Human Gastric Cancer Organoid Medium, IC-101006
Product Introduction
This product is a specialized, ready-to-use medium optimized for the establishment, culture, expansion, and cryopreservation of human gastric cancer organoids. Organoids are three-dimensional (3D) cellular structures that faithfully recapitulate the architecture, cellular heterogeneity, and functional characteristics of the original tumor tissue. human Gastric Cancer Organoid Medium provides a defined and optimized nutritional environment supplemented with essential growth factors, hormones, and signaling modulators that selectively promote the proliferation and self-organization of human gastric cancer epithelial cells while suppressing the outgrowth of stromal and non-epithelial cell populations. This medium enables researchers to generate stable and expandable organoid lines from primary human gastric tumor biopsies, providing a physiologically relevant ex vivo model for studying gastric cancer pathogenesis, drug screening, biomarker discovery, and personalized therapeutic approaches.
Product Features
1. Optimized for human Gastric Cancer: Specifically formulated to support the growth and long-term maintenance of human gastric cancer organoids from primary tumor tissue.
2. Ready-to-Use Complete Medium: Supplied as a sterile, complete formulation requiring no additional supplementation, reducing preparation time and variability.
3. High Organoid Formation Efficiency: Promotes robust organoid initiation and sustained proliferation from processed tumor tissue fragments and dissociated cell clusters.
4. Defined Serum-Free Formulation: Minimizes batch-to-batch variability associated with serum-containing media, ensuring reproducible organoid culture conditions.
5. Compatible with Downstream Applications: Organoids cultured in this medium are suitable for histological analysis, genomic and transcriptomic profiling, drug sensitivity testing, and cryopreservation.
Specifications
Size: Available in 100 mL and 500 mL
Storage and Stability
Storage Conditions: human Gastric Cancer Organoid Medium A should be stored at 4ˇăC for up to 3 months. For long-term storage, aliquot and store at -20ˇăC. Avoid repeated freeze-thaw cycles exceeding 2 times. Upon receipt, store at 4ˇăC and use within 1 month for optimal performance.
Shelf Life: The product is stable for 3 months when stored at 4ˇăC or 12 months when stored at -20ˇăC from the date of manufacture.
Protocol
Important: All procedures should be performed using aseptic technique in a certified biosafety cabinet. Pre-chill Primary Buffer B and maintain Matrigel at 0-4ˇăC at all times during handling to prevent premature gelation.
1. Tissue Pre-treatment
1.1 Materials Required
Prepare ice-cold Primary Buffer B (4ˇăC), Tissue Preservation Solution E, sampling tubes, tissue transport containers, and ice packs in advance.
1.2 Tissue Acquisition and Transport
Tissue sampling and transport represent the first and most easily overlooked step critical to successful organoid establishment. Improper tissue preservation may lead to poor cell viability, contamination, or insufficient viable cell recovery, thereby reducing organoid initiation efficiency. Place the excised tissue into Tissue Preservation Solution E within 30 minutes of excision, wash 3-5 times with Primary Buffer B to thoroughly remove surface blood, then transfer the tissue into fresh Tissue Preservation Solution E in a sampling tube and transport at 4ˇăC (within 72 hours).
2. Organoid Primary Culture (24-well plate example)
2.1 Materials Required
Prepare ice-cold Primary Buffer B (4ˇăC), Primary Tissue Digestion Solution C (37ˇăC), Matrigel (thawed at 4ˇăC for 24 hours in advance), human Gastric Cancer Organoid Medium A (room temperature or 37ˇăC), forceps (10 cm), fine ophthalmic scissors/scalpel blades, disposable 60 mm culture dishes, 1.5 mL/15 mL/50 mL centrifuge tubes, 100 µm cell strainer, 3 mL Pasteur pipettes/1000 µL pipettes, 24-well cell culture plates, metal ice box, and water bath.
2.2 Human Gastric Cancer Organoid Establishment
2.2.1 Tissue Processing
Following resection, Human gastric cancer tissue should be stored and transported at 2-8ˇăC and rapidly transferred to a clean laboratory for the organoid establishment protocol. Photograph the tissue and record detailed information.
2.2.1.1 Tissue Washing
After disinfecting the sampling tube exterior, transfer the tissue into a culture dish within a biosafety cabinet. Add Primary Buffer B and wash by gentle pipetting using a 3 mL Pasteur pipette or 1000 µL pipette tip. Repeat the washing procedure at least three times.
2.2.1.2 Tissue Dissociation and Digestion
Remove tissue impurities using ophthalmic scissors or a scalpel blade. Using forceps, transfer the tissue to a 1.5 mL tube and mechanically dissociate into tissue fragments approximately 1-3 mm³ in volume. Transfer the fragments to a 15 mL tube, add 5 mL of Primary Tissue Digestion Solution C, and incubate with shaking at 37ˇăC for 15-25 minutes. Monitor digestion progress microscopically every 10 minutes by examining a small aliquot of the digestion mixture. Proceed to the next step when abundant cell clusters under 70 µm or single cells are observed (see Figure 1 for digestion endpoint reference). For very small or biopsy specimens, perform digestion in a 1.5 mL tube using 1 mL of Primary Tissue Digestion Solution C.
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2.2.1.3 Tissue Filtration
After digestion is complete, add 3 volumes of Primary Buffer B to terminate digestion. Filter the digested tissue mixture through a 100 µm cell strainer. Rinse the retained tissue with an additional 10-20 mL of Primary Buffer B to maximize recovery of cell clusters. Centrifuge the filtrate at 300 x g for 5 minutes and discard the supernatant. Resuspend the pellet in 8-10 mL of Primary Buffer B to remove additional impurities, centrifuge at 300 x g for 5 minutes, and discard the supernatant. If the cell pellet contains erythrocytes, add 1-2 mL of Red Blood Cell Lysis Buffer for 1-2 minutes, dilute to 10 mL, centrifuge at 300 x g for 5 minutes, and discard the supernatant. If the pellet is minimal or no erythrocytes are present, proceed directly to organoid culture.
2.2.1.4 Tumor Organoid Culture
Estimate the pelleted cell volume after centrifugation and resuspend in 25 times the pellet volume of Matrigel to create a 3D culture matrix. Avoid introducing air bubbles during resuspension. Reference Figure 2 for pellet volume estimation and add 300 µL, 250 µL, 150 µL, or 100 µL of Matrigel accordingly. Seed 25-30 µL of the cell-Matrigel suspension per well of a 24-well plate, maintaining Matrigel at 0-4ˇăC throughout the entire procedure. Place the culture plate in a 37ˇăC incubator for 10-15 minutes to allow Matrigel polymerization. After gelation, add 750 µL of Human Gastric Cancer Organoid Medium A (equilibrated to room temperature) to each well and culture in a 37ˇăC incubator.
Figure 2. Centrifuged cell pellet volume estimation.
3. Organoid Passaging (24-well plate example)
3.1 Materials Required
Prepare Passage Buffer G (4ˇăC), Organoid Digestion Solution D (room temperature or 37ˇăC), Matrigel (thawed at 4ˇăC for 24 hours in advance), Human Gastric Cancer Organoid Medium A (room temperature or 37ˇăC), 1.5 mL/15 mL centrifuge tubes, 24-well cell culture plates, and ice box.
3.2 Organoid Passaging
Select organoids suitable for passaging, typically after approximately one week of culture when more than 20 organoids are visible under 10X magnification or individual organoids measure 100-200 µm in size. Aspirate the culture medium, add an equal volume of Passage Buffer G to each well, gently pipette to dissolve the Matrigel, and pool the contents of 6-8 wells into a 15 mL centrifuge tube. Incubate at 4ˇăC for 10-15 minutes.
3.2.1 Organoid Digestion
Determine whether enzymatic digestion is required based on organoid growth status. If the pellet after centrifugation is small, cells are not visible, or Matrigel shows no separation, resuspend again, increase centrifugal force, and centrifuge again. When organoid numbers are insufficient or organoids are small, centrifuge at 300 x g for 5 minutes and discard the supernatant. When organoid numbers are abundant or organoids are large, centrifuge at 300 x g for 5 minutes, discard the supernatant, and choose either enzymatic digestion or mechanical dissociation. For enzymatic digestion: add 1-2 mL of Organoid Digestion Solution D, gently resuspend the pellet, and digest at room temperature for 2-4 minutes.
Figure 3. Organoid passaging: digestion endpoint reference.
Pipette 20 times every minute and monitor microscopically until organoids reach the state shown in Figure 3A-B, then immediately add 3 volumes of Passage Buffer G relative to the Organoid Digestion Solution D volume to terminate digestion. Centrifuge at 300 x g for 5 minutes and discard the supernatant.
3.2.2 Passaged Organoid Culture
Estimate the pelleted organoid volume after centrifugation. If the pellet is very small, retain approximately 1 pellet volume of supernatant for seeding. If the pellet is abundant, remove the supernatant completely. Resuspend the pellet in 25 times the pellet volume of Matrigel; refer to Figure 2 for volume reference. Seed 25-30 µL per well of a 24-well plate, maintaining Matrigel at 0-4ˇăC throughout the procedure. Place the culture plate in a 37ˇăC incubator for 10-15 minutes to allow Matrigel polymerization. After gelation, add 750 µL of Human Gastric Cancer Organoid Medium A (equilibrated to room temperature) to each well and culture in a 37ˇăC incubator.
4. Organoid Cryopreservation (24-well plate example)
4.1 Materials Required
Prepare Passage Buffer G (4ˇăC), Organoid Freezing Solution F (4ˇăC), 15 mL centrifuge tubes, cryovials, controlled-rate freezing container, and pipettes.
4.2 Organoid Cryopreservation
Organoids not immediately required should be cryopreserved and stored in low-temperature conditions. Aspirate the culture medium, add an equal volume of Passage Buffer G to each well, gently pipette to dissolve the Matrigel, and pool the contents of 6-8 wells into a 15 mL centrifuge tube. Incubate at 4ˇăC for 10-15 minutes. Centrifuge at 300 x g for 5 minutes and discard the supernatant. For every three wells harvested, add 2 mL of Organoid Freezing Solution F, gently pipette to mix, and transfer 1 mL aliquots to cryovials. Label the cryovials appropriately, place them in a controlled-rate freezing container, transfer to a -80ˇăC freezer for 48 hours, then transfer to liquid nitrogen for long-term storage. Alternatively, incubate at 4ˇăC for 40 minutes, then transfer to -20ˇăC for 2 hours, move to -80ˇăC for 48 hours, and finally transfer to liquid nitrogen for long-term storage.
5. Organoid Thawing and Recovery Culture (24-well plate example)
5.1 Materials Required
Prepare Passage Buffer G, Human Gastric Cancer Organoid Medium A, Matrigel (thawed at 4ˇăC for 24 hours in advance), 24-well cell culture plates, ice box, 15 mL centrifuge tubes, water bath, and 3 mL Pasteur pipettes/pipettes.
5.2 Organoid Thawing and Recovery Culture
Remove cryopreserved organoids from low-temperature storage and rapidly thaw in a 37ˇăC water bath with gentle agitation to ensure complete thawing of the freezing solution within a short period. Transfer the thawed organoids immediately to a 15 mL centrifuge tube, gently pipette 6-8 times, centrifuge at 300 x g for 5 minutes, and discard the supernatant. Resuspend in an appropriate volume of Passage Buffer G, transfer to a 1.5 mL centrifuge tube, centrifuge at 300 x g for 5 minutes, and discard the supernatant. Resuspend the pellet from each cryovial in 120 µL of Matrigel. Seed 25-30 µL per well of a 24-well plate, maintaining Matrigel at 0-4ˇăC throughout the procedure. Place the culture plate in a 37ˇăC incubator for 10-15 minutes to allow Matrigel polymerization. After gelation, add 750 µL of Human Gastric Cancer Organoid Medium A (equilibrated to room temperature) to each well and culture in a 37ˇăC incubator.
6. Matrigel Usage
Thaw Matrigel overnight at 2-8ˇăC. When using Matrigel, keep it on an ice box to prevent premature gelation. Matrigel forms a gel within 20 minutes at 37ˇăC.
6.1 Matrigel Characteristics:
- Maintains good fluidity at 4ˇăC for up to 14 consecutive days
- Polymerizes within 10-15 minutes upon placement in a 37ˇăC incubator
- Resistant to damage during culture; cleanly removable from culture plates without residue
Application Scope: For Laboratory Research Use Only. Not For Use In Diagnostic Procedures.
Precautions
1. Maintain Matrigel at 0-4ˇăC at all times during handling to prevent premature gelation; pre-chill pipette tips, tubes, and culture plates on ice.
2. Use fresh tissue whenever possible and ensure rapid transfer to Tissue Preservation Solution E within 30 minutes of excision to maximize cell viability and organoid formation efficiency.
3. Avoid prolonged enzymatic digestion as over-digestion compromises cell viability and organoid initiation; monitor digestion progress microscopically at regular intervals.
4. For small tissue biopsies, adjust reagent volumes proportionally and perform processing steps in 1.5 mL tubes to minimize cell loss.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What should I do if my tissue sample contains visible red blood cell contamination after centrifugation?
A1: Add 1-2 mL of Red Blood Cell Lysis Buffer to the cell pellet, incubate for 1-2 minutes at room temperature, dilute to 10 mL with buffer, centrifuge at 300 x g for 5 minutes, and discard the supernatant. If the pellet is minimal or erythrocytes are absent, proceed directly to organoid culture.
Q2: How do I determine the appropriate amount of Matrigel to add to the cell pellet?
A2: Estimate the pelleted cell volume visually after centrifugation and add 25 times that volume of ice-cold Matrigel. For example, a pellet volume of approximately 10 µL requires 250 µL of Matrigel for resuspension.
Q3: How can I assess whether organoids are ready for passaging?
A3: Organoids are typically ready for passaging after approximately one week of culture when 10X microscopic observation reveals over 20 organoids per field or individual organoids have reached 100-200 µm in diameter.
Q4: Can this medium be frozen for long-term storage?
A4: Yes. For long-term storage, aliquot Human Gastric Cancer Organoid Medium A into single-use volumes and store at -20ˇăC for up to 12 months. Avoid repeated freeze-thaw cycles exceeding 2 times to maintain medium performance.
Q5: What are the Matrigel characteristics?
A5: Matrigel maintains good fluidity at 4ˇăC for up to 14 consecutive days, polymerizes within 10-15 minutes upon placement in a 37ˇăC incubator, is resistant to damage during culture, and can be cleanly removed from culture plates without residue.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of culture conditions using a small-scale pilot experiment is strongly recommended before processing valuable samples.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product.
5. This product is manufactured by Shanghai Jiefangkairui Biotechnology Co., Ltd. under commission.
Ordering Information
Catalog Number: IC-101006
Product Name: Human Gastric Cancer Organoid Medium
Size: 100 mL / 500 mL
Price:
100 mL: CNY ¥3600.00 / USD $360.00 / EUR €432.00 / JPY ¥64800.00
500 mL: CNY ¥14500.00 / USD $1450.00 / EUR €1740.00 / JPY ¥261000.00
2023 Version
