Methacrylated Type I Collagen Kit for Crosslinkable Hydrogels
Full Product Description
Advanced BioMatrix offers LifeInk 100, a purified methacrylated Type I bovine collagen kit, which provides native-like 3D collagen gels with the unique attributes to be prepared at various concentrations and crosslinked to provide various gel stiffness.
The LifeInk 100 kit consists of purified methacrylated Type I bovine collagen as the core component with other support reagents in the kit as well as additional syringes, couplers and caps. Table 1 provides a list of the kit components.
|Item||Catalog No.||Package Size|
|Collagen, Type I, methacrylated, lyophilized||5198-100MG||100 mg|
|Acetic Acid, 20 mM solution||5079-50ML||50 ml|
|Neutralization solution||5155-10ML||10 ml|
|Photoinitiator solution||5200-1ML||1 ml|
The methacrylated Type I collagen is produced from telo-peptide intact bovine collagen where the collagen has been modified by reacting the free amines, primarily the ?-amines groups of the lysine residues as well as the ?-amines groups on the N-termini. Approximately 40% of the total lysine residues of the collagen molecule have been methacrylated.
The collagen is extracted from bovine hide and contains a high monomer content. The collagen starting material was isolated from a closed herd and purified using controlled manufacturing processes.
The 20 mM acetic acid solution is provided to solubilize the lyophilized methacrylated collagen at concentrations ranging from 3 to 8 mg/ml.
The neutralization solution consists of an alkaline 10X phosphate buffered saline (PBS) solution which provides physiological salts and pH in the final mixture.
The photoinitiator solution consists of Irgacure 2959 as a powder to be formulated in methanol, which allows UV crosslinking of the collagen at 365 nm.
(Note – Irgacure in solution has a shelf life of 2 weeks. Only dissolve required amount of Irgacure powder in a 10% solution)
Characterization and Testing
The formulated LifeInk 100 has the following characteristics as shown in Table 2.
|Purity by SDS PAGE electrophoresis||? 98%
(with ?85% collagen containing
within alpha, beta, and gamma band.
? 15% collagen contained within
bands traveling faster than alpha).
|Gel tube assay||? 10 minutes|
|Kinetic gel assay||? 10 minutes|
|Gel Stiffness||See graph 1 below|
|Differential Scanning Calorimetry (DSC) Thermal Analysis||Characteristic|
|Endotoxin||? 10 Eu/ml|
The product ships on frozen gel packs. Upon receipt, store the collagen, acetic acid and photoinitiator at
2 to 10°C. Do not freeze. Store the neutralization solution at room temperature.
The expiration dates are printed on the product label and certificate of analysis for each specific lot as appropriate. The expiration date is applicable when product is handled and stored as directed. After solubilization of the collagen with acetic acid, the collagen solution is stable for 2 months when stored at 2 to 10°C.
The LifeInk 100 kit is designed to provide collagen gels with varying gel stiffness based on collagen concentration and crosslinking. Graph 1 shows typical gel stiffness results of LifeInk 100 at varying concentrations with and without UV crosslinking.*
Preparation and Usage
Note: Employ aseptic practices to maintain the sterility of the product throughout the preparation and handling of the collagen and other solutions.
Note: It is recommended that the collagen and other working solutions be chilled and kept on ice during the preparation of the collagen.
Note: Vortexing is not recommended at any step.
- Add volume of 20 mM acetic acid (shown below) to the lyophilized methacrylated collagen to achieve desired concentration. Recommend concentration for 3D Bioprinting is 8-10 mg/ml for higher viscosity and print resolution.Table 3:
|Desired LifeInk® 100 Concentration||Volume of 20 mM Acetic Acid|
|3 mg/ml||33.3 ml|
|4 mg/ml||25.0 ml|
|6 mg/ml||16.7 ml|
|8 mg/ml||12.5 ml|
|10 mg/ml||10 ml|
- Mix on a shaker table or rotator plate at 2-10? until fully solubilized or overnight. Avoid formation of air bubbles as possible.
Note: The higher concentrations of collagen will take longer to solubilize.
- If crosslinking is desired, first add 1 mL of neat methanol to the small amber vial of photoinitiator containing 100 mg of Irgacure, and vortex.
- Calculate the volume of the photoinitiator required by multiplying the total volume of collagen required by 0.02.
- Add the calculated volume of chilled photoinitiator to the collagen solution and mix thoroughly.
Note: Keep the collagen mixture chilled throughout this process.
Note: If air bubbles are a concern, allow to sit on ice until the bubbles come to the surface.
- Dispense the collagen mixture into the desired printing mechanism (i.e.10cc syringe).
Note: To allow gelation of the collagen, neutral pH and physiological salts are required. Ensure support media contains 1X salts and is neutral pH.
- Print LifeInk® 100 acidic solution into the support media. Gelation of collagen should occur within 2 minutes. Allow collagen to gel prior to UV crosslinking for best results.
- If crosslinking is desired, place directly under a 365 nm UV light crosslinking source.
- To incorporate cells, we recommend two options:
- Employ a dual extruder system. Cells can be suspended in our LifeInk® 200 high concentration collagen #5202-1EA.
- Post UV-Crosslinking, seed cells on the printed construct.
Tunability of LifeInk® 100 product exposed to UV for 45, 90 and 600 seconds results respectively in a 22, 53, and 75% increase in gel stiffness.
Longer exposure allows more crosslinking, though each cell type withstands different degrees of UV light and free radicals (generated by the photoinitiator) that mediate crosslinking.