Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.
Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.
The ability to harvest and culture primary organoids have been described for small intestine, colon, pancreas, liver and brain and are exciting advances germane to understanding a more physiologically representative phenomena for tissue biology1-5. The first methods describing the culture and maintenance of small intestinal organoids was reported by Sato et al. out of the lab of Hans Clevers1. Prior to this method, harvesting and culture of primary intestinal epithelial cells proved to be limited and ineffective in sustaining epithelial cell growth. Methods included dissociation of tissue via incubation with enzymes, such as collagenase and dispase, which would ultimately lead to the outgrowth of intermixed primary fibroblast cells6. These conditions would also be time restricted in sustaining the epithelial cell culture. Minimal to no epithelial cell niche would form, as the epithelial cells would enter apoptosis due to the lack of appropriate growth factors or loss of contact integrity, termed anokis7. The advent of the 3D-organoid culture system has provided a method to culture primary intestinal cells containing a spectrum of intestinal cell types in sustained culture1. These epithelial organoids have advantages over cell lines being that they are composed of several differentiated cells, and better mimic the organ they are derived from in vivo8. The process to ultimately "grow a mini gut in a dish" has proven to be a valuable tool for assessing the response of intestinal epithelium under different stimuli. Investigating the interaction of primary intestinal cells with microbial pathogen associated molecular patterns (PAMPs) is relevant to the field of immunology as these molecular patterns can regulate diverse responses from both host and microbe9. Not only can investigators now explore these interactions with mouse organoids, but they can be cultured from humans as well2. This technology has the potential to dramatically alter personalized medicine and it is tempting to speculate about advances that this technique will make possible in the near future.
The overall goal of this method is to provide a protocol for the culture, expansion, and treatment of intestinal organoids with a variety of stimuli. Such stimuli can ultimately range from vaccines, bacterial PAMPs, live pathogens, gastrointestinal (GI) and cancer therapeutics. The isolation and culture of mouse intestinal organoids has been adapted from Sato et al. Though there are slight deviations from the original method, the end product being organoid culture is still achieved when following this protocol. This method is focused on describing an adequate technique for proper normalization when working with non-homogenous cell structures, which must be taken into consideration when conducting an assay based on cell number.
All research was approved and conducted under Virginia Tech IACUC guidelines
1. Prepare R-Spondin1 Conditioned Media From HEK293T-Rspo1 Cell Line
2. Preparation of Organoid Growth Media and Reagents for Harvesting Small Intestinal Crypts
3. Harvesting Mus musculus Small Intestinal Crypts for Organoid Culture1
4. Passaging Organoids Every 7th Day
5. Plating Organoids on Day 14 for Pattern Recognition Receptor Stimulation with PAMPs and Listeria monocytogenes for Gene Expression Analysis
6. Plating Organoids on Day 14 for PAMP and L. monocytogenes Challenge for Protein Analysis in Supernatant
When following this protocol to cultivate intestinal organoids, characteristic sphere shaped organoids will be present after harvesting. The addition of R-spondin1 conditioned media daily will initiate the growth and budding of the organoids. The growth of organoids is shown in Figure 1A–F, and is representative of intestinal organoids on days 1, 2, 4, 5, 6 and day 14. Figure 1F represents the non-homogeneous growth characteristics of organoids on day 14.
Once the organoids are grown to an adequate number, they can be replated and challenged with various PAMPs and/or microbes. Expression analysis can be performed via standard techniques. This is represented in Figure 2A-C which shows the mRNA expression of inflammatory cytokines IL-18, IL-6, and TNFα which were analyzed following a 24 hr challenge of organoids with heat killed L. monocytogenes and the PAMP flagellin. The rationale for evaluating the mRNA expression cytokines IL-18, IL-6, and TNFα was that these were good candidates for being modulated by epithelial cells in response to pathogenic challenge, and modulation of these inflammatory cytokines would demonstrate the effectiveness of the technique.
Figure 3A–C demonstrates the relative nuclear staining of intestinal organoids with nuclear staining dye following fixation with minimal background staining of debris in the resident protein matrix. This method of fixation and staining can be applied to normalize assays, which will account for differing cell numbers in each well. This is apparent in Figure 4 when generating a standard curve of serial dilutions of Caco-2 cells plated in protein matrix, then fixed and stained with nuclear staining dye. The R2 value of 0.89 indicates a linear relationship between cell number and mean fluorescent intensity, and the linear equation can be used to normalize organoids to cell number. The standard curve depicted in Figure 4 begins to reach the saturation limit beyond 30,000 cells per well. A titration of cells above 30,000 cells per well has been shown in Figure 4 to include the saturation range of nuclear staining dye. Increased accuracy of normalization will be obtained with a cell number that reflects the linear range of the nuclear staining dye before the saturation limit is reached.
Figure 1: Small Intestinal Organoid Growth. Time course of growth for murine small intestine derived organoids following isolation. (A) Day 1. (B) Day 2. (C) Day 4. (D) Day 5. (E) Day 6. (F) Day 14. Images are representative of organoid growth for each given day. Scale bars equal 200 μm in the left image for A, B, C, D, and E. Scale bars equal 100 μm in the right image for A, B, C, D, and E. Scale bars equal 1,000 μm and 200 μm for left and right images for F, respectively. Please click here to view a larger version of this figure.
Figure 2: mRNA Expression of Inflammatory Cytokines Following 24 hr PAMP Challenge. Relative mRNA expression of wild-type intestinal organoids challenged for 24 hr with PAMPs and heat killed Listeria monocytogenes. (A–C) represent fold change of the inflammatory cytokines IL-18, IL-6 and TNFα respectively. Error bars represent Standard Deviation (S.D.) Please click here to view a larger version of this figure.
Figure 3: Relative Fluorescent Staining of Organoids for Normalization. Nuclear staining of organoids with following fixation. (A) Bright field of organoids. (B) Fluorescent staining of organoids with nuclear staining dye. (C) Merged image of bright field and fluorescent staining. Scale bars equal 400 μm in all images. Please click here to view a larger version of this figure.
Figure 4: Standard Curve generated from Caco-2 Cells. Standard curve generated from triplicate wells. Cell seeding had a range starting at 60,000 cells per well with dilutions down to 2,500 cells per well. Please click here to view a larger version of this figure.
Figure 5: Overview of the Protocol Diagram. 1-Top Panel of the Figure illustrates harvesting the R-spondin1 conditioned media. 2-Middle Panel of the Figure illustrates aspects of protocol to harvest and culture mouse small intestinal organoids. 3-Bottom Panel of the Figure illustrates: (A) The plating of 14 day cultured organoids. (B) Challenge with PAMPs/ L. monocytogenes, and leaving unoccupied wells to generate a standard curve. (C) Following the PAMP challenge, the plating of Caco-2 cells in the previously unoccupied wells to generate a standard curve. (D) Following the fixation and addition of a nuclear staining dye, measuring the excitation/ emission of the wells and normalizing against a standard curve. Please click here to view a larger version of this figure.
The culture and maintenance of intestinal organoids is a procedure that can be mastered by any individual with adequate tissue culture technique. There are subtleties in passaging when compared to growing cells in a more conventional monolayer, but these subtleties are not difficult to overcome. The critical steps of this method involve being able to grow the organoids to a high enough density for optimal seeding. Experiments must be scaled down with organoids as large seeding densities that can commonly be achieved with cell lines are not practical. This becomes especially apparent when there are multiple treatment groups.
This protocol is intended to provide a step-by-step method to study host-pathogen interactions of the intestinal epithelium with a variety of different bacterial, viral, and fungal pathogens, as well as address difficulties using this system with respect to normalization. Reports are available that describe the interactions of intestinal organoids with the bacterial pathogen Salmonella, yet do not address normalization methods when measuring secreted cytokines13.
There are several difficulties that are encountered when normalizing organoid cultures by different methods. Normalizing secreted protein via bicinchoninic acid assay (BCA) is an option; however, the growth components required for organoid culture (N2 and/or Vitamin B27) interfere with the BCA assay (data not shown). Normalizing via cellular viability, such as a modified MTT assay has been described14; however, a treatment that will alter the mitochondrial metabolic activity of the organoids will introduce an inaccurate method for normalization via this technique as MTT is based on reduction by the action of mitochondrial dehydrogenases15. It is also necessary to remove N-acetyl-cysteine (NAC) from the media, not only if normalization via the MTT technique is desired, but if treating with a bacterial pathogen as NAC can inhibit bacterial growth16.
Advantages of this technique are that secreted cytokines and protein products from organoids can now be normalized against a standard curve of Caco-2 cells. Limitations of this technique are that the normalization is correlative because nuclear staining of non-transformed primary epithelial organoids are normalized against the nuclear staining of a colon cancer cell line. The authors find that fixing with methanol and staining nuclei with nuclear staining dye is an effective normalization technique against a standard curve of Caco-2 cells. Although the growth characteristics of this colon cancer cell line do not exactly mimic the growth characteristics of intestinal organoids, using a nuclear stain and measuring mean fluorescence intensity is a good strategy to normalize against total cell number.
Taken together, the technique described here provides a good starting point to mimic host-pathogen interactions with adequate normalization that is essential for making accurate interpretations using this model system. The significance of this technique with respect to alternative methods is that proper normalization must be performed when conducting any evaluation of secreted protein, such as ELISA based assays.
The authors have nothing to disclose.
The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Fetal Bovine Serum (FBS) | Atlanta Biologicals | S11050 | (Section 1,3,6) Or equivalent brand |
Sorvall Legend XTR Centrifuge | Thermo | (Section 1,3) | |
DMEM | GE Healthcare | Sh30243.01 | (Section 1,6) For Caco-2 and HEK293 Rspondin1 cells |
HEK293T-Rspondin1 secreting cell line | (Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia. | ||
50 ml conical tube | Falcon | 352070 | (Section 1) Or equivalent brand |
T-175 Flask | Corning | 431079 | (Section 1) Or equivalent brand |
Protein Matrix | Corning | 356231 | (Section 2,3,4,5,6) Matrigel Growth Factor Reduced |
HyClone Dulbecco's (DPBS) | GE Healthcare | SH30264.01 | (Section 2,3) |
DMEM/F12 | Life Technologies | 12634-010 | (Section 2,3) Advanced DMEM/F12 |
Corning 24 Well TC Plates | Corning | 3524 | (Section 2) |
N2 Supplement 100x | Life Technologies | 17502-048 | (Section 2) |
B27 without vitamin A 50x | Life Technologies | 12587-010 | (Section 2) |
Trizol | Life Technologies | 15596-026 | (Section 2) |
Glutamine Supplement (Glutamax) | Life Technologies | 35050-061 | (Section 2) Can Combine with Advanced DMEM/ F12 |
HEPES (1 M) | Life Technologies | 15630-080 | (Section 2) Can Combine with Advanced DMEM/ F12 |
10ml Serological Pipet | Falcon | 357551 | (Section 2) Or equivalent brand |
Murine Noggin | Peprotech | 250-38 | (Section 2) Stock = 100 mg/ml |
N-Acetyl-L-cysteine | Sigma-Aldrich | A9165 | (Section 2) Stock = 1M |
Recombinant Mouse EGF | Biolegend | 585608 | (Section 2) Stock = 500 mg/ml |
Rocker Variable | Bioexpres | (Section 3) | |
dissecting scissors | (Section 3) | ||
forceps | (Section 3) | ||
glass slides | (Section 3) | ||
dissecting tweezers | (Section 3) | ||
25 ml Serological Pipet | Falcon | (Section 3) | |
EDTA | Sigma-Aldrich | SLBB9821 | (Section 3) 0.5M or alternative TC grade EDTA |
Sterile Petri Dish 100mm x 15mm | Fisher | FB0875712 | (Section 3) Or equal sized TC dish |
1ml Syringe | Becton Dickinson | 309659 | (Section 4) |
Precision Glide Needle | Becton Dickinson | 305120 | (Section 4) 23G x 1 1/4 (0.6mm x 30mm) |
Flagellin from Bacillus subtilis | Invivogen | tlrl-bsfla | (Section 5,6) |
Listeria monocytogenes | ATCC | 19115 | (Section 5,6) (Murray et al.) |
Hemocytometer | Sigma-Aldrich | Z359629-1EA | (Section 5,6)Or equivalent brand |
BBL Brain Heart Infusion Agar | Becton Dickinson | 211065 | (Section 5) |
Bacto Brain Heart Infusion | Becton Dickinson | 237500 | (Section 5) |
Caco-2 | ATCC | HTB-37 | (Section 6) |
Trypsin | gibco | 25200056 | (section 6) |
Methanol | Fisher | A412-4 | (Section 6) |
SpectraMax M5 | Molecuar Devices | (Section 6) | |
96 Well Assay Plate | Corning | 3603 | (Section 6) Black Plate, Clear Bottom TC treated |
Nuclear Staining Dye | Life Technologies | H1399 | (section 6) Hoechst 33342 |
T-75 Flask | Corning | 430641 | (Section 6) Or equivalent brand |
15 ml conical tube | Falcon | 352096 | (Section1,3) Or equivalent brand |
1.7 ml polypropylene tube | Bioexpress | C-3262-1 | Or equivalent brand |
Quick-RNA MiniPrep | Zymo Research | R1054 | Or equivalent brand |
TNF-alpha | Applied Biosystems | Mm 00443260_g1 | Taqman gene expression assay kit |
IL-6 | Applied Biosystems | (Mm 00446190_m1 | Taqman gene expression assay kit |
IL-1beta | Applied Biosystems | Mm 00434228_m1 | Taqman gene expression assay kit |
IL-18 | Applied Biosystems | Mm 00434225_m1 | Taqman gene expression assay kit |
18s | Applied Biosystems | Hs 99999901_s1 | Taqman gene expression assay kit |
7500 Fast Real Time PCR System | Applied Biosystems | ||
Nexus gradient Mastercycler | Eppendorf | ||
TaqMan Fast Universal PCR Master Mix | Life Technologies | 4352042 | |
High Capacity cDNA Reverse Transcription Kit | Life Technologies/Applied Biosystems | 4368814 | |
Fast Optical 96-Well Reaction Plate, 0.1 mL | Life Technologies/Applied Biosystems | 4346907 | |
Recombinant Mouse R-Spondin 1 Protein | R&D Systems | 3474-RS-050 | 500 ng/ml |
chloroform | Sigma-Aldrich | C7559 |