Skripsi

Analisis Histologi Organoid SHiNTA: Jumlah, Ukuran, dan Distribusi Sel = Histological Analysis of SHiNTA Organoid: Cell’s Count, Size, and Distribution.

Latar Belakang : Organoid SHiNTA merupakan hati bioartifisial menggunakan organoid hati yang dibentuk dengan sel punca yang dikultur dalam perancah hati biologis terdeselularisasi yang dikembangkan oleh Fakultas Kedokteran Universitas Indonesia. Pada 2021, dibentuk organoid SHiNTA, tetapi setelah percobaan didapatkan permasalahan dimensi yang kurang adekuat saat dilakukan perfusi. Untuk menangani hal ini, dilakukan ko-kultur hepatosit dengan sel nonparenkimal lainnya, peningkatan dimensi perancah, dan peningkatan kecepatan perfusi bioreaktor. Penelitian ini bertujuan untuk menganalisis histologis dan menilai profil sel pada organoid SHiNTA yang dibentuk dengan metode baru ini. Metode : Slide preparat organoid SHiNTA yang diwarnai dengan pewarnaan Hematoksilin Eosin untuk dilakukan analisis histologis dan distribusi sel serta perhitungan jumlah sel, pewarnaan Masson’s Trichrome untuk analisis histologis dan menilai fraksi area kolagen, pewarnaan imunofluoresen dengan marker HNF4α untuk menilai maturitas sel mirip hepatosit dan distribusi sel. Selain itu, akan dilakukan juga perhitungan jumlah dan konfluensi sel menggunakan Biotek Cytation1 Cell Counting Imaging. Ukuran sel dihitung melalui hasil foto Biotek Cytation1 Cell Counting Imaging. Hasil : Hasil analisis histologi menunjukkan organoid SHiNTA tampak seperti pulau-pulau akibat fragmentasi setelah perfusi. Inti sel yang terpulas memiliki bentuk yang bervariasi dan terdistribusi menyebar di pinggir perancah. Dapat terlihat adanya sel-sel yang melekat pada matriks ekstraseluler kolagen. Hasil imunofluoresen dengan marker HNF4α menunjukkan maturitas sel mirip hepatosit yang berhasil melekat pada perancah. Sel positif kuat HNF4α cenderung soliter dan sel positif lemah HNF4α cenderung berkelompok. Diameter sel yang teridentifikasi sebesar 25.59±3.88 µm. Hasil perhitungan konfluensi sel sebesar 34.3%. Kesimpulan : Organoid SHiNTA pada penelitian ini memiliki karakteristik bentuk kepulauan dengan matriks ekstraseluler kolagen intak, sel-sel matur yang berhasil melekat pada perancah dengan jumlah dan konfluensi lebih tinggi jika dibandingkan dengan studi sebelumnya, serta ukuran sel yang dapat indikatif terhadap ukuran hepatosit.
Kata Kunci: Histologi, organoid SHiNTA, sel punca.


Introduction : SHiNTA organoids are bioartificial livers using liver organoids formed with stem cells cultured in a decellularized biological liver scaffold developed by the Faculty of Medicine, University of Indonesia. In 2021, SHiNTA organoids were formed, but after experiments, dimensional problems were found that were not adequate when perfusion was performed. To deal with this, co-culture of hepatocytes with other nonparenchymal cells was carried out, scaffolding dimensions were increased, and the perfusion rate of bioreactors was increased. This study aims to analyze histologically and assess the cell profile of SHiNTA organoids formed by this new method. Method : SHiNTA organoid preparation slides were stained with Hematoxylin Eosin staining for histological analysis and cell distribution and cell count calculation, Masson's Trichrome staining for histological analysis and assessing collagen area fractions, immunofluorescence staining with HNF4α marker to assess hepatocytelike cell maturity and cell distribution. In addition, the number and confluence of cells will also be calculated using Biotek Cytation1 Cell Counting Imaging. Cell size was calculated through image from Biotek Cytation1 Cell Counting Imaging. Results : The results of histological analysis showed that the SHiNTA organoids looked like islands due to fragmentation after perfusion. The nucleus of the strained cell has a varied shape and is distributed on the edge of the scaffold. The presence of cells attached to the extracellular matrix of collagen can be seen. The results of immunofluorescence with the marker HNF4α showed the maturity of hepatocyte-like cells that successfully attached to the scaffold. Strong positive cells of HNF4α tend to be solitary and weak positive cells of HNF4α tend to be clustered. The identified cell diameter was 25.59±3.88 μm. The result of the calculation of cell confluence was 34.3%. Conclusion : The SHiNTA organoid in this study has the characteristics of an archipelago shape with intact extracellular matrix of collagen, mature cells that successfully attach to the scaffold with a higher number and confluence when compared to previous studies, and the cell size that can be indicative of hepatocyte size.
Keywords: Histology, SHiNTA organoids, stem cells.