Journal of Microbiology and Biotechnology

  • 제32권2호
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  • Pages.238-247
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  • 2022
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Three-Dimensional Skin Tissue Printing with Human Skin Cell Lines and Mouse Skin-Derived Epidermal and Dermal Cells

  • Jin, Soojung (Core-Facility Center for Tissue Regeneration, Dong-Eui University) ;
  • Oh, You Na (Core-Facility Center for Tissue Regeneration, Dong-Eui University) ;
  • Son, Yu Ri (Core-Facility Center for Tissue Regeneration, Dong-Eui University) ;
  • Kwon, Boguen (Core-Facility Center for Tissue Regeneration, Dong-Eui University) ;
  • Park, Jung-ha (Core-Facility Center for Tissue Regeneration, Dong-Eui University) ;
  • Gang, Min jeong (Biopharmaceutical Engineering Major, Division of Applied Bioengineering, College of Engineering, Dong-Eui University) ;
  • Kim, Byung Woo (Biopharmaceutical Engineering Major, Division of Applied Bioengineering, College of Engineering, Dong-Eui University) ;
  • Kwon, Hyun Ju (Core-Facility Center for Tissue Regeneration, Dong-Eui University)
  • 투고 : 2021.11.24
  • 심사 : 2021.12.13
  • 발행 : 2022.02.28
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초록

Since the skin covers most surfaces of the body, it is susceptible to damage, which can be fatal depending on the degree of injury to the skin because it defends against external attack and protects internal structures. Various types of artificial skin are being studied for transplantation to repair damaged skin, and recently, the production of replaceable skin using three-dimensional (3D) bioprinting technology has also been investigated. In this study, skin tissue was produced using a 3D bioprinter with human skin cell lines and cells extracted from mouse skin, and the printing conditions were optimized. Gelatin was used as a bioink, and fibrinogen and alginate were used for tissue hardening after printing. Printed skin tissue maintained a survival rate of 90% or more when cultured for 14 days. Culture conditions were established using 8 mM calcium chloride treatment and the skin tissue was exposed to air to optimize epidermal cell differentiation. The skin tissue was cultured for 14 days after differentiation induction by this optimized culture method, and immunofluorescent staining was performed using epidermal cell differentiation markers to investigate whether the epidermal cells had differentiated. After differentiation, loricrin, which is normally found in terminally differentiated epidermal cells, was observed in the cells at the tip of the epidermal layer, and cytokeratin 14 was expressed in the lower cells of the epidermis layer. Collectively, this study may provide optimized conditions for bioprinting and keratinization for three-dimensional skin production.

키워드

과제정보

This research was supported by a Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry of Education (2020R1A6C101A201, 2021R1A6C103B395).

참고문헌

  1. Baroni A, Buommino E, De Gregorio V, Ruocco E, Ruocco V, Wolf R. 2012. Structure and function of the epidermis related to barrier properties. Clin. Dermatol. 30: 257-262. https://doi.org/10.1016/j.clindermatol.2011.08.007
  2. Benitez JM, Montans FJ. 2017. The mechanical behavior of skin: Structures and models for the finite element analysis. Comput. Struct. 190: 75-107. https://doi.org/10.1016/j.compstruc.2017.05.003
  3. Schmuth M, Feingold KR, Elias PM. 2020. Stress test of the skin: The cutaneous permeability barrier treadmill. Exp. Dermatol. 29: 112-113. https://doi.org/10.1111/exd.14055
  4. Piquero-Casals J, Morgado-Carrasco D, Granger C, Trullas C, Jesus-Silva A, Krutmann J. 2021. Urea in dermatology: A review of its emollient, moisturizing, keratolytic, skin barrier enhancing and antimicrobial properties. Dermatol. Ther. 11: 1905-1915. https://doi.org/10.1007/s13555-021-00611-y
  5. Koster MI. 2009. Making an epidermis. Ann. N. Y. Acad. Sci. 1170: 7-10. https://doi.org/10.1111/j.1749-6632.2009.04363.x
  6. Iizuka H. 1994. Epidermal turnover time. J. Dermatol. Sci. 8: 215-217. https://doi.org/10.1016/0923-1811(94)90057-4
  7. Downing DT. 1992. Lipid and protein structures in the permeability barrier of mammalian epidermis. J. Lipid Res. 33: 301-313. https://doi.org/10.1016/S0022-2275(20)41520-2
  8. Yagi M, Yonei Y. 2018. Glycative stress and anti-aging: 7. Glycative stress and skin aging. Glycative Stress Res. 5: 50-54.
  9. Hwa C, Bauer EA, Cohen DE. 2011. Skin biology. Dermatol. Ther. 24: 464-470. https://doi.org/10.1111/j.1529-8019.2012.01460.x
  10. Kadoya K, Sasaki T, Kostka G, Timpl R, Matsuzaki K, Kumagai N, et al. 2005. Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis. Br. J. Dermatol. 153: 607-612. https://doi.org/10.1111/j.1365-2133.2005.06716.x
  11. Hunter JA. 1973. Diseases of the skin. Structure and function of skin in relation to therapy. Br. Med. J. 4: 340. https://doi.org/10.1136/bmj.4.5992.340
  12. Amsden BG, Goosen M. 1995. Transdermal delivery of peptide and protein drugs: an overview. AIChE J. 41: 1972-1997. https://doi.org/10.1002/aic.690410814
  13. Askari M, Naniz MA, Kouhi M, Saberi A, Zolfagharian A, Bodaghi M. 2021. Recent progress in extrusion 3D bioprinting of hydrogel biomaterials for tissue regeneration: a comprehensive review with focus on advanced fabrication techniques. Biomater. Sci. 9: 535-573. https://doi.org/10.1039/D0BM00973C
  14. Singh M, Haverinen HM, Dhagat P, Jabbour GE. 2010. Inkjet printing-process and its applications. Adv. Mater. 22: 673-685. https://doi.org/10.1002/adma.200901141
  15. Guillotin B, Souquet A, Catros S, Duocastella M, Pippenger B, Bellance S, et al. 2010. Laser assisted bioprinting of engineered tissue with high cell density and microscale organization. Biomaterials 31: 7250-7256. https://doi.org/10.1016/j.biomaterials.2010.05.055
  16. Park JY, Jang J, Kang H. 2018. 3D Bioprinting and its application to organ-on-a-chip. Microelectron. Eng. 200: 1-11. https://doi.org/10.1016/j.mee.2018.08.004
  17. Xu J, Zheng S, Hu X, Li L, Li W, Parungao R, et al. 2020. Advances in the research of bioinks based on natural collagen, polysaccharide and their derivatives for skin 3D bioprinting. Polymers 12: 1237. https://doi.org/10.3390/polym12061237
  18. Nair K, Gandhi M, Khalil S, Yan KC, Marcolongo M, Barbee K, et al. 2009. Characterization of cell viability during bioprinting processes. Biotechnol. J. Healthc. Nutr. Technol. 4: 1168-1177.
  19. Kim BS, Kwon YW, Kong J, Park GT, Gao G, Han W, et al. 2018. 3D cell printing of in vitro stabilized skin model and in vivo pre-vascularized skin patch using tissue-specific extracellular matrix bioink: a step towards advanced skin tissue engineering. Biomaterials 168: 38-53. https://doi.org/10.1016/j.biomaterials.2018.03.040
  20. Kim BS, Lee J, Gao G, Cho D. 2017. Direct 3D cell-printing of human skin with functional transwell system. Biofabrication 9: 025034. https://doi.org/10.1088/1758-5090/aa71c8
  21. Kim BS, Gao G, Kim JY, Cho D. 2019. 3D cell printing of perfusable vascularized human skin equivalent composed of epidermis, dermis, and hypodermis for better structural recapitulation of native skin. Adv. Healthc. Mater. 8: 1801019. https://doi.org/10.1002/adhm.201801019
  22. Baltazar T, Merola J, Catarino C, Xie CB, Kirkiles-Smith NC, Lee V, et al. 2020. Three dimensional bioprinting of a vascularized and perfusable skin graft using human keratinocytes, fibroblasts, pericytes, and endothelial cells. Tissue Eng. Part A 26: 227-238. https://doi.org/10.1089/ten.tea.2019.0201
  23. Kang H, Lee SJ, Ko IK, Kengla C, Yoo JJ, Atala A. 2016. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat. Biotechnol. 34: 312-319. https://doi.org/10.1038/nbt.3413
  24. Adib AA, Sheikhi A, Shahhosseini M, Simeunovic A, Wu S, Castro CE, et al. 2020. Direct-write 3D printing and characterization of a GelMA-based biomaterial for intracorporeal tissue engineering. Biofabrication 12: 045006. https://doi.org/10.1088/1758-5090/ab97a1
  25. Kim HB, Jung S, Park H, Sim DS, Kim M, Das S, et al. 2021. Customized 3D-printed occluders enabling the reproduction of consistent and stable heart failure in swine models. Bio-Des. Manuf. 4: 833-841. https://doi.org/10.1007/s42242-021-00145-4
  26. Neff EP. 2017. Printing cures: Organovo advances with 3D-printed liver tissue. Lab Anim. 46: 57. https://doi.org/10.1038/laban.1203
  27. Netzlaff F, Lehr C, Wertz PW, Schaefer UF. 2005. The human epidermis models EpiSkin, SkinEthic and EpiDerm: An evaluation of morphology and their suitability for testing phototoxicity, irritancy, corrosivity, and substance transport. Eur. J. Pharm. Biopharm. 60: 167-178. https://doi.org/10.1016/j.ejpb.2005.03.004
  28. Bas A, Burns N, Gulotta A, Junker J, Drasler B, Lehner R, et al. 2021. Understanding the development, standardization, and validation process of alternative in vitro test methods for regulatory approval from a researcher perspective. Small 17: e2006027.
  29. Pfuhler S, van Benthem J, Curren R, Doak SH, Dusinska M, Hayashi M, et al. 2020. Use of in vitro 3D tissue models in genotoxicity testing: strategic fit, validation status and way forward. Report of the working group from the 7th International Workshop on Genotoxicity Testing (IWGT). Mut. Res. Genet. Toxicol. Environ. Mutagen. 850: 503135. https://doi.org/10.1016/j.mrgentox.2020.503135
  30. Deyrieux AF, Wilson VG. 2007. In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line. Cytotechnology 54: 77-83. https://doi.org/10.1007/s10616-007-9076-1
  31. Nobusawa A, Sano T, Negishi A, Yokoo S, Oyama T. 2014. Immunohistochemical staining patterns of cytokeratins 13, 14, and 17 in oral epithelial dysplasia including orthokeratotic dysplasia. Pathol. Int. 64: 20-27. https://doi.org/10.1111/pin.12125
  32. Roy RR, Shimada K, Murakami S, Hasegawa H. 2021. Contribution of transglutaminases and their substrate proteins to the formation of cornified cell envelope in oral mucosal epithelium. Eur. J. Oral Sci. 129: e12760.
  33. Pedde RD, Mirani B, Navaei A, Styan T, Wong S, Mehrali M, et al. 2017. Emerging biofabrication strategies for engineering complex tissue constructs. Adv. Mater. 29: 1606061. https://doi.org/10.1002/adma.201606061
  34. Wang R, Wang Y, Yao B, Hu T, Li Z, Huang S, et al. 2019. Beyond 2D: 3D bioprinting for skin regeneration. Int. Wound J. 16: 134-138. https://doi.org/10.1111/iwj.13003
  35. Huang J, Fu H, Li C, Dai J, Zhang Z. 2017. Recent advances in cell-laden 3D bioprinting: materials, technologies and applications. J. 3D Print. Med. 1: 245-268. https://doi.org/10.2217/3dp-2017-0010
  36. Ma X, Liu J, Zhu W, Tang M, Lawrence N, Yu C, et al. 2018. 3D bioprinting of functional tissue models for personalized drug screening and in vitro disease modeling. Adv. Drug Deliv. Rev. 132: 235-251. https://doi.org/10.1016/j.addr.2018.06.011
  37. McGrath JA, Eady R, Pope FM. 2004. Anatomy and organization of human skin. y: Burns T, Breathnach S, Cox N, Griffiths C (Eds.), Rook's textbook of dermatology.
  38. Proksch E, Brandner JM, Jensen J. 2008. The skin: an indispensable barrier. Exp. Dermatol. 17: 1063-1072. https://doi.org/10.1111/j.1600-0625.2008.00786.x
  39. Akiyama M, Takeichi T, McGrath JA, Sugiura K. 2018. Autoinflammatory keratinization diseases: an emerging concept encompassing various inflammatory keratinization disorders of the skin. J. Dermatol. Sci. 90: 105-111. https://doi.org/10.1016/j.jdermsci.2018.01.012
  40. Herrmann H, Bar H, Kreplak L, Strelkov SV, Aebi U. 2007. Intermediate filaments: from cell architecture to nanomechanics. Nat. Rev. Mol. Cell Biol. 8: 562-573. https://doi.org/10.1038/nrm2197
  41. Quinlan RA, Schiller DL, Hatzfeld M, Achtstatter T, Moll R, Jorcano JL, et al. 1985. Patterns of expression and organization of cytokeratin intermediate filaments. Ann. N. Y. Acad. Sci. 455: 282-306. https://doi.org/10.1111/j.1749-6632.1985.tb50418.x
  42. Moll R, Divo M, Langbein L. 2008. The human keratins: biology and pathology. Histochem. Cell Biol. 129: 705. https://doi.org/10.1007/s00418-008-0435-6
  43. Hohl D, Olano BR, de Viragh PA, Huber M, Detrisac CJ, Schnyder UW, et al. 1993. Expression patterns of loricrin in various species and tissues. Differentiation 54: 25-34. https://doi.org/10.1111/j.1432-0436.1993.tb00656.x
  44. Pillai S, Bikle DD. 1991. Role of intracellular-free calcium in the cornified envelope formation of keratinocytes: Differences in the mode of action of extracellular calcium and 1, 25 dihydroxyvitamin D3. J. Cell. Physiol. 146: 94-100. https://doi.org/10.1002/jcp.1041460113
  45. Rahn E, Thier K, Petermann P, Rubsam M, Staeheli P, Iden S, et al. 2017. Epithelial barriers in murine skin during herpes simplex virus 1 infection: the role of tight junction formation. J. Invest. Dermatol. 137: 884-893. https://doi.org/10.1016/j.jid.2016.11.027
  46. Jean J, Bernard G, Duque-Fernandez A, Auger FA, Pouliot R. 2011. Effects of serum-free culture at the air-liquid interface in a human tissue-engineered skin substitute. Tissue Eng. Part A 17: 877-888. https://doi.org/10.1089/ten.tea.2010.0256