4 Notes
1. Gelatin (type A or type B) from any source and with any
molecular weight can be used. Similarly, the methacrylation
reaction can be performed with any type of hyaluronic acid,
independently of its molecular weight.
2. For the synthesis of photocurable PEGylated fibrinogen,
PEG-diacrylate (PEGDA) at different molecular weights can
be used. Typically, 4, 6 or 20 kDa PEGDA are used in the
synthesis. Such polymers can be prepared by reacting PEG with
acryloyl chloride in anhydrous conditions (for more details see
Cruise et al. [22]) or bought from local suppliers.
3. The bioink solution can be prepared in any container, but care
must be taken to protect the solution from light after photoinitiator addition.
4. Bioink formulation can vary from one experiment to another.
In particular, its formulation is generally influenced by the cell
type used in the experiment. Therefore, in order to provide the
best microenvironment to the cells for their growth, proliferation, and differentiation, bioinks can contain a single photocurable biopolymer or a combination of them.
5. We recommend using a 19G outer needle and a 25G inner
needle. While coaxial needle assemblies can be bought from
specialized providers, lab-built solutions can easily be assembled starting from conventional blunt tip needles.
6. Two pumps are always needed to supply the bioink and the
crosslinking solution (CaCl 2 ), respectively, to the inner and
outer needle of the coaxial system. Additional pumps may be
required to supply multiple bioinks simultaneously.
7. All the instructions are meant for 1 mL of bioink solution, but
the quantities can be scaled up for larger volumes. For small
volumes (i.e., less than 2 mL), we recommend using 1.5 mL or
2 mL tubes. For larger volumes (> 2 mL), we recommend
using screw-cap glass vials.
8. We suggest the following concentration ranges: GelMA
1Ä10% w/w, HAMA 1Ä3% w/w, CS-AEMA 2Ä10% w/w,
and PEG-fibrinogen 0.8Ä1.5% w/w.
9. Bioink buffer can be also formulated without FBS.
10. Cell density may vary according to the cell type and the application. Typically, cell density values in the range
5Ä50 Â 10
6 cells/mL are recommended. However, a large
cell number can lead to difficulties in cell resuspension and
formation of cell aggregates.
52
Marco Costantini et al.
1. Gelatin (type A or type B) from any source and with any
molecular weight can be used. Similarly, the methacrylation
reaction can be performed with any type of hyaluronic acid,
independently of its molecular weight.
2. For the synthesis of photocurable PEGylated fibrinogen,
PEG-diacrylate (PEGDA) at different molecular weights can
be used. Typically, 4, 6 or 20 kDa PEGDA are used in the
synthesis. Such polymers can be prepared by reacting PEG with
acryloyl chloride in anhydrous conditions (for more details see
Cruise et al. [22]) or bought from local suppliers.
3. The bioink solution can be prepared in any container, but care
must be taken to protect the solution from light after photoinitiator addition.
4. Bioink formulation can vary from one experiment to another.
In particular, its formulation is generally influenced by the cell
type used in the experiment. Therefore, in order to provide the
best microenvironment to the cells for their growth, proliferation, and differentiation, bioinks can contain a single photocurable biopolymer or a combination of them.
5. We recommend using a 19G outer needle and a 25G inner
needle. While coaxial needle assemblies can be bought from
specialized providers, lab-built solutions can easily be assembled starting from conventional blunt tip needles.
6. Two pumps are always needed to supply the bioink and the
crosslinking solution (CaCl 2 ), respectively, to the inner and
outer needle of the coaxial system. Additional pumps may be
required to supply multiple bioinks simultaneously.
7. All the instructions are meant for 1 mL of bioink solution, but
the quantities can be scaled up for larger volumes. For small
volumes (i.e., less than 2 mL), we recommend using 1.5 mL or
2 mL tubes. For larger volumes (> 2 mL), we recommend
using screw-cap glass vials.
8. We suggest the following concentration ranges: GelMA
1Ä10% w/w, HAMA 1Ä3% w/w, CS-AEMA 2Ä10% w/w,
and PEG-fibrinogen 0.8Ä1.5% w/w.
9. Bioink buffer can be also formulated without FBS.
10. Cell density may vary according to the cell type and the application. Typically, cell density values in the range
5Ä50 Â 10
6 cells/mL are recommended. However, a large
cell number can lead to difficulties in cell resuspension and
formation of cell aggregates.
52
Marco Costantini et al.