Challenges in Obtaining Good Metaphase Spread of Rat Full-term Amniotic Fluid Stem Cell Line (R3) for karyotyping Analysis

Mathandaver, Devasharmini (2020) Challenges in Obtaining Good Metaphase Spread of Rat Full-term Amniotic Fluid Stem Cell Line (R3) for karyotyping Analysis. [Project Paper] (Submitted)

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Abstract

Stem cells have been shown to be the promising cells for various therapeutic applications including for cell replacement therapy. Their ability to undergo self- renewability permits them to be preserved as stem cell lines for prolonged culture. Hence, making them as a reliable source for continuous supply of cells for therapy, namely as the cell source for transplantation. One such stem cell line is the newly in-house established rat full-term amniotic fluid stem cell (AFSC) line, known as R3. The line has been cultured up to 100 passages without losing its differentiation potential. However, its chromosomal stability has not been assessed before. Stable stem cell line is important before they could be used in transplantation. One of the ways is to check their chromosomal stability via karyotyping analysis. Nonetheless, it is not an easy process since the process relies on acquiring a good quality metaphase spread of the chromosomes, which is a tedious process as there is no standard karyotyping protocol attributed to AFSC line, albeit the protocol is well established for primary blood and amniotic fluid cells. Objective: In this study we aimed to evaluate the important variables in obtaining good metaphase spread of rat full-term AFSC line (R3) cultured at passage 37 (P37) and 38 (P38). Hypothesis: It is hypothesized that acquiring a good quality metaphase spread is dependent on different factors associated to karyotyping steps. Methodology: R3 was propagated to its respective passage number and karyotyping was performed to assess the effect of fixative drying time, the effect of adding colcemid after 34 hours instead of 18 hours and the effect of cell suspension dropping height on the quality of metaphase spread. Result: The good quality metaphase spread was not observed in this study despite of several attempts. However, we could observe the presence of clumped chromosomes and high number of chromosome spreads at a single area that might be due to the speed of fixative drying time. In addition, interphase-like cells were also observed that might be caused by the colcemid inefficiency. Structural disorganization of the chromosomes which could be attributed by a high dropping height of R3 cell suspension was also detected. Conclusion: Proper optimization for fixative drying time, colcemid incubation duration and suitable dropping height of cell suspension are essential elements in acquiring a good quality metaphase spread for full-term AFSC line. The specific optimized conditions could provide valuable parameters towards initial checking of R3 stability. The stability of R3 upon prolonged culture is essential as this finding could be further translated to human counterpart as the prospective cells for transplantation from a source that is merely discarded, that is the human full-term amniotic fluid.

Item Type:	Project Paper
Faculty:	Faculty of Medicine and Health Science
Depositing User:	Ms. Nor Safa'aton Saidin
Date Deposited:	23 Aug 2023 00:38
Last Modified:	23 Aug 2023 00:38
URI:	http://psaspb.upm.edu.my/id/eprint/1285

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