Protocol to assess human glioma propagating cell migration on linear micropatterns mimicking brain invasion tracks

Abstract

Glioblastoma (GBM) cells invade the brain by following linear structures like blood vessel walls and white matter tracts by using specific motility modes. In this protocol, we describe two micropatterning techniques allowing recapitulation of these linear tracks in vitro: micro-contact printing and deep UV photolithography. We also detail how to maintain, transfect, and prepare human glioma propagating cells (hGPCs) for migration assays on linear tracks, followed by image acquisition and analysis, to measure key parameters of their motility. For complete details on the use and execution of this protocol, please refer to Monzo et al. (2016) and Monzo et al. (2021a).

Keywords: Biophysics; Biotechnology and bioengineering; Cancer; Cell Biology; Cell culture; Cell-based Assays; Microscopy.

Graphical abstract

Figure 1

Microcontact printing (A) Preparation of PDMS (soft lithography). (a) elastomer base and cross-linking agent are mixed on a weighing board at 10:1 ratio. (b) PDMS is degassed under vacuum for 30 min. (c) PDMS is poured on the silicon wafer. (d) PDMS is degassed under vacuum for 30 min. (e) PDMS is cured for 2 h at 70°C. (f) PDMS is unmolded from the silicon wafer with a scalpel. (B) Details of the various steps involved in microcontact printing. (a) PDMS is unmolded from the silicon wafer. (b) Stamps are cut in 1 cm² squares and plasma-cleaned for 3 min. (c) Stamps are coated with laminin for 30 min. (d) stamp is blown with an air-gun to remove the excess of laminin. (e) Stamp is leant and removed from the substrate to perform microcontact printing. (C) Main steps of microcontact printing: (a) The mixture of elastomer base and cross-linking agent (PDMS 10:1, previously degased) is poured on the silicon wafer (which has been silanized). (b) The PDMS on the silicon wafer is kept in a vacuum desiccator to remove all bubbles. (c) After curing, the PDMS is unmolded and cut into small stamps. The inset shows the gridded micropattern used in (Monzo et al., 2021a). (d) Stamps are plasma cleaned. (e) After incubation with laminin, the stamps are gently dried with an air-gun. (f) PDMS stamps are gently pressed onto the surface of the imaging dish to print laminin micropatterns (see inset). Scale bars are 100 μm.

Figure 2

Deep-UV photolithography (A) Principles of deep-UV lithography. PEGylated coverslips are placed on the chrome mask, bubbles are removed and the PLL-g-PEG is burned through the micropatterns drawn on the photomask, by deep UV, for 10 min. The coverslips are then mounted either on an imaging chamber or on a 35-mm dish with a hole, and coated with laminin at 37°C for 1 h. (B) Main steps of deep-UV lithography: (a) acid washed and air-dried coverslips are placed in a coverslip holder for plasma cleaning. (b) plasma cleaned coverslips are coated with PEG-g-PLL. (c) PEGylated coverslips are placed onto a drop of milliQ water covering the micropatterns on the chrome mask. (d) The chrome mask is placed in the deep-UV machine coverslips facing down. (e) The coverslips are lifted from the chrome mask using milliQ water. (f) Micropatterned coverslips are mounted in magnetic imaging chambers.

Figure 3

hGPC maintenance and transfection (A) hGPC tumor-spheres as seen under the microscope (10× objective) after 5 days in culture. (B) hGPCs after trituration (no spheres are present). (C) hGPCs as a confluent monolayer grown on a laminin-coated dish. (D) Neon pipette and Neon tip. (E) Neon Electroporator setup. (F and G) hGPCS transfected with GFP control vector 24 h after electroporation (GFP channel and phase contrast). Scale bars are 100 μm.

Figure 5

From manual tracks to mean speed and persistence calculation (A–F) visual instructions for mean speed, persistence and plot at origin representation. Error bars are S.D.

Figure 4

hGPC imaging and manual tracking (A and B) Setup used for time-lapse acquisition. (C and D) Manual tracking plugin in ImageJ/Fiji and tracks. Bars are 100 μm. (E) Maximum intensity projection: the images of each slice of the timelapse are overlaid on top of each other with ImageJ: Image>Stack> Z-project>MAX intensity. (F) Resulting maximum intensity projection of the temporal stack of hGPCs migrating on the gridded micropattern over 7 h movie. Bar is 100 μm.