Design & Manufacturing

Korean Society of Die & Mold Engineering (한국금형공학회)
권호 표지

Fabrication and validation study of a 3D tumor cell culture system equipped with bloodvessle-mimik micro-channel

혈관모사 마이크로채널이 장착된 3D 종양 세포 배양 시스템의 제작 및 검증 연구

  • Park, Jeong-Yeon (Shape Manufacturing R&D Department, Korea Institute of Industrial Technology) ;
  • Koh, Byum-seok (Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology) ;
  • Kim, Ki-Young (Therapeutics & Biotechnology Division, Korea Research Institute of Chemical Technology) ;
  • Lee, Dong-Mok (Safety System R&D Group, Korea Institute of Industrial Technology) ;
  • Yoon, Gil-Sang (Shape Manufacturing R&D Department, Korea Institute of Industrial Technology)
  • 박정연 (한국산기술연구원 뿌리산업기술연구소 형상제조연구부문) ;
  • 고범석 (한국화학연구원 신약파이프라인연구단 신약기반기술연구센터) ;
  • 김기영 (한국화학연구원 신약파이프라인연구단 신약기반기술연구센터) ;
  • 이동목 (한국생산기술연구원 대경본부 안전시스템연구그룹) ;
  • 윤길상 (한국산기술연구원 뿌리산업기술연구소 형상제조연구부문)
  • Received : 2021.03.03
  • Accepted : 2021.06.30
  • Published : 2021.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Recently, three-dimensional (3D) cell culture systems, which are superior to conventional two-dimensional (2D) vascular systems that mimic the in vivo environment, are being actively studied to reproduce drug responses and cell differentiation in organisms. Conventional two-dimensional cell culture methods (scaffold-based and non-scaffold-based) have a limited cell growth rate because the culture cannot supply the culture medium as consistently as microvessels. To solve this problem, we would like to propose a 3D culture system with an environment similar to living cells by continuously supplying the culture medium to the bottom of the 3D cell support. The 3D culture system is a structure in which microvascular structures are combined under a scaffold (agar, collagen, etc.) where cells can settle and grow. First, we have manufactured molds for the formation of four types of microvessel-mimicking chips: width / height ①100 ㎛ / 100 ㎛, ②100 ㎛ / 50 ㎛, ③ 150 ㎛ / 100 ㎛, and ④ 200 ㎛ / 100 ㎛. By injection molding, four types of microfluidic chips were made with GPPS (general purpose polystyrene), and a 100㎛-thick PDMS (polydimethylsiloxane) film was attached to the top of each microfluidic chip. As a result of observing the flow of the culture medium in the microchannel, it was confirmed that when the aspect ratio (height/width) of the microchannel is 1.5 or more, the fluid flows from the inlet to the outlet without a backflow phenomenon. In addition, the culture efficiency experiments of colorectal cancer cells (SW490) were performed in a 3D culture system in which PDMS films with different pore diameters (1/25/45 ㎛) were combined on a microfluidic chip. As a result, it was found that the cell growth rate increased up to 1.3 times and the cell death rate decreased by 71% as a result of the 3D culture system having a hole membrane with a diameter of 10 ㎛ or more compared to the conventional commercial. Based on the results of this study, it is possible to expand and build various 3D cell culture systems that can maximize cell culture efficiency by cell type by adjusting the shape of the microchannel, the size of the film hole, and the flow rate of the inlet.

Keywords

Acknowledgement

본 연구는 한국산업기술연구원의 기관주요사업(바이오 부품 적용을 위한 선택적 투과성의 다층 3차원 기저막 구조 제작 금형기술개발, Kitech EO-200070)과 NST 프로그램(역생공학 기법을 이용한 3차원 세포배양 기반 신약평가 기술개발, CAP-15-10-KRICT)의 재정적 지원에 의해 수행되었습니다.

References

  1. Sourla, A., Doillon, C., & Koutsilieris M., "Three dimensional type I collagen gel system containing MG-63 osteoblastslike cells as a model for studying local bone reaction caused by metastatic cancer cells", Anticancer Research, 16(5A), pp. 2773-2780, 1996.
  2. Dhandayuthapani, B., Yoshida, Y., Maekawa, T., & Kumar, D. S., "Polymeric scaffolds in tissue engineering application: A review", International Journal of Polymer Science, Volume 2011, pp. 1-19, 2011. https://doi.org/10.1155/2011/290602
  3. Bergenstock, M. K., Balaburski, G. M., Lau, W., Sun, W., Sun, Q., & Liu, Q., "Polystyrene scaffolds: A novel tool for in vitro three-dimensional cancer models", The First AACR International Conference on Frontiers in Basic Cancer Research, October pp. 8-11, 2009.
  4. Fisher, K. E., Pop, A., Koh, W., Anthis, N. J., Saunders, W. B., & Davis, G. E., "Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling", Molecular Cancer, 5(69), 2006.
  5. Torkian, B. A., Yeh, A. T., Engel, R., Sun, C. H., Tromberg B. J., & Wong, B. J., "Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy", Archives of Facial Plastic Surgery, 6(3), pp. 180-187, 2004. https://doi.org/10.1001/archfaci.6.3.180
  6. Kobayashi, K., Yoshida, A., Ejiri, Y., Takagi, S., Mimura, H., Hosoda, M., Matsuura, T., & Chiba K., "Increased expression of drug-metabolizing enzymes in human hepatocarcinoma FLC-4 cells cultured on micro-space cell culture plates", Drug Metabolism Pharmacokinetics, 27(5), pp. 478-485, 2015. https://doi.org/10.2133/dmpk.DMPK-12-RG-016
  7. Kermanizadeh, A., Lohr, M., Roursgaard, M., Messner, S., Gunness, P., Kelm, J. M., Moller, P., Stone, V., & Loft, S., "Hepatic toxicology following single and multiple exposure of engineered nanomaterials utilising a novel primary human 3D liver microtissue model", Part Fibre Toxicol, 11(56), 2014.
  8. Guzman, A., Ziperstein, M. J., & Kaufman, L. J., "The effect of fibrillar matrix architecture on tumor cell invasion of physically challenging environments", Biomaterials, 35(25), pp. 6954-6963, 2014. https://doi.org/10.1016/j.biomaterials.2014.04.086
  9. Vinci, M.; Gowan, S., Boxall, F., Patterson, L., Zimmerman, M., Court, W., Lomas, C., Mendiola, M., Hardisson, D., & Eccles, S. A., "Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation", BMC Biology, 10, (29), 2012.
  10. Jang, M., Neuzil, P., Volk, T., Manz, A., & Kleber, A., "On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro", Biomicrofluidics, 9(3), 034113, 2015. https://doi.org/10.1063/1.4922863
  11. Wan, C. R., Chung, S., & Kamm, R. D., "Differentiation of embryonic stem cells into cardiomyocytes in a compliant microfluidic system", Annals of Biomedical Engineering, 39 (6), pp. 1840-1847, 2011. https://doi.org/10.1007/s10439-011-0275-8
  12. https://www.corning.com/worldwide/en/products/life-sciences/products/permeable-supports/transwell-guidelines.html
  13. Zimmerman, M., Court, W., Lomas, C., Mendiola, M., Hardisson, D., & Eccles, S. A., "Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation", BMC Biology, 10, (29), 2012.
  14. Jang, M., Neuzil, P., Volk, T., Manz, A., & Kleber, A., "On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro", Biomicrofluidics, 9(3), 034113, 2015. https://doi.org/10.1063/1.4922863
  15. Wan, C. R., Chung, S., & Kamm, R. D., "Differentiation of embryonic stem cells into cardiomyocytes in a compliant microfluidic system", Annals of Biomedical Engineering, 39 (6), pp. 1840-1847, 2011. https://doi.org/10.1007/s10439-011-0275-8