SCIEnTIFIC REPORtS | (2018) 8:4556 | DOI:10.1038/s41598-018-22598-x
Origami-based self-folding of co-
cultured NIH/3T3 and HepG2 cells
into 3D microstructures
, Takaharu Okajima
, Hiroaki Onoe
, Agus Subagyo
, Kazuhisa Sueoka
This paper describes an origami-inspired self-folding method to form three-dimensional (3D)
microstructures of co-cultured cells. After a conuent monolayer of broblasts (NIH/3T3 cells) with
loaded hepatocytes (HepG2 cells) was cultured onto two-dimensional (2D) microplates, degradation
of the alginate sacricial layer in the system by addition of alginate lyase triggered NIH/3T3 cells
to self-fold the microplates around HepG2 cells, and then 3D cell co-culture microstructures were
spontaneously formed. Using this method, we can create a large number of 3D cell co-culture
microstructures swiftly with ease in the same time. We nd that HepG2 cells conned in the 3D cell
co-culture microstructures have an ability to enhance the secreted albumin compared to 2D system in
a long culture period. The result indicates that the origami-based cell self-folding technique presented
here is useful in regenerative medicine and the preclinical stage of drug development.
A challenge for regenerative medicine and drug development is to fabricate in vitro 3D structures that mimic
tissues in vivo. Several strategies have been developed to fabricate in vitro 3D cell-laden structures using a
, which involves micro-sized 3D cell-laden microstructures such as blocks
. is approach allows one to control the size and shape of these microstructures, so that they can be
easily handled and assembled to mimic in vivo tissue.
3D microstructures with dierent types of cells have been intensively investigated to mimic in vivo tissues with
a heterogeneous structure
. In this research, we applied an origami based-technique called cell origami
produce many 3D cell co-culture microstructures swily with ease at the same time. e process of producing 3D
cell co-culture microstructures using the cell origami is as simple as that for conventional cell culture in 2D dishes
(Fig.1). e cells are grown on engineered microplates xed to a at surface. e microplates are then detached
from the surface by degrading an alginate sacricial layer under the plates using alginate lyase. is allows the
cells to pull the plates using their traction force and self-fold around other types of cells and create a 3D culture
condition. Unlike other techniques such as microuidic devices, any extra equipment including tubes and micro
pumps, is not necessary in the cell origami technique.
Other advantages of using the cell origami technique for forming 3D cell co-culture microstructures are that
it can provide both at and 3D culture conditions depending on the cell types and increase the area of interaction
between co-culture cells. No other technique with these advantages has been previously developed. It is impor-
tant to consider dierent culture conditions to retain the functions of dierent cell types during co-culture
Previous researches showed that broblasts and endothelial cells can proliferate and retain their function on a
at substrate. Conversely, hepatocytes and pancreatic cells prefer 3D culture conditions such as in spheroids. It
has also shown that interactions between dierent types of cells facilitates an increase in their functions
successful co-culture technique, therefore, requires the ability to i) culture one type of cell on a at substrate, ii)
culture another type of cell in 3D conditions, and iii) provide sucient interactions between these two types of
cells. ese can be achieved using the cell origami technique.
Here, we produced the 3D cell co-culture microstructures with broblasts (NIH/3T3) and hepatoma cells
(HepG2) simply and rapidly using the cell origami technique. is 3D cell co-culture microstructure provides
Institute for the Advancement of Higher Education, Hokkaido University, Sapporo, Japan.
Graduate School of
Information Science and Technology, Hokkaido University, Sapporo, Japan.
Department of Mechanical Engineering,
Faculty of Science and Technology, Keio University, Tokyo, Japan.
Creative Research Institution Sousei, Hokkaido
University, Sapporo, Japan. Correspondence and requests for materials should be addressed to K.K.-S. (email:
Received: 19 June 2017
Accepted: 26 February 2018
Published: xx xx xxxx