Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

Amanda Mizukamia, Maristela D. Orellanaa, Sâmia R. Carusoa, Karen de Lima Prataa, Dimas T. Covasa, Kamilla

Swiecha,b*

a Hemotherapy Center of Ribeirão Preto, Faculty of Medicine of Ribeirão Preto, University of São Paulo. Tenente Catão Roxo street, 2501, CEP 14051-140, Ribeirão Preto-SP, Brazil.

b Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. Café av w/n. CEP 14040-903, Ribeirão Preto-SP, Brazil.

*Corresponding author at Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo. Café av w/n. CEP 14040-903, Ribeirão Preto-SP, Brazil. Phone: (5516) 36020243 E-mail address: [email protected]

ABSTRACT

The need for efficient and reliable technologies for clinical-scale expansion of mesenchymal stromal cells (MSC)

has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood-

derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0x107 cells, after 7

days of culture, it was possible to produce of 4.2(±0.8)x108 cells, which represents a fold increase of 7.0 (±1.4).

After enzymatic retrieval from Fibra-Cell disks, the cells were able to maintain their potential for differentiation into

adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90 and CD105). The

results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel

approach presents several advantages over the current expansion systems, based on culture flasks or

microcarrier-based spinner flasks and represents a key element for MSC cellular therapy according to GMP

compliant clinical-scale production system.

Key words: mesenchymal stromal cell, cell expansion, disposable culture system.

1. Introduction

Therapies based on Mesenchymal stromal cells have been emerging as a leading alternative for a number

of diseases due to the potential to differentiate and replenish specialized tissues1. To meet this high demand,

efficient, reliable and cost effective expansion methods should be developed2,3. MSC has been traditionally

expanded in single or multilayered culture flasks. The expansion procedure for this technology is based on

enzymatic passages, done manually, and re-seeded into larger number of flasks. Although effective, this

Cell Culture and Tissue Engineering Biotechnology ProgressDOI 10.1002/btpr.1707

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article asdoi: 10.1002/btpr.1707

© 2013 American Institute of Chemical Engineers Biotechnol ProgReceived: Oct 01, 2012; Revised: Feb 06, 2013; Accepted: Feb 11, 2013

technology has limited scale-up potential due to its open nature, associated safety risk, high labor costs and

requirement for expensive infrastructure3.

A fixed bed-based culture system is a type of bioreactor that presents several advantages regarding cell

culture: cell–cell or cell–matrix interactions are possible providing a better mimic of the in vivo intricate structure4

and a low shear stress environment with a high aeration rate and high surface area is provided for cell growth. In

this paper, we demonstrated the feasibility of a disposable fixed bed culture system for efficient expansion of

human cord blood-derived MSC (hCBMSC). It consists of an immobilized scaffold (Fibra-Cell disks, New

Brunswick Scientific) in a disposable sterile bottle wherein cells are seeded. This is an automated system, which

slowly raises or lowers the medium level in the bottles by collapsing or expanding the bellows at the base of the

bottle. During compression, the culture medium flows to the cells providing nutrients and removing unwanted

metabolites. On expansion, the culture medium returns to the bottle, allowing an adequate gas exchange to the

cells. The main advantage of this bioreactor is being disposable, single use and thus facilitates the regulation and

quality assurance of the expansion process5.

In this study we investigated, for the first time, the feasibility of hCBMSC expansion in a disposable fixed

bed culture system (FibraStage®, New Brunswick Scientific) that fulfills the requirements for production of cells for

therapy purposes.

2. Material and Methods

2.1 Cell culture

Human umbilical cords were collected (n = 5) from full-term newborns (39 – 40 weeks) after Cesarean

births, after informed consent from the person legally responsible for the donor. The mesenchymal cell isolation

protocol from umbilical cords was described by de Lima Prata et al., 20126. Ethical approval was obtained from

the Institutional Ethical Review Board (Hospital das Clínicas da Faculdade de Medicina de Ribeirao Preto -

Universidade de São Paulo, protocol HCRP number 14906/2010).

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2.2 Monolayer cultivation

hCBMSC were expanded on T-flasks (75 cm2) prior to fixed bed inoculation using Alpha Minimum

Essential Medium (α-MEM) (GIBCO, Grand Island, New York) containing 10% of fetal bovine serum (v/v) (FBS)

(HyClone, Canada), 4.5 mM HEPES (GIBCO, Burlington, Canada), 26.2 mM sodium bicarbonate (MERCK,

Darmstadt, Germany) and 1% penicillin/streptomycin (GIBCO, Burlington, Canada). The cells were grown at

37°C under a 5% CO2 humidified atmosphere. In each passage, cells were detached using TrypleTM Express

(GIBCO, Grand Island, New York).

2.3 Fixed bed culture system

After obtaining 6.0x107 cells by growing on T-flasks, cells at the passage 5 were inoculated in the

FibraStage bottle (presterilized and preloaded with 10 g of Fibra-Cell disks by manufacturer) with 500 mL of

culture medium α-MEM (1.2x105 cells/mL). The seeding phase lasted 6 hours. To evaluate the cell adhesion in

Fibra-Cell disks, supernatant samples were taken at each two hours to verify cell density and viability in

suspension. The fixed bed culture system was kept in incubator at 37 °C, 5% CO2 and 85% of humidity. The

experiments were carried out using the parameter Up 1.0 mm/sec – Top Hold Time: 10 seconds; Down 1.0

mm/sec - Bottom Hold Time: 30 minutes during culture time. Media changes (50%) were performed every day

starting on day 5. Samples of Fibra-Cell disks and supernatant were taken every day and assessed for cell

growth and metabolism characterization, respectively. After 7 days of cultivation, the bed-containing cells were

washed twice with PBS for 10 minutes and 300 mL of TrypleTM were then added for 30 minutes. During this

process the equipment parameters were set up to: Up: 1.0 mm/sec – Top Hold Time: 5 minutes; Down 1.0

mm/sec - Bottom Hold Time: 0 sec. The number of cells recovered after the harvesting procedure was estimated

by Neubauer hemacytometer.

2.4 Cell proliferation and metabolism analysis

Cell growth on Fibra-Cell disks was detected by MTT assay (Sigma-Aldrich, Saint Louis, USA). Therefore,

samples of 10 disks were placed with 200 µL α-MEM without phenol red and incubated with 20 µL of MTT (3-

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(4,5-dimethylthiazol-2-yil)-2,5-diphenyltetrazolium bromide) (5 mg/mL in PBS) at 37 °C for 4 hours. The

absorbance at 570 nm was recorded under a microplate reader (Multiskan FC, Thermo Scientific).

Glucose, lactate and glutamine concentrations were determined in the supernatant samples collected

throughout the experiments using an automatic analyzer (YSI 2700, Yellow Springs Instruments, Ohio, USA).

Ammonia concentrations were determined in the supernatant samples using ion selective electrode (Thermo

Scientific, ORION 720A+).

2.5 Flow cytometry

hCBMSC cells were harvested using TrypleTM and divided into aliquots of 100mL of PBS with 2x105cells.

Immunolabelling was performed according to standard indirect immunocytochemistry protocols, using primary

antibodies Anti- Anti-CD13–APC, Anti-CD14–PE, Anti-CD29–APC, Anti-CD31–FITC, Anti-CD34–APC, Anti-

CD44–FITC, Anti-CD49e–PE, Anti-CD73-PE, Anti-CD90–PE, Anti-CD105-PerCP, Anti-CD166–PE and Anti–

HLA–DR (PerCP) (all from Becton Dickinson, San Jose, CA, USA). The cells were analyzed on a FACScaliburTM

Flow cytometer (Becton Dickinson, San Jose, CA, USA). Ten thousand events were captured and analyzed using

CELLQuestTM (Becton Dickinson, San Jose, CA, USA).

2.6 Differentiation of hCBMSC

4x104 cells were seeded in 24-well plates and cultivated in expansion medium (α-MEM plus 7.5% FBS)

until almost confluence reached. The following day (adipogenic differentiation), the medium was switched to

differentiation medium consisting of α-MEM plus 15% FBS supplemented with 400 µL insulin (Sigma-Aldrich,

Saint Louis, USA), 100 µL indomethacin (Sigma-Aldrich, Saint Louis, USA) and 2 µL dexamethasone (Sigma-

Aldrich, Saint Louis, USA) and cultivated for 3 days. The medium was changed at each 2 days with medium

described above until day 21. For osteogenic differentiation, the same method was applied and in the following

day, the medium was switched to differentiation medium consisting of α-MEM plus 7.5% FBS supplemented with

1 mL β-glycerol phosphate (Sigma-Aldrich, Saint Louis, USA), 1 mL L-ascorbic acid (Sigma-Aldrich, Saint Louis,

USA) and 2 µL dexamethasone and cultivated for 3 days. The medium was changed at each 2 days with medium

described above until day 28.

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2.7 Scanning Electron Microscopy (SEM)

To observe morphology of cells attached to the Fibra-Cell disks, 3 cell-disks complexes were fixed in 2.5%

(v/v) glutaraldehyde (Sigma-Aldrich, Saint Louis, USA) and 4.0% (v/v) paraformaldehyde for 1 hour. After that, the

samples were washed three times with cacodilate buffer (0.2 M, pH 7.2) (Sigma-Aldrich, Saint Louis, USA) for 10

minutes and were immersed in osmium tetroxide (1%) (Sigma-Aldrich, USA) for 1 hour. Then, the samples were

dehydrated through a graded ethanol series (50%, 70%, 95%) for 15 min each and twice at 100%. The samples

were dried by using the critical-point-dryer CPD030 (BAL-TEC AG, Balzers, Liechtenstein). The dried samples

were sputter coated with gold and examined with a scanning electron microscope High Vacuum JSM-6610LV

(JEOL).

3. Results and discussion

3.1 Cell expansion

With an initial seeding of 6.0x107 cells, hCBMSC were inoculated in the fixed bed culture system, using α-

MEM medium supplemented with 10% FBS (v/v). The growth characteristics of MSC on Fibra-Cell disks were

monitored by MTT assay during cultivation (n=3, three different donors) (Fig. 1A). After an inoculation phase of 6

hours, with seeding efficiency higher than 95%, it was observed an intense cell growth (µmax=0.21 d-1, r2=0.985),

without the common 24 hour lag phase observed in microcarrier-based expansion systems7-12. The cell viabilities

remained above 95% and the pH in the optimal range (7.2-7.4) during entire cultivation. The amount of

suspended cells was lower than 10% of the total of adhered cells. At day 7, the maximal cell density of

4.15(±0.810)x108 cells was obtained, corresponding to 7.0(±1.4)-fold increase approximately (Fig. 1B). Despite

the high expansion level, it was possible at the end of culture to retrieve only 18% (±0.77) of the cells. It was

observed after 2 cycles of TrypleTM treatment that the majority of the cells remained attached to Fibra-Cell disks.

This low cell recovery may be attributed to insufficient time of TrypleTM treatment and to gentle platform motion

which prevents efficient enzyme diffusion throughout the bed. Parameters such as the type of enzyme used,

needed for chelating agent, treatment temperature and time must be tested to improve the cell harvest. As well

as in cell cultivation on microcarriers, it is crucial to identify an optimal enzymatic protocol to maximize the

quantity and the quality of cells at the harvest13. The optimization of the culture conditions, which includes new

harvest protocols, will be further developed.

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The morphology of the hCBMSC during proliferation was shown by SEM (Scanning Electron Microscopy).

It was observed a gradual increase in Fibra-Cell disks occupancy by the cells during the days 3, 5 and 7 after

inoculation (Fig. 1C).

3.2 Metabolism

In order to prevent metabolic limitations, the feeding regimen of the experiments consisted of removing

50% of the exhausted culture medium every day after day 5 and replacing with fresh medium. The concentration

of nutrients (glucose and glutamine) and metabolites (lactate and ammonia) in the supernatant was measured

throughout cultivation (Fig. 2). During the period in culture, glucose and glutamine concentrations did not

decrease below 0.26 and 0.08 mM, respectively (Fig. 2A and B). As a result of glucose and glutamine

consumption, lactate and ammonia were produced, reaching a maximum concentration of 10.4 and 1.3 mM at

day 5, respectively. The inhibitory concentrations for human MSC are reported to be 28 mM for lactate14 and

4mM for ammonia15, and these values were not observed in the experiment.

Metabolic quotients provide an efficient way of characterizing cell metabolism patterns in terms of

consumption and production rates per viable cells. Table 1 presents the metabolic parameters, glucose (qgluc) and

glutamine (qglut) specific consumption rates; lactate (qlac) and ammonia (qammo) specific production rates and

apparent yield of lactate from glucose (Ylact./gluc.) estimated during exponential growth phase. As reported in

literature for MSC expansion on microcarriers2,9,15, qgluc was considerably higher than qglut. Consequently, qlact was

also higher than qammo. The Ylact./gluc. provides an estimate of the fraction of glucose converted into lactate via

glycolysis14. The Ylact./gluc. value (1.09 mollac./molgluc.) was lower than theoretical maximum (2.0 mollac./molgluc.) and

also lower than reported for microcarrier-based cultures2,8,15, indicating a more efficient energy metabolism.

3.3 Characterization of the cells after harvesting

After cultivation in fixed bed culture system, the cells were enzymatically harvested from the disks and

characterized regarding differentiated potential and immunophenotype analysis. The results of immunophenotype

analysis revealed that these expanded cells were positive for several markers common to MSC. No significant

difference in the percentage of positive cells was observed before and after cell expansion (p < 0.05) (Fig. 3A).

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Furthermore, hCBMSC expanded were able to differentiate into adipogenic (Fig. 3B) and osteogenic (Fig. 3C)

lineages.

4. Conclusion

In this study, we have successfully demonstrated the feasibility of hCBMSC expansion in a disposable

fixed bed culture system, while retaining immunophenotypic characteristics and differentiation potential. This

alternative approach enables reliable and high-level MSC expansion in only one homogenous culture bottle,

avoiding the use of several static cultures flasks. The amount of cells expanded (4.15x108 cells) are sufficient to

infuse 6 patients (70 Kg) assuming 1x106 cells/kg patient and to produce the same amount of cells it will be

necessary to use 120 75 cm2 culture flasks.

Acknowledgments

This work was financed by FINEP (0108059100) and BNDES (09.2.0706.1). We thank Patricia Vianna

Bonini Palma and Danielle Aparecida Rosa de Magalhães for their technical assistance.

References

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List of figures

Fig. 1. Cell proliferation of hCBMSC (n=3, mean±SD). (A) Growth profile; (B) Cell expansion in terms of fold increase in total cell number and (C) Scanning electron microscopic (SEM) photographs (150x) of hCBMSC on Fibra-Cell disks at 3, 5 and 7 days after inoculation. Fig. 2. Nutrient and by-product profiles during the culture of hCBMSC. 50% of the medium was changed every day from day 5.

Fig. 3. (A) Immunophenotype analysis and (B and C) differentiated potential of hCBMSC after

expansion in Fibrastage culture system. (B) Adipocytes at day 21 and (C) osteocytes at day 28 post differentiation induction.

List of table

Table 1. Metabolic parameters for expansion of hCBMSC.

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Parameter Value (mean±SD)

qglu (pmol/cell.day) -2.90 ± 0.32

qlact (pmol/cell.day) 3.16 ± 0.22

qglut(pmol/cell.day) -0.14 ± 0.43

qammo(pmol/cell.day) 0.40 ± 0.16

Ylac/gluc(mmol/mmol) 1.09 ± 0.020

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Cell proliferation of hCBMSC (n=3, mean±SD). (A) Growth profile; (B) Cell expansion in terms of fold increase in total cell number and (C) Scanning electron microscopic (SEM) photographs (150x) of hCBMSC

on Fibra-Cell disks at 3, 5 and 7 days after inoculation. 115x76mm (300 x 300 DPI)

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Nutrient and by-product profiles during the culture of hCBMSC. 50% of the medium was changed every day from day 5.

172x71mm (300 x 300 DPI)

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(A) Immunophenotype analysis and (B and C) differentiated potential of hCBMSC after expansion in Fibrastage® culture system. (B) Adipocytes at day 21 and (C) osteocytes at day 28 post differentiation

induction

173x85mm (300 x 300 DPI)

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Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

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