Gangguan fungsi sitoskeleton pada proses vitrifikasi keratinosit primer manusia

Indra Kusuma, Restu Syamsul Hadi, Yurika Sandra


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


Keratinosit; Sitoskeleton; Vitrifikasi; Kriopreservasi

Full Text:



Balci D, Can A. The Assessment of Cryopreservation Conditions for Human Umbilical Cord Stroma-Derived Mesenchymal Stem Cells towards a Potential Use for Stem Cell Banking. Current stem cell research & therapy. Bentham Science Publishers; 2013;8(1):60–72.

Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nature cell biology. Nature Publishing Group; 2001;3(4):339–45.

Desai N, Xu J, Tsulaia T, Szeptycki-Lawson J, AbdelHafez F, Goldfarb J, et al. Vitrification of mouse embryo-derived ICM cells: a tool for preserving embryonic stem cell potential? Journal of assisted reproduction and genetics. Springer; 2011;28(2):93–9.

Djuwantono T, Wirakusumah FF, Achmad TH, Sandra F, Halim D, Faried A. A comparison of cryopreservation methods: Slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells. BMC research notes. BioMed Central Ltd; 2011;4(1):371.

Freshney RI. Culture of animal cells, a manual of basic technique. John Wiley and Sons, inc;

Grinnell K, Bickenbach J. Skin keratinocytes pre-treated with embryonic stem cell-conditioned medium or BMP4 can be directed to an alternative cell lineage. Cell proliferation. Wiley Online Library; 2007;40(5):685–705.

Isachenko E, Isachenko V, Katkov II, Rahimi G, Sch?ndorf T, Mallmann P, et al. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Human Reproduction. ESHRE; 2004 a;19(4):932–9.

Isachenko V, Isachenko E, Katkov II, Montag M, Dessole S, Nawroth F, et al. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biology of reproduction. Soc Study Reprod; 2004 b;71(4):1167–73.

Kusuma I, Hadi RS. Geraniin suplementation increases human keratinocyte proliferation in serum-free culture. Universa Medicina. 2013;32(1):3–10.

Norman LL, Brugés J, Sengupta K, Sens P, Aranda-Espinoza H. Cell blebbing and membrane area homeostasis in spreading and retracting cells. Biophysical journal. Elsevier; 2010;99(6):1726–33.

Reubinoff B, Pera M, Vajta G, Trounson A. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Human Reproduction. ESHRE; 2001;16(10):2187–94.

Selman H. Vitrification versus conventional cryopreservation technique. Middle East Fertility Society Journal. 2005;10(3).

Str?m S, Holm F, Bergstr?m R, Str?mberg A-M, Hovatta O. Derivation of 30 human embryonic stem cell lines—improving the quality. In Vitro Cellular & Developmental Biology-Animal. Springer; 2010;46(3-4):337–44



  • There are currently no refbacks.

Copyright (c) 2017 Jurnal Kedokteran YARSI

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.


Copyright of Jurnal Kedokteran YARSI.

Powered by OJS.

Creative Commons License

This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License.