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INTRODUCTION
The prostate is a gland in the male reproductive system that produces seminal fluid. The pseudostratified epithelium present in the prostate consists of luminal, basal and rare neuroendocrine cells. Androgen receptor (AR) signaling is essential in prostate development and homeostasis, and in the development and progression of prostate cancer. As is well recognized (1,2), prostate cancer research is hampered by the lack of suitable in vitro model systems. Although powerful in vivo models are available, they are often expensive, time-consuming, and technically challenging. Most in vitro research is done with cell lines derived from neoplastic lesions and most of them do not have an intact AR signaling pathway, making them poor representatives of healthy prostate and prostate cancer tissues. We have developed a testosterone-sensitive prostate organoid culture system obtained from primary prostate tissue and advanced prostate cancer tissue (3,4), opening new avenues for studying homeostasis and prostate cancer. In this protocol, we provide more details on setting up and testing these culture systems.
Comparison with other methods
The culture system described here was developed by adapting and optimizing the culture conditions previously used to establish cultures of murine and human small and large intestine organoids (5,6). We added various compounds and growth factors to generic organoid medium (containing epidermal growth factor (EGF), noggin, and R-spondin 1 (Table 1) (5)) to create culture conditions that support growth in long-term studies of human and prostate tissue Mouse. and advanced prostate cancer. The composition of the medium we used is summarized in Table 1. Using this culture system, we have shown that: (i) both the luminal and basal lineages harbor multipotent progenitor cells and can be propagated for long-term growth, (ii) organoids functionally recapitulate AR signaling, (iii) organoids, organoids derived from mouse models of prostate cancer recapitulate mouse phenotypes, and (iv) organoids derived from human prostate cancer genetically and phenotypically mimic the tumor from which they are derived. derived (3.4).
Several groups have demonstrated primary prostate tissue growth in vitro. However, unlike our prostate organoid cultures, most of these models only support short-term growth, mainly basal cell growth, and do not allow for full luminal differentiation (limiting androgen reactivity). (7-10). In addition, these methods do not allow for efficient growth of prostate cancer tissue. A study by Liu et al. developed protocol. (11) allows reprogrammed prostate epithelial cells to grow indefinitely. Interestingly, the Rho-associated kinase (ROCK) inhibitor Y-27632 and the presence of feeder cells (producing factors that may also be present in our defined prostate culture medium) are essential in this system. However, cells grown under these conditions do not closely resemble the prostate in vivo and, moreover, androgen reactivity in this system is limited. Finally, Chua et al. (12) recently demonstrated a culture system that exclusively allows the growth of organoids from single luminal cells, albeit with a lower seeding efficiency than that reported with our method (0.2–0.3%, reported by Chua et al. ( 12) compared to 1-2% using our method (3)). Under these conditions, organoids resembling the prostate arise in vivo. However, organoids derived from basal cells cannot be propagated over a long period of time. Also, unlike our method, the medium is not fully defined. Undefined media supplements (e.g. fetal calf serum) may contain growth factors present in our defined medium. To date, no study has investigated whether prostate cancer tissue can spread under these conditions.
Limitations of the method
With an average division ratio of 1:2 every 2 weeks, the proliferation rate of sorted human luminal cells is not very high. Organoid lines derived from advanced prostate cancer can have a similarly low average proliferation rate, although this rate can vary widely between cultures derived from different donors.
Box 1 | Preparation of R-spondin 1 conditioned medium (b) TIME 10-11 days Materials required but not provided 293T-HA-RspoI-Fc cell line (19) (available from CalvinKuo Laboratory, Stanford University) ! CAUTION: The cell lines used must be checked regularly to ensure that they are authentic and not infected with mycoplasma. Procedure 1. Grow cell line 293T-HA-RspoI-Fc (19) (low passage cells (up to 15 passages should be used)) in DMEM + 10% (v/v) FBS + penicillin/streptomycin + Zeocin (300 [micro]g/mL) in 175 cm2 flask to confluency.2. Pass the confluent flask (split ratio ~1:6) and grow cells in DMEM + 10% (v/v) FBS + penicillin/streptomycin, without Zeocin.3. When cells reach confluency (after 3–4 days), replace DMEM+ 10% (v/v) FBS + penicillin/streptomycin medium with adDMEM/F12 +/+/+.4. After one week, collect the medium in a 50 mL Falcon tube.5. Centrifuge the tube for 5 minutes at 4°C and 450 g.6. Pass the medium through a Stericup GP with a 0.22 µm filter. (a) CRITICAL STEP It is important that all 293T cells are removed from the R-spondin 1.7 conditioned medium. Store R-Spondin 1 conditioned medium at -20°C for up to 6 months. Avoid repeated freeze-thaw cycles.
Furthermore, our culture protocol has so far failed to grow organoids derived from primary prostate cancers. This is probably because tumor cells in our prostate culture medium have no selective advantage over normal cells. In contrast, the normal prostate cells present in each sample appear to outgrow the tumor cells.
Experimental design
Summary of the procedure. The following step-by-step procedure describes how to harvest mouse and human prostate organoids from healthy prostate tissue and advanced prostate cancer. The main procedure (steps 1-3) describes the dissection and digestion of the tissue, followed by cell plating and organoid passage. Organoids are plated on Matrigel and cultured in a defined prostate culture medium that is renewed every 2 to 3 days. Table 2 describes how to culture organoids specifically derived from basal Luminaland lines. Separation of the luminal and basal lineages requires antibody staining and FACS separation. We also describe protocols for cryopreservation of the organoids once established (Step 4A), RNA isolation and cDNA production (Step 4B), and genomic DNA isolation (Step 4C). Organoids are genetically and phenotypically stable and can be genetically engineered using many different genome editing systems (3,4,13-16), making them an extremely useful tool for studying tissue homeostasis and cancer.
culture medium. In this protocol, mouse and human prostate organoids are grown in a defined prostate culture medium containing R-spondin1. Medium conditioned with R-spondin 1 (see Table 1 for preparation) or recombinant R-spondin 1 (R&D Systems) can be used at a final concentration of 500 ng/mL. Using a prostate culture medium containing medium conditioned with R-spondin 1 or recombinant R-spondin 1, we observed no difference in organoid establishment, maintenance or morphology. stored at 4°C for no longer than 1 week) and use growth factors and chemical compounds that are effective and properly stored.
Box 2 | Establishment of light- and base-derived organoid cultures (b) TIME 14 days Antibodies required CD26-FITC-conjugated antibody (anti-human 1:200, M-A261, eBioscience) CD49f-Alexa Fluor 647-conjugated antibody (anti-human). /mouse 1:200, GoH3, BD Biosciences)CD49f-PE antibody conjugate (anti-human/mouse 1:200, GoH3, BDBiosciences)CD24-Alexa Fluor 647 antibody conjugate (anti-mouse 1:200, 30 -F1 ,eBioscience) Procedure1 . Prewarm tissue culture plates at 37°C overnight.2. Digest mouse (Fig. 3a) or human (Fig. 3b) prostate tissue enzymatically as described in step 2A(i-xii) or step 2B(i-v) of the main procedure, respectively.3. Wash the digested single cells in 10 mL blocking solution in a 15 mL Falcon tube.4. Centrifuge the tube for 5 minutes at 4°C and 200 g.5. Aspirate the supernatant.6. Resuspend cells in 1 mL blocking solution.7. Pass the resuspended cells through a cell strainer of a 5 mL polystyrene round-bottom tube.8. Incubate the cells on ice for 15 minutes.9. Centrifuge the cells at 200 g for 5 minutes at 4 °C and aspirate the supernatant. Reserve a few cells (about a tenth of the total) as an unstained negative control. Use the remaining cells for antibody staining (as described in step 10 below).10. Incubate cells with 500 µL of staining solution for 60 minutes on ice in the dark with CD26-FITC-conjugated antibody (M-A261.1:200) and CD49f-Alexa Fluor 647-conjugated antibody (GoH3, 1:200) for human organoids or CD24-Alexa Fluor 647 conjugated antibody (30-F1, 1:200) and CD49f-PE conjugated antibody (GoH3, 1:200) for mouse organoids.11. Wash all aliquots of cells from steps 9 and 10 with 10 mL of adDMEM/F12 +/+/+ plus Y-27632 dihydrochloride (10 μM) plus dihydrotestosterone (1 nM).12. Centrifuge the cells at 200 g, 4 °C for 5 minutes.13. Repeat steps 11 and 12.14. Resuspend cells in staining solution (no antibody added).15. Add DAPI to each cell sample (to obtain a final concentration of 1.0 µg/mL).16. Add DNaseI to each cell sample (to obtain a final concentration of 0.5–1.0 U/μL). SOLVE PROBLEMS17. Isolate cells using FACS (Fig. 3). On average, 200 mg of prostate tissue should contain about 150,000 lumen cells and 600,000 basal cells. SOLVE PROBLEMS18. Plate luminal and basal cells as described in step 2A (xiv-xvii) (for mouse cells) and step 2B (viii-x) (for human cells) of the main procedure (Fig. 3).19. Refresh the medium every 2-3 days with Y-27632 dihydrochloride and check the organoid growth under a light microscope. After 2–3 days, organoids should form from the basal cells, while small organoids should form from the luminal cells, after 5–7 days. For cultures longer than 7 days, do not add Y-27632 dihydrochloride to the medium. From 7, medium without Y-27632.20 dihydrochloride should be used. After approximately 14 days, pass the organoids as described in PROCEDURE Step 3A (mouse) and Step 3B (human).
age of the mouse. To establish mouse prostate organoids, prostates must be isolated from mice that are at least 8 weeks old. The maximum age of mice for efficient organoid establishment has not been carefully considered. However, we have successfully established organoids from mice as young as 2 years of age.
Control S. The separation of luminal and basal cells from mouse and human prostate tissue (Table 2) is based on double antibody staining for marker gene expression, followed by FACS. To specifically stain luminal cells, we used conjugated antibodies against CD26 and CD24 (human and mouse, respectively). For basal cell-specific staining, we used a CD49f-specific conjugated antibody for both humans and mice. An isotype-matched control antibody derived from the same species as the target antibody can be used as a negative control. In addition, single color staining controls (staining for basal or luminal markers) can also be included.
To verify that the organoids are indeed derived from prostate tissue, (q-)RT-PCR for prostate-specific gene expression can be performed (see EXPECTED RESULTS and Karthaus et al. (3)). In addition, to confirm the growth of prostate tumor organoids, whole genome sequencing can be performed to analyze mutational spectra (4). In this protocol, we also describe procedures for isolating RNA, preparing cDNA, and isolating genomic DNA from organoids.
MATERIALS
REAGENTS
* Collagenase Type II (Life Technologies, cat # 17101-015)
* TrypLE Express (Life Technologies, Kat.-Nr. 12605-010)
* DMEM (Life Technologies, Kat.-Nr. 31966)
* Advanced DMEM/F12 (adDMEM/F12; Life Technologies, Kat.-Nr. 12634-034)
* GlutaMAX, 100x (Life Technologies, Kat.-Nr. 35050-068)
* Penicillin-Streptomycin (Life Technologies, Kat. Nr. 15140-122)
* HEPES (Life Technologies, Kat.-Nr. 15630-056)
* Zeocin (Life Technologies, Kat.-Nr. R250-01)
* PBS
* Matrigel, growth factor reduced, without phenol red (BDBiosciences, cat. # 356231)
* Supplement B27, 50x (Life Technologies, cat # 17504-044).
Store at -20°C until the expiry date.
* Nicotinamide (Sigma-Aldrich, Cat. No. N0636)
* N-Acetyl-L-Cystein (Sigma-Aldrich, Kat.-Nr. A9165)
* A83-01 (Tocris Bioscience, Kat.-Nr. 2939)
* Y-27632 Dihydrochlorid (Abmole Bioscience, Kat. Nr. M1817)
* Human Fibroblast Growth Factor (FGF)-10 (PeproTech, Kat. Nr. 100-26)
* Human FGF-2 (PeproTech, Cat# 100-18B)
* Human Epidermal Growth Factor (EGF) (PeproTech, Cat. No. AF-100-15)
* Recombinant Human Noggin (Peprotech, Cat# 120-10C)
*Medium conditioned with R-spondin 1 (see Table 1 for preparation instructions) or recombinant R-spondin 1 protein (R&D Systems, cat #4645-RS-025)
* Prostaglandina E2 (Tocris Bioscience, Cat.-Nr. 2296)
* SB202190 (Sigma-Aldrich, Kat.-Nr. S7076)
* (DiHydro)Testosteron (5α-Androstan-17β-ol-3-on) (Sigma-Aldrich, Kat.-Nr. A8380)
* FBS (Sigma-Aldrich, Kat.-Nr. F7524)
* DNase I) from bovine pancreas (Sigma-Aldrich, Cat. No. D5025)
* DAPI (Life Technologies, Kat.-Nr. D1306)
* RosetteSep Human CD45 Depletion Cocktail (StemCell Technologies, Cat. Nr. 15122)
* Ficoll-Paque PLUS (GE Healthcare Life Sciences, Kat.-Nr. 17-1440-02)
* Recovery Cell Culture Freezing Medium (Life Technologies, Kat. Nr. 12648-010)
* RNeasy Mini-Kit (Qiagen, Kat.-Nr. 74104)
* Reliaprep gDNA Mini Tissue Prep System (Promega, Kat. Nr. A2052)
* GoScript Reverse Transkriptase (Promega, Kat.-Nr. A5003)
* Oligo[(dT)sub.15] Primer (Promega, Kat.# C1101)
* Recombinant RNasin-RNase-Inhibitor (Promega, Cat. Nr. N2511)
* Mouse or human material (see reagent setup for more details on suitable sources)
EQUIPMENT
* Falcon tubes, 15 ml
* Falcon tubes, 50 ml
* SepMate-50 (StemCell Technologies, Cat.-Nr. 15450)
* Polystyrene round bottom tubes with cell filter caps, 5mL (Falcon)
* Microcentrifuge tubes, 1.5mL
* Vibrationsplattform, 37°C
* Plates, 6 wells (Greiner Bio-One, Cat. No. 657 160)
* Plates, 12-well (Greiner Bio-One, Cat. No. 665 180)
* Plates, 24-well (Greiner Bio-One, Cat. No. 662 160)
*Plates, 48-well (Greiner Bio-One, Cat. No. 677 180)
* 100 x 20 mm cell culture dishes (Greiner Bio-One, cat. no. 664160)
* Glasstic slide with hemocytometer counting grid (KovaInternational, cat. no. 87144E)
* Glass Pasteur pipettes (VWR, Cat. No. 612-1701)
* Optical microscope (Nikon, Eclipse TS100)
* Dissecting microscope (Leica, MZ75)
* Dissection tools (NeoLab)
* FACS machine (DaKo MoFlo)
* Disposable scalpels (Swann-Morton, code 0501)
* Centrifuge for 15 and 50 ml Falcon tubes (Eppendorf, Cat. No. 5810R)
* Microcentrifuge (Eppendorf, Cat. No. 5424)
* Incubator C[O2] (5 % C[O2], 37°C)
* Biosafety cabin
* CoolCell freezer container (BioCision)
* Stericup-GP, 0.22 µm, polyestersulfone, 500 mL, radioactively sterilized (Millipore, Cat # SCGPU05RE)
REAGENTS SETUP
Human and mouse material The method can be applied to whole mouse prostate, human prostate tissue pieces (minimum size 1 [mm3]), human metastatic biopsy pieces (minimum size 1 [mm3]) or blood samples. from patients with advanced prostate cancer (requires 8 ml of blood). We have successfully grown organoids from all of these starting materials (3,4). Although fresh material should preferably be used, we were able to establish organoids from human and mouse tissues stored overnight at 4°C in adDMEM/F12 +/+/+ (i.e. adDMEM/F12 with penicillin/streptomycin, 10 mM) HEPES and 2mM GlutaMAX (100x diluted). !WARNING All experiments on mice must comply with local animal welfare laws and policies. ! CAUTION Informed consent must be obtained from all subjects. Studies must comply with all relevant institutional and governmental regulations.
Mouse Prostate Culture Medium The final composition of the medium is summarized in Table 1. To prepare the medium, combine 1.0 mL of B27, 125.0 µL of N-acetyl-L-cysteine (500 mM in PBS), and 5.0 µL of EGF (0.5 mg/mL in PBS + 0.1% (w/v) BSA), 2.0 µl A83-01 (5 mM in DMSO), 50.0 µl noggin (100 µg/ml in PBS + 0.1% (w/v) BSA) , 50.0 µl R-Spondin 1 (500 µg/ml in PBS + 0.1% (w/v) BSA or 10% conditioned medium) and 50.0 µl dihydrotestosterone (1 µM inethanol). Make up to 50 ml with adDMEM/F12 +/+/+. (a) CRITICAL As described in the PROCEDURE, post cell passage, Y-27632 dihydrochloride (ROCK inhibitor) should be included in the culture medium to a final concentration of 10 μM. To prepare this medium, add 5.0 µL of 100 mM Y-27632 dihydrochloride to 50 mL of mouse prostate culture medium. (a) CRITICAL Culture medium should not be stored longer than 1 week at 4°C.
Human Prostate Culture Medium The final composition of the medium is summarized in Table 1. To prepare the medium, combine 1.0 mL of B27, 500 µL of nicotinamide (1 M in PBS) and 125.0 µL of N-acetyl-L-cysteine (500 mM in PBS), 0.5 µL of EGF (0.5 mg/ml in PBS + 0.1% BSA), 5.0 µl A83-01 (5 mM in DMSO), 50.0 µl noggin (100 [micro]g/ml in PBS + 0.1% (wt. /vol) BSA), 50.0 [micro]l R-Spondin 1 (500 [micro]g/ml in PBS + 0.1% (w/v) BSA or 10% conditioned medium), 50, 0 µL dihydrotestosterone (1 µM in ethanol), 5.0 µL FGF2 (50 µg/mL in PBS + 0.1% (w/v) BSA), 5.0 [microliters FGF10 (0.1 mg/mL in PBS + 0.1% (w/v) BSA), 5.0 microliters prostaglandin E2 (10mM in DMSO) and 16.7 microliters SB202190 (30mM in DMSO). Make up to 50 ml with adDMEM/F12 +/+/+. (a) CRITICAL As described in the PROCEDURE, post cell passage, Y-27632 dihydrochloride must be included in the culture medium to a final concentration of 10 μM. To prepare this medium, add 5.0 µL of 100 mM Y-27632 dihydrochloride to 50 mL of human prostate culture medium.
(a) CRITICAL Culture medium should not be stored longer than 1 week at 4°C.
Reverse transcription mix For each reaction, combine 4.0 µl 5x GoScript reaction buffer, 4.0 µl 25 mM Mg[Cl2], 1.0 µl PCR nucleotide mix 10 mM, 0.5 µl. l recombinant RNasinRNase Inhibitor and 1.0 µl GoScript Reverse Transcriptase. Make up to 15.0 µl with RNase-free [H.sub.2]O. Add 15.0 µl reverse transcription mix to 5.0 µl RNA/oligo[(dT)15] mix. The reverse transcription mix must be freshly prepared.
Thaw the original Matrigel vial overnight at 4°C on ice. Mix well by pipetting and divide the Matrigel into 1 mL aliquots in 1 mL cryovials. Aliquots can be stored at -20°C until the expiration date.
R-Spondin 1 medium Prepare the medium as described in Box 1. N-Acetyl-L-cysteine Dissolve 81.5 mg/mL [H.sub.2]O to make a 400 x 500 mM stock solution for up to 1 month.
FGF-10 Dissolve 500 µg in 5 mL PBS + 0.1% BSA to make a 10,000 x 0.1 mg/mL stock solution for up to 1 month.
Nicotinamide: Dissolve 1.2g in 10mL PBS to make a 100x 1M stock solution. Store aliquots at -20°C until the expiration date.
Human EGF: Dissolve 1 mg in 2 mL PBS + 0.1% (w/v) BSA to make a 10,000 x 0.5 mg/mL stock solution for up to 1 month.
recommended Human noggin Dissolve 100 µg in 1 mL PBS + 0.1% (w/v) BSA to make a 1000x stock solution for up to 1 month.
Y-27632 dihydrochloride Dissolve 50 mg in 1.5 mL of [H.sub.2]O to make a 10,000 x 100 mM stock solution for up to 1 month.
SB202190 Dissolve 25mg in 2.75mL DMSO to make a 30mM3000x stock solution for up to 1 month.
A83-01 Dissolve 10 mg in 950 µL DMSO to make a 25 mM stock solution, 50,000x for up to 1 month.
Dissolve FGF2 50 µg in 100 µl 5 mM Tris, pH 7.6 (0.5 mg/ml). Dilute to 10,000 x 50 µg/mL stock solution by adding 900 µL PBS + 0.1% (w/v) BSA to 100 µL 0.5 mg/mL solution for up to 1 month.
PGE2 Dissolve 10 mg in 2.84 mL DMSO to make a 10,000 x 10 mM stock solution for up to 1 month.
Dissolve DHT 1 mg in 3.44 mL of 100% ethanol to make a 1 mM solution. Dilute 1000x in 100% ethanol to make a 1000x 1 µM stock solution.
Store at -20°C until the expiry date.
Collagenase Type II Dissolve 5 mg of Collagenase Type II in 1 mL of adDMEM/F12 +/+/+ to make a 5 mg/mL solution. Add Y-27632 dihydrochloride to a final concentration of 10 μM and dihydrotestosterone to a final concentration of 1 nM. The 5 mg/ml collagenase solution must be freshly prepared.
Blocking Solution Add 2.5 mL FBS to 47.5 mL adDMEM/F12 +/+/+ to obtain a 5% (v/v) Blocking Solution. Add Y-27632 dihydrochloride to a final concentration of 10 μM and dihydrotestosterone to a final concentration of 1 nM. The blocking solution must be freshly prepared.
Staining Solution Add 25.0 µL FBS to 49.975 mL adDMEM/F12 +/+/+ to make a 0.05% (v/v) blocking solution. Add Y-27632 dihydrochloride to a final concentration of 10 μM and dihydrotestosterone to a final concentration of 1 nM. The staining solution must be freshly prepared.
PROCEDURE
Establishment and cultivation of prostate organoid cultures.
1| Prewarm tissue culture plates at 37 °C overnight.
2| If culturing mouse prostate organoids, follow option A. For human prostate organoid cultures from human prostate tissue, follow option B. For organoids from prostate cancer metastatic biopsies, follow option C.
For circulating prostate tumor cell organoids, follow option D.
(A) Establishment of mouse prostate organoid cultures (b) TIME 7 d
(i) Euthanize a <8 week old male mouse and then isolate the urogenital system (Fig. 1a).
(ii) Remove the seminal vesicles by breaking or cutting through the blood vessels and connective tissue and making an incision at the base of the urethra (Fig. 1b,c; for a detailed mouse prostate isolation protocol see ref. 17).
(iii) Remove the vas deferens by cutting it close to the prostate (Fig. 1d).
(iv) Remove the bladder by cutting it near the base of the urethra (Fig. 1e).
(v) Remove the remaining vesicles and adipose tissue by gentle cutting (Fig. 1f).
(vi) remove the urethra; Gently pull on the lobules of the prostate so that they are no longer attached to the urethra (Fig. 1g).
(a) CRITICAL STEP Do not isolate the ampulla gland which lies between the two lobes of the anterior prostate (Fig. 1i,j) but is not considered part of the prostate.
(vii) isolate each lobe individually (anterior prostate, ventral prostate and dorsolateral prostate); Alternatively, proceed to the entire prostate (Fig. 1g,h,k).
(viii) Use a scalpel to mince the entire prostate or lobules of the prostate into small pieces (~1 [mm3]) in a 10 cm culture dish.
(ix) Digest the prostate in 5 mg/mL collagenase type II with 10 µM Y-27632 dihydrochloride in a 15 mL Falcon tube for 1-1.5 hours at 37°C on a shaking platform. Use 1mL of 5mg/mL Collagenase Type II per approximately 50mg minced tissue.
[FIGURE 1 ESSENTIAL]
(x) Wash once by making up to 10 mL with adDMEM/F12 +/+/+.
(xi) Centrifuge the tube at 4°C and 150g for 5 minutes.
(xii) Aspirate supernatant and resuspend pellet in 1 ml TrypLE containing 10 μM Y-27632 dihydrochloride, then digest at 37°C for approximately 15 minutes.
(a) CRITICAL STEP Pipette up and down every 5 minutes with a P1000 pipette to ensure efficient digestion.
? TROUBLESHOOTING
(xiii) Wash once by making up to 10 mL with adDMEM/F12 +/+/+, then centrifuge the tube at 150 g for 5 minutes at 4 °C.
(xiv) Count cells with a hemocytometer and plate 20,000 cells in a 40 µl droplet in the center of a well of a 24-well tissue culture plate (Figure 2a and Table 2). On average, a whole prostate produces 25 drops.
(a) CRITICAL STEP Work quickly to ensure the matrigel does not solidify before planting.
(a) CRITICAL STEP To ensure an effective coating, Matrigel must not be diluted too much. The average ending percentage of Matrigel should be ~75%.
(xv) Place the tissue culture plate upside down in the C[O2] incubator (5% C[O2], 37°C) for 15 minutes to allow the Matrigel to solidify.
(a) CRITICAL STEP The plate should be placed upside down in the incubator to prevent it from sticking to the bottom of the plate.
(xvi) Prewarm the mouse prostate culture medium in a 37°C water bath for approximately 10 minutes before adding it to the organoids.
(xvii) Gently pipet 500 μL of prewarmed (37 °C) mouse prostate culture medium plus 10 μM Y-27632 dihydrochloride into each well, then place the plate on the C[O2] incubator (5% C[O2] , 37 °C).
(xviii) Refresh the medium every 2-3 days with 10 µM Y-27632 dihydrochloride. From 7 days after the first sowing, a medium without Y-27632 dihydrochloride should be used. Examples of the typical morphology of prostate organoids after 1 and 7 days of culture are shown in Figure 2b.
(B) Establishment of human prostate organoid cultures (b) TIME up to 14 days
(i) Use a scalpel to mince human prostate tissue into small pieces (~1–5 [mm3], Fig. 3) in a 10 cm culture dish.
(ii) Digest tissue overnight in 5 mg/mL collagenase type II with 10 µM Y-27632 dihydrochloride in a 15 mL Falcon tube at 37 °C on a shaking platform. Use 1mL of 5mg/mL Collagenase Type II per approximately 50mg minced tissue.
(iii) Wash once and make up to 10 mL with adDMEM/F12 +/+/+.
(iv) Centrifuge the tube at 200g for 5 minutes at 4°C.
(v) Resuspend the pellet in 1 ml TrypLE containing 10 µMY-27632 dihydrochloride and then digest at 37°C for approximately 15 minutes.
(a) CRITICAL STEP Pipette up and down every 5 minutes to ensure efficient digestion (P1000 pipette).
? TROUBLESHOOTING
(vi) Wash once by making up to 10 mL with adDMEM/F12 +/+/+.
(vii) Centrifuge the tube at 200g for 5 minutes at 4°C.
(viii) Count cells with a hemocytometer and plate approximately 20,000 cells in a 40 µl droplet in the center of a well of a 24-well plate (Figure 2a and Table 2).
(a) CRITICAL STEP Work quickly to ensure the matrigel does not solidify before planting.
(a) CRITICAL STEP To ensure an effective coating, Matrigel must not be diluted too much. The average ending percentage of Matrigel should be ~75%.
[FIGURE 2 ESSENTIAL]
[FIGURE 3 ESSENTIAL]
(ix) Place the plate in the C[O2] incubator (5% C[O2], 37°C) for 15 minutes to allow MatrigeL to solidify.
(a) CRITICAL STEP Place the plate upside down in the incubator to prevent it from sticking to the bottom of the plate.
(x) Gently pipet 500 µL of pre-warmed human prostate culture medium (approximately 10 minutes in a 37 °C water bath) plus 10 µL of MY-27632 dihydrochloride into each well and place the plate in a CO2 solution incubator (5th %C[O2], 37°C).
(xi) Replace the medium with medium containing 10 µM Y-27632 dihydrochloride every 2-3 days and continue culturing for 7 days.
(xii) At the next culture change after the first 7 days of culture, replace the medium with a medium that does not contain Y-27632 dihydrochloride. Proceed to the next step (organoid passage) 1–2 weeks after starting culture.
(a) CRITICAL STEP Human organoids should be split 1:2 every 1 to 2 weeks. The exact timing depends on the density and whether the organoids are from luminal or basal lineages. For example, the 7-day from the base and 14-day from light organoids shown in Fig. 3 do not have the size and density that they need to go through.
(C) Establishment of organoids from prostate cancer metastatic biopsies (b) TIME up to 14 days
(i) Use a scalpel to mince human advanced prostate cancer biopsy tissue (minimum size ~1 [mm3]) into small pieces (~1-5 [mm3], Fig. 3b).
(ii) Digest tissue in 5 mg/mL collagenase type II containing 10 µM Y-27632 dihydrochloride in a 15 mL Falcon tube for 1 hour at 37°C. Use 1mL of 5mg/mL Collagenase Type II per approximately 50mg minced tissue.
(iii) Wash once with 10 mL of adDMEM/F12 +/+/+.
(iv) Centrifuge the tube at 200g for 5 minutes at 4°C.
(v) Resuspend the pellet in 1 ml TrypLE containing 10 µMY-27632 dihydrochloride and then digest at 37°C for approximately 10 minutes.
(a) CRITICAL STEP Pipette up and down every 5 minutes to ensure digestion (P1000 pipette).
(vi) Wash once with 10 mL of adDMEM/F12 +/+/+.
(vii) Centrifuge the tube at 200g for 5 minutes at 4°C.
(viii) Repeat the washing procedure (step 2C(vi, vii)).
(ix) Count cells with a hemocytometer and then plate approximately 50,000 cells in a 40 microliter drop in the center of a well of a 24-well plate (Fig. 2a and Table 2).
(a) CRITICAL STEP Work quickly to ensure the matrigel does not solidify before planting.
(a) CRITICAL STEP To ensure an effective coating, Matrigel must not be diluted too much. The average ending percentage of Matrigel should be ~75%.
(a) CRITICAL STEP Seed high density prostate cancer cells (~50,000 cells in 40 µL Matrigel).
(x) Place the plate in the C[O2] incubator (5% C[O2], 37°C) for 15 minutes to allow the Matrigel to solidify.
(a) CRITICAL STEP Place the plate upside down in the incubator to prevent it from sticking to the bottom of the plate.
(xi) Gently pipet 500 μL of pre-warmed (~10 minutes at 37 °C) human prostate culture medium plus 10 μM Y-27632 dihydrochloride into each well, then place the plate on a Co. sub.2] incubator (5% C [O2], 37°C).
(xii) Replace the medium every 2-3 days with medium containing Y-27632 dihydrochloride and check the growth of the organoids under a light microscope. From 7 days after the first sowing, a medium without Y-27632 dihydrochloride should be used.
After 5-7 days, small organoids should appear.
(xiii) After approximately 14 days, continue with organoid passage (step 3).
(D) Establishment of organoids from circulating prostate tumor cells (CTCs) (b) TIME up to 14 days
(i) Obtain 8 ml of blood from a patient with advanced prostate cancer.
(a) CRITICAL STEP The minimum total number of CTCs should be approximately 50 in 8 mL of blood. A CTC count can be performed using the Cellsearch Circulated Tumor Cell Kit (http://www.cellsearchctc.com).
(ii) Incubate the blood sample with 400 µl RosetteSephuman CD45 depletion cocktail for 20 minutes at room temperature (~20°C).
(iii) Pipet 15 mL of Ficoll into a 50 mL SepMate tube.
(iv) Gently add the blood to the Ficoll in the SepMate tube so that it forms a layer on top of the Ficoll.
(v) Centrifuge the tube at 1200g for 20 minutes at room temperature.
(a) CRITICAL STEP To keep the layers intact, turn without the brake.
(vi) Collect the interface with the desired cells in a 15 mL Falcon tube.
(vii) Add 10 ml PBS + 2% (v/v) FBS and mix by inverting the tube.
(viii) Centrifuge the tube at 200g for 5 minutes at 4°C.
(ix) Repeat washing as described in step 2D (vii, viii).
(x) Wash once by making up to 10 mL with adDMEM/F12 +/+/+.
(xi) Place cells in 30 µL of ice-cold Matrigel and pipette up and down 5 to 10 times to mix.
(a) CRITICAL STEP Work quickly to ensure Matrigel does not solidify before planting.
(a) CRITICAL STEP To ensure an effective coating, Matrigel must not be diluted too much. The average ending percentage of Matrigel should be ~75%.
(xii) Plate CTCs in a 30 µl drop in the center of a well of a 24-well plate.
(xiii) Place the plate in the C[O2] incubator (5% C[O2], 37°C) for 15 minutes to allow the Matrigel to solidify.
(a) CRITICAL STEP Place the plate upside down in the incubator to prevent it from sticking to the bottom of the plate.
(xiv) Gently pipette 500 μL of pre-warmed (37 °C) human prostate culture medium plus 10 μM Y-27632 dihydrochloride into each well and then incubate the cells on plates in a C[O.sub.2] incubator (5% C [O2], 37°C).
(xv) Replace medium every 2-3 days with medium containing Y-27632 dihydrochloride. Check organoid growth every 2 days with a light microscope. After 5-7 days, small organoids should appear.
(xvi) After approximately 14 days, pass the organoids as described in step 3.
3| If culturing mouse tissue, follow option A; If you are growing human tissue, follow option B.
(A) Passage of mouse tissue-derived organoids (b) Time variable
(i) Collect the organoids (approximately 7 days after starting the culture; Fig. 2b) in the culture medium in the well containing the organoids and transfer the organoids from the same sample to a 15 mL tube.
? TROUBLESHOOTING
(ii) Dissociate the organoids by grinding them with a fire-polished glass pipette. After polishing, the glass pipette should have an opening of about 0.5–1 mm in diameter.
(a) CRITICAL STEP By crushing with a fire-polished glass pipette, the organoids are broken up into small clumps of cells.
(iii) Pipette up and down 15-20 times.
(iv) Add 5 mL of ice-cold adDMEM/F12 +/+/+ to dissolve any residual Matrigel.
(v) Centrifuge the tube at 150g for 5 minutes at 4°C.
(vi) Aspirate and discard the supernatant.
(vii) Resuspend the pellet in 160 µl of Matrigel and place a drop of 40 µl of Matrigel in the center of a well of a 24-well plate (Table 2 and Figure 2a), splitting each organoid culture 1:4.
(viii) Place the plate in the C[O2] incubator (5% C[O2], 37°C) for 15 minutes to allow the Matrigel to solidify.
(a) CRITICAL STEP Place the plate upside down in the incubator to prevent it from sticking to the bottom of the plate.
(ix) Gently pipet 500 μL of pre-warmed (37 °C) mouse prostate culture medium into each well and place in a C[O2] (5% C[O2]) incubator. , 37[deg]C).
(x) Replace media every 2-3 days.
(a) CRITICAL STEP At this stage of the culture, addition of Y-27632 dihydrochloride to the medium is not required. Y-27632 dihydrochloride is required in the culture medium only when the organoids have been freshly passaged with TrypLE. TrypLE treatment provides a high percentage of single cells and Y-27632 dihydrochloride promotes single cell growth after seeding.
(B) Passage of organoids from human tissue. (b) TIME variable
(i) Collect the organoids in the culture medium in the well containing the organoids and transfer the organoids from the same sample to a 15 mL tube.
? TROUBLESHOOTING
(ii) Dissociate organoids enzymatically with 0.5 mL of Y-27632 dihydrochloride containing 10 µM TrypLE on a shaking platform for 5 minutes at 37 °C.
(a) CRITICAL STEP The best method for dividing human organoids is TrypLE. However, if the organoids are small but the density is high, we do not recommend digesting with TrypLE but instead use a fire-polished pipette as described in step 3A for mouse organoids.
(iii) Inactivate TrypLE by adding ~5 mL of adDMEM/F12 +/+/+ with 5% (v/v) FBS.
(iv) Centrifuge the tube at 200g for 5 minutes at 4°C.
(v) Aspirate and discard the supernatant.
(vi) Resuspend the pellet in 80 µl Matrigel. Plate40-μL is dropped into the center of each well of a 24-well plate, splitting each organoid culture 1:2 (two wells total).
(vii) Place the plate in the C[O2] incubator (5% C[O2], 37°C) for 15 minutes to allow the Matrigel to solidify.
(a) CRITICAL STEP Place the plate upside down in the incubator during this incubation to prevent it from sticking to the bottom of the plate.
(viii) Gently pipette 500 μL of prewarmed (37 °C) human prostate culture medium plus 10 μM Y-27632 dihydrochloride into each well and place the plate in a C[O. sub.2] incubator (5% C[O2], 37°C).
(ix) Replace media every 2-3 days. Replace the medium with medium containing Y-27632 dihydrochloride for the first 7 days. After 7 days, medium without Y-27632 dihydrochloride should be used.
Downstream Tests
4| At appropriate times during culturing, cultures can be cryopreserved (option A), RNA isolated and cDNA produced (option B), or genomic DNA isolated (option C).
(A) Cryopreservation of prostate organoids (b) TIME 30 min
(i) Aspirate the medium from the Matrigel disc.
(ii) Use a P1000 pipette to resuspend the Matrigel disc in 1 mL of TrypLE plus 10 µM Y-27632 dihydrochloride.
(iii) Transfer the suspension to a 15 mL Falcon tube.
(iv) Incubate the tube at 37°C for 5-10 minutes. Pipette up and down as needed to break up the organoid structures after incubation (using a P1000 pipette).
(v) Add 10 mL of ice-cold adDMEM/F12 +/+/+.
(vi) Centrifuge the tube at 300g for 5 minutes at 4°C.
(vii) Resuspend the organoids from one well of a 24-well plate in 500 µL of recovery cell culture freezing medium and transfer to a 1.5 mL cryotube. Freeze cells at -80 °C using a CoolCell freezing container (BioCision) or comparable freezing container.
(c) PAUSE POINT Cells can be stored at -80°C for up to 1 month. For long-term storage (>1 month), transfer the cryotubes to liquid nitrogen.
(viii) If you wish to thaw the cells, thaw the cells in the cryovial in a 37°C water bath.
(ix) Transfer the thawed cells to a 15 mL Falcon tube and add 10 mL of adDMEM/F12 +/+/+ dropwise to wash out the freezing medium.
(x) Centrifuge the tube at 200g for 5 minutes at 4°C.
(xi) Aspirate the supernatant and collect the cell pellet in the desired volume of Matrigel.
(xii) Plate the organoids as described in step 3 above.
(B) RNA isolation and cDNA production from prostate organoids (b) TIME 2 h
(i) Aspirate the prostate culture medium from the Matrigel disk.
(ii) Harvest organoids (a minimum of 50 µL of Matrigel-containing organoids is required to obtain sufficient RNA for most applications) directly into 350 µL of RLT buffer (Qiagen) (preferably add β-mercaptoethanol, but not essential).
(iii) Incubate the tube on a rocking platform at room temperature for 15 minutes.
(iv) Add 350 µl of 70% (v/v) ethanol and mix by pipetting.
(v) Transfer the mixture to an RNeasy column (Qiagen).
(vi) Centrifuge for 30 s at 8000 g and discard the continuous flow.
(vii) Add 700 µl RW1 buffer (Qiagen).
(viii) Centrifuge for 30 s at 8000 g and discard the continuous flow.
(ix) Add 500 µl RPE buffer (Qiagen).
(x) Centrifuge at 8000 g for 30 s and discard the continuous flow.
(xi) Add 500 µl RPE buffer.
(xii) Centrifuge at 8000g for 2 minutes and discard the continuous flow.
(xiii) Place the column in a clean collection tube and centrifuge at full speed for 1 minute.
(xiv) Elute RNA with 30 µl RNase-free [H.sub.2]O.
? TROUBLESHOOTING
(xv) Pipette 100-500 ng of RNA into a 1.5 mL microcentrifuge tube without RNase.
(xvi) Add 1 µl of oligo[(dT)15] and bring the mixture to a total volume of 5 µl with RNase-free [H2]O.
(xvii) Incubate the tube at 70°C for 5 minutes and place once.
(xviii) Add 15.0 µl Reverse Transcription Mix (see Reagent Setup) to the RNA/Oligo[(dT)15] mix.
(xix) Perform reverse transcription with the following incubations:
Step Temperature Time1 25[deg]C 5 min2 42[deg]C 60 min3 70[deg]C 15 min
(xx) Use the cDNA product for specific (q-)RT-PCR analyzes of interest.
(C) Isolation of genomic DNA from organoids (b) TIME 90 min
(i) Collect the organoids (a minimum of 50 µl of Matrigel-containing organoids is required to obtain sufficient DNA for most applications) in the culture medium covering the organoids and transfer to a 10-20 microcentrifuge tube. 1.5ml .
(ii) Centrifuge the tube at 750g for 5 minutes at 4°C.
(iii) Aspirate the supernatant.
(iv) Resuspend the pellet in 160 µl PBS.
(v) Add 20 µl Proteinase K solution (Promega).
(vi) Add 200 ml Cell Lysis Buffer (CLB; Promega) to the tube.
(vii) Mix by vortexing thoroughly.
(viii) Incubate the tube at 56°C for 1 hour; Vortex every 15 minutes.
(ix) Add 20 µl RNaseA solution (Promega).
(x) Mix thoroughly on a vortex.
(xi) Incubate the tube at 56°C for 10 minutes.
(xii) Add 250 µl Binding Buffer (BBA; Promega) and mix by vortexing.
(xiii) Transfer the solution to a ReliaPrep binding column (Promega).
(xiv) Centrifuge at maximum speed for 1 minute and discard the continuous flow.
(xv) Add 500 µl of column wash solution (Promega) to the column.
(xvi) Centrifuge at maximum speed for 2 minutes and discard excess liquid.
(xvii) Repeat step 4C (xv, xvi) twice (steps are performed three times in total).
(vxiii) Place the column in a clean 1.5mL microcentrifuge tube.
(xix) Add 50 µl of nuclease-free [H.sub.2]O to elute genomic DNA from the column.
(xx) Centrifuge for 1 minute at 20,000g at room temperature. The continuous flow contains genomic DNA that can be stored at -20 °C.
? TROUBLESHOOTING
See Table 3 for troubleshooting tips.
(b) TIME
Steps 1-3, establishment and culture of prostate organoid cultures: variable; up to ~2 weeks Step 4, subsequent attempts: variable
Box 1, R-Spondin 1 conditioning medium preparation: 10-11 days
Box 2, Establishment of organoid cultures derived from basal cells and luminal cells: 14 days
EXPECTED RESULTS
This protocol describes an efficient method for establishing organoid cultures from human and mouse prostate tissue.
The establishment efficiency of these cultures is >95%, although the establishment efficiency of organoids from advanced prostate cancer is much lower (~15–20%), mainly due to the small amount of input material. After sorting, basal cells have ~70% organoid-forming capacity (of which >95% are solid), while ~1-2% of the sorted luminal cells give rise to organoids (>95% are cystic). To achieve this efficiency and maintain growth, it is important to use fresh media (stored at 4 °C for no more than a week) and to use potent and properly stored growth factors and chemicals. Prostate organoids can be genetically engineered (3) and can therefore be used to study gene involvement in prostate homeostasis and cancer. We failed to culture organoids derived from primary prostate cancers, most likely due to overgrowth of normal prostate epithelium in each sample.
When culturing human and mouse organoids, small organoids can usually be detected within 2–3 days after seeding (Fig. 2b and 3a,b). Mouse organoids are generally cystic (Figs. 2b and 3a), whereas unsorted newly established human organoid cultures consist mainly of solid organoids derived from basal cells during the first passages. After 5–7 days, small cystic organoids can be observed from ordered luminal cells (Fig. 3b). The morphology of organoids derived from advanced prostate cancer can vary widely between patients (eg, cystic versus solid structures) and even within cultures derived from the same patient due to tumor heterogeneity (4).
To confirm that the organoids are indeed derived from prostate tissue, the expression of prostate-specific genes, such as those encoding prostate-specific antigen (PSA), can be determined. Expression analysis of specific luminal markers (e.g. AR cytokeratin 8, cytokeratin 18, probasin, PSA) and specific basal markers (e.g. p63, cytokeratin 5) should be used to confirm the presence of both lineages in established crops (3rd ).
In specimens derived from advanced prostate cancer, growth rate and morphology vary widely (from weekly split ratios of 1:2 to 1:3 to monthly split ratios of 1:2 to 1:3). Confirmation of the cancerous origin of these organoids can be achieved by genomic analysis using whole genome sequencing or by comparative genomic hybridization (4).
In addition, genitourinary sinus mesenchymal recombination (UGSM) assays (described in detail in Xin et al. (18)) can be used, in which single organoid cells of the prostate are mixed with mesenchymal cells derived from the urogenital sinus of mouse embryos under the renal capsule placed. to confirm that healthy organoids or tumor organoids give rise to prostate gland and neoplastic growth, respectively, in vivo.
ACKNOWLEDGMENTS We thank the members of the contributing laboratories for their support. We are grateful for support from: The Netherlands Organization for Scientific Research (NWO-ZonMw) VENI grant to J.D.(91614138); Koningin Wilhelmina Fonds (Dutch Cancer Foundation) to W.R.K. (2015-7545); the Prostate Cancer Foundation (C.L.S., Y.C.); and the program CancerGenomics.nl (NWO Gravitation).
CONTRIBUTIONS BY AUTHOR J.D., W.R.K., Y.C., C.L.S. and H.C. designed the study. J.D., W.R.K., D.G. and E.D. wrote the manuscript.
Competing Financial Interests The authors declare that they have no competing financial interests.
Reprint and permission information is available online at http://www.nature. com/reprints/index.html. 1 2 3 4 5 6
(1.) Pienta, K.J. et al. The current status of preclinical animal models for prostate cancer. Prostate 68, 629-639 (2008).
(2.) Toivanen, R., Taylor, RA., Pook, D.W., Ellem, S.J. & Risbridger, G.P. Overcoming a hurdle in prostate cancer research: an update on human model systems. J.Steroid Biochem. Mol. Biol. 131, 122-131 (2012).
(3.) Karthaus, W.R. et al. Identification of multipotent luminal progenitor cells in cultured human prostate organoids. Cell 159, 163-175 (2014).
(4.) Gao, D. et al. Organoid cultures from patients with advanced prostate cancer. Cell 159, 176-187 (2014).
(5.) Sato, T. et al. Individual Lgr5 stem cells form cryptvillus structures without a mesenchymal niche in vitro. Nature 459, 262-265 (2009).
(6.) Sato, T. et al. Long-term expansion of human colon epithelial organoids, adenomas, adenocarcinomas, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).
(7.) Xin, L., Lukacs, R.U., Lawson, D.A., Cheng, D., and Witte, O.N. Self-renewal and multilineage differentiation in vitro from murine prostate stem cells. Stem Cells 25, 2760-2769 (2007).
(8.) Garraway, IP et al. Human globular prostate cells represent a subset of basal epithelial cells capable of glandular regeneration in vivo. Prostate 70, 491-501 (2010).
(9.) Niranjan, B. et al. Primary culture and propagation of human prostate epithelial cells. Methods Mol. Biol. 945, 365-382 (2013).
(10.) Hofner, T. et al. Defined conditions for the isolation and expansion of mouse basal prostate progenitor cells of human origin. Stem Cell Reports 4, 503-518 (2015).
(11.) Liu, X. et al. ROCK inhibitor and feeder cells induce conditional reprogramming of epithelial cells. Am. J Pathol. 180, 599-607 (2012).
(12.) Chua, C.W. et al. Single luminal epithelial progenitors can generate prostatic organoids in culture. nat. cell biology 16, 951-961 (2014).
(13.) Koo, B.K. et al. Controlled gene expression in primary cultures of Lgr5 organoids. nat. Methods 9, 81-83 (2011).
(14.) Schwank, G., Andersson-Rolf, A., Koo, B.K., Sasaki, N. & Clevers, H. Generation of BAC transgenic epithelial organoids. PLoS ONE 8,e76871 (2013).
(15.) Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9in organoids from intestinal stem cells from patients with cystic fibrosis. Cell Stem Cell 13, 653-658 (2013).
(16.) Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43-47 (2015).
(17.) Lukacs, RU, Goldstein, A.S., Lawson, DA, Cheng, D., and Witte, O.N. Isolation, culture and characterization of adult murine prostate stem cells. nat. Protocol 5, 702-713 (2010).
(18.) Xin, L., Ide, H., Kim, Y., Dubey, P., and Witte, O.N. In vivo regeneration of the murine prostate from dissociated cell populations of the postnatal epithelium and mesenchyme of the urogenital sinus. proc. National Academy Sci. USA 100, 11896-11903 (2003).
(19.) Kim, K.A. et al. Mitogenic influence of human R-Spondin1 on the intestinal epithelium. Science 309, 1256-1259 (2005).
Jarno Drost [1,2,6], Wouter R Karthaus [3,6], Dong Gao [3], ElseDriehuis [1,2], Charles L Sawyers [3], Yu Chen [3-5] and Hans Clevers, 2
[1] Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and University Medical Center (UMC) Utrecht, Utrecht, Netherlands. [2] Cancer Genomics Netherlands, UMC Utrecht, Utrecht, The Netherlands. [3] Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA [4] Department of Medicine, Weill Cornell School of Medicine and New York Presbyterian Hospital, New York, New York, USA [5] Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA [6] These authors contributed equally to this work. Correspondence should be addressed to J.D. be judged. (j.drost@hubrecht.eu) or H.C. (h.clevers@hubrecht.eu).
Published online January 21, 2016; doi: 10.1038/nprot.2016.006
TABLE 1 | Overview of culture medium components for mouse and human prostate organoids. 10% 500 ng/mL or 10% conditioned medium A83-01 conditioned medium 200 nM 500 nMFGF10 - 10 ng/mLFGF2 - 5 ng/mL Prostaglandin E2 - 1 [micro]M Nicotinamide - 10 mMSB202190 - 10 [micro] MDHT 1 nM 1 nMY -27632 dihydrochloride (a) 10 [micro]M 10 [micro]M(a) Y-27632 dihydrochloride is added to the medium only during culture establishment and after passage through organoids with TrypLE.TABLE 2 | Plate format and volumes of culture medium used for organoid culture. -5 10006 wells 40 10-15 2000 TABLE 3 | Troubleshooting Table. Step Problem Possible Cause Solution 2A(xii), Large Tissue Ineffective to increase digestion time 2B(v) Pieces remain in digestion time. After digestion, the duration of the enzymatic digestion varies and depends on the original tissue size. Pipette up and down with a fire-polished glass pipette after digestion3A(i), No, little or insufficient. Change medium each 3B(i) small organoid growth factor 2-3 subject. Allow new activity to appear in the medium to ensure that the culture medium and growth factors in the culture medium are active. outgrowth4B(xiv) No or low yield. The amount of RNA organoids used for isolation. Use more organoid insulation as the input was too low. Organoids were not efficiently frozen. Quick-freeze samples in liquid nitrogen lysate after step 4B(iii) for more efficient lysis 2 . Cell Suspension High Add more DNasel to step 16. The genomic DNA suspension remains viscous in the suspension after the addition of DNasel. Markers (e.g. Epcam). Cells (e.g. check immune cell culture) Conditions used (not on sorted epithelial cells if the population does not grow under the described prostate culture conditions)
FAQs
What is an organoid in cancer research? ›
(OR-guh-noyd) A 3-dimensional, mini-organ-like structure made by growing a person's tumor cells or stem cells (cells from which other types of cells develop) in the laboratory. Organoids contain many types of cells and closely mimic the structure, organization, and some of the functions of human tissues and organs.
Is prostate cancer epithelial tissue? ›This study demonstrates that both primary human basal and luminal epithelial cells are cells of origin for prostate cancer through the use of a prostate organoid culture system.
What epithelial tissue is in prostate? ›The prostate epithelium has two cell layers: the luminal layer, which is composed of columnar epithelial cells, and the basal layer, which is composed of cuboidal epithelial cells. Either epithelial cell type can become transformed and give rise to prostate cancer.
What are the problems with organoid? ›Despite the promising features of organoids, their broad utility is tempered by a variety of limitations yet to be overcome, including lack of high-fidelity cell types, limited maturation, atypical physiology, and lack of arealization, features that may limit their reliability for certain applications.
Why are organoids better than cell lines? ›Compared with immortalized cell lines, organoids are considered superior in recapitulating the 3D architecture, heterogeneity, and cell functions of the primary tissues and hence, are more physiologically relevant for modeling human diseases and predicting drug response.
How much does an organoid cost? ›| Culture Type | Total Price |
|---|---|
| 10ml HVO organoids medium | $59.21 |
| 3D HVO per/well | $55.52 |
| 1 frozen vial of HVO line | $456.45 |
| Generation of HVO line per/sample | $1,160.56 |
Lack of a vascular system
The human body is dynamic; blood constantly flows through the blood vessels to exchange nutrients, oxygen, and waste ensuring cell survival. However, organoids lack a vascular system and are cultured through static methods.
On top of this, organoids retain a fundamental advantage of cell lines: because the organoids can be grown and expanded in culture, then divided to give abundant identical samples, it is still possible to run high throughput screens using them.
What are 2 cancers derived from epithelial tissue? ›There are different types of epithelial cells and these can develop into different types of carcinoma. These include: squamous cell carcinoma. adenocarcinoma.
What is the tissue test for prostate cancer? ›A biopsy is a procedure that can be used to diagnose prostate cancer. A biopsy is when a small piece of tissue is removed from the prostate and looked at under a microscope to see if there are cancer cells. A Gleason score is determined when the biopsy tissue is looked at under the microscope.
What cells become cancerous in prostate cancer? ›
Adenocarcinoma of the prostate
Adenocarcinomas develop in the gland cells that line the prostate gland and the tubes of the prostate gland. Gland cells make prostate fluid. Adenocarcinomas are the most common type of prostate cancer. Nearly everyone with prostate cancer has this type.
The thinness of the epithelial barrier facilitates these processes. Simple epithelial tissues are generally classified by the shape of their cells. The four major classes of simple epithelium are: 1) simple squamous; 2) simple cuboidal; 3) simple columnar; and 4) pseudostratified.
What are the different types of epithelial cells in the prostate? ›These criteria have led to the notion that prostate glands contain three unique epithelial cell types: basal, luminal, and neuroendocrine (NE).
What are cancers of the epithelial tissues? ›Carcinoma is cancer that forms in epithelial tissue. Epithelial tissue lines most of your organs, the internal passageways in your body (like your esophagus), and your skin. Most cancers affecting your skin, breasts, kidney, liver, lungs, pancreas, prostate gland, head and neck are carcinomas.
What is the life span of organoid? ›An important drawback of organoid systems is the limited time span for which they can be maintained in culture. Epithelial organoids have lifespans on the order of one week, which is often insufficient to robustly differentiate ASCs into the full set of differentiated cell types expected in vivo.
What are the pros and cons of using organoids? ›Organoids offer superior morphology if you are studying a glandular tissue, but they are not appropriate for studying stratified tissues, such as skin. Originally organoid cultures were grown to investigate normal cellular differentiation in the prostate3 and breast.
How often should you passage organoids? ›We typically passage BTC organoids once every 7-10 days and split cells at ratios 1:5-1:10.
Can organoids replace organs? ›Although patient-derived organoids exhibit substantial variability, they could eventually become useful for personalized medicine applications [9, 26]. A major prospect for organoids is their potential use as transplantable organs (Fig. 2A).
Is 3D cell culture the same as organoid? ›Organoids are a promising 3D culture system between 2D cultures and in vivo models that allow the manipulation of signaling pathways and genome editing of cells in a body-like environment but lack the many disadvantages of a living system.
How long does it take to grow an organ from stem cells? ›Tengion takes some of your cells and grows them in culture for five to seven weeks around a biodegradable scaffold. When the organ is ready, it can be transplanted without the need to suppress the patient's immune system (because the organ was grown from the patient's own cells, it carries no risk of rejection).
Can humans be studied by organoids? ›
Organoids allow investigators to recapitulate morphogenetic events in human development that lead to tissue and organ formation. Organoids can be used to study mechanisms of disease acting within human tissues, generating knowledge and tools applicable to preclinical studies, including drug testing.
How many types of organoids are there? ›There are two main types of organoids based upon the choice of stem cells. The first is derived from PSCs that include both embryonic stem cells (ESCs) and iPSCs and the second type is derived from organ-specific adult stem cells (ASCs) [22].
What are the ethical concerns of organoids? ›The main concerns on brain organoids revolve around the fact that these miniature organs constitute neural entities of human origin and whether they could obtain human characteristics, cognitive abilities or be sentient.
What are the ethical issues of organoid research? ›Research on and use of cerebral organoids raise a host of ethical issues ranging from traditional research ethics questions, including informed consent, biobanking, and data protection, to issues of clinical translation concerning, for example, the uncertain evidence basis for first-in-human trials or the risk of ...
What are the ethical implications of organoids? ›Ethical considerations of organoid models. Organoids are likely to affect policies for research using animals and human embryos. They also have implications for biobanking and patient consent policies and require particular responsibility in communicating results to the public.
What are organoids in cancer treatment? ›Organoids in cancer research and personalized medicine. Tumor organoid biobanks can be established from genetically modified noncancerous organoids or tumor organoids. These biobanks enable drug efficacy testing, drug screening, and toxicity testing, to facilitate personalized clinical medicine.
Why are organoids better? ›Organoids Recapitulate Cell-Cell Interactions
As such, they offer a unique opportunity to better understand the multicellular signaling networks and behaviors, whether tumor and stroma or host and pathogen, as they interact in the human body during disease71-74.
Bottomline, organoids lack the full complement of cells and factors found in a patient's tumor. Tumoroids, in comparison, are created directly from fresh patient tumor tissue and retain the full architecture of the tumor microenvironment (TME) and extracellular matrix (ECM).
What is the most common malignant tumor of the epithelial tissue? ›Carcinoma. Carcinoma refers to a malignant neoplasm of epithelial origin or cancer of the internal or external lining of the body. Carcinomas, malignancies of epithelial tissue, account for 80 to 90 percent of all cancer cases.
Why do so many cancers start in epithelial tissue? ›Epithelial cells turnover rapidly and mutations naturally accumulate throughout life. Most epithelial cancers arise from this normal mutation rate.
What is the most common epithelial cell tumor? ›
Serous epithelial ovarian cancer is the most common type. Your doctor examines the cancer under the microscope. They grade your cancer depending on how much the tumour cells look like normal tissue.
What is the best test to rule out prostate cancer? ›If your doctor determines you should undergo screening, he or she will most likely recommend the PSA test. For more than 30 years, the PSA test has been the gold standard in prostate cancer screening. This simple blood test measures how much prostate-specific antigen is in your blood.
What is the most aggressive form of prostate cancer? ›Small cell carcinoma, the most aggressive type of neuroendocrine cancer in the prostate that develops in small round cells of the neuroendocrine system. Squamous cell carcinoma, a very rare, fast-growing form of prostate cancer that starts in the flat cells that cover the prostate glands.
What is the best biopsy for prostate cancer? ›Transrectal ultrasound (TRUS)-guided systematic biopsy of the prostate. TRUS-guided systematic biopsy of the prostate is considered to be the gold standard for the diagnosis of prostate cancer. The procedure may be done after sedation in most cases.
At what age is prostate cancer most aggressive? ›If you're black or you have a close relative with prostate cancer, they jump up at 40. About two-thirds of all prostate cancers are diagnosed in men age 65 and older. But the older you are, the less aggressive the disease is, especially after 70.
What makes prostate cancer aggressive? ›Other risk factors for prostate cancer diagnosis and worse outcomes are social and environmental factors—particularly a diet that is low in vegetables and high in processed meat and saturated fat—and lifestyle. Men who are overweight or obese are at greater risk of developing an aggressive form of prostate cancer.
What is the least aggressive form of prostate cancer? ›The scale, used to determine tumor aggressiveness, gives out ratings from 6 to 10 depending on severity. A Gleason 6 tumor is the least aggressive form of prostate cancer. In fact, this cancer is so slow growing and nonaggressive that many doctors think it shouldn't be called cancer at all.
What does it mean when the epithelial cells are positive? ›This means that the sample contains cells from another part of the body. This can happen if you do not clean your genital area well enough when collecting your urine sample with the clean catch method.
How do you identify epithelial tissue? ›There are three basic shapes used to classify epithelial cells. A squamous epithelial cell looks flat under a microscope. A cuboidal epithelial cell looks close to a square. A columnar epithelial cell looks like a column or a tall rectangle.
What are the names of the 3 types of epithelial tissue? ›Epithelial cells may be squamous, cuboidal, or columnar in shape and may be arranged in single or multiple layers. Simple cuboidal epithelium is found in glandular tissue and in the kidney tubules.
What are the epithelial cells or stromal cell of prostate? ›
The prostate is a tubulo-alveolar gland composed of epithelial tissues embedded in stromal components. The main cell types in the prostate stroma include fibroblasts, myofibroblasts and smooth muscle cells.
Why do 90% of all cancers occur in epithelial tissues? ›Carcinoma
Carcinomas, malignancies of epithelial tissue, account for 80 to 90 percent of all cancer cases since epithelial tissues are most abundantly found in the body from being present in the skin to the covering and lining of organs and internal passageways, such as the gastrointestinal tract.
Epithelial ovarian cancer — the most common ovarian cancer — develops in tissue surrounding your ovaries. It includes fallopian tube cancer and primary peritoneal cancer. High-grade serous ovarian cancer (HGSOC) occurs most often and tends to spread before detection.
What is carcinoma of epithelial tissue called? ›Cancer arising from the epithelial tissues of internal organs and glands is referred as melanoma.
How gut organoids are cultured? ›Tissue is dissociated into small epithelial fragments that are embedded into extracellular matrix “domes” using basement membrane extract (BME) and then supplemented with medium containing growth factors for organoid expansion. Small intestine and colon biopsies are both suitable organoid culture sources.
What is organoid culture? ›Organoids are tiny, self-organized three-dimensional tissue cultures that are derived from stem cells. Such cultures can be crafted to replicate much of the complexity of an organ, or to express selected aspects of it like producing only certain types of cells.
What is media for organoid culture? ›Some of the most important components of organoid media are growth factors such as R-Spondins, Noggin, and Wnt-3a, which need to display high levels of activity, batch-to-batch consistency, and be free of contaminants to ensure that they provide optimal, consistent organoid growth.
What is culture medium for organoids? ›IntestiCult™ organoid culture media are complete growth media that support establishment, expansion, long-term maintenance, and further differentiation of intestinal organoid cultures from human or mouse intestinal crypts.
Are organoids 2D or 3D? ›Organoids are in-vitro derived 3D cell aggregates derived from primary tissue or stem cells that are capable of self-renewal, self-organization and exhibit organ functionality.
What are the different types of organoids? ›There are two main types of organoids based upon the choice of stem cells. The first is derived from PSCs that include both embryonic stem cells (ESCs) and iPSCs and the second type is derived from organ-specific adult stem cells (ASCs) [22].
What is the difference between organoids and cell cultures? ›
Organoids developed using HUB protocols are of epithelial origin. Primary cell cultures on the other hand consist largely of differentiated cells, and the culture conditions can vary between cultures making it more challenging to establish a robust culture from some cancer types.
Which media is best for plant tissue culture? ›Growing plant tissue in the laboratory may include seed, meristem, callus, and bud culture, and requires specialized plant culture media. Murashige and Skoog (also called MS media, MSO, or MS0) and Gamborg's B5 medium are two of the most essential media formulations used for culturing plants.
How many cells are in an organoid? ›Organoids can arise either from round colonies generated by single cells7,34 or from initial multicellular structures such as intestinal crypts29,62, cell aggregates23 or micropatterned cells25.
What are the three types of culture medium used in tissue culture technique? ›The important culture media used for all purpose experiment and Murashige and Skoog medium (MS medium), Gamborg medium (B5 medium), White medium (W medium) and Nitsch medium.
What is the importance of organoid culture? ›Human organoids have been used to study infectious diseases, genetic disorders and cancers through the genetic engineering of human stem cells, as well as directly when organoids are generated from patient biopsy samples.
What is three dimensional organoid cultures? ›Organoids are three-dimensional (3D) multi-cellular, microtissues derived from stem cells that are designed to closely mimic the complex structure and functionality of human organs like the lung, liver or brain.