Sains Malaysiana 48(9)(2019): 1855–1865

http://dx.doi.org/10.17576/jsm-2019-4809-06 

Pengkulturan dan Pencirian Penanda Molekul Sel Stem Manusia daripada Pulpa Gigi Susu (SHED) dan Gigi Kekal (DPSC)

(Culture and Molecular Markers Characterization of Stem Cells from Human Deciduous (SHED) and Permanent (DPSC) Teeth Pulp)

SHAHRUL HISHAM ZAINAL ARIFFIN¹*, THANALETCHUMI MANOGARAN¹, INTAN ZARINA ZAINOL ABIDIN², ZAIDAH ZAINAL ARIFFIN³ & ROHAYA MEGAT ABDUL WAHAB⁴

¹Pusat Bioteknologi dan Makanan Berfungsi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor Darul Ehsan, Malaysia

²Centre for Research and Postgraduate Studies, Cyberjaya University College of Medical Sciences, 63000 Cyberjaya, Selangor Darul Ehsan, Malaysia

³Pusat Pengajian Biologi, Fakulti Sains Gunaan, Universiti Teknologi MARA, 40450 UiTM Shah Alam, Selangor Darul Ehsan, Malaysia

⁴Unit Ortodontik, Pusat Kesihatan Pergigian Keluarga, Fakulti Pergigian, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Wilayah Persekutuan, Malaysia

Received: 25 February 2019/Accepted: 25 June 2019

ABSTRAK

Sel stem pulpa gigi manusia yang dipencilkan daripada tisu pulpa gigi merupakan sel stem dewasa bersifat multipoten. Objektif kajian ini adalah untuk mengenal pasti teknik pengkulturan in vitro sel stem pulpa gigi susu (SHED) dan gigi kekal (DPSC) melalui penentuan pasaj, kesan eraman tripsin-EDTA dan potensi proliferasi serta untuk mencirikan kedua-dua sel ini melalui profil penanda molekul. Kaedah pencernaan enzim digunakan pada tisu pulpa gigi susu dan gigi kekal masing-masing untuk pemencilan sel SHED dan DPSC. Kedua-dua sel dikulturkan daripada pasaj 1 hingga 5 dan pewarnaan tripan biru digunakan untuk memperoleh lengkuk pertumbuhan bagi menentukan masa penggandaan populasi sel (PDT) pada setiap pasaj. Kesan tripsin-EDTA terhadap kedua-dua sel dikaji menggunakan pewarnaan Alamar biru untuk menentukan masa eraman yang optimum semasa proses pengsubkulturan. Potensi proliferasi in vitro bagi kedua-dua sel selama 21 hari ditentukan melalui asai 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromida (MTT). Pencirian kedua-dua sel melalui pengekspresan penanda biologi molekul ditentukan melalui pendekatan RT-PCR. Morfologi kedua-dua sel didapati menyerupai sel fibroblas pada kesemua pasaj. Sel SHED dan DPSC pada pasaj 3 menunjukkan PDT terendah iaitu masing-masing 43 ± 2.3 dan 63 ± 3.1 jam. Pendedahan SHED terhadap tripsin-EDTA menunjukkan penurunan peratus sel viabel berbanding DPSC. Pertumbuhan sel SHED didapati ~2.3 kali ganda lebih tinggi berbanding DPSC. Pencirian molekul kedua-dua sel menunjukkan pengekspresan penanda sel stem mesekima dan bukannya penanda sel stem hematopoietik. Kesimpulannya, morfologi sel yang homogenus dan nilai PDT terendah yang ditunjukkan oleh sel daripada pasaj 3 menjadikannya pasaj terbaik untuk menentukan potensi proliferasi sel dan pengekpresan penanda molekul. SHED didapati mampu berproliferasi dengan lebih baik berbanding DPSC. Walau bagaimanapun, DPSC lebih rentan terhadap tripsin-EDTA berbanding SHED. Pencirian molekul pula mendapati kedua-dua sel merupakan sel stem jenis mesenkima.

Kata kunci: Kesan tripsin-EDTA; masa penggandaan sel; potensi proliferasi; profil penanda biologi molekul; sel stem pulpa gigi manusia

 ABSTRACT

Human dental pulp stem cells are adult multipotent stem cells isolated from dental pulp tissue. Our objective was to determine in vitro culture technique for stem cells from deciduous tooth (SHED) and permanent tooth (DPSC) through cell passage identification, the effect of trypsin-EDTA and proliferation potential, and to characterize both cells using molecular markers profile. Enzyme digestion method was used on dental pulp tissue from deciduous and permanent tooth for SHED and DPSC isolation, respectively. Both cells were cultured at passage 1 until 5 and trypan blue assay was used to obtain growth curve in determining cell population doubling time (PDT) for each passage. Effect of trypsin-EDTA on both cells were studied using Alamar blue assay to determine optimum incubation time for subculturing process. Cell proliferation potential for both cells within 21 days was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell characterization through molecular biology markers was performed using RT-PCR. Both cells at all passages appeared fibroblast-like. SHED and DPSC at passage 3 exhibited the lowest PDT with 43 ± 2.3 and 63 ± 3.1 h, respectively. SHED exposed to trypsin-EDTA showed decrease in cell viability percentage compared to DPSC. Cell growth for SHED was ~2.3-fold higher than DPSC. Both cells expressed mesenchymal stem cell markers and not hematopoietic stem cell markers. In conclusion, homogenous morphology and lowest PDT value indicated that cells at passage 3 are the best to determine proliferation potential and molecular markers expression. SHED proliferated better than DPSC. However, DPSC was more resistant to trypsin-EDTA than SHED. Based on molecular marker profile, both cells are mesenchymal stem cells.

Keywords: Cell doubling time; human dental pulp stem cells; molecular biology markers profile; proliferation potential; trypsin-EDTA effect

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*Corresponding author; email: hisham@ukm.edu.my