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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+epidermal+growth+factor+(EGF)+on+cell+proliferation+of+the+gastrointestinal+mucosa+in+rodents.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+crypt+formation+and+juvenile+polyposis+on+BMP+inhibition+in+mouse+intestine.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-spondin1,+a+novel+intestinotrophic+mitogen,+ameliorates+experimental+colitis+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+BAC+transgenic+epithelial+organoids.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+stress+causes+rapid+loss+of+intestinal+epithelial+stemness+through+activation+of+the+unfolded+protein+response.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+murine+leukemia+virus+DNA+depends+on+mitosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5+homologues+associate+with+Wnt+receptors+and+mediate+R-spondin+signalling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+enhancement+of+retroviral+mediated+gene+transfer.">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 border="0" cellpadding="0" cellspacing="0" width="539">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Reagent</td>
			<td style="width:91px;">Company&#160;</td>
			<td style="width:103px;">Cat. No.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Polyethylene imine</td>
			<td style="width:91px;">polysciences</td>
			<td style="width:103px;">23966-2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">DMEM medium</td>
			<td style="width:91px;">Lonza</td>
			<td style="width:103px;">BE12-614F</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Fetal calf serum</td>
			<td style="width:91px;">Lonza</td>
			<td style="width:103px;">DE14-801F</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Penicillin-streptomycin</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">15140-122</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Glutamin</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">25030-024</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">matrigel</td>
			<td style="width:91px;">BD</td>
			<td style="width:103px;">BD 356231</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">Advanced DMEM-F12</td>
			<td style="width:91px;">Gibco</td>
			<td style="width:103px;">12634-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">N2</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">17502-048</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">B27</td>
			<td style="width:91px;">Invitrogen</td>
			<td style="width:103px;">17504-044</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:345px;">N-acetyl cysteine</td>
			<td>Sigma</td>
			<td>A9165-1G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse Egf</td>
			<td>Invitrogen</td>
			<td>PMG8045</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes 1M</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glutamax 100x</td>
			<td>Invitrogen</td>
			<td>35050-038</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chir 99021</td>
			<td>axon</td>
			<td>1386</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632&#160;</td>
			<td>Sigma</td>
			<td>Y0503-5MG</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Sigma</td>
			<td>107689</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Lonza</td>
			<td>BE02-007E</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">puromycin</td>
			<td>sigma</td>
			<td>P 7255</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74106</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">b-mercaptoethanol</td>
			<td>Merck</td>
			<td>8,057,400,250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ovation Pico WTA system</td>
			<td>NuGen</td>
			<td>3300-12</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma</td>
			<td>252549-1L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glass vial conical 12mm x 75mm 5ml</td>
			<td>VWR</td>
			<td>LSUKM12</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eosin Yellowish</td>
			<td>VWR</td>
			<td>1,159,350,025</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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30.4K visualizações.  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. 

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

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

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 ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

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 ...