This protocol make it possible to see different types of cultures and observe their interactions with extra-cellular matrix. The main advantage of this method is that the matrix you obtain is regenerated by fibroblast and it will be quite similar to the ones generated in vivo. We believe that this sickness come irrelevant when using fibroblast obtaining from baking dishes.
Establishing a matrix from these cells could enlighten us how it can effect, for instance, persistent situation. These methods can also be applied to, for example, physical studies to survivors from matrix proprieties. Visualization of this protocol it's important to observe a pipetting technique of the steps that you have to regent.
When trying these methods for the first time it will probably take more time than expected. Our advice is to prepare as much as you can in advance and using duration periods for getting ready for next steps. Begin by preparing 0.2%gelatin, 1%glutaraldehyde and one molar ethanolamine solutions according to the manuscript directions.
Making sure to run each solution through a 0.22 micrometer filter prior to use. Next, prepare ascorbic acid and lysis buffer. Dilute the PBS pen-strep as described in the manuscript and prepare DMEM with 10%FBS.
Prepare heat-denatured 2%BSA by adding 2 grams of BSA to 100 milliliters of sterile water and warming it in a boiling water bath for seven minutes. Then, supplement FBS with penicillin, streptomycin, gentamicin, and amphotericin B.Culture recombinant telomeres transfected immortalized human foreskin fibroblasts and DMEM with 10%FBS and maintained them at 37 degrees Celsius and 5%carbon dioxide. To establish the primary fibroblast culture, cut the tissue samples into small pieces of approximately two to three milliliters cubed and seed them in FPS with a high concentration of antibiotics.
When the first fibroblasts appear, replace the medium with FBM medium for cell maintenance. Add two milliliters of 0.2%gelatin solution to each well of a six-well plate and incubate it for one hour for 37 degrees Celsius or overnight at four degrees Celsius. After the incubation, aspirate the gelatin and wash each well with two milliliters of PBS.
Add two milliliters of 1%of glutaraldehyde to each well and incubate the plate at room temperature for thirty minutes which will cross-link the gelatin. Aspirate the glutaraldehyde and wash the wells in two milliliters of PBS for five minutes. To block the remaining glutaraldehyde, add two milliliters of one molar ethanolamine and incubate the plate at room temperature for thirty minutes.
Aspirate the ethanolamine and repeat the washes with PBS. Add one milliliter of DMEM with 10%FPS. If the medium immediately turns pink, remove it, wash the wells once with two milliliters of PBS and add another milliliter of DMEM.
Seed one milliliter of fibroblast suspension with 5 x 10 to the fifth cells in each well and culture the cells until 100%confluence is reached. Then remove the medium and replace it with DMEM with 10%FBS and 50 micrograms per milliliter ascorbic acid. Replace the medium every two days for six days.
Two days after the last ascorbic acid treatment remove the medium and wash the wells with two milliliters of PBS. After the washes, slowly add one milliliter of preheated lysis buffer and incubate the plate at room temperature for five to ten minutes until the fibroblasts are lysed. Without removing the lysis buffer, carefully add two milliliters of PBS to each well and then aspirate approximately 2.5 milliliters.
Repeat this process twice for a total of three washes. After the final wash, add two milliliters of PBS with pen-strep. Seal the plate with film and keep it at four degrees Celsius for up to three months.
Grow HUVEC cells and EMB-2 with 2%FBS until maximum confluence. Then replace the medium with EBM-2 without FBS for eight hours. To prepare the matrices for cell seeding, remove them from the refrigerator and leave them at room temperature for one hour.
Block the matrices by add two milliliters of heat-denatured 2%BSA and incubate them at 37 degrees Celsius for one hour. Then, aspirate the BSA and wash them with two milliliters of PBS. Seed 2 x 10 to the fifth HUVEC cells to each well of the plate coated with fibroblast-derived matrices.
Incubate the cells at 37 degrees Celsius for 16 hours then examine the tube-like structures under a standard bright field microscope at 20-40x magnification. This protocol was used to examine the affects of PDGF treatment on fibroblast-derived matrix formation. The BGH third fibroblasts that were incubated with PDGF showed a more aligned cell distribution in matrices than those incubated without PDGF.
Collagen 1 and fibronectin protein expression was increased in matrices derived from PDGF-stimulated fibroblasts and consequently matrix thickness was increased. Moreover, directionality histograms demonstrate that collagen 1 and fibronectin show parallel patterns. When HUVEC cells were seeded on to decellularized matrices derived from PDGF-stimulated fibroblasts they developed more capillary-like structures than under none stimulated conditions.
Confirming that matrices derived from PDGF-BB stimulated fibroblasts promote endothelial cells activation. When performing this procedure, it's important to prepare solutions in advance and make sure the cells are ready before starting. Besides endothelial cells studies matrices can also be seeded with epithelial cells and the reaction to external factors like condition in media or cytotoxic drugs can be observed.
Generation of extra-cellular matrix is crucial in the design of tumor macroenvironment experiments.
