Tesis

Efek simpan beku berulang konsentrat Trombosit kadaluarsa terhadap karakteristik Sel punca asal jaringan lemak manusia = Effect of repeated freezed-thawed outdated thrombocyte concentrates on the characteristics on human adipose-derived stem cells.

Pemrosesan konsentrat trombosit kadaluwarsa untuk mendapatkan lisat platelet sebagai pengganti fetal bovine serum pada medium kultur sel punca asal jaringan lemak manusia (SPAJLM) dapat dilakukan dengan cara simpan-beku berulang. Tidak terdapat informasi apakah variasi ulangan simpan-beku (-20oC) 1, 2, 3, 5, dan 10 kali pada konsentrat trombosit kadaluwarsa memberikan efek yang sama terhadap karakterisasi dan proliferasi SPAJLM. Studi ini dilakukan untuk mengetahui karakterisasi dan proliferasi SPAJLM yang dikultur pada medium mengandung konsentrat trombosit kadaluwarsa simpan-beku berulang. Jumlah sel hidup diukur pada hari ke-2, hari ke-4, hari ke-7, hari ke-10, dan hari ke-13 dan dikonfirmasi dengan pengukuran population doubling time (PDT). Korelasi antara jumlah trombosit pecah dengan jumlah sel dihitung menggunakan regresi linier multivariat dan uji korelasi Spearman. Diferensiasi kondrosit, adiposit, dan osteosit pada SPAJLM dilakukan secara induksi maupun spontan. Ekspresi marka permukaan positif (CD90, CD73, CD105) dan negatif (CD34, CD45, CD11b, CD19, HLA-DR) diukur. Hasil penelitian menunjukkan SPAJLM memiliki potensi diferensiasi yang sama dan memperlihatkan pola grafik proliferasi sel yang saling berhimpitan dengan poin nilai yang berdekatan, terkonfirmasi dengan hasil pengukuran PDT yang tidak berbeda signifikan (P>0.05), serta tidak ditemukan korelasi jumlah trombosit pecah dengan jumlah sel (P>0.05; R=32%, r < 1). Ekspresi marka permukaan positif > 80% dan negatif < 2% (P>0.05). Terdapat kecenderungan ekspresi CD105 yang menurun seiring bertambahnya siklus simpan-beku. Kesimpulannya, SPAJLM yang dikultur pada konsentrat trombosit kadaluwarsa simpan-beku berulang sesuai dengan kriteria sel punca mesenkimal menurut International Society for Cell Therapy (ISCT).
Kata kunci: konsentrat trombosit kadaluwarsa, siklus simpan-beku, platelet lysate, sel punca asal jaringan lemak manusia.


Processing of outdated thrombocyte concentrate (TC) to get platelet lysate as fetal bovine serum substitute on human adipose derived-stem cells (hADSCs) culture medium can be done by various freeze-thaw cycles. Lack of knowledge whether various freezed-thawed (1, 2, 3, 5, and 10 times) numbers of TC gave the same characterizations and proliferation on hADSCs. The aim of this study was to conduct hADSCs’ characterizations and proliferations when expanded in variously processed TC. MTS-proliferation assay was measured on day-2, day-4, day-7, day-10, and day13 confirmed by PDT and measurement. Correlation between lysed platelet and cells· numbers was analyzed using multivariate linier regression and Spearman correlation test. Cells were differentiated into chondrocyte, adipocyte, and osteocyte either induced or spontaneous. Surface marker expression were analyzed where CD90, CD73, CD105 as positive markers, CD34, CD45, CD11b, CD19, HLA-DR as negative markers. The result showed various freezed-thawed TC gave same differentiation potential and has coincided pattern of proliferation’s trend line with adjacent point value, this also confirmed by PDT (P>0.05) and there was no correlation between lysed platelet and cells· numbers (P>0.05; R=32%, r < 1). All positive surface marker expression levels are above 80% and all negative surface marker expression was below 2% (P>0.05). The surface marker CD105 tend to decrease simultaneous with the numbers of freezed-thawed cycles. It concluded that hADSCs cultured in various freezed-thawed cycles outdated-TC correspond with mesenchymal stem cells according to the requirement of International Society for Cell Therapy (ISCT).
Keywords: outdated thrombocyte concentrate, freezed-thawed cycles, platelet lysate, human adipose-derived stem cells.