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

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