Spheroid preparation and immunofluorescence protocol

Last edited Mon 09 Sep 2024

3D  in vitro  culture models better recapitulate physiological conditions than 2D cell cultures and can serve as a representative transitional stage between  in vitro  and  in vivo  experimental studies.

Spheroids are self-aggregated sphere-like formations that mimic certain aspects of the tissue architecture and microenvironment. Spheroids recapitulate essential aspects of cell-cell interactions and cell-extracellular matrix properties.

Spheroids can be prepared from a wide range of cells, such as tumors, embryonic cells, neural tissues, hepatocytes, mammary gland cells, etc., using different methods (non-adherent plates, hanging drop, microfluidics, spinners, agarose molds, bioprinting, etc.). They are increasingly used in drug screening, cancer research, toxicology and tissue bioengineering.

Stage 1 - Spheroid preparation in a 3D environment

Spheroids are 3D in vitro aggregates created using a wide range of methods. The type of 3D culture method will depend on the origin of the tissue/cells, cost, and purpose of the study. Independent of their formation method, spheroids can be included in different extracellular scaffolds to provide an environment that better mimics native tissue architecture. Spheroids can also be analyzed scaffold-free.

Materials required

• Non-adherent tissue culture plates (96-well plates or other)
• Sterile PBS 1%  Bovine Serum Albumin (BSA)
• Imaging grade plates (96-well plates or other)
• 3D scaffold

Steps

A wide range of methods can be used to create spheroids. For the round-bottom non-adherent tissue culture plate method, seed cells in wells.

Incubate at 37ºC in a cell culture incubator and verify good spheroid formation visually.

Recover the spheroids using wide-bore ice-cold tips (or cut tips with clean scissors) and place them into ice-cold tubes.

• Tubes should be precoated overnight with sterile 1% Bovine Serum Albumin (BSA)/phosphate-buffered saline (PBS) to reduce cell adhesion to the tubes.

Spin the tubes for 20 seconds at 4ºC (20 x g).

Discard the medium carefully by aspiration.

Add Matrigel™ or another 3D scaffolding material (synthetic scaffolds, collagen, hydrogels etc) to each tube.

Mix very carefully by gently pipetting up and down two times using wide bore tips.

Dispense the scaffold and spheroid mix into the wells of a PDL-coated imaging-grade plate (eg 96-well format).

Incubate for 15 min at 37ºC in a cell culture incubator.

Add warm culture medium (37ºC).

Incubate for 90 min at 37ºC in a cell culture incubator.

Stage 2 - Spheroid fixation

Like in 2D immunocytochemistry, fixation is an essential step in 3D immunofluorescence. The right fixation method preserves and stabilizes the cell morphology and architecture of the spheroid, allowing for the best results. Spheroid fixation deactivates proteolytic enzymes that could degrade the sample and protects the sample against decomposition from microorganisms.

Materials required

• PBS (ab285410)
• 4% paraformaldehyde and/or 100% methanol (chilled at -20°C)

Steps

Remove the culture medium and wash the samples three times using PBS.

Remove PBS and add cell fixative (preferably under a chemical fume hood). Among others, the two following methods could be used:

• 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature
• 100% methanol (chilled at -20°C) at 4°C for 5 min

Remove the fixative and wash plates three times with PBS.

Stage 3 - Permeabilization

Incubating the spheroids with a detergent allows antibodies to penetrate cell membranes, which is necessary for immunostaining intracellular targets. Different detergents have different permeabilization properties, some more efficient than others for a particular protein of interest. For example, proteins localized in the mitochondria and/or the nucleus often require the use of a detergent such as Triton X-100 for their detection.

For 3D biological samples in the extracellular matrix, the detergent Triton X-100 is recommended. The detergent used, the optimal percentage and the incubation time will depend on the protein of interest and should be optimized. Methanol fixation will also permeabilize the cells and higher antibody penetration may be achieved using this fixation method.

Materials required

• Permeabilization buffer: PBS 0.5% Triton X-100, PBS 2% Triton X-100, or PBS 10% Triton X-100

Steps

Remove PBS and add permeabilization buffer for one hour at room temperature using a flat shaker.

Stage 4 - Blocking, immunostaining, and imaging

A blocking step is necessary to reduce non-specific antibody binding. Serum and/or an abundant protein buffer is used to saturate potential non-specific binding sites. Ideally, the serotype included in the blocking buffer should match the species of the secondary antibody to achieve the best results. For spheroid immunostaining, all blocking, washing, and immunostaining steps should be performed with constant gentle mixing, eg, using a flat shaker at room temperature.

Materials required

• PBS containing 0.1% Tween, 1% Bovine Serum Albumin, 22.52 mg/ml Glycine, and 10% Goat Serum ab7481
• PBS
• Wash buffer: PBS containing 0.1% Tween
• Primary antibodies
• Secondary antibodies
• PBS containing 0.1% sodium azide

Steps

Remove the permeabilization buffer and add PBS containing 0.1% Tween, 1% Bovine Serum Albumin, 22.52 mg/ml Glycine, and 10% Goat Serum.

• Incubate overnight at room temperature on a flat shaker.

Wash the samples with PBS containing 0.1% Tween (wash buffer).

Add primary antibodies (unconjugated or conjugated) at the desired concentration.

• Titration of the primary/conjugated antibodies may be required to achieve satisfactory immunostaining. Incubate according to the manufacturer’s protocol.

Wash the samples four times with Wash buffer (four washes of one hour each).

If required, add secondary antibodies and/or a nuclear stain (eg, DAPI or Hoechst). Incubate overnight according to the manufacturer’s protocol.

Wash the samples four times with Wash buffer (four washes of one hour each).

PBS and add mounting media or storage buffer (PBS containing 0.1% sodium azide).

Store in the dark at 4ºC until required for imaging.

Image the samples using the appropriate excitation/emission filter sets and/or lasers.