Yasuyuki Sakai
Yasuyuki Sakai, Ph.D.
Professor, Department of Chemical System Engineering,
Graduate School of Engineering, University of Tokyo
7-3-1 Hongo, Bunky-ku, Tokyo 113-8656, Japan
Tel/Fax, +81-3-5841-7073
Vice Director, International Research Center on Integrative Biomedical Systems (CIBiS),
Institute of Industrial Science, University of Tokyo.
Project Professor, Max Planck - The University of Tokyo Center for Integrative
Inflammology, University of Tokyo
4-6-1 Komaba, Megur-ku, Tokyo 153-8505, Japan
Tel, +81-3-5452-6352; Fax, +81-3-5452-6353; E-mail: sakaiyas@iis.u-tokyo.ac.jp


Dr. Sakai received Ph.D. in chemical engineering from University of Tokyo in 1993 and stated his work at Institute of Industrial Science, University of Tokyo.  In 1997-1998, he stayed in University of Rochester, as a visiting scientist investigating 3D culture of bone marrow cells (Prof. David Wu's Lab). In 2003-2008, he worked as an associate professor of Regenerative
Medical Engineering Laboratory at the Center for Disease Biology and Integrative Medicine (CDBIM), Graduate School of Medicine, University of Tokyo. He returned to IIS as a professor
and then moved to the current position in 2015. During his research carrier, he got several scientific awards such as young investigator award of Society of Chemical Engineers, Japan, publication awards of Society for Bioscience and Bioengineering, Japan and Japanese Society for Alternatives to Animal Experiments.  He recently became a fellow of American Institute for Medical and Biological Engineering (AIMBE).

His current research topics are engineering of multi-scale 3D tissues/organs for clinical applications and cell-based pharmacological or toxicological assays.  He has been
placing particular importance on simultaneous realization of good mass transfers and 3D organization of stem/progenitor cells together with various micro-technologies.

1. Comparison of the transcriptomic profile of hepatic human induced pluripotent stem like cells cultured in Petri and in a 3D microscale dynamic environment, Leclerc, E. et al., Genomics, 109(1), 16-26 (2017).
2. Alteration of pancreatic carcinoma and promyeloblastic cell adhesion in liver microvasculature by co-culture of hepatocytes, hepatic stellate cells and endothelial cells in a physiologically-relevant model, Danoy, M. et al. Integ. Biol., 9, 350-361 (2017). 
3. Novel integrative methodology for engineering large liver tissue equivalents based on
three-dimensional scaffold fabrication and cellular aggregate assembly, Pang, Y. et a al.,  Biofabrication, 8(3):035016 (2016).
4. Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture, Horiguchi, I and Sakai, Y., Biotechnol. Prog., 32(4):1009-16 (2016).
5. Establishment of a new liver tissue model by hierarchically coculturing primary rat hepatocytes with liver sinusoidal endothelial cells on a gas-permeable membrane, Xiao, W. et al., Integ. Biol., 7, 1412-22 (2015).
6. Combination of microwell structures and direct oxygenation enables efficient and size-regulated aggregate formation of an insulin-secreting pancreatic b-cell line, M. Shinohara, M. et al., Biotechnol. Prog., 30, 178-187 (2014).
7. Development of bioactive hydrogel capsules for the 3D expansion of pluripotent stem cells in bioreactors, Tabata, Y. et al., Biomat. Sci., 2, 176-183 (2014).
8. Three-dimensional culture of fetal mouse, rat and porcine hepatocytes, Y. Sakai et al., in "Fetal Tissue Transplantation", etd. by N. Bhattacharya and P. Stubblefield, Springer-Verlag UK, pp.47-63 (2013).
9. Enhanced bile canaliculi formation enabling direct recovery of biliary metabolites of hepatocytes in 3D collagen gel microcavities, H. Matsui et al., Lab on-a-Chip, 12, 1857-1864  (2012).
10. Direct oxygen supply with polydimethylsiloxane (PDMS) membranes induces a spontaneous organization of thick heterogeneous liver tissues from rat fetal liver cells in vitro, Hamon, M et al., Cell Transplant., 21, 401-410 (2012).
11. Engineering of implantable liver tissues, Y. Sakai et al., in "Liver Stem Cells" etd. by T. Ochiya, Hamana Press, pp.189-216 (2011).


Oxygen-permeable membranes for aerobic organization and culture of liver tissues

Statement of Purpose: In vitro oxygenation of highly metabolic hepatocytes has been a fundamental problem, particularly in static culture where oxygen is supplied only by diffusion through the culture medium layer above the cells cultured at the bottom surface of plates (Fig. 1).  Although this limitation of oxygen supply was first pointed out by Steven in 1965 [1], it has not yet been overcome completely even now.  Therefore, we have been investigating the feasibility of direct oxygenation from the bottom surface using highly oxygen-permeable polydimethylsiloxane (PDMS) membranes [2].

Figure 1.  Oxygen supply to hepatocytes cultured at the bottom of TCPS and PDMS plates in static conditions [2].
Enhanced spontaneous organization: A Proliferative liver cell line grew spontaneously to form multilayered seudo-3D tissues in PDMS plates (Vessel Inc. Fukuoka, Japan) [3].  Even in such multilayered tissues, the cells took aerobic respiration on PDMS membranes.  In fact, oxygen consumptions of hepatocytes in sandwich culture clearly took two respiration circuits, anaerobic in tissue-culture-treated polystyrene (TCPS) plates and aerobic in PDMS plates [4].  Such aerobic respiration produce stoichiometrically 20 times higher ATP on a one-mole-glucose basis and thus it is expected to enable higher spontaneous organization in vitro. Oxygen concentrations strongly influenced fetal liver tissue development; initially low but later high oxygen concentration formed highly developed and functional liver tissues consisting of higher emergence of hepatocyte progenitors with well-developed stromal cell layers and extracellular matrix (ECM), which has never been observed before in vitro [5].  In the case of mature hepatocytes in sandwich culture, 10% oxygen was the best for various hepatic functions [4].

Integration with microfabrication technologies: When we combined such PDMS membranes with honeycomb microwell structures, aggregate formation were accelerated even in very high
inoculum density [6] and this method was very effective in forming cocultured aggregates [7]. Collagen gel microwells (60-80 mm in diameter) with PDMS membranes helped small hepatocyte colonies that produce and accumulate bile acid at the center of each colonies, enabling direct recovery of the bile acid fractions by a capillary syringe for further pharmacological analyses [8].

3D hierarchical cocultures for advanced physiological assays: Using PDMS membrane, complete double layers of hepatocytes and fibroblasts [9] or liver sinusoidal endothelial cells [10] was simply obtained by stacking the second cells after monolayers of the first cells completed.  In contrast in TCPS, both two cell populations tried to occupy the same culture surfaces and finally hepatocytes formed island-like structures surrounded by the endothelial cells on the bottom
surfaces.  Vertical and intimate cell-cell contacts in individual cell basis with accumulated ECM remarkably improve various hepatocyte functions and expression of transporters of both apical and basolateral sides of the hepatocytes.  Further inclusion of liver stellate cells showed the formation of stable and quiescent tissue against inflammatory stimulation, indicating robust and better physiological status is realized [11].

Conclusions:  These results shows the importance of aerobic respiration in in vitro cell culture, particularly cultures of cells have a high oxygen consumption. PDMS plate culture allows cells to take this in vivo-like efficient energy production. As evidenced by the results, it can serve as a simple and better physiologically-relevant organotypic hepatic model in various applications such as not only tests for efficacy/toxicity of drugs/chemicals but also as in vitro disease models.

1. Stevens KM, Nature. 1965;206:199.
2. Sakai Y et al. in Liver Stem Cells etd by Ochiya T. Hamana Press, New York, 2011, pp.189-216.
3. Evenou F et al. Tissue Eng C. 2010;16:311-318.
4. Xiao W, Biotechnol Prog. 2014;30:1401-1410.
5. Hamon M et al. Cell Transplant. 2012;21:401-410.
6. Shinohara M et al, Biotechnol Prog. 2014;30:178-187.
7. Pang Y et al. Biofab. 2012;4:045004.
8. Matsui H et al. Lab Chip, 2012;12:1857-1864.
9. Nishikawa M et al. J Biotechnol. 2008;99:1472-1481.
10. Xiao W et al. Integ Biol. 2015;7:1412-22.
11. Danoy M. et al. Integ Biol. 2017;9:350-361.  

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