Growth and Functional Harvesting of Human Mesenchymal Stromal Cells

Cultured on a Microcarrier-Based System

Samia R. Caruso, Maristela D. Orellana, Amanda Mizukami, and Taisa R. FernandesHemotherapy Center of Ribeir~ao Preto, School of Medicine of Ribeir~ao Preto, University of S~ao Paulo, Tenente Cat~ao Roxo Street,2501, CEP 14051-140, Ribeir~ao Preto-SP, Brazil

Aparecida M. FontesHemotherapy Center of Ribeir~ao Preto, School of Medicine of Ribeir~ao Preto, University of S~ao Paulo, Tenente Cat~ao Roxo Street,2501, CEP 14051-140, Ribeir~ao Preto-SP, Brazil

Dept. of Genetics, School of Medicine of Ribeir~ao Preto, Caf�e av w/n. CEP 14040-903, Ribeir~ao Preto-SP, Brazil

Claudio A. T. SuazoDept. of Chemistry Engineering, Federal University of S~ao Carlos, Washington Lu�ıs road, km 235, S~ao Carlos, Brazil

Viviane C. OliveiraDept. of Dental Materials and Prosthodontics, Faculty of Odontology of Ribeir~ao Preto, University of S~ao Paulo, Caf�e av w/n. CEP14040-903, Ribeir~ao Preto-SP, Brazil

Dimas T. CovasHemotherapy Center of Ribeir~ao Preto, School of Medicine of Ribeir~ao Preto, University of S~ao Paulo, Tenente Cat~ao Roxo Street,2501, CEP 14051-140, Ribeir~ao Preto-SP, Brazil

Dept. of Medical Clinic, Faculty of Medicine of Ribeir~ao Preto, Caf�e av w/n. CEP 14040-903, Ribeir~ao Preto-SP, Brazil

Kamilla SwiechDept. of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences of Ribeir~ao Preto, University of S~ao Paulo, Caf�e av w/n. CEP14040-903, Ribeir~ao Preto-SP, Brazil

DOI 10.1002/btpr.1886Published online in Wiley Online Library (wileyonlinelibrary.com)

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engi-neering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses ofhMSCs necessary for infusion, scalable and cost-effective technologies for in vitro cell expansionare needed to produce MSCs while maintaining their functional, immunophenotypic and cytoge-netic characteristics. Microcarrier-based culture systems are a good alternative to traditional sys-tems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess forthe expansion of human bone marrow mesenchymal stromal cells (hBM-MSCs) on microcarriers tooptimize growth and functional harvesting. In general, the results obtained demonstrated the feasi-bility of expanding hBM-MSCs using microcarrier technology. The maximum cell concentration (n5 5) was �4.82 6 1.18 3 105 cell mL21 at day 7, representing a 3.9-fold increase relative to theamount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability 595%). Cell metabolism analysis revealed that there was no depletion of important nutrients such asglucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed atinhibitory concentrations. The cells that were recovered after the expansion retained their immuno-phenotypic and functional characteristics. These results represent an important step toward theimplementation of a GMP-compliant large-scale production system for hMSCs for cellular therapy.VC 2014 American Institute of Chemical Engineers Biotechnol. Prog., 000:000–000, 2014Keywords: mesenchymal stromal cells, cell expansion, microcarrier culture, spinner flasks,functional harvest

Introduction

Human mesenchymal stromal cells (hMSCs) have greatpotential for various therapeutic applications due to the ease

with which they can be isolated and expanded in cell culture

and their ability to differentiate into specialized cell types of

different lineages and produce immunomodulatory effects.1–3

However, due to the low availability of hMSCs in tissues (as

example, in bone marrow 1:104 cells in young ages, decay-

ing with age)4 and the high doses necessary for an infusion

(about 106 cells kg21 patient5, the key to these successfulCorrespondence concerning this article should be addressed to

K. Swiech at kamilla@fcfrp.usp.br.

VC 2014 American Institute of Chemical Engineers 1

cell-based therapies relies on the development of robust, effi-

cient and scalable processes that maintain the biological

properties of these cells while controlling the cost and time

required to produce a sufficient quantity of cells.

Human mesenchymal stromal cells have been expanded intraditional two-dimensional cultures in static single or multi-layered culture flasks. Although efficient and reliable, suchmethods involve a heterogeneous culture environment, arevery labor intensive, space and time-consuming, and havelimited scale-up potential.6 To expand hMSCs in a scalableand cost-effective manner, a large cell surface is needed toenable cell attachment in a controlled and homogenous way,thereby minimizing supply costs. In addition, a closed cul-ture system is necessary for the production of cells inaccordance with good manufacturing practice (GMP).Microcarrier-based technology meets these criteria and couldbe a realistic option for the next generation of scaled-uphMSC production processes.6

Microcarriers are small, spherical beads (<200 lm diame-ter) with a large surface area and can be maintained in suspen-sion in a stirred bioreactor.6–9 This technology has beensuccessfully used for the growth of anchorage-dependent celllineages for the production of vaccines and biopharmaceuticalsas well as to expand primary cells. Many microcarriers, bothcommercially available and those developed for specific appli-cations, with different characteristics are now in use for celltherapy and tissue engineering applications.10 Commerciallyavailable microcarriers are porous or nonporous and are com-posed of gelatin, glass, collagen, or cellulose with a diameterof 170–6.000 lm. To further improve cell-culture perform-ance, these microcarriers can be functionalized with differentcoating materials (e.g., ECM proteins and small molecules).11

This culture system also provides a high ratio of growth sur-face to medium volume. As an example, 1 g of microcarrier(Cytodex 1) provides a surface area of 4400 cm2, which repre-sents approximately fifty-eight 75-cm2 static flasks.10

The expansion of hMSCs in this culture system has beenwidely reported.12–15 Although these studies have demon-strated the feasibility of hMSC expansion in microcarrier-based systems, the performance of this approach is relativelylow and highly variable compared with conventional staticcultures. Therefore, further improvements are needed beforethis technology can be implemented for the robust and repro-ducible production of hMSCs.6 Thus, the aim of the presentwork was to develop culture conditions using a Cytodex 3microcarrier for optimized growth and functional harvest ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs). We employed 7- and 10-day cultures and measuredthe harvest efficiency and the functional characteristics ofthe cells at the end of the culture.

Materials and Methods

Monolayer cell cultivation

The bone marrow used in this study was extracted fromthe iliac crest of five healthy donors (n 5 5) after providinginformed consent as approved by the ethics committee of theUniversity of S~ao Paulo, Ribeir~ao Preto, Brazil. The mononu-clear cells from the bone marrow were separated using theFicoll-Paque (GE Healthcare, BioSciences, Uppsala, Sweden)1.077 density gradient centrifugation technique and were sus-pended in alpha-minimum essential medium (a-MEM—GIBCO, New York, EUA) supplemented with 15% fetal

bovine serum (v/v) (FBS) (HyClone, Canada), 1% penicillin/streptomycin (GIBCO, NY), and 2 mM glutamine (GIBCO,New York, EUA). All cultures were maintained at 37�C in a5% CO2 humidified incubator, and non-adherent cells wereremoved from cultures after 5 days. The monolayer cultureswere maintained for 3 weeks, and the medium was exchangedevery 3 days. Upon reaching 90% confluence, the cells weretrypsinized with 0.2% trypsin-EDTA (Sigma–Aldrich, MO)and subcultured for further experiments. Cells at passages 3–5were used for microcarrier cultivation.

Microcarrier cultivation in spinner flaks

For microcarrier cultures, 125-mL siliconized spinnerflasks (Techne, United Kingdom) were used with a workingvolume of 50 mL and a stirring speed of 50 rpm. The cul-tures were maintained in a humidified atmosphere of 5%CO2 at 37�C. The appropriate quantities of the Cytodex 3microcarrier (GE Healthcare, BioSciences, Sweden) wereweighed (3.0 g L21), hydrated with PBS and sterilized byautoclaving as recommended by the manufacturer. To seedthe microcarriers, passage 3–5 cells were harvested from tis-sue culture flasks. A total of 6.25 3 106 cells were inocu-lated on microcarriers at 1/3 of the final working volume.The stirring regime comprised a 2-min stirring phase at 30rpm, followed by a rest phase (0 rpm) for 30 min. After 6 h,the medium was added to a final working volume of 50 mL,followed by continuous stirring at 50 rpm. During the cultureperiod, 50% of the medium was replaced with fresh mediumevery day starting from day 2. The experiments were con-ducted for 7 and 10 days. To monitor cell growth and metab-olism in the microcarrier cultures, samples were removeddaily. The results are expressed as the mean 6 SD of fiveindependent experiments (n 5 5).

Cell growth and metabolism analysis

Cell culture samples were collected daily. The concentra-tion of cells in the suspension was determined using a hemo-cytometer after diluting the samples with Turk’s solution (adye composed of acetic acid and gentian violet) under aphase contrast microscope (Olympus, IX31). The concentra-tion of cells attached to the microcarriers was also evaluatedby counting with a hemocytometer after treatment withTryple

VR

Express (GIBCO, NY, EUA) for 15 min to detachcells. Cell viability was determined using a FACScaliburTM

Flow cytometer (Becton Dickinson, San Jose, CA) with pro-pidium iodide.

Analyses of glucose, glutamine, lactate and amino acidswere performed by high-performance liquid chromatography(HPLC). For glucose and lactate, an Aminex HPX-87H col-umn (Bio-Rad, Richmond, CA) was used with 5 mM H2SO4

(Sigma, St. Louis, MO, EUA) as the eluent. Analyses wereperformed on a Waters chromatography system (WatersAssociates, Milford, MA) with detection by refractive indexfor carbohydrates (W410) and 210 nm UV for lactate. Theequipment was operated at a temperature of 65�C and a flowrate of 0.6 mL min21.

Amino acids analyses were based on the method proposedby Henrikson and Meredith (1984). A pico-tag column(Waters, Milford, MA) was used as the stationary phase, anda gradient consisting of mobile phases A and B was used asthe eluent. Phase A contained sodium acetate trihydrate

2 Biotechnol. Prog., 2014, Vol. 00, No. 00

(Sigma, St. Louis, MO, EUA), triethylamine (Sigma, St.Louis, MO, EUA), deionized water and acetonitrile (Sigma,St. Louis, MO, EUA). Phase B contained only deionizedwater and acetonitrile. The chromatography operating condi-tions were as follows: a temperature of 36�C, a 45-min runtime and a gradient with predetermined flow. The peakswere detected at a wavelength of 254 nm.

The ammonia concentrations in the supernatant sampleswere determined with an ion-selective electrode (ThermoScientific, ORION 720A1).

The glucose- and glutamine-specific consumption (qglc andqgln) and lactate and ammonia production (qlac and qammo)rates were estimated during the exponential growth phaseand calculated as a mean value for each time interval usingthe following equation: qmet 5 (Dmet/Dt).Xv, where Dmet isthe change in the nutrient/metabolite concentration duringthe time interval (Dt) and Xv is the average viable cell num-ber during the same time period.1

Cell harvesting

To harvest the cells, the cell-microcarrier suspension wasremoved from the spinner flask and rinsed twice with PBS.Treatment with Tryple

VR

Express (GIBCO, NY, EUA) wasthen performed for 15 min in a 37�C water bath. Occasionalflicking was performed to facilitate the detachment of cells.The suspension was then passed through a 100-mM cellstrainer (BD Biosciences, Bedford, MA), centrifuged andresuspended in fresh medium for further analysis.

The harvesting efficiency was determined based on thenumber of cells in the sample and the number of cells recov-ered at the end of the culture.

Cell characterization

Flow Cytometry. At the third passage before and aftergrowth on microcarriers, cell-surface antigens were analyzed inthe hBM-MSC samples by staining with specific monoclonalantibodies. Immunolabeling was performed according to stand-ard indirect immunocytochemistry protocols using the primaryantibodies CD 73-PE (SH3/SH4), HLA class 1-PE, CD90-PE(Thy-1), CD 105-FITC (SH2), CD 146-FITC (MCAM) EP-CD49e (Integrin a5-), CD29-PE (b1-integrin), CD13-PE, PE-class II HLA, HLA-DR-FITC, CD31-FITC (PECAM), CD34-PE, CD45-FITC, and CD14-PE (all from Becton Dickinson,San Jose, CA), which represent markers of mesenchymal cells,hematopoietic cells and endothelial cells. The cells were ana-lyzed on a FACScaliburTM flow cytometer (Becton Dickinson,San Jose, CA). Ten thousand events were captured and ana-lyzed using CELLQuestTM (Becton Dickinson, San Jose, CA).

Multilineage Differentiation. Cells cultured on staticflasks (before microcarrier expansion) and cells harvested frommicrocarriers were seeded at 4 3 104 cells well21 in 24-wellcell culture plates containing 1 mL of culture medium. After 24h, adipocyte differentiation was induced in the cultures in a-MEM medium supplemented with 15% FBS, 1 mM dexametha-sone (Sigma-Aldrich, Saint Louis, USA), 2.5 mg mL21 insulin(Sigma–Aldrich, Saint Louis, USA) and 100 mM indomethacin(Sigma–Aldrich, Saint Louis, USA). For osteogenic differentia-tion, the same method was applied, except that the differentia-tion medium consisted of a-MEM plus 7.5% FBSsupplemented with 1 M b-glycerol phosphate (Sigma–Aldrich,Saint Louis, USA), 20 mM L-ascorbic acid (Sigma-Aldrich,Saint Louis, USA) and 0.1 mM dexamethasone. Control cul-

tures of 5 3 103 cells/well were grown in parallel in a-MEMplus 7.5% FBS without additives. The medium was replacedtwice each week. Adipogenesis was observed over a period of14 days, and osteogenesis was observed after 21 days. The cul-tures were then stained with Sudan II-Scarlet for fat dropletsand von Kossa for mineralized bone matrix deposition.

Inhibition of T-lymphocyte Proliferation. The potentialinhibition of T-lymphocyte proliferation was assessed beforeand after culture on the microcarriers. Peripheral blood mono-nuclear cells (PBMCs) stained with 2.5 mM CFSE (carboxy-fluorescein diacetate succinimidyl ester, Molecular Probes,USA) and stimulated with 1 mg/mL phytohemagglutinin(PHA) (Sigma-Aldrich, Saint Louis, USA) were co-culturedwith different hBM-MSC concentrations (1:2, 1:5, and 1:10)for 5 days. At the end of the experiment, the cells were stainedwith CD3-APC (Becton Dickinson, San Jose, CA) and ana-lyzed by flow cytometry. As a control, we used unlabeledPBMCs, labeled PBMCs and labeled, stimulated PBMCs.Triplicate samples were removed from each culture condition.

Cytogenetic Analysis. The cytogenetic profile of thehBM-MSCs (n 5 5) was evaluated after culture on microcar-riers. The cells were maintained in static flasks until �70% con-fluent. Colcemid (Sigma–Aldrich, Saint Louis, USA) was usedto disrupt the cell cycle in metaphase. The cells were fixed andprepared from at least five layers of each culture. The bandingprocess after staining with Giemsa was performed on the bladesthat presented adequate spreading of the chromosomes andgood-quality GTG banding (G-banding trypsin-Giemsa), withat least 400 bands (ideal, 550 bands) analyzed by conventionaloptical microscopy. For each sample, images of at least 50(fifty) cells in metaphase were captured and analyzed using thesoftware EXPO BANDView

VR

(Applied Spectral Imaging, CA).The karyotypes were described according to international crite-ria for chromosome nomenclature (International System Cyto-genetic for Human Nomenclature—ISCN, 2009).

Scanning Electron Microscope (SEM). To observe themicrocarrier occupancy by hBM-MSCs, cell-microcarriercomplexes were fixed in 2.5% (v/v) glutaraldehyde (Sigma-Aldrich, Saint Louis, USA) and 4.0% (v/v) paraformaldehydefor 1 h. The samples were then washed three times with caco-dylate buffer (0.2 M, pH 7.2) (Sigma–Aldrich, Saint Louis,USA) for 10 min and immersed in osmium tetroxide (1%)(Sigma–Aldrich, USA) for 1 h. Next, the samples were dehy-drated through a graded ethanol series (50, 70, and 95%) for15 min each and twice at 100%. The samples were dried usingthe critical-point-dryer CPD030 (BAL-TEC AG, Balzers,Liechtenstein), coated with gold and examined with a HighVacuum JSM-6610LV scanning electron microscope (JEOL).

Statistical analysis

The Mann–Whitney test with a nonparametric andunpaired method was used to analyze statistical differencesin all experiments. The Wilcoxon test was used with a non-parametric method for paired samples to analyze inhibitionof T-lymphocyte proliferation. In these tests, P < 0.05 wasconsidered statistically significant.

Results and Discussion

hBM-MSC expansion on the Cytodex 3 microcarrier

hBM-MSCs from five different donors (n 5 5) were cul-tured on Cytodex 3 microcarriers for 7 and 10 days in

Biotechnol. Prog., 2014, Vol. 00, No. 00 3

spinner flasks. Figure 1 presents the cell growth profileobtained in these experiments (mean 6 SD). The growthprofiles of the 7- and 10-day cultures were very similar andincluded a lag phase of �48 h followed by an exponentialphase (until day 5) with a high growth rate (lmax 5 0.036 6

0.004 h21, tD 5 19.2 h for the 7-day culture and lmax 5

0.042 6 0.002 h21, tD 5 16.5 h for the 10-day culture),leading to maximum cell concentrations at day 7 of 4.82 6

1.18 3 105 cells mL21 (7-day culture) and 4.23 6 0.65 3

105 cells mL21 (10-day culture), representing 3.9- and 3.4-fold increases (based on the amount of cells inoculated)respectively. This specific growth rate (lmax 5 0.036 6

0.004 h21 for the 7-day culture and lmax 5 0.042 6 0.002h21 for the 10-day culture), which was calculated based onthe average cell concentration, was higher than that of previ-ous studies (0.02 h21 for goat BM-MSCs cultured on theCytodex 1 microcarrier6; 0.013 h21 for hMSCs cultured onCultispher S12; 0.029 h21 for hMSCs cultured on CultispherS15). During the culture, a low concentration of suspendedcells was detected, and a stationary phase or death phasewas not observed until day 7. After day 7, a death phase wasobserved in the 10-day culture. In both the 7- and 10-daycultures, a high growth variation between donors (n 5 5)was observed, which might be attributable to cellular hetero-geneity among the donor population18–21 nonhomogenousaggregation.

The microcarrier occupancy by hBM-MSCs during prolif-eration was determined by SEM (Figure 2). The formationof cell-microcarrier aggregates began at day 5 and continueduntil the end of the culture period. As the number of daysincreased, the size and density of these aggregates increased,and the beads became completely populated with cells.Frauenschuh et al., Weber et al., and Schop et al. alsodescribed the formation of these cell-microcarrier aggregates.According to Schop et al., these aggregates formed due to anincrease in the number of cells/microcarrier. When no freshmicrocarriers are added to the culture, the cells will seek outother surfaces on which to grow, and consequently, they willadhere partly on one microcarrier and partly on anothermicrocarrier, causing cell aggregation. Aggregation mightalso be caused by cell–cell connections aided by the forma-tion of an extracellular matrix.22 Stirred cultures promote the

formation of aggregation because the microcarriers collidefrequently and the extracellular matrix acts like an adhesivematerial to connect the microcarriers, leading to cell aggre-gation. Aggregation could be minimized by adding freshmicrocarriers during the culture and increasing the stirringrate. The end of exponential growth was observed aroundday 5 of culture (Figure 1), possibly due to the formation oflarge cell aggregates, as shown in Figure 2; the cells trappedwithin these aggregates would no longer be able to receivesufficient nutrients and oxygen.

Several studies have also demonstrated satisfactory growthon Cytodex 3 microcarriers of human,12–14,23 murine,7,24 rab-bit,8 goat,6 porcine25 MSCs and embryonic stem cells.26–29

However, the majority of the studies did not describe theamount of cells harvested after expansion, which is anextremely important variable for the development of a scal-able and cost-effective expansion method.

Because the objective of this work was to establish cultureconditions leading to a better balance between cell growthand efficient harvest of functional cells, the cells were har-vested at the end of the culture period, and the efficiency ofthe procedure was measured. After 7 and 10 days of culture,87.2% (viability 5 95%) and 77.5% (viability 5 95%) ofthe expanded cells were harvested, respectively. For the 10-day culture, both the final cell concentration and the

Figure 1. Cell proliferation of hBM-MSCs cultivated on theCytodex 3 microcarrier in spinner flasks for culturedurations of 7 and 10 days. The results areexpressed as the mean 6 SD of five independentexperiments (n 5 5).

Figure 2. Scanning electron microscopic (SEM) photographs(3150) of the Cytodex 3 microcarrier without cells(A and B) and with hBM-MSCs at 3 (C and D), 7 (Eand F) and 10 days (G and H) after inoculation.

4 Biotechnol. Prog., 2014, Vol. 00, No. 00

harvesting efficiency were lower than for the 7-day culture,possibly due to a higher level of cell-microcarrieraggregation.

hBM-MSC metabolism

The metabolism analysis was performed only for the 7-day culture due to the better balance of cell growth and har-vesting efficiency. The concentrations of nutrients (glucoseand glutamine) and metabolites (lactate and ammonia) in thesupernatant were measured throughout the culture period(Figure 3), and specific consumption (qglc and qgln) and pro-duction (qlac and qammo) rates during the exponential growthphase were estimated. The concentrations of glucose andglutamine did not decrease beyond 2.1 and 1.4 mM, respec-tively; these concentrations are higher than the respectiveMonod constants, KS (�0.1–0.4 for glucose and 0.08–0.15mM for glutamine), for mammalian cells due to the replace-ment of the medium.12 Glucose and glutamine consumptionresulted in the production of lactate and ammonia. The maxi-mum concentrations of lactate (15 mM) and ammonia (0.7mM) were obtained on day 5. The reported inhibitory con-centrations for hMSCs are 35.4 mM for lactate and 2.4 mMfor ammonia6; these values were not observed in the experi-ments. In addition, throughout the culture period, no aminoacid exhaustion was observed (data not shown).

Metabolic quotients are an efficient means of characteriz-ing cellular metabolic patterns in terms of consumption andproduction rates per cell. During the exponential growthphase, qglc was 1.86 pmol/cell/day, which was significantlyhigher than the qgln value of 0.17 pmol/cell/day, in accord-ance with previous reports.6,12,27 According to Schop et al.,glutamine is not an important energy source for MSCs; theseauthors observed that the rate at which glutamine disap-peared from the medium was similar to the rate of spontane-ous decomposition. Consistent with this result, we observedthat qlact (4.04 pmol/cell/day) was also higher than qammo

(0.71 pmol/cell/day).

Characterization of microcarrier-expanded MSCs

The differentiation and inhibition of T-lymphocyte prolifer-ation potential and the immunophenotypic characteristics ofthe expanded cells were assessed after 7 days of culture; theexpanded cells were also subjected to cytogenetic analysis.

As expected, the microcarrier-expanded cells maintainedtheir characteristic immunophenotype (>90% positive forCD73, CD90, and CD105); no significant difference (P <0.05) was observed before and after expansion (n 5 5) (Fig-ure 4A). The cells also retained their osteogenic and adipo-genic differentiation potential (Figure 4B). Differentiationinto osteocytes was confirmed by accumulation of crystals ofcalcium oxalate stained with von Kossa and could bedetected at day 15 after differentiation induction. Adipocyteswere characterized by the formation of fat droplets, whichwere stained by Sudan II-Scarlet. There was no qualitativedifference in the differentiation into adipocytes and osteo-cytes of the hBM-MSC samples before and after cultivationon microcarriers.

We also examined the karyotype of the microcarrier-expanded hBM-MSCs to assess potential genetic instability.No clones with chromosomal abnormalities were identified.

The assay of T-lymphocyte proliferation inhibition in vitrois a functional test because hMSCs possess immunomodula-tory properties.30 MSCs exert an immunosuppressive effecton mixed lymphocyte cultures; high proportions of MSCssuppress T cell reactivity and reduce lymphocyte prolifera-tion in response to specific stimuli. Different concentrationsof MSCs/lymphocytes (1:2, 1:5, 1:10) were evaluated, andwe observed that the MSCs were able to inhibit lymphocyteproliferation efficiently under all conditions. The mean per-centage of lymphocyte inhibition by hBM-MSCs (n 5 5)before and after cultivation was 81.45 and 88.27% (P 5

0.06), respectively (Figure 5). There was a significantincrease in the potential for inhibition of lymphocyte prolif-eration in samples taken after microcarrier expansion, imply-ing a greater immunosuppressive capacity of these cells afterculture. This study is the first to use this functional test toevaluate the function of cultured hMSCs.

Conclusion

We have successfully demonstrated the growth and func-tional harvesting of hBM-MSCs cultivated on a Cytodex 3microcarrier. This culture system enables MSC expansion ina single homogenous culture flask, thereby avoiding the useof several static flasks, while retaining immunophenotypiccharacteristics, differentiation potential, genetic stability aswell as inhibition of cell proliferation potential. On the basis

Figure 3. Metabolic profiles of the hBM-MSCs cultured on Cytodex 3 microcarriers in spinner flasks for 7 days. The results areexpressed as the mean 6 SD of five independent experiments (n 5 5).

Biotechnol. Prog., 2014, Vol. 00, No. 00 5

of our data, we obtained a total cell production of 2.1 3 107

cells (considering a concentration of 4.82 6 1.18 3 105 cellmL21 in 50 mL at day 7 and a harvesting efficiency of87.2%), and thus we estimate that scaling up to a 1-L stirredbioreactor will enable the production of �4.2 3 108 cells,the amount required to infuse 2 70-kg patients with a celldose of 2 3 106 cell kg21. However, scaling up this methodis not straightforward, mainly due to shear stress. Theseresults represent an important step toward the implementa-tion of a GMP-compliant large-scale production system forMSCs for cellular therapy.

Acknowledgments

The authors acknowledge the financial support of theNational Institute of Science and Technology in Stem Celland Cell Therapy (CNPq 573754-2008-0; FAPESP 2008/578773) and FINEP 0108059100 – BNDES 09.2.0706.1.They also thank Karina Solano Candido, Mario Soares deAbreu Neto, Patricia Viana Bonini, Rose Eli Grassi, andMaria Dolores Seabra Ferreira for their technical assistance.

Figure 4. (A) Immunophenotypic analysis and (B) differentiation potential (adipogenic and osteogenic) of hBM-MSCs after expansionon the Cytodex 3 microcarrier.

Figure 5. Inhibition of the proliferation potential of hBM-MSCs after expansion on the Cytodex 3 microcar-rier. * Indicates a statistically significant difference.

6 Biotechnol. Prog., 2014, Vol. 00, No. 00

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