Establishment and Culture of Patient-Derived Breast Organoids

Human patient-derived organoids are three-dimensional in vitro model systems that represent both patient diversity and cellular heterogeneity of the tumors. This protocol and video demonstration provide a detailed, hands-on guide to establishing patient-derived breast tumor and normal organoids. Patient-derived breast tumor organoids are exciting new models, but difficult to establish.

The comprehensive protocol we provide here should aid in preparing the researchers attempting to develop breast organoids and acquaint them with the expected challenges. Every PDO line derived from a different patient is unique in morphology and growth rate. Unlike two 2D cell line systems, organoids grow better when plated at a high density, allowing for better intercellular interactions.

Demonstrating the procedure will be Disha Aggarwal, a graduate student in my laboratory. To begin, thaw a bottle of basement membrane matrix on ice or overnight at four degrees Celsius. Transfer the resected tissue to a 10-centimeter sterile Petri dish.

Examine the tissue macroscopically and make a note if it appears morphologically fatty, vascularized, or necrotic. Additionally, record the size and shape of the tissue and take a picture of the tissue with a ruler in view. Mince the tissue into small pieces with a sterile number 10 scalpel and transfer it into a 50-milliliter conical tube.

Add 10 milliliters of two milligrams per milliliter collagenase IV solution and seal the tube. Place the tube on an orbital shaker at 37 degree Celsius at 140 RPM for 30 to 90 minutes at a 30-degree angle. During the incubation, place an aliquot of complete medium to pre-warm at 37 degrees Celsius in a bead or water bath.

Every 15 minutes, resuspend the tissue by mixing up and down vigorously using a five-milliliter, sterile, pre-coated serological pipette. Monitored dissociation over time by observing the tube under the microscope at a 5X or higher magnification. Once the tissue is dissociated, centrifuge at 400 G for five minutes, aspirate the supernatant, and add 10 milliliters of AdDF+Again, centrifuge and carefully aspirate the supernatant, as tissue pellets can occasionally be loose.

If the tissue pellet is partially red, add two milliliters of red blood cell lysis buffer and incubate for five minutes at room temperature. After incubation, add 10 milliliters of AdDF+to the tube, centrifuge at 400 G for five minutes, and discard the supernatant. Resuspend the pellet in 50 to 300 microliters of undiluted cold basement membrane matrix and mixed by pipetting carefully to avoid forming bubbles.

Using a six-well tissue culture plate that is pre-warmed in the incubator overnight, plate 300 microliters of basement membrane matrix dome containing organoids in each well. Leave the plate undisturbed in the hood for five minutes before placing it at 37 degrees Celsius for 20 to 30 minutes for the basement membrane matrix dome to fully solidify. At the end of incubation, add three milliliters of pre-warmed complete medium dropwise to each well and incubate at 37 degrees Celsius and 5%carbon dioxide.

After incubation, capture images of the organoids using a 5X objective on an inverted bright-field microscope. Lift the basement membrane matrix dome into the medium in the well using a cell scraper or a one-milliliter pipette tip. Using a pre-coated pipette tip, transfer the floating dome of the organoids with medium to a 15-or 50-milliliter conical tube, depending on the number of wells being harvested.

Then add DPBS to increase the volume to at least five milliliters. Spin the tubes at 400 G for five minutes. The basement membrane matrix with organoids forms a layer at the bottom.

After aspirating the supernatant, add 5 to 20 milliliters of DPBS, depending on the number of wells pooled in a tube. Mix the organoid basement membrane matrix pellet in DBPS using a pre-coated sterile disposable pipette. Again, centrifuge and discard the supernatant.

Using a coated pipette tip, add cell dissociation reagent at three times the volume of the basement membrane matrix and resuspend the organoids. Place the tube on an orbital shaker at 37 degrees Celsius at 140 RPM for 8 to 15 minutes in an angled position. Monitor the tube by observing it under the microscope every five minutes to ensure the organoids are broken into smaller clusters.

Add AdDF+at a volume equal to or greater than the cell dissociation reagent and pipette to mix the organoids. Spin at 400 G for five minutes to obtain an organoid pellet. Once a white organoid pellet with no undissolved basement membrane matrix is obtained, discard the supernatant and resuspend the pellet in one milliliter of AdDF+Make up the volume to 10 milliliters with AdDF+Spin the tube for the wash step and discard the supernatant.

Add the required amount of basement membrane matrix to the digested organoids based on the appropriate split ratio. Mix by gently pipetting up and down to avoid creating bubbles and immediately place on ice. Plate 300-microliter domes of organoids resuspended in a basement membrane matrix in a pre-warmed six-well plate.

Leave the plate undisturbed in the hood for five minutes before placing it at 37 degrees Celsius with 5%carbon dioxide for 20 to 30 minutes for the domes to solidify. At the end of incubation, and three milliliters of pre-warmed complete medium to each well and place the plate back in the incubator. Add fresh complete medium every five to seven days.

The various patient-derived breast tumor organoid lines differ in morphology and growth rate. The normal breast organoids and the few early ductal carcinomas in C2-derived organoids resembled the normal breast structure, with a central lumen surrounded by ductal cells. Organoids derived from invasive lobular carcinoma tend to form loosely-attached grape bunch-like structures.

Meanwhile, organoids derived from invasive ductal carcinomas tend to form dense, large, and round organoids. The growth of organoids was measured using a luminescent cell viability assay on days three, six, nine, and 12, with a baseline reading on day one after plating. The bright-field images of the same organoids expanded over time are shown here.

Some patient-derived organoid lines have a doubling time of two days, while some take five days. It is important to be patient with particular organoid lines that are slow to establish. In our experience, increasing plating density or filtering debris out promotes the growth of PDOs.

Patient-derived organoids, or PDOs, are excellent models for drug screening. They model cell-cell as well as cell-extracellular matrix interactions that are key to studying cancer pathophysiology. Additionally, PDOs can undergo genetic manipulation and can be used to develop xenografts in co-culture systems, making them great models for mechanistic studies.