Biomaterials Lab 4: Collagen Hydrogel Application

Course Information

• Course Title: Biomaterials
• Course Number: ENGR-UH 4810
• Professor: Jeremy Teo
• Instructor: Shafiya Sabah
• Semester: Fall 2024

Lab 4: Fabrication of Collagen Hydrogel and its Use as a Cell Culture System

Collagen and the Extracellular Matrix

• Collagen is the most abundant structural protein in the extracellular matrix (ECM).
• The ECM is a complex network of proteins and macromolecules surrounding cells in tissues.
• ECM provides structural support for cells.
• ECM helps cells attach to and communicate with nearby cells.
• ECM plays a role in cell growth and movement.

Mimicking the Extracellular Matrix

• Creating in-vitro tissue models that mimic the structural, mechanical, and physical properties of the ECM is desirable.
• This helps better understand cell behavior.
• It enables the study of ECM changes contributing to disease progression.
• Mimicking native tissue, hydrogels are a key material in developing synthetic ECM analogs for regenerative medicine.

3D Cell Culture Systems

• Cells in 3D culture environments differ morphologically and physiologically from cells in 2D culture environments.
• 3D cell culture systems more accurately represent the microenvironment found in tissues.

Collagen Hydrogels

• Collagen hydrogels are used as 3D cell culture substrates, scaffolds in tissue engineering, and to study tumor progression.
• Collagen type I is the primary type in most hydrogels, readily extracted from animal sources (bovine, porcine, rat tail tendon, fish).
• Collagen hydrogel polymerization rate is slow in low temperatures, resulting in thicker fibers; at 37°C, a uniform diameter is obtained.

Collagen Hydrogel Preparation

• 3D collagen matrices with variable fiber thickness and length can be prepared in vitro by altering polymerization conditions (e.g., collagen concentration, pH, temperature).
• Higher collagen concentration leads to denser fibers and smaller pores.
• Increasing pH leads to thinner collagen fibers.

Lab Protocol

• Preparation:
  • Prepare ice containers.
  • Label microcentrifuge tubes and petri dishes with concentration (3mg/ml and 1mg/ml) and group number.
  • Place coated coverslips on petri dishes.
  • Add solutions to the tubes in the specified order:
    • Column 1: Coll (mg/ml)
    • Column 2: 0.1% Acetic Acid
    • Column 3: Coll
    • Column 4: Buffer
• Procedure:
  • Pipette gel solutions onto coverslips (50µL per coverslip).
  • Place petri dishes in a 37°C incubator for 40-45 minutes.
  • Remove petri dishes from the incubator; the gels appear turbid (white), indicating gelation.

Coverslip Preparation

• Glass coverslips are coated with APTES (3-aminopropyl)triethoxysilane and glutaraldehyde (GA).
• The APTES-GA coating immobilizes hydrogels on coverslip surfaces through covalent bonding.
• Calculations for creating 1% and 0.5% GA solutions are provided for APTES and GA coating.

Results

• 1 mg/mL collagen concentration hydrogels have larger pores than 3 mg/mL collagen hydrogels.
• 3 mg/mL collagen hydrogels exhibit higher fiber density.
• Cell morphology and cytoskeleton structures differ in 1 mg/ml and 3 mg/ml environments.