3D bioprinting is an innovative and time-saving method to precisely generate cell-laden 3D structures for clinical and research applications. Ejected cell number and cell distribution are two key technical parameters for evaluation of the bioprinter performance. In this paper, a modified droplet imaging system is used to study cell-size fluorescent particle number and distribution within droplets ejected from a microvalve-based 3D bioprinter. The effects of droplet dispensing physics (dosing energy Ed), ink properties (Z number—the inverse of the Ohnesorge number and particle sedimentation velocity), and input particle concentration are considered. The droplet imaging system demonstrates a strong capability in analyzing bioprinting performance for seeded concentrations less than 3×106 particles/ml. The printed particle number increases near-linearly under increasing dosing energy and Z number. It was found that for 7<Z<21 and seeded particle concentration no less than 3×106 particles/ml, particles within the visualized droplets approached a homogeneous distribution in the 2D images. It was also determined that the particle sedimentation velocity within the ink has a positive relationship to the ejected particle number over time—with the particle distribution approaching a homogeneous state over increasing sedimentation time.

1.
M.
Mobaraki
,
M.
Ghaffari
,
A.
Yazdanpanah
,
Y.
Luo
, and
D. K.
Mills
, “
Bioinks and bioprinting: A focused review
,”
Bioprinting
18
,
e00080
(
2020
).
2.
H.
Gudapati
,
M.
Dey
, and
I.
Ozbolat
, “
A comprehensive review on droplet-based bioprinting: Past, present and future
,”
Biomaterials
102
,
20
(
2016
).
3.
E.
Saygili
,
A. A.
Dogan-Gurbuz
,
O.
Yesil-Celiktas
, and
M. S.
Draz
, “
3D bioprinting: A powerful tool to leverage tissue engineering and microbial systems
,”
Bioprinting
18
,
e00071
(
2020
).
4.
S.
Chameettachal
,
S.
Yeleswarapu
,
S.
Sasikumar
,
P.
Shukla
,
P.
Hibare
,
A. K.
Bera
,
S. S. R.
Bojedla
, and
F.
Pati
, “
3D bioprinting: Recent trends and challenges
,”
J. Indian Inst. Sci.
99
,
375
(
2019
).
5.
P.
Datta
,
M.
Dey
,
Z.
Ataie
,
D.
Unutmaz
, and
I. T.
Ozbolat
, “
3D bioprinting for reconstituting the cancer microenvironment
,”
npj Precis. Oncol.
4
,
1
13
(
2020
).
6.
W. L.
Ng
,
J. M.
Lee
,
M.
Zhou
,
Y. W.
Chen
,
K. X. A.
Lee
,
W. Y.
Yeong
, and
Y. F.
Shen
, “
Vat polymerization-based bioprinting—Process, materials, applications and regulatory challenges
,”
Biofabrication
12
,
022001
(
2020
).
7.
S.
Vanaei
,
M.
Parizi
,
S.
Vanaei
,
F.
Salemizadehparizi
, and
H.
Vanaei
, “
An overview on materials and techniques in 3D bioprinting toward biomedical application
,”
Eng. Regener.
2
,
1
18
(
2021
).
8.
B.
Yilmaz
,
A.
Al Rashid
,
Y. A.
Mou
,
Z.
Evis
, and
M.
Koç
, “
Bioprinting: A review of processes, materials and applications
,”
Bioprinting
23
,
e00148
(
2021
).
9.
Y.
Zhang
,
P.
Kumar
,
S.
Lv
,
D.
Xiong
,
H.
Zhao
,
Z.
Cai
, and
X.
Zhao
, “
Recent advances in 3D bioprinting of vascularized tissues
,”
Mater. Des.
199
,
109398
(
2021
).
10.
W. L.
Ng
,
J. M.
Lee
,
W. Y.
Yeong
, and
M.
Win Naing
, “
Microvalve-based bioprinting-process, bio-inks and applications
,”
Biomater. Sci.
5
,
632
647
(
2017
).
11.
H.
Xu
,
J.
Casillas
, and
C.
Xu
, “
Effects of printing conditions on cell distribution within microspheres during inkjet-based bioprinting
,”
AIP Adv.
9
,
095055
(
2019
).
12.
S.
Yamaguchi
,
A.
Ueno
,
Y.
Akiyama
, and
K.
Morishima
, “
Cell patterning through inkjet printing of one cell per droplet
,”
Biofabrication
4
,
045005
(
2012
).
13.
C. J.
Ferris
,
K. J.
Gilmore
,
S.
Beirne
,
D.
McCallum
,
G. G.
Wallace
, and
M.
In Het Panhuis
, “
Bio-ink for on-demand printing of living cells
,”
Biomater. Sci.
1
,
224
230
(
2013
).
14.
V.
Tran
and
X.
Wen
, “Rapid prototyping technologies for tissue regeneration,” in
Rapid Prototyping of Biomaterials
(Woodhead Publishing, 2014), pp. 97–155.
15.
K.
Dubbin
,
Y.
Hori
,
K. K.
Lewis
, and
S. C.
Heilshorn
, “
Dual-stage crosslinking of a gel-phase bioink improves cell viability and homogeneity for 3D bioprinting
,”
Adv. Healthcare Mater.
5
,
2488
2492
(
2016
).
16.
W.
Ng
,
W.
Yeong
, and
M.
Naing
, “
Polyvinylpyrrolidone-based bio-ink improves cell viability and homogeneity during drop-on-demand printing
,”
Materials
10
,
190
(
2017
).
17.
F. J.
O’Brien
, “
Biomaterials & scaffolds for tissue engineering
,”
Mater. Today
14
,
88
(
2011
).
18.
M.
Dey
and
I. T.
Ozbolat
, “
3D bioprinting of cells, tissues and organs
,”
Sci. Rep.
10
,
14023
(
2020
).
19.
V.
Goranov
,
T.
Shelyakova
,
R.
De Santis
,
Y.
Haranava
,
A.
Makhaniok
,
A.
Gloria
,
A.
Tampieri
,
A.
Russo
,
E.
Kon
,
M.
Marcacci
,
L.
Ambrosio
, and
V. A.
Dediu
, “
3D patterning of cells in magnetic scaffolds for tissue engineering
,”
Sci. Rep.
10
,
2289
(
2020
).
20.
Y. K.
Kim
,
J. A.
Park
,
W. H.
Yoon
,
J.
Kim
, and
S.
Jung
, “
Drop-on-demand inkjet-based cell printing with 30-μm nozzle diameter for cell-level accuracy
,”
Biomicrofluidics
10
,
064110
(
2016
).
21.
P.
Shi
,
Y. S. E.
Tan
,
W. Y.
Yeong
,
H. Y.
Li
, and
A.
Laude
, “
A bilayer photoreceptor-retinal tissue model with gradient cell density design: A study of microvalve-based bioprinting
,”
J. Tissue Eng. Regen. Med.
12
,
1297
1306
(
2018
).
22.
N.
Okubo
,
A. J.
Qureshi
,
K.
Dalgarno
,
K. L.
Goh
, and
S.
Derebail
, “
Cost-effective microvalve-assisted bioprinter for tissue engineering
,”
Bioprinting
13
,
e00043
(
2019
).
23.
B. E.
Grottkau
,
Z.
Hui
, and
Y.
Pang
, “
A novel 3D bioprinter using direct-volumetric drop-on-demand technology for fabricating micro-tissues and drug-delivery
,”
Int. J. Mol. Sci.
21
,
3482
(
2020
).
24.
Y. W.
Lin
,
K. L.
Tsou
,
C. D.
Fay
,
X.
Liu
,
J. H.
Chung
,
D.
Sharma
,
A.
Jeiranikhameneh
,
P. H.
Kuo
,
C. K.
Tzeng
,
G. G.
Wallace
,
C. Y.
Wu
,
M. D.
Ker
,
J. I.
Chao
, and
Y. T.
Cheng
, “
A microvalve cell printing technique using riboflavin photosensitizer for selective cell patterning onto a retinal chip
,”
Bioprinting
20
,
e00097
(
2020
).
25.
M.
Klinger
,
C.
Laske
,
M.
Graeve
,
M.
Thoma
, and
A.
Traube
, “
A sensor for the in-flight detection of single fluorescent microbodies in nanoliter droplets
,”
IEEE Sens. J.
20
,
5809
5817
(
2020
).
26.
W. L.
Ng
,
C. K.
Chua
, and
Y. F.
Shen
, “
Print me an organ! Why we are not there yet
,”
Prog. Polym. Sci.
97
,
101145
(
2019
).
27.
E.
Cheng
,
H.
Yu
,
A.
Ahmadi
, and
K. C.
Cheung
, “
Investigation of the hydrodynamic response of cells in drop on demand piezoelectric inkjet nozzles
,”
Biofabrication
8
,
015008
(
2016
).
28.
P.
Benítez
,
J. C.
Miñano
,
P.
Zamora
,
R.
Mohedano
,
A.
Cvetkovic
,
M.
Buljan
,
J.
Chaves
, and
M.
Hernández
, “
High performance Fresnel-based photovoltaic concentrator
,”
Opt. Express
18
,
A25
(
2010
).
29.
H. Y.
Lo
,
Y.
Liu
, and
L.
Xu
, “
Mechanism of contact between a droplet and an atomically smooth substrate
,”
Phys. Rev. X
7
,
021036
(
2017
); arXiv:1703.01419.
30.
F.
Wetzel
,
S.
Rönicke
,
K.
Müller
,
M.
Gyger
,
D.
Rose
,
M.
Zink
, and
J.
Käs
, “
Single cell viability and impact of heating by laser absorption
,”
Eur. Biophys. J.
40
,
1109
1114
(
2011
).
31.
D. A.
Guertin
and
D. M.
Sabatini
, “Cell size control,” in Encyclopedia of Life Sciences (John Wiley & Sons, New York, 2006.)
32.
A. K.
Bryan
,
A.
Goranov
,
A.
Amon
, and
S. R.
Manalis
, “
Measurement of mass, density, and volume during the cell cycle of yeast
,”
Proc. Natl. Acad. Sci. U.S.A.
107
,
999
1004
(
2010
).
33.
J.
Fowler
and
M.
Litorja
, “
Geometric area measurements of circular apertures for radiometry at NIST
,”
Metrologia
40
,
S9
(
2003
).
34.
E.
Senchenko
and
Y.
Chugui
, “
Shadow inspection of 3D objects in partially coherent light
,”
Meas. Sci. Rev.
11
,
104
107
(
2011
).
35.
F.
Yu
and
B. D.
Shaw
, “
Interpretation of backlit droplet images from ISS droplet combustion experiments
,”
Gravit. Space Res.
2
(1),
82
–93 (
2014
).
36.
R. C.
Bradshaw
,
D. P.
Schmidt
,
J. R.
Rogers
,
K. F.
Kelton
, and
R. W.
Hyers
, “
Machine vision for high-precision volume measurement applied to levitated containerless material processing
,”
Rev. Sci. Instrum.
76
,
125108
(
2005
).
37.
E.
Gorges
,
L. M.
Racz
,
A.
Schillings
, and
I.
Egry
, “
Density measurements on levitated liquid metal droplets
,”
Int. J. Thermophys.
17
,
1163
1172
(
1996
).
38.
T. R.
Jones
,
I. H.
Kang
,
D. B.
Wheeler
,
R. A.
Lindquist
,
A.
Papallo
,
D. M.
Sabatini
,
P.
Golland
, and
A. E.
Carpenter
, “
Cellprofiler analyst: Data exploration and analysis software for complex image-based screens
,”
BMC Bioinformatics
9
,
482
(
2008
).
39.
A. E.
Carpenter
,
T. R.
Jones
,
M. R.
Lamprecht
,
C.
Clarke
,
I. H.
Kang
,
O.
Friman
,
D. A.
Guertin
,
J. H.
Chang
,
R. A.
Lindquist
,
J.
Moffat
,
P.
Golland
, and
D. M.
Sabatini
, “
CellProfiler: Image analysis software for identifying and quantifying cell phenotypes
,”
Genome Biol.
7
,
R100
(
2006
).
40.
M. R.
Lamprecht
,
D. M.
Sabatini
, and
A. E.
Carpenter
, “
CellProfiler™: Free, versatile software for automated biological image analysis
,”
BioTechniques
42
,
71
75
(
2007
).
41.
N.
Yang
,
J.
Boselli
, and
I.
Sinclair
, “
Simulation and quantitative assessment of homogeneous and inhomogeneous particle distributions in particulate metal matrix composites
,”
J. Microsc.
201
,
189
200
(
2001
).
42.
A.
Ayyar
,
G. A.
Crawford
,
J. J.
Williams
, and
N.
Chawla
, “
Numerical simulation of the effect of particle spatial distribution and strength on tensile behavior of particle reinforced composites
,”
Comput. Mater. Sci.
44
,
496
506
(
2008
).
43.
Z.
Zhang
,
Y.
Jin
,
J.
Yin
,
C.
Xu
,
R.
Xiong
,
K.
Christensen
,
B. R.
Ringeisen
,
D. B.
Chrisey
, and
Y.
Huang
, “
Evaluation of bioink printability for bioprinting applications
,”
Appl. Phys. Rev.
5
,
041304
(
2018
).
44.
Y.
Liu
and
B.
Derby
, “
Experimental study of the parameters for stable drop-on-demand inkjet performance
,”
Phys. Fluids
31
,
032004
(
2019
).
45.
J. N.
Chung
, “
The motion of particles inside a droplet
,”
J. Heat Transfer
104
,
438
445
(
1982
).
46.
Y.
Wei
,
W.
Deng
, and
R. H.
Chen
, “
Effects of internal circulation and particle mobility during nanofluid droplet evaporation
,”
Int. J. Heat Mass Transf.
103
,
1335
1347
(
2016
).
47.
F.
Wang
,
J.
Li
,
Y.
Wang
,
W.
Bao
,
X.
Chen
,
H.
Zhang
, and
Z.
Wang
,
Proc. SPIE
11041
,
110410L
(
2019
).

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