Abstract

Recently, 3D bioprinting techniques have been broadly recognized as a promising tool to fabricate functional tissues and organs. The bioink used for 3D bioprinting consists of biological materials and cells. Because of the dominant gravitational force, the suspended cells in the bioink sediment resulting in the accumulation and aggregation of cells. This study primarily focuses on the quantification of cell sedimentation-induced cell aggregation during and after inkjet-based bioprinting. The major conclusions are summarized as follows: (1) as the printing time increases from 0 min to 60 min, the percentage of the cells forming cell aggregates at the bottom of the bioink reservoir increases significantly from 3.6% to 54.5%, indicating a severe cell aggregation challenge in 3D bioprinting, (2) during inkjet-based bioprinting, at the printing time of only 15 min, more than 80% of the cells within the nozzle have formed cell aggregates. Both the individual cells and cell aggregates tend to migrate to the vicinity of the nozzle centerline mainly due to the weak shear-thinning properties of the bioink, and (3) after the bioprinting process, the mean cell number per microsphere increases significantly from 0.38 to 1.05 as printing time increases from 0 min to 15 min. The maximum number of cells encapsulated within one microsphere is ten, and 29.8% of the microspheres with cells encapsulated have contained small or large cell aggregates at the printing time of 15 min.

References

1.
Zhang
,
B.
,
Luo
,
Y.
,
Ma
,
L.
,
Gao
,
L.
,
Li
,
Y.
,
Xue
,
Q.
,
Yang
,
H.
, and
Cui
,
Z.
,
2018
, “
3D Bioprinting: An Emerging Technology Full of Opportunities and Challenges
,”
Bio-Des. Manuf.
,
1
(
1
), pp.
2
13
.
2.
Chen
,
R.
,
Chang
,
R. C.
,
Tai
,
B.
,
Huang
,
Y.
,
Ozdoganlar
,
B.
,
Li
,
W.
, and
Shih
,
A.
,
2020
, “
Biomedical Manufacturing: A Review of the Emerging Research and Applications
,”
ASME J. Manuf. Sci. Eng.
,
142
(
11
), p.
110807
.
3.
Thakare
,
K.
,
Jerpseth
,
L.
,
Qin
,
H.
, and
Pei
,
Z.
,
2021
, “
Bioprinting Using Algae: Effects of Extrusion Pressure and Needle Diameter on Cell Quantity in Printed Samples
,”
ASME J. Manuf. Sci. Eng.
,
143
(
1
), p.
014501
.
4.
Li
,
J.
,
Chen
,
M.
,
Fan
,
X.
, and
Zhou
,
H.
,
2016
, “
Recent Advances in Bioprinting Techniques: Approaches, Applications, and Future Prospects
,”
J. Transl. Med.
,
14
(
1
), p.
271
.
5.
Gudapati
,
H.
,
Dey
,
M.
, and
Ozbolat
,
I.
,
2016
, “
A Comprehensive Review on Droplet-Based Bioprinting: Past, Present and Future
,”
Biomaterials
,
102
, pp.
20
42
.
6.
Li
,
X.
,
Liu
,
B.
,
Pei
,
B.
,
Chen
,
J.
,
Zhou
,
D.
,
Peng
,
J.
,
Zhang
,
X.
,
Jia
,
W.
, and
Xu
,
T.
,
2020
, “
Inkjet Bioprinting of Biomaterials
,”
Chem. Rev.
,
120
(
19
), pp.
10793
10833
.
7.
Nakamura
,
M.
,
Kobayashi
,
A.
,
Takagi
,
F.
,
Watanabe
,
A.
,
Hiruma
,
Y.
,
Ohuchi
,
K.
,
Iwasaki
,
Y.
,
Horie
,
M.
,
Morita
,
I.
, and
Takatani
,
S.
,
2005
, “
Biocompatible Inkjet Printing Technique for Designed Seeding of Individual Living Cells
,”
Tissue Eng.
,
11
(
11–12
), pp.
1658
1666
.
8.
Gungor-Ozkerim
,
P. S.
,
Inci
,
I.
,
Zhang
,
Y. S.
,
Khademhosseini
,
A.
, and
Dokmeci
,
M. R.
,
2018
, “
Bioinks for 3D Bioprinting: An Overview
,”
Biomater. Sci.
,
6
(
5
), pp.
915
946
.
9.
Wang
,
S.
,
Lee
,
J. M.
, and
Yeong
,
W. Y.
,
2015
, “
Smart Hydrogels for 3D Bioprinting
,”
Int. J. Bioprinting
,
1
(
1
), pp.
3
14
.
10.
Lee
,
A.
,
Hudson
,
A.
,
Shiwarski
,
D.
,
Tashman
,
J.
,
Hinton
,
T.
,
Yerneni
,
S.
,
Bliley
,
J.
,
Campbell
,
P.
, and
Feinberg
,
A.
,
2019
, “
3D Bioprinting of Collagen to Rebuild Components of the Human Heart
,”
Science
,
365
(
6452
), pp.
482
487
.
11.
De Melo
,
B. A.
,
Jodat
,
Y. A.
,
Cruz
,
E. M.
,
Benincasa
,
J. C.
,
Shin
,
S. R.
, and
Porcionatto
,
M. A.
,
2020
, “
Strategies to Use Fibrinogen as Bioink for 3D Bioprinting Fibrin-Based Soft and Hard Tissues
,”
Acta Biomater.
,
117
, pp.
60
76
.
12.
Parak
,
A.
,
Pradeep
,
P.
,
du Toit
,
L. C.
,
Kumar
,
P.
,
Choonara
,
Y. E.
, and
Pillay
,
V.
,
2019
, “
Functionalizing Bioinks for 3D Bioprinting Applications
,”
Drug Discov. Today
,
24
(
1
), pp.
198
205
.
13.
Jin
,
Y.
,
Xiong
,
R.
,
Antonelli
,
P. J.
,
Long
,
C. J.
,
McAleer
,
C. W.
,
Hickman
,
J. J.
, and
Huang
,
Y.
,
2021
, “
Nanoclay Suspension-Enabled Extrusion Bioprinting of Three-Dimensional Soft Structures
,”
ASME J. Manuf. Sci. Eng.
,
143
(
12
), p.
121004
.
14.
Thakare
,
K.
,
Jerpseth
,
L.
,
Pei
,
Z.
, and
Qin
,
H.
,
2022
, “
Applying Layer-by-Layer Photo-Crosslinking in Green Bioprinting: Shape Fidelity and Cell Viability of Printed Hydrogel Constructs Containing Algae Cells
,”
ASME J. Manuf. Sci. Eng.
,
144
(
9
), p.
094502
.
15.
Kesti
,
M.
,
Müller
,
M.
,
Becher
,
J.
,
Schnabelrauch
,
M.
,
D’Este
,
M.
,
Eglin
,
D.
, and
Zenobi-Wong
,
M.
,
2015
, “
A Versatile Bioink for Three-Dimensional Printing of Cellular Scaffolds Based on Thermally and Photo-Triggered Tandem Gelation
,”
Acta Biomater.
,
11
, pp.
162
172
.
16.
Patenaude
,
M.
, and
Hoare
,
T.
,
2012
, “
Injectable, Mixed Natural-Synthetic Polymer Hydrogels With Modular Properties
,”
Biomacromolecules
,
13
(
2
), pp.
369
378
.
17.
Gao
,
G.
,
Schilling
,
A. F.
,
Yonezawa
,
T.
,
Wang
,
J.
,
Dai
,
G.
, and
Cui
,
X.
,
2014
, “
Bioactive Nanoparticles Stimulate Bone Tissue Formation in Bioprinted Three
-
Dimensional Scaffold and Human Mesenchymal Stem Cells
,”
Biotechnol. J.
,
9
(
10
), pp.
1304
1311
.
18.
Xu
,
C.
,
Chai
,
W.
,
Huang
,
Y.
, and
Markwald
,
R. R.
,
2012
, “
Scaffold
-
Free Inkjet Printing of Three
-
Dimensional Zigzag Cellular Tubes
,”
Biotechnol. Bioeng.
,
109
(
12
), pp.
3152
3160
.
19.
Faulkner-Jones
,
A.
,
Fyfe
,
C.
,
Cornelissen
,
D.-J.
,
Gardner
,
J.
,
King
,
J.
,
Courtney
,
A.
, and
Shu
,
W.
,
2015
, “
Bioprinting of Human Pluripotent Stem Cells and Their Directed Differentiation Into Hepatocyte-Like Cells for the Generation of Mini-Livers in 3D
,”
Biofabrication
,
7
(
4
), p.
044102
.
20.
Hockaday
,
L.
,
Kang
,
K.
,
Colangelo
,
N.
,
Cheung
,
P.
,
Duan
,
B.
,
Malone
,
E.
,
Wu
,
J.
,
Girardi
,
L.
,
Bonassar
,
L.
, and
Lipson
,
H.
,
2012
, “
Rapid 3D Printing of Anatomically Accurate and Mechanically Heterogeneous Aortic Valve Hydrogel Scaffolds
,”
Biofabrication
,
4
(
3
), p.
035005
.
21.
Saunders
,
R. E.
,
Gough
,
J. E.
, and
Derby
,
B.
,
2008
, “
Delivery of Human Fibroblast Cells by Piezoelectric Drop-On-Demand Inkjet Printing
,”
Biomaterials
,
29
(
2
), pp.
193
203
.
22.
Saunders
,
R. E.
, and
Derby
,
B.
,
2014
, “
Inkjet Printing Biomaterials for Tissue Engineering: Bioprinting
,”
Int. Mater. Rev.
,
59
(
8
), pp.
430
448
.
23.
Wang
,
Z.
, and
Belovich
,
J. M.
,
2010
, “
A Simple Apparatus for Measuring Cell Settling Velocity
,”
Biotechnol. Prog.
,
26
(
5
), pp.
1361
1366
.
24.
Xu
,
H.
,
Zhang
,
Z.
, and
Xu
,
C.
,
2019
, “
Sedimentation Study of Bioink Containing Living Cells
,”
J. Appl. Phys.
,
125
(
11
), p.
114901
.
25.
Parsa
,
S.
,
Gupta
,
M.
,
Loizeau
,
F.
, and
Cheung
,
K. C.
,
2010
, “
Effects of Surfactant and Gentle Agitation on Inkjet Dispensing of Living Cells
,”
Biofabrication
,
2
(
2
), p.
025003
.
26.
Chahal
,
D.
,
Ahmadi
,
A.
, and
Cheung
,
K. C.
,
2012
, “
Improving Piezoelectric Cell Printing Accuracy and Reliability Through Neutral Buoyancy of Suspensions
,”
Biotechnol. Bioeng.
,
109
(
11
), pp.
2932
2940
.
27.
Zhang
,
Z.
,
Xu
,
C.
,
Xiong
,
R.
,
Chrisey
,
D. B.
, and
Huang
,
Y.
,
2017
, “
Effects of Living Cells on the Bioink Printability During Laser Printing
,”
Biomicrofluidics
,
11
(
3
), p.
034120
.
28.
Lee
,
A.
,
Sudau
,
K.
,
Ahn
,
K. H.
,
Lee
,
S. J.
, and
Willenbacher
,
N.
,
2012
, “
Optimization of Experimental Parameters to Suppress Nozzle Clogging in Inkjet Printing
,”
Ind. Eng. Chem. Res.
,
51
(
40
), pp.
13195
13204
.
29.
Ringeisen
,
B. R.
,
Othon
,
C. M.
,
Barron
,
J. A.
,
Young
,
D.
, and
Spargo
,
B. J.
,
2006
, “
Jet-Based Methods to Print Living Cells
,”
Biotechnol. J.
,
1
(
9
), pp.
930
948
.
30.
Wu
,
D.
, and
Xu
,
C.
,
2018
, “
Predictive Modeling of Droplet Formation Processes in Inkjet-Based Bioprinting
,”
ASME J. Manuf. Sci. Eng.
,
140
(
10
), p.
101007
.
31.
Xu
,
C.
,
Zhang
,
M.
,
Huang
,
Y.
,
Ogale
,
A.
,
Fu
,
J.
, and
Markwald
,
R. R.
,
2014
, “
Study of Droplet Formation Process During Drop-On-Demand Inkjetting of Living Cell-Laden Bioink
,”
Langmuir
,
30
(
30
), pp.
9130
9138
.
32.
Xu
,
H.
,
Casillas
,
J.
, and
Xu
,
C.
,
2019
, “
Effects of Printing Conditions on Cell Distribution Within Microspheres During Inkjet-Based Bioprinting
,”
AIP Adv.
,
9
(
9
), p.
095055
.
33.
Xu
,
H.
,
Liu
,
J.
,
Zhang
,
Z.
, and
Xu
,
C.
,
2022
, “
Cell Sedimentation During 3D Bioprinting: A Mini Review
,”
Bio-Des. Manuf.
, pp.
1
10
.
34.
Cheng
,
E.
,
Yu
,
H.
,
Ahmadi
,
A.
, and
Cheung
,
K. C.
,
2016
, “
Investigation of the Hydrodynamic Response of Cells in Drop on Demand Piezoelectric Inkjet Nozzles
,”
Biofabrication
,
8
(
1
), p.
015008
.
35.
Zhou
,
J.
, and
Papautsky
,
I.
,
2020
, “
Viscoelastic Microfluidics: Progress and Challenges
,”
Microsyst. Nanoeng.
,
6
(
1
), p.
113
.
36.
Yang
,
S.
,
Lee
,
S. S.
,
Ahn
,
S. W.
,
Kang
,
K.
,
Shim
,
W.
,
Lee
,
G.
,
Hyun
,
K.
, and
Kim
,
J. M.
,
2012
, “
Deformability-Selective Particle Entrainment and Separation in a Rectangular Microchannel Using Medium Viscoelasticity
,”
Soft Matter
,
8
(
18
), pp.
5011
5019
.
37.
Xu
,
H.
,
Martinez Salazar
,
D. M.
, and
Xu
,
C.
,
2022
, “
Investigation of Cell Concentration Change and Cell Aggregation Due to Cell Sedimentation During Inkjet-Based Bioprinting of Cell-Laden Bioink
,”
Machines
,
10
(
5
), p.
315
.
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