Sains Malaysiana 48(1)(2019): 137–144

http://dx.doi.org/10.17576/jsm-2019-4801-16

Long Term Effect of Cryopreservation on Primary Human Skin Cells

(Kesan Jangka Panjang Pengawetan Krio pada Sel Kulit Primer Manusia)

ISHAK, M.F.¹, MANIRA, M.¹, NG, M.H.¹, KHAIRUL, B.², GARGY, L.², AMINUDDIN, B.S.³ & RUSZYMAH, B.H.I.^4*

¹Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Center, Jalan Yaacob Latif, 56000 Kuala Lumpur, Federal Territory, Malaysia

²Cell Tissue Technology Sdn Bhd, Kuala Lumpur, Federal Territory, Malaysia

³Ampang Puteri Specialist Hospital, 68000 Ampang, Selangor Darul Ehsan, Malaysia

⁴Departments of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Kuala Lumpur, Federal Territory, Malaysia

Received: 30 March 2018/Accepted: 5 September 2018

ABSTRACT

Cryopreservation is essential for tissue engineering and regenerative medicine. This study was carried out to assess the effect of cryopreservation on skin cells and evaluate the performance of cells after 12 months of cryopreservation. Redundant skin tissue samples were obtained from surgery with consent from patients. The tissue was cleaned, processed and cultured until passage 3. Upon confluency, cells were trypsinised and total cell yield and viability were determined before and after being cryopreserved. Sterility and immunocytochemistry analysis for collagen type I (Col-1) and cytokeratin 14 (CK14) antibodies were also performed on cells cryopreserved for one, three, six and twelve months. There is no significant difference in growth rates for cryopreserved cells for 1 to 12 months, except for fibroblasts at 6 months. Cell viability for both keratinocytes and fibroblasts decreased with time (65%± 3.5% - 89%± 4.5%). Sterility testing showed no contamination after 12 months of cryopreservation. Immunocytochemistry analysis showed positive expression for CK14 (keratinocytes) and Col -1 (fibroblasts) after 12 months of cryopreservation. Morphologically, keratinocytes and fibroblasts were able to retain its phenotype. The loss in viability is consistent in all samples and possibly due to thermal-cycling effect. Immunocytochemistry and consistent cell growth analysis showed that keratinocytes and fibroblasts were able to retain their characteristics in cryopreservation condition. These preliminary findings show that primary skin cells can be stored via cryopreservation and still retain their characteristics. However, further investigations using longer periods of cryopreservation (24 months, 48 months) should be conducted.

Keywords: Cell proliferation; cryopreservation; human skin cells; gene expression

ABSTRAK

Pengawetan krio adalah penting bagi bidang kejuruteraan tisu dan penjanaan semula perubatan. Kajian ini dijalankan untuk menilai kesan pengawetan krio pada sel kulit dan prestasi sel selepas pengawetan krio selama 12 bulan. Sampel tisu kulit berlebihan daripada pembedahan diambil dengan kebenaran pesakit. Tisu dibersihkan, diproses dan dikultur sehingga subkultur 3. Selepas mencapai kepadatan sesuai, sel dituai dan jumlah keseluruhan sel berserta keviabelan sel ditentukan sebelum dan selepas pengawetan krio. Ujian kesterilan dan imunositokimia menggunakan antibodi kolagen jenis I (Col-1) dan sitokeratin-14 (CK14) dijalankan terhadap sel bagi bulan pertama, ketiga, keenam dan kedua belas. Tidak terdapat perbezaan signifikan bagi kadar pertumbuhan sel yang telah diawet bagi bulan pertama dan kedua belas, kecuali bagi sel fibroblas pada bulan keenam. Keviabelan sel bagi keratinosit dan fibroblas menurun mengikut peningkatan masa (65%± 3.5% - 89%± 4.5%). Ujian kesterilan menunjukkan tiada sebarang pelumusan selepas dua belas bulan pengawetan krio. Secara morfologi, keratinosit dan fibroblas mampu mengekalkan fenotipnya. Pengurangan keviabelan adalah konsisten bagi semua sampel dan ini mungkin berlaku akibat daripada kesan kitaran terma. Analisis imunositokimia dan pertumbuhan sel yang tekal menunjukkan bahawa keratinosit dan fibroblas berupaya untuk mengekalkan cirinya setelah pengawetan krio. Keputusan awal ini menunjukkan sel kulit primer boleh disimpan melalui proses pengawetan krio dan cirinya dikekalkan. Namun, kajian lanjutan menggunakan tempoh pengawetan krio yang lebih lama (24 bulan, 48 bulan) perlu dijalankan.

Kata kunci: Pengawetan krio; pengekspresan gen; sel kulit manusia; sel pembiakan

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*Corresponding author; email: ruszyidrus@gmail.com