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Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2006) 30, 521–524 (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


Abstract

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.


1 Introduction

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 1000nasa units (Fig. 1B). Two hundred milliliters of venous whole blood from three donors (males 26 and 38 years old, female 64 years old) was taken into the large bag with glass beads and incubated at room temperature for 30min under gently stirring conditions to prepare serum (Fig. 1C). Approximately 100ml of serum from each donor could be separated from clot by centrifugation at 2500rpm for 10min (Fig. 1D, E). After the air in the bag with glass beads had been transferred into another large bag, the serum was divided into three attached small bags using a plasma extractor (Fig. 1F, G), and stored at −80°C until use.


Fig. 1

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 30min. (D) Centrifugation of the bags at 2500rpm for 10min. (E) Separated serum. (F) Plasma extractor by which serum in the large bag was transferred into three small bags. (G) The large bags with clot left and three small bags containing the serum.


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 2×108cells/100mm tissue culture dish and maintained in 10ml of DMEM supplemented with 100units/ml penicillin, 100μg/ml streptomycin and 10% HAS (MHAS). The rest of each cell preparation was seeded under the same conditions and maintained in 10ml of DMEM supplemented with the antibiotics and 10% FCS (MFCS). Three days after seeding, floating cells were removed and the medium was replaced by fresh MHAS and MFCS. Thereafter, the attached cells were cultured with fresh MHAS and MFCS every 3 days. Passages were performed when cells were approaching confluence. hMSC were seeded in 100mm plastic tissue culture dishes and incubated in 5% CO2/95% air at 37°C. hMSC obtained from the cultures at the third passages were used for the following experiments.

2.3 Cell proliferation

Each hMSC was seeded at a density of 3×104cells/35mm plastic tissue culture plates and maintained in MHAS or MFCS. Cell numbers were counted with a hemacytometer at the indicated times.

2.4 Osteoblastic induction

hMSC in cultures at the third passages were harvested and seeded at a density of 2×104cells/35mm plastic culture plates in MHAS or MFCS. One day later, the cells were incubated in DMEM supplemented with 10nM β-glycerophosphate, 100mM dexamethasone, 0.25mM l-ascorbic acid, and 10% HAS or 10% FCS (Tsutsumi et al., 2001). The media were replaced twice a week. After 3 weeks, mineralized deposition was identified by von Kossa staining.

2.5 Adipose induction

hMSC in cultures at the third passages were harvested and seeded at a density of 1.5×105cells/35mm plastic culture plates in MHAS or MFCS and they became confluent in 3–5 days. After confluence, the cells were incubated in Alpha Minimal Essential Medium supplemented with 5μg/ml insulin, 10nM dexamethasone, 50μM 5,8,11,14-eicosatetraynoic acid, and 10% HAS or 10% FCS (Tsutsumi et al., 2001). Medium was replaced twice a week. After 3 weeks, hMSC were fixed in 10% neutral buffered formalin for 25min and stained with Oil Red O.

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;100ml of human serum. Although the serum obtained with the bags had not been 0.22-μm-filtered, hMSC could be maintained in the medium containing the serum for long terms without contamination. Furthermore, bacteria, fungus, mycoplasma and endotoxin were not detected in the HAS-contained medium where hMSC had been cultured (data not shown).

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).


Fig. 2

Cell proliferation of hMSC cultures in HAS or FCS. hMSC-1, -2 and -3 were seeded and maintained as described in Section 2. The cell number was counted on days 3, 6 and 9. Values are means±S.D. of three cultures. Differences were significant (*P<0.01) between numbers of hMSC cultured in HAS and those in FCS on the same day.


Fig. 3

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.

Acknowledgements

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 2006

doi:10.1016/j.cellbi.2006.01.010


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)