Page 135 - IJB-10-3

International Journal of Bioprinting New challenges in liver tissue engineering

Microfluidic systems allow the cells being physiologically the mechanical properties of the hydrogel increased from
stimulated under a continuous flow of cell culture media. healthy liver values (0.5 kPa) to those of cirrhotic liver
As previously described, a microfluidic bioreactor (15 kPa). HepG2 cells were less viable in the stiff scaffolds
133
was interfaced with a bioprinter to fabricate 3D hepatic and had a significantly reduced expression of liver-
constructs of spheroids encapsulated in photocrosslinkable specific markers, indicating that the cirrhotic mechanical
GelMA hydrogel. The cultured spheroids were viable environment plays a significant role in liver function.
and active for 30 days and responded to acetaminophen GelMa was used to model liver fibrosis for the co-
treatment in a similar way to those described in other culture of HepaRG and LX-2 cells. The shear storage
137
published studies on animal or in vitro models.
modulus of the hydrogels was controlled by the Gel
Bioprinting can be used to build complex 3D concentration, from values ranging from 0.05 kPa to 2.12
structures that mimic the liver lobule microarchitecture kPa for 2.5% and 15% solutions, respectively. The printed
for drug toxicity experiments. In a previous study, the structures were viable for 1 month, and HepaRG cells were
134
authors propose a physiologically relevant in vitro liver able to differentiate in three dimensions in the absence of
model formed by hepatic sinusoids with alternate cords dimethylsulfoxide. HSCs activated in response to TGF-b,
of hepatocytes and non-parenchymal cells obtained by depositing fibrillar collagen, suggesting the system’s
extrusion bioprinting. The bioink used was a mixture of suitability for modeling liver fibrosis.
Gel, silk fibroin, and liver dECM. The authors demonstrated
that the co-culture of hepatocytes and non-parenchymal Although 3D bioprinting has allowed the cost-
cells is more accurate than hepatocyte monoculture in effective rapid manufacture of 3D models, there are still
predicting hepatotoxicity. limitations and challenges to address before they can
be applied to clinical applications. For instance, current
6.2.2. Other diseases bioprinting resolution is not accurate enough to reproduce
Bioprinting can be used to develop precise liver disease the complex hepatic microenvironment that includes
models to study the underlying mechanisms and test the abundant vascularization to ensure long-term hepatotoxic
121
potential of drugs under development (see the recent experiments or research on liver diseases. Most of the
advances in Table 2). Bioprinted Gel-Alg hydrogels were studies reviewed proposed successful bioinks with cell lines.
used to culture primary HCC cells obtained from six different However, the bioink characteristics need to be improved
patients. The cells retained the expression of specific HCC to allow the survival of PHHs and the maintenance of
biomarkers as well as the genetic alterations and expression the cells’ original functionality and morphology, which is
profiles of their original tumors. The model was used to still a challenge with the current bioinks. Many bioink
121
evaluate the efficacy of candidate drugs for HCC. HCC crosslinking methods rely on UV exposure, which is
135
models were also developed with photocrosslinkable liver suspected of being cytotoxic, so that there is a need to develop
dECM bioprinted through a rapid light-based process to bioinks able to crosslink under visible light. Additional
138
tune the mechanical properties of the hydrogels. The research is needed before the optimal bioprinting method,
136
dECM was mixed with photocrosslinkable GelMA to composition, and crosslinking mechanism are obtained to
produce the bioink. By changing the light exposure time, put the technique into clinical practice.

Table 2. Applications of bioprinting for disease modeling
Ink Cell source Printing 3D model Outcomes Refs
composition method
DILI
Pluronic F127 HepG2/C3A Pneumatic Square + – 18.4 kPa of storage modulus after crosslinking. 129
and Alg extrusion inner grid – Homogeneous cell distribution and maintenance of viability (>95%).
– Increased urea and albumin production after 7 days.
– Upregulation of CYP1A2 expression.
– Increased sensitivity to APAP-induced hepatotoxicity.
Col I and HA Lx2 cells/HSCs Pneumatic Circle + – Printable bioinks form stable constructs. 130
and PHHs extrusion inner grid – Maintenance of viability (>80%).
– Col-I rich bioinks displayed improvements in cell morphology (Lx2
and HSCs).
– Hepatotoxicity assessment of APAP-induced toxicity (urea and
albumin production).
(Continued...)

Volume 10 Issue 3 (2024) 127 doi: 10.36922/ijb.2706