Protocols

Seeding cells into the Random or Aligned Mimetix scaffold in a 12-well plate format.

• Precondition

The Mimetix scaffold needs to be wetted with ethanol in order to allow a cell suspension to access the pores. For best results, add 1 mL 20% ethanol to each well and allow the ethanol to soak into the membrane. This should wet the scaffolds evenly.

• Wash

Aspirate ethanol carefully from the edge of the insert, being careful not to touch the membrane, wash the scaffold 2x with PBS. Leave scaffold in cell culture medium until seeding.

• Seed

Add the cell suspension. We recommend seeding 50,000 to 100,000 cells suspended in 1 mL as a general guideline; or alternatively, whichever cell number the experiment to be conducted requires.

• Exchange Medium

For long-term experiments, the medium should be exchanged every 3 days. Half-dilutions of the medium (old with new) can be applied to keep the environmental change to the cells to a minimum.

Seeding cells into the Mimetix scaffold in a 6-well plate format with hanging inserts.

• Precondition

The Mimetix scaffold needs to be wetted with ethanol in order to allow a cell suspension to access the pores. For best results, add 1 mL 20% ethanol on top of each insert and allow for it to pass through the membrane. This should wet the scaffolds evenly. Discard ethanol at the bottom of the wells.

• Wash

Carefully add 1 ml cell culture medium and allow it to pass through the membrane; repeat twice while being careful to not let the scaffold dry out. Discard medium at the bottom of the wells.

• Seed

Add the cell suspension to the membrane. We recommend seeding between 50,000 to 100,000 cells suspended in 1 mL of medium per insert as a general guideline; or alternatively, whichever cell number the experiment to be conducted requires. Next, add sufficient media into the bottom of the well through the side gaps of the insert to reach the level of the scaffold, and add another 1 mL of medium on top of the scaffold in case the medium has passed through.

• Exchange Medium

For long-term experiments, the medium should be exchanged every 3 days. Half-dilutions of the medium (old with new) can be applied to keep the environmental change to the cells to a minimum.

Seeding cells into the Random or Aligned Mimetix scaffold in a 96-well plate format

• Precondition

The Mimetix scaffold needs to be wetted with ethanol in order to allow a cell suspension to access the pores. For best results, add 200 μL to each well and allow the ethanol to soak into the membrane. This should wet the scaffolds evenly.

• Wash

Aspirate ethanol carefully from the edge of the insert, being careful not to touch the membrane, wash the scaffold 2x with PBS. Leave scaffold in cell culture medium until seeding.

• Seed

Add the cell suspension. We recommend seeding 10,000 cells suspended in 100-200 μL medium as a general guideline; or alternatively, whichever cell number the experiment to be conducted requires.

CellTiter Blue assay (Promega)

• Add 10 µl of CellTiter Blue reagent per well of a 96-well plate.
• Incubate for 90 minutes at 37˚C.
• Place plate into plate reader and shake vigorously for several seconds.
• Measure fluorescence at 580-620 nm (excite at 530-570 nm) according to the manufacturer’s instructions.
• Please note: The Mimetix scaffold does not cause background fluorescence.

CellTiter Glo assay (Promega)

• Add 100 µl of CellTiter Glo reagent per well of a 96-well plate.
• Incubate for 10 minutes at 37˚C.
• Place plate into plate reader and shake vigorously for several seconds.
• Measure luminescence according to the manufacturer’s instructions.
• Please note: It is not necessary to use the “CellTiter Glo 3D” assay kit. The scaffold thickness is only 50 µm and thereby compatible with the standard CellTiter Glo assay kit.

MTT/MTS/WST-1 assay (various suppliers)

• Add 10-20 µL of the MTT/MTS/WST-1 assay reagent and incubate for 2-4 hours (refer to the respective instructions for further guidance).
• MTT only: Carefully aspirate the supernatant and add 200 µL DMSO or other recommended solvent.
• Shake vigorously for 2-5 min until a homogeneous solution is obtained and all crystals have dissolved (MTT only).
• Transfer 100-200 µL of the content of each well into a fresh, clear plastic tissue culture plate: the scaffold gives rise to a background signal in absorbance-based assays!
• Measure absorbances at 540 nm (MTT), 490 nm (MTS) and 450 nm (WST-1), respectively.

Confocal microscopy is a useful tool to obtain high-resolution images of cells within the Mimetix^® scaffold and to investigate their depth profile. But cells can be easily observed using a “classic” fluorescence microscope too.

In the example protocol in the next paragraph, nuclei are stained with TO-PRO^® 3 and F-actin with Alexa Fluor^® 488 Phalloidin (both from Life Technologies). The Mimetix scaffold used here is a bespoke product and has Rhodamine 6G incorporated into the fibres. Please contact us if this could also be of interest for your research.

For a typical experiment using Mimetix^® in a 12-well plate format, prepare a cell suspension at a concentration of 10⁵ cells/mL for short experiments (1-3 days) or 5 * 10⁴ cells/mL for longer experiments (up to 7 days) and add 1 mL into each well. Allow cells to adhere for 24 hours or overnight.

• To fix the cells in the scaffolds, aspirate medium and add 0.5 mL pre-warmed 4% paraformaldehyde in PBS to each well.
• Incubate for 15-30 min, then aspirate paraformaldehyde, wash 2x with PBS and add 0.5 mL 0.1% Triton X-100 in PBS to each well to permeabilize cell membranes (only necessary when performing Alexa Fluor® 488 Phalloidin staining).
• Incubate for 5 min, then aspirate Triton X-100, wash 2x with PBS and add 0.5 mL 0.5% BSA in PBS to each well to act as a blocking agent (only necessary when performing Alexa Fluor® 488 Phalloidin staining).
• Incubate for 30-60 min, then aspirate 0.5% BSA in PBS, wash 2x with PBS and add 200 µL Alexa Fluor® 488 Phalloidin working solution into each sample.

• Incubate for 30-60 min, then aspirate Alexa Fluor® 488 Phalloidin, wash 2x with PBS and add 1 mL TO-PRO® 3 working solution (1:10,000 in PBS) to each dish.
• Incubate for 10 min, then aspirate TO-PRO® 3, wash 2x with PBS and 1x with water to prevent the formation of salt crystals.
• Remove scaffold discs from wells and place on a glass slide, cell-seeded side facing up. Add a drop of mounting medium, place cover slip on top (dry off excess mounting medium with paper towel) and seal edges with nail varnish.
• Image stained cells within Mimetix® by exciting Alexa Fluor® 488 Phalloidin at 488 nm using an Argon laser and detecting at 500-550 nm, Rhodamin 6G-labelled fibres at 543 nm using a He-Ne laser and detecting at 550-615 nm, and TO-PRO® 3 at 633 nm using a He-Ne laser and detecting at 650-710 nm.

Cell recovery by trypsinisation

Cells can be removed from the Mimetix scaffold by trypsinisation and agitation. In general, longer incubation times and more rigorous shaking is required compared to a 2D culture.

Please optimise your trypsinisation protocol for each cell type to achieve full cell recovery.

Cell lysis for RNA extraction

• Add 60 µl RLT (RNeasy kit, Qiagen) per well.
• Leave the plate on ice for 5 minutes.
• Shake the plate vigorously for 2 minutes in a plate shaker.
• Leave the plate on ice for a further 30 minutes.
• Carry on with the RNA isolation or freeze down the samples for later use (-80˚C).

Cell lysis for protein extraction

• Add 30-60 µl RIPA buffer and incubate for 5 min.
• Shake the plate vigorously for 2 min.
• Incubate the plate on ice for 20 min.
• Centrifuge the samples at 4˚C for 10 min and transfer to a clean tube.

Please note: Shaking gives better results than pipetting up and down. Shaking the plate for 1 min or less is not sufficient. Tearing the scaffold off with a pipette tip does not lead to higher protein yield.