3D bioprinting of dECM-incorporated hepatocyte spheroid for simultaneous promotion of cell-cell and -ECM interactions

Min Kyeong Kim; Wonwoo Jeong; Seunggyu Jeon; Hyun-Wook Kang

doi:10.3389/fbioe.2023.1305023

3D bioprinting of dECM-incorporated hepatocyte spheroid for simultaneous promotion of cell-cell and -ECM interactions

Front Bioeng Biotechnol. 2023 Nov 13:11:1305023. doi: 10.3389/fbioe.2023.1305023. eCollection 2023.

Authors

Min Kyeong Kim^#^{1

2}, Wonwoo Jeong^#^{1

3}, Seunggyu Jeon^{1

3}, Hyun-Wook Kang¹

Affiliations

¹ Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.
² Center for Scientific Instrumentation, Korea Basic Science Institute, Chungbuk, Republic of Korea.
³ Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, United States.

^# Contributed equally.

Abstract

The cell spheroid technology, which greatly enhances cell-cell interactions, has gained significant attention in the development of in vitro liver models. However, existing cell spheroid technologies still have limitations in improving hepatocyte-extracellular matrix (ECM) interaction, which have a significant impact on hepatic function. In this study, we have developed a novel bioprinting technology for decellularized ECM (dECM)-incorporated hepatocyte spheroids that could enhance both cell-cell and -ECM interactions simultaneously. To provide a biomimetic environment, a porcine liver dECM-based cell bio-ink was developed, and a spheroid printing process using this bio-ink was established. As a result, we precisely printed the dECM-incorporated hepatocyte spheroids with a diameter of approximately 160-220 μm using primary mouse hepatocyte (PMHs). The dECM materials were uniformly distributed within the bio-printed spheroids, and even after more than 2 weeks of culture, the spheroids maintained their spherical shape and high viability. The incorporation of dECM also significantly improved the hepatic function of hepatocyte spheroids. Compared to hepatocyte-only spheroids, dECM-incorporated hepatocyte spheroids showed approximately 4.3- and 2.5-fold increased levels of albumin and urea secretion, respectively, and a 2.0-fold increase in CYP enzyme activity. These characteristics were also reflected in the hepatic gene expression levels of ALB, HNF4A, CPS1, and others. Furthermore, the dECM-incorporated hepatocyte spheroids exhibited up to a 1.8-fold enhanced drug responsiveness to representative hepatotoxic drugs such as acetaminophen, celecoxib, and amiodarone. Based on these results, it can be concluded that the dECM-incorporated spheroid printing technology has great potential for the development of highly functional in vitro liver tissue models for drug toxicity assessment.

Keywords: 3D bioprinting; cell-ECM interaction; dECM-incorporated hepatocyte spheroid; decellularization; liver tissue engineering.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A2C2012808 and NRF- 2018K1A4A3A01063890).