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

REFERENCES

Adha, P.R., Chua, K.H., Mazlyzam, A.L., Low, K.C., Aminuddin, B.S. & Ruszymah, B.H. 2008. Usage of allogeneic single layered tissue engineered skin enhance wound treatment in sheep. Med. J. Malaysia 63: 30-31.

Alam, H., Sehgal, L., Kundu, S.T., Dalal, S.N. & Vaidya, M.M. 2011. Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells. Mol. Biol. Cell. 22(21): 4068-4078.

Alfaqeh, H., Norhamdan, M.Y., Chua, K.H., Chen, H.C., Aminuddin, B.S. & Ruszymah, B.H. 2008. Cell based therapy for osteoarthritis in a sheep model: Gross and histological assessment. Med. J. Malaysia 63: 37-38.

Aminuddin, B.S. & Ruszymah, B.H. 2008. Tissue engineering research in developing countries, the significant and differences as compared to the developed countries. Med. J. Malaysia 63: 47-48.

Bocker, W. 2002. WHO classification of breast tumors and tumors of the female genital organs: Pathology and genetics. Verh. Dtsch. Ges. Pathol. 86: 116-119.

Brockbank, K.G., Heacox, A.E. & Schenke-Layland, K. 2011. Guidance for removal of fetal bovine serum from cryopreserved heart valve processing. Cells Tissues Organs 193(4): 264-273.

Chen, S.J., Yuan, W., Mori, Y., Levenson, A., Trojanowska, M. & Varga, J. 1999. Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: Involvement of Smad 3. J. Invest. Dermatol. 112(1): 49-57.

Chowdhury, S.R., Aminuddin, B.S. & Ruszymah, B.H. 2012. Effect of supplementation of dermal fibroblasts conditioned medium on expansion of keratinocytes through enhancing attachment. Indian J. Exp. Biol. 50(5): 332-339.

Frey, N.V., Lazarus, H.M. & Goldstein, S.C. 2006. Has allogeneic stem cell cryopreservation been given the ‘cold shoulder’? An analysis of the pros and cons of using frozen versus fresh stem cell products in allogeneic stem cell transplantation. Bone Marrow Transplant 38(6): 399-405.

Gao, D. & Critser, J.K. 2000. Mechanisms of cryoinjury in living cells. ILAR J. 41(4): 187-196.

Gurtovenko, A.A. & Anwar, J. 2007. Modulating the structure and properties of cell membranes: The molecular mechanism of action of dimethyl sulfoxide. J. Phys. Chem. B 111(35): 10453-10460.

Hunt, C.J. 2011. Cryopreservation of human stem cells for clinical application: A review. Transfus. Med. Hemother. 38(2): 107-123.

Hunt, C.J., Pegg, D.E. & Armitage, S.E. 2006. Optimising cryopreservation protocols for haematopoietic progenitor cells: A methodological approach for umbilical cord blood. Cryo Letters 27(2): 73-86.

Hunt, C.J., Armitage, S.E. & Pegg, D.E. 2003. Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing. Cryobiology 46(1): 76-87.

Ibnubaidah, M.A., Chua, K.H., Mazita, A., Azida, Z.N., Aminuddin, B.S., Ruszymah, B.H. & Lokman, B.S. 2008. Derivation of cochlea hair cell for in vitro expansion and characterization. Med. J. Malaysia 63: 115-116.

Ishak, M.F., Chua, K.H., Asma, A., Saim, L., Aminuddin, B.S., Ruszymah, B.H. & Goh, B.S. 2011. Stem cell genes are poorly expressed in chondrocytes from microtic cartilage. Int. J. Pediatr. Otorhinolaryngol. 75(6): 835-840.

Kalyanaraman, B. & Boyce, S.T. 2009. Wound healing on athymic mice with engineered skin substitutes fabricated with keratinocytes harvested from an automated bioreactor. J. Surg. Res. 152(2): 296-302.

Katayama, S., Skoog, T., Jouhilahti, E.M., Siitonen, H.A., Nuutila, K., Tervaniemi, M.H., Vuola, J., Johnsson, A., Lonnerberg, P., Linnarsson, S., Elomaa, O., Kankuri, E. & Kere, J. 2015. Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control. BMC Genomics 16: 476.

Kim, M.S., Song, H.J., Lee, S.H. & Lee, C.K. 2014. Comparative study of various growth factors and cytokines on type I collagen and hyaluronan production in human dermal fibroblasts. J. Cosmet. Dermatol. 13(1): 44-51.

Law, J.X., Musa, F., Ruszymah, B.H., El Haj, A.J. & Yang, Y. 2016. A comparative study of skin cell activities in collagen and fibrin constructs. Med. Eng. Phys. 38(9): 854-861.

Liu, G., Zhou, H., Li, Y., Li, G., Cui, L., Liu, W. & Cao, Y. 2008. Evaluation of the viability and osteogenic differentiation of cryopreserved human adipose-derived stem cells. Cryobiology 57(1): 18-24.

Manira, M., Khairul Anuar, K., Seet, W.T., Ahmad Irfan, A.W., Ng, M.H., Chua, K.H., Mohd Heikal, M.Y., Aminuddin, B.S. & Ruszymah, B.H. 2014. Comparison of the effects between animal-derived trypsin and recombinant trypsin on human skin cells proliferation, gene and protein expression. Cell Tissue Bank 15(1): 41-49.

Mazlyzam, A.L., Aminuddin, B.S., Saim, L. & Ruszymah, B.H. 2008a. Human serum is an advantageous supplement for human dermal fibroblast expansion: Clinical implications for tissue engineering of skin. Arch. Med. Res. 39(8): 743-752.

Mazlyzam, A.L., Aminuddin, B.S., Saim, L. & Ruszymah, B.H. 2008b. Living bilayered human skin equivalent: Promising potentials for wound healing. Med. J. Malaysia 63: 32-33.

Mazlyzam, A.L., Aminuddin, B.S., Fuzina, N.H., Norhayati, M.M., Fauziah, O., Isa, M.R., Saim, L. & Ruszymah, B.H. 2007. Reconstruction of living bilayer human skin equivalent utilizing human fibrin as a scaffold. Burns 33(3): 355-363.

Mazlyzam, A.L., Aminuddin, B.S., Lokman, B.S., Isa, M.R., Fuzina, H., Fauziah, O. & Ruszymah, B.H. 2004. Quality evaluation analysis of bioengineered human skin. Med. J. Malaysia 59: 39-40.

Muhd Fakhruddin, B.H., Aminuddin, B.S., Mazlyzam, A.L. & Ruszymah, B.H. 2004. The effects of age on monolayer culture of human keratinocytes for future use in skin engineering. Med. J. Malaysia 59: 182-183.

Naaldijk, Y., Johnson, A.A., Friedrich-Stockigt, A. & Stolzing, A. 2016. Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants. BMC Biotechno. 16(1): 85.

Norhayati, M.M., Mazlyzam, A.L., Asmah, R., Fuzina, H., Aminuddin, B.S., Ruszymah, B.H. & Fauziah, O. 2004. Collagen fibers an important entity in skin tissues remodeling. Med. J. Malaysia 59: 184-185.

Ortiz-Lopez, L., Gonzalez-Olvera, J.J., Vega-Rivera, N.M., Garcia-Anaya, M., Carapia-Hernandez, A.K., Velazquez- Escobar, J.C. & Ramirez-Rodriguez, G.B. 2017. Human neural stem/progenitor cells derived from the olfactory epithelium express the TrkB receptor and migrate in response to BDNF. Neuroscience 355: 84-100.

Pasch, J., Schiefer, A., Heschel, I. & Rau, G. 1999. Cryopreservation of keratinocytes in a monolayer. Cryobiology 39(2): 158-168.

Rheinwald, J.G. & Green, H. 1975. Formation of a keratinizing epithelium in culture by a cloned cell line derived from a teratoma. Cell 6(3): 317-330.

Roseeuw, D., De Coninck, A., Neven, A.M., Vandenberghe, Y., Kets, E., Verleye, G. & Rogiers, V. 1991. Fresh and cryopreserved cultured keratinocyte allografts for wound healing. Toxicol. in Vitro 5(5-6): 579-583.

Rowley, S.D., Bensinger, W.I., Gooley, T.A. & Buckner, C.D. 1994. Effect of cell concentration on bone marrow and peripheral blood stem cell cryopreservation. Blood 83(9): 2731-2736.

Rubinstein, P., Dobrila, L., Rosenfield, R.E., Adamson, J.W., Migliaccio, G., Migliaccio, A.R., Taylor, P.E. & Stevens, C.E. 1995. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc. Natl. Acad. Sci. US A 92(22): 10119-10122.

Ruszymah, B.H. 2008. Tissue engineering provides the potential to replace and regenerate. Med. J. Malaysia 63: 27-28.

Ruszymah, B.H., Chua, K.H., Mazlyzam, A.L. & Aminuddin, B.S. 2011. Formation of tissue engineered composite construct of cartilage and skin using high density polyethylene as inner scaffold in the shape of human helix. Int. J. Pediatr. Otorhinolaryngol. 75 (6): 805-810.

Saim, L., Aminuddin, B.S., Munirah, S., Chua, K.H., Izuddin Fahmy, A., Fuzina, N.H., Isa, M.R. & Ruszymah, B.H. 2004. Formation of tissue-engineered human auricular cartilage via tissue engineering technique for future use in ear surgery. Med. J. Malaysia 59: 192-193.

Schiozer, W.A., Gemperli, R., Muhlbauer, W., Munhoz, A.M. & Ferreira, M.C. 2013. An outcome analysis and long-term viability of cryopreserved cultured epidermal allografts: Assessment of the conservation of transplantable human skin allografts. Acta Cir. Bras. 28(12): 824-832.

Seet, W.T., Manira, M., Khairul Anuar, K., Chua, K.H., Ahmad Irfan, A.W., Ng, M.H., Aminuddin, B.S. & Ruszymah, B.H. 2012. Shelf-life evaluation of bilayered human skin equivalent, MyDerm. PLoS One 7(8): e40978.

Stolzing, A., Naaldijk, Y., Fedorova, V. & Sethe, S. 2012. Hydroxyethylstarch in cryopreservation - Mechanisms, benefits and problems. Transfus. Apher. Sci. 46(2): 137-147.

Teepe, R.G., Kreis, R.W., Koebrugge, E.J., Kempenaar, J.A., Vloemans, A.F., Hermans, R.P., Boxma, H., Dokter, J., Hermans, J. & Ponec, M. 1990. The use of cultured autologous epidermis in the treatment of extensive burn wounds. J. Trauma 30(3): 269-275.

Udoh, Y., Yanaga, H., Tai, Y., Kiyokawa, K. & Inoue, Y. 2000. Long-term viability of cryopreserved cultured epithelial grafts. Burns 26(6): 535-542.

Woods, E.J., Perry, B.C., Hockema, J.J., Larson, L., Zhou, D. & Goebel, W.S. 2009. Optimized cryopreservation method for human dental pulp-derived stem cells and their tissues of origin for banking and clinical use. Cryobiology 59(2): 150-157.

Yanaga Hiroko, Yukihiro Udoh, Misa Yamamoto, Satoko Yoshii, Seiichiro Mori, Toshihiko Yamauchi, Kensuke Kiyokawa, Mika Koga & Katsu Yanaga. 2017. Cryopreserved cultured epithelial allografts for pediatric deep partial dermal burns: Early wound closure and suppression of scarring. Regenerative Therapy 6: 74-82.

Yazid, A.G., Anuar, A., Onhmar, H.T., Ng, A.M., Ruszymah, B.H. & Amaramalar, S.N. 2008. Sourcing different neuro-progenitor cell for the use of nerve construct. Med. J. Malaysia 63: 113-114.

Zhou, C.Q., Mai, Q.Y., Li, T. & Zhuang, G.L. 2004. Cryopreservation of human embryonic stem cells by vitrification. Chin. Med. J. (Engl) 117(7): 1050-1055.

 

*Corresponding author; email: ruszyidrus@gmail.com

 

 

 

 

previous