Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium

Podsumowanie

Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy.

Streszczenie

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

Wprowadzenie

Organoids are miniature three-dimensional (3D) cellular structures that resemble the architecture and functionality of the organs from which they were derived^1,2. These lab-grown models are created by cultivating stem cells or tissue-specific cells in a controlled environment that allows these cells to self-organize and differentiate into various cell types^1,2,3. One of the key advantages of organoids is their ability to recapitulate human biology more closely than traditional two-dimensional (2D) cell cultures^1,2,3. In particular, human organoids have been shown to maintain the genetic diversity of their tissue of origin^3,4,5. Organoids offer a unique opportunity to study human organ development, model diseases, and test potential therapeutics in a controlled laboratory setting. Furthermore, organoids can be derived from individual patient samples, enabling personalized medicine approaches and the potential development of individualized treatments^3,6,7.

Researchers have used human gastric organoids to investigate various aspects of gastric biology and pathology. Prominent examples include the use of patient-derived organoids (PDOs) to predict gastric cancer chemotherapy responses^8,9,10 and model the epithelial response to Helicobacter pylori infection^11,12,13. Human gastric organoids consist of various cell types found in the stomach, including neck cells, pit cells, and other supporting cells^11,14. Gastric organoids can either be generated from induced pluripotent stem cells (iPSCs) or stem cells directly isolated from gastric tissue obtained via biopsies or from gastric resection specimens^11,14. The isolation of gastric stem cells from gastric tissue is commonly done by isolating and culturing gastric glands or enzymatically digesting tissue samples to liberate single cells^9,13,15. Importantly, the differentiation of cells within gastric organoids generated using either of these techniques has been shown to be similar¹³. The protocol described herein focuses on a single-cell digest.

Organoids represent a scientific innovation that bridges the gap between traditional cell culture and whole organs. As research in the field continues to progress, organoids are poised to contribute to the development of more effective treatments and therapies for a wide range of applications. Given the rising utilization of gastric PDOs, there is a timely need for a standardized approach to their generation. Here, the protocol for generating human gastric PDOs from single cells isolated from benign gastric biopsy tissue acquired during upper endoscopy is described. Importantly and uniquely, a standardized number of single cells is determined for seeding to reliably generate gastric PDOs and allow subsequent characterization. Using this technique, reliable differences in the formation and growth of organoids generated from biopsies of either the gastric body or gastric antrum are demonstrated.

Protokół

All human tissue utilized in this protocol was collected from individuals who provided informed consent for tissue collection through a gastric tissue collection study approved by the University of Pennsylvania Institutional Review Board (IRB #842961). Participants in this study were required to undergo an upper endoscopy as part of their routine care, be at least 18 years old, and be able to provide informed consent. All research conducted adhered to the guidelines set forth by the University of Pennsylvania.

1. Experimental preparation

1. Prepare conditioned L-WRN media as previously described¹⁶. Although not mandatory, the concentrations of Wnt-3A, R-spondin, and noggin contained in the conditioned media can be checked using a commercially available ELISA kits following the manufacturer's instructions (see Table of Materials).
2. Prepare RPMI-Antibiotics (RPMI-ABX), PBS-Antibiotics (PBS-ABX), PBS-Dithiothreitol (PBS-DTT), digestion buffer, and gastric organoid media (see Supplementary Table 1 for detail composition). Gastric organoid media is stable for 1 week at 4 °C.
3. Autoclave forceps, fine dissection scissors, and 1.5 mL tubes.
4. Begin thawing the basement membrane matrix (Matrigel) on ice.
5. Begin thawing the digestion buffer and Trypsin-EDTA in a 37 °C water bath.
6. Set an incubator orbital shaker to 37 °C and 200 rpm.

2. Isolating single cells from the biopsy tissue

1. Collect gastric biopsies during an upper endoscopy¹⁷ following the institutional clinical protocol, likely involving the use of jumbo forceps. Utilize a blunt-tipped needle to extract tissue samples from the forceps and place them in a 15 mL conical tube containing RPMI-ABX media. Keep the tube on ice for rapid transfer to the laboratory.
   NOTE: At minimum, 2-4 biopsies should be collected.
2. Allow the biopsy tissue to settle at the bottom of the conical tube. Use a pipette to aspirate the media and wash the biopsies twice with 1 mL of PBS-ABX buffer. There is no need for centrifugation, as the tissue pieces settle naturally in the conical tube.
3. Transfer a minimum of 20 mg of biopsy tissue to a 1.5 mL tube containing 1 mL of PBS-DTT.
4. Use fine dissection scissors to cut the tissue into pieces that are 1-2 mm or smaller.
5. Utilize a tabletop minicentrifuge for 15 s to aggregate tissue pieces at the tube's bottom and aspirate as much supernatant as possible. A small residual volume of PBS-DTT is acceptable.
6. Add 5 mL of freshly warmed digestion buffer to a 50 mL conical tube. To transfer the small tissue pieces without losing tissue, take 500 µL of the digestion buffer and add it to the 1.5 mL tube containing the tissue. Next, modify the tip of a 1000 µL pipette tip with scissors to increase the tip's diameter, allowing for easy aspiration and transfer of tissue pieces to the 50 mL conical tube containing the digestion buffer.
7. Incubate the digestion buffer and tissue mix at 37 °C for 30 min with orbital shaking at 200 rpm.
8. Add 5 mL of warmed trypsin with 0.25% EDTA to the digestion buffer and incubate for an additional 10 min at 37 °C with orbital shaking at 200 rpm.
9. Neutralize the digestion buffer and trypsin by adding an equal volume of Advanced (Adv.) DMEM/F12 media and pass the solution through a 70 µm cell strainer.
10. Pellet the cells by centrifugation at 1400 x g for 4 min at 4 °C. Depending on the initial size of the biopsy tissue, a small cell pellet may or may not be visible.
11. Resuspend the cell pellet in 1 mL of Adv. DMEM/F12 media and count the number of viable cells using Trypan Blue and a hemocytometer.
12. Pellet the cells again through centrifugation at 1400 x g for 4 min at 4 °C and remove the supernatant.
    ​NOTE: Figure 1 presents a schematic representation of the process for isolating single cells from the biopsy tissue.

3. Embedding single cells in a basement membrane matrix "dome"

1. Based on the counted number of viable cells, calculate the volume of basement membrane matrix required to achieve a final concentration of 10⁵ viable cells per 50 µL of basement membrane matrix.
2. Perform the following swiftly to prevent the basement membrane matrix from polymerizing before plating. Remove the thawed basement membrane matrix from ice, add the previously calculated volume of basement membrane matrix to the cells, and gently mix by pipetting up and down for approximately 10 s.
   NOTE: It is crucial to avoid creating bubbles during this step. Do not dilute the basement membrane matrix.
3. Rapidly pipette 50 µL aliquots of the basement membrane matrix/cell mixture into the center of individual wells in a 24-well tissue culture plate.
4. Immediately cover the 24-well plate and, in a single smooth motion, flip the plate upside-down. Place the inverted plate in a 37 °C tissue culture incubator for 35 min to allow the basement membrane matrix to polymerize.
   NOTE: Inverting the plate is essential to prevent the cells from sinking to the bottom of the plate and to enable the basement membrane matrix to polymerize into a 3D "dome" shape.
5. Add 500 µL of pre-warmed gastric organoid media to each well, ensuring that the top of each "dome" is fully submerged in the media. Dispense the media down the side of each well to avoid disturbing the "dome."
6. Change the media every 2-3 days.

4. Routine passaging of organoids  via fragmentation

1. Once the organoids are ready for passaging, remove the media from each designated well.
   NOTE: To determine the appropriate time for passaging, please refer to the Representative Results section.
2. Dispense 1 mL of ice-cold Adv. DMEM/F12 media directly onto a basement membrane matrix "dome." This should readily initiate the breakup of the "dome." Continue aspirating and dispensing until all fragments of the "dome" detach from the plate.
3. Transport both the media and the fragmented basement membrane matrix to the next well, repeating this process for all wells slated for passaging. After disassembling the final basement membrane matrix "dome," dispense the media and the mixture of organoids and basement membrane matrix into a 1.5 mL tube.
4. Attach a 1000 µL tip to a P1000 pipette, and then insert this tip into a 200 µL tip. This will create a pipette tip with a small enough diameter to fragment the organoids while still allowing the aspiration of a larger volume.
5. Vigorously pipette the mixture of organoids and basement membrane matrix up and down approximately 25 times to fragment the organoids into small pieces.
6. Centrifuge the fragmented organoid mixture using a tabletop centrifuge at 4 °C for 30 s at 2000 x g. This will result in a pellet of fragmented organoids separated from the basement membrane matrix and media. Use a pipette to aspirate the media and basement membrane matrix supernatant. Do not use a vacuum to aspirate the supernatant, as the pellet is loose.
7. Calculate the volume of freshly thawed basement membrane matrix required so that the number of wells/domes is split at a 1:2 ratio (50 µL/dome). Add the fresh basement membrane matrix and gently pipette up and down to mix, taking care to avoid creating bubbles.
8. Swiftly aliquot 50 µL of the mixture of organoid fragments and basement membrane matrix into individual wells of a 24-well plate.
9. Cover the plate, flip it upside down, and place it in a 37 °C tissue culture incubator for 35 min to allow the basement membrane matrix to polymerize.
10. Add 500 µL of pre-warmed gastric organoid media to each well.
    NOTE: Figure 2 presents a schematic overview of the passaging of gastric patient-derived organoids through fragmentation.

Wyniki

The subsequent representative results are derived from biopsies taken from the benign epithelium of both the gastric body and gastric antrum regions of the stomachs of five different patients undergoing upper endoscopy. Two to four "domes"/wells were plated and analyzed per patient for both gastric body and antrum biopsies. Organoids were successfully generated from the gastric body and gastric antrum biopsy tissue from all five patients. On average, 41 organoids were analyzed per "dome"/well. All images ...

Dyskusje

Herein, a detailed protocol for reliably generating human gastric organoids from single cells isolated from biopsies of benign epithelium from the gastric body and antrum is outlined. Critical steps in the protocol revolve around timing as well as handling the basement membrane matrix. To preserve viability, it is essential to initiate the protocol as soon as possible after acquiring the biopsy tissue. The aim is to start digesting the biopsy tissue within 30 min of the biopsy being performed. Handling the basement membr...

Materiały

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco	25200-056
A83-01	R&D Systems	2939
Advanced DMEM/F12	Gibco	12634-010
Amphotericin B	Invitrogen	15290018
B27	Invitrogen	17504044
BZ-X710	Keyence	n/a
cellSens	Olympus	n/a
Collagenase III	Worthington	LS004182
Dispase II	Sigma	D4693-1G
Dithiothreitol (DTT)	EMSCO/Fisher	BP1725
DPBS	Gibco	14200-075
Fungin	InvivoGen	NC9326704
Gastrin I	Sigma Aldrich	G9145
Gentamicin	Invitrogen	1570060
Glutamax	Gibco	35050-061
hEGF	Peprotech	AF-100-15
HEPES	Invitrogen	15630080
hFGF-10	Peprotech	100-26
L-WRN Cell Line	ATCC	CRL-3276
Matrigel	Corning	47743-715
Metronidazole	MP Biomedicals	155710
N2 Supplement	Invitrogen	17502048
Noggin ELISA Kit	Novus Biologicals	NBP2-80296
Pen Strep	Gibco	15140-122
RPMI 1640	Gibco	11875-085
R-Spondin ELISA Kit	R&D Systems	DY4120-05
Wnt-3a ELISA Kit	R&D Systems	DY1324B-05
Y-27632	Sigma Aldrich	Y0503