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Cell Biology International (2006) 30, 521524 (Printed in Great Britain)
Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures
Noriyoshi Mizunoa*, Hideki Shibaa, Yoshitaka Ozekia, Yoshihiro Mouria, Miyuki Niitania, Takafumi Inuia, Hideaki Hayashia, Koji Suzukib, Seishin Tanakab, Hiroyuki Kawaguchia and Hidemi Kuriharaa
aDepartment of Periodontal Medicine, Division of Frontier Medical Science, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan
bJMS Company, Limited, 12-17, Kako-machi, Naka-ku, Hiroshima 730-8652, Japan
The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.
Keywords: Human autologous serum, Human mesenchymal stem cell, Cell therapy, Cell proliferation, Multipotency.
*Corresponding author. Tel.: +81 82 257 5663; fax: +81 82 257 5664.
Therapy in which cells obtained from a patient are re-implanted into the same patient has now become feasible, and implies that the re-implanted cells replace lost and dysfunctional tissues by those with regenerated functional activity.
hMSC are able to differentiate into various cells, such as osteoblasts, chondrocytes and adipocytes in vitro and in vivo (Pittenger et al., 1999; Jaiswal et al., 1997; Negishi et al., 2000). hMSC can easily and repeatedly be obtained from a patient by bone marrow aspiration (Tsutsumi et al., 2001). From these findings, hMSC are expected to be useful for cell therapy (Noel et al., 2002; Kotobuki et al., 2004). Moreover, the reimplantation of hMSC into the source patient would give no problem of immune reaction, and would not raise ethical considerations.
In vitro expansion of hMSC isolated from a patient must be undertaken when a large number of cells need to be re-implanted. It is more appropriate to culture hMSC with HAS rather than with FCS, and human homologous serum for the expansion of hMSC in order for a patient not to be contaminated by virus, bacteria and foreign protein (Nijweide and Bauger, 1990). Therefore, a tool by which HAS can be simply, safely and efficiently obtained from a patient needed to be developed. In addition, the question is whether hMSC cultured in a medium containing HAS obtained with the developed closed bag system maintains proliferative and differentiative ability in the same way as hMSC cultured with commercially available FCS. We have examined proliferation and multipotency in these circumstances and now report our findings.
2 Materials and methods
2.1 Preparation of HAS
We have developed a completely closed bag system separating serum for cell culture. The system has two large bags and three attached small bags (Fig. 1A). These five bags are connected to each other. One of the two large bags has five glass beads for activating platelet and removal of fibrin from blood. After disinfection with antiseptic solution, the crook of a donor was covered with a sheet of sterilized paper in the clean room, air cleanness level of which was 1000
Protocol for the separation of human serum using the completely closed bag system. (A) A whole view of the closed bag system separating serum. (B) Disinfection of the crook with antiseptic solution in the clean room. (C) Gently stirring of the blood in a large bag with glass beads, being done at room temperature for 30
2.2 Isolation and expansion of hMSC
hMSC were separately isolated from the ileum bone marrow aspirates of the three donors (hMSC-1, -2 and -3) according to a protocol approved by the ethical authorities at Hiroshima University. Some of cells, including erythrocytes, from each donor were seeded at 2
2.3 Cell proliferation
Each hMSC was seeded at a density of 3
2.4 Osteoblastic induction
hMSC in cultures at the third passages were harvested and seeded at a density of 2
2.5 Adipose induction
hMSC in cultures at the third passages were harvested and seeded at a density of 1.5
2.6 Statistical analysis
Statistical analyses of the numbers of each cell cultured with HAS or FCS for 3, 6, 9 days were performed using Student's t-test.
3 Results and discussion
We have developed a completely closed bag system for separating &007E;100
HAS obtained with the device had greater activity on proliferation of hMSC-1, -2 and -3 on day 9 than FCS (Fig. 2). Furthermore, hMSC-1, -2 and -3 cultured in the HAS, like FCS, differentiated into osteoblasts and adipocytes (Fig. 3 and data not shown). These findings are consistent with the previous report of Stute et al. (2004).
Cell proliferation of hMSC cultures in HAS or FCS. hMSC-1, -2 and -3 were seeded and maintained as described in Section
Phase-contrast micrographs of hMSC exposed to various differentiation media containing HAS or FCS. (A) von Kossa staining identifying mineralized deposition in hMSC-2. (B) Oil red O staining identifying fat cells with lipid vacuoles in hMSC-2.
In conclusion, we could simply, safely, efficiently and quickly obtain human serum with our closed bag system. Furthermore, the HAS stimulated the proliferation of hMSC and maintained multipotential. The closed bag system could greatly contribute to the progress of basic research and cell therapy in which human serum is needed.
This work was supported in part by Grant-in-Aid for Scientific Research (A) (No.15390647) and Grant-in-Aid for Encouragement of Young Scientists (B) (No.16791314) from the Japan Society for the Promotion of Science, Japan.
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Received 17 June 2005/9 November 2005; accepted 30 January 2006doi:10.1016/j.cellbi.2006.01.010