2D and 3D cartilage models for drug testing and “high throughput” screening – the impact of PORCN
inhibition on cartilage development

Michael Killinger^1,2,4, Tereza Szotkowska^3, Mario Rothbauer^4, Marcela Buchtova^3, Karel
Klepárník^1


^1 Department of Bioanalytical Instrumentation, Institute of Analytical Chemistry, Czech Academy of
Science, Brno, Czech Republic

^2 Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic^

^3 Laboratory of Molecular Morphogenesis, Institute of Animal Physiology and Genetics, Czech
Academy of Science, Brno, Czech Republic

^4 Karl Chiari Lab for Orthopaedic Biology, Department of Orthopaedic and Trauma Surgery, General
Hospital of Wien, Austria^


Porcupine (PORCN) is an endoplasmic reticulum protein belonging to membrane-bound O–acyl
transferase superfamily. This molecule is necessary for the attachment of long-chain faty acids to
WNT proteins essential for their secretion. PORCN is expressed in most tissues of the body and
animals with loss of function in PORCN exhibit embryonal lethality at early developmental stages
with extensive gastrulation defects. Several dominant mutations in PORCN were described in human
patients, who demonstrated significant skeletal abnormalities.


Currently, remarkable progress in stem cell-based tissue modelling has been achieved worldwide,
allowing the introduction of human-specific, cost-effective alternatives to animal studies for the
pharmaceutical industry. Novel methods of three-dimensional (3D) cell cultures became frequently
utilized in many research fields due to their enhanced biological functions as compared to
conventional two-dimensional (2D) cultures. 3D cell spheroids or organoids can replicate tissue
functions, which enables their use both as in vitro models or as a necessary intermediate step in
tissue biofabrication approaches. The most promising tool for generating and analysis of 3D cell
structures is the recent application of miniaturized platforms or microfluidic chips.
Microfluidic technology allows controlled conditions, automatization, reduced amount of reagents
and cells, and mainly dynamic conditions for continuous perfusion of nutrients and removal
of the metabolic waste of the cells.


In this study, several in vitro 2D and 3D methods were established to study the role of PORCN in
chondrogenesis in regenerative medicine. Here, we used classical animal models such as primary cell
cultures, tissue cultures of long bones, and modern 3D cell culture approaches. Modifying the PORCN
function with the LGK-974 inhibitor showed a massive increase in cartilage mass in all model
systems. 3D cell cultures were compared to standard culture methods to reflect the benefits of
microfluidic platforms. Furthermore, our PDMS platform base can be connected to the syringe pump to
reproduce in vivo perfusion conditions and increase the value of 3D cell culture.


The research was supported by the Czech Science Foundation (GA CR project 20–00726S). M.K. is Brno
PhD Talent Scholarship Holder funded by the Brno City Municipality.