Introduction of cell culture methods in channel slides

This application note shows how to grow adherent cells in cell culture microchannels. Cell seeding, medium exchange, and optical properties will be described. In addition, the main differences between cell culture channels and standard open well formats are shown.

To demonstrate cell seeding and Î¼-Slide treatment, a demonstration was performed using Î¼-Slide VI 0.4. A cell suspension (e.g., 3 x 105 cells/ml) was prepared as in the usual method and 30 Î¼l was added to the channel. Place the tip of the pipette on the inlet of the channel and point it toward the channel as shown. Quickly allocate and fill the entire channel.

After the cells were attached, as shown in the figure above, 60 Î¼l of cell-free medium was filled into each channel. Take care to avoid air bubbles. If the filling step is performed after cell adhesion, the cells are not washed out of the channel. If you want to fill the channel immediately after cell inoculation, carefully pipette.

Î¼-Slide VI 0.4 is filled with cells and medium during microscopic observation.

Fill the Luer reservoir of Î¼-Slide VI 0.4.

2. Medium exchange
a. Continuous Media Exchange - Recommended For continuous media replacement, first remove the old media from the storage. Then, an appropriate amount of fresh medium is added to one reservoir while aspirating from the other reservoir. Carefully use the cell culture pipette. We recommend a volume of at least 3 times the channel volume. Refill the reservoir.

The medium of continuous exchange medium is 3 times the volume.

b. Complete culture fluid exchange - only for expensive liquids
If you only want to change the liquid in the channel, first empty the channel. The tip of the pipette is then placed on the channel inlet and the liquid is carefully aspirated from the channel. Use a cell culture pipette to thoroughly remove all liquids. In order to refill the channel, fresh material of the channel capacity is injected directly into the channel. Avoid creating bubbles. Refill the reservoir holes on both sides.

Clearing the channel completely may result in bubbles forming after refilling.

The channel and reservoir holes are completely replaced.

3. Channel Slides and Porous Slides <br> In the following section, we compare the characteristics of Î¼-Slide VI 0.4 (cell culture channels) with Î¼-Slide 8 wells (open format).

For seeded cells, the main difference is the height of the liquid inside the structure. The small area sections of the two systems are shown below. The distance between the bottom and top of the Î¼-Slide VI 0.4 channel is 400Î¼m. The filled Î¼-Slide 8-well slide has no top structure. In an 8-well slide, the medium was approximately 0.3 millimeters and the channel slides were 7.5 times thinner than the open format 8-well slides.

For cell seeding, different cell densities must be applied to obtain the same amount of cells on the surface. In this example, the goal is to inoculate 25 cells per unit area. Since the height of the liquid in the Î¼-Slide 8 well is 7.5 times, the applied cell concentration must be lower than the same factor.

Liquid internal Î¼-Slide VI 0.4 and Î¼-Slide 8 holes based on different heights. In the above example, the same growth area and number of cells but different volumes result in different cell concentrations.

In order to achieve a considerable degree of fusion, we recommend using 3 ... 7 x 105 cells / ml (Î¼-Slide VI 0.4) and 4 ... 9 x 104 cells / ml (Î¼-Slide 8 wells). This is the same ~7.5 times between the two geometries. After cell adhesion, the number of cells per unit area is the same.

Due to the different heights, different cell concentrations produce the same level of cellular aggregation.

4. Advantages of Channel Î¼-Slide <br> Channel slides have a strong advantage over standard aperture formats.

a. Phase contrast microscopy is better. The channel geometry is very convenient to use phase contrast microscopy. Because there is no influence of the concave surface, the whole growth area can be observed by phase contrast technique.

Unlike open porous slides, channel slides do not interfere with the beam path of the phase contrast microscope for better phase contrast.

Open perforated plates (Fig. a, Fig. b): The concave surface affects the phase difference effect. Only the center provides high quality contrast.

Channel slides (Figure c, Figure d): In the channel geometry, the beam paths are always aligned. High quality phase contrast microscopy is available throughout the field of view.

b. Evenly distributed cells
In the channel, the homogeneity of the adherent cells is much better because of the small edge effect.

In the microscopic images below, the phase difference (a) and fluorescence (b) modes show the heterogeneity of cell distribution in the open wells (1-3) and the homogeneity in the cell culture channels (4-6).

1 open porous slide, edge position imaging
2 open porous slides, random position imaging
3 open porous slides, central part
4-channel slide, edge position imaging
5 channel slides, random position imaging
6-channel slide, center position imaging

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