Ref No: BPR- IKD119261001
Date: 03-JAN-2023
Author: PT,EZ,VK. Version: 2

NEUTRALIZATION AND BIOPRINTING PROTOCOL

Coll 1 Solution
This is a suggested procedure, please adjust according to your experimental needs.
To maintain the sterility of the product, work under sterile conditions.

Protocol aim

Materials needed

The aim of this protocol is to provide instructions forneutralization and subsequent bioprinting of theneutralized Coll 1 Solution. This document covers
the bioprinting of Coll 1 droplets with embedded cells
and cell post-seeding on printed Coll 1 constructs.
The biomaterial thermally gels at 37°C.

••••••••••••••

Coll 1 Solution (10 mL at 10 mg/mL)*
Collagen Buffer (5 mL)*
Ice bath
1x PBS
1M NaOH
Container for mixing (15 mL Falcon tube or 5mL Eppendorf tube)
3 mL syringes with Luer lock connections
Female/female Luer lock adaptors*
Cells* + culture medium*
Cartridges, 3 cc*
Sterile conical bioprinting nozzles*
Temperature-controlled printhead* (optional)
BIO X*, BIO X6* or INKREDIBLE-series* 3DbioprinterWell plate or mold*The product can be purchased in the CELLINKshop at www.cellink.com/shop/.

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Protocol for neutralization
During the whole procedure keep all materials on ice. Cool down the Coll 1 solution after every component isadded.

1.

Preparing Coll 1MATERIAL
Coll 1 Solution
Collagen Buffer
Ice bath

DESCRIPTION••••

Place the vial of Coll 1 Solution and the Collagen Buffer on ice to keep them cool.
CS is the concentration of the original Coll 1 Solution (10 mg/mL).
Record the desired final volume of the bioink (VINK, mL).
Record the desired final Coll 1 concentration after neutralization (C F).

Note: CF and CS cannot be the same, otherwise the solution would not be neutralized (refer to Table 1).

Table 1. Preparation of Coll 1 biomaterial with different concentration.
VINK , mL
12.

CF , mg/mL
864

CS , mg/mL
10𝑉𝐶𝑜𝑙𝑙 1 , µL
800
600
400

𝑉𝐶𝐵 , µL
1239262

Calculations for neutralization

DESCRIPTION
CF × VINK•

Volume of needed Coll 1 Solution: 𝑉𝐶𝑜𝑙𝑙 1 (𝑚𝐿) =•Volume of Collagen Buffer: 𝑉𝐶𝐵 (𝑚𝐿) = 𝑉𝐶𝑜𝑙𝑙 1 × 0.154•

Volume of 1M NaOH: 𝑉𝑁𝑎𝑂𝐻 (𝑚𝐿) = 𝑉𝐶𝑜𝑙𝑙 1 × 0.025•Volume of 1x PBS to reach CF: 𝑉𝑃𝐵𝑆 (𝑚𝐿) = 𝑉𝐼𝑁𝐾 − 𝑉𝐶𝑜𝑙𝑙 1 − 𝑉𝐶𝐵 − 𝑉𝑁𝑎𝑂𝐻

3.

CS

Neutralization

MATERIAL
1x PBS
1M NaOH
Container for mixing
Ice bathwww.cellink.com

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𝑉𝑁𝑎𝑂𝐻 , µL
20
15
10

𝑉𝑃𝐵𝑆 , µL
57293
528

DESCRIPTION••Mix 𝑉𝐶𝑜𝑙𝑙 1 and 𝑉𝐶𝐵 in a sterile container with a suitable volume capacity by vortexing or pipetting up anddown. Be extra careful with keeping the solution cool once the 𝑉𝐶𝐵 is added as Coll 1 may self-assemble
if heated.
Add 𝑉𝑁𝑎𝑂𝐻 to the mixing container.

Note: The natural material oxidation after the vial opening may affect the material pH. Therefore, if using
previously opened vial, it is recommended to proceed by adding 𝑉𝑁𝑎𝑂𝐻 by smaller volume steps, until thecolor of the solution corresponds to a pH between 6.9-7.3 (refer to Figure 1). Note down the volume used.
Cool on ice.
•Add 𝑉𝑃𝐵𝑆 to the mixing container and homogenize by pipetting up and down or by vortexing.

Figure 1. Illustration of solution color correspondence to pH [1].
[1]https://bit.ly/3lmRoCv

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Protocol for bioprinting of neutralized Coll 1 solution
Make sure to follow the neutralization protocol above prior to following the bioprinting protocol. This bioprinting
protocol works best using the BIO X/BIO X6 equipped with the Temperature-controlled printhead. If using theINKREDIBLE system, the bioprinting procedure should be performed fast to prevent the solution from warming
and gelling in the cartridge during the experiment.

1.

Prepare Coll 1 for mixing

MATERIAL
Neutralized Coll 1 solution
3 mL syringe with Luer lock connections

DESCRIPTION•••

2.

Cool down the neutralized Coll 1 on ice for 10 min to make sure it remains in the liquid state.
Transfer the solution into a 3 mL syringe using the following procedure: remove the syringe plunger →cap the syringe with a tip cap → pour the Coll 1 solution in the syringe → insert the plunger → flip thesyringe → release the tip cap to evacuate the air.
If cells will be post-seeded on printed constructs, move directly to Step 3.

Mixing Coll 1 with cells

MATERIAL
Cell suspension in a syringe
Cooled Coll 1 solution
Female/female Luer lock adaptor

DESCRIPTION•Mix ten parts of Coll 1 solution with one part of cell suspension without introducing air bubbles to themixture. For detailed instructions see the Mixing cells with bioink Protocol.
••Attach the Coll 1 solution syringe to the syringe with cell suspension using a female/female
Luer lock adaptor.
Carefully mix the solution with the cell suspension by gently pushing them back and forth
between the syringes.

Note: Suggested cell suspension density is 5x106 cells/mL to 10x106 cells/mL.
Note: To avoid an air gap when mixing the solution and the cell suspension, carefully pre-fill the Luer lockadaptor with Coll 1 solution before attaching the syringe with the cell suspension.

3.

Preparing for print

MATERIAL
Temperature-controlled printhead (optional)
Cartridge, 3 ccColl 1 mixed with cells
Conical bioprinting nozzles

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DESCRIPTION•

If using the BIO X/BIO X6, pre-cool the Temperature-controlled printhead to 5°C. If using theINKREDIBLE-series, cool down the cartridge on ice if needed.

Note: The bioprinting temperature can be increased to 10°C or 15°C, but this will reduce the time available
for Coll 1 bioprinting process prior to its inadvertent self-assembly.
•4.

Transfer the solution (with or without cells) to the cartridge, cap it with a bioprinting nozzle of choice andplace it in the printhead.

Printing

MATERIALBIO X, BIO X6 or INKREDIBLE series bioprinterWell plate or mold

DESCRIPTION•

5.

Print droplets with the desired size in a mold or well plate.

Crosslinking

DESCRIPTION•Coll 1 can be crosslinked via thermal gelation.
••

Warm the bioprinted construct to 37°C until gelation occurs, approx. 10-15 min. The BIO X/BIO
X6 heated print bed or incubation can be alternatively used. Refer to Figure 2 for thermalgelation behavior of Coll 1 with different final concentration.
If cells were mixed with Coll 1 solution prior to bioprinting, move directly to Step 7.

Note: The crosslinking time might be adjusted based on the construct thickness.

Figure 2. Thermal gelation of neutralized Coll 1 solution with different collagen concentrations (C F) indicated asstorage moduli increase over time at 37°C.

6.

Cell post-seeding

MATERIAL
Cell suspension

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DESCRIPTION•

7.

Dispense the cell suspension in the middle of the printed hydrogel. Suggested cell suspension density:
20x103 cells/cm2 to 50x103 cells/cm2 (a highly concentrated cell suspension is suggested).

Incubation

MATERIAL
Cell culture medium

DESCRIPTION•

Add the desired medium to submerge the constructs and place in incubator.

Note: Ensure that the bioprinted constructs are crosslinked and do not dissolve in warm media.
•Incubate the constructs in cell culture medium in standard culture conditions (37°C, 5% CO 2 and 95%
relative humidity) or according to your application.

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