Protocol for generating human tonsil immune organoids with epithelial and germinal center layers using low-Matrigel culture

Summary

Tonsil immune organoids traditionally include either epithelial layers or immune compartments, failing to recapitulate the integrated physiological microenvironment of human tonsils. Here, we present a protocol to generate tonsil immune organoids that recapitulate both epithelial and germinal center-like structures using a low-concentration Matrigel transwell system. We describe organoid assembly, immune stimulation, and evaluation of epithelial architecture and germinal center formation. This platform provides a versatile tool for dissecting human immune responses, assessing vaccines, discovering antibodies, and exploring autoimmune mechanisms.

Graphical abstract

Highlights

• •Instructions for generating epithelial-immune organoids in low-concentration Matrigel
• •Preparation of L-WRN conditioned medium and optimized organoid culture medium
• •Guidance on organoid characterization by immunofluorescence and flow cytometry

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

Tonsil immune organoids traditionally include either epithelial layers or immune compartments, failing to recapitulate the integrated physiological microenvironment of human tonsils. Here, we present a protocol to generate tonsil immune organoids that recapitulate both epithelial and germinal center-like structures using a low-concentration Matrigel transwell system. We describe organoid assembly, immune stimulation, and evaluation of epithelial architecture and germinal center formation. This platform provides a versatile tool for dissecting human immune responses, assessing vaccines, discovering antibodies, and exploring autoimmune mechanisms.

Before you begin

Human tonsils provide a complex immune microenvironment where epithelial, stromal, and immune cells interact to generate effective local immune responses.^1,2 Current ex vivo models often fail to recapitulate this complexity, limiting their utility for functional studies such as vaccine adjuvant evaluation, antibody discovery, and mucosal immune response assessment.³ Here, we present a protocol to generate human tonsil immune organoids that reconstruct epithelial and germinal center–like compartments, thereby capturing a more complete immune microenvironment. While optimized for fresh tonsil tissue, the system can be adapted to other secondary lymphoid tissues and enables ex vivo studies of epithelial–immune interactions, B cell activation, germinal center organization, and functional immune responses relevant to immunotherapy and vaccine development.

Innovation statement

This protocol introduces an innovative workflow for generating a human tonsil immune organoid that simultaneously reconstructs epithelial and germinal center–like immune compartments. It advances current tonsil and lymphoid organoid models by integrating epithelial and immune components within a single culture platform. The innovation lies in modifications to conventional organoid culture methods, including the use of a low-concentration (5%) Matrigel matrix that facilitates nutrient exchange and spatial organization. The reduced Matrigel density minimizes physical barriers to immune cell migration, allowing dynamic lymphocyte aggregation, movement, and functional activation,⁴ while still supporting epithelial adhesion and polarized growth. Additionally, by optimizing the tonsil-specific organoid culture medium with L-WRN conditioned medium and defined cytokines, this protocol achieves a high establishment success rate—over 85% of donor samples reliably form organoids. These optimizations substantially improve physiological relevance and reproducibility compared with current systems that reconstruct either epithelial or immune germinal center components alone.^5,6 Furthermore, we define the temporal framework of this system: antigen introduction at Day 0 serves to accelerate structural maturation, yielding a stabilized immune-epithelial interface that is ready for functional assays or secondary evaluation (e.g., adjuvant testing) after 15–20 days culture period. Together, these modifications overcome the compartmentalization and microenvironmental limitations of conventional tonsil organoids, resulting in a more physiologically integrated model. The organoid maintains lymphocyte viability, supports B cell activation and germinal center–like differentiation, and promotes polarized epithelial architecture. Overall, this protocol establishes a physiologically relevant tonsil model suitable for studying epithelial–immune crosstalk, germinal center organization, and mucosal immune responses. It also provides a practical platform for evaluating vaccine, adjuvants and discovering neutralizing antibodies, representing a significant advancement in mucosal immunology and organoid engineering.^7,8

Institutional permissions

All human tissue samples used in this study were collected with written informed consent under protocols approved by the Research Ethics Committee of Peking University Shenzhen Hospital and the Ethics Review Committee of Tsinghua Shenzhen International Graduate School (Ethics Approval Number: 2024098). All experiments were performed in accordance with institutional and national guidelines and regulations governing the use of human biological materials.

Researchers intending to replicate this protocol will need to obtain approval from their respective institutional ethics committees and comply with all applicable regulations regarding the use of human tissue.

Preparatory steps

• 1.
  Tissue acquisition and cell preparation.

  • a.
    Collect fresh human tonsil tissue under approved ethical guidelines and transport on ice in Tissue Storage Solution.
  • b.
    Remove visible fat or necrotic tissue. Single-cell suspension will be generated during protocol execution.

Note: Gentle handling preserves lymphocyte viability and epithelial/stromal fragments necessary for organoid formation.

• 2.
  Culture environment setup.

  • a.
    Place Matrigel, PBS, and pipette tips in the 4°C refrigerator 12–16 h to pre-cool and allow Matrigel to hydrate. Maintain sterility throughout.
  • b.
    Prepare 24-well Transwell plates (0.4 μm pore size, Polyethylene terephthalate (PET) membrane) by adding complete culture medium 1–2 hours before use and place them in the incubator (37°C, 5% CO₂) to pre-equilibrate.

CRITICAL: All handling of Matrigel and reagents must be on ice and under sterile conditions to prevent premature polymerization and contamination. Pre-equilibrating plates ensures optimal temperature, pH, and CO₂ conditions for cell seeding.

• 3.
  Reagent preparation.

  • a.
    Prepare low-concentration Matrigel (2%–10%) as a 3D scaffold.
  • b.
    Prepare complete organoid culture medium including basal medium, serum, antibiotics, and conditioned medium (e.g., L-WRN supernatant) with cytokines such as IL-21, and BAFF.

Note: Step 2 (Culture environment setup) and 3 (Reagent preparation) are preparatory steps that can be performed concurrently, or in advance of the main experiment, and do not need to follow Step 1 chronologically. Only Step 1a (Tissue Collection) is the initial step, while Step 1b (Cell preparation) occurs immediately prior to the main protocol steps (Cell Seeding).

Preparation one: Human tonsil sample handling and single-cell suspension collection

Timing: 4–5 h (total)

Step 4: 0.5 h

Step 5: 0.5 h

Step 6: 1 h

Step 7: 0.5 h

Step 8–9: 1 h

Step 10: 1 h

Note: Recipes for required solutions are detailed under materials and equipment.

• 4.
  Collect tonsil samples immediately after surgical removal.

  • a.
    Immerse tissue in Tissue Storage Solution inside sterile breast milk storage bags and place the bags on ice (see troubleshooting 1).

    Note: Ensure that the tonsil tissue is fully submerged in Tissue Storage Solution to prevent desiccation and oxidative damage during transport (Figure 1).

  • b.
    Transport samples to the laboratory using a cold chain with ice packs.

    CRITICAL: Minimize transport time to preserve cell viability.

• 5.
  Surface sterilization and antimicrobial treatment.

  • a.
    Using sterile scissors and forceps, trim away necrotic or discolored tissue from the surface of the tonsil.
  • b.
    Transfer the trimmed tissue to a 60 cmm² sterile culture dish using sterile forceps.

    Note: The culture dish must be placed on a sterile ice brick, which is then rested on crushed ice to maintain the tissue temperature at 4°C throughout the trimming process, which is essential for preserving cell viability.

  • c.
    Add 1–2 mL iodophor to cover the tissue, gently rotate and flip to expose all surfaces for 5 min.
  • d.
    Wash tissue thoroughly with PBS to remove residual iodophor.

    • i.
      Carefully aspirate the iodophor solution using a sterile pipette.
    • ii.
      Gently rinse the tissue with 3mL of pre-chilled PBS by slowly adding the solution onto the tissue.
    • iii.
      Use sterile forceps to gently flip the tissue, ensuring all surfaces are rinsed by the PBS before aspirating the liquid. Repeat this washing step three times in total.

      Note: If there are many free-floating cell clumps in the liquid, these can be collected in a 15 mL centrifuge tube and centrifuged at 300 × g for 5 min to obtain a cell pellet.

  • e.
    Immerse tissue in antimicrobial medium and incubate on ice for 20 min.

    CRITICAL: Ensure complete immersion for effective decontamination.

    Note: Discard the supernatant and resuspend the free-floating cell clumps in 1 mL of antimicrobial medium. The resuspended pellet then is combined with the main tissue in the 100 mm² culture dish for the subsequent antimicrobial incubation.

• 6.
  Tissue trimming and enzymatic digestion (Figure 2).

  • a.
    Wash tissue with PBS 2–3 times to remove antimicrobial medium.
  • b.
    Transfer the tissue into a sterile 100 mm² culture dish placed on an ice block. Add 6–7 mL of pre-chilled enzymatic digestion solution.
  • c.
    Cut the main tissue pieces into small pieces (approximately 1–2 mm) directly in the enzymatic digestion solution using sterile scissors and forceps.

    Note: This process must be performed quickly and on ice to minimize mechanical and thermal damage to the cells. The free-floating cell clumps due to its size, does not require this trimming step.

  • d.
    Incubate at 37°C on a shaker at 30 g for 60 min.

    CRITICAL: Avoid over-digestion to maintain cell viability.

• 7.
  Generation of single-cell suspension.

  • a.
    Prepare a new 60 mm² sterile culture dish and place it on a metal ice brick.
  • b.
    Place a 70 μm cell strainer in the dish.
  • c.
    Using a Pasteur pipette, transfer the digested tissue and medium onto the cell strainer.
  • d.
    Using a 5 mL syringe plunger, gently grind the tissue in the cell strainer for 5–7 min on ice.

    Note: Keeping the strainer and dish on ice helps maintain cell viability during the process.

    CRITICAL: Avoid excessive force during grinding to prevent cell damage.

  • e.
    Add 2–3 mL of pre-chilled PBS to the cell strainer and gently pipette up and down 2–3 times to wash and recover remaining cells.

    Note: Repeat this wash and gentle grinding cycle as needed to maximize single-cell recovery.

  • f.
    Collect the resulting single-cell suspension from the culture dish into a sterile key using a Pasteur pipette or pipette gun. A single 50 mL centrifuge tube is typically sufficient for the volume generated from one tonsil.

    Alternatives: If multiple 15 mL tubes were used for initial collection, centrifuge all tubes, discard the supernatant, and use pre-chilled PBS to pool all cell pellets into a single 15 mL tube before performing the 300 × g final wash.

  • g.
    Centrifuge at 300 × g for 5 min at 4°C and discard the supernatant using a pipette, leaving about 5 mL of liquid to ensure the cell pellet remains undisturbed.
  • h.
    Transfer this 5 mL residual volume into a new 15 mL centrifuge tube. Add pre-chilled PBS to a final volume of 10 mL.
  • i.
    Re-centrifuge at 300 × g for 5 min at 4°C to wash cells.
• 8.
  Repeat digestion if necessary.

  • a.
    Check for undissociated tissue. If 5–8 visible tissue fragments (approximately >1 mm in size) remain, secondary digestion is required.
  • b.
    Filter the current suspension through a cell strainer to collect the dissociated cells. Store this suspension on ice to prevent over-digestion.
  • c.
    Add fresh enzymatic solution to the remaining undissociated fragments and repeat steps 3–4.
• 9.
  Red blood cell removal.

  • a.
    Resuspend pellet in 2–3 mL red blood cell lysis buffer and incubate 2 min at 20°C–25°C.

    Note: Use 2–3 mL for average pellets. Increase volume up to 3–5 mL for visibly larger cell pellets to ensure complete resuspension and efficient lysis.

    CRITICAL: Do not exceed lysis time to avoid leukocyte damage.

  • b.
    Add pre-cooled PBS to 10–12 mL and centrifuge at 300 × g for 5 min. Discard supernatant.
• 10.
  Cell collection and cryopreservation.

  • a.
    Combine cell suspensions from previous steps.
  • b.
    Assess cell viability using Trypan Blue staining and an automated cell counter; aim for >85% viable cells.

    Note: For samples with cell viability between 70% and 85%, perform a low-speed centrifugation (100–200 g for 5 min) to remove dead cells, which improves viability at the expense of total cell yield. However, if viability is <70%, the sample should be discarded, as this typically indicates excessive damage during surgical resection or transportation that will significantly reduce the success rate of organoid generation.

  • c.
    Resuspend cells in freezing medium at a concentration of 1.0–1.5 × 10⁷ cells/mL and aliquot into cryovials.
  • d.
    Place cryovials in a cell freezing container and transfer to −80°C 12–16 h. The next day, transfer vials to liquid nitrogen for long-term storage.

    Note: The cell freezing container lowers the temperature at approximately −1°C/min. Cells reach −80°C in roughly 2 h. Once frozen (minimum 3–4 h) or on the next day, transfer vials to liquid nitrogen for long-term storage.

    CRITICAL: High cell viability before freezing is essential for successful recovery after thawing.

Figure 1.

immersion of human tonsil tissue in Tissue Storage Solution

Freshly excised tonsil tissue is fully submerged in sterile Tissue Storage Solution and maintained on ice immediately after surgery. This preserves tissue hydration and cell viability prior to laboratory processing.

Figure 2.

Mechanical and enzymatic dissociation workflow for generating tonsil-derived immune organoids

(A) Fresh human tonsil tissues are placed into sterile breast milk preservation solution, ensuring complete immersion. The tissues are transported to the laboratory on ice to maintain viability.

(B) Tonsil tissues are placed on metal ice box in the biosafety cabinet for mechanical dissociation.

(C) Tonsil tissues are manually minced into small fragments using sterile surgical scissors.

(D) Following enzymatic digestion, the dissociated cell suspension is collected and centrifuged to remove debris and isolate single cells.

(E) Representative image of tonsil immune organoids cultured for 15–20 days, showing well-formed spherical structures.

Preparation two: L-WRN conditioned medium collection

Timing: 5–8 days (total)

Step 11: 1–2 days

Step 12: 1–2 days

Step 13: 3–4 days

• 11.
  L-WRN cell thawing and recovery.

  • a.
    Rapidly thaw frozen L-WRN cells in the 37°C water bath (See troubleshooting 5).
  • b.
    Resuspend in DMEM high-glucose medium and centrifuge at 300 × g for 5 min.
  • c.
    Dispose of media and resuspend the pellet in L-WRN complete DMEM medium. Seed the contents of one vial (approximately 1–2 × 10⁶ cells) into a 10 cm culture dish.

    Note: The corresponds to a seeding density of approximately 1.8–3.6 × 10⁴ cells/cm², allowing for optimal recovery.

  • d.
    Incubate at 37°C, 5% CO₂ until cells adhere and spread (about 24–48 h).
• 12.
  Selection and expansion.

  • a.
    Replace basal medium with L-WRN selection medium.
  • b.
    Perform 1–2 passages to expand cells. Passage cells when they reach about 90% confluency (typically every 2 days). Healthy L-WRN cells should exhibit a spindle-shaped, fibroblast-like morphology and be firmly adherent.

Note: If cells appear rounded, detached, or exhibit excessive vacuolization: Check for potential contamination or drug selection issues. If morphology does not improve, discard the culture and thaw a new vial.

• 13.
  Conditioned medium collection.

  • a.
    Seed L-WRN cells in 100 mm² culture dishes and culture cells until they reach high confluency (>95%).

    Note: High confluency is critical for producing high-titer conditioned medium.

  • b.
    Once cells are adherent and confluent, gently wash the monolayer with PBS to remove the medium. Add fresh complete DMEM medium and incubate for 24 h.
  • c.
    Collect supernatant (Day 1 conditioned medium) into a sterile conical tube and store at 4°C. Replace with fresh medium and repeat daily for 4 days.
  • d.
    After the fourth collection, combine Day 1–4 supernatants, mix thoroughly, and centrifuge at 3,000 × g for 5 min to remove cell debris.
  • e.
    Aliquot the clarified supernatant into 50 mL sterile centrifuge tubes and store at −80°C for use.

    Note: Conditioned medium contains R-spondin 3, Wnt3a, and Noggin, and is used in the final complete organoid medium.^9,10

Preparation three: Culture system and Matrigel preparation

Timing: 25–34 h (total)

Step 14: 12–16 h

Step 15: 1–2 h

Step 16: 12–16 h

• 14.
  Matrigel pre-cooling.

  • a.
    Place Matrigel vials and pipette tips on a metal ice brick or in a 4°C refrigerator 12–16 h.

CRITICAL: Maintain Matrigel on ice at all times to prevent premature gelation.

• 15.
  Plate pre-equilibration.

  • a.
    Add basal DMEM/F12 medium to 24-well Transwell® inserts (0.4 μm pore size, PET membrane preferred for imaging) placed in a 24-well culture plate.

    Note: The 0.4 μm pore size is critical to prevent cell migration while ensuring nutrient diffusion. While Corning inserts were used here for their membrane durability, other brands with equivalent specifications (including PC membranes) could be used.

  • b.
    Incubate in a 37°C, 5% CO₂ incubator for 1–2 h to equilibrate temperature and CO₂.
• 16.
  Culture medium preparation.

  • a.
    Thaw frozen L-WRN conditioned medium in a 4°C refrigerator 12–16 h.
  • b.
    Prepare complete tonsil organoid medium by combining basal DMEM/F12 medium with serum, antibiotics, cytokines, and L-WRN conditioned medium.

Note: Conditioned medium can be prepared in advance and stored at −80°C, mixing gently before use.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Purified anti-human CXCR4 (CD184) (IF:1/500)	BioLegend	Cat# 306501
Human CD83 recombinant rabbit antibody (IF:1/500)	Abcam	Cat# ab205343
Pan-Keratin Polyclonal antibody (IF:1/500)	proteintech	Cat# 26411-1-AP
PE anti-human CD45 Antibody (FC:1/50)	BioLegend	Cat# 304008
BV421 Mouse Anti-Human CD45 (FC:1/50)	BD Biosciences	Cat# 563879
BV510 Mouse Anti-Human CD45 (FC:1/50)	BD Biosciences	Cat# 563204
Anti-human CD20 Monoclonal Antibody (L26), eFluor™ 660 (IF:1/200)	eBioscience™	Cat# 50-0202-82
F(ab’)2-Goat anti rabbit IgG (H+L) Alexa Fluor™ plus 488 (IF:1/1000)	Invitrogen	Cat# A48282TR
F(ab’)2-Goat anti mouse IgG (H+L) Alexa Fluor™ plus 555 (IF:1/1000)	Invitrogen	Cat# A48287
FITC anti-human CD3 (FC:1/50)	BioLegend	Cat# 300440
Alexa Fluor® 700 anti-human CD19 Antibody (FC:1/50)	BD Biosciences	Cat# 363034
BD Horizon™ BV510 Mouse Anti-Human CD27 (FC:1/50)	BD Biosciences	Cat# 563090
APC anti-human CD38 Antibody (FC:1/50)	BioLegend	Cat# 303510
BV421 Mouse Anti-Human CD86 (FC:1/50)	BD Biosciences	Cat# 562432
APC anti-human CD4 (FC:1/50)	BioLegend	Cat# 317318
Biological samples
Adult tonsil tissue	Peking University Shenzhen Hospital; https://www.pkuszh.com/ENGLISH	Cat# N/A
Chemicals, peptides, and recombinant proteins
Tissue storage solution	Miltenyi	Cat# 130-100-008
DMEM/F-12 (1×)	Gibco™	Cat# 11320033
DMEM (High glucose)	Cytiva	Cat# SH30022.01
Fetal bovine serum (FBS)	Gibco™	Cat# 10270-106
Penicillin-Streptomycin (PS)	Gibco™	Cat# 15140122
Phosphate-buffered saline PBS (1×)	Gibco™	Cat# 20-012-027
Trypsin-EDTA (0.25%), phenol red	Gibco™	Cat# 25200072
Insulin-Transferrin-Selenium-Pyruvate (ITS-A) (100×)	Gibco™	Cat# 51300044
MEM (Non-essential Amino acid)	Gibco™	Cat# 11140050
GlutaMAX™	Gibco™	Cat# 35050061
HEPES (1 M)	Gibco™	Cat# 15630-080
Y-27632 (ROCK inhibitor)	MedChemExpress	Cat# HY-10071
Normocin™	InvivoGen	Cat# ant-nr-05
Nicotinamide	InvivoGen	Cat# HY-B0150
L-Ascorbic acid (Vc)	Solarbio	Cat# 50-81-7
A83-01	MedChemExpress	Cat# HY-10432
Human recombinant IL-21	PeproTech	Cat# 200-21-10UG
Recombinant human BAFF	GenScript	Cat# Z02976-1
Human HGF	GenScript	Cat# Z03229-10
Human Recombinant bFGF	STEMCELL	Cat# 78003
Collagenase Type I, Cls I	Sigma-Aldrich	Cat# C1-28-100MG
Collagenase Type II, Cls I	Sigma-Aldrich	Cat# C2-28-100MG
DNase I	Beyotime	Cat# D7076
Red blood cell lysis buffer	Solarbio	Cat# R1011
DMSO	Sigma-Aldrich	Cat# C6295
Matrigel Matrix (Growth Factor Reduced)	Corning	Cat# 356234
Trypan Blue (0.4%)	Sigma-Aldrich	Cat# 72-57-1
Iodophor	LIRCON	Cat# XY0058
G418	ThermoFisher	Cat# 10131035
Hygromycin B	ThermoFisher	Cat# 10687010
SuperKine™ Enhanced Antifade Mounting Medium with DAPI	Abbkine	Cat# BMU107
Goat serum	Abbkine	Cat# BMS0050
SARS-CoV-2 Spike N-Terminal Domain (NTD)	Sino Biological	Cat# 40591-V49H
Sucrose	Solarbio	Cat# 57-50-1
Triton X-100	ThermoFisher	Cat# 85112
Critical commercial assays
AM/PI Live/Dead Viability Assay Kit	Beyotime	Cat# C2015M
Experimental models: Cell lines
L-WRN cells (secreting Wnt3a, R-spondin 3, Noggin)	ATCC	Cat# CRL-2647
Software and algorithms
FlowJo (v10.8.1)	BD Biosciences	RRID:SCR_008520
GraphPad Prism (v10)	GraphPad	RRID:SCR_002798
ImageJ (v1.54f)	NIH	RRID:SCR_003070
Other
70 μm Cell Strainer	Falcon	Cat# 352350
Pasteur pipette	JETBIOFIL	Cat# PP102030
60 cm2 TC-treated Culture Dish	Corning	Cat# 430166
100 cm2 TC-treated Culture Dish	Corning	Cat# 430167
BeyoCool™ Cell Freezing Container	Beyotime	Cat# FCFC012
Metal Ice Box	Biosharp	Cat# BC032
Cryogenic vials (2 mL)	NEST	Cat# 607401
50 mL Centrifuge tube	Corning	Cat# 430790
15 mL Centrifuge tube	Corning	Cat# 430828
5 mL syringe plunger	Gufastore	Cat# GF22101402
0.4 μm transwell PET	Corning	Cat# 3470
Neg-50 Frozen Section Medium(OCT)	ThermoFisher	Cat# 6502B
Breast milk storage bags	Hotsale	Cat# N/A
CO2 Incubator (37°C, 5% CO2)	ESCO	Model# CCL-050B-8
Water bath	Thermo Scientific	Cat# TSGP02
Confocal laser scanning microscope	Olympus Corporation	Model# FV3000
CytoFLEX Flow Cytometer	Beckman	Model# V0-B5-R3

Materials and equipment

Antimicrobial medium

Reagent	Final concentration	Amount
DMEM/F-12 (1×)	93% (v/v)	46.4 mL
FBS	5% (v/v)	2.5 mL
PS	2% (v/v)	1 mL
Normocin™	1 mg/mL	0.1 mL
Total	N/A	50 mL

Storage: Store at 4°C for up to 2 weeks. Prepare fresh if medium shows turbidity or color change.

CRITICAL: Contains multiple antibiotics (PS, Normocin) that are heat-labile. Avoid filtration through 0.22 μm filters containing surfactants that may inactivate antibiotics. Handle under sterile conditions in a biosafety cabinet to prevent contamination.

Alternatives: Antibiotic-Antimycotic (Thermo Fisher 15240062) can replace PS + Normocin. If fungal contamination is suspected, Amphotericin B (1 μg/mL) can be added.

Enzymatic digestion solution

Reagent	Final concentration	Amount
DMEM/F-12 (1×)	93% (v/v)	46.84 mL
FBS	5% (v/v)	2.5 mL
PS	1% (v/v)	0.5 mL
Normocin™	1mg/mL	0.1 mL
Collagenase Type I	0.5 mg/mL	0.05 mL
Collagenase Type II	0.5 mg/mL	0.05 mL
DNAase	50 μg/mL	0.05 mL
Total	N/A	50 mL

Storage: Prepare fresh before each use. Enzyme activity decreases rapidly; do not store for reuse.

CRITICAL: Collagenase and DNase are bioactive enzymes; wear g appropriate PPE (Personal Protective Equipment to prevent contact. Avoid excessive agitation to maintain enzyme stability. Keep the buffer on ice before use and incubate only immediately prior to digestion.

Alternatives: Commercial tissue dissociation kits (e.g., Miltenyi Biotec Human Tumor Dissociation Kit) can substitute the enzyme mixture for reproducibility.

Complete DMEM medium

Reagent	Final concentration	Amount
DMEM (High glucose)	90% (v/v)	44.5 mL
FBS	10% (v/v)	5 mL
PS	1% (v/v)	0.5 mL
Total	N/A	50 mL

Storage: Store at 4°C for up to 1 month. Warm to 37°C before use.

CRITICAL: Freezing the complete medium is highly not recommended, as this causes precipitation and degrades essential nutrients. Protect from light to prevent phenol red oxidation. Discard medium showing precipitates or pH color change.

Alternatives: DMEM/F12 + 10% FBS + PS can be used as an alternative for lymphoid cell culture.

L-WRN selection medium

Reagent	Final concentration	Amount
DMEM (High glucose)	90% (v/v)	44.4 mL
FBS	10% (v/v)	5 mL
PS	1% (v/v)	0.5 mL
G418	0.5 mg/mL	0.05 mL
Hygromycin B	0.5 mg/mL	0.05 mL
Total	N/A	50 mL

Storage: Store at 4°C for up to 1 month. Prepare fresh selection medium weekly for optimal activity of antibiotics.

CRITICAL: Selection antibiotics are cytotoxic; handle with care and avoid inhalation or contact with skin. Always use sterile pipette tips and media containers. Confirm cell survival curve before applying new antibiotic concentrations.

Alternatives: Puromycin or Zeocin can be used as alternative selection markers depending on plasmid resistance genes.

Organoid medium

Reagent	Final concentration	Amount
DMEM/F-12 (1×)	59% (v/v)	29.465 mL
L-WRN conditioned medium (Wnt3a, R-spondin 3, Noggin)	25% (v/v)	12.5 mL
FBS	10% (v/v)	5 mL
PS	1% (v/v)	0.5 mL
GlutaMAX™	1×	0.5 mL
ITS-A (100×)	1×	0.5 mL
MEM	1×	0.5 mL
Y-27632	10 μM	0.5 mL
HEPES (1 M)	10 mM	0.05 mL
HGF (Ready-to-use)	50 ng/ml	0.05 mL
bFGF (Ready-to-use)	20 ng/ml	0.05 mL
Nicotinamide	10 mM	0.05 mL
Vc	20 μg/ml	0.05 mL
A83-01 (Ready-to-use)	20 nM	0.05 mL
Human recombinant IL-21 (Ready-to-use)	10 ng/mL	0.05 mL
Recombinant human BAFF (Ready-to-use)	100 ng/mL	0.125 mL
Normocin™	1mg/mL	0.1 mL
NTD antigen (added during plating)	4 μg/ml	0.05 mL
Total	N/A	50 mL

Note: Prepare fresh working aliquots every 2 weeks; store at 4°C for short-term use.

CRITICAL: Contains growth factors (IL-21, BAFF and NTD) that are heat-labile. Avoid repeated freeze–thaw cycles. Y-27632 is light-sensitive and should be handled under low light (See troubleshooting 6).

Alternatives: Commercial Wnt3a/R-spondin Noggin recombinant proteins can replace L-WRN conditioned medium if available.

Freezing medium

Reagent	Final concentration	Volume
FBS	90% (v/v)	45 mL
DMSO	10% (v/v)	5 mL
Total	N/A	50 mL

Storage: Store at 4°C for up to 1 month. Prepare fresh if DMSO precipitates or discoloration occurs.

CRITICAL: DMSO is toxic and can be absorbed through the skin. Handle inside a fume hood while wearing appropriate PPE. Avoid direct contact and exposure to light. Ensure cells are cooled at a controlled rate (∼1°C/min) to prevent ice-crystal formation.

Alternatives: Commercial cryopreservation media (e.g., Thermo Fisher CryoStor CS10) can be used as an alternative to the self-prepared freezing medium.

Sucrose solutions (for tissue dehydration)

Reagent	Concentration (10%)	Concentration (20%)	Concentration (30%)
Sucrose	1 g	2 g	3 g
dd H2O	9 g	8 g	7 g
Total	10 g	10 g	10 g

Storage: Store at 4°C for up to 2–3 week.

CRITICAL: Ensure sucrose is completely dissolved before use to prevent osmotic artifacts during dehydration.

Alternatives: Concentrations can be adjusted (10%–30%) according to tissue density or embedding requirements.

Blocking buffer (for immunostaining)

Reagent	Final concentration	Volume
Goat serum	5% (v/v)	0.5 mL
Triton X-100	0.1% (v/v)	0.01 mL
PBS	94.9%	9.49 mL
Total	N/A	10 mL

Storage: Store at 4°C for up to 1 month.

CRITICAL: Mix gently to avoid foaming; excessive Triton concentration may damage tissue morphology.

Alternatives: Commercial blocking solutions such as Background Sniper (Biocare Medical) or Image-iT FX Signal Enhancer (Thermo Fisher) can be used as blocking buffer. And 5%–10% Bovine Serum Albumin (BSA) can be used instead of goat serum.

Washing buffer (for immunostaining)

Reagent	Final concentration	Volume
Triton X-100	1% (v/v)	0.1 mL
PBS	99% (v/v)	9.9 mL
Total	N/A	10 mL

Storage: Store at 4°C for up to 2 weeks.

CRITICAL: Ensure buffer pH = 7.4; inappropriate pH can affect antibody affinity.

Alternatives: If cells or tissues are sensitive to detergents, 0.05%–0.1% Tween-20 in PBS can be used instead of Triton X-100 to reduce membrane permeabilization. For more stringent washing after antibody incubation, increase Triton concentration to up to 2% or include 0.5 M NaCl to minimize nonspecific binding. Alternatively, commercial immunostaining wash buffers (e.g., Abcam ab64247) can be used for consistent results across experiments.

Primary antibody incubation solution 1 (for germinal center immunostaining)

Reagent	Final concentration	Volume
Purified anti-human CXCR4 (CD184)	1:500	0.004 mL
Human CD83 recombinant rabbit antibody	1:500	0.004 mL
Blocking buffer	N/A	1.992 mL
Total	N/A	2 mL

Primary antibody incubation solution 2 (for epithelial-immune immunostaining)

Reagent	Final concentration	Volume
Pan-Keratin Polyclonal antibody	1:500	0.004 mL
Blocking buffer	N/A	1.996 mL
Total	N/A	2 mL

Storage: Prepare fresh before use.

CRITICAL: Avoid repeated freeze-thaw of antibodies. Incubate 12–16 h at 4°C to enhance signal specificity.

Secondary antibody incubation solution 1 (for germinal center immunostaining)

Reagent	Final concentration	Volume
F(ab’)2-Goat anti rabbit IgG (H+L) Alexa Fluor™ plus 488	1:1000	0.002 mL
F(ab’)2-Goat anti mouse IgG (H+L) Alexa Fluor™ plus 555	1:1000	0.002 mL
Blocking buffer	N/A	1.996 mL
Total	N/A	2 mL

Secondary antibody incubation solution 2 (for epithelial-immune immunostaining)

Reagent	Final concentration	Volume
F(ab’)2-Goat anti mouse IgG (H+L) Alexa Fluor™ plus 555	1:1000	0.002 mL
Blocking buffer	N/A	1.998 mL
Total	N/A	2 mL

Storage: Prepare immediately before use; protect from light.

CRITICAL: All steps involving fluorochrome-conjugated antibodies should be performed in the dark.

Alternatives: PBS containing 1% BSA can substitute for this buffer if fluorescence intensity is stable. For delicate antigens or when signal quenching is observed, use PBS with 0.05% Tween-20 or commercial antibody diluents (e.g., Dako Antibody Diluent, Agilent S302283-2) to reduce nonspecific binding.

Tertiary antibody incubation solution 1 (for germinal center immunostaining)

Reagent	Final concentration	Volume
Anti-human CD20 Monoclonal Antibody (L26), eFluor™ 660	1:200	0.01 mL
Blocking buffer	N/A	1.99 mL
Total	N/A	2 mL

Tertiary antibody incubation solution 2 (for epithelial-immune immunostaining)

Reagent	Final concentration	Volume
PE anti-human CD45 Antibody	1:200	0.01 mL
Blocking buffer	N/A	1.99 mL
Total	N/A	2 mL

Storage: Prepare immediately before use; protect from light.

CRITICAL: Ensure adequate washing before this step to prevent antibody cross-binding.

Flow cytometry staining buffer

Reagent	Final concentration	Volume
FBS	2% (v/v)	0.2 mL
PBS	98% (v/v)	9.8 mL
Total	N/A	10 mL

Storage: Store at 4°C for up to 2 weeks. Prepare fresh if contamination or turbidity is observed.

CRITICAL: Keep on ice during use to maintain cell viability. The buffer is used for washing and resuspending cells before antibody staining to maintain viability and reduce nonspecific binding.

Alternatives: Commercially available FACS buffers (e.g., BD Pharmingen Stain Buffer [FBS], Cat. No. 554656) can replace the homemade buffer. For experiments sensitive to serum-derived artifacts, substitute FBS with 1% BSA or 2% human serum albumin (HSA) in PBS.

Flow cytometry single-stain control solutions

Antibody/dye	Dilution/volume (μL)	FBS (μL)	Flow cytometry staining buffer (μL)	Total volume (μL)
FITC anti-human CD3	1(1:50)	1	48	50
Alexa Fluor® 700 anti-human CD19	1(1:50)	1	48	50
BV510 Mouse Anti-Human CD45	1(1:50)	1	48	50
APC anti-human CD4	1(1:50)	1	48	50
BV421 Mouse Anti-Human CD45	1(1:50)	1	48	50
PI	1(1:1000)	N/A	999	1000
blank	N/A	1	49	50

Storage: Prepare fresh before use; protect from light.

Note: Surrogate antibodies for compensation. To ensure accurate compensation matrices, high-expression lineage markers were used as single-stain controls for channels detecting lower-abundance antigens. Specifically, CD45 was used for BV510 and BV421, and CD4 was used for APC.

CRITICAL: Keep all single-stain control samples on ice and in the dark during staining and acquisition. Acquire within 1 h of staining to ensure fluorochrome and viability dye stability.

Alternatives: Use DAPI or 7-AAD as alternative live/dead dyes if PI is not compatible with your laser configuration. Commercial staining buffers (e.g., BioLegend Staining Buffer, Cat. No. 420201) can replace PBS + 2% FBS for consistent performance across replicates.

Fluorescence minus one as controls

Antibody/dye	FMO
FITC anti-human CD3	1 μL
Alexa Fluor® 700 anti-human CD19 Antibody	1 μL
BD Horizon™ BV510 Mouse Anti-Human CD27	N/A
APC anti-human CD38 Antibody	N/A
BV421 Mouse Anti-Human CD86	N/A
FBS	1 μL
Flow cytometry staining buffer	47 μL
Total	50 μL

Note: FMO controls are used to define gating boundaries for each marker by including all antibodies in the panel except the one being measured. Prepare fresh, keep on ice, and protect from light. Adjust volumes proportionally if total staining volume differs.

Cell antibody staining buffer (for flow cytometry)

Reagent	Final concentration	Volume
FITC anti-human CD3	2% (v/v)	0.04 mL
Alexa Fluor® 700 anti-human CD19 Antibody	2% (v/v)	0.04 mL
BD Horizon™ BV510 Mouse Anti-Human CD27	2% (v/v)	0.04 mL
APC anti-human CD38 Antibody	2% (v/v)	0.04 mL
BV421 Mouse Anti-Human CD86	2% (v/v)	0.04 mL
FBS	2% (v/v)	0.04 mL
Flow cytometry staining buffer	88% (v/v)	1.76 mL
Total	N/A	2 mL

Storage: Prepare fresh before use; protect from light.

CRITICAL: Keep samples on ice and in the dark during staining to prevent fluorochrome bleaching.

Alternatives: Depending on experimental design, alternative fluorophore combinations may be selected to match cytometer laser configurations. Alternatively, use commercial flow antibody diluents (e.g., BioLegend Staining Buffer, Cat. No. 420201) for improved consistency across replicates.

Cell Live/Dead viability staining buffer (for flow cytometry)

Reagent	Final concentration	Volume
PI	0.1% (v/v) (1:1000)	0.01 mL
Detecting buffer from AM/PI kit	99.9% (v/v)	9.99 mL
Total	N/A	10 mL

Storage: Prepare fresh before use; protect from light.

CRITICAL: Keep stained samples on ice and in the dark. Perform flow cytometry analysis within 1 h after staining to ensure accurate viability assessment.

Alternatives: Other viability dyes such as 7-AAD, Zombie Aqua, or LIVE/DEAD Fixable Dead Cell Stain Kits (Thermo Fisher) can be used depending on instrument laser configuration and fixation requirements.

Flow cytometer configurations

Beckman Coulter CytoFLEX configuration

Channel	Laser	Bandpass filter
FL1	Violet – 405 nm	450/45
FL1	Violet – 405 nm	525/40
FL2	Blue – 488 nm	488/8
FL2	Blue – 488 nm	585/42
FL2	Blue – 488 nm	525/40
FL2	Blue – 488 nm	690/50
FL2	Blue – 488 nm	780/60
FL3	Red – 638 nm	660/10
FL3	Red – 638 nm	712/25
FL3	Red – 638 nm	780/60

Step-by-step method details

Generation of human tonsil immune organoids

Timing: 15–20 days (total)

Step 1: 1–2 h

Step 2–4: 2–3 h

Step 5: 1–2 h

Step 6: 15–20 days

This step describes the preparation and seeding of human tonsil cells into a 3D matrix to generate tonsil organoids that reconstruct epithelial and germinal center–like compartments. Proper cell density and matrix handling are essential to ensure uniform organoid formation and viability.

• 1.
  Preparation of transwell Plates.

  • a.
    Pre-cool tonsil organoid medium on ice.
  • b.
    Add 100 μL of basal DMEM/F-12 medium (commercial product, e.g., Gibco™ Cat# 11320033) to the upper chamber and 500 μL to the lower chamber, avoiding bubbles.
  • c.
    Incubate in a 37°C, 5% CO₂ incubator for 1–2 h to equilibrate temperature and CO₂.
  • d.
    After equilibration, gently aspirate and discard the medium from both chambers.
  • e.
    Add 600 μL of complete tonsil organoid medium to the lower chamber, taking care to avoid air bubbles, which may affect nutrient exchange and cell viability.
• 2.
  Thawing and preparation of tonsil cells.

  • a.
    Prepare 10 mL of PBS in a 15 mL centrifuge tube and keep on ice.
  • b.
    Retrieve frozen tonsil cell vials from liquid nitrogen storage and rapidly thaw in a 37°C water bath until most of the freezing medium is thawed.
  • c.
    Transfer cells to the pre-prepared PBS tube, centrifuge at 300 × g for 5 min, and discard the supernatant.
  • d.
    Resuspend the cell pellet in 1 mL of tonsil organoid medium and mix thoroughly.
  • e.
    Count cells using 20 μL of suspension mix with 20 μL trypan blue to determine live cell density in the cell counter.
  • f.
    Add 5 mL PBS to cells, centrifuge at 300 × g for 5 min, and discard the supernatant.
  • g.
    Keep the cell pellet on ice in a 15 mL metal ice box until further use.
• 3.
  Preparation of Low-Concentration Matrigel Solution.

  • a.
    Prepare 5% (v/v) Matrigel solution by diluting Matrigel with tonsil organoid medium.

    • i.
      Example: 50 μL Matrigel + 950 μL tonsil organoid medium for 1 mL of 5% solution.

      Note: For stimulated conditions, supplement the medium with 4 μg/mL NTD antigen or other antigen of interest before mixing with Matrigel. For unstimulated controls, use medium without antigen.

  • b.
    Keep the solution on ice until use.
• 4.
  Preparation of Cell-Matrix Suspension.

  • a.
    Resuspend cells in the prepared 5% Matrigel-containing medium (with or without antigen), and adjust cell concentration to achieve 2 × 10⁶ live cells per 100 μL final suspension (See troubleshooting 2).
  • b.
    Mix gently by pipetting up and down to avoid bubbles.

Note: Matrix-scaffold and low-concentration extracellular matrix approaches follow earlier organoid culture guidelines.¹¹

• 5.
  Seeding Cells into transwell Insert.

  • a.
    Add 100 μL of the cell–matrix suspension into the upper chamber of each transwell insert.
  • b.
    Gently tap or shake the plate to distribute the suspension evenly.
  • c.
    Transfer the plate to a 37°C, 5% CO₂ incubator for culture.
• 6.
  Maintenance and Culture.

  • a.
    Carefully aspirate and replace the full volume (600 μL) of complete tonsil organoid medium (without antigen) in the lower chamber every 2 days.
  • b.
    Germinal center-like structures initiate around Day 7, maturing by Day 15 (Figure 3).
  • c.
    Continue culture for 15–20 days to generate tonsil organoids with both epithelial layers and germinal center structures.

Note: Do not add NTD antigen during medium changes. Antigen stimulation is performed only during the initial seeding.

CRITICAL: Maintain Matrigel and cell suspension on ice at all times to prevent premature polymerization. Handle cells gently to preserve viability.

Figure 3.

Immunofluorescence characterization of germinal centers

Confocal images (20×) showing CD20⁺B cell (yellow), dark zone CXCR4⁺DZ (green), light zone CD83⁺LZ (red) and DAPI (blue) structures are visible within germinal centers. Scale bars, 100 μm.

Observation and maintenance of tonsil organoids

Timing: 20 days (total)

Step 7: 20 days

Step 8: 1 h

This step ensures the healthy growth of tonsil organoids and allows early detection of abnormal morphology or contamination.

• 7.
  Monitor organoid formation.

  • a.
    Examine organoids every 2 days using a phase-contrast or inverted light microscope.
  • b.
    Record cell aggregation, cluster formation, and overall morphology (Figure 4).
• 8.
  Mechanical dispersion.

  • a.
    On approximately Day 13 post-seeding, gently pipette organoid clusters using a disposable transfer pipette to break large clusters into smaller units. This step is crucial to prevent the formation of necrotic cores caused by hypoxia in oversized organoids.
  • b.
    Take care to avoid introducing air bubbles during pipetting.

Note: Regular monitoring ensures organoid integrity, reproducibility, and consistency for downstream analyses.

Figure 4.

Bright-field imaging of tonsil organoid formation over time

Representative optical images (5×) showing the morphological development of tonsil organoids at days 1, 2, 4, 6, 8, 10, 12, 14, 17 and 20. Scale bars, 200 μm.

Immunofluorescence staining of tonsil immune organoids

Timing: 2 days (total); 3–4 h for step 9; 2 days for step 10

This section describes the fixation, cryo-embedding, sectioning, and immunofluorescence staining procedures for human tonsil immune organoids.

These steps enable the visualization and analysis of the epithelial and lymphoid compartments, including germinal center markers, within the organoid structure.

• 9.
  Fixation and cryo-embedding.

  • a.
    Gently harvest organoids from the transwell using a wide-bore pasteur pipette (or a 1000 μL tip with the end trimmed) and transfer them into 1.5 mL microcentrifuge tubes on Day 15 of culture.

    Note: Allow organoids to settle by gravity down 1.5 mL microcentrifuge tubes, and carefully remove as much supernatant as possible using a P10 pipette tip, being careful not to disturb the organoids.

  • b.
    Fix organoids by adding 200 μL of 4% paraformaldehyde (PFA) into the same tube at 20°C–25°C for 15–20 min.

    CRITICAL: Dispense the PFA slowly along the inner wall of the tube to avoid turbulence.

    Optional: Add 4% PFA directly into the upper chamber of the transwell insert and fix at 20°C–25°C for 15–20 min.

  • c.
    Remove the fixative and wash organoids 1–2 times with PBS.
  • d.
    Sequentially dehydrate the samples in the same tube by aspirating the supernatant and adding 200 μL of 10%, 20%, and 30% Sucrose solutions (w/v) respectively. Incubate for 10–20 min at 20°C–25°C for each step.
  • e.
    Transfer organoids to cryomolds (approximately 0.9 × 0.9 cm), fill with OCT compound (avoid bubbles), and freeze at −80°C (See troubleshooting 7).
  • f.
    Cut organoids at 10 μm thickness using a cryostat and store slides at −80°C until use.
• 10.
  Immunofluorescence staining (see Figures 3 and 5).

  • a.
    Re-fix sections in 4% PFA for 10 min at 20°C–25°C.
  • b.
    Outline the tissue area with a hydrophobic barrier pen.
  • c.
    Add 200 μL of PBS directly onto the tissue area outlined by the hydrophobic barrier. Wash slides on a shaker (30 g, 3–5 min) to remove residual OCT.

    CRITICAL: Do not allow slides to dry after this step.

  • d.
    Block the sections with blocking buffer for 2 h at 20°C–25°C.
  • e.
    Add Primary antibody incubation solution 1 or 2 (depending on the experimental panel) to each section and incubate 12–16 h at 4°C in a humidified chamber.

    Note: Keep slides moist throughout incubation.

  • f.
    Wash slides 2–3 times in washing buffer (3–5 min per wash, 30 g).
  • g.
    Incubate with fluorophore-conjugated secondary antibody incubation solution 1 or 2 (depending on the experimental panel) for 1 h at 20°C–25°C in the dark.

    CRITICAL: Protect fluorophore-labeled samples from light to prevent photobleaching.

    Note: Avoid excessive secondary antibody or prolonged incubation to reduce background.

  • h.
    Wash slides again 3 times in washing buffer (3–5 min per wash).
  • i.
    Add Tertiary antibody incubation solution 1 or 2 (depending on the experimental panel) to each section and incubate 3h at 20°C–25°C in a humidified chamber.
  • j.
    Wash slides again 3 times in washing buffer (3–5 min per wash).
  • k.
    Mount slides with antifade mounting medium containing DAPI (≈20 μL) and gently place coverslips from one edge to another to minimize bubbles.

    Note: Press gently between dust-free papers or books to ensure flat mounting. Seal coverslip edges with clear nail polish and image immediately using a confocal laser scanning microscope. If imaging cannot be performed immediately, store at 4°C and image within one week.

Figure 5.

Immunofluorescence characterization of lymphocyte cell and epithelial organization

Confocal images (20×) showing lymphocyte cell (CD45⁺), and epithelial (anti-Pan-CK⁺) distributions. Scale bars, 100 μm.

Flow cytometric analysis of tonsil organoids

Timing: 4–7 h (total)

Step 11: 1–2 h

Step 12–13: 2–3 h

Step 14: 1–2 h

This procedure enables quantitative assessment of immune cell composition and activation states within the tonsil organoids. Flow cytometry allows detailed profiling of lymphocyte subsets and activation markers, providing insights into immune organization and germinal center formation within the 3D structure.

• 11.
  Organoid dissociation.

  • a.
    Collect the medium from the upper chamber of the transwell into a sterile 1.5 mL microcentrifuge tube on Day 15 of culture.
  • b.
    Add 50 μL of 0.25% trypsin to the upper chamber and incubate at 37°C for 3–5 min.

    Note: Avoid over-digestion (>5 min) during trypsinization to prevent loss of surface epitopes. Trypsin should cover the membrane surface without immediate vigorous mixing.

  • c.
    After incubation, gently pipette up and down to detach the cells, then collect the digested suspension into the same tube.
  • d.
    After collecting the suspension, rinse the upper chamber 1–2 times with PBS to recover any residual cells, and combine the washes with the collected suspension.
  • e.
    Pass the cell suspension through a 100 μm cell strainer to obtain a single-cell suspension.
  • f.
    Centrifuge at 300 × g for 5 min and discard the supernatant.
• 12.
  Surface marker staining (Figure 6).

  • a.
    Resuspend the washed cells in fluorochrome-conjugated antibodies buffer including single-stain control solutions, Fluorescence Minus One (FMO) and Cell antibody staining buffer for the surface markers of interest (See troubleshooting 3).
  • b.
    Incubate for 30 min at 4°C in the dark.
  • c.
    Wash cells 2–3 times with cell staining buffer to remove unbound antibodies.
• 13.
  Viability staining with PI.

  • a.
    Resuspend the cell pellet in Cell Live/Dead Viability staining buffer (See troubleshooting 4).

    Alternatives: Fixable Viability Staining (Crucial for Intracellular Staining) If the experiment involves downstream fixation and permeabilization (e.g., for intracellular cytokines), Propidium Iodide (PI) cannot be used as it is not fixable. Instead, use an amine-reactive Fixable Viability Dye (e.g., Zombie dyes or LIVE/DEAD Fixable Dead Cell Stains).

  • b.
    Incubate on ice for 10–15 min in the dark.

    Note: Keep samples on ice and protected from light after adding PI. Acquire data as soon as possible, ideally within 1 h, and no longer than 4 h to preserve staining accuracy. Do not fix cells before PI staining (e.g., with paraformaldehyde or ethanol), as fixation compromises membrane integrity and causes all cells to become PI-positive, preventing live/dead discrimination.

    CRITICAL: Always stain cells with surface marker antibodies first, and add PI as the last step before acquisition. After adding PI, do not wash the cells, as removing PI can reduce staining intensity and affect live/dead discrimination.

• 14.
  Flow cytometric acquisition and analysis.

  • a.
    Acquire data on a flow cytometer within immediately (within 1–4 h) after staining.

    Note: If acquisition must be delayed (up to 24 h), store the cells in cell staining buffer at 4°C in the dark without PI. Add PI 10–15 min prior to acquisition. Or fixed samples can be stored at 4°C and analyzed within 24 h for Fixable Viability Dyes.

  • b.
    Analyze immune cell subsets markers using FlowJo software.

    CRITICAL: Keep cells on ice whenever possible to preserve viability. Perform gentle pipetting during washing to minimize cell loss. Protect all samples from light after the addition of fluorescent dyes.

Figure 6.

Flow cytometric quantification of immune cell subsets within tonsil organoids

Flow cytometric quantification of immune cell subsets in tonsil organoids cultured with or without NTD and IL-21 stimulation. Tonsil-derived organoids (Organoid group) are compared with unstimulated tonsil cell cultures (Control group), using samples from 4 individual donors (n = 4). Organoid: Stimulated tonsil organoids (cultured with NTD and IL-21). Control: Unstimulated tonsil cells (without NTD and IL-21).

(A) Naïve B cells (CD19⁺CD27^-CD38^-) are significantly reduced in organoids compared to control, indicating activation and differentiation under antigen stimulation.

(B) Pre-germinal center B cells (CD19⁺CD27^-CD38⁺) are enriched in organoids, suggesting enhanced early GC commitment.

(C) Plasmablasts (CD19⁺CD27⁺⁺CD38⁺⁺) display higher abundance in organoid cultures, reflecting active antibody-secreting cell differentiation.

(D) Activated B cells (CD19⁺CD86⁺) show increased frequency in organoids compared to control.

(E) Germinal center B cells (CD19⁺CD27⁺CD38⁺) do not exhibit significant differences between groups.

(F) Memory B cells (CD19⁺CD27⁺CD38^-) are reduced in organoids, consistent with immune activation dynamics. Data are shown as mean ± SEM. Statistical significance was determined by unpaired t-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant.

Expected outcomes

This protocol is expected to reliably produce human tonsil immune organoids that recapitulate key structural and functional features of the tonsil microenvironment. Within 5–7 days of culture, organoids form compact 3D spheroid structures observable by brightfield microscopy. By 15–20 days, organoids develop epithelial layers on the exterior and germinal center-like regions enriched in immune cell subsets in the interior.

Immunofluorescence staining enables visualization of germinal center, including dark zone and light zones, as well as the spatial distribution of epithelial and immune cells within the tonsil organoids. Bright, distinct immunofluorescence signals reveal the organization of the epithelial layer and the localization of major immune cell populations. In parallel, flow cytometry provides quantitative measurements of germinal center formation and the proportions of major immune subsets such as various germinal center B cells, offering a complementary functional readout of immune activity within the organoids.

Collectively, the brightfield morphology, immunofluorescence patterns, and quantitative analyses demonstrate successful reconstruction of tonsil-like immune microenvironments in vitro. These organoids can serve as a robust platform to study immune cell interactions, germinal center biology, and the effects of immunomodulatory interventions.

Quantification and statistical analysis

Flow cytometry data were analyzed using FlowJo (version 10 or higher). Immune cell subsets were identified based on standard gating strategies incorporating forward and side scatter, viability dye exclusion, and lineage markers. Results are expressed as the percentage of each subset among total live cells.

To ensure the accuracy and reproducibility of scale bars in bright-field images captured under 5× magnification using a smartphone attached to the microscope, a calibration image (hemocytometer) was captured using the same microscope, objective lens (5×), and smartphone camera under identical acquisition parameters (resolution, focus, zoom, and exposure) and within the same optical field. All images were obtained without resizing, cropping, or software compression to ensure consistent pixel-to-micrometer ratios across the dataset. These conditions allowed the pixel length (measured from the hemocytometer grid) to be directly used for scale bar calibration in ImageJ. In ImageJ, the 1,000 μm square grid was selected, and five independent line measurements were drawn along one edge to obtain pixel lengths (Length). The average pixel value was used to calculate the conversion ratio between pixels and micrometers. This calibration was applied in ImageJ (Analyze - Set Scale) with the “Global” option selected to ensure consistent scaling across all images. Subsequently, a 200 μm scale bar was added to bright-field images (Analyze -Tools -Scale Bar). All calibration parameters and raw images were recorded to ensure quantitative reproducibility and transparency.

Statistical analyses were conducted in GraphPad Prism. Data are presented as mean ± SEM. Statistical significance was assessed using unpaired two-tailed t-tests or one-way ANOVA with appropriate post hoc tests. A p-value < 0.05 was considered statistically significant, and significance is indicated using the following notation: ∗∗∗∗, p < 0.0001; ∗∗∗, p < 0.001; ∗∗, p < 0.01; ∗, p < 0.05; ns, not significant. All acquired samples were included in the analysis.

Limitations

This protocol reproducibly generates human tonsil immune organoids that recapitulate both epithelial–stromal and germinal center–like immune compartments. However, several limitations should be considered. The success of organoid formation depends on the quality and viability of the tonsil tissue; samples with prolonged transport or low initial lymphocyte viability may not form well-organized immune structures. Donor-to-donor variation in immune cell composition can also affect reproducibility. The culture period required for full organoid maturation is relatively long (15–20 days), which may reduce experimental throughput and increase the risk of contamination or nutrient depletion if medium changes are not performed carefully. Finally, the use of low-concentration Matrigel improves nutrient exchange but reduces structural rigidity, making handling during long-term culture more delicate.

Troubleshooting

Problem 1 (step 4a of preparation one)

If tonsil tissue is not promptly placed on ice in tissue storage solution after surgical removal, or if transport to the laboratory is delayed before dissociation into single cells, or if only a small fragment of tissue is collected, overall cell viability can drop significantly, with B cells particularly affected. Under normal conditions, B cells constitute approximately 40%–70% of tonsil lymphocytes, but they are highly sensitive to temperature and ischemia. Improper handling can compromise organoid formation and downstream immune analyses.

Potential solution

• •Tissue preservation and ischemia prevention: Immediately place freshly resected tonsil tissue on ice in a pre-cooled tissue preservation medium to minimize ischemic and thermal damage. This is critical for maintaining lymphocyte viability, especially for temperature-sensitive B cells.
• •Transport and processing time: Minimize transport and processing time. Ideally, dissociate the tissue within 4 hours of surgical removal while maintaining the cold chain (4°C). Under no circumstances should the total time from resection to dissociation exceed 48 hours, as prolonged delays may result in substantial loss of immune cell viability and altered cell composition.
• •Tissue size and sampling strategy: Avoid using excessively small tissue fragments. If only small samples are available, consider pooling multiple fragments from the same donor to ensure sufficient cellular yield. If the tonsil tissue from a donor is too small to support organoid generation, the sample can be excluded from dissociation.
• •Temperature control during dissociation: Maintain all dissociation and handling steps on ice, except for enzymatic digestion steps that require 37°C incubation. Constant low temperature helps preserve lymphocyte viability and functionality, particularly of B cells, which are highly temperature-sensitive.
• •Pre-seeding cell viability assessment: Assess cell viability before proceeding with organoid seeding. Ensure that the post-thaw or freshly dissociated single-cell suspension has a viability greater than 80%. Low-viability samples (<70%) may compromise organoid formation efficiency and immune cell compartmentalization.

Problem 2 (step 4a of step-by-step method details)

The success and reproducibility of tonsil organoid formation are highly dependent on optimal cell seeding density and matrix consistency. Variations in the number of plated cells, the type of culture plate used (e.g., 6-well, 24-well, or 96-well), or the batch of extracellular matrix (ECM) such as Matrigel or tissue-derived ECM can significantly affect organoid morphology, immune compartmentalization, and growth rate. These inconsistencies may lead to poor reproducibility.

Potential solutions

• •Optimize seeding density for each plate format: Determine the optimal cell number empirically for each plate type. Typically, 6 × 10⁶ cells per well for a 6-well plate, 2 × 10⁶ cells per well for a 24-well plate and 1 × 10⁶ cells per well for a 96-well plate yield reproducible tonsil organoid formation. Maintaining comparable cell-to-matrix ratios across plate types ensures similar nutrient and cytokine gradients, promoting consistent tissue organization.
• •Avoid under- or over-seeding: Low seeding density may result in incomplete organoid formation and poor cell–cell interaction, whereas excessive density can cause necrotic cores or abnormal morphology. Perform preliminary titration experiments to establish an optimal range and maintain it consistently across biological replicates.
• •Use the same ECM batch for all related experiments: Different batches of Matrigel or tissue-derived ECM may vary in protein composition, stiffness, and growth factor content. To minimize batch effects, use the same ECM batch for all experiments within the same study. If new ECM batches are introduced, perform a side-by-side comparison using the same cell preparation to ensure consistency.
• •Standardize plating procedures: Maintain uniform mixing of cell suspension and ECM and consistent handling temperature to prevent premature gelation. When possible, use pre-cooled pipette tips and plates. Uneven mixing or temperature fluctuation can lead to variable gel structure and uneven cell distribution.

Problem 3 (step 12a of step-by-step method details)

Incorrect fluorophore combinations or neglecting instrument-specific laser and detector configurations can result in severe spectral overlap, false-negative signals, or misinterpretation of co-localization and cell population data. This issue is particularly relevant for both multicolor confocal imaging and flow cytometry assays.

Potential solutions

• •Fluorophore selection and spectral separation: Choose fluorophores with well-separated excitation and emission spectra according to the available laser lines of the specific confocal or flow cytometer. For example, avoid pairing fluorophores that share similar excitation/emission profiles (e.g., FITC and Alexa Fluor 488) or those overlapping within the same detector channel.
• •Instrument-specific panel design: Design antibody panels based on the laser and filter configuration of your instrument (e.g., 405 nm, 488 nm, 561 nm, 640 nm). Many cytometers and confocal systems provide built-in spectral viewers or online tools (e.g., BD Spectrum Viewer) that help optimize fluorophore combinations and minimize spillover.
• •Perform single-stain and compensation controls: Always include single-stained controls for each fluorophore to establish compensation and confirm the absence of spectral bleed-through. Use fluorescence-minus-one (FMO) controls for precise gating in multicolor flow cytometry.
• •Validate antibody–fluorophore compatibility: Before performing large-scale staining, test each antibody–fluorophore pair on pilot samples to confirm stable binding and adequate signal intensity under your specific imaging or flow settings.

Problem 4 (step 13a of step-by-step method details)

Incorrect use of propidium iodide (PI) in flow cytometry can lead to inaccurate viability assessment. If PI is added before or during antibody staining, it may produce high background, interfere with antibody binding, or cause all cells to appear dead if cell membranes are compromised, leading to false-negative or false-positive results.

Potential solutions

• •Sequential staining: Always stain cells with surface marker antibodies first, then add PI immediately prior to acquisition. Do not wash after adding PI to preserve live/dead discrimination.
• •Alternative for Intracellular Staining: If the experiment involves fixation and permeabilization (e.g., for intracellular cytokines), do not use PI as it is not fixable and will wash out or stain all cells. Instead, use an amine-reactive Fixable Viability Dye (e.g., Zombie dyes™ or LIVE/DEAD™ Fixable Stains).
• •Minimize light exposure: Keep PI-stained samples on ice and protected from light to prevent photobleaching and maintain fluorescence intensity.
• •Acquisition time: Acquire PI-stained samples as soon as possible, ideally within 1 hour, but no longer than 4 hours. Delayed acquisition can reduce signal stability and accuracy of viability measurement.
• •Safety precautions: Handle PI in a biosafety cabinet, wear appropriate personal protective equipment (PPE) including gloves and lab coat, and dispose of PI-containing waste according to institutional guidelines for mutagenic/carcinogenic chemicals.

Problem 5 (step 11a of preparation two)

Using conventional cryopreservation medium (90% FBS + 10% DMSO) results in low viability and poor recovery of L-WRN cells after thawing.

Potential solutions

• •Use 92% FBS + 8% DMSO for optimal cryoprotection. This composition maintains high cell viability and recovery rates after thawing.
• •Controlled-rate freezing: Freeze cells slowly (−1°C/min) using an isopropanol freezing container or programmable freezer before transferring to liquid nitrogen for long-term storage.
• •Rapid thawing and gentle handling: Thaw cells rapidly in a 37°C water bath and immediately dilute DMSO with PBS to minimize cytotoxicity. Avoid excessive pipetting to reduce mechanical stress.

Problem 6 (organoid medium of materials and equipment)

Repeated freezing and thawing of cytokines such as BAFF, IL-21, and protein antigens (e.g., NTD antigen) significantly reduces their biological activity and can lead to inconsistent immune responses in organoid cultures.

Potential solutions

• •Aliquot reagents: Prepare single-use aliquots of cytokines and antigens before initial freezing. Avoid multiple thawing cycles.
• •Storage conditions: Store cytokines and protein reagents at −80°C. Use low-protein-binding tubes to minimize adsorption and loss of active molecules.
• •Quality verification: If cytokine activity is in doubt, validate function using a short-term stimulation assay (e.g., pSTAT3 activation for IL-21) before applying to organoid cultures.

Problem 7 (step 9e of step-by-step method details)

Due to their small size, tonsil organoids may be lost during cryo-sectioning or difficult to locate for immunofluorescence staining, resulting in incomplete or missing data.

Potential solutions

• •Use colored OCT compound: Embed organoids in colored OCT to improve visibility during sectioning. This helps locate the organoid under the stereomicroscope and ensures correct trimming.
• •Microscopic confirmation before staining: Observe cryosections under bright-field microscopy to confirm the presence of organoid structures before proceeding with staining.
• •Whole-mount staining option: As an alternative, perform whole transwell immunofluorescence staining directly without sectioning. This approach preserves spatial architecture and minimizes loss of organoids.

Resource availability

Lead contact

Further information and requests for resources, reagents, or additional details related to this protocol should be directed to and will be fulfilled by the lead contact, Xiao Liu (liuxiao@sz.tsinghua.edu.cn).

Technical contact

Technical questions or requests for guidance on executing this protocol, including tissue dissociation, organoid culture, and immunostaining procedures, should be directed to and will be answered by the technical contact, Dandan Meng (mdd23@mails.tsinghua.edu.cn).

Materials availability

• •This study did not generate new unique reagents or cell lines.
• •All human tonsil samples were obtained from consenting donors in accordance with institutional ethical guidelines.
• •All commercially available reagents and materials used in this protocol, including organoid culture media and supplements, are listed in the key resources table. Researchers interested in obtaining specific components should refer to their respective suppliers.

Data and code availability

This protocol did not generate new software or code. All data reported are available from the lead contact upon request.

Acknowledgments

This work was supported by funding from the Shenzhen Science and Technology Program (WDZC20220819134430002), the Scientific Research Start-up Funds (QD2021005N), and Department of Chemical Engineering-iBHE Special Cooperation Joint Fund (DCE-iBHE-2025-2).

We thank all members of the Institute of Biopharmaceutical and Health Engineering (iBHE), Tsinghua Shenzhen International Graduate School, for their valuable discussions and technical assistance. We also acknowledge the support of the Tsinghua University Core Facilities for Life Sciences for providing access to flow cytometry and confocal microscopy platforms. The graphical abstract was created with BioRender.com.

Author contributions

D.M.: conceptualization, investigation, data curation, formal analysis, visualization, and writing – original draft; H.D.: investigation, methodology, and formal analysis; Q.D.: investigation and validation; X. Li: writing – review and editing; Y.T.: writing – review and editing; X. Liu: supervision, funding acquisition, project administration, and writing – review and editing.

Declaration of interests

The authors declare no competing interests.