Sains Malaysiana 41(9)(2012): 1099–1107

 

 

In vitro Proliferation of Mononucleated Suspension and Adherent Cells from Mouse and Human Peripheral Blood System

(Proliferasi In vitro Sel Ampaian dan Melekat Mononukleus daripada Sistem Darah Periferi

Mencit dan Manusia)

 

Shahrul Hisham Zainal Ariffin*, Muhammad Dain Yazid, Ruzanna Ab Kadir & Shabnam Kermani

Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi,

Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor D.E. Malaysia

 

Rohaya Megat Abdul Wahab

Jabatan Ortodontik, Fakulti Pergigian, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz

53000 Kuala Lumpur, Malaysia

 

Diserahkan: 21 Disember 2011 / Diterima: 21 Mei 2012

 

ABSTRACT

Primary cells have a limited proliferative capacity with a finite number of times as compared with cell line which can grow indefinitely. Therefore, this study was carried out to identify the proliferative capacity of primary mononucleated cells from mouse and human. The mononucleated cells were isolated from mouse and human peripheral blood by density gradient centrifugation using Ficoll-Paque™ Plus. The two types of cells i.e. suspension and adherent forms were obtained after culturing the isolated mononucleated cells for 4 days in the complete medium consists of Alpha Minimal Essential Medium, 10% newborn calf serum and 2% penicillin/streptomycin. The cells were then cultured for another 10 days to observe cell viability using trypan blue exclusion assay (suspension form) and MTT assay (adherent form). NSO and MC3T3-E1 cell lines were selected as control cell for suspension and adherent cells, respectively. Our results showed that the proliferation rate of mouse suspension mononucleated cells increased from 1.31 ± 0.24 cells/day (day 5) to 2.69 ± 0.42 cells/day (day 10) whilst, for human suspension cells, the proliferation rate slightly increased from 0.56 ± 0.20 cells/day (day 5) to 0.76 ± 0.29 cells/day (day 10). However, the proliferation rate of mouse adherent mononucleated cells decreased from 0.23 ± 0.02 cells/day (day 5) to 0.17 ± 0.01 cells/day (day 10). Meanwhile, human adherent cells maintained proliferation rate at approximately 0.67 ± 0.18 cells/day. In conclusion, adherent primary mononucleated cells from both mouse and human have limited capacity to generate more cells in vitro as compared with suspension mononucleated cells.

 

Keywords: Adherent cells; peripheral blood; proliferation; suspension cells

 

ABSTRAK

Sel primer mempunyai keupayaan yang terhad untuk berproliferasi berbanding titisan sel yang berupaya untuk berproliferasi tanpa had. Oleh itu, kajian ini dilakukan untuk menentukan keupayaan proliferasi sel primer mononukleus daripada mencit dan manusia. Sel mononukleus diasingkan daripada darah periferi mencit dan manusia dengan menggunakan larutan Ficoll-Paque™ Plus melalui kaedah pengemparan kecerunan ketumpatan. Terdapat dua jenis sel iaitu ampaian dan melekat yang diperoleh selepas pengkulturan sel mononukleus diasingkan selama 4 hari di dalam medium lengkap yang mengandungi ‘Alpha Minimal Essential Medium’, 10% serum anak lembu dan 2% penisilin-streptomicin. Sel ini kemudiannya dikultur selama 10 hari untuk analisis viabiliti menggunakan pengasaian tripan biru (sel ampaian) dan MTT (sel melekat). Sel titisan NSO and MC3T3-E1 masing-masing dipilih sebagai sel kawalan untuk sel ampaian dan melekat. Hasil kami menunjukkan bahawa kadar proliferasi sel ampaian mononukleus mencit meningkat daripada 1.31 ± 0.24 sel/hari (hari ke-5) kepada 2.69 ± 0.42 sel/hari (hari ke-10) manakala, kadar proliferasi sel ampaian manusia meningkat sedikit daripada 0.56 ± 0.20 sel/hari (hari ke-5) kepada 0.76 ± 0.29 sel/hari (hari 10). Walau bagaimanapun, kadar proliferasi sel melekat mononukleus mencit menurun daripada 0.23 ± 0.02 sel/hari (hari ke-5) kepada 0.17 ± 0.01 sel/hari (hari ke-10). Manakala, kadar proliferasi sel melekat mononukleus manusia kekal pada 0.67 ± 0.18 sel/hari. Kesimpulannya, sel primer melekat mononukleus daripada mencit dan manusia mempunyai keupayaan terhad untuk menghasilkan sel secara in vitro berbanding sel ampaian mononukleus.

 

Kata kunci: Darah periferi; proliferasi; sel ampaian; sel melekat

REFERENCES

 

Ariffin, S.H.Z., Wan Omar, W.H.H., Ariffin, Z.Z., Safian, M.F., Senafi, S. & Megat Abdul Wahab, R. 2009. Intrinsic anticarcinogenic effects of Piper sarmentosumethanolic extract on a human hepatoma cell line. Cancer Cell International 9: 6.

Berridge, M.V., Herst, P.M. & Tan, A.S. 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review 11: 127-152.

Campisi, J. 2003. Cellular senescence and apoptosis: how cellular responses might influence aging phenotypes. Experimental Gerontology 38(1-2): 5-11.

de Magalhães, J.P. & Toussaint, O. 2004. Telomeres and telomerase: a modern fountain of youth? Rejuvenation Research 7(2): 126-133.

Demsey, A. & Grimley, P.M. 1976. Characteristics of a surface-adherent subline derived from friend erythroleukemia cells in continuous suspension culture. Cancer Research 36: 384-393.

Freshney, R.I. 2011. Culture of animal cells: A manual of basic technique. In Cytotoxicity. NY: John Wiley & Sons, Inc.

Golubev, A., Khrustalev, S. & Butov, A. 2003. An in silicoinvestigation into the causes of telomere length heterogeneity and its implications for the Hayflick limit. Journal of Theoretical Biology 225(2): 153-170.

Hayflick, L. & Moorhead, P.S. 1961. The serial cultivation of human diploid cell strains. Experimental Cell Research 25(3): 585-621.

Iamaroon, A., Tait, B. & Diewert, V. 1996. Cell proliferation and expression of EGF, TGF-α, and EGF receptor in the developing primary palate. Journal of Dental Research 75(8): 1534-1539.

Kennedy, A.L., McBryan, T., Enders, G.H., Johnson, F.B., Zhang, R. & Adams, P.D. 2010. Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci. Cell Division 5:16.

Lennon, D.P., Edmison, J.M. & Caplan, A.I. 2001. Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis. Journal of Cellular Physiology 187: 345-355.

Marciniak-Czochra, A., Stiehl, T. & Wagner, W. 2009. Modeling of replicative senescence in hematopoietic development. Aging 1(8): 723.

Morgan, D.O. 2007. The Cell Cycle: Principal of Control. London: New Science Press Ltd.

Ryu, J.H., Oh, D.J., Choi, C.Y. & Kim, B.S. 2003. Suspension culture of anchorage-dependent animal cells using nanospheres of the biodegradable polymer, poly (lactic-co-glycolic acid). Biotechnology Letters 25(16): 1363-1367.

Soti, C., Sreedhar, A.S. & Csermely, P. 2003. Apoptosis, necrosis and cellular senescence: chaperone occupancy as a potential switch. Aging Cell 2(1): 39-45.

Sulic, S., Panic, L., Dikic, I. & Volarevic, S. 2005. Deregulation of cell growth and malignant transformation. Croatian Medical Journal 46(4): 622-638.

Tresini, M., Lorenzini, A., Frisoni, L., Allen, R.G. & Cristofalo, V.J. 2001. Lack of Elk-1 phosphorylation and dysregulation of the extracellular regulated kinase signaling pathway in senescent human fibroblast. Exp. Cell Res. 269(2): 287-300.

van Zglinicki, T., Saretzki, G., Docke, W. & Lotze, C. 1995. Mild hyperoxia shortens telomeres and inhibits proliferation: a model for senescence? Experimental Cell Research 220: 186.

Zhang, W. & Liu, H. T. 2002. MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Research 12(1): 9-18.

Zhang, Z., Tong, J., Lu, R., Scutt, A., Goltzman, D. & Miao, D. 2008. Therapeutic potential of non-adherent BM-derived mesenchymal stem cells in tissue regeneration. Bone Marrow Transplantation 43(1): 69-81.

 

*Pengarang surat-menyurat; email: hisham@ukm.my

 

 

 

sebelumnya