Ratih Rinendyaputri, Holy Arif Wibowo

Ekspresi gen Bax pada kultur in vitro Inner Cell Mass (ICM) mencit

Introduction

Ekspresi gen bax pada kultur in vitro inner cell mass (icm) mencit. Kaji efektifitas vitrifikasi untuk simpan beku Inner Cell Mass (ICM) mencit, sumber sel punca embrionik. Analisis viabilitas dan ekspresi gen Bax pasca vitrifikasi pada kultur in vitro.

Abstract

Inner Cell Mass (ICM) merupakan sumber sel punca embrionik (ESC, embryonic stem cell). Inner Cell Mass dapat diisolasi dari embrio tahap blastosis yang kemudian dikultur secara in vitro untuk mendapatkan koloni ESC. Penelitian-penelitian untuk menggali potensi ESC terus dilakukan, mengingat terbatasnya sumber tersebut maka perlu pengembangan teknologi penyimpanan sel punca atau (stem cell banking) seperti vitrifikasi. Efektifitas vitrifikasi pada embrio memberikan peluang terhadap penggunaan metode ini untuk simpan beku pada ICM. ICM merupakan sekumpulan beberapa sel sehingga viabilitas pasca simpan beku merupakan hal penting. Penelitian ini bertujuan untuk mengetahui efektifitas metode vitrifikasi pada ICM dengan mengamati viabilitas dan ekspresi gen Bax ICM pasca vitrifikasi. Penelitian dilakukan di Laboratorium stem cell Pusat Biomedis dan Teknologi Dasar Kesehatan Badan Litbangkes. Kegiatan diawali dengan melakukan isolasi ICM dari embrio blastosis, vitrifikasi menggunakan ethylen glycol (EG) dan dimethyl sulphoxide (DMSO) masing-masing 15% dalam mPBS FBS 20% + sukrosa 0,5 M. Thawing dilakukan secara bertahap menggunakan menggunakan 0,5 M, 0,25 M dan 0,125 masing-masing selama 2 menit. Selanjutnya ICM dikultur dan diamati ekspresi gen Bax dengan melakukan ekstraksi mRNA terhadap gen Bax dilanjutkan dengan real time PCR. Hasil menunjukkn bahwa viabilitas ICM pasca vitrifikasi lebih rendah dibandingkan kontrol (p<0,05) dan terjadi peningkatan ekspresi gen Bax pasca vitrifikasi pada hari ke 7 kultur (p>0,05). Pada penelitian ini vitrifikasi dapat digunakan untuk simpan beku ICM.

Review

This study investigates the efficacy of vitrification as a cryopreservation method for mouse Inner Cell Mass (ICM), a critical source of embryonic stem cells (ESC). Given the immense potential of ESCs and the challenges associated with their limited supply, the development of robust stem cell banking technologies, such as vitrification, is of paramount importance. The authors aimed to assess the impact of vitrification on ICM viability and the expression of the apoptotic gene Bax, thereby contributing valuable insights into the feasibility of cryopreserving ICM for future research and therapeutic applications. The methodology involved isolating ICMs from blastocyst-stage embryos, followed by a vitrification protocol using a combination of ethylene glycol and dimethyl sulfoxide, along with sucrose, prior to a stepwise thawing process. Post-thawing, ICMs were cultured, and viability was assessed. Furthermore, Bax gene expression was quantified using mRNA extraction and real-time PCR on day 7 of culture. The results indicated a statistically significant reduction in ICM viability following vitrification compared to the control group (p<0.05). Additionally, an increase in Bax gene expression was observed in vitrified ICMs on day 7 of culture, though this increase did not reach statistical significance (p>0.05). Despite these findings, the authors conclude that vitrification holds promise for the cryopreservation of ICM. While this study offers an important initial exploration into vitrifying ICM, the findings highlight areas requiring further optimization. The significant reduction in post-vitrification viability is a critical concern that warrants refinement of the vitrification and thawing protocols to enhance cell survival. Although the observed increase in Bax gene expression was not statistically significant, its biological implication as an indicator of apoptosis suggests potential cellular stress, which could be detrimental to the long-term health and stemness of the ICM. Future research should focus on optimizing cryoprotectant concentrations, exposure times, and thawing procedures to improve viability and mitigate apoptotic responses. Additionally, a more comprehensive assessment of ICM quality post-vitrification could include markers for pluripotency and differentiation potential to ensure the functional integrity of the cryopreserved cells for effective stem cell banking.

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