Gangguan fungsi sitoskeleton pada proses vitrifikasi keratinosit primer manusia

Indra Kusuma, Restu Syamsul Hadi, Yurika Sandra

Abstract


Basal keratinocytes retained a multipotency capacity which required a serum-free system to avoid spontaneous differentiation during expansion culture. Keratinocytes isolation and culture enable research and therapeutical exploration of bioengineered skin comprised of an epidermal and dermal layer. Vitrification method for crypreservation expected to protect keratinocytes viability and function in a serum-free condition. Foreskin sample of 7 school-age children undergone the khitan procedure were collected with informed consent provided by their parents. Keratinocytes isolation used a combined enzymatic approach using dispase and trypsin/EDTA. Viability and cellular proliferation measured using tryphan blue exclusion method and WST-1 reagent at 450 nm respectively. Resulting data were analyze using student t-test in Microsoft Excel 2015. Cryopreservation using vitrification method result in 80% post thaw viability without significance difference (p is more than 0.05) with standard slow-freezing method with serum as one of the cryomedium component. However, only 30% of those cells retained the ability to grow attachment to plastic culture vessel. This result was significantly different with those preserve using the standard slow-freezing method with atttacment measured at 70% (p is less than 0.05). Post-thaw fotomicrograph shows that some cells have a blebbing morphology which indicate a cytoskeletal disfunction commonly caused by hyperosmotic shock. Cellular preservation using vitrification method caused changes in viability, attachment and cellular proliferation capacity. Hyperosmotic shock was considered to be the caused of cytoskeletal disfunction that leads to decrease in post-thaw cellular attachment dan proliferative capacity of vitrified keratinocytes


Keywords


Keratinosit; Sitoskeleton; Vitrifikasi; Kriopreservasi

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References


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DOI: https://doi.org/10.33476/jky.v25i2.118

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