Controlled Cell Adhesion With ibidi Micropatterning

The ibidi µ-Patterning technology enables spatially defined cell adhesion for various 2D and 3D cell culture applications.

Miniaturized adhesive patterns (e.g., lines, squares, or dots) are irreversibly printed on the non-adhesive Bioinert surface of the ibidi Polymer Coverslip, allowing for precisely controlled cell adhesion. The µ-Patterns are dry-stable, sterile, and ready to use.

The µ-Pattern, the Bioinert surface, and the ibidi Polymer Coverslip are all optimized for high resolution imaging and microscopy.

ibidi's Ready-to-Use µ-Patterning Products

µ-Slides With Single-Cell
µ-Pattern

For various single cell assays (e.g., CAR-T cell activity assay)

µ-Slides With Multi-Cell
µ-Pattern

For culturing 3D cell aggregates (e.g., spheroids or organoids) or cell patch monolayers

µ-Slides With Test µ-Patterns

For determining the most suitable micropatterning geometry for the respective application

The Bioinert Surface

• Long-term stable, and biologically inert surface
• Superior to the standard ultra-low attachment (ULA) surfaces: no cell or protein adhesion (full passivation)
• Layered onto the ibidi Polymer Coverslip—the highest optical quality for imaging

Learn more here.

Specifications of µ-Patterns

Application Examples

Single-Cell Arrays

The size of the µ-Pattern can be adapted to the morphology of the cell type of interest, so that an array of single cells can be conveniently analyzed using applications such as high-resolution imaging.

Single-cell array with RCC26 tumor cells. µ-Patterning was done with RGD adhesion spots on the Bioinert surface. Spot size 40 µm x 40 µm. Phase contrast microscopy, 10x objective lens.

Multi-Cell Arrays

By using different geometries and sizes of the µ-Patterning, multi-cell arrays can be performed with defined adhesion for various applications, such as high-resolution imaging.

Multi-cell array with Rat1 cells. µ-Patterning was done with RGD adhesion spots on the Bioinert surface. Spot size 200 µm x 200 µm. Phase contrast microscopy, 4x objective lens.

Spheroid Generation

Defined adhesion spots, surrounded by Bioinert, are able to catch all adherent single cells from a cell suspension. Bioinert is fully non-cell-attachable. This forces all cells to aggregate to each other at the adhesion spots, thus forming spheroids in a defined and controllable way.

Suspension of NIH-3T3 cell line seeded on 200 µm adhesion spots, 64 hours live cell imaging, phase contrast, 4x objective lens.

Spheroid and Organoid Culture

The size and the cell adhesion properties of the µ-Patterning can be adjusted to hold 3D spheroids and microtissues in position. Using this setup, cells in 3D can be studied during proliferation, differentiation, invasion, and migration.

NIH-3T3 3D spheroids (left) plated on adhesive, Cy3-labeled spots (right) with a 300 µm diameter. 10x objective lens.

Generation of 3D spheroids from HT-1080, MCF-7, and NIH-3T3 cells. The spheroids were generated using the agarose assay, then put on 300 µm adhesive spots for positioning. Phase contrast, 4x objective lens.

Immunofluorescence staining of spheroids from RCC26 (left) and Rat1 cells (right) in in the µ-Slide VI ^0.4 Luer, patterned with 200 µm circles at 600 µm distance. The cells were stained with phalloidin (green) and alpha-tubulin (red). Nuclei were stained with DAPI (blue). Widefield fluorescence microscopy, 4x and 10x objective.

Spheroid Culture Under Constant Media Circulation

By creating micropatterns within a µ-Slide VI ^0.4 Luer, aggregates on the pattern can be constantly supplied with fresh media by perfusing the system with the ibidi Pump System.

Fibroblasts (3T3) under flow conditions at 3 dyn/cm² compared to the static control, cultured in the µ-Slide VI ^0.4 Luer for 14 days.

By applying a constant media flux around the cell aggregates, spheroids become more compact and round.

Fibroblast (3T3) aggregates under flow conditions at 3 dyn/cm² form a compact and round spheroid over time.

3T3 fibroblasts were seeded on a patterned µ-Slide VI ^0.4. A shear stress of 3 dyn/cm² was applied 7 days after cell seeding. 15 h time lapse microscopy, 4x objective.

CAR-T Cell Killing Assays

CAR-T cells represent a promising new cancer therapy tool. Live cell imaging allows to analyze T cell/cancer cell interaction in real time with single cell resolution. However, analysis of confluent cell layers is very time-consuming and therefore not possible in high throughput screens. To facilitate high throughput label-free analysis of T cell potency in a live cell imaging setup, we generated arrays of homogenously distributed cancer cells. By combining optical analysis and advanced image processing, cytotoxic T cell activity over time on a single cell level can be evaluated without the use of any labeling.

Single Cells in a 2D Environment

Time lapse microscopy of a CAR-T cell killing assay with RCC-26 tumor cells and JB4 T cells on a single cell pattern. Data were analyzed using FastTrack AI by MetaVi Labs.

Multicell Spots in a 3D Collagen Matrix

RCC-26 cancer cells immobilized on multi cell pads. Effector T cells applied in a collagen I matrix (Collagen Type I, Rat Tail) induce apoptotic body formation of cancer cells.

Scientific Posters

Micropatterned Adhesion Sites for Spheroid Cultivation Under Flow (PDF)

Presented at the Annual Meeting of the Biophysical Society 2020, San Diego, USA.

A Micropatterning-Supported High Throughput CAR-T Cell Potency Assay With Single Cell Resolution (PDF)

Presented at the ASCB|EMBO Meeting 2019, Washington DC, USA.

Controlled Cell Adhesion With ibidi Micropatterning (PDF)

Presented at the µTAS Conference 2019, Basel, Switzerland.

Application Notes

AN 65: Cell Adhesion on ibidi μ-Patterns: Parameters and Optimization (PDF)

Movies

MV 41: Advanced ibidi Technologies: Micropatterning, 3D Cell Culture and Flow Applications