4 Notes
1. Alginate solution can be prepared in several ways. If using a
centrifuge tube, use the ultrasonic bath to fasten the process. If
using a glass beaker, make sure the glass beaker is covered and
put into a mixer overnight at 180 rpm and at room temperature.
2. The present procedure has been optimized for Fab@Home
(https://reprap.org/wiki/RUG/Pennsylvania/State_Col
lege/Printers/PSU_Fab@Home) printing hardware and Seraph studio (https://github.com/SeraphRobotics) printing
software. However, the procedure can easily be adapted to
different printing setups.
3. In the present experiments, we have used a sturdy carcinoma
cell lines. However, biofabrication using alginate can also be
used with more sensitive cells. To work with more sensitive
cells, we recommend the following modifications:
(a) Lower the percentage of alginate. This will result in less
mechanical strength of the structure, which means it will
not be possible to print structures that are more than a few
millimeters high. When lowering the percentage of alginate, also lower the concentration of the primary CaCl 2
crosslinking solution. When working with a 4% w/v alginate solution, use a 40 mM primary crosslinking solution.
(b) Use water for embryo transfer for the preparation of the
alginate solution. As lab-grade DIW can get quite acidic,
we use water for embryo transfer with our more delicate
cells. This increases cell survival after printing. The water
can also be used for the preparation of the crosslinking
solutions.
(c) When exploring different cell concentration levels, take
into account that the cells will have an influence on the
stability of the structure, especially for very high cell
concentrations.
4. Take care that the medium used to create the cell suspension in
Subheading 3.4 does not contain any large quantity of cations
that might react with the alginate.
5. This staining protocol has been optimized for application with
our alginate structures. When using commercial live/dead
assays containing ethidium homodimer-1 and calcein, we
found that our structures were less stable, or even dissolved,
after staining.
6. When designing structures make sure the thickness for the
structures is at least twice the printing path to avoid errors
while generating the printing sequence. The minimum thickness the structure will have after printing is roughly 1 mm due
to swelling of alginate after cross-linking.
Bioprinting of Complex Alginate Constructs
147
1. Alginate solution can be prepared in several ways. If using a
centrifuge tube, use the ultrasonic bath to fasten the process. If
using a glass beaker, make sure the glass beaker is covered and
put into a mixer overnight at 180 rpm and at room temperature.
2. The present procedure has been optimized for Fab@Home
(https://reprap.org/wiki/RUG/Pennsylvania/State_Col
lege/Printers/PSU_Fab@Home) printing hardware and Seraph studio (https://github.com/SeraphRobotics) printing
software. However, the procedure can easily be adapted to
different printing setups.
3. In the present experiments, we have used a sturdy carcinoma
cell lines. However, biofabrication using alginate can also be
used with more sensitive cells. To work with more sensitive
cells, we recommend the following modifications:
(a) Lower the percentage of alginate. This will result in less
mechanical strength of the structure, which means it will
not be possible to print structures that are more than a few
millimeters high. When lowering the percentage of alginate, also lower the concentration of the primary CaCl 2
crosslinking solution. When working with a 4% w/v alginate solution, use a 40 mM primary crosslinking solution.
(b) Use water for embryo transfer for the preparation of the
alginate solution. As lab-grade DIW can get quite acidic,
we use water for embryo transfer with our more delicate
cells. This increases cell survival after printing. The water
can also be used for the preparation of the crosslinking
solutions.
(c) When exploring different cell concentration levels, take
into account that the cells will have an influence on the
stability of the structure, especially for very high cell
concentrations.
4. Take care that the medium used to create the cell suspension in
Subheading 3.4 does not contain any large quantity of cations
that might react with the alginate.
5. This staining protocol has been optimized for application with
our alginate structures. When using commercial live/dead
assays containing ethidium homodimer-1 and calcein, we
found that our structures were less stable, or even dissolved,
after staining.
6. When designing structures make sure the thickness for the
structures is at least twice the printing path to avoid errors
while generating the printing sequence. The minimum thickness the structure will have after printing is roughly 1 mm due
to swelling of alginate after cross-linking.
Bioprinting of Complex Alginate Constructs
147