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Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 www.elsevier.com/locate/jbiosc
Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering Hirokazu Akiyama,1 Asako Kobayashi,2 Masaki Ichimura,1 Hiroshi Tone,3 Masaru Nakatani,1 Minoru Inoue,2 Arinobu Tojo,2 and Hideaki Kagami2, 4, * Medical Device Development Laboratories, Kaneka Corporation, 1-8 Miyamae-cho, Takasago-cho, Takasago, Hyogo 676-8688, Japan,1 Tissue Engineering Research Group, Division of Molecular Therapy, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,2 Technology Management Department, Medical Devices Division, Kaneka Corporation, 2-3-18 Nakanoshima, Kita-ku, Osaka 530-8288, Japan,3 and Department of Oral and Maxillofacial Surgery, Matsumoto Dental University School of Dentistry, 1780 Hirookagobara, Shiojiri, Nagano 399-0781, Japan4 Received 27 December 2014; accepted 16 March 2015 Available online xxx
The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Automated cell culture system; Bone marrow stromal cells; Osteogenic potential; Bone tissue engineering; Regenerative medicine]
Substantial efforts in tissue engineering have provided innovative methods to repair or regenerate damaged tissues and organs. Yet, although many clinical trials in this field have shown promising results (1e6), several problems remain that need to be addressed before these techniques find wider clinical application in regenerative medicine. One major problem is that current clinical cell processing methods, in addition to their high cost, are dependent on manual operations, which are vulnerable to human error and microbial contamination. To achieve stable and economical cell processing, automated systems have gained interest (7). To date, several automated cell culture systems have become commercially available (8e11), such as Auto Culture (Kawasaki Heavy Industries) (8) and CompacT SelecT (The Automation Partnership) (9e11), in which cells are cultured using open culture vessels manipulated by robotic arms incorporated into the aseptically controlled chamber. Because the robotic arm can replicate manual operations, this type of system has broad applicability for various types of cells. By contrast, the automated systems P 4C S (Kaneka, Osaka, Japan) and Quantum (Terumo BCT) (12) employ closed culture vessels, which confer enormous advantages in
* Corresponding author at: Tissue Engineering Research Group, Division of Molecular Therapy, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: þ81 3 5449 5120; fax: þ81 3 5449 5121. E-mail address: [email protected] (H. Kagami).
terms of compactness and the relatively low cost of machinery. The P 4C S was developed based on the prototype system reported by Kato et al. (13) and performs cell culture using an original disposable tubing set containing a single culture flask. One of the characteristic features of this culture system is the repeated use of a single culture flask even after cell passage, which is advantageous both for the compactness of the machinery and for stable continuous culture. Although the feasibility of this system for primary cultures of BMSCs and fibroblasts was reported, the detailed characters of cells cultured with this system have not been investigated. Recently, bone tissue engineering has attracted significant attention because it is less invasive than autologous bone grafting and may be more effective than artificial bone substitutes (14). Preliminary results from clinical studies demonstrated the usefulness of this approach for severe atrophy of alveolar bone (15). However, for the wide acceptance and commercialization of bone tissue engineering, an automated culture system remains desirable. In this study, we examined the feasibility of replacing the manual culture of BMSCs in the clinical setting of alveolar bone tissue engineering with an automated procedure using the P 4C S. Emphasis was placed on comparing the manually cultured cells with cells from the automated culture system. Importantly, we also examined the safety concerns regarding the microbiological contamination of automated culture and the oncogenic transformation of the cultured cells.
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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Automated cell culture system The P 4C S (Kaneka) was developed based on the prototype system reported by Kato et al. (13). The photographs of the P 4C S are shown in Fig. 1A. The system was designed to perform cell culture in an enclosed system using a specially designed disposable tubing set containing a culture flask (culture area, 490 cm2), air filters, and solution bags. The dimensions of the machinery are 110 cm (W) 62 cm (D) 63 cm (H). The upper side of the machinery consists of the cooler and the incubation units. The cooler unit maintains the media and cell detachment solution at 5 C; the incubation unit maintains the culture environment at 37 C in an atmosphere of 5% CO2. On the lower side of the machinery, peristaltic pumps and valves are arranged to control the flow of solutions and air. The system is operated by an external personal computer. The detailed view of the whole culture system is shown in Fig. 1B. The solution bags in the tubing set consist of a sample material bag for cell loading, medium bags for storing the culture media, a cell-detachment solution bag for storing protease (e.g., trypsin), a saline solution bag for washing cells, waste bags for storing waste liquid, and a cell collection bag for collecting the cell suspension after cultivation. The system performs the typical cell manipulations of cell loading, medium exchange, and cell harvest. For cell loading, sample materials in the sample material bag are poured into the culture flask after which medium is supplied from the medium bags. For medium exchange, the spent medium in the culture flask is discarded by pumping it into the waste bag and fresh medium is then supplied. For cell harvest, the spent medium is discarded and the cells are repeatedly washed in saline solution. Protease is supplied from the cell-detachment solution bag and the cells are incubated for a specified period of time. Thereafter, fresh medium is added to stop the protease activity and the detached cells are pumped into the cell collection bag. During culture, fresh air (5% CO2) is periodically supplied to the culture flask through air filters in the incubation unit. In addition, images of multiple fixed positions within the culture flask are captured daily by a complementary metal-oxide-semiconductor camera arranged underneath the culture flask in the incubation unit. These manipulations are automatically performed according to the computer program. The detailed methods of these manipulations are described by Kato et al. (13).
J. BIOSCI. BIOENG., Media Fill test Potential microbial contamination in the automated cell culture process was estimated using a Media Fill test (16,17). The disposable tubing set, consisting of a sample material bag, medium bags, and a cell-detachment solution bag, all of which were filled with trypticase soy broth (Sysmex, Kobe, Japan) as microbial growth medium, was prepared using a clean bench with a grade A environment (class 100) in a clean room. The tubing set was then transferred to the machine, located in a conventional laboratory without any air filtration system. The culture period was set to 21 days, considered to represent the worst-case scenario (longest period of machine culture). After the simulation of the 21-day machine culture and following the 14-day manual culture (35-day culture in total), the medium was sampled. Thereafter, the harvested sample was incubated for 7 days at 22e25 C followed by an additional incubation for 7 days at 30e35 C. Subsequently, the harvested medium sample was qualitatively examined for evidence of microbial growth or turbidity during incubation and at the end of the 14-day incubation. The experiment was repeated three times. Manual and automated cultures Fresh human bone marrows, obtained from three healthy donors (25- and 28-year-old males and a 38-year-old female), were purchased from AllCells (Emeryville, CA, USA). Each bone marrow was divided into two 20-mL groups, and the manual and automated primary cultures of BMSCs were performed in parallel using the clinically acceptable protocols described below. For manual culture, 20 mL of bone marrow aspirate was diluted four-fold with serum-free alpha-minimum essential medium (a-MEM; Life Technologies, Carlsbad, CA, USA) supplemented with 50 mg gentamicin (Fuji Pharma, Tokyo, Japan)/mL and 2.5 mg amphotericin B (Fungizone; Bristol-Myers Squibb, New York, NY, USA)/mL. Then, diluted bone marrow was plated into four T-150 flasks (Corning, New York, NY, USA) so that each flask contained 5 mL of bone marrow. After 4 days of cultivation in a 37 C, 5% CO2 incubator, the culture medium was replaced with a-MEM supplemented with 10% fetal bovine serum (FBS; Thermo Scientific, Waltham, MA, USA) and 1 ng of basic fibroblast growth factor (bFGF; Kaken Pharmaceutical, Tokyo, Japan)/mL. Thereafter, the cells were fed twice a week with a-MEM supplemented with FBS and bFGF until they reached sub-confluence, at which time they were harvested using TrypLE Select (Life Technologies). For automated culture using the P 4C S, 20 mL of bone marrow aspirate was poured into the sample material bag. Cell loading was then carried out to transfer
FIG. 1. Automated cell culture system. (A) Photographs of the P 4C S. The P 4C S was designed to culture cells in a closed system by using a single-use disposable tubing set containing a cell culture flask, air filters, and solution bags. (B) A detailed view of the whole culture system.
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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bone marrow and the culture medium (a-MEM supplemented with FBS and bFGF) from the medium bag to a culture flask. The medium was exchanged according to the same schedule used for manual culture. The cells were harvested at subconfluence by supplying TrypLE Select from the cell detachment solution bag.
determined. At the end of the observation period, the mice were evaluated by an examiner. The grafts were resected, fixed, embedded in paraffin, serially sectioned, and stained with H&E. The serial sections (covering the whole sample) were analyzed under the microscope for tumor formation.
Measurements of cell number and viability The harvested cells were stained with trypan blue solution and the numbers of viable and dead cells were counted using a hemacytometer. Cell viability was calculated as the number of viable cells divided by the total number of cells.
Statistical analysis All data were expressed as means standard deviation (SD). Statistical comparison in in vivo osteogenic potential assay was evaluated using an unpaired t-test, because the differences among the transplantation sites of each graft made it difficult to presume the paired groups. The others were evaluated using a paired t-test. p < 0.05 was considered significantly different.
Flow cytometric analysis The harvested cells were fixed in 4% paraformaldehyde in PBS, washed in PBS, and then incubated with following fluorescence isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated antibodies: CD14-FITC, CD29-PE, CD34-PE, CD44-PE, CD45-FITC, CD73-PE, CD79aPE, CD90-FITC, CD166-PE, HLA-DR-FITC (all from BD Pharmingen, San Diego, CA, USA), and CD105-FITC (Ancell, Bayport, MN, USA). The labeled cells were analyzed using a FACS Calibur flow cytometer and CellQuest software (BD Biosciences, San Jose, CA, USA). A fluorescence-conjugated IgG (BD Pharmingen) served as the negative control. Cell proliferation and alkaline phosphatase activity assays Cell proliferation and alkaline phosphatase (ALP) activity after osteogenic induction were analyzed as described previously (18). The cells were seeded into the wells of a 12well plate (Corning) at a density of 2 104 cells/well and cultured with serumcontaining a-MEM. After 1 day of culture, the medium was replaced with osteogenic induction medium (serum-containing a-MEM with 10 nM dexamethasone, 100 mM ascorbic acid, and 10 mM glycerol-2-phosphate disodium salt hydrate) and cultivation was continued. As a control, the cells were cultured without osteogenic supplements. After 4 days of osteogenic induction, cell metabolic activity, as an index of cell proliferation, was measured using a commercially available kit based on the WST-8 assay (Cell Counting Kit 8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. ALP activities of noninduced and induced BMSCs were then measured with a commercially available p-nitrophenyl phosphate tablet set (SigmaeAldrich, St. Louis, MO, USA). In vivo osteogenic potential and tumorigenic risk assays All animal experiments in this study were approved by the Animal Ethics Screening Committee of IMSUT, The University of Tokyo. The in vivo osteogenic potential and tumorigenic risk assay of the cultured cells were analyzed using an ectopic transplantation model, as described previously (18). BMSCs (5 105 cells) were mixed with 50 mg of b-tricalcium phosphate (b-TCP) granules (G1 type Osferion Olympus Terumo Biomaterials, Tokyo, Japan) in a 14-mL round-bottom polypropylene tube (BD Biosciences) to which serum-containing aMEM had been added. After 1 day of culture, the culture medium was replaced with osteogenic induction medium and cultivation was continued for 2 weeks. The medium was exchanged twice a week. After 2 weeks of osteogenic induction, the mixtures of cells and b-TCP granules were transplanted into the subcutaneous space on the backs of 6-week-old female BALB/CAJc1-nu/nu mice (Nihoncrea, Tokyo, Japan) under general anesthesia with pentobarbital sodium. To assay the in vivo osteogenic potential, the mice were killed with an overdose of anesthesia at 4 weeks post-transplantation and the grafts were resected. Harvested grafts were fixed in 10% formalin and embedded in paraffin. Five-micron-thick sections were then cut and stained with hematoxylin and eosin (H&E). The number of areas of histologically defined new ectopic bone as seen on the H&E-stained sections was counted under the microscope. For tumorigenic risk assay, BMSCs from two different donors were independently prepared and transplanted into the nude mice as describe above. Four samples from one donor were transplanted into two nude mice (each receiving two grafts) and observed for 3 and 6 months each. From the other donor, four samples were also transplanted and at least the results at 3 months were
RESULTS Media Fill test Prior to the cell culture experiment, potential microbial contamination in the automated cell culture process was analyzed using a Media Fill test (16,17), in which the whole cell culture process is simulated using microbial growth medium instead of the cells and conventional culture medium. In three runs, there was no evidence of turbidity, indicative of microbial contamination. Comparison of cell yields and viabilities The bone marrow was divided into two 20-mL groups for manual and automated primary cultures of BMSCs performed in parallel using clinically acceptable protocols. In three independent experiments using bone marrow from different donors, the cells in both cultures reached sub-confluence (60e95% confluence) after 11e14 days, at which time they were simultaneously harvested and both cell numbers and viability were measured using a trypan blue dye exclusion method (Fig. 2). The yield of the automated cell culture was 1.69e2.24 107 cells (Fig. 2A) with a viability of 96.6e99.1% (Fig. 2B), which was not statistically different from the manual culture. In addition, the cell yields of the automated culture were almost equivalent to those obtained in manual culture performed as part of a clinical alveolar bone tissue engineering study (unpublished data), indicating that the P 4C S is capable of producing clinically relevant cell yields for bone tissue engineering. Comparison of cell surface marker expression profiles The expression of cell surface markers by the harvested BMSCs from the manual and automated cultures was analyzed using a flow cytometer (Fig. 3). As shown in Fig. 3A, the harvested cells from both cultures were positive for the mesenchymal stem cell (MSC) markers CD29, CD44, CD73, CD90, CD105, and CD166. Moreover, the percentages of cells positive for these markers were almost identical, indicating the equal distribution of subpopulations. These percentages and those of other surface markers (CD14, CD34, CD45, CD79a, and HLA-DR) are shown in Fig. 3B. Only a limited number of cells in both groups were positive for CD14, CD34, CD45, and CD79a. The HLA-DR-positive fraction was
FIG. 2. Comparison of cell numbers and viability. BMSCs in manual and automated cultures were harvested at the same time after reaching sub-confluence. Cell numbers (A) and viability (B) were measured using the trypan blue dye-exclusion method. The data represent the mean SD from three independent experiments using bone marrows from three different donors. The p-values are 0.16 (cell numbers) and 0.39 (cell viability), respectively.
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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J. BIOSCI. BIOENG.,
FIG. 3. Comparison of surface marker expression profiles. The expression of surface marker by BMSCs from the manual or automated cultures was analyzed using a flow cytometer. (A) Histograms of CD29, CD44, CD73, CD90, CD105, and CD166, which are positive markers for MSCs. (B) Expression percentages of surface markers including CD14, CD34, CD45, CD79a, and HLA-DR. A representative result of two independent experiments using bone marrow from three different donors is shown.
similar in the two groups (the difference was not significant). These results indicated the similar expression profiles of the manually and automated cell cultures. Comparison of in vitro osteogenic potential It was previously reported that cell proliferation in osteogenic induction culture correlated with bone-forming potential (18), in which the upregulation of ALP activity after osteogenic induction (ALP ratio ¼ ALP activity of induced cells/noninduced cells) may also play a role (19). Therefore, to compare the in vitro osteogenic potential of the BMSCs obtained by manual and automated cultures, the harvested cells were cultured in osteogenic induction medium for 4 days, after which cell proliferation was determined using the WST-8 assay (Fig. 4A). The ALP ratio was analyzed using a colorimetric p-nitrophenyl phosphate assay (Fig. 4B). In both analyses, no statistical differences were found between the manual and the automated cell culture groups, indicative of the comparable in vitro osteogenic potentials of the BMSCs of the two cultures. Comparison of in vivo osteogenic potential The in vivo osteogenic potential of the cultured cells was investigated using an ectopic transplantation model. Representative micrographs of the H&E-stained sections of the transplants are shown in Fig. 5A. Ectopic bone formations were histologically confirmed in both the manual and the automated groups. Moreover, the number of areas of bone formation in the sections was not statistically different (Fig. 5B), indicating similar in vivo osteogenic abilities of the BMSCs obtained from the two cultures.
Tumorigenic risk assay of cells obtained from automated culture To confirm the safety of the BMSCs obtained with this system, their tumorigenic risk was assayed. During the observation period, the mice were in good health and did not show any signs of tumor formation at either 3 or 6 months (data not shown). DISCUSSION Currently, clinical cell processing is a manual operation, which implies process instability because of human errors and individual differences in the required skills. The use of highly skilled personnel means higher costs, which can pose a major barrier preventing the widespread clinical use of cultured cells. Accordingly, the automation of cell processing is of interest. In the present study, we focused on BMSCs, one of the most widely used cell sources for tissue engineering, and compared the cells obtained from manual and automated cell cultures. The results showed no significant differences in cell yields, viabilities, cell populations, and ALP activities. Importantly, mixtures of the cells and b-TCP granules transplanted into the subcutaneous space of nude mice revealed no differences in their ability to induce ectopic bone formation. These results suggested that the BMSCs from the two culture methods were comparable in terms of bone regeneration and strongly supported the possibility of replacing manual cultures with automated procedure using the P 4C S. Cells from manual and automated cultures have been evaluated and compared in several reports (8e11). For example, manual and automated cell cultures of cardiac stem cells were directly compared with respect to cell growth rate, gene expression, cell
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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FIG. 4. Comparison of in vitro osteogenic potential. BMSCs obtained by manual or automated culture were cultured in osteogenic induction medium for 4 days. Cell proliferation (A) was determined using the WST-8 assay, followed by measurement of ALP activity (B) using a colorimetric p-nitrophenyl phosphate assay to determine the ALP ratio (ALP activity of induced cells/noninduced cells). The data represent the mean SD of three independent experiments using bone marrows from three different donors. The p-values are 0.21 (cell proliferation) and 0.26 (ALP ratio), respectively.
surface profiles, and genomic DNA stability (8). However, most of those studies used automated culture systems that simply replaced the human procedure with robotics. In the present study, we employed a unique culture system in which all manipulations are performed in a single culture vessel. Although a small size culture dish is usually required for the primary culture, because of the limited number of expandable cells, the results from this study show the feasibility of the single culture vessel system even for primary culture.
Other features of the P 4C S also contribute to the success of this culture system. For example, the cells are dispersed uniformly throughout the culture flask by a specialized shaking motion based on an optimized particle dispersion simulation (13). In this study, we could confirm the uniform and highly reproducible distribution of the BMSCs. This is one of the key advantages allowing the successful and stable primary culture of BMSCs, whereas in manual culture carried out by less skilled technicians this may not be possible. Additionally, during cultivation, the automated system
FIG. 5. Comparison of in vivo osteogenic potential. The grafts were retrieved at 4 weeks post-transplantation and subjected to histological examination. (A) Representative images of H&E-stained cross-sections of the grafts. The arrowheads indicate histologically defined new ectopic bones. Scale bar ¼ 200 mm. (B) The number of bone formation areas on the H&E-stained cross-sections of the graft. One representative result (mean SD; n ¼ 4) of two independent experiments using bone marrows from three different donors is shown. The p-value is 0.38.
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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periodically supplies an amount of air determined by calculating the oxygen consumption of the cultured cells, which is essential to promote their stable growth in a closed culture system. These functions may have contributed to the cell yields and properties comparable to those of cells obtained from manual culture using conventional open culture vessels. The previously reported robotic systems (8e11) use open culture vessels in an aseptically controlled chamber, whereas the automated culture system described herein uses a closed culture system, which has enormous advantages in terms of compactness and cost compared with robotic systems. Theoretically, a closed culture system does not require a clean environment, neither for the working space nor for inside the machinery. This was confirmed by placing the machinery in a conventional laboratory without any air filters and then testing for the microbial contamination of the automated culture process using a Medium Fill simulation test, which did not show any evidence of contamination. There was also no evidence of contamination of the automated cell cultures throughout this study (data not shown). These results demonstrate the robustness of this system against bacterial contamination, supporting the use of this system even outside of a specialized clean facility, which is currently required for open culture vessels (20). For clinical applications of cell-based therapy, safety is one of the most important concerns. The safety of cultured cells should be confirmed by: (i) microbial contamination and (ii) oncogenic transformation. The results of the Media Fill test confirmed that the automated process is able to produce cells without microbial contamination. In addition, the BMSCs obtained by automated culture showed no signs of tumor formation over a period of 6 months after their transplantation into nude mice. This observation is in agreement with the many reports of the resistance of MSC populations to oncogenic transformation, despite the culture methods or duration of culture (21e23), and supports the safety of BMSCs expanded with an automated system. Global gene expression profile is an important focus for the comparison of manual and automated cultures. In our preliminary experiment, we performed RNA sequencing for global gene expression profiling for the two BMSC groups obtained from independent automated cultures from different donors and compared their profiles. The result from this analysis showed significant variability in many numbers of genes, indicating the difficulty for comparing the profiles using limited samples. Numerous numbers of samples are required for precise comparison of two cultures. Although we believe that we could demonstrate here at least minimum criteria for the replacement of manual with automated cultures in a clinical setting, it is essential for comparing gene expression profiles between two cultures for a scientific aspect. Therefore we would like to perform this analysis by increasing samples in our future studies. In conclusion, in this work, we demonstrated that the BMSCs obtained by an automated cell culture system using clinically acceptable protocols were comparable to those generated by manual culture in terms of cell yields, viability, surface marker profile, and in vitro and in vivo osteogenic abilities. We were able to convincingly show that our automated process is safe in terms of microbial contamination and oncogenic transformation. In the clinical setting of bone tissue engineering, these results support the replacement of manual culture protocols with an automated culture system. ACKNOWLEDGMENTS This work was supported in part by a research grant from the Kaneka Corporation, Health Labor Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan, and the
J. BIOSCI. BIOENG., Translational Research Network Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). Hirokazu Akiyama, Masaki Ichimura, Hiroshi Tone, and Masaru Nakatani are employees of the Kaneka Corporation. Asako Kobayashi, Minoru Inoue, Arinobu Tojo, and Hideaki Kagami declare that there is no conflict of interest regarding the work described herein.
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Clinical-grade production of human mesenchymal stromal cells: occurrence of aneuploidy without transformation, Blood, 115, 1549e1553 (2010). 22. Bernardo, M. E., Zaffaroni, N., Novara, F., Cometa, A. M., Avanzini, M. A., Moretta, A., Montagna, D., Maccario, R., Villa, R., Daidone, M. G., Zuffardi, O., and Locatelli, F.: Human bone marrow derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms, Cancer Res., 67, 9142e9149 (2007). 23. Vilalta, M., Dégano, I. R., Bagó, J., Gould, D., Santos, M., García-Arranz, M., Ayats, R., Fuster, C., Chernajovsky, Y., García-Olmo, D., Rubio, N., and Blanco, J.: Biodistribution, long-term survival, and safety of human adipose tissue-derived mesenchymal stem cells transplanted in nude mice by high sensitivity non-invasive bioluminescence imaging, Stem Cells Dev., 17, 993e1003 (2008).
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering Hirokazu Akiyama,1 Asako Kobayashi,2 Masaki Ichimura,1 Hiroshi Tone,3 Masaru Nakatani,1 Minoru Inoue,2 Arinobu Tojo,2 and Hideaki Kagami2, 4, * Medical Device Development Laboratories, Kaneka Corporation, 1-8 Miyamae-cho, Takasago-cho, Takasago, Hyogo 676-8688, Japan,1 Tissue Engineering Research Group, Division of Molecular Therapy, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan,2 Technology Management Department, Medical Devices Division, Kaneka Corporation, 2-3-18 Nakanoshima, Kita-ku, Osaka 530-8288, Japan,3 and Department of Oral and Maxillofacial Surgery, Matsumoto Dental University School of Dentistry, 1780 Hirookagobara, Shiojiri, Nagano 399-0781, Japan4 Received 27 December 2014; accepted 16 March 2015 Available online xxx
The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering. Ó 2015, The Society for Biotechnology, Japan. All rights reserved. [Key words: Automated cell culture system; Bone marrow stromal cells; Osteogenic potential; Bone tissue engineering; Regenerative medicine]
Substantial efforts in tissue engineering have provided innovative methods to repair or regenerate damaged tissues and organs. Yet, although many clinical trials in this field have shown promising results (1e6), several problems remain that need to be addressed before these techniques find wider clinical application in regenerative medicine. One major problem is that current clinical cell processing methods, in addition to their high cost, are dependent on manual operations, which are vulnerable to human error and microbial contamination. To achieve stable and economical cell processing, automated systems have gained interest (7). To date, several automated cell culture systems have become commercially available (8e11), such as Auto Culture (Kawasaki Heavy Industries) (8) and CompacT SelecT (The Automation Partnership) (9e11), in which cells are cultured using open culture vessels manipulated by robotic arms incorporated into the aseptically controlled chamber. Because the robotic arm can replicate manual operations, this type of system has broad applicability for various types of cells. By contrast, the automated systems P 4C S (Kaneka, Osaka, Japan) and Quantum (Terumo BCT) (12) employ closed culture vessels, which confer enormous advantages in
* Corresponding author at: Tissue Engineering Research Group, Division of Molecular Therapy, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: þ81 3 5449 5120; fax: þ81 3 5449 5121. E-mail address: [email protected] (H. Kagami).
terms of compactness and the relatively low cost of machinery. The P 4C S was developed based on the prototype system reported by Kato et al. (13) and performs cell culture using an original disposable tubing set containing a single culture flask. One of the characteristic features of this culture system is the repeated use of a single culture flask even after cell passage, which is advantageous both for the compactness of the machinery and for stable continuous culture. Although the feasibility of this system for primary cultures of BMSCs and fibroblasts was reported, the detailed characters of cells cultured with this system have not been investigated. Recently, bone tissue engineering has attracted significant attention because it is less invasive than autologous bone grafting and may be more effective than artificial bone substitutes (14). Preliminary results from clinical studies demonstrated the usefulness of this approach for severe atrophy of alveolar bone (15). However, for the wide acceptance and commercialization of bone tissue engineering, an automated culture system remains desirable. In this study, we examined the feasibility of replacing the manual culture of BMSCs in the clinical setting of alveolar bone tissue engineering with an automated procedure using the P 4C S. Emphasis was placed on comparing the manually cultured cells with cells from the automated culture system. Importantly, we also examined the safety concerns regarding the microbiological contamination of automated culture and the oncogenic transformation of the cultured cells.
1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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Automated cell culture system The P 4C S (Kaneka) was developed based on the prototype system reported by Kato et al. (13). The photographs of the P 4C S are shown in Fig. 1A. The system was designed to perform cell culture in an enclosed system using a specially designed disposable tubing set containing a culture flask (culture area, 490 cm2), air filters, and solution bags. The dimensions of the machinery are 110 cm (W) 62 cm (D) 63 cm (H). The upper side of the machinery consists of the cooler and the incubation units. The cooler unit maintains the media and cell detachment solution at 5 C; the incubation unit maintains the culture environment at 37 C in an atmosphere of 5% CO2. On the lower side of the machinery, peristaltic pumps and valves are arranged to control the flow of solutions and air. The system is operated by an external personal computer. The detailed view of the whole culture system is shown in Fig. 1B. The solution bags in the tubing set consist of a sample material bag for cell loading, medium bags for storing the culture media, a cell-detachment solution bag for storing protease (e.g., trypsin), a saline solution bag for washing cells, waste bags for storing waste liquid, and a cell collection bag for collecting the cell suspension after cultivation. The system performs the typical cell manipulations of cell loading, medium exchange, and cell harvest. For cell loading, sample materials in the sample material bag are poured into the culture flask after which medium is supplied from the medium bags. For medium exchange, the spent medium in the culture flask is discarded by pumping it into the waste bag and fresh medium is then supplied. For cell harvest, the spent medium is discarded and the cells are repeatedly washed in saline solution. Protease is supplied from the cell-detachment solution bag and the cells are incubated for a specified period of time. Thereafter, fresh medium is added to stop the protease activity and the detached cells are pumped into the cell collection bag. During culture, fresh air (5% CO2) is periodically supplied to the culture flask through air filters in the incubation unit. In addition, images of multiple fixed positions within the culture flask are captured daily by a complementary metal-oxide-semiconductor camera arranged underneath the culture flask in the incubation unit. These manipulations are automatically performed according to the computer program. The detailed methods of these manipulations are described by Kato et al. (13).
J. BIOSCI. BIOENG., Media Fill test Potential microbial contamination in the automated cell culture process was estimated using a Media Fill test (16,17). The disposable tubing set, consisting of a sample material bag, medium bags, and a cell-detachment solution bag, all of which were filled with trypticase soy broth (Sysmex, Kobe, Japan) as microbial growth medium, was prepared using a clean bench with a grade A environment (class 100) in a clean room. The tubing set was then transferred to the machine, located in a conventional laboratory without any air filtration system. The culture period was set to 21 days, considered to represent the worst-case scenario (longest period of machine culture). After the simulation of the 21-day machine culture and following the 14-day manual culture (35-day culture in total), the medium was sampled. Thereafter, the harvested sample was incubated for 7 days at 22e25 C followed by an additional incubation for 7 days at 30e35 C. Subsequently, the harvested medium sample was qualitatively examined for evidence of microbial growth or turbidity during incubation and at the end of the 14-day incubation. The experiment was repeated three times. Manual and automated cultures Fresh human bone marrows, obtained from three healthy donors (25- and 28-year-old males and a 38-year-old female), were purchased from AllCells (Emeryville, CA, USA). Each bone marrow was divided into two 20-mL groups, and the manual and automated primary cultures of BMSCs were performed in parallel using the clinically acceptable protocols described below. For manual culture, 20 mL of bone marrow aspirate was diluted four-fold with serum-free alpha-minimum essential medium (a-MEM; Life Technologies, Carlsbad, CA, USA) supplemented with 50 mg gentamicin (Fuji Pharma, Tokyo, Japan)/mL and 2.5 mg amphotericin B (Fungizone; Bristol-Myers Squibb, New York, NY, USA)/mL. Then, diluted bone marrow was plated into four T-150 flasks (Corning, New York, NY, USA) so that each flask contained 5 mL of bone marrow. After 4 days of cultivation in a 37 C, 5% CO2 incubator, the culture medium was replaced with a-MEM supplemented with 10% fetal bovine serum (FBS; Thermo Scientific, Waltham, MA, USA) and 1 ng of basic fibroblast growth factor (bFGF; Kaken Pharmaceutical, Tokyo, Japan)/mL. Thereafter, the cells were fed twice a week with a-MEM supplemented with FBS and bFGF until they reached sub-confluence, at which time they were harvested using TrypLE Select (Life Technologies). For automated culture using the P 4C S, 20 mL of bone marrow aspirate was poured into the sample material bag. Cell loading was then carried out to transfer
FIG. 1. Automated cell culture system. (A) Photographs of the P 4C S. The P 4C S was designed to culture cells in a closed system by using a single-use disposable tubing set containing a cell culture flask, air filters, and solution bags. (B) A detailed view of the whole culture system.
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bone marrow and the culture medium (a-MEM supplemented with FBS and bFGF) from the medium bag to a culture flask. The medium was exchanged according to the same schedule used for manual culture. The cells were harvested at subconfluence by supplying TrypLE Select from the cell detachment solution bag.
determined. At the end of the observation period, the mice were evaluated by an examiner. The grafts were resected, fixed, embedded in paraffin, serially sectioned, and stained with H&E. The serial sections (covering the whole sample) were analyzed under the microscope for tumor formation.
Measurements of cell number and viability The harvested cells were stained with trypan blue solution and the numbers of viable and dead cells were counted using a hemacytometer. Cell viability was calculated as the number of viable cells divided by the total number of cells.
Statistical analysis All data were expressed as means standard deviation (SD). Statistical comparison in in vivo osteogenic potential assay was evaluated using an unpaired t-test, because the differences among the transplantation sites of each graft made it difficult to presume the paired groups. The others were evaluated using a paired t-test. p < 0.05 was considered significantly different.
Flow cytometric analysis The harvested cells were fixed in 4% paraformaldehyde in PBS, washed in PBS, and then incubated with following fluorescence isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated antibodies: CD14-FITC, CD29-PE, CD34-PE, CD44-PE, CD45-FITC, CD73-PE, CD79aPE, CD90-FITC, CD166-PE, HLA-DR-FITC (all from BD Pharmingen, San Diego, CA, USA), and CD105-FITC (Ancell, Bayport, MN, USA). The labeled cells were analyzed using a FACS Calibur flow cytometer and CellQuest software (BD Biosciences, San Jose, CA, USA). A fluorescence-conjugated IgG (BD Pharmingen) served as the negative control. Cell proliferation and alkaline phosphatase activity assays Cell proliferation and alkaline phosphatase (ALP) activity after osteogenic induction were analyzed as described previously (18). The cells were seeded into the wells of a 12well plate (Corning) at a density of 2 104 cells/well and cultured with serumcontaining a-MEM. After 1 day of culture, the medium was replaced with osteogenic induction medium (serum-containing a-MEM with 10 nM dexamethasone, 100 mM ascorbic acid, and 10 mM glycerol-2-phosphate disodium salt hydrate) and cultivation was continued. As a control, the cells were cultured without osteogenic supplements. After 4 days of osteogenic induction, cell metabolic activity, as an index of cell proliferation, was measured using a commercially available kit based on the WST-8 assay (Cell Counting Kit 8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. ALP activities of noninduced and induced BMSCs were then measured with a commercially available p-nitrophenyl phosphate tablet set (SigmaeAldrich, St. Louis, MO, USA). In vivo osteogenic potential and tumorigenic risk assays All animal experiments in this study were approved by the Animal Ethics Screening Committee of IMSUT, The University of Tokyo. The in vivo osteogenic potential and tumorigenic risk assay of the cultured cells were analyzed using an ectopic transplantation model, as described previously (18). BMSCs (5 105 cells) were mixed with 50 mg of b-tricalcium phosphate (b-TCP) granules (G1 type Osferion Olympus Terumo Biomaterials, Tokyo, Japan) in a 14-mL round-bottom polypropylene tube (BD Biosciences) to which serum-containing aMEM had been added. After 1 day of culture, the culture medium was replaced with osteogenic induction medium and cultivation was continued for 2 weeks. The medium was exchanged twice a week. After 2 weeks of osteogenic induction, the mixtures of cells and b-TCP granules were transplanted into the subcutaneous space on the backs of 6-week-old female BALB/CAJc1-nu/nu mice (Nihoncrea, Tokyo, Japan) under general anesthesia with pentobarbital sodium. To assay the in vivo osteogenic potential, the mice were killed with an overdose of anesthesia at 4 weeks post-transplantation and the grafts were resected. Harvested grafts were fixed in 10% formalin and embedded in paraffin. Five-micron-thick sections were then cut and stained with hematoxylin and eosin (H&E). The number of areas of histologically defined new ectopic bone as seen on the H&E-stained sections was counted under the microscope. For tumorigenic risk assay, BMSCs from two different donors were independently prepared and transplanted into the nude mice as describe above. Four samples from one donor were transplanted into two nude mice (each receiving two grafts) and observed for 3 and 6 months each. From the other donor, four samples were also transplanted and at least the results at 3 months were
RESULTS Media Fill test Prior to the cell culture experiment, potential microbial contamination in the automated cell culture process was analyzed using a Media Fill test (16,17), in which the whole cell culture process is simulated using microbial growth medium instead of the cells and conventional culture medium. In three runs, there was no evidence of turbidity, indicative of microbial contamination. Comparison of cell yields and viabilities The bone marrow was divided into two 20-mL groups for manual and automated primary cultures of BMSCs performed in parallel using clinically acceptable protocols. In three independent experiments using bone marrow from different donors, the cells in both cultures reached sub-confluence (60e95% confluence) after 11e14 days, at which time they were simultaneously harvested and both cell numbers and viability were measured using a trypan blue dye exclusion method (Fig. 2). The yield of the automated cell culture was 1.69e2.24 107 cells (Fig. 2A) with a viability of 96.6e99.1% (Fig. 2B), which was not statistically different from the manual culture. In addition, the cell yields of the automated culture were almost equivalent to those obtained in manual culture performed as part of a clinical alveolar bone tissue engineering study (unpublished data), indicating that the P 4C S is capable of producing clinically relevant cell yields for bone tissue engineering. Comparison of cell surface marker expression profiles The expression of cell surface markers by the harvested BMSCs from the manual and automated cultures was analyzed using a flow cytometer (Fig. 3). As shown in Fig. 3A, the harvested cells from both cultures were positive for the mesenchymal stem cell (MSC) markers CD29, CD44, CD73, CD90, CD105, and CD166. Moreover, the percentages of cells positive for these markers were almost identical, indicating the equal distribution of subpopulations. These percentages and those of other surface markers (CD14, CD34, CD45, CD79a, and HLA-DR) are shown in Fig. 3B. Only a limited number of cells in both groups were positive for CD14, CD34, CD45, and CD79a. The HLA-DR-positive fraction was
FIG. 2. Comparison of cell numbers and viability. BMSCs in manual and automated cultures were harvested at the same time after reaching sub-confluence. Cell numbers (A) and viability (B) were measured using the trypan blue dye-exclusion method. The data represent the mean SD from three independent experiments using bone marrows from three different donors. The p-values are 0.16 (cell numbers) and 0.39 (cell viability), respectively.
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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J. BIOSCI. BIOENG.,
FIG. 3. Comparison of surface marker expression profiles. The expression of surface marker by BMSCs from the manual or automated cultures was analyzed using a flow cytometer. (A) Histograms of CD29, CD44, CD73, CD90, CD105, and CD166, which are positive markers for MSCs. (B) Expression percentages of surface markers including CD14, CD34, CD45, CD79a, and HLA-DR. A representative result of two independent experiments using bone marrow from three different donors is shown.
similar in the two groups (the difference was not significant). These results indicated the similar expression profiles of the manually and automated cell cultures. Comparison of in vitro osteogenic potential It was previously reported that cell proliferation in osteogenic induction culture correlated with bone-forming potential (18), in which the upregulation of ALP activity after osteogenic induction (ALP ratio ¼ ALP activity of induced cells/noninduced cells) may also play a role (19). Therefore, to compare the in vitro osteogenic potential of the BMSCs obtained by manual and automated cultures, the harvested cells were cultured in osteogenic induction medium for 4 days, after which cell proliferation was determined using the WST-8 assay (Fig. 4A). The ALP ratio was analyzed using a colorimetric p-nitrophenyl phosphate assay (Fig. 4B). In both analyses, no statistical differences were found between the manual and the automated cell culture groups, indicative of the comparable in vitro osteogenic potentials of the BMSCs of the two cultures. Comparison of in vivo osteogenic potential The in vivo osteogenic potential of the cultured cells was investigated using an ectopic transplantation model. Representative micrographs of the H&E-stained sections of the transplants are shown in Fig. 5A. Ectopic bone formations were histologically confirmed in both the manual and the automated groups. Moreover, the number of areas of bone formation in the sections was not statistically different (Fig. 5B), indicating similar in vivo osteogenic abilities of the BMSCs obtained from the two cultures.
Tumorigenic risk assay of cells obtained from automated culture To confirm the safety of the BMSCs obtained with this system, their tumorigenic risk was assayed. During the observation period, the mice were in good health and did not show any signs of tumor formation at either 3 or 6 months (data not shown). DISCUSSION Currently, clinical cell processing is a manual operation, which implies process instability because of human errors and individual differences in the required skills. The use of highly skilled personnel means higher costs, which can pose a major barrier preventing the widespread clinical use of cultured cells. Accordingly, the automation of cell processing is of interest. In the present study, we focused on BMSCs, one of the most widely used cell sources for tissue engineering, and compared the cells obtained from manual and automated cell cultures. The results showed no significant differences in cell yields, viabilities, cell populations, and ALP activities. Importantly, mixtures of the cells and b-TCP granules transplanted into the subcutaneous space of nude mice revealed no differences in their ability to induce ectopic bone formation. These results suggested that the BMSCs from the two culture methods were comparable in terms of bone regeneration and strongly supported the possibility of replacing manual cultures with automated procedure using the P 4C S. Cells from manual and automated cultures have been evaluated and compared in several reports (8e11). For example, manual and automated cell cultures of cardiac stem cells were directly compared with respect to cell growth rate, gene expression, cell
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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FIG. 4. Comparison of in vitro osteogenic potential. BMSCs obtained by manual or automated culture were cultured in osteogenic induction medium for 4 days. Cell proliferation (A) was determined using the WST-8 assay, followed by measurement of ALP activity (B) using a colorimetric p-nitrophenyl phosphate assay to determine the ALP ratio (ALP activity of induced cells/noninduced cells). The data represent the mean SD of three independent experiments using bone marrows from three different donors. The p-values are 0.21 (cell proliferation) and 0.26 (ALP ratio), respectively.
surface profiles, and genomic DNA stability (8). However, most of those studies used automated culture systems that simply replaced the human procedure with robotics. In the present study, we employed a unique culture system in which all manipulations are performed in a single culture vessel. Although a small size culture dish is usually required for the primary culture, because of the limited number of expandable cells, the results from this study show the feasibility of the single culture vessel system even for primary culture.
Other features of the P 4C S also contribute to the success of this culture system. For example, the cells are dispersed uniformly throughout the culture flask by a specialized shaking motion based on an optimized particle dispersion simulation (13). In this study, we could confirm the uniform and highly reproducible distribution of the BMSCs. This is one of the key advantages allowing the successful and stable primary culture of BMSCs, whereas in manual culture carried out by less skilled technicians this may not be possible. Additionally, during cultivation, the automated system
FIG. 5. Comparison of in vivo osteogenic potential. The grafts were retrieved at 4 weeks post-transplantation and subjected to histological examination. (A) Representative images of H&E-stained cross-sections of the grafts. The arrowheads indicate histologically defined new ectopic bones. Scale bar ¼ 200 mm. (B) The number of bone formation areas on the H&E-stained cross-sections of the graft. One representative result (mean SD; n ¼ 4) of two independent experiments using bone marrows from three different donors is shown. The p-value is 0.38.
Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011
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periodically supplies an amount of air determined by calculating the oxygen consumption of the cultured cells, which is essential to promote their stable growth in a closed culture system. These functions may have contributed to the cell yields and properties comparable to those of cells obtained from manual culture using conventional open culture vessels. The previously reported robotic systems (8e11) use open culture vessels in an aseptically controlled chamber, whereas the automated culture system described herein uses a closed culture system, which has enormous advantages in terms of compactness and cost compared with robotic systems. Theoretically, a closed culture system does not require a clean environment, neither for the working space nor for inside the machinery. This was confirmed by placing the machinery in a conventional laboratory without any air filters and then testing for the microbial contamination of the automated culture process using a Medium Fill simulation test, which did not show any evidence of contamination. There was also no evidence of contamination of the automated cell cultures throughout this study (data not shown). These results demonstrate the robustness of this system against bacterial contamination, supporting the use of this system even outside of a specialized clean facility, which is currently required for open culture vessels (20). For clinical applications of cell-based therapy, safety is one of the most important concerns. The safety of cultured cells should be confirmed by: (i) microbial contamination and (ii) oncogenic transformation. The results of the Media Fill test confirmed that the automated process is able to produce cells without microbial contamination. In addition, the BMSCs obtained by automated culture showed no signs of tumor formation over a period of 6 months after their transplantation into nude mice. This observation is in agreement with the many reports of the resistance of MSC populations to oncogenic transformation, despite the culture methods or duration of culture (21e23), and supports the safety of BMSCs expanded with an automated system. Global gene expression profile is an important focus for the comparison of manual and automated cultures. In our preliminary experiment, we performed RNA sequencing for global gene expression profiling for the two BMSC groups obtained from independent automated cultures from different donors and compared their profiles. The result from this analysis showed significant variability in many numbers of genes, indicating the difficulty for comparing the profiles using limited samples. Numerous numbers of samples are required for precise comparison of two cultures. Although we believe that we could demonstrate here at least minimum criteria for the replacement of manual with automated cultures in a clinical setting, it is essential for comparing gene expression profiles between two cultures for a scientific aspect. Therefore we would like to perform this analysis by increasing samples in our future studies. In conclusion, in this work, we demonstrated that the BMSCs obtained by an automated cell culture system using clinically acceptable protocols were comparable to those generated by manual culture in terms of cell yields, viability, surface marker profile, and in vitro and in vivo osteogenic abilities. We were able to convincingly show that our automated process is safe in terms of microbial contamination and oncogenic transformation. In the clinical setting of bone tissue engineering, these results support the replacement of manual culture protocols with an automated culture system. ACKNOWLEDGMENTS This work was supported in part by a research grant from the Kaneka Corporation, Health Labor Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan, and the
J. BIOSCI. BIOENG., Translational Research Network Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). Hirokazu Akiyama, Masaki Ichimura, Hiroshi Tone, and Masaru Nakatani are employees of the Kaneka Corporation. Asako Kobayashi, Minoru Inoue, Arinobu Tojo, and Hideaki Kagami declare that there is no conflict of interest regarding the work described herein.
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Please cite this article in press as: Akiyama, H., et al., Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.03.011