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      Organoid Culture Guide

      Introduction to Organoids

      Organoids refer to tissue-like structures that are formed through the 3D culture of adult stem cells or pluripotent stem cells outside the body. Although organoids are not actual human organs, they can mimic the structure and function of real organs, simulating in vivo tissue architecture and function to the greatest extent, and can be stably passaged and cultured over the long term.

      Types of Organoids

      History of Organoids


      As early as the 1980s, the term "organoid" had already been proposed. In 1907, Professor Wilson from the University of Bakroreina in the United States discovered that mechanically separated sponge cells could re-aggregate and self-organize into new sponge organisms with normal functions, which was the source of the future development of organoid technology. Wilson's research proved that adult organisms possess complete information and can successfully develop into new organisms without external assistance and without starting from specific anatomical stages. The contemporary development of organoids has mainly concentrated in the last decade. In 2009, Hans Clevers' laboratory used single mouse LGR5+ intestinal stem cells to self-organize into intestinal organoids with crypt-villus structures in vitro, opening a new chapter in the development of organoids.

      At present, organoids of various organs have been successfully constructed, including those of the small intestine, stomach, colon, lung, bladder, brain, liver, pancreas, kidney, ovary, esophagus, heart, etc., encompassing not only normal organ tissue organoids but also corresponding tumor tissue organoids. In recent years, the number of publications involving organoid research has been rising sharply, including numerous articles in top-tier journals such as CNS. The global ranking of China's organoid publications has jumped from sixth place (2009-2019) to second place (2020), following the United States. In 2021, organoids were listed as a key special project in the "14th Five-Year Plan" for national key research and development plans. With continuous in-depth research, China's organoid technology level will be further improved, and the marketization of organoids will be increasingly enhanced under the active layout of related enterprises.

       

      Development Process of Organoid Research

      Comparison of Organoid Models with Other Models


      At present, the main models used for clinical drug screening are primarily divided into 2D cell models, 3D organoids, and patient-derived xenograft (PDX) models. For cancer drug screening models, they need to meet three aspects: providing drug sensitivity test results in a short period of time, having high drug screening throughput, and accurate predictive effects. In comparison with other drug screening methods, organoids have shown strong advantages in these three aspects.

      Comparison of Drug Screening Models(Source: China.org Medical Channel)
      Feature 2D Cell Model Organoid PDX
      Physiology Limited Model Semi-physiological Model Physiological Model
      Construction Time Short Moderate Long
      Modeling Success Rate High High Low
      Drug Testing Throughput Very High High Low
      Cost Low Moderate High
      Sample Size Requirement Small Small Biopsy/Surgical Tissue Large Biopsy/Surgical Tissue
      Vascularization, Immune System Not Available Not Available Available
      Operational Difficulty Easy Easy Difficult
      Genetic Editing Easy Easy Difficult
      Tumor Heterogeneity Difficult to Achieve Retainable Difficult to Achieve
      Clinical Relevance Low High High
      Biobank Establishment Feasible Feasible Difficult to Achieve


      Organoid Culture


      Using the culture of intestinal organoids as an example, the operation method is as follows:

      Experimental Materials:

      Name

      Parameters

      Notes

      Matrix Gel

       

      Thaw in advance

      Basic Culture Medium

      B27 50x; N2 100x; N-Acetyl-L-cysteine 1mM; Glutamax 100x; HEPES 10mM; Primocin 500x; Advanced DMEM/F12, ADMEM; Cytokines: Mouse-EGF (Mouse epidermal growth factor) 50ng/ml; Mouse R-spondin1 500ng/ml; Mouse-noggin (BMP inhibitor) 100ng/ml

      Do not prepare too much culture medium at once, it is recommended to prepare 20ml at a time. After preparation, filter with a 0.22um filter (the purpose of filtering is sterilization). It can be stored at 4°C for 4 weeks.

      24-Well Cell Culture Plate

       

       

      PBS

       

      Sterilized, pre-cooled, and supplemented with double antibodies


      Experimental Method

      Crypt Isolation and Plating:

      1. Dissect a 6-8 week old mouse, take approximately 10cm of the small intestine below the stomach, remove the mesentery with tweezers, and cut open the intestine along its length with scissors. Wash with pre-cooled PBS 3-5 times, carefully scrape off the villi from the surface of the small intestine using a glass slide (examine under a microscope), and place the scraped intestine into a 50ml centrifuge tube, keeping it on ice.

      2. Transfer the small intestine to a biosafety cabinet and wash with PBS containing double antibiotics 3-5 times. Transfer the small intestine to a 25ml solution of 2mmol/L EDTA and incubate at 4°C for 30 minutes.

      3. After digestion, transfer the small intestine to a 50ml centrifuge tube and wash twice with PBS containing double antibiotics.

      4. Add 25ml of PBS and gently shake 30-50 times, take a portion of the suspension for microscopic examination. Filter the suspension through a 70um filter screen into a new 50ml centrifuge tube.

      5. Repeat step 4, and centrifuge the collected filtrate at 800rpm for 5 minutes to collect the precipitate.

      6. Discard the supernatant, resuspend the pellet with 2ml of ADMEM, and count an appropriate amount of suspension; the optimal number of crypts is controlled at 5-15 per ul.

      7. Take an appropriate volume of the suspension and add it to a 1.5ml centrifuge tube, centrifuge at 800rpm for 5 minutes at room temperature.

      8. Discard the supernatant and resuspend with pre-cooled matrix gel (ensure thorough mixing).

      9. Plate the cells, add 50ul of the resuspended liquid to each well of a 24-well plate (note: the matrix gel should be placed in the center of the well to avoid adhesion to the wall).

      10. Place the plated wells in an incubator for 20-30 minutes.

      11. Add 500ul of complete culture medium to each well, and change the medium every 2-3 days, passage the cells every 5-7 days.

      Note: All operations should avoid contamination, and tissues should be kept on ice as much as possible.


      Small Intestine Organoid Culture Procedure Flowchart
      Organoid Passaging:

      1. Place the crypts to be passaged on ice for 5-10 minutes.

      2. Carefully aspirate the supernatant and add 1ml of pre-cooled ADMEM. Use a large pipette tip to gently disrupt the matrix gel, then adjust to a 700ul volume and pipette up and down about 30 times to ensure thorough mixing. Avoid creating bubbles during this process. You can take an appropriate amount of the suspension for observation until no large clumps of crypts are visible.

      3. Transfer the suspension to a 1.5ml centrifuge tube and centrifuge at 800rpm at room temperature for 5 minutes.

      4. Follow the plating steps 8-11 as described in the crypt plating procedure.

      Note: Theoretically, you can expand 3-5 times during each passage, but due to losses during the procedure, it is generally only possible to expand 3 times.

      Cryopreservation and Thawing:

      Use 10% DMSO, 20% FBS, and supplement with ADMEM. After transferring the suspension to a cryovial, place it in a freezing box and store it in a -80°C freezer for 24 hours. For long-term storage, transfer to liquid nitrogen after 24 hours. Thawing should follow standard cell recovery methods.

      Cancer Organoid Culture Method Using Colon Cancer as an Example:

      Experimental Materials

      Product Name

      Item NO.

      GFR OrganoGel Phenol red free

      abs9495

      Human Colorectal cancer Organoid Culture Medium Kit

      abs9445


      Composition

      Component

      Name

      Size

      Storage

      Component A

      Colorectal Cancer Organoid Buffer

      100ml

      4℃

      Component B

      Colorectal Cancer Organoid Basal Medium

      100ml

      4℃

      Component C

      organo Primary Enzymatic Digestion Liquid

      25ml

      4℃

      Component D

      organo Digestion Liquid

      25ml

      4℃

      Component E

      organo Primary Enzyme Termination Liquid

      100ml

      4℃

      Component F

      Tissue Preservation Liquid

      100ml

      4℃

      Component G

      organo Cryopreservation Liquid

      25ml

      4℃


      Experimental Method


      1. Primary Culture

      (1) Place the tissue in a tissue sampling vial containing special tissue preservation liquid F and keep it in a cooler box at 4°C. Take a photo and register the information.

      (2) Sterilize the sampling vial, place the tissue in a culture dish, and wash it three times with colorectal cancer organoid buffer A before mincing it in the culture dish into tissue pieces approximately 1mm^3 in size.

      (3) Digest the cancerous tissue with organo primary enzymatic digestion liquid C at 37°C with shaking for 10-20 minutes (observe the digestion process at any time), and then add three times the volume of organo primary enzyme termination liquid E to terminate the digestion.

      (4) Filter through a sieve with a pore size of 100μm, take a small amount of the filtrate for observation under a microscope, where distinct tissue pieces should be visible. Collect the filtrate and after centrifugation at 300g for 3-5 minutes to remove the supernatant, resuspend and centrifuge again.

      (5) Discard the supernatant, resuspend with an appropriate amount of matrix gel (abs9495) (for example, 30μl per well in a 24-well plate), then plate (operate at 4°C) and take a photo for tracking and positioning.

      (6) Place the plated wells in a 37°C incubator for 10-15 minutes to allow the gel to set, then add culture medium B (returned to room temperature) for cultivation.


      Organoid Culture Procedure (Matrix Gel Method)
      2. Organoid Passaging Culture

      (1) Aspirate the culture medium and add Organoid Buffer A at 4°C, let it sit for one minute.

      (2) Gently pipette the matrix gel, collect it in a 15ml centrifuge tube, and let it stand at 4°C for 5 minutes.

      (3) Centrifuge for 5 minutes, discard the liquid, resuspend with an appropriate amount of Organoid Buffer A, transfer to a 1.5ml centrifuge tube, and centrifuge at 300g for 5 minutes to discard the liquid, or (add an appropriate amount of Digestion Liquid D for 90-120 seconds to terminate, centrifuge for 5 minutes and discard the mixture).

      (4) After collecting the organoids, add an appropriate amount of Organoid Cryopreservation Liquid G, and cryopreserve at a density of 500 per/ml (or proceed to step 5).

      (5) After collecting the organoids, resuspend with matrix gel, and plate 30μl of matrix gel per well in a 24-well plate, let it sit in the incubator for 10 minutes before adding 500μl of Colorectal Cancer Organoid Culture Medium B.


      3、Organoid Thawing

      (1) Take 10ml and place it in a 15ml centrifuge tube.

      (2) Remove the frozen tumor organoid cells from the liquid nitrogen tank and quickly place them in a 37°C water bath to thaw.

      (3) During the water bath thawing process, gently shake the frozen vial to ensure that the cryopreservation liquid is completely thawed within 1-2 minutes.

      (4) Quickly transfer the thawed organoid cells to a 15ml sterile centrifuge tube, gently pipette 3 times, centrifuge at 300g for 3 minutes, then discard the supernatant and collect the organoid cell pellet. Resuspend with an appropriate amount of Colorectal Cancer Organoid Buffer A, transfer to a 1.5ml centrifuge tube, and centrifuge at 300g for 5 minutes.

      (5) Resuspend with matrix gel, plate 20μl of matrix gel per well in a 24-well plate, let it sit in the incubator for 10 minutes before adding 500μl of Colorectal Cancer Organoid Culture Medium B.



      Culture Medium for Culturing Colorectal Cancer Organoids: Day 1 to Day 11 Culture Photos

        Item NO.

      Product Name

      Size

      abs9514

      Mouse Intestinal Organoid Culture Medium Kit

      1kit

      abs9516

      Mouse Liver Organoid Culture Medium Kit

      1kit

      abs9515

      Mouse Lung Organoid Culture Medium Kit

      1kit

      abs9446

      Human Breast Cancer Organoid Culture Medium Kit

      1kit

      abs9448

       Human Endometrial carcinoma Organoid Culture Medium Kit

      1kit

      abs9445

      Human Colorectal cancer Organoid Culture Medium Kit

      1kit

      abs9443

      Human Lung Cancer Organoid Culture Medium Kit

      1kit

      abs9449

      Human Gastric Cancer Organoid Culture Medium Kit

      1kit

      abs9447

      Human Pancreatic Cancer Organoid Culture Medium Kit

      1kit

      abs9558

      Human Ovarian Cancer Organoid Culture Medium Kit

      1kit

      abs9544

      Human Liver Organoid Culture Medium Kit

      1kit

      abs9546

      Human Lung Organoid Culture Medium Kit

      1kit

      abs9549

      Mouse Gastric Cancer Organoid Culture Medium Kit

      1kit

      abs9550

      Mouse Lung Cancer Organoid Culture Medium Kit

      1kit

      abs9551

      Mouse Breast Cancer Organoid Culture Medium Kit

      1kit

      abs9552

      Mouse Hepatocarcinoma Organoid Culture Medium Kit

      1kit

      abs9553

      Mouse Pancreatic Cancer Organoid Culture Medium Kit

      1kit



      Matrigel

        Item NO.

      Product Name

      Size

      abs9410

        Ready to use Matrigel

        100ml

      abs9490

      Standard OrganoGel with Phenol red

        1.5ml*8

      abs9491

      Standard OrganoGel Phenol red free

        1.5ml*8

      abs9492

      HC OrganoGel with Phenol red

        1.5ml*8

      abs9493

        HC OrganoGel Phenol red free

        1.5ml*8

      abs9494

      GFR OrganoGel with Phenol red

        1.5ml*8

      abs9495

        GFR OrganoGel Phenol red free

        1.5ml*8

      abs9496

        IPS-qualified OrganoGel Phenol red free

        1.5ml*4

      abs9497

      HC&GFR OrganoGel with Phenol red

        1.5ml*8

      abs9498

        HC&GFR OrganoGel Phenol red free

        1.5ml*8


       

      参考文献:

      [1] CORNING. Using Organoids for Disease Modeling. Nov. 11, 2019.

      [2] Lee, J. et al. Nat Commun 11, 4283 (2020).

      [3] SCIENCING. Types of Sea Sponges. 2019.

      [4] Wilson, H. V. Development of sponges from dissociated tissue cells.

      [5] Sato, T. et al. Nature 459, 262–265 (2009).

      [6] Corrò C et al. Am J Physiol Cell Physiol. 2020 Jul 1;319(1):C151-C165.

       

       

      Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

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