IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

We detailed a protocol to establish a 3D system using an extracellular metrics along with collagen one for analyzing formation and gene expression through osteogenesis. Extracellular metrics can increase the transparency of the collagen one form gel and it is suitable for exploring the dendrite formation for imaging techniques. To begin slowly pipette 0.9 milliliters of basement membrane matrix into a new 15 milliliter centrifuge tube on ice and keep it aside.

Next, remove a T25 floss of IDG SW3 cultured cells in four mililiters of complete medium supplemented with interferon gamma from the incubator. After removing the culture medium, wash the cells with PBS pH 7.4 with a pipette. Then trypsanize the cells with 0.5 milliliters of 0.25%trypsin EDTA at 37 degrees Celsius for 30 seconds.

Inactivate trypsin by adding 3.5 milliliters of complete medium. Transfer four milliliters of the Celsius pension to a 15 milliliter centrifuge tube. Spin down the cell suspension at 300 G for five minutes at four degrees Celsius.

After discarding the supernatant, resuspend the cell palette in one milliliter of the complete medium on ice. Count the cells using a hemo cytometer and adjust the final cell density with the complete medium to four times 10 to the fifth cells per milliliter. Add 0.1 milliliters of the prepared cell suspension to the 0.9 milliliters of the extracellular matrix kept on ice.

Mix well by gently pipetting up and down. Gently mix one milliliter of the cell matrix mixture and one milliliter of the previously prepared collagen one by pipetting up and down on the ice. Pipette 0.5 milliliters of the final mixture into each well of a 24 well plate and incubate at 37 degrees Celsius for one hour to form a cell gel mixture.

After incubation, add 0.5 milliliters of osteogenic medium to the cell gel mixture in each well. To start the osteogenic differentiation at 37 degrees Celsius, consider this as day zero. Every two days, replace half the medium with fresh osteogenic medium and continue the culturing for 35 days.

Remove the calcium AM and ethidium one stock solutions from the freezer and allow them to warm to room temperature. Add four microliters of the two millimolar ethidium one stock solution and five microliters of the four millimolar calcium AM stock solution to two milliliters of DPBS and mix well to get two micromolar calcium AM and four micromolar ethidium one as a working solution. To perform cell staining on day one and day seven of culturing, pipette 0.5 milliliters of the working solution into the wells of the 24 well plate and incubate for 30 to 45 minutes at room temperature to stain the cell gel matrix.

Add approximately 0.5 milliliters of fresh DPBS to a new 35 millimeter glass bottom culture dish and cover the dish to prevent contamination or drying of the samples. Using elbow tipped forceps, carefully transfer the stained cell gel matrix into the DPBS containing culture dish without damaging or shearing the cell gel matrix. View the labeled cells under a laser confocal fluorescence microscope.

Place the culture dish containing the stained cell gel matrix on the microscope stage. Select the regions of the cell gel matrix to be scanned through the eyepiece using a 10x objective. To set the microscope scanning, select the optical filters.

View calcium as green with a standard fluorescent band pass filter and ethidium one as red with filters for propidium iodide or Texas red dye. Select the line mode for scanning, then click the acquisition mode to set the airy units to one au. Select 8-bit data depth and an image resolution of 1024 by 1024 pixels.

Then select the line step and set scan speed to five. To collect the Z stack of images, define the top and bottom positions of the cell gel matrix to be scanned by focusing through the sample while continuously scanning according to the green channel signal. Once the top and bottom of the sample have been specified, select the desired scanning frame and start scanning.

The sample is scanned from top to bottom with the selected settings generating a gallery of images. To identify the viable cells, select one image slice. The green and red channels indicate live and dead cells respectively.

To identify the cell dendrite, select the single green channel to generate 3D reconstructions with the image acquisition software, a series of rainbow pseudo color images indicate the depth information. The dendrites at the she's bottom are displayed in red, whereas those at the top are displayed in blue. On different days of culturing, remove the culture medium and wash the gels in the plate twice with DPBS.

Add 0.5 milliliters or 4%paraformaldehyde in DPBS to the well to fix the cell gel matrix in the plate for 10 minutes at room temperature. Remove the paraformaldehyde in the well and wash the cell gel matrix two more times with DPBS in the plate as demonstrated previously, and leave 0.5 milliliters of DPBS in each well for imaging. Place the plate on the stereo microscope stage and select the optimal position through the eyepiece using a 0.5x objective.

Image, the full field view of the cell gel matrix in the plate individually using automatic exposure under the bright field. On day 35 of culturing, check the liquid nitrogen levels and start the XRF system. Open the sample room and transfer the gels with elbow tipped forceps from the plate to the middle of the sample stage of the instrument.

Close the sample room and wait for 30 minutes to allow the instrument to cool before use. Set the exposure time to 35 milliseconds. The spectrum range to zero to 40 kilo electron volts and the electric current to 770 microamps for acquisition set up.

Moving the sample stage, choose three to five scanning sites for analysis. Select the elements calcium and phosphorus. Start the detection and export the results.

On different days of culturing. Wash the culture medium removed cell gel matrix twice with ice cold DPBS as demonstrated previously. Extract the total RNA from the cell gel matrix using the phenol chloroform isoamyl alcohol method.

Then reverse transcribe two micrograms of total RNA to CDNA with random hexamer primers. Place the tube on a thermocycler and perform PCR amplification. After PCR analyze the fold changes with the two delta CT method.

The live dead cell staining visualized under a confocal laser microscope showed all the cells were calcium AM positive and almost no ethidium one positive cells indicating that the gel system is highly suitable for osteocytogenesis. The cellular dendrites gradually extended into a network in the osteogenic medium by day seven. A pseudo color image of the day seven culture displays the cell androids at different depths at the bottom and at the top of the gel.

Full field images of the gel with cells and gel without cells in a 24 well plate at the indicated times are imaged. The transparency of the gel matrix continued to decline until it became opaque at day 35 unlike the cell-free gel matrix. The XRF spectrum of the opaque gel on day 35 indicated that the gel was completely filled with calcium and phosphorus deposits.

The expression of several marker genes analyzed by real-time PCR showed that the mRNA levels of podoplanin and dentine matrix protein one continually increased from day one until day 21 and the mRNA level decreased after day 21. The mRNA levels of fibroblast growth factor 23 and sclerostin continually increased during all stages. Collagen one and the basement membrane metrics gel quickly and room temperature.

Therefore, all the tips and tubes used must be pre chilled unless otherwise indicated. One can observe any interesting events during osteogenesis by imaging techniques. It is easier to localize the molecule action during dendrites formation and elongation.

Journal (Bioengineering)

Journal (Bioengineering)

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

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