Biomaterials Lab 4: Collagen Hydrogel Application

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Questions and Answers

What is the primary role of the extracellular matrix (ECM)?

To store energy for cell metabolism

To provide structural support for the cells (correct)

To provide oxygen to the cells

To enhance DNA replication

Which collagen type is predominantly used in the preparation of collagen hydrogels?

Type I Collagen (correct)

Type IV Collagen

Type III Collagen

Type II Collagen

What happens to collagen fibers in hydrogels as the concentration of collagen increases?

The fibers become thinner and more fragile.

The fibers become thicker and less dense.

The fibers remain unchanged in structure.

The fibers become denser and pore size decreases. (correct)

What advantage do 3D cell culture environments have over 2D culture environments?

They more accurately replicate the actual microenvironment found in tissues. Signup and view all the answers

How does an increase in pH affect the diameter of collagen fibers?

The diameter decreases. Signup and view all the answers

How do hydrogels contribute to tissue engineering?

By mimicking the biophysical and biochemical characteristics of native tissues Signup and view all the answers

At what temperature does collagen hydrogel polymerization achieve a uniform fiber diameter?

37 °C Signup and view all the answers

What role does the ECM play in relation to cell attachment?

It provides a framework for cell attachment. Signup and view all the answers

What is the physiological pH value at which the hydrogel polymerization will be initiated?

7.4 Signup and view all the answers

Why is it important to study how changes in the ECM contribute to disease progression?

To understand the relationship between cells and their surrounding environment. Signup and view all the answers

What is observed in collagen hydrogels with low temperatures during polymerization?

Thicker fibers result from slower polymerization. Signup and view all the answers

In which application are collagen hydrogels NOT typically used?

As a source of nutrients for cell growth Signup and view all the answers

What step is included in the lab protocol before the preparation of collagen hydrogels?

Label and prepare tubes and petri dishes on ice. Signup and view all the answers

What is a critical factor in the design of synthetic ECM analogs using hydrogels?

Mimicking both biophysical and biochemical characteristics of native tissues Signup and view all the answers

Which type of collagen is specified for use in the hydrogel polymerization experiment?

Type I collagen from rat tail. Signup and view all the answers

What effect does increasing the polymerization temperature generally have on the fiber diameter?

The diameter decreases. Signup and view all the answers

What is the purpose of adding APTES and GA to the glass coverslips?

To immobilize hydrogels on the surface through covalent bonding Signup and view all the answers

How long should the petri dishes remain in the incubator for collagen hydrogel formation?

40-45 minutes Signup and view all the answers

What indicates the completion of gelation in the collagen solution?

The solution appears turbid (white) Signup and view all the answers

What was observed regarding pore size in collagen hydrogels of different concentrations?

1 mg/ml concentration showed bigger pores than 3 mg/ml Signup and view all the answers

What feature was the highest fiber density observed in?

3 mg/ml collagen hydrogels Signup and view all the answers

What is the effect of working on ice during the gel solutions preparation?

It slows down the polymerization Signup and view all the answers

Which component is excluded in the preparation of the collagen hydrogels?

Sodium Chloride Signup and view all the answers

What concentration of collagen hydrogel was specifically mentioned as part of the results?

1 mg/ml Signup and view all the answers

Study Notes

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.

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Description

This quiz focuses on the fabrication of collagen hydrogel and its significance as a cell culture system. Learn about collagen's role in the extracellular matrix and how mimicking these structures can advance tissue engineering and disease research.