LifeInk 200 purified, highly concentrated Type I Collagen bio-ink was purchased from Advanced Biomatrix (San Diego, California). The cell-laden bio-ink was realized in accordance with the manufacturer’s instructions. Briefly, highly concentrated collagen was first neutralized by connecting the provided syringe through a sterile coupler to a 3 ml syringe containing 200 μl of sterile 1 M hydrochloric acid (HCl; Fisher Scientific Italia, Rodano, Italy), carefully avoiding introducing air. The plungers of the two syringes were then pushed back and forth at least 40 times to ensure thorough mixing of the collagen and HCl. The same procedure was repeated with the collagen syringe connected to a 3 ml syringe containing 200 μl of sterile 1 M sodium hydroxide (NaOH; Carlo Erba, Milan, Italy). After neutralization, the cell addition process was performed by dispensing concentrated chilled MSCs (38 × 106) to a 3 ml sterile syringe, connected through a sterile coupler to the neutralized collagen syringe. The two plungers were then pushed back and forth 40 times to ensure thorough mixing. The collagen bio-ink was kept chilled at all times.
Lifeink 200
Manufactured by Advanced BioMatrix
Sourced in United States
LifeInk 200 is a bioprinting instrument designed for the fabrication of 3D cell-based constructs. It utilizes a pneumatic-based extrusion system to deposit biomaterials and living cells in a precise and controlled manner. The device features a temperature-controlled print bed and supports the use of various bioinks to create complex tissue structures.
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Lab products found in correlation
4 protocols using lifeink 200
1
Neutralized Collagen Bio-ink for MSC Encapsulation
2
Biomimetic Scaffold Construction with Aligned Nanofibers
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The construction of the biomimetic scaffold was based on incorporating multilayers of aligned PCL/CNT nanofibrous mats between the bioprinted hydrogel layers. The insertion of nanofibrous layers was performed during the hydrogel bioprinting process. For the printing of hydrogel layers, a highly concentrated type 1 collagen bioink (Lifeink® 200, Advanced BioMatrix) was homogeneously blended with Phosphate Buffered Saline (PBS) (5:2 volume ratio of Lifeink® 200:PBS) to obtain a bioink at a final concentration of 25 mg·mL−1 as recommended by the manufacturer.
3D bioprinting was conducted using an extrusion 3D bioprinter (BioEdPrinterV4, BioEdTech) with the following optimized parameters: 4°C printhead temperature, 37°C printing platform, 6 mm·s−1 nozzle movement speed, 0.26 mm nozzle inner diameter, 0.2 mm layer height, and 40% infill density. After the bioprinting process, the scaffolds were kept at 37 °C for 45–60 min for the collagen hydrogel crosslinking.
To compare the effect of aligned nanofibrous mats between the hydrogel, scaffolds were constructed based on different ratios of nanofiber layers: one nanofiber mat layer to two bioprinted hydrogel layer (hydrogel:nanofibers 2:1), one nanofiber mat layer to four layers of bioprinted hydrogel (hydrogel:nanofibers 4:1), bioprinted hydrogel without nanofibers (hydrogel:nanofibers 1:0).
3D bioprinting was conducted using an extrusion 3D bioprinter (BioEdPrinterV4, BioEdTech) with the following optimized parameters: 4°C printhead temperature, 37°C printing platform, 6 mm·s−1 nozzle movement speed, 0.26 mm nozzle inner diameter, 0.2 mm layer height, and 40% infill density. After the bioprinting process, the scaffolds were kept at 37 °C for 45–60 min for the collagen hydrogel crosslinking.
To compare the effect of aligned nanofibrous mats between the hydrogel, scaffolds were constructed based on different ratios of nanofiber layers: one nanofiber mat layer to two bioprinted hydrogel layer (hydrogel:nanofibers 2:1), one nanofiber mat layer to four layers of bioprinted hydrogel (hydrogel:nanofibers 4:1), bioprinted hydrogel without nanofibers (hydrogel:nanofibers 1:0).
3
Collagen Type I Bioink Preparation
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All collagen type I bioink (LifeInk 200, Advanced Biomatrix). was prepared as previously described13 (link). Briefly, the stock 35 mg/mL LifeInk was mixed with syringes in a 2:1 ratio with 0.24 M acetic acid to produce a 23.33 mg/mL acidified collagen bioink. The bioink was then centrifuged at 3000g for 5 min to remove bubbles. For printing, the collagen bioink was transferred to a 2.5 mL gastight syringe (Hamilton Company).
4
Chondrogenic Differentiation of hNCs
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hNCs were trypsinized and resuspended in a defined serum-free chondrogenic media
(SFM) composed of DMEM, 100 U/ml penicillin and streptomycin with 2 mM
L-glutamine (Life Technologies, all), 100 mM HEPES, insulin-transferrin-selenium
(ITS) + 1, 0.1 µM dexamethasone, 0.1 mM ascorbic acid 2-phosphate, and 0.1 mM
L-proline at a concentration of 0.875 × 107 cells/ml. The cell
suspension was diluted in a 1:10 ratio with type I collagen gel (3.5 wt%,
Lifeink 200, Advanced Biomatrix, LOT: 5202-1KIT, USA) to create a final
concentration of 8.75 × 106 cells/ml. The resulting cell-laden bioink
is a neutralized type I collagen solution that is thermoresponsive and can
polymerize at 37°C.
(SFM) composed of DMEM, 100 U/ml penicillin and streptomycin with 2 mM
L-glutamine (Life Technologies, all), 100 mM HEPES, insulin-transferrin-selenium
(ITS) + 1, 0.1 µM dexamethasone, 0.1 mM ascorbic acid 2-phosphate, and 0.1 mM
L-proline at a concentration of 0.875 × 107 cells/ml. The cell
suspension was diluted in a 1:10 ratio with type I collagen gel (3.5 wt%,
Lifeink 200, Advanced Biomatrix, LOT: 5202-1KIT, USA) to create a final
concentration of 8.75 × 106 cells/ml. The resulting cell-laden bioink
is a neutralized type I collagen solution that is thermoresponsive and can
polymerize at 37°C.
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