, Volume 70, Issue 1, pp 13–29 | Cite as

Comparison of biophysical properties characterized for microtissues cultured using microencapsulation and liquid crystal based 3D cell culture techniques

  • Chin Fhong Soon
  • Kian Sek Tee
  • Soon Chuan Wong
  • Nafarizal Nayan
  • Sargunan Sundra
  • Mohd Khairul Ahmad
  • Farshid Sefat
  • Naznin Sultana
  • Mansour Youseffi
Methods paper


Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications.


3D cell culture Biophysical properties Liquid crystal Microtissues Keratinocytes Microencapsulation 



The authors are grateful to the research financial support (Science Fund Vot No.: 0201-01-13-SF0104 or S024) awarded by Malaysia Ministry of Science and Technology (MOSTI) and IGSP Grant Vot No. U679 awarded by Universiti Tun Hussein Onn Malaysia. We acknowledge the help of Arina Basyirah Ismail for routine cell sub-culture.

Author’s contribution

CFS produced the conception of the paper and prepared the manuscript of the paper. KST designed and fabricated the flicker machine. SCW performed the microencapsulation of 3D cells and quantification of microtissues. CFS conducted the 3D cell culture and staining experiments of the microtissues. NN performed the FE-SEM imaging of the microtissues. MKA conducted the FTIR experiments and NS analyzed the FTIR results. FS helped to analyze and interpret the cells staining results. MY synthesized and prepared the liquid crystals. SA/LS prepared the samples for the histological sectioning.

Compliance with ethical standards

Conflict of interest

None of the authors have any competing interest in the manuscript.

Ethics approval

Cell lines were used in the experiments. Not applicable.


  1. Amy YH, Yi-Chung T, Xianggui Qu, Lalit RP, Kenneth JP, Shuichi T (2012) 384 hanging drop arrays give excellent Z-factors and allow versatile formation of co-culture spheroids. Biotechnol Bioeng 109:1293–1304. CrossRefGoogle Scholar
  2. Antoni D, Burckel H, Josset E, Noel G (2015) Three-dimensional cell culture: a breakthrough in vivo. Int Mol Sci 16:5517–5527. CrossRefGoogle Scholar
  3. Discher D, Janmey P, Y-l Wang (2005) Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139–1143. CrossRefGoogle Scholar
  4. Edmondson R, Broglie JJ, Adcock AF, Yang L (2014) Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 12:207–218. CrossRefGoogle Scholar
  5. Fang Y, Elgen RM (2017) Three-dimensional cell cultures in drug discovery and development. SLAS Discov 1:1–17. Google Scholar
  6. Feingold K (2007) The role of epidermal lipids in cutaneous permeability barrier homeostasis. J Lipid Res 48:2531–2546. CrossRefGoogle Scholar
  7. Godugu C, Patel AR, Desai U, Andey T, Sams A, Singh M (2013) AlgiMatrix™ based 3D cell culture system as an in vitro tumor model for anticancer studies. PLoS ONE 8:e53708. CrossRefGoogle Scholar
  8. Griffith CK, Miller C, Sainson RC, Calvert JW, Jeon NL, Hughes CC, George SC (2005) Diffusion limits of an in vitro thick prevascularized tissue. Tissue Eng 11:257–266. CrossRefGoogle Scholar
  9. Hendze M, Nishioka W, Raymond Y, Allis C, Bazett-Jones D, TnJ PH (1998) Chromatin condensation is not associated with apoptosis. J Biol Chem 273:24470–24478. CrossRefGoogle Scholar
  10. Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart L (2010) Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148:3–15. CrossRefGoogle Scholar
  11. Hoath SB, Leahy DG (2003) The organization of human epidermis: functional epidermal units and phi proportionality. J Invest Dermatol 121:1440–1446. CrossRefGoogle Scholar
  12. Hongisto V, Jernstrom S, Fey V (2013) High-throughput 3D screening reveals differences in drug sensitivities between culture models of JIMT1 breast cancer cells. PLoS ONE 8:e77232. CrossRefGoogle Scholar
  13. Jackson M, Mantsch H (1995) The use and misuse of FTIR spectroscopy in the determination of protein structure. Crit Rev Biochem Mol Biol 30:95–120. CrossRefGoogle Scholar
  14. James Nelson W (2009) Remodeling epithelial cell organization: transitions between front–rear and apical–basal polarity. Cold Spring Harb Perspect Biol 1:a000513. Google Scholar
  15. Kundu AK, Khatiwala CB, Putnam AJ (2009) Extracellular matrix remodeling, integrin expression, and downstream signaling pathways influence the osteogenic differentiation of mesenchymal stem cells on poly(lactide-co-glycolide) substrates. Tissue Eng Part A 15:273–283. CrossRefGoogle Scholar
  16. Kunz-Schughart L, Freyer J, Hofstaedter F, Ebner R (2014) The use of 3-D cultures for high throughput screening: the multicellular spheroid model. J Biomol Screen 9:273–285. CrossRefGoogle Scholar
  17. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000) Molecular cell biology, 6th edn. Freeman & Co Ltd, W.HGoogle Scholar
  18. Luca A, Mersch S, Deenen R (2013) Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines. PLoS ONE 8:e59689. CrossRefGoogle Scholar
  19. Malek K, Wood BR, Bambery KR (2014) FTIR imaging of tissues: techniques and methods of analysis. In: Malgorzata B (ed) Optical spectroscopy and computation methods in biology and medicine, vol 14. Springer, Dordrecht, pp 419–474CrossRefGoogle Scholar
  20. Movasaghi Z, Rehman S, Rehman Iu (2008) Fourier transform infrared (FTIR) spectroscopy of biological tissues. Appl Spectrosc Rev 43:134–179. CrossRefGoogle Scholar
  21. Nagpal M, Singh SK, Mishra D (2013) Synthesis characterization and in vitro drug release from acrylamide and sodium alginate based superporous hydrogel devices. Int J Pharm Investig 3:131–140. CrossRefGoogle Scholar
  22. Nirmalanandhan VS, Duren A, Hendricks P, Vielhauer G, Sittampalam GS (2010) Activity of anticancer agents in a three-dimensional cell culture model. Assay Drug Dev Technol 8:581–590. CrossRefGoogle Scholar
  23. Norlen L, Plasencia G, Simonsen A, Descouts P (2007) Human stratum corneum lipid organization as observed by atomic force microscopy on langmuir–blodgett films. J Struct Biol 158:386–400. CrossRefGoogle Scholar
  24. Sarker B, Papageorgiou DG, Silva R, Boccaccini AR (2013) Fabrication of alginate-gelatin crosslinked hydrogel microcapsules and evaluation of the microstructure and physico-chemical properties. J Mater Chem B 2:1470–1482. CrossRefGoogle Scholar
  25. Soon CF, Youseffi M, Berends RF, Blagden N, Denyer MC (2013) Development of a novel liquid crystal based cell traction force transducer system. Biosens Bioelectron 39:14–20. CrossRefGoogle Scholar
  26. Soon CF et al (2014) Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals. Micron 56:73–79. CrossRefGoogle Scholar
  27. Soon CF et al (2016) A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces. Biotech Histochem 91:283–295. CrossRefGoogle Scholar
  28. Souza GR et al (2010) Three-dimensional tissue culture based on magnetic cell levitation. Nat Nanotechnol 5:291–296. CrossRefGoogle Scholar
  29. Therese Andersen PA-E, Dornish M (2015) Review 3D cell culture in alginate hydrogels. Microarrays 4:133–161. CrossRefGoogle Scholar
  30. Thoma C, Stroebel S, Rösch N, Calpe B, Krek W, Kelm J (2013) A high-throughput-compatible 3D microtissue co-culture system for phenotypic RNAi screening applications. J Biomol Screen 18:1330–1337. CrossRefGoogle Scholar
  31. Tomaro-Duchesneau C, Saha S, Malhotra M, Kahouli I, Prakash S (2013) Microencapsulation for the therapeutic delivery of drugs, live mammalian and bacterial cells, and other biopharmaceutics: current status and future directions. J Pharm 2013:1–17. Google Scholar
  32. Vantangoli MM, Madnick SJ, Huse SM, Weston P, Boekelheide K (2015) MCF-7 human breast cancer cells form differentiated microtissues in Scaffold-free hydrogels. PLoS ONE 10:e0135426. CrossRefGoogle Scholar
  33. Wang S, Wong Po Foo C, Warrier A, Poo MM, Heilshorn SC, Zhang X (2009) Gradient lithography of engineered proteins to fabricate 2D and 3D cell culture microenvironments. Biomed Microdev 11:1127–1134. CrossRefGoogle Scholar
  34. Wong SC, Soon CF, Leong WY, Tee KS (2016) Flicking technique for microencapsulation of cells in calcium alginate leading to the microtissue formation. J Microencapsul 33:162–171. CrossRefGoogle Scholar
  35. Yamada KM, Cukierman E (2007) Modeling tissue morphogenesis and cancer in 3D. Cell 130:601–610. CrossRefGoogle Scholar
  36. Ziboh V, Dreize M (1975) Biosynthesis and hydrolysis of cholesteryl esters by rat skin subcellular fractions. Biochem J 152:281–289. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Chin Fhong Soon
    • 1
  • Kian Sek Tee
    • 1
  • Soon Chuan Wong
    • 1
  • Nafarizal Nayan
    • 1
  • Sargunan Sundra
    • 1
  • Mohd Khairul Ahmad
    • 1
  • Farshid Sefat
    • 3
  • Naznin Sultana
    • 2
  • Mansour Youseffi
    • 3
  1. 1.Biosensor and Bioengineering Lab, MiNT-SRC, Faculty of Electrical and Electronic EngineeringUniversiti Tun Hussein Onn MalaysiaParit Raja, Batu PahatMalaysia
  2. 2.Faculty of Biosciences and Medical EngineeringUniversiti Teknologi MalaysiaSkudaiMalaysia
  3. 3.Faculty of Engineering and Informatics, Medical and Healthcare Technology DepartmentUniversity of BradfordBradfordUK

Personalised recommendations