Generation and Genetic Manipulation of Human Cervical Organoids

Summary

We present a detailed protocol to establish and genetically manipulate human cervical organoids.

Abstract

Cervical cancer is the fourth most common cancer in women and most frequently affects the uterine ectocervix. The uterine ectocervix is lined by stratified squamous epithelium, comprising basal cells and differentiating parabasal and suprabasal cells. The regeneration observed after cervical conization suggests the presence of robust stem cell activity. However, our understanding of the identity and regulatory mechanisms of cervical stem cells and their malignant transformation process has been limited due to the restricted access to human cervical tissues and the lack of optimal systems to assess stem cell activity in the uterine ectocervix. Recently established human cervical organoids can help overcome these limitations and offer new opportunities to study cervical physiology and pathology. However, no standardized method exists for isolating and establishing human cervical organoids. Here, we describe a method for establishing human ectocervical organoids. Specifically, we present a protocol to isolate single cells from the human ectocervical epithelium. By incubating cervical tissue in an optimized enzyme solution, we peel off the cervical epithelium to maximize cell yield while minimizing fibroblast contamination. We further outline the conditions for culturing human cervical organoids using an optimized medium formulation. In addition, we detail the procedures for passaging and genetically manipulating cervical organoids. This protocol provides a highly efficient approach for establishing human cervical organoids and utilizing them to study cervical stem cells and diseases.

Introduction

The uterine ectocervix is the most common site for cervical cancer^1,2. Although cervical cancer has been decreasing after the introduction of human papillomavirus (HPV) vaccination in developed countries, globally it still remains the fourth most common cancer among women^3,4. Also, the incidence of cervical precancerous lesions continues to increase in some countries^5,6. While low-grade squamous intraepithelial lesion (LSIL) often regresses on its own, high-grade squamous intraepithelial lesion (HSIL) carries a risk of progression to invasive cancer and typically requires surgical intervention, such as excision or conization. These procedures can shorten the ectocervix and increase the risk of miscarriage or preterm delivery^7,8. A deeper understanding of cervical biology, coupled with the development of regenerative therapy, could help address these challenges but requires robust and physiologically relevant model systems.

Animal models have significantly advanced our understanding of cervical homeostasis, disease mechanisms, and therapeutic strategies. However, their inherent differences from human biology and pathology limit their translational value. As an alternative, organoids have emerged as powerful platforms for modeling human biology and diseases. Organoids are stem cell-derived 3-dimensional microstructures that recapitulate the histology and molecular signatures of their tissue of origin^9,10,11. They have been employed to identify diverse human tissue stem cells across multiple tissues, including the trachea, esophagus, and cervix^12,13,14. Moreover, organoids enable genetic manipulation, allowing for the modeling of the pathogenesis of diverse diseases, including cancers and fibrosis^15,16,17. Additionally, organoids serve as an effective platform for screening the efficacy and toxicity of drugs or cell therapies^18,19,20,21.

Recently, two independent groups established human cervical organoid systems^22,23. Building on these advances, we significantly improved the efficiency of cervical organoid culture and identified human cervical stem cells¹³. Furthermore, by peeling off the cervical epithelium, we minimized the inclusion of fibroblasts underlying the cervical epithelium. By employing cervical organoids with genetic manipulation, we uncovered the role of microbial metabolites and their downstream pathways in maintaining cervical homeostasis and preventing precancerous progression. Thus, a standardized and optimized protocol for generating, maintaining, and genetically modifying cervical organoids will serve as an essential tool to study cervical biology and pathology.

In this article, we present a detailed and optimized protocol for generating and passaging human cervical organoids. We also describe a protocol for their genetic manipulation via electroporation.

Protocol

Cell isolation and organoid culture using human cervical tissues were approved and conducted in accordance with all relevant ethical regulations by the internal review boards of DGIST, Daegu, Korea and Chilgok Kyungpook National University Hospital, Gyeongsangbuk-do, Korea (DGIST-20210401-BR-112-01 and KNUCH-2020-12-020-001). Human ectocervical tissues were provided by the Department of Obstetrics and Gynecology at the Chilgok Kyungpook National University Hospital. Healthy cervical tissues were obtained from patients who underwent total laparoscopic hysterectomy for their benign uterine diseases. Informed consent was obtained from all patients.

1. Single-cell isolation from ectocervical tissues

NOTE: This protocol begins with a human ectocervix biopsy sample approximately 5 mm × 5 mm × 5 mm in size (Figure 1).

1. Place the biopsied tissue in a 10 cm Petri dish and wash it once with ice-cold DPBS (without calcium and magnesium) to remove blood and debris.
   NOTE: Human cervical tissues were transported to the laboratory within 3 h after surgery in ice-cold Advanced DMEM/F12 and processed immediately upon arrival.
2. Transfer the tissue to a 1.5 mL tube containing 1 mL of Dispase solution (2.4 U/mL) and incubate for 1 h at 37 °C with gentle agitation on a tube rotator (20 rpm).
   NOTE: The Dispase solution (2.4 U/mL) was freshly prepared immediately before use by dissolving Dispase powder to the working concentration in ice-cold DPBS.
3. Transfer the sample to a new 10 cm Petri dish. Carefully separate the white epithelial layer from the rest of the tissue using forceps.
4. Use sharp forceps to gently peel off the epithelial surface as an intact sheet. Immediately transfer the isolated epithelial layer into a 50 mL conical tube kept on ice.
   NOTE: If the surface epithelium is not easily peeled off, scrape the surface with blunt-ended forceps.
5. Add 1 mL of a recombinant trypsin-based dissociation solution to the peeled or scraped sample and transfer the entire mixture to a 50 mL conical tube.
6. Keep the remaining tissue from the previous step in the other 50 mL conical tube.
7. Incubate the 50 mL conical tube containing cervical epithelium at 37 °C for 5 min with gentle agitation on a tube rotator (20 rpm).
8. Add 10 mL of freshly prepared ice-cold Blocking Buffer (PBS with 2% bovine calf serum (BCS)) to the cell suspension to stop the enzymatic reaction.
9. Centrifuge the tube at 450 × g for 5 min at 4 °C. Carefully remove and discard the supernatant.
   NOTE: If blood is visually observed in the dissociated tissue or the tissue appears sticky, proceed with the following step. Otherwise, jump to step 1.13.
10. Add 1 mL of 1X RBC lysis buffer and 20 µL of DNase (10 U/µL stock) to the pellet. Incubate at room temperature for 5 min to lyse red blood cells and break down sticky DNA.
11. Add 10 mL of ice-cold Blocking Buffer (PBS with 2% bovine calf serum (BCS)) to the cell suspension to stop the enzymatic reaction.
12. Centrifuge the tube at 450 × g for 5 min at 4 °C. Carefully remove and discard the supernatant.
13. Wash the cell pellet with ice-cold Blocking Buffer (PBS with 2% bovine calf serum (BCS)).
14. Centrifuge the tube at 450 × g for 5 min. Carefully remove and discard the supernatant.
15. Resuspend the cell pellet in 200-500 µL of organoid culture medium.
16. Count the cell number by using hemocytometer with trypan blue solution. The expected cell yield is between 50,000 and 500,000 cells per biopsy sample.

2. Ectocervical organoid seeding and culture

1. Resuspend 500-2,000 viable cells in a final volume of 50 µL of organoid culture medium (composition described in Material list) in a 1.5 mL microcentrifuge tube. Cell viability was assessed by trypan blue exclusion prior to seeding.
2. Add 50 µL of a basement membrane matrix (e.g., Matrigel) to the cell suspension. Gently mix on ice to avoid bubble formation, and dispense the 100 µL mixture into the interior of a cell culture insert placed in a 24-well plate. All procedures involving basement membrane matrix handling were performed on ice or at 4 °C to prevent premature gelation unless otherwise specified.
3. Incubate the plate for 20-30 min at 37 °C until the basement membrane matrix-cell mixture solidifies.
4. Once the mixture has solidified, add 400 µL of organoid culture medium to the well below the insert.
5. Culture the organoids for 1-2 weeks until they reach an average diameter of 150-250 µm (The maximal diameter is about 500 µm).
   NOTE: When the organoids reach an appropriate size and display the concentric lamellar pattern of onion-skin-like microstructures, consider passaging if needed.

3. Organoid dissociation and reseeding

1. Add 1 mL of Dispase solution (2.4 U/mL) on top of cell culture insert and gently dissociate the basement membrane matrix using a pipette.
2. Transfer Dispase-basement membrane matrix mixture to a 15 mL tube and incubate for 1 h at 37 °C.
3. Add 10 mL of freshly prepared ice-cold Blocking Buffer (PBS with 2% bovine calf serum (BCS)) to the cell suspension to stop the enzymatic reaction.
4. Centrifuge the tube at 450 × g for 5 min at 4 °C.
5. Remove the supernatant and resuspend the pellet in 200 µL of TrypLE Express.
6. Incubate for 5 min at 37 °C.
7. Add 1 mL of ice-cold blocking buffer (PBS with 2% bovine calf serum (BCS)) and pipette gently to neutralize the enzyme.
8. Filter the suspension through a 40 µm cell strainer to obtain single cells.
9. Centrifuge the tube at 450 × g for 5 min at 4 °C. Carefully remove and discard the supernatant.
10. Resuspend the cell pellet in 200-500 µL of organoid culture medium.
11. Count the cell number by using hemocytometer with trypan blue solution. The expected cell yield is between 50,000 and 500,000 cells per well of 24-well cell culture insert.
12. Then resuspend 500-2,000 cells in 50 µL of organoid culture medium (composition described in Material list). Add 50 µL of basement membrane matrix to the cell suspension. Mix gently, avoid bubble formation, and dispense the 100 µL mixture into the interior of a cell culture insert placed in a 24-well plate.
13. Incubate the plate for 20-30 min at 37 °C until the basement membrane matrix-cell mixture solidifies.
14. Once the mixture is fully solidified (typically after 20-30 min at 37 °C), add 400 µL of organoid culture medium to the well below the insert.
15. Culture the organoids for 1-2 weeks in organoid culture medium (composition described in Material list) using a 3D extracellular matrix-embedded method. Maintain cultures at 37 °C, 5% CO₂ in a humidified incubator. Replace the medium every 2-3 days and monitor growth until organoids reach an average diameter of 150-250 µm.
16. Passage the organoids for the next round when they reach an appropriate size and display the concentric lamellar pattern of onion-skin-like microstructures, if needed.

4. Genetic manipulation of human cervical organoids by electroporation

NOTE: This protocol is based on using a Lonza Amaxa 4D-Nucleofector and the P4 Primary Cell 4D-Nucleofector X Kit (#V4XP-4032).

1. After isolation from human cervical cell suspension, transfer 100,000-200,000 cells to each 1.5 mL tube.
2. Centrifuge the tubes at 450 × g for 3 min at 4 °C. Carefully remove and discard the supernatant.
3. Resuspend the cell pellet in 20 µL of the Nucleofector Solution/Supplement mixture from the P4 Primary Cell 4D-Nucleofector X Kit. The mixture is prepared by combining 16.4 µL of Nucleofector Solution and 3.6 µL of Supplement per tube.
4. Add 300 nM of either control or target siRNA oligo to each tube containing the resuspended cells. Mix gently by pipetting.
   NOTE: The volume of the siRNA oligo should not exceed 2 µL per tube.
5. Carefully transfer the nucleofection mixture (20 µL of cells resuspended in Nucleofector Solution/Supplement mixture + up to 2 µL siRNA; total 20-22 µL) into the electroporation cuvette Vessels from the P4 Primary Cell 4D-Nucleofector X Kit.
6. Gently tap the Nucleocuvette Vessels to ensure the sample covers the bottom of the cuvette.
7. Close the lid of the Nucleocuvette Vessels and place it into the retainer of a powered-on 4D-Nucleofector unit.
   NOTE: Ensure the correct orientation of the Nucleocuvette Vessels in the retainer.
8. Initiate the nucleofection process. Set the EA-125 pulse code for each cuvette with the P4 primary cell process.
9. After completion, carefully remove the Nucleocuvette Vessels from the retainer.
10. Incubate the Nucleocuvette Vessels at room temperature for 10 min to allow the cells to stabilize.
11. Open the lid of the Nucleocuvette Vessels, add 180 µL of 37 °C pre-warmed organoid culture medium to each cuvette, and gently transfer the cell suspension to a new 1.5 mL tube for resuspension.
12. Take a volume of cell suspension containing 500-2,000 cells and resuspend it in organoid culture medium to a final volume of 50 µL.
    NOTE: Do not discard the remaining cells from step 4.12. They will be used in step 4.14.
13. Culture the organoids by following the protocol in Method 2: Ectocervix Organoid Seeding and Culture. Compare the effects of the siRNA by assessing diverse factors, including the number, size, and structure of the resulting organoids.
14. Dispense the remaining cells from step 4.12 into a 96-well ultralow attachment plate and incubate for 48 h at 37 °C.
15. After 48 h, transfer the cells to a 1.5 mL tube and centrifuge at 450 × g for 3 min. Discard the supernatant.
16. Use the cell pellet for real-time PCR experiments to confirm the knockdown of the target genes and to assess any changes in gene expression.
    NOTE: All human cervical tissues and cell suspensions should be handled as potentially infectious material. Perform all procedures involving primary human specimens in a certified biosafety cabinet under BSL-2 practices and in accordance with institutional ethical and biosafety regulations. Wear appropriate personal protective equipment (lab coat, gloves, and eye protection) and disinfect work surfaces and equipment after use with an institution-approved disinfectant. Enzymatic reagents (e.g., dispase and dissociation enzymes such as TrypLE Express) may cause skin and eye irritation and can generate aerosols during pipetting; avoid aerosol generation, keep tubes capped whenever possible, and rinse immediately with water in case of contact while following institutional exposure procedures and manufacturer's SDS guidance. Dispose of all biological waste (including matrix-containing materials and contaminated plastics) according to institutional guidelines. For siRNA electroporation, follow the manufacturer's electrical safety instructions: ensure that cuvettes are properly seated and the lid is fully closed before initiating pulses, avoid operating the device with wet gloves or near spills, inspect the device and accessories for damage, and avoid direct contact with the cuvette electrodes immediately after pulsing.

Results

3D Human cervical organoid culture

Freshly isolated human ectocervical tissues were dissociated into single cells in the order mentioned above, which were then plated on top of 24-well cell culture inserts and cultured to form cervical organoids as outlined in this protocol (Figure 1). Human cervical organoids grow up to 500 µm in diameter from single cells over 2 weeks (Figure 2A). The success rate of generating orga...

Discussion

In this manuscript, we describe the protocol for establishing and passaging human cervical organoids. We also provide a protocol for manipulating them genetically, which significantly advances the study of cervical biology and pathology. Although we optimized the protocol specifically for human uterine cervical organoids, our protocol can be applied to other organoid systems with slight modifications.

The essential information to establish and optimize organoid culture includes a detailed medi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
P4 Primary Cell 4D-Nucleofector X Kit	Lonza	V4XP-4032
96-well ultralow attachment plate	SPL	39796
Cell strainer 40 µm	SPL	93040
50 mL conical tube 500/box	SLP	SPL50050
15 ml Conical tube	Sarstedt	62.554.502
CryoPure Tube 1.8 mL -white	Sarstedt	72.379P
37 °C, 5 % CO2 incubator	Eppendorf	9734IQ905476
Cell culture insert	SPL	37024
Amaxa 4D-Nucleofector	Lonza	AAF-1003X
Blocking buffer (2% BCS in DPBS)	Gibco / Welgene	BCS: 26010074; DPBS: LB001-02
Dispase II powder	Gibco	17105041
DNAse I	Enzynomics	M059L
Matrigel	Corning	356231
RBC lysis buffer	Biosesang	R2015
TrypLE Express	Gibco	12605
Wash buffer (HBSS)	JBI	BL003-1
Advanced DMEM/F12	Gibco	12634028
GlutaMAX supplement	Gibco	35050-061
Penicillin–streptomycin	Biowest	L0022-100
HEPES	Gibco	15630-080
B27 supplement	Gibco	17504044
Noggin	PeproTech	118214
N-acetyl-l-cysteine	Sigma	A9165
Y-27632	STEMCELL Technologies	72307
FGF2	PeproTech	100-18B-50UG
FGF7	PeproTech	AF-100-19-250UG
A83-01	Selleckchem	S7692
Forskolin	Sigma	F6886
Neuregulin 1	PeproTech	100-03-50UG
NGF	PeproTech	AF-450-01-100UG
R-spondin	Sinobiological	11083-HNAS
HGF	Peprotech	100-39H-250ug
FGF10	Peprotech	100-26
Petridish 35 mm	SPL	10035
Organoid media recipe
Name	Company	Catalog Number	concentration
Advanced DMEM/F12	Gibco	12634028
GlutaMAX supplement	Gibco	35050-061	1x
Penicillin–streptomycin	Biowest	L0022-100	1x
HEPES	Gibco	15630-080	10 mM
B27 supplement	Gibco	17504044	1x w/o vitamin A
Noggin	PeproTech	118214	100 ng/ml
N-acetyl-l-cysteine	Sigma	A9165	1 mM
Y-27632	STEMCELL Technologies	72307	10 uM
FGF2	PeproTech	100-18B-50UG	50 ng/ml
FGF7	PeproTech	AF-100-19-250UG	25 ng/ml
FGF10	Peprotech	100-26	100 ng/ml
A83-01	Selleckchem	S7692	500 nM
Forskolin	Sigma	F6886	10 μM
Neuregulin 1	PeproTech	100-03-50UG	50 ng/ml
NGF	PeproTech	AF-450-01-100UG	20 ng/ml
R-spondin	Sinobiological	11083-HNAS	100 ng/ml
HGF	Peprotech	100-39H-250ug	25 ng/ml