18. Protect the plate from light with aluminum foil. To facilitate
diffusion of the working solution into the tissue construct, it
can also be incubated at 37
C or for a longer incubation time.
19. The excitation and emission wavelengths for calcein AM are
~495 nm/~515 nm. The excitation and emission wavelengths
for ethidium homodimer-1 are ~495 nm/~635 nm.
20. ImageJ or Fiji software can be used for cell quantification
automatically or manually. Expected viability values are in the
range of 85–95%.
References
1. Langer R, Vacanti JP (1993) Tissue engineering. Science 260:920–926. https://doi.org/
10.1126/science.8493529
2. Khademhosseini A, Langer R, Borenstein J,
Vacanti JP (2006) Microscale technologies for
tissue engineering and biology. Proc Natl Acad
Sci 103:2480–2487. https://doi.org/10.
1073/pnas.0507681102
3. Kolesky DB, Truby RL, Gladman AS, Busbee
TA, Homan KA, Lewis JA (2014) 3D bioprinting of vascularized, heterogeneous cell-laden
tissue constructs. Adv Mater 26:3124–3130.
https://doi.org/10.1002/adma.201305506
4. Baranski JD, Chaturvedi RR, Stevens KR,
Eyckmans J, Carvalho B, Solorzano RD, Yang
MT, Miller JS, Bhatia SN, Chen CS (2013)
Geometric control of vascular networks to
enhance engineered tissue integration and
function. Proc Natl Acad Sci 110:7586–7591.
https://doi.org/10.1073/pnas.1217796110
5. Lee KY, Peters MC, Anderson KW, Mooney
DJ (2000) Controlled growth factor release
from synthetic extracellular matrices. Nature
408:998–1000.
https://doi.org/10.1038/
35050141
6. Fischbach C, Mooney DJ (2007) Polymers for
pro- and anti-angiogenic therapy. Biomaterials
28:2069–2076. https://doi.org/10.1016/j.
biomaterials.2006.12.029
7. Richardson TP, Peters MC, Ennett AB,
Mooney DJ (2001) Polymeric system for dual
growth factor delivery. Nat Biotechnol
19:1029–1034.
https://doi.org/10.1038/
nbt1101-1029
8. Levenberg S, Rouwkema J, Macdonald M,
Garfein ES, Kohane DS, Darland DC,
Marini R, van Blitterswijk CA, Mulligan RC,
D’Amore PA, Langer R (2005) Engineering
vascularized skeletal muscle tissue. Nat Biotechnol 23:879–884. https://doi.org/10.
1038/nbt1109
9. Caspi O, Lesman A, Basevitch Y, Gepstein A,
Arbel G, Huber I, Habib M, Gepstein L,
Levenberg S (2007) Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circ Res 100:263–272.
https://doi.org/10.1161/01.RES.
0000257776.05673.ff
10. Koike N, Fukumura D, Gralla O, Au P, Schechner JS, Jain RK (2004) Tissue engineering:
creation of long-lasting blood vessels. Nature
428:138–139.
https://doi.org/10.1038/
428138a
11. Chen X, Aledia AS, Ghajar CM, Griffith CK,
Putnam AJ, Hughes CCW, George SC (2009)
Prevascularization of a fibrin-based tissue construct accelerates the formation of functional
anastomosis with host vasculature. Tissue Eng
Part A 15:1363–1371. https://doi.org/10.
1089/ten.tea.2008.0314
12. Bertassoni LE, Cecconi M, Manoharan V,
Nikkhah M, Hjortnaes J, Cristino AL,
Barabaschi G, Demarchi D, Dokmeci MR,
Yang Y, Khademhosseini A (2014) Hydrogel
bioprinted microchannel networks for vascularization of tissue engineering constructs.
Lab Chip 14:2202–2211. https://doi.org/
10.1039/c4lc00030g
13. Kolesky DB, Homan KA, Skylar-Scott MA,
Lewis JA (2016) Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad
Sci U S A 113:3179–3184. https://doi.org/
10.1073/pnas.1521342113
14. Miller JS, Stevens KR, Yang MT, Baker BM,
Nguyen D-HT, Cohen DM, Toro E, A a C, P
a G, Yu X, Chaturvedi R, Bhatia SN, Chen CS
(2012) Rapid casting of patterned vascular networks for perfusable engineered threedimensional tissues. Nat Mater 11:768–774.
https://doi.org/10.1038/nmat3357
15. Jia W, Gungor-Ozkerim PS, Zhang YS, Yue K,
Zhu K, Liu W, Pi Q, Byambaa B, Dokmeci MR,
Shin SR, Khademhosseini A (2016) Direct 3D
172
Wei Zhu et al.
diffusion of the working solution into the tissue construct, it
can also be incubated at 37
C or for a longer incubation time.
19. The excitation and emission wavelengths for calcein AM are
~495 nm/~515 nm. The excitation and emission wavelengths
for ethidium homodimer-1 are ~495 nm/~635 nm.
20. ImageJ or Fiji software can be used for cell quantification
automatically or manually. Expected viability values are in the
range of 85–95%.
References
1. Langer R, Vacanti JP (1993) Tissue engineering. Science 260:920–926. https://doi.org/
10.1126/science.8493529
2. Khademhosseini A, Langer R, Borenstein J,
Vacanti JP (2006) Microscale technologies for
tissue engineering and biology. Proc Natl Acad
Sci 103:2480–2487. https://doi.org/10.
1073/pnas.0507681102
3. Kolesky DB, Truby RL, Gladman AS, Busbee
TA, Homan KA, Lewis JA (2014) 3D bioprinting of vascularized, heterogeneous cell-laden
tissue constructs. Adv Mater 26:3124–3130.
https://doi.org/10.1002/adma.201305506
4. Baranski JD, Chaturvedi RR, Stevens KR,
Eyckmans J, Carvalho B, Solorzano RD, Yang
MT, Miller JS, Bhatia SN, Chen CS (2013)
Geometric control of vascular networks to
enhance engineered tissue integration and
function. Proc Natl Acad Sci 110:7586–7591.
https://doi.org/10.1073/pnas.1217796110
5. Lee KY, Peters MC, Anderson KW, Mooney
DJ (2000) Controlled growth factor release
from synthetic extracellular matrices. Nature
408:998–1000.
https://doi.org/10.1038/
35050141
6. Fischbach C, Mooney DJ (2007) Polymers for
pro- and anti-angiogenic therapy. Biomaterials
28:2069–2076. https://doi.org/10.1016/j.
biomaterials.2006.12.029
7. Richardson TP, Peters MC, Ennett AB,
Mooney DJ (2001) Polymeric system for dual
growth factor delivery. Nat Biotechnol
19:1029–1034.
https://doi.org/10.1038/
nbt1101-1029
8. Levenberg S, Rouwkema J, Macdonald M,
Garfein ES, Kohane DS, Darland DC,
Marini R, van Blitterswijk CA, Mulligan RC,
D’Amore PA, Langer R (2005) Engineering
vascularized skeletal muscle tissue. Nat Biotechnol 23:879–884. https://doi.org/10.
1038/nbt1109
9. Caspi O, Lesman A, Basevitch Y, Gepstein A,
Arbel G, Huber I, Habib M, Gepstein L,
Levenberg S (2007) Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circ Res 100:263–272.
https://doi.org/10.1161/01.RES.
0000257776.05673.ff
10. Koike N, Fukumura D, Gralla O, Au P, Schechner JS, Jain RK (2004) Tissue engineering:
creation of long-lasting blood vessels. Nature
428:138–139.
https://doi.org/10.1038/
428138a
11. Chen X, Aledia AS, Ghajar CM, Griffith CK,
Putnam AJ, Hughes CCW, George SC (2009)
Prevascularization of a fibrin-based tissue construct accelerates the formation of functional
anastomosis with host vasculature. Tissue Eng
Part A 15:1363–1371. https://doi.org/10.
1089/ten.tea.2008.0314
12. Bertassoni LE, Cecconi M, Manoharan V,
Nikkhah M, Hjortnaes J, Cristino AL,
Barabaschi G, Demarchi D, Dokmeci MR,
Yang Y, Khademhosseini A (2014) Hydrogel
bioprinted microchannel networks for vascularization of tissue engineering constructs.
Lab Chip 14:2202–2211. https://doi.org/
10.1039/c4lc00030g
13. Kolesky DB, Homan KA, Skylar-Scott MA,
Lewis JA (2016) Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad
Sci U S A 113:3179–3184. https://doi.org/
10.1073/pnas.1521342113
14. Miller JS, Stevens KR, Yang MT, Baker BM,
Nguyen D-HT, Cohen DM, Toro E, A a C, P
a G, Yu X, Chaturvedi R, Bhatia SN, Chen CS
(2012) Rapid casting of patterned vascular networks for perfusable engineered threedimensional tissues. Nat Mater 11:768–774.
https://doi.org/10.1038/nmat3357
15. Jia W, Gungor-Ozkerim PS, Zhang YS, Yue K,
Zhu K, Liu W, Pi Q, Byambaa B, Dokmeci MR,
Shin SR, Khademhosseini A (2016) Direct 3D
172
Wei Zhu et al.