Conditioned Medium From Bone Marrow-Derived Mesenchymal Stem Cells For Ex Vivo Expansion Of Cardiac Stem Cells

• Main Author: Umar Fuaad, Mimi Zulaikha
• Format: Thesis
• Language: English
• Published: 2016
• Subjects: RK1-715 Dentistry
• Online Access: http://eprints.usm.my/32201/1/MIMI_ZULAIKHA_UMAR_FUAAD_24%28NN%29.pdf; http://eprints.usm.my/32201/

Sel induk mesenkima (MSC) daripada sumsum tulang merembes faktorfaktor parakrin yang mampu merangsang pengaktifan sel induk endogen jantung (CSC) dan memperbaiki kefungsian jantung. Faktor-faktor ini boleh dijana melalui pelaziman secara in vitro. Tesis ini bertujuan untuk mengoptimumkan kondisi pertumbuhan MSC dan perumusan medium yang sesuai untuk menjana medium terlazim (CdM) yang mempunyai ciri-ciri “cytoprotective” terhadap CSC. MSC dipencilkan daripada tibia dan femura mencit (C57BL/6N) yang berusia 3-5 minggu secara pembilasan sumsum tulang atau dengan menghancurkan tulang berkenaan dan dicirikan menggunakan sitometri aliran. Tempoh pre-pelaziman, dan kepadatan sel sebelum memulakan pelaziman (Fasa I), perumusan media, oksigen, tempoh pelaziman, kesan pengumpulan berulang, dan kepekatan pengolahan (Fasa II) dinilai dan dioptimum berdasarkan kesan CdM yang dijana terhadap pertumbuhan CSC secara in vitro. Seterusnya, CdM yang telah dioptimumkan itu diuji terhadap kadar migrasi CSC. CdM dipekatkan sebanyak 8 kali kepekatan (Fasa III) dan diuji terhadap pertumbuhan CSC, dibandingkan dengan CdM yang tidak dipekatkan. Data dianalisa menggunakan Analisis varians (ANOVA) dan Ujian-T. Bone marrow-derived mesenchymal stem cells (MSCs) have been shown to secrete paracrine factors which can stimulate activation of endogenous cardiac stem cells (CSCs) and ameliorate infarcted heart function. These factors can be harvested through conditioning of MSCs in vitro. This thesis aimed to optimise MSC growth conditions and medium formulation for generating conditioned medium (CdM) with CSC-cytoprotective properties. Murine MSCs were isolated from tibia and femur bones of 3-5 weeks old C57BL6/N mice using flushed marrow or crushed bone and was characterised by flow cytometry. Preconditioning time and seeding density before initiation (Phase I), medium formulation, oxygen, conditioning time, effects of repeated harvesting, and treatment concentration (Phase II) were assessed and optimised based on the effects of the produced CdM on CSCs survival in vitro. Then, the optimized CdM were tested on CSC migration. To reduce metabolic waste, CdM were concentrated 8 times (Phase III) and tested on CSC survival, and compared to the crude CdM. All data were analysed using ANOVA and t-test