Simple method to isolation and culture of neuron progenitor cells (NPCs) from whole brain post-natal rat - Masagus Zainuri; Ratih Rinendyaputri; Ariyani Noviantari; Ni Ketut Susilarini (Health Science Journal of Indonesia, 2018)
Latar Belakang: Neurobiologi dipelajari menggunakan sel neuron dari kultur primer atau menggunakancell line tergantung pada tujuan penelitian yang akan dilakukan. Berbagai metode dikembangkan untukmendapatkan sel neuron pada bagian korteks, hipokampus atau dari semua jaringan otak dari otak fetusatau tikus yang baru lahir. Sel neuron tidak mampu berproliferasi sehingga perlu dikembangkan isolasineuron progenitor cells (NPCs). Penelitian ini bertujuan untuk mengembangkan metode isolasi NPCs darijaringan utuh otak tikus yang baru lahir secara mudah dan praktis. Metode: Jaringan otak diperoleh dari tikus Sprague Dawley umur 2 hari. Eksperimen dilakukan dalam duatahap yaitu memasukkan jaringan otak dalam tripsin 0,05% diinkubasi selama 10 menit, menambahkanmedium kultur, disaring dengan pori membran dan sentrifugasi selama 10 menit. Tahap selanjutnya membuang supernatan, tambahkan dengan HBSS-glukosa, dimasukkan ke dalam larutan Ficoll 35% dan 65% kemudian sentrifugasi, selanjutnya supernatan ditanam di cawan kemudian dipindahkan lagi pada cawan yang telah dilapisi dengan poly-D-lysine. Karakterisasi dilakukan dengan imunositokimia penanda neuron (NeuN danmicrotubule-associated protein 2-MAP2) dan flow cytometry (PSANCAM+ and A2B5-). Hasil: Dalam waktu kurang dari satu jam dengan menggunakan metode ini dapat diperoleh NPCs. Hasilmenunjukkan bahwa diperoleh lebih dari 95% sel dengan PSANCAM+ dan A2B5-. Setelah dikultur selama4 hari, sel positif terhadap NeuN and MAP2. Kesimpulan: Telah berhasil dikembangkan metode isolasi NPCs dari jaringan utuh otak tikus baru lahiryang mudah dan praktis dengan viabilitas dan kemurnian tinggi. (Health Science Journal of Indonesia2018;9(2):63- 9) Kata kunci: tikus baru lahir, neuron progenitor cells (NPCs), isolasi Abstract Background: Neurobiology is studied by neuron cells from primary cultures or cell lines dependingon the purpose of the research. Various methods were developed to obtain neuron cells in the cortex,hippocampus or from all brain tissue from the fetal brain or newborn mice. Neuron cells are unable toproliferate therefore the isolation of neuron progenitor cells (NPCs) needs to be developed. This studyaims to develop a method of isolating NPCs from intact tissue of newborn mouse brains easily and practically. Methods: Brain tissue was obtained from Sprague Dawley rats aged 2 days. Experiments were carried out in two stages which included add trypsin 0,05% to brain tissue and then incubated for 10 minutes, adding culture medium, then filtered with pore size membrane and centrifuging for 10 minutes. The next step is to remove the supernatant then add with HBSS-glucose, put it in a 35% and 65% Ficoll solution and centrifugation, then the supernatant is planted in dish and then transferred again to the dish with poly-D-lysine cup. Characterization of neuron marker was carried out by immunocytochemistry (NeuN and microtubule-associated protein 2-MAP2) and flow cytometry (PSANCAM + and A2B5-). Results: In this study, our result show that this method does not take longer than one hours and more than95% cells that obtained are expressing PSANCAM+ and A2B5-. After 4 days culture, cells exhibit positivefor neuron marker as MAP2 and NeuN. Conclusion: Successfully developed the easy and practical method to isolate NPCs from the whole brainof post-natal rat with high viability and purity. (Health Science Journal of Indonesia 2018;9(2):63-9) Keyword: post-natal rat, neuron progenitor cells (NPCs), isolation