on these Matrigel‐coated dishes containing complete mTeSR1 medium (StemCell Technologies). Cells were passaged every 4 days as small cell clumps via enzymatic and mechanical treatment. Briefly, cultures were washed once with DMEM/F12 (Invitrogen) and then treated with Dispase (1 mg/mL, StemCell Technologies). After incubating the culture dishes for 7‐9 minutes at 37°C, the hiPSC colonies started to curl‐up around the edges and Dispase was removed followed by two washes with DMEM/F12. Colonies were scraped from the bottom of the culture dishes and broken into smaller cell clumps by trituration using a 1mL pipette tip. Cell clumps were then replated onto Matrigel‐coated dishes in mTeSR1 medium at a 1:6 split ratio. hiPSC inoculation in the bioreactor Inoculation conditions were previously reported to have a significant effect on cell survival and growth characteristics during the initial stages of suspension culture in bioreactors. In this study, we explored three types of hiPSC inoculum: (1) single cells, (2) cell clumps, and (3) sphere‐like hiPSC aggregates. hiPSC aggregate formation We developed three different methods of generating hiPSC aggregates for suspension culture in bioreactors: Method 1: Aggregates were formed on Poly‐D‐Lysine (PDL)‐coated dishes in mTeSR1 medium supplemented with 1‐3μM Y‐27632. For the preparation of PDL‐coated dishes, 35mm or 60mm culture dishes were treated with PDL (1‐3μg/mL, Millipore) for 2‐3 hours at room temperature followed by three washes with DMEM/F12. Cells maintained in the Matrigel/mTeSR1 culture system were enzymatically dissociated into single cells using Accutase (1mg/mL, StemCell Technologies) supplemented with 5μM Y‐27632. Single cells were transferred to the PDL‐coated dishes in mTeSR1 medium supplemented with 1‐3μM Y‐27632. Small hiPSC aggregates were formed overnight and these aggregates were
6 loosely attached to the PDL‐coated surface. Detachment of the cell aggregates from the culture dishes was achieved by introducing a stream of medium across the culture surface Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
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with a 1mL pipette. Cell aggregates were then transferred into bioreactors with a working volume of 100 mL. Culture medium in the bioreactors was supplemented with 10μM Y‐ 27632. Method 2: Dissociated single cells were grown in static suspension culture condition where cells are cultured in non‐coated 35mm or 60mm petri dishes containing complete mTeSR1 medium supplemented with 10μM Y‐27632. After 3‐4 hours of static suspension cultures, hiPSC aggregates were observed, and these aggregates were pipetted up and down several times to generate smaller aggregates (25‐50μm) before inoculating them in 100 mL bioreactors. Method 3: Dissociated single cells were cultured in 15ml centrifuge tube containing complete mTeSR1 medium supplemented with 10μM Y‐27632. After 3‐4 hours, hiPSC aggregates were observed, and these aggregates were inoculated in 100 mL bioreactors. Several steps were taken to achieve the optimized size of aggregates. First, the single cell density we employed for aggregate formation in static condition was around 106 cells/cm2 in mTeSR1 medium supplemented 10µM Y‐27632. Second, we incubate the suspension cultures in a standard cell culture incubator for 3‐4 hours, depending on different cell lines. Third, after incubation, we gently pipetted the aggregates about 10 times and break them into smaller ones. Then randomly measure 30‐60 aggregates under microscope using measuring software. Aggregate‐forming efficiency To determine the effectiveness of the three methods in forming hiPSC aggregates, we estimated the percentage of viable hiPSCs that participated in aggregate formation by using this following equation: (Starting Cell Number –Number of Dead Cells after Seeding)/Starting Cell Number × 100%. Dead cells were stained positive for Trypan Blue and counted with a hemocytometer. For Method 1, dead cells were observed floating in the culture medium and some were also attached to the culture surface. Both viable aggregates and dead cells attached to the
7 culture surface were detached by introducing streams of culture media across the surface with a pipette. Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
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Cell suspension was collected and then filtered through a 40μm cell strainer. The flow‐ through contained dead cells that were calculated after centrifugation and re‐suspension. For Method 2 and 3, the dead cells were observed floating in the culture medium. Cell suspension was collected and then filtered through 40μm cell strainer. The flow‐through contained dead cells that were calculated after centrifugation and re‐suspension. Removal of dead cells prior to inoculation Cell suspension containing hiPSC aggregates and dead cells were collected into a 15mL centrifuge tube and centrifuged at 300rpm for 3 minutes. The supernatant that contained the dead cells were aspirated, leaving the hiPSC aggregates at the bottom of the tube for downstream inoculation in bioreactors. Suspension culture in stirred bioreactors For single cell inoculation, single hiPSCs were seeded in stirred suspension bioreactors at low (1×105cells/mL), medium (2.5×105cells/mL), and high (5×105cells/mL) cell densities, which were calculated from the starting cell number minus the number of dead cells. For other inoculation conditions, either cell clumps or cell aggregates were seeded in the bioreactors at low (~ 2×104cells/mL), medium (~ 4×104cells/mL), and high (~ 8×104cells/mL) densities, which were estimated from the starting cell number minus the number of single cells remaining in the culture dish after cell aggregation. First, we estimated the percentage of viable hiPSCs that participated in aggregate formation by using this following equation: (Starting Cell Number –Number of Dead Cells after Seeding)/Starting Cell Number × 100%. Dead cells were stained positive for Trypan Blue and counted with a hemocytometer. Second, we estimated live cell density in the form of cell aggregates by using this following equation:
8 (Starting Cell Number × Percentage of Viable Cells (%)/Cell Suspension Volume (mL) Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
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Suspension cultures were passaged every five days via enzymatic dissociation with Accutase and the hiPSC inoculation processes were repeated new cultures initiated through the same single cells and aggregation process, respectively as described above for each passage. Agitation speeds were set at 80, 100, and 120rpm for low, medium, and high, respectively. Determination of hiPSC number and viability from bioreactor suspension cultures Bioreactors were removed from the incubator and briefly swirled to even out the distribution of aggregates before withdrawing 2.0 mL of cell suspension for downstream cell count and viability assessment. Each collected sample was placed in a 35mm culture dish for observation and photomicrographs. Then, the cell aggregates in the dish were treated with 0.25% Trypsin‐EDTA (Invitrogen) for 5‐7 minutes at 37°C for dissociation into single cells. Subsequently, two 100μL samples were used for cell counting with a hemocytometer and cell viability was determined using the Trypan blue exclusion assay. The fold expansion at each passage was determined by the cell number at the time of harvest divided by the starting cell number. pH value measurements It has been well documented that pH in the culture environment affects cell metabolism and growth. In general, an alkalescent environment supports cell growth whereas acidic environment induces apoptosis. For each culture period, we monitored the pH of the culture media and used it as an indicator of medium replacement. Beginning from the third day (low and medium densities) or the second day (high density) after cell inoculation, a 5‐mL sample was taken from each bioreactor each day. When collecting medium sample, transferred the bioreactor into the cell culture hood, let it stand still for several minutes, gently aspirated a 5‐mL sample from the supernatant medium and then transferred it into a 50 mL tube. A digital pH meter with a probe (VWR SympHony B10P pH Meter) was used for the pH measurement of the culture media.
9 In vitro and in vivo hiPSC differentiation For in vitro differentiation, aggregates grown in suspension culture were dissociated into Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
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single cells and re‐plated on 35mm agar‐coated dishes in a differentiation medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 10μM Y‐27632,1 mM L‐GlutaMax, 0.1 mM β ‐mercaptoethanol, and 1mM nonessential amino acids (all reagents were from Life Technologies). After 8‐10 days, the cells aggregated and formed cystic embryoid bodies (EBs), which were then collected and replated in the same medium without Y‐27632 onto gelatin‐treated four‐well plates. After 6 days, the EBs were fixed and stained for immunocytochemical analysis of early differentiation markers of the three germ layers. For in vivo differentiation, hiPSC aggregates were harvested and injected into the rear leg muscles of 6‐ to 8‐week‐old SCID‐beige mice (1‐2×106 cells per injection). Teratomas consisting of all three germ layers were removed from injection sites after 10~12 weeks, fixed overnight in 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and examined histologically after staining with eosin and haematoxylin. Immunocytochemistry Cells were fixed in 4% PFA for 15 minutes at room temperature, washed three times with 1X phosphate buffered saline (PBS), permeabilized with 0.1% Triton‐x 100 (Sigma Aldrich) for 15 minutes at room temperature, and then washed three more times with PBS before blocking with 10% normal serum or 3% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature to minimize non‐specific binding of antibodies. Fixed hiPSCs were incubated with primary antibodies (Millipore) against pluripotency markers: Nanog, Oct4, SSEA‐4, TRA‐1‐60, and TRA‐ 1‐81 at 1:100 dilution, 4°C overnight. Differentiated EBs were incubated with primary antibodies (Sigma Aldrich) against β‐tubulin III (ectoderm marker), smooth muscle actin (mesoderm marker), and α‐fetoprotein (endoderm marker) at 1:400 dilution, 4°C overnight. The next day, cells were incubated with the appropriate secondary antibodies, Alexa Fluor 546 goat anti‐mouse and Alexa Fluor 594 donkey anti‐rabbit (1:200; Invitrogen) for at least two hours at room temperature and then washed 3 times with 1XPBS before imaging with confocal microscope.
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10 Flow cytometry The expression of pluripotency markers, Oct4, Nanog, and SSEA‐4 of hiPSCs grown in Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (DOI: 10.1089/scd.2017.0090) This paper has been peer‐reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Stem Cells and Development Optimizing Human Induced Pluripotent Stem Cell Expansion in Stirred Suspension Culture (doi: 10.1089/scd.2017.0090) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Downloaded by Gothenburg University Library from online.liebertpub.com at 10/12/17. For personal use only.
stirred bioreactors was analyzed by fluorescence‐activated cell sorting (FACS). Cell aggregates were dissociated into single cells via trypsinization, fixed with 4% PFA, washed three times with PBS, permeabilized with 0.75% Saponin (Sigma Aldrich). After two washes with PBS, cells were then resuspended in PBS containing 3% BSA for 30 minutes at 37°C. Re‐suspended cells were incubated with the following antibodies (Millipore) for at least 1 hour at 37°C: anti‐Oct4 (Alexa Fluor 488 conjugate; 1:50), anti‐Nanog (FITC conjugate; 1:50), anti‐SSEA‐4 (PE conjugate, 1:50 dilution). Mouse IgG Alexa Fluor 488, mouse IgG1 FITC, mouse IgG3PE were used as isotope controls for the primary antibodies. Flow cytometric analysis was performed using BD FACSAria III. Karyotype analysis Karyotype analysis of hiPSCs was carried out using G‐banding method. Briefly, hiPSCs were incubated with culture medium containing 0.1 mg/mL of colcemid (Sigma Aldrich) at 37°C for an hour, trypsinized into single cells, resuspended, and incubated in 68 mM KCl for 20 minutes at room temperature, and then fixed with fixative (3:1 methanol: glacial acetic acid), and dropped to make the spread of chromosomes on the slides. The slides were dried at 37°C on slide warmer overnight, baked at 80°C for 90 min, treated with 0.05% trypsin (Life Technologies) for 45 seconds to 1 minutes, and then stained with the Giemsa and Leishman’s solution (Sigma Aldrich). At least 10 metaphase spreads were analyzed for each of hiPSC lines grown in stirred bioreactors. Statistical analysis Statistical analysis on data was carried out using unpaired Student’s t‐test for the comparison of two groups with a minimal significance of p
