This work was supported by grants from the National Institute of Health (R01DK102943, R03CA182679, R03CA191621), the Maryland Stem Cell Research Fund (2015-MSCRFE-1759, 2017-MSCRFD-3934), the American Lung Association, the Allegheny Health Network - Johns Hopkins Research Fund and the Hopkins Digestive Diseases Basic Research Core Center.

This protocol was approved by the Johns Hopkins Medical Institutions Animal Care and Use Committee (IACUC). This protocol is modified from a previously published methods10,11,12,13.
1. Preparation of Reagents
<ol>
	<li>Prepare fresh 293T medium several hours in advance and warm to 37 &#176;C in a water bath for at least 10 min before use (Table 1).</li>
	<li>Prepare plasmid DNA2,13,14 for viral packaging. (Table 2).</li>
	<li>Acquire all other required materials (Table of Materials).</li>
</ol>
2. Lentivirus or Retrovirus Particle Production
<ol>
	<li>Human embryo kidney (HEK) 293T cell seeding (Day 1)
	<ol>
		<li>Prepare one 150 mm culture dish by coating with 50 &#181;g/mL poly-D-lysine dissolved in phosphate buffered saline (PBS; 10 mL/dish) for 1 h at room temperature (RT).</li>
		<li>Remove the phosphate buffered saline (PBS)/poly-D-lysine and wash the coated dish twice with 5 mL PBS.</li>
		<li>Seed 293T cells (8&#8210;10 x 106) in 293T medium (Table 1) to a total volume of 15 mL.</li>
		<li>Culture 293T cells overnight in a standard tissue culture incubator (37 &#176;C, 5% CO2).</li>
	</ol>
	</li>
	<li>HEK 293T cell transfection (Day 2) 
	<ol>
		<li>Perform transfection once cells have reached 70-80% confluence (usually about 24 h after seeding 8&#8210;10 x 106 cells).</li>
		<li>Prepare transfection mixture using an efficient approach such as a commercial cationic liposome formulation (Tables 1&#8210;2, Table of Materials)2.
		<ol>
			<li>Dilute lentivirus DNA constructs12 (total ~24 &#181;g plasmid DNA, Table 2) in 1.2 mL of transfection medium and incubate for 5 min at RT in 1.5 mL tubes (Table of Materials).</li>
			<li>Dilute transfection regent (36 &#956;L) in 1.2 mL of transfection medium (Table of Materials) and incubate in 5-mL tubes for 5 min according to the manufacturer&#8217;s instructions.</li>
			<li>Add the lentivirus reagent (step 2.2.2.1) to the transfection reagent (step 2.2.2.2) and gently mix by slow pipetting up and down using a 5 mL pipet.</li>
			<li>Incubate the mixture for 20 min at RT.</li>
		</ol>
		</li>
		<li>Gently wash 293T cells with 5 mL of transfection medium and replace with 10 mL of transfection medium.</li>
		<li>Add the DNA-lipid complexes (from step 2.2.2.3) dropwise to the medium of 293T cells and carefully mix the media in the culture dishes by moving in horizontal and vertical directions to ensure equal distribution of the DNA-lipid complexes in each dish.</li>
		<li>Incubate for 6 h in a standard tissue culture incubator (37 &#176;C, 5% CO2).</li>
		<li>After incubation, replace the media with 20 mL fresh virus collecting medium (Table 1).</li>
	</ol>
	</li>
	<li>Virus collection (Days 3&#8210;5)
	<ol>
		<li>Collect virus media in 50 mL tubes, and store in a 4 &#176;C refrigerator for further concentration.</li>
		<li>Replace 20 mL of fresh virus collecting medium every 24 h and culture overnight (~24 h). Repeat media collection over the next 2 days (Days 4&#8210;5).</li>
		<li>Ensure that the total volume of medium collected after day 5 is ~ 60 mL (20 mL/day x 3 days).</li>
	</ol>
	</li>
	<li>Virus concentration (Day 5)
	<ol>
		<li>Centrifuge the collected media (60 mL) for 5 min at 400 x g. Then, pass the supernatant through a filter (0.45 &#181;m pores) to remove any cellular debris.</li>
	</ol>
	</li>
	<li>Concentrate the virus by adding 15 mL of filtered virus media in a centrifugal filter unit (Table of Materials). Centrifuge at 2,500 x g for 15 min at 4 &#176;C. Because the virus cannot pass through this filter, it will be concentrated in the upper chamber of the filter.
	<ol>
		<li>Aspirate the flow-through from the tube (bottom chamber) and add another 15 mL of remaining viral collection media supernatant to the same centrifugal filter unit. Centrifuge as above (2,500 x g for 15 min at 4 &#176;C) to concentrate additional virus from the supernatant.</li>
		<li>Repeat the process using the same filter for 60 mL media from a single transduced plate until the desired concentration is reached (~100-fold). 
		Note: We typically concentrate ~60 mL of virus collection media to ~600 &#181;L.</li>
		<li>Remove the concentrated virus from the upper chamber of the filter using a 1 mL pipet, then aliquot and store in 1.5 mL tubes (50&#8210;60 &#181;L/tube) at -80 &#176;C for later use. Store concentrated particles for up to 6&#8210;12 months.</li>
	</ol>
	</li>
</ol>
3. Isolating Crypts
<ol>
	<li>Euthanize mice using CO2 according to the local IACUC guidelines.</li>
	<li>Place the euthanized animal on its back and wash the abdomen by spraying with 70% ethanol.</li>
	<li>Perform a longitudinal midline incision from the sternum to the groin, incising the skin first, and then the subcutaneous tissue.</li>
	<li>Remove the small intestine from the stomach to the cecum.</li>
	<li>Identify regions of interest within the small intestine; crypts can be isolated from the duodenum, jejunum and ileum.</li>
	<li>Using a 10 mL syringe, flush the isolated small intestine with crypt dissociation buffer (pre-chilled PBS containing 1 mM dithiothreitol (DTT), 1% penicillin/streptomycin (no Ca2+ and Mg2+)) in a 10 cm tissue culture dish (Table 1)</li>
	<li>Remove peripheral fat tissue, and open or &#8220;fillet&#8221; the intestinal tissue longitudinally on a sterile glass plate (15 cm x 15 cm).</li>
	<li>Gently scrape off the intestinal epithelial villi using a cell scraper.</li>
	<li>Cut the small intestine into 2&#8210;3 cm length-wise sections.</li>
	<li>Transfer the tissue to a 15 mL tube containing pre-chilled PBS using flat forceps (116 mm). Wash the tissue fragments by shaking vigorously by hand for ~30 s (moving the tube in opposite directions ~60 times).</li>
	<li>Refresh the PBS and repeat wash until the PBS becomes clear. 
	Note: We typically wash the fragments 2&#8210;3 times.</li>
	<li>Incubate the tissue in a 15 mL conical tube containing 10 mL of crypt dissociation buffer (Table 1) for 10 min at 4 &#176;C on an orbital shaker at medium speed once the PBS is clear.</li>
	<li>Vigorously shake the tube by hand for ~30 s (opposite directions &#8776;60 times) and transfer the tissue using flat forceps to another 15 mL conical tube containing 10 mL of crypt dissociation buffer. Incubate this fraction on ice. Do not use the first fraction for organoid culture because it contains primarily villi.</li>
	<li>Repeat steps 3.11&#8210;3.13 for 3&#8210;4 times, collecting each fraction and placing them on ice.</li>
	<li>Select the fraction that is enriched with the highest percentage of intestinal crypts by scanning 200 &#181;L samples from each fraction under an inverted microscope (4x). Identify crypts by the typical morphology as described previously (Figure 1A)10. 
	NOTE: They will appear round or oval in shape and contain granulated Paneth cells. In contrast, villi are finger-like structures lacking the granular Paneth cells (Figure 1B).</li>
	<li>Pass the selected fraction through a 40 &#181;m cell strainer to obtain crypts of similar size if required. Alternatively, isolate Lgr5+ stem cells based on flow cytometry for green fluorescent protein (GFP) if mice are crossed onto the EGFP-Lgr5+ background or another suitable model that enables identification of Lgr5+ cells2.</li>
	<li>Count the total number of crypts in the selected fraction as follows.
	<ol>
		<li>Pipette 50 &#181;L of the selected fraction into a hemocytometer and count the number of crypts using an inverted light microscope (4x). Place ~100 crypts per well when using a 48-well plate to allow for the transduction experiments in which 3&#8210;6 wells will be transduced per experimental condition for gene silencing or overexpression.</li>
		<li>Based on the number of crypts per 50 &#181;L, calculate the volume of crypt dissociation buffer needed and transfer that volume into a 1.5 mL tube. 
		Note: For example, 10 crypts per 50 &#181;L are counted, 6 x 50 &#181;L or 300 &#181;L are needed for 300 crypts.</li>
	</ol>
	</li>
	<li>Centrifuge the crypts in the 1.5-mL tubes at 300 x g for 5 min.</li>
	<li>Carefully discard the supernatant by gently pipetting off the upper liquid layer and resuspend the pellet in 100 &#181;L of growth factor reduced basement membrane matrix on ice (Table of Materials).</li>
	<li>Seed the matrix-containing crypts into a 37 &#176;C pre-warmed 48-well plate (30 &#181;L/well, ~100 crypts/well). Incubate the plate in a standard tissue culture incubator for 5&#8210;15 min to allow for matrix gelation (37 &#176;C, 5% CO2).</li>
	<li>Overlay each gel with 250 &#181;L organoid culture (ENR) medium (Table 1) and place the 48-well plates back into a standard tissue culture incubator (37 &#176;C, 5% CO2). Check the cultures for crypt organization into small, round, cystic shapes after 24 h; buds will form after 2&#8210;5 days.</li>
	<li>Gently replace ENR media every 3 days. Remove old ENR media with gentle suction, taking care not touch the matrix when replacing media.</li>
	<li>Passage organoid cultures every 4&#8210;7 days as previously described11.</li>
</ol>
4. Organoid Fragment Preparation
<ol>
	<li>Transduce organoids once they form (within 1&#8210;2 weeks) or after being passaged. For a single transduction experiment, prepare 2&#8210;3 wells of cultured organoids in a 48-well plate per condition with ~100&#8210;200 organoids/well or ~200&#8210;600 organoids per experimental transduction condition.</li>
	<li>Exchange ENR with 250 &#181;L transduction media (Table 1) and culture in the transduction media for 3 or more days or until the organoids adopt a cystic morphology. Include both Wnt3a and ROCK inhibitor (Y27632) in the transduction medium to increase the number of stem and Paneth cells; Nicotinamide (Nic) improves culture efficiency (see transduction medium in Table 1).</li>
	<li>Mechanically rupture the dome-like basement membrane matrix structure with media and a pipet tip using a 1 mL pipet.</li>
	<li>Transfer the organoids and media to a sterile 1.5 mL tube.</li>
	<li>Mechanically disrupt the matrix further by pipetting with a 200 &#181;L pipet ~10&#8210;15 times.</li>
	<li>Centrifuge the organoid fragments at RT at 500 x g for 5 min.</li>
	<li>Discard the supernatant carefully using a pipette and resuspend the pellet in 1 mL DMEM/F12 medium (Table 1).</li>
	<li>Add Dispase I (6 &#181;L at 10 mg/mL) and DNase I (2.5 &#181;L at 10 mg/mL). Mix well by pipetting gently using a 1 mL pipet.</li>
	<li>Incubate organoids at 37 &#176;C for 20 min in the 1.5 mL tube.</li>
	<li>Add 500 &#181;L of ENR media to terminate the dissociation reaction; the serum in the ENR terminates the reaction.</li>
	<li>Pass the organoid cells through a 20 &#181;m cell strainer and centrifuge the organoid fragments at 400 x g for 5 min.</li>
	<li>Resuspend organoid fragments with 150 &#181;L transduction medium (Table 1).</li>
</ol>
5. Genetic Engineering of Organoids or Crypt Cells by Viral Transduction
NOTE: See Figure 2.
<ol>
	<li>Seed organoid cell clusters with 200 &#181;L transduction medium/well in a 48-well plate and incubate in a standard tissue culture incubator (37 &#176;C, 5% CO2). Alternatively, place freshly isolated crypt cells (~1,000 crypts) in 200 &#181;L transduction medium/well in a 48-well plate and incubate in a standard tissue culture incubator (37 &#176;C, 5% CO2).</li>
	<li>Thaw vials of virus for transduction allowing for ~50 &#181;L of concentrated virus for transduction of each well in 48-well plates or ~100 &#181;L of concentrated virus per well in 24-well plates.</li>
	<li>Incubate virus with 12 &#181;L of magnetic nanoparticle solution for 15 min at RT in a 1.5 mL tube (Table 3).</li>
	<li>Add the magnetic nanoparticle solution/virus mixture to the cells to be transduced.</li>
	<li>Place the cell culture plate on a magnetic plate and incubate for at least 2 h (and up to ~6 h) in a standard tissue culture incubator (37 &#176;C, 5% CO2).</li>
</ol>
6. Seeding of Infected Organoid Fragments
<ol>
	<li>Transfer the infected organoid cell clusters and transduction media from each well into a 1.5 mL tube.</li>
	<li>Centrifuge at 500 x g for 5 min.</li>
	<li>Discard the supernatant with gentle suction and cool the tube containing the pellet on ice for 5 min.</li>
	<li>Add 120 &#181;L of basement membrane matrix and resuspend the pellet by pipetting slowly up and down.</li>
	<li>Seed 30 &#181;L drops of the matrix-cell mixture into a new 48-well plate.</li>
	<li>Incubate the plate at 37 &#176;C for 5&#8210;15 min until the matrix solidifies.</li>
	<li>Add transduction medium to each well and incubate in a standard tissue culture incubator for 3&#8210;4 days (37 &#176;C, 5% CO2).</li>
	<li>After 3&#8210;4 days, inspect cultures under a light microscope (10x) to ensure organization of cell clusters into organoid structures. Then, gently replace transduction media with 250 &#181;L ENR medium.</li>
	<li>Replace media every 3&#8210;4 days.</li>
</ol>
7. Selection (If Applicable)
<ol>
	<li>After 2&#8210;3 days, add relevant antibiotics or hormones for selection to the transduction medium if appropriate. 
	NOTE: We used puromycin (2 &#181;g/mL) for selection of the lentivrial transduction because plasmids harbored a puromycin resistance gene2,14.</li>
</ol>
8. Confirmation of Successful Transduction and Gene Expression or Silencing
<ol>
	<li>If using FUGW lentivirus2,14, estimate transduction efficiency by measuring GFP signals via fluorescent microscope or flow cytometry.</li>
	<li>Validate gene overexpression or silencing using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for quantitative comparison of mRNA in the control and experimental organoid cultures.</li>
	<li>Confirm protein levels for protein-coding gene expression or silencing by Western Blot or immunostaining2.</li>
</ol>
9. Organoid Cryosection in Basement Membrane Matrix
NOTE: See Figure 3.
<ol>
	<li>Remove ENR medium by gentle suction, being careful not to perturb the basement membrane matrix and gently wash once with 500 &#181;L of PBS.</li>
	<li>Fix organoids with 1.0 mL of 4% paraformaldehyde (PFA) solution (Table of Materials) at RT for 30 min.</li>
	<li>Remove PFA by suction, and gently wash twice with 1 mL PBS.</li>
	<li>Remove PBS by suction and add 1.0 mL of 30% sucrose buffer to each sample. Incubate fixed organoids in sucrose for 1 h at 4 &#176;C in a cold room, refrigerator, or on ice to dehydrate samples.</li>
	<li>Remove sucrose buffer by suction and add just enough embedding compound (Table of Materials) to cover the matrix layer (~300 &#181;L/well) in each well.</li>
	<li>Incubate at RT for 5 min.</li>
	<li>Place samples in a -80 &#176;C freezer for 10 min, or until the embedding compound turns solid and white.</li>
	<li>Place the dish with frozen embedding compound at RT to allow for minimal melting of the compound along the edges. Use a scalpel to separate the block from the walls of the well.</li>
	<li>Remove the matrix-embedding compound block using forceps and place it in a specimen block (e.g. cryomold), working quickly to prevent melting.</li>
	<li>Fill the mold completely with embedding compound and freeze at -80 &#176;C for 30 min.</li>
	<li>Use the block is for sectioning or storage in -80 &#176;C freezer for further use.</li>
</ol>

<ol><li>Cheung, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TIGAR+is+required+for+efficient+intestinal+regeneration+and+tumorigenesis%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">TIGAR is required for efficient intestinal regeneration and tumorigenesis.</a> Dev Cell. 25 (5), 463-477 (2013).</li>
<li>Xian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HMGA1+amplifies+Wnt+signalling+and+expands+the+intestinal+stem+cell+compartment+and+Paneth+cell+niche%5BTitle%5D)+AND+%22Nature+Comm%22%5BJournal%5D)">HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche.</a> Nature Comm. , (2017).</li>
<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>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>Kabiri, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stroma+provides+an+intestinal+stem+cell+niche+in+the+absence+of+epithelial+Wnts%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.</a> Development. 141, 2206-2215 (2014).</li>
<li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li>
<li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Forskolin-induced+Swelling+in+Intestinal+Organoids%3A+An+In+Vitro+Assay+for+Assessing+Drug+Response+in+Cystic+Fibrosis+Patients%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients.</a> J Vis Exp. (120), (2017).</li>
<li>Muñoz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Lgr5+intestinal+stem+cell+signature%3A+robust+expression+of+proposed+quiescent+%27%2B4%27+cellmarkers%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent '+4' cellmarkers.</a> EMBO J. 31 (14), 3079-3091 (2012).</li>
<li>Schwank, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+repair+of+CFTR+by+CRISPR%2FCas9+in+intestinal+stem+cell+organoids+of+cystic+fibrosis+patients%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients.</a> Cell Stem Cell. 13 (6), 653-658 (2014).</li>
<li>Andersson-Rolf, A., Fink, J., Mustata, R. C., Koo, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+video+protocol+of+retroviral+infection+in+primary+intestinal+organoid+culture%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A video protocol of retroviral infection in primary intestinal organoid culture.</a> J Vis Exp. (90), e51765(2014).</li>
<li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+mouse+small+intestinal+epithelial+cell+cultures%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Primary mouse small intestinal epithelial cell cultures.</a> Methods Mol Biol. 945, 319-328 (2013).</li>
<li>Ye, Z., Yu, X., Cheng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lentiviral+gene+transduction+of+mouse+and+human+stem+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Lentiviral gene transduction of mouse and human stem cells.</a> Methods Mol Biol. 430, 243-253 (2008).</li>
<li>Cheung, K. J., Gabrielson, E., Werb, Z., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collective+invasion+in+breast+cancer+requires+a+conserved+basal+epithelial+program%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Collective invasion in breast cancer requires a conserved basal epithelial program.</a> Cell. 155 (7), 1639-1651 (2013).</li>
<li>Tesfaye, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+High-Mobility+Group+A1+gene+up-regulates+Cyclooxygenase-2+expression+in+uterine+tumorigenesis%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">The High-Mobility Group A1 gene up-regulates Cyclooxygenase-2 expression in uterine tumorigenesis.</a> Cancer Res. 67 (9), 3998-4004 (2007).</li>
<li>Haim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synchronized+infection+of+cell+cultures+by+magnetically+controlled+virus%5BTitle%5D)+AND+%22J.+Virol%22%5BJournal%5D)">Synchronized infection of cell cultures by magnetically controlled virus.</a> J. Virol. 79 (1), 622-625 (2005).</li>
<li>Xue, X., Shah, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+organoid+culture+of+primary+mouse+colon+tumors%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vitro organoid culture of primary mouse colon tumors.</a> J Vis Exp. (75), e50210(2013).</li>
</ol>

The authors have nothing to disclose

Primary culture of adult intestinal epithelium as organoids provides a powerful tool to study molecular mechanisms involved in stem cell function, intestinal epithelial homeostasis, and pathology1,2,3,4. Although CRISPR/Cas9 technology can be used to genetically engineer organoids9, it is limited by the need for extensive screening and selection based on sequence analysi...

The ability to culture mouse or human crypts cells as three dimensional (3D) organoids from the small intestines or colon over prolonged time periods provided a major breakthrough because these cultures display defining features of intestinal epithelium in vivo1,2,3. Organoids derived from primary crypts are capable of self-renewal and self-organization, exhibiting cellular functions similar to their tissues of origin. Indeed, organoids recapitulate not only the structural organization of crypts in vivo, but also many molecular features, thus providing useful tools to study normal biology and disease states. To illustrate, organoid studies have revealed novel molecular pathways involved in tissue regeneration1,2,3,4,5 as well as drugs that could enhance function in pathologic settings6,7.
The study of intestinal stem cells is of particular interest because the intestinal lining is among the most highly regenerative mammalian tissues, renewing itself every 3-5 days to protect the organism from bacteria, toxins, and other pathogens within the intestinal lumens. Intestinal stem cells (ISCs) are responsible for this remarkable regenerative capability and thus provide a unique paradigm for studying adult stem cell function1,2. Lineage-tracing experiments in mice demonstrated that isolated Lgr5-positive stem cells can be cultured to generate 3D organoids or &#39;mini-guts&#39; in vitro where they closely mirror their in vivo counterparts. Organoid cultures can also be derived from intestinal crypt cell isolates comprised of progenitors, ISCs, and Paneth cells, the latter of which constitute the epithelial niche cells in vivo. In fact, organoid culture from primary intestinal crypt cells has evolved into a relatively routine technique that is easy to implement in most laboratories using widely available reagents. This model is also amenable to quantitative analysis of gene expression by RNA-sequencing (RNA-Seq) and proteins by mass spectrometry, immunohistochemistry, or immunofluorescent staining2,4,8. In addition, functional genetics can be studied using gain-of-function (gene overexpression or expression of an activating mutant gene) or loss-of-function (gene silencing or expression of a loss-of-function mutant) approaches2.
Importantly, low efficiency and high toxicity of standard plasmid DNA or viral transduction protocols with polybrene remain a major hurdle in the field. Although CRISPR/Cas9 technology allows for precise genome editing, this approach requires time-consuming selection followed by sequence validation9. Here, we present a viral transduction protocol for primary intestinal organoids that optimizes delivery of viral particles by conjugation to magnetic nanoparticles and application of a magnetic field. Key modifications to prior protocols4,5,10,11,12,13 and recommendations to enhance efficiency are provided. We also describe an approach to generate frozen sections from intact organoids cultured in 3D matrigel (henceforth referred to as basement membrane matrix or matrix) for further analysis with immunohistochemistry or immunofluorescent staining.

<table><tbody><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>11965092</td><td>Base medium for 293T cells</td></tr><tr><td>DMEM/F12+</td><td>Thermo Fisher Scientific</td><td>12634010</td><td>Base medium for organoid culture medium and organoid digestion buffer</td></tr><tr><td>OPTI-MEM</td><td>Thermo Fisher Scientific</td><td>11058021</td><td>Virus plasmids transfection medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-011-CV</td><td>Component of virus collection medium and 293T medium</td></tr><tr><td>Pen/Strep</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Component of organoid culture medium and crypt dissociation buffer</td></tr><tr><td>PBS (without Ca&lt;sup&gt;2+,&lt;/sup&gt; Mg&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Thermo Fisher Scientific</td><td>10010049</td><td>A wash buffer and component of crypt dissociation buffer</td></tr><tr><td>Mem-NEAA</td><td>Thermo Fisher Scientific</td><td>11140050</td><td>Component of organoid culture medium</td></tr><tr><td>GlutamaxII</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Component of organoid culture medium</td></tr><tr><td>HEPES</td><td>Thermo Fisher Scientific</td><td>15630080</td><td>Component of organoid culture medium</td></tr><tr><td>EGF</td><td>Millipore Sigma</td><td>E9644</td><td>Component of organoid culture medium</td></tr><tr><td>Noggin</td><td>Peprotech</td><td>250-38 B</td><td>Component of organoid culture medium</td></tr><tr><td>R-spondin</td><td>R&amp;amp;D</td><td>7150-RS-025/CF</td><td>Component of organoid culture medium</td></tr><tr><td>Human recombinant insulin</td><td>Millipore Sigma</td><td>I9278-5ml</td><td>Component of organoid culture medium</td></tr><tr><td>Nicotinamide</td><td>Millipore Sigma</td><td>N3376-100G</td><td>Component of Transduction medium</td></tr><tr><td>Wnt3A</td><td>R&amp;amp;D</td><td>5036-WN-010</td><td>Component of Transduction medium</td></tr><tr><td>Y27632</td><td>Millipore Sigma</td><td>Y0503-1MG</td><td>Component of Transduction medium</td></tr><tr><td>0.5 M EDTA</td><td>Thermo Fisher Scientific</td><td>15575020</td><td>Component of Crypts dissociation buffer</td></tr><tr><td>DTT (dithiothreitol)</td><td>Thermo Fisher Scientific</td><td>R0861</td><td>Component of Crypts dissociation buffer</td></tr><tr><td>Dispase I</td><td>Millipore Sigma</td><td>D4818-2MG</td><td>Component of organoid digestion buffer</td></tr><tr><td>DNase I</td><td>Millipore Sigma</td><td>11284932001</td><td>Component of organoid digestion buffer</td></tr><tr><td>matrigel(Growth factor reduced)</td><td>Corning</td><td>356231</td><td>Used as a matrix to embed organoids</td></tr><tr><td>Opti-MEM</td><td>Thermo Fisher Scientific</td><td>31985070</td><td>Medium for transfection in viral production</td></tr><tr><td>ViralMag R/L</td><td>Oz Biosiences</td><td>RL40200</td><td>Magnetic particles of viral transduction</td></tr><tr><td>Magnetic plate</td><td>Oz Biosiences</td><td>MF10000</td><td>Magnetic plate to facilitate viral transduction</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668019</td><td>Transfection agent in viral production</td></tr><tr><td>Poly-D-Lysine</td><td>Millipore Sigma</td><td>A-003-E</td><td>Coating for plates before seeding 293T cells</td></tr><tr><td>4% Formaldehyde Solution</td><td>Boster</td><td>AR1068</td><td>Solution to fix organoids</td></tr><tr><td>O.C.T embedding compound</td><td>Thermo Fisher Scientific</td><td>4583S</td><td>For embedding of the the organoids</td></tr><tr><td>5 mL Falcon polystyrene tubes</td><td>Corning</td><td>352054</td><td></td></tr><tr><td>50 mL Falcon Tubes</td><td>Sarstedt</td><td>62.547.100</td><td></td></tr><tr><td>Orbitron rotator II Rocker Shaker</td><td>Boekel Scientific</td><td>260250</td><td></td></tr><tr><td>Olympus Inverted microscop CK30</td><td>Olympus</td><td>CK30</td><td>for scanning and counting crypts</td></tr><tr><td>Zeiss Axiovert 200 inverted fluorescence</td><td>Nikon</td><td>Axiovert 200</td><td>for viewing fluorescence in the crypts</td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter unit with Ultracel-100 membrane</td><td>Milipore Sigma</td><td>UFC910024</td><td>For concentrating viruses</td></tr><tr><td>pluriStrainer 20 &amp;micro;m (Cell Strainer)</td><td>pluriSelect</td><td>SKU 43-50020</td><td>For preparing organoid fragments</td></tr><tr><td>Falcon Cell Strainer</td><td>Fisher Scientific</td><td>352340</td><td>For preparing cyrpts of similar size after crypt isolation</td></tr><tr><td>Greiner CELLSTAR&amp;nbsp;multiwell culture plates 48 wells (TC treated with lid)</td><td>Millipore Sigma</td><td>M8937-100EA</td><td>ForD2:D37+D16:D37g organoid fragments</td></tr><tr><td>Animal strain: C57BL/6J</td><td>Jackson Lab</td><td>#000664</td><td>For organoid culture</td></tr></tbody></table>

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.

Here, we describe a rapid and highly efficient transduction technique which harnesses magnetic nanoparticles exposed to a magnetic field to deliver lentivirus to cells of interest. With readily available tools, we have applied this approach not only to transduce freshly isolated crypt cells (Figure 1A), but also for organoids (Figure 2) and other cells that are refractory to more routine transduction approaches. Lentiviral partic...

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

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 ...

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Research

JoVE Journal

Developmental Biology

8.2K Views.  Johns Hopkins University School of Medicine. Omdat organoïden de inheemse structurele organisatie samenvatten, evenals vele moleculaire kenmerken van darmeptheel in vivo, stelt deze techniek ons in staat om normale biologie en ziektetoestanden in het laboratorium te bestuderen. Het belangrijkste voordeel van deze techniek is het vermogen om organoïden genetisch te manipulatie met behulp van een zeer efficiënte transductiebenadering met lage cytotoxiciteit, waardoor functionele studies van deze genetisch gemanipuleerde organoïden mog...

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

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Bioengineering

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

Cancer Research

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Medicine

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

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 ...

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 ...

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 ...

Biology

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.

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 ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Author Spotlight: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

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

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