Cell proliferation of human bone marrow mesenchymal stem cells on
biodegradable microcarriers enhances in vitro differentiation potential
L.-Y. Sun*, D.-K. Hsieh†, W.-S. Syu‡, Y.-S. Li‡, H.-T. Chiu* and T.-W. Chiou‡
*Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, †Department of Applied Chemistry, Chaoyang University of Technology, Taichung, Taiwan, and ‡Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan
Received 17 October 2009; revision accepted 12 January 2010
Abstract
Objectives: For reasons of provision of
highly-specific surface area and three-dimensional culture,
microcarrier culture (MC) has garnered great interest
for its potential to expand anchorage-dependent stem
cells. This study utilizes MC for in vitro expansion
of human bone marrow mesenchymal stem cells
(BMMSCs) and analyses its effects on BMMSC
proliferation and differentiation.
Materials and methods: Effects of semi-continuous
MC compared to control plate culture (PC) and serial
bead-to-bead transfer MC (MC bead-T) on human
BMMSCs were investigated. Cell population growth
kinetics, cell phenotypes and differentiation potential
of cells were assayed.
Results: Maximum cell density and overall fold
increase in cell population growth were similar
between PCs and MCs with similar starting
condi-tions, but lag period of BMMSC growth differed
substantially between the two; moreover, MC cells
exhibited reduced granularity and higher CXCR4
expression. Differentiation of BMMSCs into
osteo-genic and adipoosteo-genic lineages was enhanced after
3 days in MC. However, MC bead-T resulted in
changes in cell granularity and lower osteogenic and
adipogenic differentiation potential.
Conclusions: In comparison to PC, MC supported
expansion of BMMSCs in an up-scalable
three-dimensional culture system using a semi-continuous
process, increasing potential for stem cell homing
ability and osteogenic and adipogenic differentiation.
Introduction
Bone marrow mesenchymal stem cells (BMMSCs) have been recognized to constitute a powerful tool in regenera-tive medicine due to their multi-lineage differentiation ability (1,2) and their capacity for tissue repair (3–5). However, as a successful cell treatment requires at least 109functional cells per human adult patient (6,7), human BMMSC therapies would not be a viable option without a practical and up-scalable bioprocess that allows expansion and recovery of high-quality cells.
Extended expansion of mesenchymal stem cells (MSCs) in two-dimensional plate culture (PC) does not produce sufficient numbers of cells for therapeutic appli-cations and leads to their senescence and loss of multipo-tency (8–10). Recently, microcarriers in a suspension culture system have been shown to facilitate expansion of MSCs, that require attachment, and to provide an environ-ment that can be easily controlled and monitored (11–13).
MSCs can attach and proliferate on Cytodex 1, a gen-eral-purpose non-biodegradable microcarrier for anchor-age-dependent cell lines (11). However, highly efficient separation of the cells from Cytodex 1 after enzyme treat-ment is difficult to achieve and Cytodex 1 cannot be degraded by the human body (14). Thus, biodegradable substances such as gelatin-based CultiSpher microcarriers have elicited more interest and have been extensively studied for MSC culture (13,15).
Recently, multipotency of rat MSCs has been shown to be preserved in cultures with Cultispher-S (8· 105beads⁄ g) microcarriers using semi-continuous (13) or bead-to-bead transfer (15) process. In comparison with Cultispher-S, CultiSpher-G (1· 106beads⁄ g) pro-vides a higher specific surface area (surface area per
Correspondence: T.-W. Chiou, PhD, Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, No. 1, Sec. 2, Da Hsueh Rd., Shou-Feng, Hualien, Taiwan. Tel.: 886-3-8633638; Fax: 886-3-8630398; E-mail: twchiou@mail. ndhu.edu.tw
H.-T. Chiu, PhD, Department of Biological Science and Technology, National Chiao Tung University, No. 75, Po-Ai St., Hsinchu, Taiwan. Tel.: 886-3-5131595; Fax: 886-3-5719605; E-mail: chiu@mail.nctu.edu.tw
gram) and it is composed of cross-linked porcine gelatin. These microcarriers can be dissolved with trypsin-EDTA or collagenase and almost all cells grown on them can be recovered for cell transplantation or tissue engineering (14).
The aim of this study was to analyse different charac-teristics of human BMMSCs after CultiSpher-G microcar-rier culture (MC) and enzyme treatment compared to human BMMSCs maintained in PC. We investigated influence of MC on cell proliferation, morphology, popu-lation size, surface marker expression and differentiation potential of human BMMSCs.
Materials and methods
Control plate culturePhenotypic characteristics of human BMMSCs main-tained in PC have been described previously (4,16). Here, cells between passages 10 and 12 were used for all experi-ments. For control PCs, BMMSCs were seeded at three initial cell densities (6.7 · 103
, 1.1· 104
and 2.2· 104
cells⁄ cm2
) in six-well plates (BD FalconTM; BD Biosciences, Mississauga, ON, Canada) with BMMSC expansion medium (16) for 7 days (Table 1). Beginning on day 3, medium would be completely chan-ged every 3 days. All cultures were maintained at 37C in a humidified 5% CO2incubator.
Microcarrier culture
For MCs, 100- and 500-ml spinner flasks (Bellco Glass Inc., Vineland, NJ, USA) were siliconized with Sigmacote (Sigma, St Louis, MO, USA). CultiSpher-G (130–380 lm in diameter; HyClone, Logan, UT, USA) was weighed, hydrated and sterilized by autoclaving as recommended by the manufacturer (15 min, 121C), to obtain final concentrations designated for each culture condition.
Microcarriers were equilibrated in culture medium over-night prior to cell addition to maximize cell attachment (data not shown).
BMMSCs were added to 50 ml expansion medium in 100-ml spinner flasks at the two seeding densities and microcarrier densities, as indicated in Table 1, for 7 days. Cultures were stirred intermittently (25 rpm for 30 min, followed by a 10-min rest) using an external magnetic stir-ring system (Bell-ennium 5-Position Digital Display Magnetic Stirrer, Bellco Glass) for 2 h to improve cell attachment (data not shown). After this period, cultures were stirred constantly at 25 rpm. Starting on day 3, med-ium changes were performed every 3 days. Before chang-ing the medium, microcarriers were allowed to settle for 5 min; 50% supernatant was then discarded and new med-ium was added. All cultures were maintained at 37C in a humidified, 5% CO2incubator.
The semi-continuous MC method was compared to a serial bead-to-bead transfer method (MC bead-T) to exam-ine cellular characteristics of BMMSCs maintaexam-ined by a feeding regime for a total of nine days. BMMSCs were added to 50 ml of expansion medium in 100-ml spinner flasks at initial cell density of 5.0· 104
cells⁄ ml and mi-crocarrier concentration of 3.0 mg⁄ ml (final initial cell density around 1.1· 104
cells⁄ cm2
), see Table 1. Starting on day 3, equal amounts of expansion medium with 3.0 mg⁄ ml microcarrier were added every 3 days. When volume of medium was larger than 100 ml, MC of BMMSCs was transferred to a 500-ml spinner flask. Sche-matic diagram of the three culture processes is shown in Fig. 1.
To monitor location and proliferation of cells on microcarriers, 0.5-ml samples were removed from MCs, rinsed in PBS, fixed for 10 min in 10% formalin at 25C and stained by using Hoechst 33342 dye (Invitrogen-Molecular Probes, Carlsbad, CA, USA) in the dark for 2 min to detect nuclei. Samples were then rinsed three times in PBS to eliminate background fluorescence,
trans-Table 1. Initial cell density and available surface area for the control plate and microcarrier culture conditions
Culture type Fed CG⁄ ml
Initial cell density
Medium volume (ml) Available area (cm2) 104 cells⁄ ml 104cells ⁄ cm2 PC – – 10.66 2.22 2 9.6 5.33 1.11 3.22 0.67 MC – 3.0 10.00 2.22 50 225 5.00 1.11 5.0 5.00 0.67 50 375 MC Bead-T 0 3.0 5.00 1.11 50 225 1 7.35 ± 1.89 1.63 ± 0.42 100 450 2 7.20 ± 0.82 1.60 ± 0.18 200 900 CG, CultiSpher-G; MC, microcarrier culture; PC, plate culture.
ferred to slides and visualized immediately by a fluores-cence microscope (IX70; Olympus, Tokyo, Japan). Analysis of cell population growth kinetics
For daily cell count of PCs, cells were detached with trypsin-EDTA (Invitrogen-Gibco, Carlsbad, CA, USA) using a cell scraper (BD Biosciences). For daily cell count of MCs, 1.0-ml samples were removed from the spinner flask and allowed to settle for 5 min. Supernatant was removed and the culture was rinsed twice in PBS; cells were incubated in trypsin-EDTA (1 ml) for 10–15 min at 37C to dissolve the microcarriers. They were then counted in triplicate using a haemocytometer. Cell viabil-ity was measured by trypan blue (Sigma) exclusion.
Cell population growth rate, which is the reciprocal of generation time, was calculated by number of cell dou-blings per unit time during the specified time interval (16). In this study, calculated maximal cell population growth rate (lmax) represents growth rate of the exponential phase
during the culture process and average growth rate (lavg)
represents number of doublings per unit time before death phase.
Supernatant samples were collected throughout cell growth kinetics experiments. After centrifugation, glucose and lactate concentrations in supernatant were measured directly using a glucose⁄ lactate analyser (Model YSI 2700; Yellow. Springs Instrument Co., Yellow Springs, OH, USA). Glucose and lactate concentrations were used for calculation of yield of lactate from glucose (YLac⁄ Glc) by
the following equation: YLac⁄ Glc= DLactate⁄ DGlucose,
where DGlucose is change in glucose over the time period, and DLactate is the change in lactate over the time period.
Cell size, granularity and surface markers
To study their morphology and cell population (including cell size, granularity, and surface antigen phenotype), BMMSCs were detached by trypsinization for 10 min, stained with anti-human CXC chemokine receptor 4 fluo-rescein isothiocyanate (FITC)-conjugated antibodies (CXCR4; R&D Systems, Minneapolis, MN, USA) and analysed using flow cytometry (Cytomics FC500; Beck-man Coulter, Fullerton, CA, USA). BMMSCs stained with anti-mouse IgG antibodies FITC-conjugated (Dako, Car-pinteria, CA, USA) were used as negative control. Cell size and granularity of BMMSCs from the different cul-ture types were analysed using CytomicsCXP software (Beckman Coulter) at days 0, 3 and 6. Variations in sur-face marker density (CXCR4) of PC, MC and MC bead-T were analysed using CytomicsCXP software on days 0, 6 and 9.
Osteogenic and adipogenic differentiation
Osteogenesis and adipogenesis were induced by estab-lished protocols previously described (16). Cells recov-ered from PCs and MCs on day 3 and from MCs bead-T on day 6 were replated on to 35-mm dishes (BD Bio-sciences) at initial cell density of 104cells⁄ cm2
. Osteo-genic differentiation potential of BMMSCs was evaluated by measuring alkaline phosphatase (ALP) activity on days 3, 7 and 14. Adipogenic differentiation potential of BMMSCs was evaluated by Oil red O staining and Nile Red flow cytometry, measuring accumulation of lipid vesicles on days 3 and 7. ALP staining and Oil red O staining were carried out as described previously (16).
Figure 1. Three culturing conditions used to grow bone marrow mesenchymal stem cells (BMMSCs). Culture protocols are shown for control plate culture (PC), microcarrier culture (MC) and MC with serial bead-to-bead transfer (MC bead-T). Characterization of BMMSCs recovered from PC, MC and MC bead-T was performed before cell death phase of the cultures.
Quantification of ALP activity
Cells were detached by trypsinization for 10 min, resus-pended in deionized water and vortexed for 10 min to disrupt cell membranes. ALP activity was then assayed using an ALP liquicolor kit (HUMAN GmbH, Wiesba-den, Germany) in a 37C water bath. Absorbance of p-nitrophenol product was measured at 405 nm using a spectrophotometer (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). Specific ALP activity was expressed in U⁄ l ⁄ cell.
Nile Red flow cytometry
To determine percentage of BMMSCs in each sample that had undergone adipogenic differentiation, 105cells were detached by trypsinization for 10 min and stained using Nile Red fluorescent dye (Sigma, 0.5 lg Nile Red in 1 ml of 3:1 glycerol⁄ water mixture) for 5 min. Undif-ferentiated BMMSCs from PCs were also stained with Nile Red fluorescent dye to serve as negative control. Percentage of BMMSCs that had undergone adipo-genic differentiation was analysed using CytomicsCXP software.
Total RNA isolation and semi-quantitative reverse transcriptase-polymerase chain reaction
Isolation of total RNA, cDNA synthesis and amplification reactions were carried out as described previously (17,18). To analyse differences in expression levels of osteogene-sis-related and adipogeneosteogene-sis-related genes in differentiated BMMSCs, cells from PCs and MCs were replated on to
35-mm dishes at initial density of 104cells⁄ cm2
and were treated with osteogenic medium for 7 or 14 days, or with adipogenic medium for 3 or 7 days. The cDNA was amplified by PCR with gene-specific primers, as listed in Table 2. Genes amplified included osteopontin (OPN), parathyroid hormone receptor type 1 (PTH-R1), ALP, core binding factor a1 (cbfa1), CCAAT⁄enhancer-binding pro-tein a (C⁄ EBPa), proliferator-activated receptor c2 (PPARc2) and b-actin. To determine levels of mRNA expression, band intensities were analysed using ImageJ software (ImageJ 1.40g, National Institutes of Health, Bethesda, MD, USA). Gene expression levels were normalized to b-actin, which was amplified as internal control.
Statistical analyses
Statistical analyses of cell proliferation, cell population, surface marker density, ALP activity and gene expression level for each group were carried out using Microsoft Excel data analysis program for t-test analysis; P-value <0.05 was considered statistically significant. Experi-ments were performed at least twice. Results are expressed as the mean ± SD (n = 3).
Results
Comparison of cell population growth kinetics and metabolic parameters from different human BMMSC culturing conditions
To study cell populations of BMMSCs and their growth in MC, cells were seeded at three different culture conditions
Table 2. Primers used for RT-PCR
Gene name Primer sequence Product (bp)
b-actin F: 5¢-CGCCAACCGCGAGAAGAT-3¢ 168 R: 5¢-CGTCACCGGAGTCCATCA-3¢ ALP F: 5¢-TGGAGCTTCAGAAGCTCAACACCA-3¢ 454 R: 5¢-ATCTCGTTGTCTGAGTACCAGTCC-3¢ cbfa1 F: 5¢-CTCACTACCACACCTACCTG-3¢ 320 R: 5¢-TCAATATGGTCGCCAAACAGATTC-3¢ OPN F: 5¢-CATCTCAGAAGCAGAATCTCC-3¢ 313 R: 5¢-CCATAAACCACACTATCACCTC-3¢ PTH-R1 F: 5¢-CACAGCCTCATCTTCATGG-3¢ 357 R: 5¢-GCATCTCATAGTGCATCTGG-3¢ C⁄ EBPa F: 5¢-GTCGGTGGACAAGAACAGC-3¢ 234 R: 5¢-ATGGCCTTGACCAAGGAGC-3¢ PPARc2 F: 5¢-GCTGTTATGGGTGAAACTCTG-3¢ 351 R: 5¢-ATAAGGTGGAGATGCAGGCTC-3¢
ALP, alkaline phosphatase; C⁄ EBPa, CAAT ⁄ enhancer-binding protein a; cbfa1, core binding factor alpha 1; F, forward; OPN, osteopontin; PPARc2, proliferator-activated receptor c2; PTH-R1, parathyroid hormone receptor type 1; R, reverse.
(Table 1). Optically sectioned gelatin beads from cultures on days 1, 3 and 6 were visualized using Hoechst staining. Surface-adherent cells were present on day 1 and their numbers increased by day 6, based on the macroporous structures (Fig. 2a).
Initially, proliferation of BMMSCs appeared to be affected by stirring and the three-dimensional environ-ment from MC, but there were no significant differences in cell densities observed on day 6 (Table 3). Lag phase of BMMSC population growth was substantially different between PC and MC growth conditions (Fig. 2b). When cells were plated at initial density of 2.2· 104cells⁄ cm2, cell density of MCs did not increase by day 2; however, density of PCs increased to 4· 105cells⁄ cm2 over the same time (Fig. 2b). The longer lag period of BMMSCs in MC resulted in lower average growth rates as compared to PCs, but maximal population growth rate in MCs was higher than that in PCs with the same initial cell density during log phase (Table 3). Three different initial cell
densities of 2.2· 104
, 1.1· 104
and 6.6· 103
cells⁄ cm2
resulted in fold increases in MCs of 2.84, 4.85 and 6.94 respectively, and similar fold increases in PCs of 2.42, 4.77 and 7.99 respectively (Table 3).
As surface area of microcarriers limits cell expansion, we tested serial bead-to-bead transfer in which fresh beads were added when cells become 60% confluent during the log phase. Viable cell density of MC bead-T derived BMMSCs did not differ much before day 3 (Fig. 2c) as compared to cells grown in PC or MC. Cell density decreased from 3.2–3.0· 104to 1.6–1.5· 104cells⁄ cm2 upon addition of fresh medium and microcarriers on day 3. An additional lag period of about 1 day was observed after each feeding. After isolating cells from PCs, MCs or MC bead-T by trypsin-EDTA, greater than 92% of cells remained viable as assessed by trypan blue staining, at most time points (Fig. 2b,c). This is in agreement with low frequency of cell death observed after cell retrieval by trypsin-EDTA.
(a) (b)
(c)
Figure 2. Cell proliferation of BMMSCs grown in MC as compared to cells grown in PC by semi-continuous method. (a) Proliferation of BMMSCs from MCs grown in semi-continuous culture for 7 days at different initial cell densities (top, in cells⁄ ml) and CultiSpher-G concentrations (bottom, in g⁄ l), then stained using Hoechst 33342 dye. Scale bar = 500 lm. (b) Growth kinetics of BMMSCs comparing PCs and MCs with different initial cell densities of 2.22· 104
, 1.11· 104
and 6.7· 103
cells⁄ cm2
(n = 3). Data presented in semi-log linear charts by Double-Y plot that includes viable cell density (block lines) and cell viability (blue lines). (c) Viable cell density of BMMSCs in MC bead-T as compared to cells grown in PC and MC at initial cell density of 1.11· 104
cells⁄ cm2
. Data displayed in linear chart by Double-Y plot that includes viable cell density (block lines) and cell viability (blue lines).
To monitor cell metabolism, glucose and lactate con-sumption were measured. Yield of lactate from glucose (YLac⁄ Glc) is associated with inefficient metabolism of
glu-cose (Table 3). During the semi-continuous method of culture, yield of lactate from glucose was in the order of 1.04–1.32 g⁄ g for PC and 0.97–1.14 g ⁄ g for MC catego-ries. These differences were not statistically significant except for the group with initial cell density of 1.1· 104
cells⁄ cm2
(P < 0.05). Yield of lactate from glu-cose increased with initial cell density. Slightly higher YLac⁄ Glc yields were obtained from PC as compared to
MC when cells were plated at the same initial cell density. For initial cell density of 1.1· 104
cells⁄ cm2
and with bead-to-bead transfer, YLac⁄ Glc yields of 0.68–1.49 were
obtained at each feeding. Although higher YLac⁄ Glc was
obtained after the second feeding, total YLac⁄ Glc for this
method (0.96 ± 0.02) was still lower than that of the other groups.
Effects of MC on cell morphology, cell population and immunophenotype
BMMSCs are mostly spindle-shaped while grown at a low density (19). Their morphology after 3 days of PC or MC, or after 6 days of MC bead-T was similar (Fig. 3a). On day 3, there were no significant changes in morphol-ogy of BMMSCs from MCs as compared to those from PCs, but some non-spindle-shaped cells (red arrows, Fig. 3a) were observed in PC; these were rarely observed after 3 days in stirred MC with semi-continuous method. However, non-spindle-shaped cells were observed after day 6 in MC bead-T (red arrows, Fig. 3a). We hypothesize that some BMMSCs reached confluency on old beads, resulting in these non-spindle-shaped cells.
BMMSCs grown under different culture conditions were assayed for size and granularity by their forward and side scatter properties on flow cytometry. In PCs at day 3, there were two populations of BMMSCs: A divi-sion and B dividivi-sion (Fig. 3b). Percentage of A dividivi-sion was increased and percentage of B division was reduced after 3 days in MC (Table 4). Although the percentage of A division in PCs and MCs both increased after 6 days in culture (when cells reached confluence), differ-ences in the percentage of A division and B division in PCs and MCs were still significant (P-value < 0.05). In addition, there was a concomitant decrease in percentage of granular cells (C2 plus C4 division) over time (Table 4). However, after 3 days in MC, percentage of small and agranular cells (C3 division) was significantly higher than that of cells in PC (P-value <0.005). After 6 days in MC bead-T, percentage of granular cells (C2 plus C4 division) was higher than that in PC (day 3) and MC (day 6), but percentage of A division in MC bead-T was significantly lower than that in PC or MC at day 6 (Table 4). Our data suggest that cell size and cell popula-tion of BMMSCs varied depending on the different cul-ture conditions.
BMMSCs were allowed to recover at different inter-vals after PC and MC, and they were then assayed for sur-face markers using FITC-coupled antibodies against human CXCR4, by flow cytometry (Fig. 3c). Expression levels of CXCR4 on BMMSCs from PCs and MCs stea-dily decreased over the course of the 6-day culture period, but BMMSCs from MCs maintained significantly higher CXCR4 density levels and higher ratios of CXCR4-posi-tive cells (CXCR4+: 95.4 ± 6.4%; X-Mean: 44.5 ± 3.7) than did those from PCs (CXCR4+: 86.9 ± 0.5%; X-Mean: 12.9 ± 1.9) at day 3 (log phase; Fig. 3c,
Table 3. Growth kinetics and metabolic parameters obtained from PCs and MCs at day 6 and MC Bead-T at day 9
Culture type Fed
Initial cell density Maximal cell density
lavg a (h)1) lmax b (h)1) YLac⁄ Glcc Fold increase 104cells⁄ ml 104cells⁄ cm2 104cells⁄ ml 104cells⁄ cm2 PC – 10.66 2.22 25.77 ± 0.29 5.16 ± 0.59 0.047 ± 0.001 0.025 ± 0.009 1.04 ± 0.03 2.42 5.33 1.11 25.44 ± 0.29 5.09 ± 0.59 0.038 ± 0.001 0.027 ± 0.002 1.18 ± 0.04 4.77 3.22 0.67 25.59 ± 2.23 5.33 ± 0.47 0.020 ± 0.001 0.036 ± 0.003 1.32 ± 0.05 7.99 MC – 10.00 2.22 26.90 ± 3.61 5.98 ± 0.80 0.012 ± 0.002 0.034 ± 0.001 0.97 ± 0.06 2.84 5.00 1.11 24.20 ± 0.94 5.38 ± 0.21 0.016 ± 0.001 0.033 ± 0.009 1.02 ± 0.05 4.85 5.00 0.67 34.70 ± 3.16 4.63 ± 0.42 0.019 ± 0.004 0.041 ± 0.001 1.14 ± 0.16 6.94 MC Bead-T 0 5.00 1.11 14.70 ± 3.78 3.27 ± 0.84 0.022 ± 0.004 0.043 ± 0.014 0.68 ± 0.02 2.95 1 7.35 ± 1.89 1.63 ± 0.42 14.40 ± 1.65 3.20 ± 0.37 0.013 ± 0.005 0.028 ± 0.013 0.80 ± 0.03 5.79 2 7.20 ± 0.82 1.60 ± 0.18 13.90 ± 0.79 3.09 ± 0.17 0.015 ± 0.003 0.023 ± 0.002 1.49 ± 0.12 11.18
CG, CultiSpher-G; MC, microcarrier culture; PC, plate culture.
aAverage growth rate (l
avg) represents the number of doublings per unit time before the death phase. bMaximal growth rate (l
max) represents the growth rate during the log phase. cY
Table 4). BMMSCs in MC for 3 days did not, however, show significant changes in other surface markers includ-ing CD13, CD14, CD29, CD34, CD44, CD45, CD49b, CD49d, CD73, CD90, CD105, HLA-ABC and HLA-DR (data not shown). At day 6, BMMSCs from MCs bead-T maintained significantly higher levels of CXCR4 expres-sion as compared to BMMSCs from PCs or MCs, but intensity of CXCR4 expression density and ratio of CXCR4-positive cells in MCs bead-T were lower than those found at day 0 (Table 4).
Comparison of osteogenic differentiation potential of BMMSCs derived by different culturing conditions Cells of PC (day 3), MC (day 3), and MC bead-T (day 6) were replated on to 35-mm dishes under osteogenic or adi-pogenic conditions to examine the effect of microcarriers on BMMSC differentiation potential. For osteogenic dif-ferentiation, BMMSCs plated at density of 104cells⁄ cm2 were induced under osteogenic conditions for 14 days. During this period, BMMSCs exhibited dramatic change
(a) (b) (c)
Figure 3. Effects of different cell culture methods on morphology and surface marker density of BMMSCs. (a) Photomicrographs of BMMSCs recovered from PCs and MCs at day 3 and from MCs bead-T at day 6 and replated on to 35-mm dishes for one day (red arrows, non-spindle-shaped cells). (b) Change in cell size and cell population of BMMSCs grown under the different culture conditions analysed by flow cytometry using forward scatter (FS) versus side scatter (SS) density plots. The density plots were divided into A division (red ring) and B division (green ring) based on two pop-ulations of BMMSCs present in PCs at day 3 or into C1–C4 divisions based on major poppop-ulations of BMMSCs in MCs at day 3. Density level threshold, 50 cells, and density levels of 1–5 indicated by colour (blue to red). (c) Variation of surface marker density (CXCR4) of BMMSCs from different culture protocols shown in histogram plots derived from flow cytometry at days 0 (blue), 3 (red), 6 (green) and 9 (purple). Black-line histogram indicates back-ground signal; coloured histograms represent positive reactivity with the CXCR4 antibody.
in cell morphology and displayed significant increase in ALP activity, which was confirmed by ALP staining (Fig. 4a) and quantified by spectrophotometry (Fig. 4b). ALP is required in the mineralization process and hydro-lyses phosphate-containing substrates to increase local phosphate concentration (20). During the osteogenic per-iod, some cells appeared to be ALP positive and activity of ALP-positive cells from MCs was significantly higher than that from PCs at day 14, but activity of ALP-positive cells from MC bead-T was slightly lower than that from PCs and significantly lower than that from MCs (Fig. 4b). Comparison of adipogenic differentiation potential of BMMSCs derived from different culturing conditions To evaluate adipogenic potential, we plated BMMSCs at density of 104cells⁄ cm2and induced them under adipo-genic conditions for 7 days. At days 3 and 7 post induc-tion, cells from PC, MC and MC bead-T exhibited varying
degrees of adipogenesis based on changes in visible accu-mulation of neutral lipid vacuoles observed by Oil red O staining and quantified by Nile Red flow cytometry (Fig. 5). In contrast to cells from PCs (39.2%), BMMSCs from MCs yielded 83.6% adipocytes that contained lipid droplets at day 3, indicating high level of adipogenic potential. BMMSCs recovered from MCs bead-T yielded 32.8% adipocytes (day 3) and 46.5% (day 7), which was lower than the number of adipocytes in BMMSCs recov-ered from PCs and MCs.
Osteogenic and adipogenic gene expression in BMMSCs from MC and PC
BMMSCs were recovered from PC and MC (day 3) and then cultured in osteogenic medium. At days 7 and l4, total RNA was isolated and then used for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of osteogenesis-related genes (Fig. 6a,c). On
(a) (b)
Figure 4. Osteogenic differentiation potential of BMMSCs recovered from PC, MC and MC bead-T. (a) Photomicrographs of ALP activity of BMMSCs grown under different culture conditions as shown by ALP staining. Scale bar = 500 lm. (b) Quantification of specific ALP activity shown by spectrophotometry (O.D. = 405 nm). Results represented as mean ± SD of triplicate cultures from one representative experiment. *P < 0.05; †P < 0.01.
Table 4. The effects of different cell culturing methods on BMMSC cell population and CXCR4 expression level
Characteristic Day 0
Day 3 Day 6 Day 9
PC MC PC MC MC Bead-T MC Bead-T A division 82.5 ± 1.4% 78.5 ± 7.3% 90.5 ± 2.8%† 64.2 ± 1.0% 78.8 ± 1.2%‡ 83.9 ± 0.3%‡ 80.0 ± 1.8% B division 13.3 ± 1.3% 18.4 ± 6.1% 7.7 ± 2.7%* 33.6 ± 1.3% 21.2 ± 0.7%‡ 8.5 ± 1.0%‡ 14.1 ± 0.6% C3 division 81.6 ± 1.4% 85.1 ± 1.0% 95.6 ± 0.1%‡ 91.6 ± 6.5% 94.1 ± 4.0% 74.0 ± 1.6%* 82.8 ± 3.2% CXCR4+ 99.2 ± 0.2% 86.9 ± 0.5% 95.4 ± 6.4%‡ 44.1 ± 3.4% 44.0 ± 3.0% 95.7 ± 1.2%‡ 79.4 ± 2.1% X-Mean 47.4 ± 0.6 12.9 ± 1.9 44.5 ± 3.7* 5.8 ± 0.2 5.81 ± 0.1 41.1 ± 1.0‡ 7.4 ± 0.6
Cell population and CXCR4 expression level of BMMSC from MC or MC Bead-T was compared with that from PC for t-test analysis. X-Mean: mean fluorescence (FITC) intensity of CXCR4-positive cells.
day 7, expressions of PTH-R1, ALP, cbfa1, and OPN in BMMSCs from PC were up-regulated. At day 7, BMMSCs from MC showed a similar expression pattern, albeit with a significantly higher level of expression for PTH-R1. On day 14, expressions of PTH-R1, ALP and OPN in cells from PC were further up-regulated and expression of PTH-R1 in cells from MC was still signifi-cantly higher than that from PC.
BMMSCs were recovered from PC and MC (day 3) and then cultured in adipogenic medium. At days 3 and 7, total RNA was isolated and then used for semi-quantita-tive RT-PCR analysis of adipogenesis-related genes (Fig. 6b,d). On day 3, expressions of C⁄ EBPa and PPARc2 in cells from PC and MC were up-regulated, but expressions of C⁄ EBPa in cells from MC were in the order of 3-fold higher than that in cells from PC. On day 7, expressions of C⁄ EBPa and PPARc2 in cells from PC and MC were further up-regulated, but expression of PPARc2 in cells from MC was about 2-fold higher than that in cells from PC.
Discussion
MSCs can retain multipotency and proliferate rapidly at relatively low densities (21–23), but extended expansion
of MSCs with low cell densities in two-dimensional PC does not produce sufficient numbers of cells for therapeu-tic applications. Thus, we used different seeding densities and different CultiSpher-G concentrations to examine cul-ture parameters for optimal expansion of BMMSCs in MC. Cell population kinetics of BMMSC growth in MC differed from their growth kinetics in PC. In PC, BMMSC expansion began with a 1-day lag phase and then entered log phase of exponential growth, although growth rates of cells in the three different seeding densities were almost identical during the lag phase. In contrast, length of lag phase was inversely proportional to initial seeding density for MC. This phenomenon resulted in lower average growth rate for BMMSCs expanded in MCs, as compared to those in PCs. With the same seeding densities, maxi-mum growth rate was higher in MC, although maximaxi-mum cell density and fold increase at the end of the experiment were similar between the two groups.
Accumulation of lactate is associated with inefficient glucose metabolism (24). To prevent cell growth inhibi-tion by accumulainhibi-tion of metabolites such as lactate and⁄ or deficiency in nutrients such as glucose, feeding regimen should be optimized (25,26). In our study, a slightly higher YLac⁄ Glc of 1.04–1.32 g⁄ g was obtained from PC
as compared to YLac⁄ Glc of 0.97–1.14 g⁄ g from MC,
Figure 5. Adipogenic differentiation potential of BMMSCs recovered from PC, MC and MC bead-T. Photomicrographs of lipid spheres in BMMSCs grown in the different culture conditions as shown by Oil red O staining. Scale bar = 500 lm. Quantification of adipo-differentiated BMMSCs (A division) in the different culture types, expressed as percentage of total cells, as derived from Nile Red flow cytometry. Open histogram indicates background signal (undifferentiated BMMSCs stained by Nile Red); blue histogram indicates staining for Nile Red.
(a)
(b)
(c)
(d)
Figure 6. Effect of MC on osteogenic and adi-pogenic gene expression in BMMSCs. Electro-phoresis of reverse transcriptase-polymerase chain reaction (RT-PCR) products of (a) osteo-differentiated and (b) adipo-differentiated BMMSCs. BMMSC lane indicates undifferenti-ated BMMSCs from PC (day 3). To examine changes in expression of (c) osteogenic and (d) adipogenic genes with time respectively, cells were harvested on indicated days and subjected to semi-quantitative RT-PCR followed by aga-rose gel electrophoresis and ethidium bromide staining. b-actin was amplified as internal con-trol. RT-PCR amplification products quantified and values represent fold change relative to day 7 of PC (osteogenesis) or day 3 of PC (adipogene-sis). Data points represent mean value ± SD (n = 3). *P < 0.05. †P < 0.01. ‡P < 0.005.
suggesting that BMMSCs cultured in stirred MC have a higher rate of oxidative phosphorylation than do cells grown in plate culture. Additionally, lower initial cell den-sity resulted in higher YLac⁄ Glc. In agreement with this,
higher YLac⁄ Glc from MC bead-T was obtained after the
second feeding, which may suggest that adaptation of BMMSCs to these conditions caused higher metabolic stress.
Increased expression of stromal cell-derived factor-1 by ischaemic myocardium attracts CXCR4 positive bone marrow-derived stem cells (27). In addition, higher CXCR4 expression in MSCs after transduction with retro-viral vector containing CXCR4 increases cell migration toward stromal cell-derived factor-1 (28). Culturing MSCs for more than two passages results in reduced expression of adhesion molecules as well as loss of chemokine recep-tors including CXCR4 (29,30). In our analysis, BMMSCs cultured in PC and MC showed reduced levels of CXCR4 expression over time. This result confirms previous studies that showed down-regulation of CXCR4 expres-sion during in vitro culture processes (29,30). Although reduced, BMMSCs from MCs had higher expression levels of CXCR4 than those from PCs at day 6. This result suggests that the MC process promotes culture of BMMSCs and also helps to maintain CXCR4 expression, which is involved in homing ability of stem cells.
Recently, up-scalability of MSCs in MC has been expanded with (serial) bead-to-bead transfer (15,31). Based on use of bead-to-bead transfer, the available sur-face area for cell population growth can be extended and culturing of anchorage-dependent cells can be prolonged as free cells from old beads colonize fresh beads (32). In our study, use of bead-to-bead transfer did, however, result in MSC confluence on old beads (data not shown) and the percentage of granular cells (C2 plus C4 division) was higher than that observed in PC or MC. However, activity of ALP-positive cells (BMMSC-derived osteo-blasts) and percentage of cells containing lipid droplets (BMMSC-derived adipocytes) in differentiated BMMSCs from MCs bead-T were lower than for those from PCs and were also significantly lower than for those from MCs. In comparison with PC or MC bead-T, MC using a semi-con-tinuous process enhanced both osteogenic and adipogenic differentiation of BMMSC.
MSCs are heterogeneous in morphology (22,33,34), and differentiation potential of MSCs may vary depending on their source (35) or variable culture conditions includ-ing differences in initial cell density (19,36). In BMMSC cultures, large cells are considered to be mature MSCs and smaller cells as recycling stem cells (22). Recycling stem cells replicate and give rise to mature MSCs, which become the predominant cell type as cultures approach senescence (23). In our study, BMMSCs recovered from
PCs at day 3 displayed a variety of cell morphologies, whereas BMMSCs recovered from MCs at day 3 were much more homogeneous in size (Fig. 3a). By analysing cell size and granularity, we infer that MC increased per-centage of small and agranular cells in the total BMMSC culture (Fig. 3b, C3 division), but MC also selectively reduced the subpopulation of very small and agranular cells among total BMMSC culture (Fig. 3b, B division). These different subpopulations of cells may account for differences between osteogenic and adipogenic differenti-ation potential among cells from PC and MC at day 3 and from MC bead-T at day 9.
In conclusion, stirred MC systems of CultiSpher-G provide higher specific surface area, ease of monitoring, and ability to scale-up human BMMSC culture without loss of differentiation potential. To achieve more homo-geneous morphology and better differentiation potential for BMMSCs, semi-continuous MC of BMMSCs may be more beneficial than PC or MC bead-T methods.
References
1 Lysy PA, Campard D, Smets F, Malaise J, Mourad M, Najimi M et al. (2008) Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells. Cell Prolif. 41, 36–58.
2 Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.
3 Abdallah BM, Kassem M (2008) Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther. 15, 109–116. 4 Chang YJ, Liu JW, Lin PC, Sun LY, Peng CW, Luo GH et al. (2009)
Mesenchymal stem cells facilitate recovery from chemically induced liver damage and decrease liver fibrosis. Life Sci. 85, 517–525. 5 Otto WR, Rao J (2004) Tomorrow’s skeleton staff: mesenchymal
stem cells and the repair of bone and cartilage. Cell Prolif. 37, 97–110.
6 Badet L, Benhamou PY, Wojtusciszyn A, Baertschiger R, Milliat-Guittard L, Kessler L et al. (2007) Expectations and strategies regarding islet transplantation: metabolic data from the GRAGIL 2 trial. Transplantation 84, 89–96.
7 Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W et al. (2006) Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomized controlled trial. Lancet 367, 113–121.
8 Banfi A, Bianchi G, Notaro R, Luzzatto L, Cancedda R, Quarto R (2002) Replicative aging and gene expression in long-term cultures of human bone marrow stromal cells. Tissue Eng. 8, 901–910. 9 Derubeis AR, Cancedda R (2004) Bone marrow stromal cells
(BMSCs) in bone engineering: limitations and recent advances. Ann. Biomed. Eng. 32, 160–165.
10 Javazon EH, Beggs KJ, Flake AW (2004) Mesenchymal stem cells: paradoxes of passaging. Exp. Hematol. 32, 414–425.
11 Frauenschuh S, Reichmann E, Ibold Y, Goetz PM, Sittinger M, Ringe J (2007) A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Biotechnol. Prog. 23, 187–193. 12 Rubin JP, Bennett JM, Doctor JS, Tebbets BM, Marra KG (2007)
Collagenous microbeads as a scaffold for tissue engineering with adi-pose-derived stem cells. Plast. Reconstr. Surg. 120, 414–424.
13 Yang Y, Rossi FM, Putnins EE (2007) Ex vivo expansion of rat bone marrow mesenchymal stromal cells on microcarrier beads in spin cul-ture. Biomaterials 28, 3110–3120.
14 Malda J, Frondoza CG (2006) Microcarriers in the engineering of cartilage and bone. Trends Biotechnol. 24, 299–304.
15 Sart S, Schneider YJ, Agathos SN (2009) Ear mesenchymal stem cells: an efficient adult multipotent cell population fit for rapid and scalable expansion. J. Biotechnol. 139, 291–299.
16 Sun LY, Hsieh DK, Yu TC, Chiu HT, Lu SF, Luo GH et al. (2009b) Effect of pulsed electromagnetic field on the proliferation and differ-entiation potential of human bone marrow mesenchymal stem cells. Bioelectromagnetics 30, 251–260.
17 Lin PC, Chen YL, Chiu SC, Yu YL, Chen SP, Chien MH et al. (2008) Orphan nuclear receptor, Nurr-77 was a possible target gene of butylidenephthalide chemotherapy on glioblastoma multiform brain tumor. J. Neurochem. 106, 1017–1026.
18 Sun LY, Hsieh DK, Lin PC, Chiu HT, Chiou TW (2009a) Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation. Bioelectromagnetics 31, 209– 219.
19 Tropel P, Noel D, Platet N, Legrand P, Benabid AL, Berger F (2004) Isolation and characterization of mesenchymal stem cells from adult mouse bone marrow. Exp. Cell Res. 295, 395–406.
20 Robison R (1923) The possible significance of hexosephosphoric esters in ossification. Biochem. J. 17, 286–293.
21 Bobis S, Jarocha D, Majka M (2006) Mesenchymal stem cells: char-acteristics and clinical applications. Folia Histochem. Cytobiol. 44, 215–230.
22 Colter DC, Class R, DiGirolamo CM, Prockop DJ (2000) Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc. Natl. Acad. Sci. USA 97, 3213– 3218.
23 Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ (1999) Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br. J. Hae-matol. 107, 275–281.
24 Ozturk SS, Palsson BO (1991) Growth, metabolic, and antibody pro-duction kinetics of hybridoma cell culture: 2. Effects of serum con-centration, dissolved oxygen concon-centration, and medium pH in a batch reactor. Biotechnol. Prog. 7, 481–494.
25 Hassell T, Gleave S, Butler M (1991) Growth inhibition in animal cell culture. The effect of lactate and ammonia. Appl. Biochem. Bio-technol. 30, 29–41.
26 Lao MS, Toth D (1997) Effects of ammonium and lactate on growth and metabolism of a recombinant Chinese hamster ovary cell culture. Biotechnol. Prog. 13, 688–691.
27 Brunner S, Winogradow J, Huber BC, Zaruba MM, Fischer R, David R et al. (2009) Erythropoietin administration after myocardial infarction in mice attenuates ischemic cardiomyopathy associated with enhanced homing of bone marrow-derived progenitor cells via the CXCR-4⁄ SDF-1 axis. FASEB J. 23, 351–361.
28 Bhakta S, Hong P, Koc O (2006) The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc. Revasc. Med. 7, 19–24.
29 Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM, Silberstein LE (2006) Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells 24, 1030–1041.
30 Son BR, Marquez-Curtis LA, Kucia M, Wysoczynski M, Turner AR, Ratajczak J et al. (2006) Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 24, 1254–1264. 31 Schop D, Janssen FW, Borgart E, de Bruijn JD, van
Dijkhuizen-Rad-ersma R (2008) Expansion of mesenchymal stem cells using a micro-carrier-based cultivation system: growth and metabolism. J. Tissue Eng. Regen. Med. 2, 126–135.
32 Ohlson S, Branscomb J, Nilsson K (1994) Bead-to-bead transfer of Chinese hamster ovary cells using macroporous microcarriers. Cyto-technology 14, 67–80.
33 Mets T, Verdonk G (1981) In vitro aging of human bone marrow derived stromal cells. Mech. Ageing Dev. 16, 81–89.
34 Zohar R, Sodek J, McCulloch CA (1997) Characterization of stromal progenitor cells enriched by flow cytometry. Blood 90, 3471–3481. 35 Tsai MS, Hwang SM, Chen KD, Lee YS, Hsu LW, Chang YJ et al.
(2007) Functional network analysis of the transcriptomes of mesen-chymal stem cells derived from amniotic fluid, amniotic membrane, cord blood, and bone marrow. Stem Cells 25, 2511–2523.
36 Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM (2001) Purification and ex vivo expansion of postnatal human mar-row mesodermal progenitor cells. Blood 98, 2615–2625.
