aggregation will be included within the suspension, although
constant stirring at 14 RCF will be needed immediately after. If
alginate inclusion results in aggregations after 1 h of stirring, a
sterile-autoclaved spatula can be used to break down alginate
aggregations. Mixing will then need to be resumed for further
2 h.
4. After methylcellulose addition and stirring with a sterile spatula, some methylcellulose aggregates will still be visible. Overnight storage at 4
C will facilitate full methylcellulose
inclusion, resulting in a homogeneous amber-colored biopaste
ready to be printed.
5. SSCs expansion and density number will depend on donor,
passage, and culture time. We strongly recommend using
SSCs at low passage numbers (Passage 1 or Passage 2) to
preserve SSCs stemness and avoid uncontrolled differentiation
prior to the experiment.
6. Incubation time will vary depending on specific SSCs and
donor used. Forty-five minutes is the average time of SSCs
incubation within the collagenase solution.
7. Confirmation of full detachment should be performed with a
light microscope.
8. Cell density for inclusion in bioinks is still debated. We have
previously used 1 Â 10
6 cells/g, but this value can be increased
or lowered depending on the experimental plan (viability, proliferation, in vivo).
9. This procedure is carried out to obtain a paste that can be easily
loaded and cells can be quickly mixed. Fast spinning is
recommended.
10. Mixing must be carried out gently. Using a sterile stainless steel
spatula, stem cells can be easily mixed within paste by slow
clockwise bottom-up mixing. This procedure is critical for
post-printing viability and must be performed carefully.
11. A sterile spatula can be used to scoop 3-3-3 from the beaker
and slowly adding it to the syringe barrel. For a piston-driven
printing process, using a Luer-lock disposable syringe, it is
recommended that biopaste should not be loaded more than
¾ of the syringe total volume.
12. Conical-shaped disposable sterile nozzles are preferable when
printing living cells [17]. Flow-dynamic force field representing shear stresses found during extrusion indicates higher
forces applied to nozzle boundaries when a cylindrical extruder
is used. Conical nozzles show a lower degree of shear applied
along the extrusion direction, resulting in a clear advantage in
preserving cell viability.
70
Gianluca Cidonio et al.
constant stirring at 14 RCF will be needed immediately after. If
alginate inclusion results in aggregations after 1 h of stirring, a
sterile-autoclaved spatula can be used to break down alginate
aggregations. Mixing will then need to be resumed for further
2 h.
4. After methylcellulose addition and stirring with a sterile spatula, some methylcellulose aggregates will still be visible. Overnight storage at 4
C will facilitate full methylcellulose
inclusion, resulting in a homogeneous amber-colored biopaste
ready to be printed.
5. SSCs expansion and density number will depend on donor,
passage, and culture time. We strongly recommend using
SSCs at low passage numbers (Passage 1 or Passage 2) to
preserve SSCs stemness and avoid uncontrolled differentiation
prior to the experiment.
6. Incubation time will vary depending on specific SSCs and
donor used. Forty-five minutes is the average time of SSCs
incubation within the collagenase solution.
7. Confirmation of full detachment should be performed with a
light microscope.
8. Cell density for inclusion in bioinks is still debated. We have
previously used 1 Â 10
6 cells/g, but this value can be increased
or lowered depending on the experimental plan (viability, proliferation, in vivo).
9. This procedure is carried out to obtain a paste that can be easily
loaded and cells can be quickly mixed. Fast spinning is
recommended.
10. Mixing must be carried out gently. Using a sterile stainless steel
spatula, stem cells can be easily mixed within paste by slow
clockwise bottom-up mixing. This procedure is critical for
post-printing viability and must be performed carefully.
11. A sterile spatula can be used to scoop 3-3-3 from the beaker
and slowly adding it to the syringe barrel. For a piston-driven
printing process, using a Luer-lock disposable syringe, it is
recommended that biopaste should not be loaded more than
¾ of the syringe total volume.
12. Conical-shaped disposable sterile nozzles are preferable when
printing living cells [17]. Flow-dynamic force field representing shear stresses found during extrusion indicates higher
forces applied to nozzle boundaries when a cylindrical extruder
is used. Conical nozzles show a lower degree of shear applied
along the extrusion direction, resulting in a clear advantage in
preserving cell viability.
70
Gianluca Cidonio et al.