PROGRESS REPORT
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Scaffold-Free Bio-3D Printing Using Spheroids as “Bio-Inks”
for Tissue (Re-)Construction and Drug Response Tests
Daiki Murata,* Kenichi Arai, and Koichi Nakayama
In recent years, scaffold-free bio-3D printing using cell aggregates (spheroids)
as “bio-inks” has attracted increasing attention as a method for 3D cell
construction. Bio-3D printing uses a technique called the Kenzan method,
wherein spheroids are placed one-by-one in a microneedle array (the
“Kenzan”) using a bio-3D printer. The bio-3D printer is a machine that was
developed to perform bio-3D printing automatically. Recently, it has been
reported that cell constructs can be produced by a bio-3D printer using
spheroids composed of many types of cells and that this can contribute to
tissue (re-)construction. This progress report summarizes the production and
effectiveness of various cell constructs prepared using bio-3D printers. It also
considers the future issues and prospects of various cell constructs obtained
by using this method for further development of scaffold-free 3D cell
constructions.
1. Introduction
In recent years, the production of new “bio-inks” suitable for 3D
bioprinting has attracted increasing attention.[1,2] 3D bioprinting
is a process involving the creation of cell patterns in a limited
space using printer technology. The number of additive man-
ufacturing techniques used in the process increases annually,
with current examples based on the use of “bio-ink,” includ-
ing inkjet techniques, laser forward transfer, and lithography
techniques.[3,4] As the number of technologies increases, the
number of types of “bio-ink” also increases. The term “bio-ink”
was first used in 2003;[5,6] the definition of the term can now
be viewed as relatively broad due to the recent diversification of
“bio-inks.”[3]
The term “bio-ink” has become independent of the technol-
ogy used for bioprinting and is generally defined as follows “a
formulation of cells suitable for processing by an automated
Prof. D. Murata, Prof. K. Arai, Prof. K. Nakayama
Center for Regenerative Medicine Research
Faculty of Medicine
Saga University
Honjo-machi, Saga 840-8502, Japan
E-mail: st0358@cc.saga-u.ac.jp
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.201901831
© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co.
KGaA, Weinheim. This is an open access article under the terms of the
Creative Commons Attribution License, which permits use, distribution
and reproduction in any medium, provided the original work is properly
cited.
DOI: 10.1002/adhm.201901831
Adv. Healthcare Mater. 2020, 9, 1901831 1901831 (1 of 18)
biofabrication technology that may also
contain biologically active components and
biomaterials.”[3] Based on this definition,
“bio-inks” can be divided into two broad
categories, namely “bio-inks” consisting
of biomaterials containing cells, and “bio-
inks” consisting only of cells.[4] Until
recently, a “bio-ink” was simply a biomate-
rial for 3D bioprinting that consisted of a
scaffold with cells.[4] However, as research
using scaffolds has progressed, various
problems and limitations have emerged in
the application of such materials, including
immune response, risk of infection, and
the potential for disease transmission from
“bio-inks” composed of biomaterials.[7]
However, if it is possible to fabricate a
3D cell construct without a scaffold, the
problems mentioned above can be fundamentally solved. In
terms of a tissue engineering method that does not use a
scaffold material, the formation of a cell sheet by pouring cells
into a template has been attempted.[8] The usefulness of many
types of cells for such a process has also been confirmed,[8,9]
and the application of various tissues as tools in regenera-
tive medicine has been reported such as skin,[10] cornea,[11,12]
esophagus,[13,14] heart muscle,[15] blood vessel,[16–19] nerve,[20]
liver,[21] pancreas,[22] bone,[23] articular cartilage,[24] periodontal
ligament,[25] lung,[26] thyroid,[27] and urinary bladder.[28] How-
ever, none of the aforementioned methods have achieved 3D
cell constructions of varying shapes, and numerous technical
difficulties remain. The use of scaffold-free “bio-ink” consisting
only of cells is now attracting increased attention as a bioprint-
ing method for the 3D construction of cells,[7] whereby the
application of spheroid constructs is eagerly anticipated.[7]
More specifically, spheroids are composed of cells, and they
exhibit properties suitable for “bio-inks” when brought into con-
tact with one another.[4,7] However, several restrictions exist in
the application of spheroids as “bio-inks.” For example, spheroids
with different compositions could not be placed in arbitrary po-
sitions, and spheroids placed in predetermined positions could
not be maintained.[7] To remove the above limitations, co-author,
Nakayama studied a method of 3D laminating spheroids us-
ing microneedles.[7] He discovered a phenomenon in which
spheroids fused on the needle. Spheroids, composed of tens of
thousands of cells, are skewered onto microneedles, forcing them
to directly touch one another and fuse together.[7] From this, he
created a needle array presenting a large number of micronee-
dles and achieved automation of the above process, i.e., spheroids
were automatically placed at any position in the microneedle
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array (the “Kenzan” (see below)).[4,7] This led to the successful de-
Daiki Murata is an assistant
velopment of a robot capable of performing the operation, which
professor at the Center for Re-
was more versatile than existing biomaterial dependent 3D bio-
generative Medicine Research
printers. This robot was named a “Bio-3D Printer” (Regenova;
at Saga University. He has
Cyfuse Biomedical K. K., Tokyo, Japan).[4] The method employed
worked on research in ortho-
for the lamination of spheroids on a needle array is known as
pedic diseases of humans and
the Kenzan method, and a series of laminating processes using
animals. His current research
the Kenzan method in bio-3D printers are referred to as bio-3D
is to develop scaffold-free cell
printing.[4] Details regarding the structure and system of the bio-
constructs that can be applied
3D printer will be described later, although it should be men-
to regenerative medicine for
tioned that the Kenzan method promotes cell interactions, and
cartilage damage, osteoarthri-
produces significant amounts of extracellular matrix (ECM). Us-
tis, bone fracture, meniscus
ing this method, a scaffold-free cell construct with a specific tis-
tear, tendon rupture, and liga-
sue structure and mechanical robustness can be produced. We
ment injury.
succeeded in providing the cells with an opportunity to organize
in three dimensions and to impart biological properties to the cell Kenichi Arai is also an assis-
construct using an additional culture after removing the “Ken- tant professor at the Center
zan.” Several reports regarding the production of a variety of cell for Regenerative Medicine Re-
constructs by the bio-3D printer using spheroids composed of search at Saga University. His
many types of cells and their subsequent contributions to tissue work is to develop a scaffold-
reconstruction have recently been presented. In this report, we free cardiac construct that
summarize the production and effectiveness of scaffold-free cell can be applied to regenerative
constructs prepared by the bio-3D printer that employs spheroids medicine and drug discovery.
as “bio-inks.” This work considers current problems, future is- His current research is mainly
sues, and future prospects to ensure that this printer will con- to verify the drug response by
tribute to further development in the area of 3D cell construc- analyzing the contractile force
tions. of fabricated cardiac constructs
when a model drug is added to
the construct.
2. Bio-3D Printing Using Spheroids as “Bio-Inks”
(The Kenzan Method) Koichi Nakayama is the chair-
man and professor at the Cen-
The equipment introduced here comprises each part of the bio-
ter for Regenerative Medicine
3D printer (Figure 1). A clean bench is used for the outer shell
Research at Saga University,
of the printer, and a worktable for the printer is installed in the
a board member of the Inter-
workspace. The printer worktable is broadly divided into two ar-
national Society for Biofabri-
eas, namely a management area and a printing area. The manage-
cation, and the co-founder of
ment area contains a plate management and transport unit, while
Cyfuse Biomedical K.K. He has
the printing area contains an image capture unit, a spheroid suc-
worked on research in ortho-
tion and placement unit, and a Kenzan holder stand (Figure 1).
pedic surgery and regenerative
A computer unit is installed under the workspace.
medicine. He invented bio-
3D printer, Regenova, and has
2.1. Equipment Required for the Kenzan Method pioneered scaffold-free bio-
fabrication. He leads research on regenerative medicine for
2.1.1. Plate Management and Transport Unit many kinds of diseases using scaffold-free cell constructs.

As the plate management and transport unit of the bio-3D

printer (Figure 1), a storage magazine collects up to ten spheroid-
containing multiwell plates, and a waste magazine is provided for
the spheroid-inspected and used plates (Figure 1). The plates set
in the storage magazine are automatically taken out by the plate
carrier and carried to the printing area (Figure 1).
2.1.2. Image Capture Unit and Image Analysis System
In the image capture unit, two cameras (upper and lower) are
installed in the printing area (Figure 1). The tops of the micronee-
dles in the Kenzan holder are captured using the upper camera,
while the spheroids in the plate wells and the tip of the spheroid
suction nozzle are captured with the lower camera (Figure 1).
The photographic images obtained using the two cameras are
analyzed by an image analysis system to confirm the positions of
the microneedles in the Kenzan, to measure the size and shape
of the spheroids to evaluate the quality and properties of the
spheroid before printing, and to check the condition of the noz-
zle tip (Figure 1). Additionally, the smoothness of the spheroid
surface and spheroid density can be measured using the camera
system.[29,30]

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Figure 1. Equipment employed in bio-3D printer (Regenova) for the Kenzan method. This printer has a plate management and transport unit, an image
capture unit, a spheroid suction and placement unit, and Kenzan holder stand on the worktable. The plate management and transport unit has a storage
magazine, a waste magazine, and a plate carrier. The image capture unit has a upper camera and a lower camera. The spheroid suction and placement
unit has a 26-guage nozzle. The Kenzan holder stand is a stand for Kenzan holder which can fix Kenzan.

Figure 2. “Kenzan” types. Kenzan is a needle array and now four types are available. A) 9 × 9 of Kenzan was used for urinary bladder reconstruction in
rat,[72] liver-like construct for drug response test,[106] and tendon/ligament-like construct for in vitro study.[114] B) 9 × 9 (circle) of Kenzan was adopted
to peripheral nerve reconstruction in rat,[40] osteochondral reconstruction in minipig,[60] blood vessel-like construct for rat,[41] heat-like construct for in
vitro study,[30] trachea-like construct for rat,[85] and esophagus-like construct for rat.[92] C) 13 × 13 (circle) of Kenzan was employed for osteochondral
reconstruction in minipig.[60] D) 26 × 26 of Kenzan was applied to heart-like construct for rat[77] and liver-like construct for rat.[29] E) 34 × 34 (circle) of
Kenzan was applied to blood vessel reconstruction in minipig,[36] peripheral nerve reconstruction in dog,[43] and diaphragm reconstruction in rat.[42]

2.1.3. Spheroid Suction and Placement Unit 2.1.4. Kenzan

The spheroid suction and placement unit contain a 26-gauge
stainless-steel nozzle with an inner diameter of 250 µm and an
outer diameter of 460 µm (Figure 1). The nozzle with a needle
length of 14 mm and whose entire length is 30 mm is connected
to a compressor with a pipeline and is equipped with a bidirec-
tional air circulation system for inhalation and exhalation that
can be switched using negative and positive pressures. In addi-
tion, the nozzle is installed on a movable arm of the printer, which
is capable of 3D positioning control with microlevel accuracy. In
the printing area, the nozzle picks up spheroids one by one from
each well of a multiwell plate and places them onto the micronee-
dles of the Kenzan (Figure 1).
The most important device in the bio-3D printing system,
the Kenzan (Figure 1), is a needle array with 160 µm diame-
ter stainless-steel microneedles arranged at 400 µm intervals
(Figure 2). The design of the Kenzan requires spheroids of
≈600 µm diameter to be produced to ensure their direct contact
with one another. The larger number of cells and the larger
the spheroids, the lower the diffusion of oxygen and nutrients.
Therefore, it is paramount that the size of the spheroids, the
diameter of the needle, and the distance between the needles are
kept uniform in the Kenzan. Currently, four types of Kenzan are
available, i.e., 9 × 9 (Figure 2A,B), 13 × 13 (Figure 2C), 26 × 26
(Figure 2D), and 34 × 34 (Figure 2E).

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Figure 3. Spheroids formation as bio-inks and Scaffold-free cell construct fabrication using Kenzan method. A,B) Spheroids are prepared with only cells.
A) Three types of cells are prepared for spheroid formation. B) Spheroid was formed and release extracellular matrix. C–G) Spheroids are employed to
fabricate scaffold-free cell construct. C) Kenzan are prepared for bio-3D printing. D) Spheroids are skewered onto Kenzan automatically using bio-3D
printer. E) Spheroids are cultured on the microneedles of Kenzan to fuse with each other. F) Scaffold-free cell construct is retrieved from Kenzan. G) Cell
construct are cultured on tube for further maturation.
2.1.5. Kenzan Holder
The Kenzan holder is a container that can fix the Kenzan in place
(Figure 1). It should be filled with a transparent medium or phos-
phate buffer saline and installed on the Kenzan holder stand (Fig-
ure 1).
2.1.6. Computer Unit
The computer unit controls all the operations for the dynamic
unit. The above-described image analysis system is also a part of
this unit. The most important role of this unit is to coordinate
a series of printing operations. Owing to this role, it is possible
to place the spheroids at any position on the Kenzan quickly and
accurately according to the 3D design produced previously us-
ing dedicated computer design software (Bio 3D Designer; Cy-
fuse Biomedical K. K., Tokyo, Japan).
2.2. Spheroids and Other Materials for the Kenzan Method
2.2.1. Spheroids as “Bio-Inks”
The properties of cell constructs produced by bio-3D printing are
highly dependent on the quality of the spheroids employed as the
“bio-inks” (Figure 3). Spheroids are generally pre-prepared with
only cells using a nonadhesive round-bottomed multiwell tissue
culture plate (Figure 3A,B). As mentioned above, since the dis-
tance between the Kenzan needles is set to 400 µm in the bio-3D
printer, it is recommended to prepare spheroids with a diame-
ter of ≈600 µm. The number of cells necessary to satisfy the re-
quired spheroid size varies greatly depending on the cell type.
Additionally, the spheroid shape is related to the quality of the
spheroid, the smoothness of the spheroid surface being a criti-
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cal component of quality. Furthermore, perhaps the most impor-
tant condition is the spheroid density, since appropriate physio-
logical conditions (i.e., the diffusion of oxygen and metabolites)
are necessary for the survival of cells inside the spheroid. Thus,
spheroid formation involves a range of parameters, including
the cell size, cell cohesion, cell combination, culture period, and
medium composition. As such, these parameters must be opti-
mized for each cell type to produce suitable spheroids for bio-3D
printing.
2.2.2. Medium for Spheroid Formation
In most cases, the medium used for cell culture is continuously
adopted to spheroid formation. Depending on the properties of
the spheroids, the composition of the medium may be altered
by the addition of new growth factors or the removal of included
growth factors, while the ratio of the medium may be changed
if two or more types of medium are mixed. For example, fibrob-
last growth medium (FGM) is adopted for spheroid formation
when human fibroblasts are used, and FGM and endothelial cell
growth medium (EGM) are adopted when human fibroblasts and
human vascular endothelial cells are used. If the spheroids are
not properly formed, it is not possible to proceed to the subse-
quent cell construct production, and so optimal spheroid growth
conditions are critical to bio-3D printing.
2.3. Scaffold-Free Cell Construct Prepared Using the Kenzan
Method
2.3.1. Medium and Bioreactor for the Cell Construct
Similar to spheroid formation, an appropriate medium is also
required for cell construct fabrication. Depending on the quality
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Figure 4. Bio-3D constructs for the in vivo promotion of tissue (re-)construction. A) Recipient-derived vascular endothelial cells and vascular smooth
muscle cells migrated into the construct in constructed blood vessel.[36] B) Nerves were continuously connected, functional neurons with myelinated
axons and schwann cells were observed in reconstructed peripheral nerve.[40,43] C) Recipient-derived muscular structures and networks of blood vessels
and nerves were confirmed in reconstructed diaphragm.[42]
of the construct, it is necessary to reconsider an appropriate
medium to obtain a construct with the desired characteristics. In
our experience, there seems to be a tendency that cell constructs
exhibiting the desired qualities can be produced using a medium
in which the spheroids are properly formed. In addition, a cir-
culating culture enables an efficient supply of a medium to the
spheroids, thereby promoting the formation of the desired cell
construct compared with the case where a stationary culture
is used. For this reason, circulating cultures are commonly
employed in the preparation of cell constructs using bioreactors
immediately after bio-3D printing.
2.3.2. Scaffold-Free Cell Construct Fabrication
Spheroids are skewered onto the Kenzan automatically using
bio-3D printer (Figure 3C,D) and cultured on the micronee-
dles for several days until they have fused to each other (Fig-
ure 3D,E). The cells that make up the spheroids move to the
gap between spheroids, creating a construct from the fusion of
many spheroids with their neighbors.[31] After the spheroids fuse,
they are retrieved as a scaffold-free cell construct (Figure 3E–G)
by sliding a preinstalled removal plate in the Kenzan. The cell
construct is usually cultured in a tube for several more days for
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further maturation (Figure 3G). The cell construct produced by
bio-3D printing is not only required to exhibit mechanical prop-
erties for withstanding subsequent surgical operations and su-
turing, but it is also required to possess the necessary biologi-
cal characteristics to ensure that it exhibits the desired function
in vivo and/or in vitro. It is necessary to determine the param-
eters required for spheroid formation and construct fabrication
by conducting a detailed evaluation of the characteristics of these
components. Examples, whereby the effectiveness of spheroids
prepared from various cell types using a bio-3D printer and sub-
sequent transplant into animals have been reported, are pre-
sented in Sections 3–5. We also note that only one study has fo-
cused on the evaluation of the properties of a cell construct for
application to a drug response test, and this is also introduced in
Section 5.
3. Bio-3D Construct to Promote Tissue
(Re-)Construction In Vivo
In this section, tubular cell constructs using fibroblasts, which
function as a foundation and conduit for the migration of recip-
ient cells around the implant site, are introduced using the ex-
amples of blood vessel construction, nerve reconstruction, and
diaphragm (re-)construction (Figure 4).
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Table 1. Fabrication conditions for bio-3D constructs to promote tissue reconstruction in vivo.

Target tissue Cell source Cell number of a Kenzan type Construct Medium Culture period for spheroid and
(animal) [Ref ] [%] spheroid/spheroid size shape/size construct/medium circulation flow rate

Blood vessel hDFs (100) N.D. 34 × 34 (circle) Tubular N.D. Spheroid; 1 day
(minipig) [36] 730 ± 43 µm T; 0.5 mm Construct on Kenzan; <1 week
D; 5 mm Construct on tube; several weeks
L; 10 mm N.D.
Peripheral nerve hDFs (100) 3.0 × 104 cells 9 × 9 (circle) Tubular FGM Spheroid; 1 day
(rat) [40] 750 ± 50 µm T; 0.5 mm Construct on Kenzan; 1 week
D; 2 mm Construct on tube; N.D.
L; 8 mm N.D.
Peripheral nerve cDFs (100) 1.0 × 104 cells 34 × 34 (circle) Tubular FGM + Spheroid; 2–3 days
(dog) [43] 550 ± 50 µm T; 0.5 mm 10% FBS Construct on Kenzan; 1 week
D; 5 mm Construct on tube; N.D.
L; 8 mm N.D.
a)
Diaphragm hDFs (90) 3.0 × 104 cells 34 × 34 (circle) Tabular Combined Spheroid; 1–2 days
b)
(rat) [42] hUVECs (10) 662 ± 52 µm (rectangle) medium Construct on Kenzan; 1 week
T; 0.5 mm #1 Construct on Tube; 3 weeks
V; 12 mm N.D.
H; 8 mm
a) The
tubular construct was cut to form a rectangular patch after removing the Kenzan; b) Combined culture medium #1; FGM and EGM with a ratio of 1:1. hDFs, human
dermal fibroblasts; cDFs, canine dermal fibroblasts; hUVECs, human umbilical vein endothelial cells; T, thickness; D, inner diameter; L, length; V, vertical; H, horizontal; FGM,
fibroblast growth medium; EGM, endothelial cell growth medium; FBS, fetal bovine serum; N.D., no data.

3.1. Blood Vessel Construction
Currently, artificial blood vessels are implanted as a treatment
method to replace broken or weakened blood vessels. However,
significant problems exist relating to thrombosis, infection, and
biocompatibility, among others, pose a significant problem for
current artificial vessel design, especially in the case of small-
diameter blood vessels.[32–35] The development of scaffold-free
cellular constructs for small diameter blood vessel reconstruc-
tion using the Kenzan-based bio-3D printing technology has been
examined by Itoh et al.[36] Spheroids consisting only of human
dermal fibroblasts (hDFs) were employed, and tubular cell con-
structs were prepared using a bio-3D printer (Table 1) prior to
xenotransplantion into the jugular veins of newly developed im-
munodeficient pigs.[36] It was found that adverse events, such as
immune rejection, were not observed over 3 months, and the
tubular construct was maintained, functioning as a blood flow
path.[36] Furthermore, several minipig-derived cells such as vas-
cular endothelial cells and vascular smooth muscle cells migrated
into the construct (Figure 4A).[36] Based on the results of this re-
search, it was concluded that the produced tubular cell constructs
might serve as blood conduits in minipigs, and the vascular struc-
ture may be maintained long-term due to self-organization by the
recipient-derived cells.[36]
The only cells used in the aforementioned experiment were
hDFs (Table 1),[36] and these cells generally produced an abun-
dant ECM mainly composed of collagen, thereby imparting
mechanical strength and elasticity to the cell constructs. The use
of hDFs was considered a major advantage in bio-3D printing for
blood vessel construction, and it accounted for the construct cul-
ture period being more than 3 weeks after printing (Table 1).[36]
In addition, hDFs are abundant just under the skin over the area
of the whole body, and so can be easily collected. This ease of
collection is also considered a great advantage for subsequent
clinical applications. However, the diameters of the spheroids
used in this experiment were very large (Table 1),[36] and thus,
the cells inside of the spheroids were expected to be necrotic. As
such, evaluation of the cell viability is considered necessary, as is
verification of the relationship between the cell viability and the
effect after implantation. In addition, depending on the conclu-
sion drawn following verification, it may be necessary to work on
the production of these cell constructs by reducing the number
of cells. Although the constructs produced in this experiment
did not exhibit any vascular structure, they could withstand
normal blood pressure and maintained a tubular structure for
3 months.[36] They were also able to self-repair a puncture hole
produced by a blood collection needle.[36] No description is
available regarding the composition of the medium used in this
study, and so it is possible that a special medium may have been
employed. In the future, it may be possible to evaluate further
the effectiveness and degradation of cell constructs by examining
the cell viability inside spheroids and conducting longer-term
animal experiments. Regardless, the ECM secreted from the cells
in the construct an important attribute for use as a cellular artifi-
cial blood vessel. As such, future clinical applications are eagerly
anticipated in lieu of mitigating necrosis inside the constructs.
3.2. Peripheral Nerve Reconstruction
If the nerve gap is too large after peripheral nerve injury, autol-
ogous nerve transplantation is currently considered the first-line
treatment.[37,38] However, numerous problems exist for this kind
of treatment, such as inconsistencies and neuroma formation.[39]
Therefore, Yurie et al. attempted to develop scaffold-free cel-
lular nerve conduits aimed at peripheral nerve reconstruction
using bio-3D printing.[40] Using spheroids consisting only of
hDFs, tubular cell constructs were prepared using a bio-3D

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printer (Table 1) and implanted at the sciatic nerve (5 mm nerve
defect) of immunodeficient rats as nerve conduits.[40] The per-
formed xenotransplantation resulted in confirmation that nerves
were continuously connected 8 weeks after implantation, and
functional neurons with myelinated axons were observed in all
areas of the nerve gap (Figure 4B). Overall, it was found that
transplanted tubular cell constructs could promote functional
nerve reconstruction in a rat sciatic nerve injury model.[40]
It should be noted that only hDF cells were used in the de-
scribed experiment whereby the physical stress on the nerve con-
duits depended on joint movement. Importantly, other cell types
promote axonal remodeling more efficiently, and it may be neces-
sary to evaluate the mechanical strength of the cell construct (e.g.,
the bending strength and flexibility) using not only hDFs, but also
other types of cells. [40] It has been suggested that cell construct
composed of only hDFs induce a greater blood supply compared
to existing nerve conduits prepared from artificial materials such
as silicon. Regardless of the cell type used, evaluation of the cell
viability is necessary due to the large diameters of the spheroids
used in this study, similarly to the vascular construction experi-
ments (Table 1).[40] It is conceivable that the cell constructs had
been rejected due to xenotransplantation, due to their visual re-
gression 8 weeks after implantation.[40] In addition, the 5 mm
nerve gap may not be long enough to allow evaluation of its effec-
tiveness as a nerve conduit, and so the longer construct should
be implanted to obtain a larger gap in the future. The medium
employed for the purpose of this experiment was the FGM;[40]
however, other studies have reported the use of a mixed medium
of FGM and vascular EGM to increase the strength and elastic-
ity of the hDF construct.[41,42] Use of mixed medium should be
considered for further experiments. Moreover, following review
of the types of cell for use in such experiments, it is expected that
the effectiveness and degradation mechanisms of the cell con-
structs may be evaluated by conducting animal experiments with
a larger nerve gap and a longer follow-up period.
Mitsuzawa et al. also evaluated the effectiveness of fibroblast
constructs as nerve conduits in large animals with a view to fu-
ture clinical applications based on the concept of Yurie et al.[40,43]
Using only canine skin fibroblasts (cDFs), a tubular cell con-
struct was prepared in a manner similar to that described by
Yurie et al. (Table 1), and was employed at the ulnar nerve section
(5 mm nerve defect) of a dog.[43] The ulnar nerve was regenerated
macroscopically and was still observed 10 weeks after transplan-
tation, with the presence of neurons and schwann cells numer-
ous myelinated axons being confirmed (Figure 4B).[43] This study
revealed that the implanted cell construct promoted functional
nerve repair in a canine ulnar nerve injury model, and its effec-
tiveness has been demonstrated in large animal experiments.[43]
Since only autologous cDF (Table 1) cells were used in this
experiment,[43] the possibility that the implanted construct had
been maintained in vivo without immune rejection was consid-
ered to be extremely high. Moreover, if spheroids and constructs
can be produced using human leukocyte antigen-adopted in-
duced pluripotent stem cells that are genetically modified to over-
express neurotrophic factors, they could yield useful implants for
application in clinical practice. It should be noted that macro-
scopic regression was not confirmed in the cell construct im-
planted autologously, and no significant changes were observed
even 10 months after implantation.[43] Additionally, no immune
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rejection or inflammatory reaction was observed during the 10
months. The culture medium used in this experiment was an
FGM containing fetal bovine serum (FBS), and a culture period
of 2–3 days was required for spheroid formation (Table 1).[43]
Notably, some difference was observed between the hDFs and
cDFs spheroid formation. In the future, we expect that cell con-
structs with optimal functions could be created as nerve conduits
through the preparation of optimized spheroids and constructs
after further detailed investigations into the different cell types.
3.3. Diaphragm Reconstruction
Currently, diaphragm reconstruction using an artificial patch
has been performed as a treatment for congenital diaphrag-
matic hernia.[44,45] However, artificial materials do not grow with
newborns, resulting in recurrent hernias and secondary chest
malformations.[46,47] Zhang et al. focused on the development of
a method to create scaffold-free cellular patches for diaphragm
reconstruction using bio-3D printing.[42] Tabular cell constructs
(square-shaped) were prepared using a bio-3D printer employ-
ing spheroids consisting of hDFs and human umbilical cord vas-
cular endothelial cells (hUVECs) (Table 1), and were xenotrans-
planted into the diaphragmatic resection (a rectangle defect of
12 × 10 mm) of immunodeficient rats.[42] The rats survived for
more than 710 days after implantation, and it was revealed that
the reconstructed diaphragm contained recipient-derived muscu-
lar structures in addition to networks of both blood vessels and
nerves (Figure 4C).[42] These results indicated that the tabular cell
construct had facilitated diaphragm reconstruction in the rat di-
aphragm hernia model, and could be considered a safe and effec-
tive treatment strategy for diaphragmatic hernias.[42]
Although hDF and hUVEC cells were used in this experi-
ment (Table 1), human umbilical fibroblasts should be employed
for spheroid formation in further studies since they can be eas-
ily collected as autologous cells from newborn patients.[42] In
addition, the cell construct produced in this experiment exhib-
ited a structure in which a complex microvascular network was
formed in collagen secreted from hDFs.[42] Since hUVECs can se-
crete several angiogenesis-promoting factors, such as the vascu-
lar endothelial growth factor (VEGF) for paracrine effects,[48–50]
angiogenesis is promoted not only in the constructs but also
in the surrounding tissues before and after implantation. My-
oblasts have also been shown to promote the reorganization of
microvessels[51–53] and can be considered promising candidates
for future use. For this experiment, a mixed medium of FGM
and EGM was employed (Table 1), which provides strength and
elasticity to the hDF-based cell construct. In the future, we expect
that a clinical-grade patch for diaphragmatic hernia reconstruc-
tion could be produced by examining the cell types in more detail
and working on the production of larger cell constructs.
4. Bio-3D Construct for Tissue Reconstruction by
In Vivo Differentiation
In this section, various shapes of cell constructs were introduced
for the purpose of osteochondral and bladder reconstructions
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Figure 5. Bio-3D constructs for in vivo differentiation during tissue reconstruction. Tissue reconstruction by differentiation of bio-3D construct. A)
Mesenchymal stem cells (MSCs) differentiated to chondrocytes and osteocytes, and then regeneration of articular cartilage in surface layer and formation
of subchondral bone in deep layer were observed at the implanted site.[60] B) MSCs differentiated to smooth muscle cells, and blood vessels were
extended from the recipient tissue to the implanted site in reconstructed urinary bladder.[72]

Table 2. Fabrication conditions for the bio-3D constructs for in vivo differentiation.

Target tissue Cell source Cell number of a Kenzan type Construct Medium Culture period for spheroid and
(animal) [Ref ] [%] spheroid/spheroid size shape/size construct/medium circulation flow rate

Osteocartilage sAT-SCs 1.0 × 104 cells 13 × 13 (circle) Tubular a) Combined Spheroid; 1 day
a)
(minipig) [60] (100) T; 1.5 mm medium #2 Construct on Kenzan; 1 week
D; 2 mm Construct on tube; N/A
L; 4 mm N.D.
≈550 µm 9 × 9 (circle) Tubular
T; 1.5 mm
D; 0.5 mm
L; 4 mm
Urinary bladder rBM-SCs 4.0 × 104 cells 9×9 Tabular (square) DMEM + Spheroid; 2–4 days
(rat) [72] (100) N.D. T; 1 mm 10% FBS Construct on Kenzan; 1 week
V; 3 mm Construct on Tube; N/A
H; 3 mm
a) Combined culture medium #2; Xeno-free MSC culture medium and serum-free MSC culture medium with a ratio of 1:1. sAT-SCs, swine adipose tissue-derived stem cells;

rBM-SCs, rat bone marrow derived stem cells; T, thickness; D, inner diameter; L, length; V, vertical; H, horizontal; DMEM, Dulbecco’s modified eagle’s medium; FBS, fetal
bovine serum; N/A, not applicable; N.D., no data.

(Figure 5). Specifically, the constructs were prepared using mes-
enchymal stem cells and differentiated into the appropriate tis-
sues according to the surrounding environments at the im-
planted sites.
4.1. Osteochondral Reconstruction
Osteoarthritis (OA) is a major joint disease that causes middle-
aged and old-aged movement disorders.[54–57] Currently, osteo-
chondral reconstruction using the mosaicplasty method is per-
formed to treat OA, although issues related to the damage of
sound and limited cartilage restrict its use.[58,59] Yamasaki et al.
attempted to develop scaffold-free stem cell-based constructs for
bone and cartilage reconstruction in knee joints using bio-3D
printing.[60] Employing a spheroid consisting only of swine adi-
pose tissue-derived stem cells (sAT-SCs), large and small tubular
cell constructs were prepared using a bio-3D printer (Table 2).[60]
Subsequently, the two different types of constructs were im-
planted into the osteochondral resection site (a cylindrical shape
with a diameter of 5.2 mm) created at the femoral trochlear
groove in minipigs, whereby the small construct was inserted
into the large one.[60] Indeed, the regeneration of articular car-
tilage was observed in the surface layer of the implanted site 6
months after implantation, and the formation of subchondral
bone was confirmed in the deep layer of the site (Figure 5A).[60]

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Two large and small tubular constructs were found to contribute
directly to osteochondral reconstruction in the knee joints of
minipigs.[60]
Although sAT-SCs cells were employed for this study (Table 2),
other stem cells with higher chondrogenic differentiation abili-
ties have been reported,[61] and thus, other types of mesenchymal
stem cells may be considered potential cell sources for spheroids.
In addition, through the mixing of differentiated cells such as
chondrocytes, osteoblasts, and osteoclasts, osteochondral regen-
eration could potentially be achieved at an earlier stage. The cell
constructs produced in this experiment were filled with connec-
tive tissue consisting of abundant type I collagen[60] suggesting
that mesenchymal stem cells contributed toward improving the
strength and elasticity of the construct and the fibroblasts. In
addition, filling a cylindrical osteochondral defect by inserting a
small construct into a large one can be considered a unique im-
plantation method. However, it has not been confirmed to what
extent sAT-SCs in the construct maintained undifferentiation im-
mediately prior to implantation, and so further verification is re-
quired. Although the mixed medium used in this experiment has
not been reported for use in other research (Table 2), it could be
more widely applicable since it resulted in the production of a
construct with suitable strength and elasticity following a short-
term culture. By analyzing the components of these two media
and the properties of the cell construct in more detail, new knowl-
edge was obtained regarding the production of abundant collagen
and the maintenance of cell undifferentiation in the construct.
Soon, it will be possible to produce cell constructs exhibiting op-
timal functions as implants for osteochondral reconstruction by
examining the cell types involved, in addition to spheroid forma-
tion. We predict larger osteochondral defect models will be used
to determine the mechanism of articular cartilage regeneration
and subchondral bone formation as a function of the regenera-
tion process over time.
The use of spheroids consisting only of adipose-derived stem
cells, which are inoculated into a cylindrical mold to create
a scaffold-free cylindrical cell construct for osteochondral and
meniscal regeneration, has also been published.[60–66] While bio-
3D printing technology is not used in these studies, they may be
helpful in informing the use of bio-3D printers for locomotive
organ reconstruction.
4.2. Urinary Bladder Reconstruction
The urinary bladder has been often affected by the side effects
of pelvic radiotherapy[67,68] but there are only a few effec-
tive treatments for this radiotherapy-induced urinary bladder
damage.[69–71] Therefore, Imamura et al. worked to develop
scaffold-free stem cell-based constructs for the purpose of blad-
der wall reconstruction using the bio-3D printing technology.[72]
Using tabular cell constructs prepared by a bio-3D printer based
on spheroids consisting of only rat bone marrow derived stem
cells (rBM-SCs) (Table 2),[72] cell constructs were allografted onto
a 5 mm incision site of the radiation damage area created in the
anterior wall of the rat bladder.[72] Results revealed that the im-
planted constructs were maintained in the animal body even after
4 weeks of implantation, the rBM-SCs were differentiated into
smooth muscle cells, and blood vessels were extended from the
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recipient tissue to the construct (Figure 5B).[72] Furthermore, the
rats showed an improvement in urinary symptoms, decreased
residual urine volumes, and regular urination 4 weeks after
implantation. Overall, the tabular constructs contributed directly
to bladder wall reconstruction in the rat bladder injury model.[72]
Although only rBM-SC cells were used for the purpose of this
experiment (Table 2), it would be expected that by mixing other
types of cells, such as vascular endothelial cells and smooth mus-
cle cells, bladder wall regeneration could be achieved at an earlier
stage.[72] However, it was found that the culture for spheroid for-
mation was unstable after 2–4 days (Table 2), and the size of the
cell construct was reduced from 5 to ≈3 mm due to cultivation on
the Kenzan.[72] Considering the unstable conditions of spheroid
formation and construct fabrication, it may be possible that gen-
eral cell culture medium was not suitable. This highlights the
importance of verifying the best growth medium and possible
development of an original medium that is optimal for produc-
ing the desired cell construct. Optimization of medium nutrients
is predicted to produce more ideal cell constructs for bladder wall
regeneration.
5. Bio-3D Construct to Function In Vivo as a
Constructed Tissue In Vitro
The constituent cells and/or other accessory cells (vascular en-
dothelial cells, smooth muscle cells, fibroblasts, and/or mes-
enchymal stem cells) of various tissues have been used to fab-
ricate cell constructs that mimic tissues such as blood vessels,
the heart, trachea, esophagus, liver, and tendons. In this section,
examples of tissue reconstruction upon the implantation of such
constructs are introduced (Figure 6), and an example of a liver-
like tissue construct is evaluated as a useful tool in drug response
tests.
5.1. Blood Vessel Reconstruction Using Blood Vessel-Like
Constructs
As mentioned in Section 3.1 (blood vessel construction), numer-
ous problems exist regarding the implantation of small-diameter
artificial blood vessels.[32–35] As such, Itoh et al. worked to
develop a small-diameter blood vessel-like construct for the
purpose of blood vessel reconstruction using the bio-3D printing
technology.[41] Spheroids composed of hDFs, hUVECs, and
human aortic smooth muscle cells (hASMCs) were used to cre-
ate tubular cell constructs (Table 3), and these constructs were
xenoimplanted into the abdominal aorta of immunodeficient
rats through end-to-end anastomosis.[41] It was found that no
thrombosis or blood vessel-like tissue reconstruction (i.e., expan-
sion of the luminal area and thinning of the wall) were observed
5 days after implantation (Figure 6A).[41] In addition, donor-
derived vascular endothelial cells were observed in the luminal
surface, and reorganized tissue was also confirmed (Figure 6A).
The tubular blood vessel-like construct appears to be useful for
the reconstruction of the damaged aorta in the rat aorta injury
model.[41]
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Figure 6. Bio-3D constructs that function in vivo as tissues constructed in vitro. Tissue reconstruction by functional bio-3D construct. A) Donor-derived
vascular endothelial cells were observed in the luminal surface, and donor-derived smooth muscle cells and reorganized tissue was also confirmed
in reconstructed blood vessel.[41] B) Recipient-derived blood vessels and cardiomyocytes were observed in heart-like construct.[77] C) Blood supply
from recipient and cartilage formation was observed in trachea-like construct.[85] D) Recipient-derived esophageal epithelial cells covered the lumen
of esophagus-like constructs.[92] E) Blood vessels consisted of donor-derived endotherial cells and bile duct from recipient were observed in liver-like
construct.[106]

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Table 3. Fabrication conditions for the bio-3D constructs to function in vivo.

Target tissue Cell source [%] Cell number of a Kenzan type Construct Medium Culture period for spheroid and
(animal) [Ref ] spheroid/spheroid size shape/size construct/medium circulation flow rate

Blood vessel hDFs (50) 2.5 × 104 cells 9 × 9 (circle) Tubular Combined Spheroid; 1 day
a)
(rat) [41] hUVECs (40) 615 ± 51 µm T; 0.5 mm medium Construct on Kenzan; 4 days
hASMCs (10) D; 1.5 mm #3 Construct on tube; 4 days
L; 7 mm First 2 days on tube; 2 mL min−1
Last 2days on tube; 4 mL min−1
Blood vessel hiPSCs-SMs (91) 2.5 × 104 cells N.D. N.D. EGM Spheroid; 1 day
(N/A) [74] hiPSCs-ECs (9) ≈500 µm N.D. Construct on Kenzan; 1 week
Construct on tube; N/A
1 week on Kenzan; 4 mL min−1
Heart hiPSCs-CMs (70) 3.3 × 104 cells 26 × 26 Tabular (square) RPMI/B-27 Spheroid; 3 days
(rat) [77] hVCFs (15) 505 ± 31 µm T; 0.5 mm Construct on Kenzan; 3 days
hUVECs (15) V; 3 mm Construct on dish; 3 days
H; 3 mm 3 days on dish; 150 rounds min−1
Heart hiPSCs-CMs (50) 3.5 × 104 cells 9 × 9 (circle) Tubular Combined Spheroid; 1 week
b)
(N/A) [30] hDFs (25) ≈600 µm T; 0.5–1 mm medium Construct on Kenzan; 1 week
hUVECs (25) D; 1.5 mm #4 Construct on tube; 1 week
L; 2 mm N.D.
Trachea rRCs (70) 4.0 × 104 cells 9 × 9 (circle) Tubular Combined Spheroid; 3 days
c)
(rat) [85] rLVECs (20) N.D. T; 0.5 mm medium Construct on Kenzan; 1 week
rBM-SCs (10) D; 2 mm #5 Construct on tube; 4 weeks
L; 5.5 mm 5 weeks; 3.3 mL min−1
Esophagus hDFs (50) 2.0 × 104 cells 9 × 9 (circle) Tubular N.D. Spheroid; 3 days
(rat) [92] hBM-SCs (30) N.D. T; 0.6 mm Construct on Kenzan; 1 week
hESMCs (20) D; N.D. Construct on Tube; 3 weeks
L; N.D. 4 weeks; 3.3 mL min−1
Liver hHs (50) 2.0 × 104 cells 26 × 26 Tabular (rectangle) Medium Spheroid; 2 days
d)
(rat) [29] hUVECs (35) N.D. T; 0.5 mm combination Construct on Kenzan; 3 days
hBM-SCs (15) V; 6 mm Construct on tube; N/A
H; 4 mm 3 days on Kenzan; 2 mL min−1
Liver hHs (50) 2.0 × 104 cells 9×9 Tabular (square) Combined Spheroid; 3 days
e)
(N/A) [106] mFs (50) N.D. (3 × 3 was used T; 0.5 mm medium Construct on Kenzan; 4 days
out of 9 × 9) V; 1 mm #6 Construct on tube; >2 weeks
H; 1 mm N.D.
Tendon hDFs (100) 2.5 × 104 cells 9×9 Tubular (ring) DMEM + Spheroid; 1.5 days
(N/A) [114] 500–600 µm T; 0.5 mm 10% FBS Construct on Kenzan; 8 days
D; 4.5 mm Construct in stress; 8 weeks
L; 1 mm N.D.

a) Combined culture medium #3; EGM, SMGM, and FGM with a ratio of 1:1:1; b) Combined culture medium #4; iCMM, EGM, and FGM with a ratio of 2:1:1; c) Combined
cultra medium #5; CGM and EGM with a ratio of 1:1; d) Medium combination; The medium was changed for spheroid formation and construct fabrication. For detailed
information, please review in ref. [29]; e) Combined culture medium #6; WME and DMEM + PHMS + FBS. hDFs, human dermal fibroblasts; hUVECs, human umbilical vein
endothelial cells; hASMCs, human aortic smooth muscle cells; hiPSCs-SMs, human induced pluripotent stem cell-derived smooth muscle cells; hiPSCs-ECs, human induced
pluripotent stem cell-derived endothelial cells; hiPSCs-CMs, human induced pluripotent stem cell-derived cardiomyocytes; hVCFs, human ventricular cardiac fibroblasts;
rRCs, rat rib chondrocytes; rLVECs, rat lung microvessel endotherial cells; rBM-SCs, rat bone marrow derived stem cells; hESMCs, human esophagus smooth muscle cells;
hBM-SCs, human bone marrow derived stem cells; hHs, human hepatocytes; mFs, mouse fibroblasts; T, thickness; D, inner diameter; L, length; V, vertical; H, horizontal;
EGM, endothelial cell growth medium; SMGM, smooth muscle cell growth medium; FGM, fibroblast growth medium; RPMI/B-27, Roswell Park Memorial Institute cell media
supplemented with B-27; iCMM, iCell maintenance medium; CGM, chondrocyte growth medium; WME, Williams’s medium E; DMEM, Dulbecco’s modified eagle’s medium;
PHMS, primary hepatocyte maintenance supplements; FBS, fetal bovine serum; N/A, not applicable; N.D., no data.

To create the tubular cell construct, hDF, hUVEC, and hASMC
cells were employed, resulting in abundant collagen production
mainly from fibroblasts (Table 3).[41] ECM contributes to cell
organization within the construct, in addition to improving the
strength and elasticity of the cell construct. Previous reports
prepare tubular cell constructs using a 1:5:14 ratio of hDFs to
human aortic endothelial cells and hASMCs.[73] In contrast, Itoh
et al. used a much higher ratio of hUVECs in the spheroids
(Table 3),[41] suggesting that the construct formation was pro-
moted by endothelialization. The resulting constructs had
endothelial cells on the surface of the lumen after implantation
instead of recipient-derived endothelial cells. However, due to
the absence of elastic fibers in the construct after implantation,
it was considered necessary to increase the cell ratio of hASMCs
and/or to reexamine the culture conditions during the construct
maturation process. Itoh et al. used a mixture of cell culture
media to support all three cell types (Table 3),[41] including EGM
and FGM. In terms of the circulating culture employed for

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the maturation of the cell constructs, the bioreactor flow rate
was varied in two stages (Table 3).[41] Therefore, constructs ex-
hibiting good mechanical strength and elasticity were produced
despite the short culture period.[41] Future work should focus
on the creation of larger cell constructs and a more detailed
examination of the cell types and ratios. Additional studies in
larger experimental animal models with long-term assessment
of durability, patency, and antithrombogenicity should also be
attempted before verification at the clinical stage.
In another example, Moldovan et al. created a spheroid
consisting of human-induced pluripotent stem cell-derived
smooth muscle-forming cells (iPSCs-SMFCs) and human-
induced pluripotent stem cell-derived vascular endothelial
colony-forming cells (iPSCs-ECFCs) to a prepare tubular cell
construct for blood vessel reconstruction using a bio-3D printer
(Table 3).[74] The resulting construct presented a layered dis-
tribution of alpha smooth muscle actin (𝛼-SMA)-positive cells
and extracellular matrix deposits.[74] The iPSC-derived vascular
constituent cells were promising cellular components for the
production of vascular-like tissues.[74] In the future, it will
be necessary to evaluate the effectiveness of the construct in
vivo using vascular injury animal models after assessing the
mechanical strength of the construct.
5.2. Heart Construction Using Heart-Like Constructs
Current therapies for heart failure only relieve symptoms and
slightly extend the patient lifespan, with no cure having yet been
established to reconstruct functional heart tissue.[75,76] As such,
Ong et al. focused on the development of heart-like constructs
to reconstruct failing hearts using bio-3D printing technology.[77]
Tabular cell constructs were fabricated using spheroids con-
sisting of human-induced pluripotent stem cell-derived car-
diomyocytes (hiPSC-CMs), human ventricular cardiac fibrob-
lasts (hVCFs), and hUVECs (Table 3), and spontaneous pulsa-
tion of the construct was confirmed.[77] Following xenoimplanta-
tion onto the heart surface of immunodeficient rats, engraftment
and angiogenesis of the construct were confirmed one week after
implantation (Figure 6B).[77] Overall, the tabular cell constructs
were found to contribute directly to the construction of the rat
hearts.[77]
Three types of cells were used for the purpose of this experi-
ment, including hiPSC-CMs (Table 3).[77] Based on previous stud-
ies, spheroids cannot be formed using only hiPSC-CMs, and re-
quire co-culturing with fibroblasts (15% minimum) and vascular
endothelial cells.[77,78] Upon the application of this system, tabu-
lar cell constructs were observed 3 days after bio-3D printing.[77]
The resulting cell constructs were further cultured for an addi-
tional 3 days, after which the presence of ventricular myocyte-like
action potential waveforms in the construct and uniform electri-
cal conduction throughout the construct was identified.[77] Ad-
ditionally, angiogenesis was observed in the construct, and the
presence of connexin 43, a major gap junction protein in the ven-
tricular myocardium, was also observed at the cell boundary.[77]
Furthermore, >90% cell viability was maintained in the
construct.[77] Although the medium used in this experiment was
designed for general suspension cell culture (Table 3), no previ-
ous reports its use were found. Considering that the culture pe-
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riod was short compared to those of other studies, it may be pru-
dent for other groups to consider the use of this medium. How-
ever, it should be noted that a contractile force and conduction
velocity similar to those of ventricular myocardial tissue have not
yet been obtained,[77] and the construct size must be increased.
Future studies should focus on the maturation of larger cell con-
structs as ventricular myocardial tissues to demonstrate their ef-
fectiveness on a clinical scale. After in vitro optimization, long-
term in vivo experiments would provide information on durabil-
ity and immune response.
Arai et al. also reported the use of hiPSCs-CMs, hDFs, and
hUVECs to produce tubular cell constructs for heart reconstruc-
tion using a bio-3D printer (Table 3) and confirmed its self-
organization.[30] It was also shown that applying electrical stim-
ulation to the cell construct increases the beat rate, and upon
removal of the stimulation, the beat rate dropped to its initial
value.[30] These results support maturation of the tubular cell con-
struct into a functional heart-like tissue.[30] Future studies should
focus on the effectiveness of the constructs by implantation into
heart disease animal models after evaluating its mechanical prop-
erties and suture strength during the transplantation operation.
As an alternative application for the construct, its usefulness in a
drug response test should be considered.
Furthermore, the production of constructs without the use of
the bio-3D printing technology and myocardial spheroids has
been reported,[79,80] and such studies may be useful during the
preparation of myocardial constructs using bio-3D printers.
5.3. Tracheal Reconstruction Using Trachea-Like Constructs
Currently, airway stenosis caused by various diseases is treated by
costal cartilage transplantation. However, this treatment method
has a number of issues, and a standardized method for clinical
treatment has yet to be established.[81–84] Recently, Taniguchi et al.
developed a trachea-like construct for the purpose of tracheal re-
construction using bio-3D printing technology.[85] Tubular cell
constructs were fabricated by a bio-3D printer using spheroids
consisting of rat rib chondrocytes (rRCs), rat lung microvascu-
lar endothelial cells (rLVECs), and rBM-SCs (Table 3).[85] Con-
structs containing silicone stents were allografted to the excised
part of three tracheal cartilages prepared in rat trachea.[85] As a
result, the cell constructs were maintained at the transplant sites
even 23 days after implantation with the support of the silicon
stent.[85] Moreover, the recipient blood supply produced by an-
giogenesis was observed in the construct, and reorganization of
the construct into tracheal tissue was also detected (Figure 6C).[85]
These results indicated that the tubular cell construct contributed
directly to the reconstruction of tracheal tissue in a rat tracheal
injury model and was useful as a tracheal graft.[85]
Consistent blood supply has been cited as the main challenge
for tracheal reconstruction.[81,86] A recent study reported the
maintenance of functional vasculature a long period of time
when mesenchymal stem cells are transplanted with endothe-
lial cells.[87] Spheroids consisting of rLVECs and rBM-SCs
have also been shown to avoid ischemia of the construct after
implantation,[85] which is considered a particularly important
development in this field of research. The cell constructs ob-
tained from these spheroids presented cartilage formation and
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angiogenesis after 4 weeks of culture, and showed maturation
as a tracheal graft.[85] Furthermore, the maximum amount
of cartilage tissue was observed in the construct 23 days af-
ter implantation, and no acute rejection was seen without
immunosuppression.[85] However, inappropriate granulation
tissue formation was observed after stent implantation, and
complete epithelialization was not verified.[85] Considering these
issues, stent removal may be necessary for long-term follow-up
in the future. We also note that the culture medium used in this
experiment was a 1:1 mixture of growth media for chondrocytes
and endothelial cells (Table 3).[85] The culture parameters for
the spheroids and cell constructs employed in this experiment,
including the flow rate of the circulating culture and the cul-
ture period, were optimized in previous studies (Table 3). In
long-term follow-up studies after implantation, stent removal
may be required, and observation of epithelialization should
be performed in addition to a detailed analysis of cartilage for-
mation and vascularization in the reconstructed tracheal tissue.
Moreover, implant experiments using large animal models will
be necessary to demonstrate clinical grade efficacy.
In 2019, Macino et al. created cartilage spheroids consisting of
human articular chondrocytes, hUVECs, and human bone mar-
row derived stem cells (hBM-SCs), as well as fibrous spheroids
consisting of UVECs, hBM-SCs, and hDFs.[88] The two types of
spheroids were alternately stacked one by one onto a Kenzan to
produce a trachea-like construct.[86] Xenotransplantation with a
silicon stent was performed on the excised part of four tracheal
cartilages prepared in the trachea of immunodeficient rats.[88]
However, the goal of this study has yet to be reached,[88] with ex-
amination of the cells and culture methods required to obtain a
matured trachea-like construct being necessary.
5.4. Esophageal Construction Using Esophagus-Like Constructs
Currently, reconstruction of the esophagus is often required
when an esophagectomy is performed for various esophageal
diseases, but numerous problems exist, including the possi-
bility of complications and a high mortality rate.[88–91] To ad-
dress these issues, Takeoka et al. focused on the development
of esophagus-like constructs for esophageal reconstruction using
bio-3D printing technology.[92] Spheroids consisting of hDFs, hu-
man esophageal smooth muscle cells (hESMCs), and hBM-SCs
were prepared, and tubular cell constructs were fabricated using
a bio-3D printer (Table 3).[92] The constructs containing a silicone
stent were allografted to bypass the incision made in a rat esopha-
gus and stomach.[92] Consequently, the cell constructs supported
by the silicon stent were confirmed to have been engrafted in
the rats 30 days after implantation, and it was also found that
food successfully passed through the tubular constructs.[92] In
addition, hESMCs were maintained in the construct, and the
recipient-derived esophageal epithelial cells had covered the lu-
men of the constructs (Figure 6D).[92] These results demonstrate
that the tubular cell constructs contributed directly to esophageal
construction in rat esophagus-gastric bypass models, and there-
fore exhibited promise for application as implants in esophagec-
tomy procedures.[92]
In the described experiment, the highest priority was given
to producing cell constructs with mechanical and suture reten-
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tion strength, with hDFs, hESMCs, and hBM-SCs being used in
a ratio optimized through prior studies (Table 3).[92] Although
the spheroids created in this experiment did not contain vas-
cular endothelial cells, which are known to play an important
role in angiogenesis, the cell construct strongly expressed 𝛼-
SMA and produced abundant elastic fibers, as well as abundant
VEGF due to the co-culture of hESMC and hBM-SCs.[92] In addi-
tion, it was confirmed that after implantation, the lumen of the
constructs had been covered with recipient-derived esophageal
epithelial cells even in the case of the inserted stents.[92] Fur-
thermore, the cell constructs exhibited resistance to gastric acid
stimulation since food residues were contained inside the cell
construct.[92] However, details regarding the medium employed
in this study are unknown, possibly suggesting a special medium
was used. Orthotopic implantation results were also obtained,
and ectopic implantation was the first step to further research
development.[92] Future work should focus on orthotopic implan-
tation and long-term tracking using a large animal model to rep-
resent a significant step toward clinical application.
5.5. Liver-Like Constructs for Liver Reconstruction and Drug
Response Tests
Orthotopic liver transplantation has been performed for half a
century as a treatment for end-stage liver disease, but the results
have been limited due to a lack of organ donors.[93] Therefore,
Yanagi et al. worked to develop liver-like constructs for the pur-
pose of liver reconstruction using bio-3D printing technology.[29]
Using spheroids consisting of human hepatocytes (hHs), hU-
VECs, and hBM-MSCs, tabular cell constructs were fabricated by
a bio-3D printer (Table 3).[29] The constructs were xenoimplanted
onto the hepatectomy site of immunodeficient rats. Blood vessels
consisting of donor-derived endothelial cells and bile duct from
the recipient were observed in the construct, which resulted in
the reconstruction of functional livers (Figure 6E).[29] The study
indicated that tabular cell constructs contributed directly to the
reconstruction of liver tissue in the rat liver injury models.[29]
Spheroids produced using only hHs are limited to a diame-
ter of 100–150 µm due to the restricted diffusion of oxygen and
nutrient supply to the core.[94–96] Using a combination of hU-
VECs and MSCs has been reported to promote angiogenesis by
releasing the VEGF and the hepatocyte growth factor (HGF) un-
der hypoxic conditions.[97,98] Spheroids for liver constructs were
therefore produced using three types of cells, namely hHs, hU-
VECs, and hBM-SCs (Table 3).[29] The hHs were situated on the
surface layer of the spheroids to avoid the hypoxic state, and the
hUVECs and hBM-SCs were located in the center as a result.[29]
The mixture of hUVECs and hBM-SCs with hHs was not suc-
cessful, indicated by declining liver function in the early stage
of spheroid formation,[29] resulting in the microvascular net-
work and bile duct structures failing to form in the construct. It
has been shown that recipient-derived microvascular networks
and bile duct-like structures may be formed in liver-like con-
structs after implantation.[29] However, it is important that the
structures of the blood vessels and bile ducts are formed in the
construct prior to implantation to ensure the fabrication of a
functioning liver. Other cell types should be considered, such
as hepatic progenitor cells, which can differentiate into both
© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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hepatocytes and bile duct cells,[99–101] vascular endothelial cells,
and hepatic stellate cells. In addition, different media were em-
ployed for spheroid formation and cell construct production (Ta-
ble 3), a previously unreported tactic. Future work should focus
on the detailed reexamination of cell types and culture conditions
to produce functional liver-like constructs. Long-term follow-up
studies should also be performed using large animal models.
Currently, no liver tissue model is available that can main-
tain liver function in vitro over a long period of time for use
in pharmaceutical drug research.[102–105] A model for the evalua-
tion of drug hepatotoxicities and metabolite production is there-
fore required. As such, Kizawa et al. focused on the development
of liver-like constructs for drug discovery research using bio-3D
printing technology.[106] More specifically, tabular cell constructs
were prepared by a bio-3D printer using spheroids consisting of
hHs and mFs (Table 3).[105] As a result, hepatic drug transporters
and metabolic enzymes were observed, and bile acid secretion
was confirmed.[106] Glucose production was also observed and
was actively suppressed by insulin.[106] Furthermore, bile ducts
and sinusoidal structures were found in the constructs, thereby
suggesting that bile acid secretion occurred through this sinu-
soidal hepatocyte-bile duct pathway.[106] As described above, such
tabular cell constructs are expected to be applicable for the de-
velopment of new in vitro liver tissue models since various liver
metabolic functions were exhibited over several weeks during the
course of this study.
5.6. Tendon and/or Ligament-Like Constructs
Currently, suture surgery or autologous tendon transplantation is
performed as treatments for tendon and ligament injuries. The
re-tear and re-operation rates are notably high, and a secondary
effective treatment method has yet to be established.[107–113] Thus,
Nakanishi et al. examined the development of tendon/ligament-
like constructs for the purpose of tendon/ligament reconstruc-
tion using the bio-3D printing technology.[114] A tubular (ring-
shaped) tendon/ligament-like construct was prepared by a bio-
3D printer using spheroids composed of hDFs (Table 3), and
cultured for 8 weeks in a static tensile environment in vitro.[113]
An increase in the collagen deposition in the cell construct was
observed, while the rearrangement of spindle-shaped cells and
the distribution of Tenascin C in the tensile direction were also
confirmed.[114] These results indicated that ring-shaped cell con-
structs were successfully remodeled in vitro and that they ma-
tured into tendon-like tissues.[114] Future studies should focus on
the evaluation of the mechanical strength of the cell construct
and the post-implant efficacy using animal tendon/ligament in-
jury models.
6. Conclusion
The use of scaffold-free bio- 3D printing using cell aggregates
(spheroids) as “bio-inks” has attracted growing attention for 3D
cell construction. More specifically, cell constructs have been pro-
duced by a bio-3D printer using spheroids composed of many
types of cells for application in tissue (re-)constructions. We
wished to summarize the production and effectiveness of vari-
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ous cell constructs produced by this means while considering the
future development of scaffold-free 3D cell constructions.
More specifically, upon considering a scaffold-free cell con-
struct produced by the Kenzan method using spheroids as “bio-
inks,” the reconstruction of blood vessels,[36] nerves,[40,43] and
diaphragms[42] was examined using cell structures that function
as a foundation and conduit for the migration of recipient cells to
the implanted site. Examples of osteochondral[60] and bladder[72]
reconstruction using various shapes of cell constructs were intro-
duced, whereby these were prepared using mesenchymal stem
cells and differentiated into appropriate tissues according to the
surrounding environment at the implanted sites. Furthermore,
we described cell constructs that mimic various tissues such
as the blood vessels,[41,74] heart,[30,77] trachea,[85] esophagus,[92]
liver,[29] and tendons,[114] and also introduced examples of tissue
reconstruction by implantation of the corresponding constructs.
In addition, the use of liver-like tissue constructs was evaluated
as a useful tool in drug response tests.[106] To prepare the desired
cell constructs, many parameters, such as the cell type, the cell
number of a spheroid, the spheroid size, the “Kenzan” tailored
to the study design, the media employed for spheroid formation
and construct fabrication, the culture period, and the flow veloc-
ity must be examined in detail.
In terms of blood vessel and peripheral nerve reconstruction,
the cell constructs were composed only of fibroblasts due to the
requirement for strength and elasticity immediately after implan-
tation (Table 1).[36,40,43] Although the tabular cell construct used
for diaphragm reconstruction was mainly based on fibroblasts,
it was mixed with ≈10% of vascular endothelial cells to promote
angiogenesis and supply oxygen and nutrients soon after implan-
tation (Table 1).[42] While angiogenesis was observed in the con-
struct, no vascularization data was available. If blood vessels form
soon after implantation, they can supply blood to the cell con-
struct, and making the addition of vascular endothelial cells to
the spheroid beneficial. With the addition of endothelial cells, the
influence of their ratio to fibroblasts on the strength and elasticity
of the cell construct must be considered, like in Zhang et al.[42]
In the reconstruction of osteocartilage and urinary bladder tis-
sue, the spheroids were composed only of mesenchymal stem
cells due to the difficulty of culturing differentiated cells and
producing the target tissues in vitro (Table 2).[60,72] Stem cells
were therefore used to produce pluripotent cell constructs for
differentiation according to the surrounding environment after
implantation.[60,72] Spheroids consisting only of induced pluripo-
tent stem cell-derived chondrocytes have been reported to con-
tribute cartilage regeneration in minipigs in only one month.[115]
However, it appeared that component cells from target tissues or
differentiated cells from stem cells might also be necessary.
In the construction of tissues in vitro (Table 3), the main
component cells in the target tissue are contained in spheroids
for the majority of constructs.[29,41,74,85,92,106] The presence of
vascular endothelial cells was also found to be important to
ensure oxygen and nutrient supplies soon after implantation
(Table 3).[29,30,41,74,77] In terms of the construction of blood
vessel-like tissues, vascular endothelial cells and smooth mus-
cle cells were employed (Table 3),[41,74] although further studies
are required in this area. In the construction of heart-like tis-
sues, iPS-derived cardiomyocytes, vascular endothelial cells, and
fibroblasts were used.[30,77] Due to the differences between
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cardiac fibroblasts and dermal fibroblasts, it was considered that
cardiac fibroblasts might be favorable for producing constructs
exhibiting a strong contractile force.,[116] Chondrocytes (70%),
vascular endothelial cells (20%), and mesenchymal stem cells
(10%) were adopted for trachea-like tissue constructs (Table 3),[85]
whereby the mesenchymal stem cells were expected to differen-
tiate into other component cells. Although an attempt to repro-
duce the construct with tracheal rings in which the two types of
spheroids were arranged in stripes has been reported,[88] addi-
tional post-implant data are required for evaluation in the pa-
rameters of construct fabrication. For the esophagus-like tissue
construction, fibroblasts (50%), mesenchymal stem cells (30%),
and smooth muscle cells (20%) were employed in a suitable ra-
tio to obtain high initial strength and elasticity, as well as the
blood vessel-like construct (Table 3).[41,92] Similarly, in the liver-
like tissue construct, hepatocytes (50%), vascular endothelial cells
(35%), and mesenchymal stem cells (15%) were applied using a
similar cell ratio to that employed for the tracheal-like tissue con-
struct (Table 3).[29,85] The construct that adjusts a suitable cell ra-
tio in order of main cells, vascular endothelial cells,[117] neuronal
cells,[118] and mesenchymal stem cells[119] may serve as a model
for tissue construction.[120] In contrast, although only fibroblasts
were used for the tendon-like tissue construction (Table 3),[114]
the cell numbers and spheroid sizes of this construct exhibited
similar trends to those of the constructs for blood vessel construc-
tion, peripheral nerve reconstruction, and diaphragm reconstruc-
tion (Table 1),[36,40,42] thereby indicating the reproducible nature
of the spheroids. Although a different medium was used for the
tendon-like tissue construct, no data was available regarding its
strength, and further measurements are required in the future.
Overall, we note that the cell number and spheroid size were
optimized along with the Kenzan design, whereby it was con-
sidered ideal for preparing spheroids with the recommended
size (i.e., 500–600 µm) regardless of the construct design.
Zhang et al., Ong et al., and Arai et al. have verified those
parameters[30,42,78] with high cell viability (98.6%) of spheroids
over 650 µm in diameter and remained high even in the con-
structs (>99.2%).[42] However, other groups have not reported
size dependent parameters in other studies. Future work should
focus on the cell viability of spheroids and constructs, the rela-
tionship of these factors to the effectiveness after implantation,
and the construct viability.
Due to limitations on spheroid size, the printer resolution
of the Kenzan method may appear to have lower resolution
when compared to other 3D bioprinters.[1–7] However, it has
been reported that constituent cells can migrate effectively in-
side the spheroids of the fused construct, reconstituting the
printed material.[29,30,41,74,77,85,92,106,114] This reconstitution of the
construct increases the resolution of the printer, arguably making
it more accessible to outside cells compared with scaffold based
methods.
In terms of the required Kenzan geometry, blood vessel, and
peripheral nerve reconstruction (Table 1)[36,40,43] should use a cir-
cular cross section to supply the construct with medium after
printing. Alternatively, the largest 34 × 34 Kenzan should be used
to design large cell constructs for clinical application.[36,43] In the
reconstruction of osteocartilage and bladder tissue (Table 2),[60,72]
and in the construction of tissue-like constructs in vitro (Table 3)
[29,30,41,74,77,85,92,114]
the Kenzan type was selected according to the
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design of the cell construct, the transplantation method, and the
size of the implantation site.
A construct wall designed using a single spheroid
layer tends to exhibit a thickness of ≈0.5 mm (Table 1
and 3).[29,30,36,40,41,43,77,85,92,106,114] However, since shrinkage
has been observed in the direction of the long axis depending
on the cells and the medium, the development of an appropriate
culture method is therefore required. The cause of the shrink-
age is presumably due to the intracellular actin filaments and
the extracellular intermediate filaments, but the details of this
phenomenon have yet to be elucidated.
For blood vessel construction and peripheral nerve reconstruc-
tion, FGM was used to produce cell constructs based on fi-
broblasts, while EGM promoted vascular endothelial cells (Ta-
ble 1).[40,42,43] EGM has been found to increase the strength and
elasticity of the construct composed of only fibroblasts. In addi-
tion, it was found that different culture periods were required
for spheroid formation using different cell types, and this often
depends on the cell compatibility with the medium. Although a
culture period of one week on the Kenzan is considered reason-
able, additional culture for several weeks is required to provide
strength and elasticity to the construct (Table 1).[36,40,42,43] Further
studies into the compatibility between the cells and the media
are necessary, including in the case of bladder reconstruction and
for the production of the cell constructs transplanted into the os-
teochondral defects, whereby a unique mixed medium was em-
ployed to give a shorter culture time (Table 2).[60,72] In the con-
struction of tissue-like constructs in vitro, it was confirmed that
spheroid formation was slower when larger numbers of cell types
were seeded (Table 3).[29,30,41,74,77,85,92,106,114] Regardless, the cul-
ture period on the Kenzan was short for all constructs (i.e., sev-
eral days to one week maximum), with the observed differences
being attributed to the high oxygen and nutrient requirements
inside the construct, rather than the strength and elasticity of the
construct.
Histology confirmed that recipient cells migrated from the
surrounding tissue to the fibroblast-based cell constructs of (re-
)constructed blood vessels, peripheral nerves, and diaphragms to
build the native tissue structure (Figure 4).[36,40,42,43] In the recon-
struction of osteocartilage and bladder tissue, mesenchymal stem
cell differentiation was observed in response to the surrounding
tissue of the implanted sites (Figure 5).[60,72] Lastly, spheroid con-
structs consisting of cells that constitute the target tissue were
important in the early stages after implantation and for in vitro
drug response tests (Figure 6).[29,41,77,85,92]
Finally, we note that detailed studies must be carried out in the
future to determine and optimize the above-mentioned parame-
ters with relative urgency. Research into additional target tissues
is also necessary to contribute to the ongoing development of re-
generative medicine.
Conflict of Interest
Co-author, K.N., is a co-founder and shareholder of Cyfuse Biomedical KK.
and an inventor/developer designated on the patent for the Bio-3D printer.
Patent title: Method for Production of 3D Structure of Cells; patent num-
ber: JP4517125. Patent title: Cell Structure Production Device; patent num-
ber: JP5896104. The other authors have declared that no competing inter-
ests exist.
© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.advancedsciencenews.com
Keywords
bio-3D printers, bio-ink, scaffold-free printing, spheroids, tissue recon-
struction
Received: December 20, 2019
Revised: February 21, 2020
Published online: May 6, 2020
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