supplementary information 3-d microwell … word - demirci & kaye supplemantary...
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SUPPLEMENTARY INFORMATION 1
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3-D Microwell Array System for Culturing Virus Infected Tumor Cells 3
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Rami El Assal1, Umut Atakan Gurkan2,3, Pu Chen1, Franceline Juillard4, 5
Alessandro Tocchio1, Thiruppathiraja Chinnasamy1, Chantal Beauchemin4, 6
Sebnem Unluisler2, Serli Canikyan2, Alyssa Holman1, Srikar Srivatsa1, 7
Kenneth M. Kaye4*, Utkan Demirci1,2,5* 8
9 1 Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratories, Canary Center 10
at Stanford for Cancer Early Detection, Department of Radiology, Stanford 11
University School of Medicine, Palo Alto, CA, 94304, USA 12
13 2 Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratories, Division for 14
Biomedical Engineering, Division of Infectious Diseases, Renal Division, 15
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical 16
School, Boston, MA, 02115, USA 17
18 3 Biomanufacturing and Microfabrication Laboratory, Mechanical and Aerospace 19
Engineering Department, Biomedical Engineering Department, Department of 20
Orthopedics, Advanced Platform Technology Center, Louis Stokes Cleveland 21
Veterans Affairs Medical Center, Case Western Reserve University, Cleveland, 22
OH, 44106, USA 23
24 4 Division of Infectious Diseases, Departments of Medicine, Brigham and 25
Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA 26
27 5 Department of Electrical Engineering (by courtesy), Stanford University School 28
of Engineering, Stanford, CA, 94305, USA. 29
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* Corresponding Author’s E-mail: [email protected]; [email protected] 31
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33 Supplementary Figure 1. Mass swelling ratio of PEG hydrogels. The mass 34
swelling ratio of the hydrogels (n=10) was measured for up to 24 hours. The 35
hydrogels reached the swelling plateau in about a 1 hour incubation period. 36
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0 6 12 18 24
Mas
s Sw
ellin
g R
atio
Time (Hours)
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0 30 60 90 120
Mas
s S
wel
ling
Rat
io
Time (Min)
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47 Supplementary Figure 2. Photomicrographs showing the microwell array 48
system. The KSHV infected BJAB cells with puromycin selection were imaged 49
after 10 days in culture using bright field (a) and fluorescence (b) 50
photomicrographs. Scale bar represents 450 µm. 51
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a b
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Supplementary Figure 3. Fluorescent photomicrograph showing cell spill-78
over from a microwell. After proliferating BJAB (infected) fill a 3-D microwell, 79
cells begin to spillover the top. This image was taken on day 14. Scale bar 80
represents 100 µm. 81
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Supplementary Figure 4. Uninfected BJAB cells maintained in 3-D 450 µm 103
microwells or 2-D suspension, both with (+) and without (-) puromycin 104
selection. a) Brightfield photomicrographs of BJAB cells grown for 15 days. 105
Images were taken at 3-day intervals, beginning with the day of seeding. Growth 106
was observed for the 3-D grown cells without puromycin selection but not with 107
puromycin. b) Fluorescent photomicrographs of BJAB cells grown for 15 days. 108
We did not observe fluorescent cells, since the cells lack infection with KSHV. 109
Scale bars represent 200 µm. 110
a
b
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
3-D
3-D
- P
urom
ycin
2-D
2-D
+ P
urom
ycin
-
Pur
omyc
in
+ P
urom
ycin
-
Pur
omyc
in
+ P
urom
ycin
-
Pur
omyc
in
+ P
urom
ycin
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15
200µm
200µm
200µm
200µm
200µm
200µm
200µm
200µm
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Supplementary Table 1. Comparison of 3-D model generation methods. 111
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Method' Advantages' Disadvantages'
Hanging&Drop& + Inexpensive&+ Simple&&+ Fast&3+D&model&forma9on&&
+ Scale+up&is&difficult&+ Long+term&culture&is&difficult&+ Instability&of&the&liquid+air&
interface&+ Time+consuming&+ Labor+intensive&&
Spinner&Flasks& + Simple&&&+ Ease&to&scale+up&&+ Long+term&culture&&
+ Specialized&equipment&is&required.&
+ Varia9on&in&size/cell&number&+ High&shear&stress&&
Rotary&System& + Simple&&&+ Ease&to&scale+up&&+ Long+term&culture&
+ Specialized&equipment&is&required.&
+ Varia9on&in&size/cell&number&
Assembly&& + Fast&3+D&model&forma9on&&+ Reconfigurable&method&+ Non+invasive&method&&&
+ Specialized&equipment&is&required&(e.g.,&acous9c&generator&orµ+robo9c&system)&
+ May&require&magne9c&nanopar9cles&
Bioprin9ng& + Fast&3+D&model&forma9on.&+ Control&over&spa9al&and&
temporal&distribu9on&of&cell&seeding&&
+ Specialized&equipment&is&required&(i.e.,&bioprinter).&
+ Specialized&equipment&is&required&for&culturing&(i.e.,&bioreactor).&
+ Thermal&and&mechanical&stress.&&+ Ejector&clogging.&&&
Microfluidic&& + Dynamic&culture&&+ Resembles&the&physiological&
environment&(flow&condi9on)&
+ On+chip&analysis&&
+ Specialized&equipment&is&required&(e.g.,&laser&cuWer).&&&
+ Long+term&culture&is&difficult.&+ Shear&stress&&+ Time+consuming&
Microwell& + Simple&to&perform&&+ Ease&to&scale+up&&+ Well+controlled&3+D&model&
size&+ Designed&geometry&+ Long+term&culture&
+ Required&specialized&facility&to&fabricate&the&mold.&
+ Long&term&culture&ends&when&the&cells&outgrow&of&wells.&
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Supplementary Table 2. Determination of the microwell volume of three 116
different microwell sizes (150 µm, 300 µm, and 450 µm) based on the mask 117
design used during fabrication. 118
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Microwell)Diameter)&)Height)(μm)) Microwell)Volume)(μm3))(v="π#r2h))
150%x%150% 2.65%x%106%%
300%x%300% 2.12%x%107%
450%x%450% 7.16%x%107%