Skripsi

Culture of Oct-4 Positive Cells from Preputial Epidermis = Kultur Oct-4 Positif Sel dari Epidermis Prepusium.

The medical waste of preputial skin is easily obtained in Indonesia. The cells isolated from six samples are expected to have pluripotency and able to differentiate to other lineage for medical treatment. This research uses the epidermal layer of preputial skin to obtain keratinocytes, this cells are taken using dispase and trypsin solution overnight respectively. Then, the keratinocytes are subsequently cultured using DMEM complete high glucose to increase the number of cells. The cultured cells are then taken for immunocytochemistry (ICC) of Oct-4, since it is the marker of pluripotency. The other half of cultured samples are continued for over confluency analysis for fourteen days to observe spontaneous differentiation. This research has successfully used an alternative and cost effective culture medium for keratinocytes. It consist of DMEM high glucose, PRP 10%, heparin 1%, FBS 10%, penstrep 1%, and fungizone 1%. The result of ICC is partially positive with keratinocytes nuclear being stained dark brown in five hpf from each sample. However, spontaneous differentiation analysis using alcian blue shows negative result of chondrogenic formation from keratinocytes.
Keywords: prepuce epidermis, pluripotency, Oct-4, immunocytochemistry, isolation, culture, spontaneous differentiation, alcian blue staining.



Sampah medis berupa prepusium sangatlah mudah di peroleh di Indonesia. Sel-sel yang di dapat dari enam sampel di duga memiliki kapasitas pluripotensi dan dapat berdiferensiasi ke lini lain untuk pengobatan secara medis. Pada experiment ini, sel keratinosit di peroleh dari epidermis prepusium, sel tersebut di ambil mengunakan larutan enzymatik dispase dan tripsin masing-masing di inkubasi semalaman dengan sampel. Kemudian sel-sel tersebut di kultur dengan DMEM tinggi glukosa untuk meningkatkan jumlah sel. Setelah di kultur, sel-sel tersebut di ambil untuk pengecekan immunositokimia (ISK) Oct-4, hal ini di karenakan Oct-4 adalah penanda kapasitas pluripotensi. Sample lainnya tetap di kultur untuk eksperimen konfluensi/differensiasi spontan selama 14 hari. Riset ini telah sukses menggunakan medium kultur alternatif dan berbiaya efektif untuk menkultur keratinosit. Bahan medium tersebut yakni; DMEM tinggi glukosa, PRP 10%, heparin 1%, FBS 10%, penstrep 1%, dan fungizone 1%. Hasil dari pada ISK tersebut adalah positif parsial dengan nukleus keratinosit yang terwarnai coklat tua pada lima lapang pandang berkekuatan tinggi dari setiap sampel. Namun, analisis diferensiais spontan menggunakan alcian blue menunjukkan hasil negative dengan tidak adanya perubahan dari lini keratinosit ke kondrosit.
Kata kunci: epidermis prepusium, pluripotensi, Oct-4, immunositokimia, isolasi, kultur, differensiasi spontan, pewarnaan alcian blue.