Article introduction
In January 2016, the team of Hans Clevers of the Royal Netherlands Academy of Arts and Sciences (KNAW) was presented in the journalnature protocols（if:14.8000）An article entitled "Organoid Culture Systems for Prostate Epithelial and Cancer Tissue" was published.
This article focuses on methods for generating 3D prostate organoids from healthy mouse and human prostate, metastatic prostate cancer lesions, and circulating tumor cells. The steps and advantages of the method are described in detail, and relevant experimental details and technical guidelines are provided.
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Summary. abstract
This protocol describes a method to generate 3D prostate organoids from healthy mouse and human prostate (tissue blocks or single lumen and basal cells sorted by FACS), metastatic prostate cancer lesions, and circulating tumor cells. Starting with the culture of digested tissue material, fully grown organoids are usually obtained within 2 weeks。The culture protocol presented here is currently the only one that allows both luminal and basal prostate epithelial lines to grow, as well as advanced prostate cancer. Organoids built with this method can be used to study many different aspects of prostate biology, including homeostasis, tumorigenesis, and drug discovery.
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Experimental ideas. methods
By building a prostate organoid system, to:To study the biology of the prostate gland and the mechanism of prostate cancer。Specifically, a 3D prostate organoid system was established by isolating and culturing healthy mouse and human prostate, metastatic prostate cancer lesions, and circulating tumor cells. The system can simulate the growth and development process of prostate tissue, and can be used to study the mechanism of prostate cancer occurrence and progression. In addition, genomic analysis and cell marker analysis were used to confirm the nature and nature of organoids, so as to ensure the reliability and accuracy of the experimental results.
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Expected results. results
The expected result is that the established 3D prostate organoid system can successfully culture healthy mouse and human prostate, metastatic prostate cancer lesions, and circulating tumor cells. These organoids can mimic the growth and development of prostate tissue, and can be used to study the mechanisms of prostate cancer initiation and development. In addition, through genomic analysis and cell marker analysis, the ** and nature of organoids can be confirmed, so as to ensure the reliability and accuracy of experimental results. These results provide new ideas and methods for the research of prostate cancer, and are expected to provide new strategies and directions for the prevention and prevention of prostate cancer.
Fig.1 Establishment of mouse prostate organoids. a) Overview of isolation of the prostate from the mouse genitourinary system (see 25 for detailed isolation protocol). This procedure refers to **b) Schematic diagram of the anatomy of the mouse prostate. AP, anterior prostate, DLP, dorsal prostate, VP, ventral prostate. c) How to droplet Matrigel droplets in the wells of tissue culture plates. d) Representative organoids grown on day 1 and day 7 of mouse prostate tissue after inoculation**. Scale bar, 100.
Fig.2 Establishment of prostate organoids from mouse and human cavity and basal cells**. a) Overview of mouse prostate organoid cultures using flow cytometry to establish intraluminal and basal cells**. Cells are sorted according to the expression of CD24 (intraluminal cells) and CD49F (basal cells). Scale bar, 100. b) Overview of the establishment of human prostate organoid cultures using flow cytometry** for intraluminal and basal cells**. Cells were sorted based on the expression of CD26 (intraluminal cells) and CD49F (basal cells). Scale bar, 100.
Table 1 Review of media components for mouse and human prostate organoids. Y-27632 is added to the medium only during culture establishment and after passage of organoids using Tryple.
Table 2 Organoid culture inoculation volume and medium volume.
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Materials and equipment. materials&equipment
Please refer to the original text for the reagents, materials and equipment required for the experiment.
Reagent preparation
Mouse prostate medium.
Add 10 ml b27，125.0 L N-acetylcysteine (500 mM, PBS), 50 µl egf（0.5 mg/ml，pbs + 0.1% bsa），2.0 µl a83-01（5mm，dmso），50.0 µl noggin（100 µg/ml，pbs + 0.1% bsa），50.0 µl r-spondin 1（500 µg/ml，pbs + 0.1% BSA or 10% conditioned medium), 500 L Dihydrotestosterone (1 M, ethanol) made up to 50 ml with Addmem F12 (containing penicillin streptomycin, 10 mM HEPES and Glutamax 100 diluted). After passaging, Y-27632 is added to the medium (e.g., 5. in 50 ml mouse prostate mediumY-0 L 100 mm of 27632).
Human prostate medium.
Join 10 ml l nicotinamide (1 m, PBS.)0 L N-acetylcysteine (500 mM, PBS.)5 μl egf（0.5 mg/ml，pbs + 0.1% bsa.0 μl a83-01（5 mm，dmso.0 μl noggin（100 μg/ml，pbs + 0.1% bsa），50.0 μl r-spondin 1（500 μg/ml，pbs+ 0.1% BSA or 10% conditioned medium. 0 μl dihydrotestosterone（1 μm，ethanol.0 μl fgf2（50 μg/ml，pbs + 0.1% bsa），5.0 μl fgf10（0.1 mg/ml，pbs + 0.1% bsa） 5.0 L prostaglandin E2 (10 mM, DMSO), 167 L SB202190 (30 mM, DMSO) to 50 mL with Addmem F12 (diluted with penicillin, streptomycin, 10 mM HEPES, and Glutamax 100). After passaging, add Y-27632 to the medium (e.g., add 5Y-0 L 100 mm of 27632).
Key stepsThe medium should not be kept at 4 for more than 2 weeks.
Key stepsWhen using prostate medium containing R-Spondin 1 conditioning medium or recombinant R-Spondin 1, we have never found any difference in organoid establishment, maintenance, and morphology between them.
Preparation of R-Spondin 1 conditioned medium.
Regarding the 136 specific steps of the experimental operation in this study, the primary school club will explain it in detail in the next tweet!
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References. protoc. 2016 feb;11(2):347-58. doi: 10.1038/nprot.2016.006. epub 2016 jan 21.pmid: 26797458 pmcid: pmc4793718.
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