Protocol for 3D tumor spheroid generation, immunostaining, and imaging through comparative approaches

Summary

3D tumor spheroids are complex systems that can simulate protein localization in vivo. Here, we present a protocol for spheroid generation coupled with immunolabeling strategies. We describe steps for spheroid formation, block preparation, and immunostaining. We then detail procedures for image capturing and analysis. We provide guidance in maintaining spheroid cytoarchitecture, ensuring optimal antigen retrieval, and achieving consistent antibody accessibility. This protocol enables researchers to choose the most suitable approach for high-quality imaging to study 3D protein localization.

For complete details on the use and execution of this protocol, please refer to Adhikari et al.¹

Graphical abstract

Highlights

• •Steps for preparing 3D tumor spheroids from T47D and Huh7 cells
• •Guidance for visualizing proteins by immunofluorescence or immunohistochemistry
• •Procedures for comparative analysis of the multicellular spheroids

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

3D tumor spheroids are complex systems that can simulate protein localization in vivo. Here, we present a protocol for spheroid generation coupled with immunolabeling strategies. We describe steps for spheroid formation, block preparation, and immunostaining. We then detail procedures for image capturing and analysis. We provide guidance in maintaining spheroid cytoarchitecture, ensuring optimal antigen retrieval, and achieving consistent antibody accessibility. This protocol enables researchers to choose the most suitable approach for high-quality imaging to study 3D protein localization.

Before you begin

To understand the differential gene and protein expression in a 3D perspective, the tumor spheroid models are appropriate systems to work with. This protocol presents a comprehensive methodological framework of spheroid formation techniques with spatial protein analysis methods. Here, a systematic evaluation of each of the technique’s suitability for two different cell line systems, including scalability requirements and experimental endpoints, while simultaneously providing comprehensive guidance on visualization strategies, has been described. The comparative approaches employed in the protocol, including the pros and cons of the methodologies, will enable the researchers to make an appropriate decision based on their experimental requirements. This systematic approach would reduce optimization time, improve reproducibility, and maximize data quality in 3D tumor spheroid models.

Growth and maintenance of cells

Timing: 4 days

Day 1–4.

• 1.Thaw the frozen cell stock (T47D and Huh7) quickly by incubating it in a 37°C water bath.
• 2.Take out the content of cryovials to a 15 mL centrifuge tube.
• 3.Add 10 mL of complete RPMI 1640 media (25 mM Glucose, 10% FBS, 1X penicillin streptomycin with 1X amphotericin B, 1X Glutamax and 1X non-essential amino acids) for T47D and complete DMEM (5.5 mM Glucose, 10% FBS, 1X penicillin streptomycin with 1X amphotericin B, 1X Glutamax and 1X non-essential amino acids) for Huh7 in 15 ml centrifuge tube to the 1 mL freezing medium containing the cells.

Note: In general, T47D and Huh7 are cultured in complete RPMI 1640 media and complete DMEM media, respectively. That is why we added complete DMEM media to dilute the freezing mixture. The use of media depends on the cell type. In general, the media that is used for the culturing of a particular cell should be used here in this step.

• 4.Spin at 500×g for 5 minutes.
• 5.Aspirate the supernatant and resuspend the cell pellet in fresh growth media.
• 6.Plate the cells in a T25 flask and incubate at 37°C incubator with 5% CO₂.
• 7.Grow the cells until they reach 80% confluency, then split them in a ratio of 1:4.

Seeding of the cells for spheroid formation

Timing: 2 days

Day 5–6.

• 8.When the cells grow to a minimum confluency of 70%–80% (for T47D and Huh7 cells), take out the cells from the incubator.

Note: The percentage of confluency depends on cell type. In general, the confluency should not exceed 80%–90%.

• 9.Wash the cells with 1X phosphate-buffered saline (PBS).
• 10.Add 1 ml 0.25% trypsin and incubate for 2 to 3 minutes at 37°C.

Note: The percentage of trypsin and the duration of incubation depend on the adherent property of the cell type. For low adherent cells, 0.05% trypsin should be used.

• 11.Quench the action of Trypsin by adding 2 mL of media and flush the cells properly to detach them from the flask.
• 12.Take the cell suspension in a 15 mL centrifuge tube and centrifuge at 500×g for 5 minutes.
• 13.Aspirate the media and resuspend the cells in growth media.
• 14.Count the cells and seed about 1 million appropriate number of cells for either hanging drop, AggreWell plate or ultralow attachment plates.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
DMEM F12 Media	Gibco	12500062
EGF	Thermo Scientific	E9644
FGF	Gibco	15940877
Insulin	Gibco	RP-10908
B27 supplement	Gibco	12587-010
BSA	HiMedia	MB083-100G
0.25% Trypsin-EDTA	Gibco	25200056
Triton X-100	Sigma-Aldrich	X100-100ML
Ultralow attachment plate 6 well	Corning	3471
Spheroid culture plate	Thermo Scientific	174925
AggreWell 400 24-well plate Starter Kit	STEMCELL technologies	34450
Cell culture plate	Thermo Scientific	150464
DMEM media powder	Gibco	31600-034
RPMI 1640 media powder	Gibco	31800-022
MEM Non-essential amino Acid solution	Gibco	11140050
GlutaMAX Supplement	Gibco	35050061
Antibiotic-Antimycotic	Gibco	15240062
FBS	Gibco	10270106
DAB chromogen A	Millipore	71897
DAB Chromogen B	Millipore	71898
Paraffin	SRL	66400
PBS	Gibco	10010023
KH2PO4	Sigma-Aldrich	P5655-500G
K2HPO4	Sigma-Aldrich	P3786
Trizma base	Sigma-Aldrich	T1503
EDTA	Sigma-Aldrich	E9884-500G
HCL	Supelco, Merck	DB3D730159
Human insulin	Sigma-Aldrich	I9278-5ML
DPX mounting media	Merck	DL2DA21793
Microscopic slide	Riviera	72900–135
Coverslips	Bluestar	18mm size, #1 (0.13–0.16mm thick)
Trypan blue	HiMedia	TCL046-100ML
HEPES	Sigma-Aldrich	H4034-500G
NaHCO3	Sigma-Aldrich	S8875-1KG
Tween 20	Pro-pure	M147-1L
NP40	Merck Millipore	492016-100ML
Paraformaldehyde	Sigma-Aldrich	158127
Ethanol	Supelco	K52477883022
Xylene	SRL	54717
Hematoxylin	Sigma-Aldrich	H3136-25G
Hydrogen peroxide blocking buffer	Merck	925B-04
15 mL centrifuge tube	Thermo Scientific	339650
1.5 mL microcentrifuge tube	Tarson	500010
Glycerol mounting medium - anti-fade with DAPI	Abcam	Ab188804
100 mm cell culture dish	Thermo Scientific	150460
Agarose	Sigma-Aldrich	A9539
Deposited data NA
Experimental models: Cell lines
T47D	ATCC	HTB-133
Huh7	Merck	01042712
Antibody
Anti-Actin (working dilution 1:500)	Abcam	ab3280
Anti-H3 (working dilution 1:500)	Abcam	ab1791
Anti-GAPDH (working dilution 1:500)	Abcam	ab9485
Goat anti-rabbit IgG (H+L) Alexa Fluor 488 (working dilution 1:1,000)	Invitrogen	A32731
Goat anti-mouse IgG (H+L) Alexa Fluor 555 (working dilution 1:1,000)	Invitrogen	A32727
Software and algorithms
NIS Elements	Nikon	https://www.microscope.healthcare.nikon.com
Image J	NIH	https://imagej.net/ij/
Others
37°C 5% CO2 incubator	Eppendorf	Model: CO170S-230-1000 SL No: 41217
Centrifuge	Eppendorf	Model-5810R SL No: 5811XQ242484
Cell counter	Invitrogen	Countess 3 SL No: 2187A22064265
Heat block	Labnet International, Inc.	D1100-230V
Microwave oven	LG	MC3286BRUM
Microtome	SLEE	CUT 5062
Light microscope	Thermo Fisher Scientific	EVOS XL Core
Confocal microscope	Nikon	ECLIPSE Ti, A1R MP, Spinning disk,
SureTemp dual convection incubator	Benchmark Scientific	H2505-40
4°C refrigerator	Celfrost	NFG 450
Nutator	Benchmark Scientific	R5010
Orbital shaker	Benchmark Scientific	BT1011

Materials and equipment

Complete DMEM Media

Reagent (stock concentration)	Final concentration	Amount
DMEM powder	NA	1 pack
Glucose	5.5 mM	1 g
HEPES	15 mM	3.5 g
Sodium bicarbonate	45 mM	3.75 g
GlutaMAX Supplement (200 mM)	2 mM	10 ml
MEM Non-Essential Amino Acids Solution (100X)	1X	10 mL
Antibiotic-Antimycotic (100X)	1X	10 mL
FBS	10%	100 mL
Water	NA	870 mL

Store at 4°C for up to one month.

Complete RPMI 1640 media

Reagent (stock concentration)	Final concentration	Amount
RPMI 1640 powder	NA	1 pack
Glucose	25 mM	4 g
HEPES	15 mM	3.5 g
Sodium bicarbonate	45 mM	3.75 g
GlutaMAX Supplement (200 mM)	2 mM	10 mL
Antibiotic-Antimycotic (100X)	1X	10 mL
FBS	10%	100 mL
Water	NA	870 mL

Store at 4°C for up to one month.

Spheroid-forming medium

Reagent (stock concentration)	Final concentration	Amount
DMEM F12 Media	–	1 pack
EGF (100 μg/mL)	20 ng/mL	200 μL
FGF (50 μg/mL)	20 ng/mL	400 μL
Insulin (10 mg/mL)	5 μg/mL	500 μL
B27 supplement (50X)	1X	20 mL
BSA (10%)	0.4%	8 mL
Water	NA	971 mL

Store at 4°C for up to one month.

Tris-EDTA Buffer (pH 9)

Reagent (stock concentration)	Final concentration	Amount
Tris	10 mM	1.21 g
EDTA	1.3 mM	0.37 g
Water	NA	1000 mL

Adjust the pH to 9.0 with 1 M NaOH. Store the buffer at 20°C–25°C temperature.

CRITICAL: NaOH is harmful to skin; wear gloves.

1X PBST Buffer

Reagent (stock concentration)	Final concentration	Amount
PBS (1X)	1x	1990 mL
Tween 20 (20%)	0.1	10 mL

Store it at 20°C–25°C temperature for more than a year.

Phosphate Buffer (pH- 7.0)

Reagent (stock concentration)	Final concentration	Amount
Na2HPO4	0.5 mM	0.069 g
NaH2PO4	0.5 mM	0.060 g
Water	NA	1000 mL

Store it at 20°C–25°C temperature for more than a year.

Step-by-step method details

Spheroid formation

Timing: 6–8 days

Timing: 6 days (for step 1)

Timing: 6 days (for step 2)

Timing: 8 days (for step 3)

These steps describe the different procedures for the generation of tumor spheroids.

• 1.Hanging droplet method.
  Day 7–12.

  • a.
    After harvesting, dissolve the cells in complete DMEM culture media.
  • b.
    Dilute the cells to an appropriate density and dispense 20–50 μL droplet with 20–200 μL pipette to the inner side of the lid of a 100 mm cell culture dish.

    Note: The number of cells in a droplet depends on the doubling time of the cell. For T47D cell number should be between 1,000 and 2,000 and for Huh7, it should be between 2,000 and 3,000 cells per droplet.

  • c.
    Fill the cell culture dish with 15 mL 1X PBS.
  • d.
    Carefully place the cover with the cell droplets on top of the cell culture dish.
  • e.
    Incubate the cells in droplets in 37°C, 5% CO₂ incubator for 4 to 5 days (Figures 1A and 2A) (troubleshooting 2).
• 2.AggreWell or microwell plate.
  Day 7–12.

  • a.
    Add 500 μL of AggreWell rinsing solution in each well of a 12-well AggreWell plate and centrifuge at 2000xg for 2 minutes and incubate at 25°C temperature for 5 minutes.
  • b.
    Discard the rinsing solution and add 500 μL of spheroid-forming media.
  • c.
    Centrifuge at 2000xg for 2 minutes and discard the media.
  • d.
    Add 1 mL spheroid-forming media in each well and centrifuge at 2000xg for 2 minutes.
  • e.
    After harvesting, add an appropriate number of cells in 1 mL spheroid-forming media in each well.

    Note: The seeding cell number in each well depends on the doubling time of the cell type and also on the duration of the experimental procedure. For 5–7 days of seeding to harvesting, around 40,000 cells of T47D and 60,000 cells of Huh7 are good to start with. One should standardize the initial cell number according to their cell type, experimental duration and experimental treatment, if the experimental treatments can influence cell proliferation.

  • f.
    Centrifuge at 2000xg for 3 minutes and incubate in 37°C, 5% CO₂ incubator for 7 days for spheroid formation (troubleshooting 2).
  • g.
    Refresh 500 μL of new spheroid-forming media on each alternating day.
  • h.
    Take the spheroids with the spheroid forming medium in a 15 mL Falcon by pipetting.

    Note: The tip should be cut half a centimeter to avoid any harm to the spheroid formed.

  • i.
    Centrifuge at 500xg for 5 minutes.
  • j.
    Discard the supernatant and dissolve it in the desired cell culture media (Complete RPMI for T47D and Complete DMEM for Huh7).
  • k.
    Add 2 mL fresh cell culture media (Complete RPMI for T47D and Complete DMEM for Huh7) in each well of a 6-well ultra-low attachment plate and carefully add the spheroids dropwise with a one ml pipette (The tip should be cut half a centimeter to avoid any harm to the spheroid formed).
    Incubate in 37°C, 5% CO₂ incubator for 7 days for spheroid maturation (Figures 1B and 2B).
• 3.Low attachment plate.
  Day 7–15.

  • a.
    Add 200 μL of spheroid-forming media in each well of a 96-well ultra-low attachment plate.
  • b.
    Add appropriate number of cells in each well and mix properly with a pipette.

    Note: The seeding cell number in each well depends on the doubling time of the cell type and also on the duration of the experimental procedure. For 5–7 days of seeding to harvesting, around 1000 cells of T47D and 2000 cells of Huh7 are good to start with. One should standardize the initial cell number according to their cell type, experimental duration and experimental treatment, if the experimental treatments can influence cell proliferation.

  • c.
    Incubate in 37°C, 5% CO2 incubator for 5–7 days for spheroid formation.
  • d.
    Discard 100 μL of old media and add 100 μL of new spheroid-forming media on each alternating day.
  • e.
    After 5–7 days, take the spheroid out in a 15 mL centrifuge tube discard as much media as possible with a 200 μL pipette.

    Note: The tip should be cut half a centimeter to avoid any harm to the spheroid formed and centrifuge at 200xg for 5 min.

  • f.
    Carefully add 200 μL of fresh cell culture media in each well of a 96 well plate and incubate in 37°C, 5% CO₂ incubator for 7 days for spheroid maturation (Figures 1C and 2C) (troubleshooting 2).

Figure 1.

Different methods of T47D spheroid formation

Bright field image of spheroids of T47D cells developed following hanging droplet method (A), AggreWell method (B) and 96-well low-attachment plate method (C) imaged under 10x objective lens. Scale bar 100 μm.

Figure 2.

Different methods of Huh7 spheroid formation

Bright field image of spheroids of T47D cells developed following hanging droplet method (A), AggreWell method (B) and 96-well low-attachment plate method (C) imaged under 10x objective lens. Scale bar 100 μm.

Spheroid immunostaining

Timing: 2–5 days

Timing: 2 days (for step 4)

Timing: 2 days (for step 4a)

Timing: 2 days (for step 4b)

Timing: 5 days (for step 5)

Timing: 2 days (for step 5a)

Timing: 2 days (for step 5b)

Timing: 3 days (for step 5c)

Timing: 3 days (for step 5d)

These steps describe different methods of spheroid staining to visualize protein expression and localization in both 2D and 3D space.

• 4.
  Staining without paraffin block preparation.

  Note: These steps elaborate on the procedure of staining different proteins in an intact spheroid to visualize their localization and expression in 3D space.

  Day 15–16.

  • a.
    “In-tube staining” for immunofluorescence.

    Note: This section describes the procedure for staining multiple intact spheroids with different antibodies to study protein localization and expression in 3D space.

    • i.
      Harvest the spheroids into a 1.5 mL microcentrifuge tube, then centrifuge at 100xg to pellet them and aspirate the supernatant¹ (troubleshooting 1).

      Note: Always use a 1 mL pipette tip and cut the pipette tip to reduce damage while pipetting or collecting the spheroid in a 1.5 mL microcentrifuge tube. For hanging droplet and 96-well low-attachment plate, slowly pipette the spheroid and for AggreWell flash the well slowly 2 to 3 times and pipette it into a 1.5 mL microcentrifuge tube.

      Note: In-tube staining is compatible with all the above three mentioned spheroid-forming protocols. The yield and integrity of the spheroids are comparatively better in “low attachment 96 well plate”.

    • ii.
      Rinse the spheroid pellet two times with 1X PBS.

      Note: Always cut the pipette tip to reduce damage while pipetting.

    • iii.
      To fix the spheroid, add 4% cold (or 25°C temperature) Paraformaldehyde in PBS and incubate for 10–15 minutes in a nutator at 20 rpm at 25°C temperature.
    • iv.
      After this, wash the spheroid pellet twice with 1X PBS.

      Pause point: Can be stored in 1X PBS at 4°C for couple of days.

    • v.
      To permeabilize the spheroids, add 1% Triton-X 100 in PBS and incubate for 10 minutes in a nutator at 20 rpm at 25°C temperature.
    • vi.
      Following permeabilization, perform blocking by adding 5% BSA in PBST and keeping it at 25°C temperature for 1–2 hours in a nutator at 20 rpm.
    • vii.
      Once blocking is complete, add the primary antibody in PBST and incubate in a nutator at 20 rpm at 4°C for 12–16 hours.

      Note: The dilution of the antibody should be standardized for different proteins. The amount should be between 3 and 5μg. The dilution of any antibody should be standardized in 2D cell culture system of the same cell line from which the spheroid is made.

    • viii.
      The next day, discard the primary antibody and wash the spheroids twice with PBST for 10 minutes each.
    • ix.
      Add the secondary antibody at a dilution of 1:1500 in PBST, and incubate for 2 hours in a nutator at 20 rpm at 25°C temperature away from light.

      Note: After this step, if the secondary antibody is conjugated with light sensitive fluorophore, everything is suggested to be performed in low illumination.

    • x.
      Wash twice with PBST for 10 minutes each on a nutator set on 25 rpm speeds.
    • xi.
      Wash the spheroid once with PBS.
    • xii.
      Spin it at 150×g for 1 minute and keep 100 μL of PBS with the precipitated spheroid and discard the rest of the PBS.
    • xiii.
      Put around 50 μL of PBS containing the spheroid onto a poly-L-lysine-coated glass slide and dry out the PBS using tissue paper.
    • xiv.
      Put 10 μL of Glycerol Mounting Medium - Anti-Fade with DAPI and DABCO on the spheroid-containing glass slide and mount the cover slip.
    • xv.
      Keep it at 25°C temperature for 15 minutes and seal the edges of the cover slip with transparent nail polish.
    • xvi.
      Leave the slides at 25°C temperature for 12–16 hours for drying and store at 4°C (Figure 3) (troubleshooting 3, 4, 5, and 7).

      Note: The spheroid can also be immobilized on the coverslip before mounting on the glass slide.

  • b.
    “In-well staining” for immunofluorescence.

    Note: This section describes the procedure for staining a single intact spheroid at a time with different antibodies to study protein localization and expression in 3D space.

    • i.
      For in-well staining, spheroids should be made in a 96-well low-attachment plate.
    • ii.
      After spheroid maturation, carefully aspirate the media out and wash the spheroids with 1X PBS twice (troubleshooting 1).

      Note: Always use a 200 μL pipette tip for discarding any solution from the well.

    • iii.
      Fix the spheroid by adding cold (or 25°C temperature) 4% Paraformaldehyde in PBS and incubating for 10 minutes at 25°C temperature in an orbital shaker at 80 rpm.
    • iv.
      Discard the paraformaldehyde and wash the spheroids twice with 1X PBS at 25°C temperature in an orbital shaker at 80 rpm.

      Note: Keeping the spheroid in cold conditions helps to maintain better spheroid integrity. Otherwise, using reagents at 25°C temperature for washing and blocking can also be done.

      Pause point: Can be stored in 1X PBS at 4°C for couple of days.

    • v.
      Permeabilize the spheroids by adding 1% cold Triton-X 100 in PBS and incubating for 10 minutes at 25°C temperature in an orbital shaker at 80 rpm.
    • vi.
      For blocking, add 5% BSA in PBST and let it sit at 25°C temperature for 1-2 hours in an orbital shaker at 50 rpm.
    • vii.
      After blocking, add the primary antibody in 3% BSA in PBST at 4°C for 12–16 hours in an orbital shaker at 50 rpm.

      Note: The dilution of the antibody should be standardized for different proteins. The amount should be between 3 and 5μg.

    • viii.
      Next day, wash the spheroids twice with PBST for 10 minutes each in an orbital shaker at 80 rpm.
    • ix.
      Add the secondary antibody diluted to 1:1500 in PBST and incubate for 2 hours at 25°C temperature in an orbital shaker at 50 rpm.

      Note: After this step, everything should be done in the dark.

    • x.
      Wash again twice with PBST for 10 minutes each in an orbital shaker at 80 rpm.
    • xi.
      Wash the spheroid once with PBS.
    • xii.
      Carefully, take 4 to 5 spheroids one by one onto a poly-L-lysine-coated glass slide and dry out the extra PBS with tissue paper.
    • xiii.
      Put 10 μL of Glycerol Mounting Medium - Anti-Fade with DAPI and DABCO on the spheroid-containing glass slide and mount the cover slip.
    • xiv.
      Keep it at 25°C temperature for 15 minutes and seal the edges of the cover slip with transparent nail polish.
    • xv.
      Leave the slides at 25°C temperature 12–16 hours for drying and store them at 4°C (Figure 4) (troubleshooting 3, 4, 5, and 7).
• 5.
  Staining by paraffin block preparation.

  Note: These steps elaborate on the procedure for studying protein localization and expression in a particular plane of a 3D tumor spheroid.

  Steps a and b describe different methods of paraffin block preparation for microtome sectioning.

  Day 15–16.

  • a.
    Block preparation and sectioning by using filter paper.

    Note: These sections describe the steps of paraffin block preparation by using filter paper.

    Harvesting and fixation of the spheroids (troubleshooting 1):
    Prepare the spheroid and fix it in 4% cold paraformaldehyde following any of the above-mentioned protocols.

    Note: For monitoring the protein expression in a 3D spheroid model, a low attachment 96-well plate or hanging droplet method can be used. However, if further biochemical assays need a larger spheroid yield, the AggreWell method should be followed.

    Pause point: Can be stored in 1X PBS at 4°C for couple of days.

    Attachment of spheroids to the tissue paper and dehydration:

    • i.
      Sieve the whole PBS containing spheroids through a folded filter paper (Figure 5).

      Note: This will help to collect the spheroids in a small part of the tissue paper. Cut the filter paper in such a way that the spheroids stay in the center of the filter paper. Then fold the filter paper to keep the spheroids inside.

      Note: If the spheroid is formed and fixed in a 96-well low attachment plate, then the spheroid should be placed in a particular place of a tissue paper drop-wise with a tip-cut pipette.

    • ii.
      Keep the filter paper on a paraffin block forming cassette.

      Note: Instead of a paraffin block forming cassette, the spheroid containing filter paper can be put inside a permeable pouch for the dehydration step.

    • iii.
      For the dehydration, immerse the spheroid-containing cassette in ethanol, gradually increasing the concentration.
       60% alcohol for two times of 5 minutes each,
       70% alcohol for two times of 5 minutes each,
       80% alcohol for two times of 5 minutes each,
       90% alcohol for two times of 5 minutes each,
       And 100% alcohol for two times of 10 minutes each.
    • iv.
      After that, transfer it to xylene two times for 10 minutes each.
      Paraffin block preparation:
    • v.
      Place the dehydrated spheroid-containing cassette in the paraffin block preparation chamber and pour 65°C hot paraffin onto it.

      CRITICAL: The maintenance of temperature at 65°C is very critical. Otherwise, it may damage the integrity of the spheroids.

    • vi.
      Allow it to cool and solidify for approximately 30 minutes at 25°C temperature.
    • vii.
      Store the paraffin block either at 4°C or at 25°C temperature.

      Pause point: Can be stored at 4°C or 25°C temperature for couple of years.

      Microtome sectioning and Deparaffinization (troubleshooting 6):
    • viii.
      Take out the paraffin block and slice it into 4-8μm sections using a microtome.

      Note: The integrity and thickness of the section depend on the quality of the spheroid. It is better to cut 8 μm or thicker sections.

    • ix.
      Put the sliced sections into a 50°C water bath using a brush.
    • x.
      Place the poly-L-lysine-coated glass slide below the floating sections to mount it over the slide.

      Pause point: Can be stored at 4°C or 25°C temperature for couple of years.

    • xi.
      Deparaffinize the sections by heating the slide in a 65°C incubator for 40–50 minutes and immediately immerse the slide in xylene two times for 5 minutes each.

      CRITICAL: The incubator temperature should not exceed 65°C. Otherwise, the paraffin will evaporate, and the spheroid will get dry heat, which will reduce the staining quality.

    • xii.
      For rehydration of spheroid section,² immerse the spheroid section in alcohol and gradually reduce the alcohol concentration as follows:
       100% ethanol for 5 minutes two times,
       90% ethanol for 5 minutes,
       80% ethanol for 5 minutes,
       70% ethanol for 5 minutes,
       60% ethanol for 5 minutes,
       And finally, in water for 5 minutes two times.
      Antigen Retrieval:
    • xiii.
      For antigen retrieval, immerse the slide in a tris-EDTA buffer (pH 9.0) containing beaker.
    • xiv.
      Heat the beaker at 90°C–95°C for 1 hour.

      Note: Do not heat it at more than 100°C.

    • xv.
      After heating, allow it to cool to 25°C temperature.
  • b.
    Block preparation and sectioning by using the Agarose gel block.

    Note: These sections describe the steps of paraffin block preparation by using agarose.

    Harvesting and fixation of the spheroids (troubleshooting 1):
    Prepare the spheroid and fix it in 4% cold paraformaldehyde following any of the above-mentioned protocols.

    Note: The integrity of the spheroid is better in a low attachment 96-well plate. So, it’s better to follow that protocol.

    Pause point: Can be stored in 1X PBS at 4°C for couple of days.

    Preparation of agarose block and dehydration:

    • i.
      Harvest 10–20 spheroids in a 1.5 mL microcentrifuge tube during the last step of washing and discard all the remaining buffer (Figure 6).
    • ii.
      Prepare a 1% agarose solution and pour 200 μL over the spheroids, ensuring thorough mixing before allowing it to solidify.

      CRITICAL: Make sure the temperature of agarose does not exceed 60°C while pouring it onto the spheroids. Always use cut-tip while mixing.

    • iii.
      For the dehydration, immerse the spheroid-containing agarose block in ethanol, gradually increasing the concentration.
       60% alcohol for two times of 5 minutes each,
       70% alcohol for two times of 5 minutes each,
       80% alcohol for two times of 5 minutes each,
       90% alcohol for two times of 5 minutes each,
       And 100% alcohol for two times of 10 minutes each.
    • iv.
      After that, transfer it to xylene two times for 10 minutes each.
      Paraffin block preparation:
    • v.
      Place the solidified agarose into a paraffin block forming mold and pour 65°C hot paraffin onto it.
    • vi.
      Allow it to cool and solidify for approximately 30 minutes.
    • vii.
      Store the paraffin block either at 4°C or at 25°C temperature.

      Pause point: Can be stored at 4°C or 25°C temperature for couple of years.

      Microtome sectioning and Deparaffinization (troubleshooting 6):
    • viii.
      Take out the paraffin block and slice it into sections of desired thickness using a microtome.

      Note: The integrity and thickness of the section depend on the quality of the spheroid. It is better to cut 3 μm or thicker sections.

    • ix.
      Put the sliced sections into a 50°C water bath using a brush.
    • x.
      Place the poly-L-lysine-coated glass slide below the floating sections to mount it over the slide.

      Pause point: Can be stored at 4°C or 25°C temperature for couple of years.

    • xi.
      Deparaffinize the sections by heating the slide in a 65°C incubator for 60 minutes and immediately immerse the slide in xylene for two times, 5 minutes each.

      CRITICAL: The incubator temperature should not exceed 65°C. Otherwise, the integrity of the paraffin-embedded section may be compromised.

    • xii.
      For rehydration of spheroid section², immerse the spheroid section in alcohol and gradually reduce the alcohol concentration as follows:
       100% ethanol for 5 minutes two times,
       90% ethanol for 5 minutes,
       80% ethanol for 5 minutes,
       70% ethanol for 5 minutes,
       60% ethanol for 5 minutes,
       And finally, in water for 5 minutes twice.
      Antigen Retrieval:
    • xiii.
      For antigen retrieval, immerse the slide in a tris-EDTA buffer (pH 9.0) containing beaker.
    • xiv.
      Heat the beaker at 90°C–95°C for 1 hour.

      Note: Do not heat it at more than 100°C.

    • xv.
      After heating, allow it to cool to 25°C temperature.
  • c.
    Staining of spheroid sections for visualization: Immunohistochemistry (IHC).

    Note: This section elaborates the method of visualizing a single protein expression in a particular paraffin-embedded section of a tumor spheroid using HRP-conjugated antibody.

    Day 17–19

    • i.
      Make a circle around the spheroid sections on the glass slide with a hydrophobic pen after visualizing the sections in a bright field light microscope.

      Note: It is necessary to minimize the use of reagents.

    • ii.
      Cover the spheroid sections with 3% hydrogen peroxide blocking buffer and allow it to sit at 25°C temperature for 10 minutes.
    • iii.
      Rinse the slide with 0.5 mM phosphate buffer (pH 7.0) for 2 minutes.
    • iv.
      To block nonspecific binding, apply a solution of 5% BSA (in water) to the slide and let it rest at 25°C temperature for 10 minutes.
    • v.
      Again, wash the slide with 0.5 mM Phosphate buffer (pH 7.0) for 2 minutes.
    • vi.
      Next, cover the sectioned spheroid on the slide with a 1:200 dilution of the desired primary antibody in 1X PBST and keep it for 2 hours at 25°C temperature or 12–16 hours at 4°C.

      Note: During incubation with primary antibody, always keep the slide in a humidified chamber or place it on an elevated platform in a covered petri-dish containing water or PBS to reduce evaporation.

      Note: The dilution of the antibody should be standardized for different proteins. The amount should be between 3 and 5μg.

    • vii.
      Wash the slide twice with 1X PBST for 10 minutes each.
    • viii.
      Add the secondary antibody in a dilution of 1:1000 in 1X PBST the same way as the primary antibody and incubate for 30-40 minutes at 25°C temperature.
    • ix.
      Following this, wash the slide twice with 1X PBST for 10 minutes each and once with 0.5 mM Phosphate buffer (pH 7.0).

      Note: From this point forward, perform all subsequent steps in the dark.

    • x.
      Mix DAB and DAB substrate in a 1:5 ratio in a microcentrifuge tube.
    • xi.
      Cover the sections with it and incubate at 25°C temperature for 1–2 minutes.

      Note: Carefully monitor the DAB color formation. Rinse as soon as the color starts developing.

    • xii.
      Wash it off with 0.5 mM Phosphate buffer (pH 7.0), then rinse with running water.
    • xiii.
      For counterstaining, cover the slide with Hematoxylin and keep it at 25°C temperature for 15–20 seconds.
    • xiv.
      Finally, rinse the slide with running water.
    • xv.
      Dry the section at 25°C temperature and mount it with cover slips using DPX.
    • xvi.
      Image the sections under a bright field light microscope (Figure 7).
  • d.
    Staining of spheroid sections for visualization: Immunofluorescence (IF).

    Note: This section elaborates the method of visualizing different protein expression at the same time in a particular paraffin-embedded section of a tumor spheroid using fluorophore-conjugated antibody.

    Day 17–19.

    • i.
      Immerse the prepared slide in 1% Triton-X 100 for 10 minutes on a shaker set to 70 rpm. Repeat this step twice.
    • ii.
      Wash the slide with 1X PBS, twice.
    • iii.
      For blocking, put the slide in 5% BSA (in 1X PBST) on a shaker at 50 rpm at 25°C temperature for 1–2 hours.
    • iv.
      After blocking, make a circle around the spheroid sections on the glass slide with a hydrophobic pen after visualizing the sections in a bright field light microscope.

      Note: The hydrophobic boundary prevents the spillage of reagents from the glass slide during subsequent incubation steps.

    • v.
      Next, cover the sectioned spheroid on the slide with a 1:200 dilution of the desired primary antibody in 1X PBST and keep it 12–16 hours at 4°C.

      Note: During incubation with primary antibody, always keep the slide in a humidified chamber or place it on an elevated platform in a covered Petri dish containing water or PBS to reduce evaporation.

      Note: The dilution of the antibody should be standardized for different proteins. The amount should be between 3 and 5μg.

    • vi.
      Subsequently, wash the slide twice with 1X PBST for 5 minutes on a shaker set to 80 rpm.
    • vii.
      Add fluorophore-conjugated secondary antibody in a dilution of 1:1000 in 1X PBST, the same way as primary antibody and incubate for 60 minutes at 25°C temperature.

      Note: From this point forward, perform all subsequent steps in the dark.

    • viii.
      Wash the slide twice with 1X PBST for 10 minutes each on a shaker at 80 rpm.
    • ix.
      Cover the slide with DAPI and incubate at 25°C temperature for 5 minutes.
    • x.
      Again, wash the slide twice with PBST for 5 minutes each on a shaker at 80 rpm.
    • xi.
      Wash the slide with CuSO₄-NH₄Cl for 10 minutes at 25°C temperature on a shaker at 80 rpm speed.³
    • xii.
      Perform two final washes with 1X PBST for 5 minutes each on an orbital shaker at 80 rpm.
    • xiii.
      Put 10 μL of Glycerol Mounting Medium - Anti-Fade with DAPI and DABCO on the spheroid-containing glass slide and mount the cover slip.
    • xiv.
      Keep it at 25°C temperature for 15 minutes and seal the edges of the cover slip with transparent nail polish.
    • xv.
      Leave the slides at 25°C temperature 12–16 hours for drying and store at 4°C (Figures 8 and 9) (troubleshooting 3, 4, and 7).

Figure 3.

In Tube Immunofluorescence (IF) staining of tumor spheroids

(A) Confocal immunofluorescence image of matured T47D spheroid stained with anti-H3 and anti-Actin antibody and imaged under 10X and 100X objective lenses.

(B) 3D visualization of T47D spheroid using 10X and 100X z-stack images.

(C) Confocal fluorescence image of matured Huh7 spheroid stained with anti-H3 and anti-Actin antibody and imaged under 10X and 100X objective lenses.

(D) 3D visualization of Huh7 spheroid using 10X and 100X z-stack images.

Scale bar 20 μm.

Figure 4.

In Well Immunofluorescence (IF) staining of tumor spheroids

(A) Confocal immunofluorescence image of matured T47D spheroid stained with anti-H3 and anti-Actin antibody and imaged under 10X and 100X objective lenses.

(B) 3D visualization of T47D spheroid using 10X and 100X z-stack images.

(C) Confocal fluorescence image of matured Huh7 spheroid stained with anti-H3 and anti-Actin antibody and imaged under 10X and 100X objective lenses.

(D) 3D visualization of Huh7 spheroid using 10X and 100X z-stack images.

Scale bar 20 μm and 200 μm.

Figure 5.

Graphical representation of paraffin block preparation and microtome sectioning by filter paper method

After fixation and washing, the spheroids are placed dropwise on the top of a filter paper. The Filter paper is folded and put in a permeable pouch or in a box with multiple pores for dehydration, followed by paraffin mold preparation.

Figure 6.

Graphical representation of paraffin block preparation and microtome sectioning by agarose block method

After fixation and washing the spheroids are pulled down in a microcentrifuge tube. The agarose is boiled and cooled to 60°C and pour into the microcentrifuge tube containing the spheroids and mix gently before solidification. Then the agarose block was dehydrated and paraffinized for microtome sectioning.

Figure 7.

Immunohistochemical (IHC) staining of spheroid sectioned by filter paper method and agarose block method

(A) The 8 μm thick section of paraffin block of T47D and Huh7 spheroid made by filter paper method is stained with anti-H3 antibody and imaged under bright field light microscope.

(B) The 4 μm thick section of paraffin block of T47D and Huh7 spheroid made by agarose block method is stained with anti-H3 and anti-GAPDH antibody and imaged under bright field light microscope. The left-hand side 20X bright field image shows the architecture of spheroid after deparaffinization. The middle and right-hand side 20X bright field images show the IHC of H3 and GAPDH protein.

Scale bar 100 μm.

Figure 8.

Immunofluorescence (IF) staining of T47D spheroid sectioned by agarose block method

(A and C) The 4 μm thick section of paraffin block of T47D spheroid made by agarose block method is stained with anti-H3 and anti-Actin antibody and imaged under confocal fluorescence microscope under 10X and 60X objective lenses.

(B and D) 3D visualization of Huh7 spheroid using 10X and 60X z-stack images.

Scale bar 20 μm.

Figure 9.

Immunofluorescence (IF) staining of Huh7 spheroid sectioned by agarose block method

(A and C) The 4 μm thick section of paraffin block of Huh7 spheroid made by agarose block method is stained with anti-H3 and anti-Actin antibody and imaged under confocal fluorescence microscope under 10X and 60X objective lenses.

(B and D) 3D visualization of Huh7 spheroid using 10X and 60X z-stack images.

Scale bar 50 μm.

Imaging

Timing: 2 days

This section describes the procedure to capture the images of spheroids.

Day: 20–21.

• 6.Imaging of Intact spheroid and IHC sections.

A bright-field light microscope (e.g. EVOS XL Core) could be used for imaging the whole spheroids (with a 10X objective lens with NA 0.25) and IHC sections (with a 20X objective lens with NA 0.40).

• 7.
  Imaging of Immunofluorescence staining.

  • a.
    A confocal microscope (e.g. Spinning disk confocal microscope from Nikon with a resonant scanner) could be used for the immunofluorescence imaging of whole spheroids (using 10X objective lens with NA 0.5) or magnified image of a specific portion of the spheroid (using 100X objective lens with NA 1.40).
  • b.
    After turning the instrument on, select the channels (408 nm, 488 nm and 561 nm) according to the fluorophore used during staining the spheroid.
  • c.
    After focusing, set the laser intensity (408 nm, 488 nm and 561 nm Coherent solid-state lasers) according to the quality of staining.
  • d.
    All the images were grabbed using z-stack mode using the Ti ZDrive system with 0.35 μm step size.

CRITICAL: For larger spheroids, one may increase the z stack step size to prevent the bleaching of the fluorophore during longer laser exposure.

Expected outcomes

The comprehensive methods enable the successful formation of 3D tumor spheroids through multiple approaches, having their own merits and demerits. We have also elaborated the immunostaining analysis for studying protein expression and localization. Notably, the AggreWell method of spheroid formation is robust and can produce 300 uniform spheroids at a time, making it advantageous for monitoring spheroid growth. For visualization of a particular protein localization, the hanging droplet method and the low-attachment 96-well plate method are superior due to proper spheroid integrity. At the same time, to visualize a protein localization in a 3D space, the in-tube or in-well staining methods are recommended (Figures 3 and 4), whereas to monitor a protein expression at a particular region of a spheroid (for example whether it has differential expression in tumor core and periphery) it is advantageous to follow agarose block method (Figures 8 and 9). Hence, depending upon the experimental need, which could consist of intact spheroid morphology, high-quality immunofluorescence (IF) or immunohistochemistry (IHC) staining with minimal background, appropriate methodology can be chosen. These protocols ensure preserved spheroid architecture during paraffin embedding and sectioning. Further, the protocol ensures consistency in IHC and IF staining and provides details on the optimization steps of antigen retrieval and antibody dilution.

Limitations

This protocol works well for the spheroids originating from various cancer cell lines. The manual droplet generation in the hanging drop method limits large-scale studies, and the spheroids generated are prone to fusion as the droplets coalesce. The Aggrewell / Microwell plates require precise cell number optimization to prevent overcrowding or sparse spheroid formation, as well as the retrieval of the spheroids from these plates may compromise their structural integrity thus complicating the procedure. The limitation of using an ultra-low attachment plate is the size heterogeneity of the spheroids. For the whole mount immunostaining protocol, the antibodies fail to infiltrate the spheroids, leading to a preferred staining in the peripheral zone and an incomplete clearing, which leads to a high background autofluorescence. The paraffin sectioning of spheroids may lead to their fragmentation or compression, and the fixation-induced epitope masking necessitates aggressive antigen retrieval, risking tissue damage.

Troubleshooting

Problem 1

Irregular Spheroid morphology or its disintegration.

Potential solution

The former is probably caused due to inconsistent cell aggregation or clumping, while the latter is caused due to mechanical stress from pipetting.

Pre-coat plates with 1% agarose or use DNase I (10U/mL) in suspension media for uniformity of spheroid morphology.

If spheroids are breaking while changing media or harvesting for immune staining, use pipette tips with a wider orifice to remove the strain on the spheroids while handling. It is preferable to coat the pipette tips with 5% BSA to let the spheroids pass smoothly while handling.

If the spheroids are breaking while harvesting for the trans well plate method, then incubate the plate for a longer period of time with the nonadherent solution prior to the seeding.

If the spheroids are breaking while agarose embedding, then fix the spheroids for 20 minutes in 4% paraformaldehyde instead of 10 minutes.

Problem 2

The Core of the spheroid becomes necrotic or dead.

Potential solution

The tumor core becomes necrotic (indicated by pale, darkish concave central part of the spheroids that stop growing) is an indication that there is nutrient unavailability or induction of deep hypoxia during the spheroid culture.

It is advisable to start with an optimum cell number (for T47D: 8000-12000 cells per droplet for hanging droplet, 20000 cells per well for AggreWell plates, 200 cells per well for low attachment 96 well plates and for Huh7: 12000-15000 cells per droplet for hanging droplet, 25000 cells per well for AggreWell plates, 300 cells per well for low attachment 96 well plates) while seeding spheroids so that the excess growth of the cell mass can be restricted and less nutrient is consumed so that the core region does not become necrotic.

Spheroid culture should be closely monitored to reduce the overall spheroid diameter <300 μm or supplement the media with 10 mM HEPES to maintain the pH of the culture medium between 6.8-8.2. The latter supplementation is useful where pH fluctuations are the cause of poor cell health.

Problem 3

The antibody staining is showing a high background in immunostaining.

Potential solution

This is probably because of non-specific primary/secondary antibody binding.

To avoid high background staining, keep the spheroids in blocking buffer (5% BSA in TBST) for at least two hours, increase the percentage of Tween-20 in TBST and wash slightly longer period of time.

It is recommended to use 2 to 4 μg of primary antibody, but the dilution of primary antibody should be optimized prior to the staining, and the BSA percentage can be increased in the antibody binding buffer. It is always better to do 12-16 hour primary antibody binding at 4°C. The secondary antibody dilution should be more than 1:1000, and the duration of binding can be reduced to 30 minutes.

If the in-tube or in-well staining method is followed, then wash the spheroid with Copper Sulphate buffer for 10 minutes after secondary antibody binding.

Problem 4

The antibody staining is showing weak or no signals in immunostaining.

Potential solution

This is probably caused by over-fixation or antigen-epitope masking.

The fixation time should be reduced. The epitope masking could be alleviated by using alternative buffer systems, viz. citrate buffer pH 6.0, 95°C, 20 minutes.

Problem 5

The core of the spheroid is not staining properly.

Potential solution

Spheroids are multicellular entities, and they form a rigid structure during maturation due to the deposition of matrix components like collagen, etc. The size, rigidity and integrity of the spheroids may vary according to the cell line/ lines used to prepare spheroids. If the core of the spheroid becomes necrotic or dead, then it will appear hollow while staining. The upper and bottom parts of the Z-stack will be stained, leaving a hollow, unstained core.

In such a situation, the cell number for the spheroid formation should be reduced, and the spheroids should be harvested before the necrotic core appears.

Permeabilization with 0.5% Triton X-100 and 0.1% SDS for 1 hour can be carried out or alternatively collagenase treatment (1 mg/mL, 37°C, 30 minutes) can also be done. The percentage of Tween-20 in PBST can also be increased while antibody binding and the duration of antibody binding should be increased. If doing DAPI staining instead of using DAPI-containing mounting media, it is recommended to perform counterstaining with DAPI for 20 to 30 minutes at 4°C temperature.

To visualize the uniform distribution of a protein in different parts of a 3D spheroid, it is recommended to stain by paraffin block preparation.

Problem 6

The Microtome section of the spheroid is not forming a continuous ribbon but getting crumbled and detached from the paraffin section, leaving a hollow space in the paraffin ribbon while cutting.

Potential solution

The microtome section of a spheroid may break due to several reasons, including a necrotic/dead spheroid core or improper paraffinization.

Reduce the number of cells for spheroid formation for the former situation, while for the latter situation, keep the spheroid in 4% paraformaldehyde for 20 minutes and reduce the concentration of agarose to 0.8 to 0.9% for the proper penetration of paraffin.

To avoid the crumbling of sections while doing microtomy, use a good quality histopathology grade paraffin, and the microtome blade should not have any shattering to avoid uneven distribution of blade pressure while cutting.

If one is following the paraffin block preparation by filter paper method, it is advisable to cut between 8-14 μm sections to avoid breaking of the ribbons as the filter paper become rigid after paraffinization and tends to break while cutting. One can use a drop of eosin to stain the spheroid red before paraffinization. It will help to visualize the spheroids while cutting.

Problem 7

An observable air bubble in the mounted sections.

Potential solution

Rapid paraffin cooling or improper mounting is the possible cause for this issue.

Slow cooling protocol of the paraffin is recommended. Apply anti-fade mounting medium carefully from one edge; degas before use.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Prof. Chandrima Das (chandrima.das@saha.ac.in).

Technical contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the technical contact, Prof. Chandrima Das (chandrima.das@saha.ac.in).

Materials availability

This study did not generate new, unique reagents.

Data and code availability

• •This paper doesn’t report the original code.
• •Data and any additional information related to the data reported in this paper are available from the lead contact upon request.

Acknowledgments

C.D. acknowledges Basic and Applied Research in Biophysics and Material Science (RSI 4002) from the Department of Atomic Energy, Core Research Grant (CRG/2022/005052) from Department of Science and Technology and Grant Number BT/PR52628/MED/30/2553/2024 from the Department of Biotechnology Govt. of India. Figures were created with BioRender.com.

Author contributions

A.M.: overall protocol conceptualization, spheroid formation, spheroid staining, spheroid imaging, data analysis, and manuscript writing; S.N.: protocol conceptualization and spheroid formation; B.M.: manuscript writing; I.B.: spheroid imaging; M.W.A.D.H.: manuscript writing; C.D.: overall protocol conceptualization, data monitoring, data analysis, resource management, and manuscript writing.

Declaration of interests

The authors declare no competing interests.