3. Heated holder for the pressure-driven syringe (operating at
37
C) (see Note 2).
4. Programmable pressure controller (OB1-MK3, Elveflow,
0–8000 mbar range).
5. Petri dishes, glass slides, or other printing substrates.
6. Crosslinking solution: 25 mM CaCl 2
3 Methods
Although the procedures are described for a Pluronic/alginate
solution of selected concentration, the proposed methods can be
extended to each heat-sensitive hydrogel having a transition from
the sol to the gel phase upon increasing the temperature, at values
included between 20
C and 30
C. Information on the sol-gel
transition kinetic, gelation time under physiological conditions
should be determined for each novel printing ink. A detailed
description of all these characterization steps is out of the scope of
the present chapter. However, a representative example of the set of
analyses required for the optimization of a novel thermogel can be
found in Gioffredi et al. [8].
3.1 Preparation
of Pluronic/Alginate
Solution
The Pluronic/alginate solution can be prepared according to the
so-called “cold method” proposed by Schmolka [11].
1. In a glass vial, add 100 mg of sodium alginate to 3.9 mL of an
ice-cold 0.2Â solution of DMEM under mild stirring.
2. While keeping the solution in an ice bath, slowly disperse 1 g of
Pluronic F127 in the alginate solution under mild stirring (see
Note 3).
3. Sterilize the solution by autoclaving, and store sterilized solution at 4
C.
3.2 Printing Process
The main steps of scaffold manufacturing by direct-write deposition are summarized in Fig. 1.
Subconfluent HDFs can be routinely processed by trypsinization and centrifugation to obtain a cell pellet (see Note 4).
1. Working in a biosafety cabinet, homogeneously suspend HDFs
in the sterile Pluronic/alginate solution kept at 4
C, at a final
concentration of 1 Â 10
6 cells/mL.
2. Transfer the bioink into the pressure-driven syringe.
3. Mount the syringe on the heated holder, connect it to the
pressure controller, and bring it at 37
C (see Note 5).
4. Extrude the bioink at a pressure of 1.2 bar (see Note 6) with a
needle/substrate relative speed of 10 mm/s. Several layers can
Direct-Write Deposition of Thermogels
139
37
C) (see Note 2).
4. Programmable pressure controller (OB1-MK3, Elveflow,
0–8000 mbar range).
5. Petri dishes, glass slides, or other printing substrates.
6. Crosslinking solution: 25 mM CaCl 2
3 Methods
Although the procedures are described for a Pluronic/alginate
solution of selected concentration, the proposed methods can be
extended to each heat-sensitive hydrogel having a transition from
the sol to the gel phase upon increasing the temperature, at values
included between 20
C and 30
C. Information on the sol-gel
transition kinetic, gelation time under physiological conditions
should be determined for each novel printing ink. A detailed
description of all these characterization steps is out of the scope of
the present chapter. However, a representative example of the set of
analyses required for the optimization of a novel thermogel can be
found in Gioffredi et al. [8].
3.1 Preparation
of Pluronic/Alginate
Solution
The Pluronic/alginate solution can be prepared according to the
so-called “cold method” proposed by Schmolka [11].
1. In a glass vial, add 100 mg of sodium alginate to 3.9 mL of an
ice-cold 0.2Â solution of DMEM under mild stirring.
2. While keeping the solution in an ice bath, slowly disperse 1 g of
Pluronic F127 in the alginate solution under mild stirring (see
Note 3).
3. Sterilize the solution by autoclaving, and store sterilized solution at 4
C.
3.2 Printing Process
The main steps of scaffold manufacturing by direct-write deposition are summarized in Fig. 1.
Subconfluent HDFs can be routinely processed by trypsinization and centrifugation to obtain a cell pellet (see Note 4).
1. Working in a biosafety cabinet, homogeneously suspend HDFs
in the sterile Pluronic/alginate solution kept at 4
C, at a final
concentration of 1 Â 10
6 cells/mL.
2. Transfer the bioink into the pressure-driven syringe.
3. Mount the syringe on the heated holder, connect it to the
pressure controller, and bring it at 37
C (see Note 5).
4. Extrude the bioink at a pressure of 1.2 bar (see Note 6) with a
needle/substrate relative speed of 10 mm/s. Several layers can
Direct-Write Deposition of Thermogels
139