Collagen Concentration to the size of Pores in the Gel Matrix
Question:
Do you have any information relating collagen concentration to the size of pores in the gel matrix?
Answer:
See reference below: http://www.nature.com/labinvest/journal/v88/n2/full/3700703a.html#fig5
In summary:
Methods
Preparation of Collagen Gel:
Collagen type I gels were prepared by mixing appropriate volumes of collagen solution with 10X PBS) and 1 N NaOH in pre-cooled eppendorf tubes. The final concentrations of the collagen in the gel matrix were varied from 0.7 to 2.5 mg/ml to obtain an appropriate collagen sieve diameter. An average diameter of 5–7 μm is considered as optimal for the present application (average bacterial diameter, 2 μm and Ped-2E9 cell diameter, 10 μm). After addition of the reagents the gel solutions were kept at 37°C for 15–30 min to complete the gelation.
Determination of Gel Matrix Pore Size
by Cryo-Scanning Electron Microscopy:
To determine the pore size of the gel matrix sieves (with different collagen concentrations from 0.7 to 2 mg/ml), we performed a cryo-scanning electron microscopy (SEM) analysis with minimal
dehydration of the gel matrix. The gel preparations were mounted on a slit holder and plunged into liquid nitrogen slush. A vacuum was pulled and the samples were transferred to the Gatan Alto 2500 (Pleasanton, CA, USA) pre-chamber (cooled to approximately −60°C). After the samples were cut with a microtome with a cold knife, the samples were sublimated at −85°C for 30 min followed by sputter coating for 100 s with platinum. The samples were then transferred to the microscope cryostage (approximate temperature: −130°C) for imaging. The SEM images were taken with an FEI NOVA
nanoSEM (FEI, Hillsboro, OR, USA) using ET and TLD detectors operating at 5 kV accelerating voltage, with approximately 6 mm working distance and the
magnifications were varied between × 2500 and × 50 000.
Results
The collagen matrix microenvironment was fabricated such that the pore diameter of the gel matrix sieve is maintained between 5 and 7 μm. The cryo-SEM photographs confirmed that a collagen gel concentration of 1.5 mg/ml optimally achieves the desired pore size for the purposes of this study.
On average:
1.0 mg/ml, give ~ 10 μm pore size
1.5 mg/ml, give ~ 6 μm pore size
2.0 mg/ml, give ~ 2 μm pore size
