Harnessing endogenous signals from hepatocytes using a low volume multi-well plate

Abstract

Hepatocytes are highly differentiated epithelial cells that lose their phenotype and function when removed from the in vivo environment. Given the importance of hepatic cultures for drug toxicity, bioartificial liver assist devices and basic biology studies, considerable efforts have been focused on maintenance of hepatic function in vitro. The methods used to date include co-cultivation of hepatocytes with stromal cells, organizing these cells into spheroids and imbedding them into bioactive gels. Our team has recently demonstrated that primary rat hepatocytes confined to microfluidic channels in the absence of convection maintained epithelial phenotype through upregulation of endogenous signals including hepatocyte growth factor (HGF). The objective of the present study was to transition from microfluidic devices, which are somewhat specialized and challenging to use and towards low volume multiwell plates ubiquitous in the biology laboratories. Using a combination of 3D printing and micromolding we have constructed inserts that could be placed into standard 12-well plates and could be used to create low volume culture conditions under where primary hepatocytes maintained differentiated phenotype. This phenotype enhancement was confirmed by assays including albumin synthesis and expression. Importantly we confirmed upregulation of HGF inside the low volume culture plates and demonstrated that inhibition of HGF signaling degraded hepatic phenotype in our cell culture platform. Overall, this study outlines a new cell culture system that leverages low volume effects of microfluidic channels in a multiwell plate format. Beyond hepatocytes, such system may be of use in maintenance of other difficult-to-culture cells including stem cells and primary cancer cells.

Paper-Based Cell Culture: Paving the Pathway for Liver Tissue Model Development on a Cellulose Paper Chip

“…Furthermore, within low-volume microconfined culture conditions, hepatocytes exhibit higher functional states than when in traditional cultures, primarily due to high accumulation of endogenous factors in small volumes. 97,98 Thus, paper-patterning techniques (as described in section 3) may enable us to establish low-volume cultures in the chemically isolated culture zones (for example, see ref 82). Table 4, Figure 4, and Figure 5 summarize the few takehome ideas that could be inherited from the existing knowledge on the paper-based cell culture technology to address the challenges existing in the field.…”

“…In this regard, it would also be necessary to first understand the dynamics between different designs, patterning configurations, and cellular behavior. Furthermore, within low-volume microconfined culture conditions, hepatocytes exhibit higher functional states than when in traditional cultures, primarily due to high accumulation of endogenous factors in small volumes. , Thus, paper-patterning techniques (as described in section ) may enable us to establish low-volume cultures in the chemically isolated culture zones (for example, see ref ).…”

Liver Tissue Engineering: Challenges and Opportunities

“…The cells cultured in the microconfinements exhibited epithelial-polarized phenotype and showed an enhanced expression of hepatocytespecific functional markers. 79,80 Such microconfinements resulted in better oxygen transport and lead to the significantly higher accumulation of endogenous growth factor signal, which would further result in the enhanced differentiated phenotype of the cultured hepatocytes. 79,80 3.2.1.3.…”

“…79,80 Such microconfinements resulted in better oxygen transport and lead to the significantly higher accumulation of endogenous growth factor signal, which would further result in the enhanced differentiated phenotype of the cultured hepatocytes. 79,80 3.2.1.3. Topological Aspects of the Implantable Construct.…”

Current Advances on 3D‐Bioprinted Liver Tissue Models

“…This device creates low‐volume culture conditions under which primary HCs can maintain differentiated phenotypes. [ 63 ] Lee et al. constructed a 3D‐bioprinted liver‐on‐a‐chip using polyethylene vinyl acetate (PEVA) and a HepaRG–liver dECM bioink mixture.…”