Ref No: BPR-5212-100MG
Date: 28-MAR-2023
Author: MSL, VK. Version: 1

RECONSTITUTION AND BIOPRINTING PROTOCOLPhotoHA® Stiff
This is a suggested procedure, please adjust it according to your experimental needs.
To maintain the sterility of the product, work under sterile conditions.

Protocol aimMaterials needed

The aim of this protocol is to provide instructions on
how to reconstitute PhotoHA® Stiff (methacrylated
hyaluronic acid, also known as HAMA) to yourdesired concentration using 1x PBS as well as
mixing with cells and bioprinting. The addition of aphotoinitiator (PI) and the use of 365 or 405 nm LED
modules
ensuresstableandcontrolled
photocrosslinking of PhotoHA constructs for 3D cellculturing. This protocol has been optimized for use
with the BIO X and BIO X6 systems.

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PhotoHA® Stiff (100 mg)*
1x PBS
Photoinitiator (PI)*Two 3 mL syringes
0.22 µm sterile syringe filter
Two 15 mL Falcon tubes
Female/female Luer lock adaptors*
Cell suspension and cell culture medium
3cc amber cartridge* with tip cap
Bioprinter (BIO X or BIO X6)
22G conical bioprinting nozzle
Well plate*The product can be purchased in the CELLINKshop at www.cellink.com/shop/.

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Protocol
This protocol describes the reconstitution of 100 mg of PhotoHA® Stiff to obtain bioinks of different concentrations.
We recommend concentrations from 5 to 30 mg/mL (0.5–3.0%). The reconstitution solution contains thephotoinitiator LAP, which allows photocrosslinking of the PhotoHA at 365 or 405 nm to provide various gel stiffness.
Different LAP concentrations and light exposure times will directly affect the final stiffness of the bioink. Protect thebioinks from light when mixed with LAP. Employ aseptic practices to maintain the product’s sterility throughout thepreparation and handling of PhotoHA and other solutions.

1.

Preparation of the reconstitution solution with PIMATERIAL
1x PBS
Photoinitiator (PI), e.g., LAP
20 mL syringe
0.22 µm sterile syringe filter
Two 15 mL Falcon tubes

DESCRIPTION•
Weigh the desired amount of PI in a 15 mL Falcon tube. See Table 1 for suggested LAP concentrations.
•Add 15 mL of 1x PBS to the tube and slightly shake until dissolution.
Note: Remember to protect all PI containing solutions from light. Ensure PI is properly dissolved before
proceeding.
•Transfer the PI solution (Reconstitution agent + PI) to a 20 mL syringe and sterile filter it into anothersterile 15 mL Falcon tube using a 0.22 µm sterile syringe filter.
Table 1. Suggestions of LAP concentrations for PhotoHA bioink.

2.

LAP concentration in bioink

LAP mass for 15 mL of 1x PBS

0.10% (1 mg/mL)
0.25% (2.5 mg/mL)

15 mg
37.5 mg

Reconstitution of PhotoHA® StiffMATERIALPhotoHA® StiffPI solution

DESCRIPTION•

Add the prepared PI solution to the 100 mg PhotoHA® Stiff vial. See Table 2 for suggested bioink
concentrations.
Note: If not reconstituting the total mass of the vial, aliquot the necessary amount and store the remaining
PhotoHA® Stiff at -20°C. Adjust the PI volume solution accordingly.
•Mix on a shaker table or rotator plate until fully solubilized (30-60 minutes) at 2-10°C. Alternatively,
place the bottle in the fridge and gently rotate it a couple of times until complete dissolution.
Note: Solubilization times may vary depending on the desired concentration and volume of PBS added.
•Store the reconstituted PhotoHA solution at 2-10°C if needed.

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Table 2. Suggestions of final bioink concentrations for reconstitution of 100 mg PhotoHA® Stiff.
Concentration of PhotoHA bioink (w/v)
1% (10 mg/mL)
2% (20 mg/mL)
3% (30 mg/mL)

3.

Volume of PI solution needed
10 mL
5 mL
2 mL

Mixing PhotoHA with cells

MATERIAL
PhotoHA solution
Two 3 mL syringes
Cell suspension
Female/female Luer lock adaptor
3cc amber cartridge with tip cap

DESCRIPTION•Transfer the PhotoHA solution into a 3 mL syringe using the following procedure: remove a syringe
plunger → cap the syringe → transfer the bioink to the syringe → insert the plunger → flip the syringe →release the tip cap to evacuate the air.
•Prepare a cell suspension with the desired number of cells dispersed in cell culture medium. The
volume of the cell suspension should be 10% of bioink volume. Transfer the cell suspension to a 3 mLsyringe.
•Connect the PhotoHA solution syringe to the syringe with cell suspension using a female/female Luerlock adaptor.
•Mix ten parts of PhotoHA solution with one part of the cell suspension without introducing air bubbles tothe mixture. If using other ratios, recommended printing parameters (Table 3) should be adjusted
accordingly. For detailed instructions see the Mixing Cells and Bioink Protocol.
Note: To avoid an air gap when mixing the bioink and the cell suspension, carefully pre-fill the Luer lockadaptor with PhotoHA solution before attaching the syringe to the cell suspension.
•Mix the bioink with the cell suspension by gently pushing them back and forth between the syringes.
•Transfer the mixture to a 3cc amber cartridge by connecting the syringe to the cartridge using the Luer
lock adaptor.
•Add a tip to the amber cartridge containing the mixture and keep it at room temperature.

4.

Bioprinting PhotoHA

MATERIAL
Bioprinter (BIO X or BIO X6)
3cc cartridge with PhotoHA mixed with cells
22G conical bioprinting nozzle
Well plate

DESCRIPTION••••

Attach the 22G conical nozzle to the 3cc cartridge with the PhotoHA mixed with cells.
Place the cartridge in the printhead of the bioprinter.
Calibrate the bioprinter to the correct position of the well plate.
In the pressure settings, test the flow of the bioink. Start with low pressure and gradually increase until
bioink extrudes from the nozzle.

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Note: The initial pressure for the liquid to come out of the nozzle is higher than the pressure used during
printing. Reduce the pressure at this stage to not lose material with the first droplet during printing.
•Print droplets with the desired size. Use the suggested parameters (see Table 3) as a guide for yourprinting to achieve droplets of approximately 10-15 µL.
Note: If printability is not as desired, adjust the pressure up/down to extrude more/less material at different
extrusion times.
Note: Different nozzle sizes and concentrations of bioink can be used if desired. Adjust the parameters forprinting accordingly.

Table 3. Recommended starting parameters for droplet printing of PhotoHA at different concentrations*.
Concentration ofPhotoHA (mg/mL)

Pressure for printing(kPa)

Extrusion time (s)Initial pressure fortesting bioink flow (kPa)

10130.8

1630

170.822*Bioprinting parameters were obtained with PhotoHA prepared at a specific concentration and further mixed with cells (10:1). The
cell density and different proportionality may affect the final viscosity of the bioink. If changes are made, printing parameters need
to be adjusted accordingly.

5.

Crosslinking and incubation

MATERIAL
Well plate with printed droplets
Bioprinter (BIO X or BIO X6) with LED modules of choice for photocuring
Cell culture medium

DESCRIPTION•Photocure the droplets using the LED module. See Figure 1 for the comparative stiffness of PhotoHA
constructs at different concentrations.
Note: It is recommended to use the 405 nm photocuring module for 30 seconds instead of 365 nm, if
possible, when photocuring PhotoHA with LAP photoinitiator. Overexposure at the 405 nm and 365 nm
wavelength might damage the cells.
Note: Time and distance will directly affect the stiffness of the material and the viability of cells. Use adistance between 3 and 5 cm from the plate to the LED module, and a maximum of 30 seconds of lightexposure.
•Submerge the constructs in the cell culture medium and place them in the incubator (37°C, 5% CO 2 and95% relative humidity) for 3D cell culturing according to your application.

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Figure 1. PhotoHA Stiff stiffness at different concentrations. Rheology tests were performed on a Discovery Hybrid
Rheometer, HR 20, TA instruments, with a UV exposure at 405 nm (LAP as a PI), for 30 seconds and at 5 cm
distance from the light source.

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