Poster O15 - Exploration of Tissue Specificity Preservation in Liver Decellularized Extracellular Matrix for Guiding Hepatic Progenitor Differentiation into Functional Hepatocytes

Introduction: The dearth of high-quality donor livers presents a significant obstacle in the treatment of advanced liver diseases, highlighting the urgent need for alternative therapeutic strategies. As an alternative, the potential of transplanting cells from the hepatic lineage is notable; however, the critical challenge remains the acquisition of a sufficient quantity of functional hepatocytes for clinical use. This study aims to overcome these barriers by focusing on the development of functional substrates and scaffolds that emulate the microenvironment of the liver in vivo, thereby supporting hepatocyte viability and function. Traditional protocols for hepatic differentiation largely depend on Matrigel, which does not provide the complex signaling cues found in the native liver matrix. To address this, the current study introduces a novel protocol that utilizes decellularized liver-derived extracellular matrix (LdECM) as a differentiation substrate for hepatocytes. This investigation assessed the effects of LdECM and Matrigel on chemically derived hepatic progenitors (CdHs) during hepatic differentiation, revealing that LdECM significantly enhances the expression of mature hepatocyte markers and improves hepatic function compared to Matrigel. Moreover, this study integrates encapsulation and three-dimensional bioprinting techniques to precisely control cellular configuration, thus enabling the construction of complex liver tissue structures that closely replicate the in vivo microenvironment. In summary, the approach incorporates two principal elements: (1) analyzing the liver's extracellular matrix to identify proteins that influence the differentiation of hepatic lineage cells; (2) replicating the structural features of pre-vascularized hepatic tissue through bioprinting, offering insights into hepatocyte interactions with sinusoidal structures.

Materials and

Methods: Generation of mCdH and differentiation
To produce mCdH from primary hepatocytes, the cells were first cultured and subjected to a reprogramming medium. The cells were diluted and allocated into 96-well plates to isolate single cell-derived clones. For hepatic differentiation, the mCdHs were plated on collagen-coated dishes and cultured in a differentiation-specific medium. Subsequently, the cells were overlaid with either Matrigel or LdECM, which was diluted in differentiation medium, and further cultured..
Liver decellularization
Roughly sliced porcine liver tissue was subjected to a washing process using distilled water. After the washing process, the tissue slices were treated with a solution containing detergent. The sample was sterilized using peracetic acid in PBS solution. To remove any remaining detergent and cell debris, the tissues were washed with distilled water during the process. Lastly, the decellularized tissues were freeze-dried.
Fabrication of Pre-vascularized Liver Patch
The RX1 printer for Aspect Biosystems was utilized for the construction. The bioinks were formulated as follows: the core comprised 0.6% LdECM with mCdH, while the shell consisted of 0.6% VdECM (Vessel-dECM) and 0.2% alginate with HUVECs. The crosslinking agent employed was a 100 mM CaCl2 solution enhanced with 10% PVA, and the buffer was standardized to 1X DPBS. Throughout the printing process, all bioinks were maintained in ice-cold conditions. Subsequent to printing, the constructs were placed in tissue culture dishes and incubated at 37 ℃ with 5% CO2 for a duration of 7-days.

Results, Conclusions, and Discussions: The successful implementation of the decellularization technique yielded LdECM from native porcine sources. Distinct from Matrigel, LdECM is enriched with a comprehensive array of matrisomal proteins, notably featuring an increased concentration of liver-specific proteins. The presence of these specialized proteins in LdECM is believed to enhance hepatocyte functional signaling pathways, thereby facilitating hepatic differentiation. Comparative analyses of hepatic differentiation in Matrigel and LdECM revealed that cells within LdECM demonstrated superior indocyanine green (ICG) uptake, akin to that of mature hepatocytes. Moreover, there was a notable increase in the expression of hepatic markers in differentiated hepatocytes compared to their non-differentiated mCdH counterparts, with the most pronounced expression occurring in cells differentiated within LdECM. Histological assessments of these hepatocytes, using periodic acid-Schiff (PAS) staining and anti-albumin immunostaining, identified glycogen accumulation and albumin production, respectively, which are hallmark features of functional hepatocytes. Additionally, this study pioneered the development of a pre-vascularized hepatic sheet for transplantation purposes, designed to assess the therapeutic efficacy of hepatocytes differentiated from mCdHs. Hepatic sheets incorporating vascular structures exhibited enhanced albumin secretion capabilities. This investigation has successfully advanced the differentiation of mCdHs into mature hepatocytes using a novel LdECM-based protocol. Unlike Matrigel, which has traditionally served as a differentiation matrix, LdECM contains a robust composition of liver-specific proteins that are primarily involved in vital cellular and catalytic processes essential for hepatic functionality. The integration of these liver-specific proteins within LdECM has proven crucial in driving a highly efficient differentiation process, as evidenced by the increased expression of hepatocyte-specific markers. The methodology facilitated by LdECM offers substantial promise for the production of functional hepatocytes, presenting significant prospects for advancements in liver tissue engineering and regenerative medicine.

Acknowledgements (Optional): This work was supported by the Alchemist Project (20012378, Development of Meta Soft Organ Module Manufacturing Technology without Immunity Rejection and Module Assembly Robot System) funded by the Ministry of Trade, Industry & Energy(MOTIE, Korea), and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2023-00248438).