J. Heijmans is supported by a stipendium from the Dutch cancer foundation (KFW). G.R. van den Brink is supported by funding from the European Research Council under the European Community&#8217;s Seventh Framework Program (FP7/2007-2013)/European Research Council grant agreement number 241344 and by a VIDI grant from the Netherlands Organization for Scientific Research (GvdB).

1. Preparation of Polyethylenimine (PEI) as Transfection Reagent
<ol>
	<li>Dissolve approximately 150 mg of PEI into 100 ml of H2O.</li>
	<li>Adjust solution to pH 7.4 by adding HCl until solution becomes clear and stir until completely dissolved. This may take between 10 and 60 min and add water to an end concentration of 1 mg/ml.</li>
	<li>When clear, filter the PEI solution through sterile 0.22 &#181;m filter and store in a -80 &#176;C freezer in aliquots of 5 ml.</li>
</ol>
2. Production of Lentiviral Particles
Day 1:
<ol>
	<li>Split HEK293T cells to 60%-80% confluency in 162 cm2 flask or large petri dish in cell line culture medium (DMEM supplemented with 10% FCS, 1% penicillin/streptomycin and 2 mM glutamine supplement).</li>
</ol>
Day 2:
<ol start="2">
	<li>Prepare DNA transfection solution containing 45 &#181;g of total plasmid DNA by adding together lentiviral packaging vectors (7 &#181;g of pVSVg; 5 &#181;g of pRSV rev; 13 &#181;g of pMDL) and 20 &#181;g of lentiviral plasmid encoding the gene of interest or shRNA of interest. Adjust to a volume of 1 ml using DMEM.</li>
	<li>Prepare PEI transfection solution by adding 90 &#181;l of 1 mg/ml PEI to 930 &#181;l of DMEM and incubate for 5 min at room temperature.</li>
	<li>Add DNA transfection solution to PEI solution. Vortex or invert a number of times and incubate for 5 min at room temperature to obtain DNA transfection solution.</li>
	<li>Drip 2 ml of the DNA transfection solution onto the HEK293T cells and incubate for 4 hr in a humidified cell culture incubator on 37 &#176;C.</li>
	<li>After 4 hr, refresh the culture medium to remove PEI. It is not necessary to wash cells before adding new medium. 
	NOTE: PEI is cytotoxic and incubation times longer than 4 hr may cause harm to the HEK293T cells.</li>
</ol>
Day 4:
<ol start="7">
	<li>Replace supernatant with new culture medium. Keep supernatant (containing virus); this will be used in step 2.10.</li>
	<li>Put supernatant in 15 ml flask. To remove dead cells, centrifuge for 5 min at 500 x g.</li>
	<li>Push supernatant through 0.45 &#181;m filter using a large 60 ml syringe. Store overnight at 4 &#176;C.</li>
</ol>
Day 5:
<ol start="10">
	<li>Collect the second batch of supernatant; centrifuge and filter as in steps 2.8-2.9.</li>
	<li>Pool the supernatants from steps 2.9 and 2.10 in ultracentrifuge tubes and centrifuge at 50,000 x g in an ultracentrifuge for 90 min.</li>
	<li>Take out capsules containing the ultracentrifuge tubes very carefully and put into a laminar flow hood, remembering the orientation of the tube inside the centrifuge.</li>
	<li>Open capsule holding ultracentrifuge tube and decant medium carefully in such a fashion that the pellet is on the upper side of the tube. Since viral pellets may be difficult to visualize, remember on what side of the tube a pellet will have formed. Take a micropipette and remove last bit of medium while taking care not to agitate the opaque brown pellet that is visible on the side of the bottom of the ultracentrifuge tube.</li>
	<li>Resuspend this pellet in 500 &#181;l of organoid culture medium supplemented with 10 mM nicotinamide, 10 &#181;M Chir99021, 10 &#181;M Y27632 and 8 &#181;g/ml polybrene. Resuspension in this medium is important since high titer virus is used to transduct organoids directly.</li>
	<li>Optionally: freeze the virus in this medium aliquoted in two batches of 250 &#181;l in -80 &#176;C.</li>
</ol>
3. Lentiviral Transduction of Organoids
Day 0:
<ol>
	<li>Split a full 0.95 cm2 well of organoids two days prior to transduction into a new well, aiming to obtain approximately 50 small organoids. Split organoids according to previously published protocol1 (Figure 3A, B).</li>
	<li>Supplement organoid culture medium with 10 &#181;M Chir99021 and 10 mM nicotinamide to obtain cystic hyper proliferative crypts (Figure 3C). 
	NOTE: Cystic crypts will develop best when freshly split organoids are grown in the presence of Chir99021.</li>
</ol>
Day 2:
<ol start="3">
	<li>Harvest organoids by pipetting up and down the matrigel and medium, thereby disrupting the mixture with a p1000 micropipette. Place the mixture in a 15 ml tube.</li>
	<li>Disrupt further using Pasteur pipet in which the distal opening has been decreased by melting. Centrifuge organoids to pellet for 5 min at 100 x g. 
	NOTE: Dependent on the size of the centrifuge, use a different centrifuge speed. It is necessary to find the exact speed in which disrupted organoids are separated from the mixture.</li>
	<li>Remove the supernatant and add 500 &#181;l of pre-warmed 1x trypsin (similar to 0.25% trypsin). Resuspend organoids in trypsin and incubate 3 min in a 37 &#176;C water bath.</li>
	<li>Inactivate trypsin by adding 3.5 ml of cell line culture medium (DMEM supplemented with 10% FCS, 1% penicillin/streptomycin and glutamine). Centrifuge for 5 min at 500 x g.</li>
	<li>Remove the supernatant to leave pellet in approximately 20 &#181;l of medium. Transfer the organoids in this last small amount of medium into a well on a 48-wells plate.</li>
	<li>Add high titer lentivirus in transduction medium as described in step 2.14, resuspend and continue to step 3.10. When encountering low transduction efficacy, step 3.9 may be added. 
	NOTE: For efficient transduction, typically add one aliquot of 250 &#181;l of high titer lentivirus to one well of organoids. This represents 50% of all harvested lentivirus from a single production as described in step 2 of this protocol.</li>
	<li>Optionally: to enhance transduction efficacy, perform spinoculation by putting the 48 well plate containing organoids in a pre-warmed centrifuge on 32 &#176;C and rotate at 600 x g for 1 hr.</li>
	<li>Put organoid-virus mixture in culture incubator and incubate for 1 hr at 37 &#176;C in a culture incubator to allow transduction.</li>
	<li>Optionally: to further enhance transduction efficacy, incubate organoids for additional 3 hr at 37 &#176;C in a culture incubator.</li>
	<li>Add 1 ml of organoid culture medium and resuspend the organoid-virus mixture, transfer into a microcentrifuge tube and centrifuge in a microcentrifuge for 5 min at 850 x g to pellet organoids.</li>
	<li>Remove the supernatant and resuspend the pellet in 20 &#181;l of ice-cold matrigel. Since the material solidifies when it becomes warmer, use pipette tips that are chilled by pipetting up and down ice cold PBS for a number of times.</li>
	<li>Put the droplet in the middle of a well in a 48 wells plate and incubate in culture incubator at 37 &#176;C for 15 min to solidify.</li>
	<li>After 15 min, carefully add 250 &#181;l of organoid culture medium supplemented with 10 mM nicotinamide, 10 &#181;M Chir99021 and 10 &#181;M Y27632. Small disrupted organoid fragments will form into small cystic organoids within 24 hr.</li>
</ol>
Day 5:
<ol start="16">
	<li>Refresh medium and supplement with a selection antibiotic (for puromycin, use 4 &#181;g/ml).</li>
</ol>
Day 7:
<ol start="17">
	<li>Replace medium for standard organoid culture medium, supplemented with selection antibiotic. 
	NOTE: Budding will be complete 2-3 weeks after Chir99021 withdrawal. After selection, organoids may be grown without selection antibiotic.</li>
</ol>
4. Organoid RNA Preparation for Quantitative RT-PCR or Microarray
<ol>
	<li>For RNA preparation, use a commercial kit according to manufacturer&#8217;s protocol. Remove the medium from organoids and add 350 &#181;l of buffer RLT, supplemented with &#946;-mercaptoethanol straight on the dome of matrigel containing organoids. Resuspend the material in RLT by pipetting using p1000 micropipette.</li>
	<li>Perform further RNA prep according to manufacturer&#8217;s protocol.</li>
	<li>Optionally: increase RNA yield by amplification using Ovation Pico WTA system, requiring initial input of 50 &#181;g total RNA according to manufacturer&#8217;s protocol.</li>
	<li>Optionally: for quality control prior to RNA microarray, run samples on a 2100 bioanalyzer using an eukaryote total RNA nano chip, aiming for an RNA integrity number (RIN) of 8.5 or more (Figure 5) according to manufacturer&#8217;s protocol.</li>
</ol>
5. Processing Organoids for Paraffin Embedding and Immunohistochemistry
<ol>
	<li>Prior to initiation of organoid embedding pre-warm an aluminum block with holes fitting 12 mm diameter glass tubes in incubator at 70 &#176;C to keep paraffin liquid.</li>
	<li>Take a single well with full-grown organoids and remove the medium, leaving the embedded organoids intact.</li>
	<li>Add 1 ml of 4% paraformaldehyde in PBS straight to well and fix at 4 &#176;C anywhere from 1 hr to overnight.</li>
	<li>Replace paraformaldehyde with 1 ml of ice cold PBS. 
	Optionally: keep fixed organoids in PBS up to 1 week at 4 &#176;C after this step.</li>
	<li>Resuspend fixed organoids in 1 ml of PBS and place into glass vial.</li>
	<li>Let organoids sink to bottom for 1 min, decant PBS and replace with 70% ethanol in which a couple of droplets of eosin solution are dissolved to enable visualization of organoids throughout the embedding process.</li>
	<li>Leave organoids in 70% ethanol at room temperature. After 30 min, remove 70% ethanol by carefully decanting, being able to visualize the organoids by eye because of slight pinkish eosin color. Replace embedding solution with 96% ethanol.</li>
	<li>Repeat step 5.7, each time replacing the embedding solution with the next one. Pass organoids through the following solutions subsequently: 70% ethanol, 90% ethanol, 96% ethanol, 100% ethanol, 100% ethanol, xylene, xylene.</li>
	<li>Decant the last xylene wash and pour paraffin into the tube. Put the tube immediately in the pre warmed aluminum block at 70 &#176;C for 30 min and replace paraffin with new clean paraffin.</li>
	<li>Pour paraffin off and pipet organoids into the paraffin block mold using a pre-warmed Pasteur pipet with large opening. Keep Pasteur pipet warm using a Bunsen burner. Place all organoids in the paraffin block mold in a small layer of liquid paraffin.</li>
	<li>As much as possible, try to manipulate all organoids towards the center of the paraffin block mold using a warmed dissection needle. Keep dissection needle warm using Bunsen burner.</li>
	<li>When localization of organoids in the mold is satisfactory, chill mold slightly to solidify the paraffin layer.</li>
	<li>Finish the block by pouring more paraffin on top and add a standard histological embedding cassette.</li>
</ol>

<ol><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li>
<li>Al-Nafussi, A. I., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+epidermal+growth+factor+%28EGF%29+on+cell+proliferation+of+the+gastrointestinal+mucosa+in+rodents%5BTitle%5D)+AND+%22Virchows+Archiv.+B%2C+Cell+Pathology+Including+Molecular+Pathology%22%5BJournal%5D)">The effect of epidermal growth factor (EGF) on cell proliferation of the gastrointestinal mucosa in rodents.</a> Virchows Archiv. B, Cell Pathology Including Molecular Pathology. 40 (1), 63-69 (1982).</li>
<li>Haramis, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((De+novo+crypt+formation+and+juvenile+polyposis+on+BMP+inhibition+in+mouse+intestine%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine.</a> Science. 303 (5664), 1684-1686 (2004).</li>
<li>Zhao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((R-spondin1%2C+a+novel+intestinotrophic+mitogen%2C+ameliorates+experimental+colitis+in+mice%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice.</a> Gastroenterology. 132 (4), 1331-1343 (2007).</li>
<li>Schwank, G., Andersson-Rolf, A., Koo, B. K., Sasaki, N., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+BAC+transgenic+epithelial+organoids%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Generation of BAC transgenic epithelial organoids.</a> PLoS One. 8 (10), e76871(2013).</li>
<li>Koo, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlled+gene+expression+in+primary+Lgr5+organoid+cultures%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9 (1), 81-83 (2012).</li>
<li>Heijmans, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ER+stress+causes+rapid+loss+of+intestinal+epithelial+stemness+through+activation+of+the+unfolded+protein+response%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">ER stress causes rapid loss of intestinal epithelial stemness through activation of the unfolded protein response.</a> Cell Rep. 3 (4), 1128-1139 (2013).</li>
<li>Roe, T., Reynolds, T. C., Yu, G., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integration+of+murine+leukemia+virus+DNA+depends+on+mitosis%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Integration of murine leukemia virus DNA depends on mitosis.</a> The EMBO Journal. 12 (5), 2099-2108 (1993).</li>
<li>Lau, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lgr5+homologues+associate+with+Wnt+receptors+and+mediate+R-spondin+signalling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.</a> Nature. 476 (7360), 293-297 (2011).</li>
<li>Bahnson, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Centrifugal+enhancement+of+retroviral+mediated+gene+transfer%5BTitle%5D)+AND+%22Journal+Of+Virological+Methods%22%5BJournal%5D)">Centrifugal enhancement of retroviral mediated gene transfer.</a> Journal Of Virological Methods. 54 (2-3), 131-143 (1995).</li>
</ol>

The authors have nothing to disclose.

The current video protocol describes lentiviral transduction of organoids from primary intestinal epithelium and downstream analysis of these organoids using quantitative RNA techniques and immunohistochemistry.
Lentiviral transduction is often performed in adherent or floating cells in culture plates. Since the three-dimensional structure of organoids renders them difficult to penetrate by viral particles, a number of methods to increase efficacy are used. Pretreatment of organoids using Chir...

The intestinal epithelium is one of the most rapidly proliferating bodily tissues, which has caused it to attract wide interest from research on cancer and stem cells. In 2009 a technique was published to generate long lasting cultures of small intestinal crypts in matrigel, conserving a 3 dimensional structure1. These structures, termed intestinal organoids, can be cultured using standard techniques, with surrounding medium supplemented with a number of defined growth factors, including the Bmp-signaling pathway inhibitor noggin (Nog), the Wnt-signaling pathway enhancer rspondin 1 (Rspo1) and epidermal growth factor (Egf) all found to enhance intestinal proliferation2-4.
Organoids surpass traditional cancer cell lines in the aspects that they are non-mutated, have maintained stem cell hierarchy, display intact cellular polarization and exhibit differentiation into all cell lineages found in the nascent small intestinal epithelium. Since they can be transduced to carry transgenes or RNA interference constructs5, they are used to study specific genetic elements, outweighing experiments using transgenic mice in facets of cost and speed. Transgenic expression in organoids can be performed using either murine retroviral or lentiviral vectors6,7. Due to the limitations of murine retroviruses, capable of transducing mitotic cells exclusively8, lentiviral transduction is more frequently used for cells that are difficult to infect, such as organoids.
Virally transduced and stably expressing transgenic organoids can be used for a multitude of downstream analyses, including quantitative RNA analyses and immunohistochemistry. Taken together, culture of organoids from primary intestinal epithelial cells has evolved into a routine technique that is easy to implement without specific laboratory requirements, and has become the novel standard in cell culture in research on the intestinal epithelium.
Techniques of viral transduction and subsequent downstream analysis in organoids are tedious to perform and to aid organoid experiments we generated this video protocol, showing methods for lentiviral transduction of cultured organoids. We additionally show how correct processing of organoids can increase yield and therefore enhance performance of downstream analysis using RNA techniques or immunohistochemistry. In the protocol, organoids that are derived from small intestinal crypts were exclusively used, although the techniques described may be applied to colonic organoids as well.

<table><tbody><tr><td>Polyethylene imine</td><td>Polysciences</td><td>23966-2</td><td></td></tr><tr><td>DMEM medium</td><td>Lonza</td><td>BE12-614F</td><td></td></tr><tr><td>Fetal calf serum</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Glutamin</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>matrigel</td><td>BD</td><td>BD 356231</td><td></td></tr><tr><td>Advanced DMEM-F12</td><td>Gibco</td><td>12634-010</td><td></td></tr><tr><td>N2</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>B27</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-acetyl cysteine</td><td>Sigma</td><td>A9165-1G</td><td></td></tr><tr><td>mouse Egf</td><td>Invitrogen</td><td>PMG8045</td><td></td></tr><tr><td>Hepes 1 M</td><td>Invitrogen</td><td>15630-056</td><td></td></tr><tr><td>glutamax 100x</td><td>Invitrogen</td><td>35050-038</td><td></td></tr><tr><td>Chir 99021</td><td>Axon</td><td>1386</td><td></td></tr><tr><td>Y27632&amp;nbsp;</td><td>Sigma</td><td>Y0503-5MG</td><td></td></tr><tr><td>polybrene</td><td>Sigma</td><td>107689</td><td></td></tr><tr><td>nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>Trypsin</td><td>Lonza</td><td>BE02-007E</td><td></td></tr><tr><td>puromycin</td><td>Sigma</td><td>P 7255</td><td></td></tr><tr><td>Rneasy mini kit</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Merck</td><td>8,057,400,250</td><td></td></tr><tr><td>Ovation Pico WTA system</td><td>NuGen</td><td>3300-12</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>252549-1L</td><td></td></tr><tr><td>glass vial conical 12&amp;nbsp;mm x 75&amp;nbsp;mm 5&amp;nbsp;ml</td><td>VWR</td><td>LSUKM12</td><td></td></tr><tr><td>Eosin Yellowish</td><td>VWR</td><td>1,159,350,025</td><td></td></tr></tbody></table>

a protocol for lentiviral transduction and downstream analysis of intestinal organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

Organoid lentiviral transduction
The technique of organoid transduction using lentiviral particles depends on correct handling of organoids prior and during transduction. Organoids (Figure 3A) were cultured and they were disrupted into single crypts (Figure 3B). As previously reported, these single crypts, when cultured in the presence of the GSK3 inhibitor Chir99021 became cystic crypts9 (Figure 3C). Subsequently organoids...

Watch this Scientific Journal Video about A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids at JoVE.com

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

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Biology

30.7K Views.  Academical Medical Center. In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry. 

Video: A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Universal and Efficient Electroporation Protocol for Genetic Engineering of Gastrointestinal Organoids

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

This protocol describes an efficient electroporation method for the transfection of four different gastrointestinal organoid entities with larger plasmids (to the extent of 10 kB). It can be performed within one day and does not need extensive preparation or special, cost-intensive electroporation buffers.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the ...

Genetics

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Developmental Biology

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

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

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 ...

Immunology and Infection

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

Bioengineering

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

Author Spotlight: Investigating Viral Disruption of Intestinal Epithelial Signaling &#8211; Research Insights and Future Directions 

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Summary

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Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

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Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Generation and Manipulation of Rat Intestinal Organoids

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level