Name: Generation and Genetic Manipulation of Human Cervical Organoids (Video) | JoVE Journal
Uploaded: 2026-03-10T13:20:00.000+00:00
Duration: 10 min 29 s
Description: Learn to generate and genetically manipulate human cervical organoids using enzymatic digestion and nucleofection. This protocol enables long-term culture and functional studies of cervical stem cell biology and disease mechanisms.

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5 minutes

This work was supported by a grant from the Ministry of Health and&#160;Welfare (RS-2024-00439434 for Y. Jeong).

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 &#215; 5 mm &#215;&#160;5 mm in size (Figure 1).
<ol>
	<li>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.</li>
	<li>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 &#176;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.</li>
	<li>Transfer the sample to a new 10 cm Petri dish. Carefully separate the white epithelial layer from the rest of the tissue using forceps.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Keep the remaining tissue from the previous step in the other 50 mL conical tube.</li>
	<li>Incubate the 50 mL conical tube containing cervical epithelium at 37 &#176;C for 5 min with gentle agitation on a tube rotator (20 rpm).</li>
	<li>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.</li>
	<li>Centrifuge the tube at 450 &#215; g for 5 min at 4 &#176;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.</li>
	<li>Add 1 mL of 1X RBC lysis buffer and 20 &#181;L of DNase (10 U/&#181;L stock) to the pellet. Incubate at room temperature for 5 min to lyse red blood cells and break down sticky DNA.</li>
	<li>Add 10 mL of ice-cold Blocking Buffer (PBS with 2% bovine calf serum (BCS)) to the cell suspension to stop the enzymatic reaction.</li>
	<li>Centrifuge the tube at 450 &#215; g for 5 min at 4 &#176;C. Carefully remove and discard the supernatant.</li>
	<li>Wash the cell pellet with ice-cold Blocking Buffer (PBS with 2% bovine calf serum (BCS)).</li>
	<li>Centrifuge the tube at 450 &#215; g for 5 min. Carefully remove and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 200-500 &#181;L of organoid culture medium.</li>
	<li>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.</li>
</ol>
2. Ectocervical organoid seeding and culture
<ol>
	<li>Resuspend 500-2,000 viable cells in a final volume of 50 &#181;L 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.</li>
	<li>Add 50 &#181;L of a basement membrane matrix (e.g., Matrigel) to the cell suspension. Gently mix on ice to avoid bubble formation, and dispense the 100 &#181;L 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 &#176;C to prevent premature gelation unless otherwise specified.</li>
	<li>Incubate the plate for 20-30 min at 37 &#176;C until the basement membrane matrix-cell mixture solidifies.</li>
	<li>Once the mixture has solidified, add 400 &#181;L of organoid culture medium to the well below the insert.</li>
	<li>Culture the organoids for 1-2 weeks until they reach an average diameter of 150-250 &#181;m (The maximal diameter is about 500 &#181;m). 
	NOTE: When the organoids reach an appropriate size and display the concentric lamellar pattern of onion-skin-like microstructures, consider passaging if needed.</li>
</ol>
3. Organoid dissociation and reseeding
<ol>
	<li>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.</li>
	<li>Transfer Dispase-basement membrane matrix mixture to a 15 mL tube and incubate for 1 h at 37 &#176;C.</li>
	<li>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.</li>
	<li>Centrifuge the tube at 450 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the pellet in 200 &#181;L of TrypLE Express.</li>
	<li>Incubate for 5 min at 37 &#176;C.</li>
	<li>Add 1 mL of ice-cold blocking buffer (PBS with 2% bovine calf serum (BCS)) and pipette gently to neutralize the enzyme.</li>
	<li>Filter the suspension through a 40 &#181;m cell strainer to obtain single cells.</li>
	<li>Centrifuge the tube at 450 &#215; g for 5 min at 4 &#176;C. Carefully remove and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 200-500 &#181;L of organoid culture medium.</li>
	<li>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&#160;of 24-well cell culture insert.</li>
	<li>Then resuspend 500-2,000 cells in 50 &#181;L of organoid culture medium (composition described in Material list). Add 50 &#181;L of basement membrane matrix to the cell suspension. Mix gently, avoid&#160;bubble formation, and dispense the 100 &#181;L mixture into the interior of a cell culture insert placed in a 24-well plate.</li>
	<li>Incubate the plate for 20-30 min at 37 &#176;C until the basement membrane matrix-cell mixture solidifies.</li>
	<li>Once the mixture is fully solidified (typically after 20-30 min at 37 &#176;C), add 400 &#181;L of organoid culture medium to the well below the insert.</li>
	<li>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 &#176;C, 5% CO&#8322; in a humidified incubator. Replace the medium every 2-3 days and monitor growth until organoids reach an average diameter of 150-250 &#181;m.</li>
	<li>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.</li>
</ol>
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).
<ol>
	<li>After isolation from human cervical cell suspension, transfer 100,000-200,000 cells to each 1.5 mL tube.</li>
	<li>Centrifuge the tubes at 450 &#215; g for 3 min at 4 &#176;C. Carefully remove and discard the supernatant.</li>
	<li>Resuspend the cell pellet in 20 &#181;L of the Nucleofector Solution/Supplement mixture from the P4 Primary Cell 4D-Nucleofector X Kit. The mixture is prepared by combining 16.4 &#181;L of Nucleofector Solution and 3.6 &#181;L of Supplement per tube.</li>
	<li>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 &#181;L per tube.</li>
	<li>Carefully transfer the nucleofection mixture (20 &#181;L of cells resuspended in Nucleofector Solution/Supplement mixture + up to 2 &#181;L siRNA; total 20-22 &#181;L) into the electroporation cuvette Vessels from the P4 Primary Cell 4D-Nucleofector X Kit.</li>
	<li>Gently tap the Nucleocuvette Vessels to ensure the sample covers the bottom of the cuvette.</li>
	<li>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.</li>
	<li>Initiate the nucleofection process. Set the EA-125 pulse code for each cuvette with the P4 primary cell process.</li>
	<li>After completion, carefully remove the Nucleocuvette Vessels from the retainer.</li>
	<li>Incubate the Nucleocuvette Vessels at room temperature for 10 min to allow the cells to stabilize.</li>
	<li>Open the lid of the Nucleocuvette Vessels, add 180 &#181;L of 37 &#176;C pre-warmed organoid culture medium to each cuvette, and gently transfer the cell suspension to a new 1.5 mL tube for resuspension.</li>
	<li>Take a volume of cell suspension containing 500-2,000 cells and resuspend it in organoid culture medium to a final volume of 50 &#181;L. 
	NOTE: Do not discard the remaining cells from step 4.12. They will be used in step 4.14.</li>
	<li>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.</li>
	<li>Dispense the remaining cells from step 4.12 into a 96-well ultralow attachment plate and incubate for 48 h at 37 &#176;C.</li>
	<li>After 48 h, transfer the cells to a 1.5 mL tube and centrifuge at 450 &#215; g for 3 min. Discard the supernatant.</li>
	<li>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&#39;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&#39;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.</li>
</ol>

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</ol>

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...

The uterine ectocervix is the most common site for cervical cancer1,2. 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 women3,4. Also, the incidence of cervical precancerous lesions continues to increase in some countries5,6. 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 delivery7,8. 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 origin9,10,11. They have been employed to identify diverse human tissue stem cells across multiple tissues, including the trachea, esophagus, and cervix12,13,14. Moreover, organoids enable genetic manipulation, allowing for the modeling of the pathogenesis of diverse diseases, including cancers and fibrosis15,16,17. Additionally, organoids serve as an effective platform for screening the efficacy and toxicity of drugs or cell therapies18,19,20,21.
Recently, two independent groups established human cervical organoid systems22,23. 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.

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

generation and genetic manipulation of human cervical organoids

Generation and Genetic Manipulation of Human Cervical Organoids

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.

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...

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

Cervical cancer is the fourth most common cancer in women and most frequently affects the uterine ectocervix. The uterine ectocervix is lined by ...

We established a Schumann cervical organoid systems to study cervical biology and pathology, including genetically-engineered organoids to study gene functions and disease mechanisms. This protocol can be applied to study human cervical biology, modern cervical diseases, and screen effective drugs. In addition, this protocol can be modified to establish other epithelial organoid systems.To begin, place the biopsied human ectocervix tissue sample in a 10 centimeter Petri dish. Use ice cold Dulbecco's PBS without calcium and magnesium to wash the tissue once to remove blood and debris. Transfer the tissue to a 1.5 milliliter tube containing one milliliter of Dispase solution at 2.4 units per milliliter.Place the tube on a tube rotator set to 20 revolutions per minute and incubate for one hour at 37 degrees Celsius with gentle agitation. Then transfer the sample to a new 10 centimeter Petri dish. Using forceps, carefully separate the white epithelial layer from the rest of the tissue.Now with sharp forceps, gently peel off the epithelial surface as an intact sheet and immediately transfer the isolated epithelial layer into a 1.5 milliliter tube. Add one milliliter of recombinant trypsin-based dissociation solution to the peeled or scraped sample. Place the 1.5 milliliter conical tube containing cervical epithelium on a tube rotator set to 20 revolutions per minute and incubate at 37 degrees Celsius for five minutes.Then transfer the cell suspension to a 50 milliliter conical tube and add 10 milliliters of freshly-prepared ice cold blocking buffer composed of PBS with 2%bovine calf serum to stop the enzymatic reaction. Place the tube in a centrifuge and spin at 450 G for five minutes at four degrees Celsius. Discard the supernatant and add one milliliter of red blood cell lysis buffer to the pellet, followed by 20 microliters of deoxyribonuclease and incubate at room temperature for five minutes.Then wash the cells twice by adding 10 milliliters of ice cold blocking buffer, followed by centrifuging at 450 G for five minutes at four degrees Celsius. After two washes, re-suspend the pellet in 200 to 500 microliters of organoid culture medium. Using a hemocytometer and Trypan blue solution count the cell number.Re-suspend 500 to 2, 000 viable cells in a final volume of 50 microliters of organoid culture medium in a 1.5 milliliter micro centrifuge tube. Add 50 microliters of basement membrane matrix to the cell suspension and gently mix on ice to avoid bubble formation. Dispense the 100 microliter mixture into the interior of a cell culture insert placed in a 24 well plate.Place the plate in a 37 degrees Celsius incubator for 20 to 30 minutes until the basement membrane matrix cell mixture solidifies. Next, add 400 microliters of organoid culture medium to the well below the insert after the mixture has solidified. Culture the organoids for one to two weeks until they reach an average diameter of 150 to 250 micrometers.Add one milliliter of Dispase solution at 2.4 units per milliliter on top of the cell culture insert and gently dissociate the basement membrane matrix. Transfer the Dispase base membrane matrix mixture to a 15 milliliter tube and incubate for one hour at 37 degrees Celsius. Now add 10 milliliters of freshly-prepared ice cold blocking buffer to the cell suspension to stop the enzymatic reaction.Place the tube in a centrifuge and spin at 450 G for five minutes at four degrees Celsius. Remove the supernatant, re-suspend the pellet in 200 microliters of trypsin and incubate at 37 degrees Celsius for five minutes. Next, add one milliliter of ice cold blocking buffer and gently mix to neutralize the enzyme.Filter the suspension through a 40 micrometer cell strainer to obtain single cells. Place the tube in a centrifuge and spin at 450 G for five minutes at four degrees Celsius. After discarding the supernatant, re-suspend the cell pellet in 200 to 500 microliters of organoid culture medium.Using a hemocytometer, and Trypan blue solution, count the cell number. Re-suspend 500 to 2, 000 cells in 50 microliters of organoid culture medium in a micro centrifuge tube and add 50 microliters of basement membrane matrix to the cell suspension. Gently mix to avoid bubble formation and dispense the 100 microliter mixture into the interior of a cell culture insert placed in a 24 well plate.Place the plate in a 37 degrees Celsius incubator, for 20 to 30 minutes until the basement membrane matrix cell mixture solidifies. Add 400 microliters of organoid culture medium to the well below the insert once the mixture is fully solidified. Then, culture the organoids in organoid culture medium for one to two weeks in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide, replacing the medium every two to three days.Prepare the nucleofector solution and supplement mixture by combining 16.4 microliters of nucleofector solution with 3.6 microliters of supplement per tube. Then, re-suspend a cell pellet containing 100, 000 to 200, 000 cells isolated from the human cervical tissue sample in 20 microliters of the prepared nucleofector solution and supplement mix. Then add 300 nanomolar of either control or target small interfering RNA oligo to each tube containing the re-suspended cells and mix gently.Carefully transfer 20 to 22 microliters of the nucleofection mixture into the electroporation cuvette vessels from the transfection kit. Gently tap the Nucleocuvette vessel to ensure the sample covers the bottom of the cuvette. Close the lid of the Nucleocuvette vessel and place the vessel into the retainer of a powered-on 4D-Nucleofector unit.To initiate the nucleofection process, select the P4 primary cell process and set the pulse code to EA125 for each cuvette. After completion, carefully remove the Nucleocuvette vessel from the retainer and incubate the Nucleocuvette vessel at room temperature for 10 minutes to allow the cells to stabilize. Next, open the lid of the Nucleocuvette vessel and add 180 microliters of 37 degrees Celsius pre-warmed organoid culture medium to each cuvette.Gently transfer the cell suspension to a new 1.5 milliliter tube for re-suspension. Finally, take a volume of the cell suspension containing 500 to 2, 000 cells, re-suspend in organoid culture medium to a final volume of 50 microliters and culture the organoids as demonstrated previously. Human cervical organoids grew up to 500 micrometers in diameter from single cells over two weeks.Hematoxylin and eosin staining clearly revealed the formation of stratified squamous epithelium in the organoids. Immunofluorescent staining revealed that the basal layer was located in the outer rim of the organoids surrounding the super basal layers. Cervical organoids could be passaged at least 10 times over six months.After electroporating the human cervical epithelial cells with siRNA oligos against AKT, about 97.3%of cells were viable. Simultaneous silencing of AKT1 and AKT2 effectively silenced both genes and interfered with cervical organoid formation. This protocol enables quantitative evaluation of drug and gene function in human cervical stem cells, facilitating mechanistic studies of cervical development and disease.Peeling or scraping of small pieces of cervical epithelia in the process of single cell dissociation can be technically challenging for beginners. Histologic analysis such as H&E staining and immuno staining, transcriptomic or proteomic analysis and functional perturbation assays can be done following this procedure.

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Learn to generate and genetically manipulate human cervical organoids using enzymatic digestion and nucleofection. This protocol enables long-term culture and functional studies of cervical stem cell biology and disease mechanisms.

Generation and Genetic Manipulation of Human Cervical Organoids

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