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Abstract

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Acknowledgements

Materials

References

Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Published: May 18th, 2016

DOI:

10.3791/54033

1Department of Biomedical Sciences and Pathobiology, Virginia Maryland College of Veterinary Medicine, Virginia Tech, 2Department of Biomedical Engineering, Cornell University, 3School of Electrical and Computer Engineering, Cornell University, 4Department of Biomedical Engineering, Duke University

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 ....

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All research was approved and conducted under Virginia Tech IACUC guidelines

1. Prepare R-Spondin1 Conditioned Media From HEK293T-Rspo1 Cell Line

  1. Generation of HEK293T-Rspondin1 cells has been previously described10. Seed HEK293T-Rspondin1 secreting cells at 5-10% confluency, approximately 8 x 105-1.7 x 106 cells, in a T-175 flask with 40 ml of 1x Dulbecco's Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) as the growth media, and incubate at 37 °C +.......

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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 chara.......

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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.......

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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.

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Name Company Catalog Number Comments
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

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