Name: establishment of human epithelial enteroids and colonoids from whole tissue and biopsy
Uploaded: 2015-03-06T14:00:00
Duration: 6 min 33 s
Description: Watch this Scientific Journal Video about Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy at JoVE.com

We gratefully acknowledge Dr. Hans Clevers (Hubrecht Institute for Developmental Biology and Stem Cell Research, Netherlands) for the kind gift of TOPflash cells.
This project was supported in part by NIH-R01DK083325 (MAH); NIH P30 DK078392 (Digestive Health Center); NIH UL1RR026314 (CTSA).

NOTE: Ethics Statement: All experimentation using human tissues described herein was approved by an IRB at CCHMC (IRB #2012-2858; #2014-0427). Informed consent for tissue collection, storage, and use of the samples was obtained from the donors at CCHMC.
1. Preparation for Culture
NOTE: All reagents are listed in Table 1.
<ol>
	<li>Prepare EDTA stock solution as follows: prepare 0.5 M ethylenediaminetetraacetic acid, pH 8 (EDTA) in ultrapure H2O, filter sterilized with 0.22&#160;&#181;m filter. Optionally, store the EDTA stock solution at RT indefinitely.</li>
	<li>Prepare chelating buffer as follows: mix 2% sorbitol, 1% sucrose, 1% bovine serum albumin fraction V (BSA) and 1x Gentamicin/Amphotericin solution in Dulbecco&#8217;s Phosphate buffered saline without Ca2+ and Mg2+ (DPBS), filter sterilized with 0.22&#160;&#181;m filter. Prepare the chelating buffer fresh.</li>
	<li>Prepare Wnt-3A-conditioned medium as follows: Wnt-3A-conditioned medium is made in house using the cell line L Wnt-3A according to the manufacturer&#8217;s instructions (ATCC, CRL-2647). Supplement the medium with 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin, 1 N2 supplement, 1 B27 supplement, 1% BSA and filter sterilized with 0.22-&#181;m filter. 
	NOTE: Test every batch for Wnt activity using a TOPflash assay. Use a stable HEK293 TOPflash cell line (Hans Clevers lab) with a Renilla luciferase assay kit according to the manufacturer&#8217;s instructions. Normalize the TOPflash assay with 100 ng/ml&#160;human recombinant Wnt-3A. Confirm at least a 10 fold-change activity of the conditioned media compared to control. Divide fresh Wnt-3A-conditioned medium into 10 ml aliquots in 15 ml conical tubes and freeze at -20&#176;C for up to 6 months. Store thawed aliquots up to 5 days at 4&#176;C without loss of activity.</li>
	<li>Prepare human minigut medium as follows: Supplement Advanced DMEM/F12 medium with 2 mM glutamine, 10 mM HEPES, 100 U/mL penicillin, 100 g/mL streptomycin, 1 N2 supplement, 1 B27 supplement, 1% BSA and filter sterilized with 0.22&#160;&#181;m filter. 
	NOTE: Divide fresh human minigut medium into 10 ml aliquots in 15 ml conical tubes and freeze at -20&#176;C for up to 3 months. Store thawed aliquots up to 5 days at 4&#176;C without loss of activity.</li>
	<li>Prepare complete human minigut medium as follows: Prepare fresh before crypt culture or medium change human minigut medium (see 1.4) supplemented with 50% Wnt-3A-conditioned medium (see 1.3), 1 &#181;g/ml&#160;R-spondin 1 (1:1,000 dilution of 1 mg/ml stock), 100 ng/ml Noggin (1:1,000 dilution of 100 g/mL stock), 50 ng/mL EGF (1:10,000 dilution of 500 &#956;g/mL stock), 500 nM A-83-01 (1:1,000 dilution of 500 M stock), 10 &#181;M SB202190 (1:3,000 dilution of 30 mM stock), 10 nM [Leu]15-Gastrin 1 (1:10,000 dilution of 100 &#181;M stock), 10 mM Nicotinamide (1:100 dilution of 1 M stock) and 1 mM N-Acetylcysteine (1:1,000 dilution of 1 M stock). 
	NOTE: Store complete human minigut media up to 2 days at 4&#176;C without loss of activity.</li>
</ol>
2. Crypt Isolation from Whole Tissue
NOTE: From the tissue collection, it is essential to maintain the sample in saline. It is recommended to keep the tissue on ice during transportation. Preparation of the sample for isolation of crypt should be performed as soon as possible. 
NOTE: All reagents are listed in Table 1, tools, equipment, and consumables are listed in Table 2.
<ol>
	<li>Prepare all the reagents before beginning the experiment. Thaw the basement membrane matrix on ice and pre-incubate a 24-well plate in a CO2 incubator at 37&#176;C. 
	NOTE: Alternatively make a thin layer of basement membrane matrix using 15 &#181;l/well in the center of a 24-well plate and place it in a CO2 incubator at 37&#176;C. This facultative step keeps the basement membrane matrix as a drop during polymerization.</li>
	<li>Wash the tissue with ice-cold Dulbecco&#8217;s Phosphate buffered saline without Ca2+ and Mg2+ (DPBS). Proceed until the content of DPBS is clear.</li>
	<li>Using 0.2 mm diameter minutien pins, secure the tissue on a silicone-coated glass Petri dish filled with ice-cold DPBS. Stretch and pin the tissue flat with the mucosal side facing up.</li>
	<li>Under a dissecting microscope, remove the overlying mucosa from the submucosa and connective tissue using micro-dissecting scissors and fine point curved forceps (Figure 2A, B).</li>
	<li>Stretch and pin the dissected mucosa flat on the silicone-coated glass Petri dish with mucosal side facing up. The remaining submucosa and connective tissue can be discarded or used for further experiments (Figure 2C).</li>
	<li>Gently scrape the surface of the mucosa with curved forceps. This step is necessary to improve the quality of the preparation.
	<ol>
		<li>For small intestine, gently scrape the mucosa to remove the villi.</li>
		<li>For colon, gently scrape the mucosa to remove mucous and debris.</li>
	</ol>
	</li>
	<li>Wash the mucosa 3-4 times with ice-cold chelation buffer to remove villi and debris.</li>
	<li>Cover the mucosa with freshly prepared 2mM EDTA chelation buffer (200 &#181;l 0.5 M EDTA in 49.6 ml chelation buffer).</li>
	<li>Place the Petri dish on ice and shake gently for 30 min on a horizontal orbital shaker.</li>
	<li>Wash the tissue 3-4 times with ice-cold chelation buffer without EDTA. After washing, leave the mucosa in ice-cold chelation buffer.</li>
	<li>Process the mucosa under a dissecting microscope using curved and fine forceps. Gently scrape the mucosa to release intestinal crypts using the curved forceps.</li>
	<li>Gently remove the crypt suspension from the petri dish using a pipette and transfer it into a 50 ml conical tube. 
	NOTE: check the tissue to make sure that almost all crypts have been removed from the mucosa.</li>
	<li>Filter the crypts suspension through a 150 &#181;m mesh 2 times. 
	NOTE: check the flow-through for crypt enrichment under a microscope (Figure 2D).</li>
	<li>Centrifuge the crypt suspension 5 min at 50 xg, 4&#176;C. Discard the supernatant.</li>
	<li>Resuspend the pellet in 5 ml ice-cold chelation buffer.</li>
	<li>Count the number of crypts per 10-&#181;l drop from the suspension from step 2.15. Transfer the number of crypts needed for plating to a 5 ml round-bottom tube. Use 200 to 500 crypts per well of a 24-well dish to establish enteroids or colonoids.</li>
	<li>Centrifuge the crypt fraction for 10 min at 150 g, 4&#176;C. Remove the supernatant.</li>
	<li>Use crypts for subsequent culture.</li>
</ol>
3. Crypt Isolation from Biopsy
<ol>
	<li>Prepare all the reagents before the beginning of the experiment. Thaw the basement membrane matrix on ice and pre-incubate a 24-well plate in a CO2 incubator at 37&#176;C.</li>
	<li>Wash the biopsy with ice-cold Dulbecco&#8217;s Phosphate buffered saline without Ca2+ and Mg2+ (DPBS).</li>
	<li>Using 0.1 mm diameter minutien pins, secure the biopsy on a silicone-coated glass Petri dish filled with ice-cold DPBS. Stretch and pin the mucosa flat with the mucosal side facing up (Figure 2E).</li>
	<li>Gently scrape the surface of the mucosa with curved forceps to remove villi and debris. This step is necessary to improve the quality of the preparation.</li>
	<li>Wash the biopsy 3-4 times with ice-cold chelation buffer to remove villi and debris.</li>
	<li>Cover the biopsy with freshly prepared 2mM EDTA chelation buffer (200 &#181;l 0.5 M EDTA in 49.8 ml chelation buffer).</li>
	<li>Place the Petri dish on ice and shake gently for 30 min on a horizontal orbital shaker.</li>
	<li>Wash the biopsy 3-4 times with ice-cold chelation buffer without EDTA. After washing, leave the biopsy in ice-cold chelation buffer.</li>
	<li>Process the biopsy under a dissecting microscope using curved and fine forceps. Gently scrape the mucosa to release the intestinal crypts using curved forceps.</li>
	<li>Gently remove the crypt suspension from the petri dish using a pipette and transfer it to a 50 ml conical tube. 
	NOTE: check the tissue to make sure that almost all crypts have been removed from the mucosa.</li>
	<li>Filter the crypt suspension through a 150 &#181;m nylon mesh 2 times. 
	NOTE: check the flow-through for crypt enrichment under a microscope.</li>
	<li>Centrifuge the crypt suspension 5 min at 50 xg, 4&#176;C. Discard the supernatant.</li>
	<li>Resuspend the pellet in 1 ml ice-cold chelation buffer. Transfer the crypt suspension to a 1.5 ml microfuge tube.</li>
	<li>Centrifuge the crypt fraction for 10 min at 150 x g, 4&#176;C. Remove the supernatant.</li>
	<li>Use crypts for subsequent culture.</li>
</ol>
4. Crypt Culture in Basement Membrane Matrix
<ol>
	<li>Using pre-chilled pipet tips, resuspend the crypt pellet (from step 2.18 or 3.15) in basement membrane matrix (200 to 500 crypts/50 &#181;l basement membrane matrix).</li>
	<li>Apply 50 &#181;l of crypt suspension in basement membrane matrix per well on the pre-warmed plate. Slowly eject the basement membrane matrix in the center of the well.</li>
	<li>Place the 24-well plate in a 37&#176;C, 5% CO2 incubator for 30 min to allow a complete polymerization of the basement membrane matrix.</li>
	<li>Overlay the basement membrane matrix with 500 &#181;l of complete human minigut medium supplemented with 2.5 &#181;M CHIR99021 (1:4,000 dilution of 10 mM stock) and 2.5 &#181;M Thiazovivin (1:4,000 dilution of 10 mM stock).</li>
	<li>Incubate the plate in a 37&#176;C, 5% CO2 incubator.</li>
	<li>Replace the medium with fresh complete human minigut medium every 2 days.</li>
</ol>
5. Passaging of Cultured Enteroids and Colonoids.
NOTE: Passage Enteroids and Colonoids every 7 to 10 days after initial plating. Generally, split one well into 3 to 4 wells.
<ol>
	<li>Prepare all the reagents before the beginning of the experiment. Thaw the basement membrane matrix on ice and pre-incubate a 24-well plate in a CO2 incubator at 37&#176;C.</li>
	<li>Remove the media using sterile tips and overlay with 1 ml of ice-cold DPBS.</li>
	<li>Pipette back and forth with a 1,000 &#181;l&#160;tip. Transfer the solution in a new 15 ml conical tube.</li>
	<li>Add 2 ml of human minigut medium supplemented with 5% FBS per 1 ml of medium.</li>
	<li>Centrifuge the solution for 5 min at 50 x g, 4&#176;C. Discard the supernatant.</li>
	<li>Resuspend the pellet with 2 ml of cell dissociation enzyme supplemented with 10 &#181;M Y-27632 (1:1,000 dilution of 10 mM stock). Incubate for 5 min at 37&#176;C in a water-bath.</li>
	<li>Dissociate the cell clumps using a 3 ml Luer-Lock syringe equipped with a 18-G fill/blunt needle. Gently pipette the solution back and forth using the syringe 10 times.</li>
	<li>Centrifuge the suspension for 5 min at 500 x g, 4&#176;C. Discard the supernatant.</li>
	<li>Using pre-chilled pipet tips, resuspend the cell pellet in basement membrane matrix.</li>
	<li>Apply 50 &#181;l of crypt suspension in basement membrane matrix per well on the pre-warmed plate. Slowly eject the basement membrane matrix in the center of the well.</li>
	<li>Place the 24-well plate in a 37&#176;C, 5% CO2 incubator for 20 min to allow a complete polymerization of the basement membrane matrix.</li>
	<li>Overlay the basement membrane matrix with 500 &#181;l of complete human minigut medium supplemented with 10 &#181;M Y-27632 (1:1,000 dilution of 10 mM stock).</li>
	<li>Incubate the plate in a 37&#176;C, 5% CO2 incubator.</li>
	<li>After 2 days, replace the medium with fresh complete human minigut medium supplemented with 10 &#181;M Y-27632 (1:1,000 dilution of 10 mM stock). Thereafter, replace the medium with fresh, complete human minigut medium every other day.</li>
</ol>
6. Freezing of Cultured Enteroids and Colonoids
NOTE: Usually freeze one well into 2-3 cryovials.
<ol>
	<li>Repeat steps 5.1 to 5.8.</li>
	<li>Resuspend the pellet with cold freezing medium. Transfer 1 ml of freezing solution into a labeled cryovial. Place the cryovial in a freezing container containing 500 ml of isopropyl alcohol.</li>
	<li>Transfer the freezing container to a 80&#176;C freezer for 24 h, then transfer cryovial to liquid nitrogen storage. 
	NOTE: Store Enteroids and Colonoids for up to 1 year.</li>
</ol>

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D., 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=Brief+report:+CD24+and+CD44+mark+human+intestinal+epithelial+cell+populations+with+characteristics+of+active+and+facultative+stem+cells.">Brief report: CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells.</a> Stem Cells. 31 (9), 2024-2030 (2013).</li><li>Foulke-Abel, 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=Human+enteroids+as+an+ex-vivo+model+of+host-pathogen+interactions+in+the+gastrointestinal+tract.">Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract.</a> Exp Biol Med (Maywood). , (2014).</li><li>Dekkers, J. F., 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=A+functional+CFTR+assay+using+primary+cystic+fibrosis+intestinal+organoids.">A functional CFTR assay using primary cystic fibrosis intestinal organoids.</a> Nat Med. 19 (7), 939-945 (2013).</li><li>Kovbasnjuk, O., 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=Human+enteroids:+preclinical+models+of+non-inflammatory+diarrhea.">Human enteroids: preclinical models of non-inflammatory diarrhea.</a> Stem Cell Res Ther. 4, S3 (2013).</li><li>Fujii, M., Sato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+intestinal+stem+cells:+applications+for+colorectal+cancer+research.">Culturing intestinal stem cells: applications for colorectal cancer research.</a> Front Genet. 5, 169 (2014).</li><li>Koo, B. K., Sasselli, V., 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=Retroviral+gene+expression+control+in+primary+organoid+cultures.">Retroviral gene expression control in primary organoid cultures.</a> Curr Protoc Stem Cell Biol. 27 (Unit 5A), 6 (2013).</li></ol>

The authors have nothing to disclose.

This method provides a complete system reproducing intestinal epithelial lineages and epithelial dynamics which constitute a useful tool to study intestinal epithelial biology. The method presented herein was adapted from the original murine study by Sato and Clevers22 that efficiently results in human enteroids and colonoids. Here, we manually picked up the crypts by microdissection to avoid any cellular contaminants. This method allows a direct visualization of the crypts and leads to consistency overtime compared to the original crypt collection by &#8220;shaking&#8221;. Other groups developed similar technics using slightly different approaches especially replacing the chelation by EDTA with collagenase25. Besides the differences in crypt collection, those technics use a defined media that is required to grow the human enteroids in culture22. To increase growth efficiency at crypt seeding, we add a GSK3 inhibitor (CHIR99021) for the first two days 12.
The handling of the tissue or biopsies is important and the crypt isolation should be performed as soon as the tissue arrives in the lab. However, delayed crypt isolation and culture could be performed up to 24 hr after tissue collection (data not shown) as previously described for murine tissue26. The intestinal tissue should be placed in a conical tube completely filled with DPBS to avoid tissue disruption and should be maintained at 4&#176;C. The delayed preparation allows for tissue shipping but temperature variation should be avoided during transport. The overall time needed for the initial crypt plating is approximately 2 hr with 15 to 30 min to process the tissue and 1 to 2 hr to isolate and plate the crypt. The microdissection of the tissue is a critical determinant and predicate of a clean crypt preparation. However, the crypt release by hand-shaking as described in various protocols is possible22,23.
Despite the similarities with the murine enteroid (enteroids) system, the human enteroids require specific molecules to enhance and sustain their growth over time. The growth factors, Egf, Noggin, R-spondin are used similarly to the murine epithelial organoids. However, the use of Wnt-3A is critical. We noted that the formation as well as the growth efficiency is greater using a Wnt-3A conditioned medium than the human recombinant protein. Concomitantly, we demonstrated improved culture conditions using an inhibitor of the glycogen synthase kinase 3 (CHIR99021)12. Recombinant growth factors could be replaced by Wnt-3A, R-spondin, and Noggin conditioned-media. A Wnt-3A-expressing L-cell line is commercially available (ATCC). Other groups have developed R-spondin 1&#8211;23,27, Noggin-19 , and Wnt-3A/R-spondin3/Noggin&#8211;28 expressing cell lines. Two small molecule inhibitors are used in the culture media and are necessary for the maintenance of the culture29. A-83-01 is a selective inhibitor of transforming growth factor &#946; and Activin/Nodal receptors (activin-like kinase 4, 5, 7) and SB202190 is a p38 mitogen-activated protein kinase inhibitor (MAPK). Both inhibitors have been used respectively to sustain human induced pluripotent stem cells self-renewal and to establish human na&#239;ve pluripotent stem cells30-32. Also, nicotinamide, a precursor of nicotinamide adenine dinucleotide, is required to maintain enteroids and colonoids expansion in a long-term manner22,29.
The EDTA chelation is an important step as it determines the yield from the crypt preparation. We have been successful with 2 mM EDTA treatment. However, EDTA concentration can be modified from 2 mM to 15 mM regarding the type of tissue. In that case, time of incubation has to be empirically determined. After the initial plating, the crypt will round-up and eventually form enteroids. However, the enteroids or colonoids often demonstrate a &#8220;stem&#8221; phenotype by forming spheres with little to no differentiated cells. In that case, differentiation can be initiated by withdrawing Wnt-3A, nicotinamide and the p38 MAPK inhibitor. The use of Notch inhibitor such as DAPT or DBZ helps enhancing the differentiation within the enteroids22.
This model recapitulates the intestinal physiology with continual crypt-budding events arising from a stem cell compartment as well as villus-like epithelial domains containing both absorptive and secretory differentiated lineages. Interestingly, this system does not contain any mesenchymal cells and uses specific media conditions to meet the niche signal requirements.
Like the murine model, human enteroids can be generated from isolated intestinal epithelial cells to test the capacity of those cells to function as a stem cell. Several studies have used cluster of differentiation markers (CD44, CD24 or CD166) and EPHB2 positive cells to enrich for cells with stem properties12,23,33. Together, these studies demonstrate the utility of human enteroids cultures for testing the stemness. Other investigators are using this model to investigate intestinal diseases such as infectious diarrheal diseases, cystic fibrosis, or colorectal cancers 22,34-37. These studies demonstrate that human enteroids constitute a reliable human disease model with a possibility to move towards a personalized screening. Human enteroids can be genetically modified using DNA transfection or infection with viral particles38. This provides a powerful tool to study gene-specific functions within the human epithelial organoids or correct genetic mutations. Recently, Schwank and colleagues demonstrated the possibility to edit the genome with the CRISPR/Cas9 system and correct the mutation on the CFTR gene causing a cystic fibrosis24. Human enteroids constitute a valuable system to study intestinal stem-cell and epithelial mucosal biology and serve as a novel experimental system to study both normal and abnormal gastrointestinal physiology.

The lining epithelium of the gastrointestinal tract is in constant renewal. This process is triggered by proliferation of intestinal stem cells (ISCs) which continuously produce progeny to rapidly replace the intestinal epithelium as it turns over. The proliferative compartment comprising the ISCs is confined to the bottom of the crypts. The ISCs give rise to progeny which eventually differentiate into absorptive or secretory lineages. Moving out of the crypt and onto the villus or surface epithelium, cells differentiate progressively as they migrate upwards before exfoliation into the lumen1. ISCs give rise to all intestinal epithelial cell types including enterocytes, microfold cells, enteroendocrine cells, goblet cells, tuft cells and Paneth cells. The colon is characterized by elongated crypts composed mostly of colonocytes and goblet cells, with scattered enteroendocrine and tuft cells2.
Ex vivo culture systems constitute promising tool for studying ISC maintenance and intestinal tissue homeostasis. However, it is difficult to rely on tissue culture technologies as physiological conditions are not fully reproduced and the epithelial microenvironment often altered3,4. A major advancement in ISC field was the establishment of tissue culture techniques to maintain and expand individual murine ISCs using defined growth factors to replace the normal intestinal niche signals. Long-term culture conditions were described by Sato et al., in which single crypts or isolated stem cells from the intestinal epithelium grow to form 3-dimensional epithelial structures including multiple crypt-like domains 5-7. These tridimensional structures undergo fission events to continuously expand. Interestingly, all the intestinal cell types specific to the tissue of origin are produced and as well are extruded into a lumen8. Using modifications of this system, epithelial organoids can be generated from the stomach, small intestine and colon. More specifically, epithelial organoids from the small intestine are enteroids9, and those from the colon are colonoids9,10. These epithelial organoid culture systems have been used to test the ability of isolated single cells to function as stem cells in vitro, thus testing the &#8220;stemness&#8221; of isolated cells5,6,10-15. Other investigators have used both enteroids and colonoids to study the function of individual epithelial cells16-21. Thus, enteroid and colonoid cultures can be used to evaluate both stem and non-stem cell functions and give new insight into fundamental cellular interactions within the intestines.
In 2011, Sato and colleagues generated long-term culture of epithelial organoids derived from human small intestine and colon22,23. Besides differences in media composition, the human epithelial enteroids and colonoids exhibit the same features as their murine counterpart. Furthermore, they can be generated from diseased tissues such as Barrett&#8217;s esophagus, adenoma or adenocarcinoma, and cystic fibrosis22,24. Human enteroids constitute a valuable system to study intestinal stem-cell and epithelial mucosal biology and serve as a novel experimental system to study both normal and abnormal gastrointestinal physiology3.
Here we describe methods to establish enteroids and colonoids from human small intestine and colon crypts (Figure 1). In this methodological review, we emphasize the crypt collection from whole tissue and biopsies. We recapitulate the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids and the possible experimental strategies carried out by this model.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:480px;">Name of Reagent</td>
			<td style="width:195px;">Company</td>
			<td style="width:129px;">Catalog Number</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dulbecco&#8217;s Phosphate buffered saline Ca2+, Mg2+ free (DPBS)</td>
			<td>Life technologies; Gibco</td>
			<td>14190-144</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Fischer Scientific</td>
			<td>BP439-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Fischer Scientific</td>
			<td>BP220-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA) Fraction V</td>
			<td>Fischer Scientific</td>
			<td>BP1600-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gzntamycin/Amphotericin B solution</td>
			<td>Life technologies; Gibco</td>
			<td>R-015-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wnt-3A conditionned medium</td>
			<td>in house</td>
			<td>-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Advanced DMEM/F12</td>
			<td>Life technologies; Gibco</td>
			<td>12634-028</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES 1M</td>
			<td>Life technologies; Gibco</td>
			<td>15630-080</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX (glutamine)</td>
			<td>Life technologies; Gibco</td>
			<td>35050-061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies; Gibco</td>
			<td>15140-148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Life technologies; Gibco</td>
			<td>17502-048</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement</td>
			<td>Life technologies; Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotidamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel, GFR, Phenol free (basement membrane matrix)</td>
			<td>Corning</td>
			<td>356231</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant Noggin</td>
			<td>R&#38;D</td>
			<td>6057-NG/CF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant R-Spondin</td>
			<td>Preprotech</td>
			<td>120-38</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant EGF</td>
			<td>Sigma-Aldrich</td>
			<td>E9644-.2MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503-1MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067-5MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human [Leu]15-Gastrin 1</td>
			<td>Sigma-Aldrich</td>
			<td>G9145-.1MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thiazovivin</td>
			<td>Stemgent</td>
			<td>04-0017</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express Enzyme (1X), phenol red (cell dissociation enzyme)</td>
			<td>Life technologies; Gibco</td>
			<td>12605-010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Life technologies; Gibco</td>
			<td>10082-147</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CTS Synth-a-Freeze Medium (freezing medium)</td>
			<td>Life technologies; Gibco</td>
			<td>A13713-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L Wnt-3A cell line</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Renilla luciferase assay</td>
			<td>Promega</td>
			<td>E2710</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human recombinant Wnt-3A</td>
			<td>R&#38;D</td>
			<td>5036-WN/CF</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">HEK293 TOPflash cell line</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

establishment of human epithelial enteroids and colonoids from whole tissue and biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Figure 2D shows a typical example of freshly isolated crypts from whole tissue (Figure 2D). The number of crypts isolated from a biopsy is lower than in whole tissue. Using standard capacity biopsy forceps with needle, we usually perform two biopsy bites on a single pass. Each biopsy bite results in a 10 mm2 surface with an average of 50 to 100 crypts per biopsy (Figure 2F).
After culture in basement membrane matrix, the crypts round up to form enterospheres for small intestine and colonospheres for colon. The crypt budding usually occurs within 5 to 6 days, after seeding. However it is not unusual to see either enteroids (enteroids) or colonoids (colonoids) forming spheres in the basement membrane matrix (Figure 3A-C; Movie 1). The passaging can be done after 7 days, depending on the size of the enteroids. The enteroids or colonoids established from biopsies undergo the same development in culture. However, as the crypt density at seeding is lower, the passaging is usually done after 10 to 12 days in culture (Figure 4a,b). Enteroids and colonoids cultures expand in a reproducible manner.
Both enteroids and colonoids present a luminal side and are lined with an epithelium (Figure 5A, B). Proliferative cells can be observed within the enteroids and are located within the bud tips (Figure 5C, D). Confocal imaging of enteroids stained with E-cadherin (Ecad) shows the epithelial cells (Figure 5E).
Both enteroids and colonoids can be established from tissue obtained from patients with genetic/congenital disorders. Figure 6 shows representative enteroids growing from a patient with cystic fibrosis (Figure 6A) and a tufting enteropathy due to a congenital mutation in the epithelial cell adhesion molecule gene (EpCAM) (Figure 6B). Beside the genetic defect, the enteroids do not exhibit differences in basal condition.
<img alt="Figure 1" src="/files/ftp_upload/52483/52483fig1highres.jpg" /> 
Figure 1.&#160;Workflow of crypts dissociation and generation of human enteroids and colonoids in culture. Crypts (from human small intestine or colon) are isolated by EDTA chelation. Cultured crypts form enteroids for the small intestine and colonoids for the colon. <a href="https://www.jove.com/files/ftp_upload/52483/52483fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52483/52483fig2highres.jpg" /> 
Figure 2.&#160;Dissection process for the crypt isolation. (A) The small intestine specimen is stretched and pinned flat into a silicone-coated Petri dish. (B) The mucosa is separated from the underlying submucosa. (C) The dissected mucosa is stretched and pinned flat into a silicone-coated Petri dish. (D) After EDTA chelation, the crypts are isolated from the tissue. (E) One biopsy is stretched and pinned flat into a silicone-coated Petri dish. (F) After EDTA chelation, crypts are isolated from the biopsy. <a href="https://www.jove.com/files/ftp_upload/52483/52483fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52483/52483fig3highres.jpg" /> 
Figure 3.&#160;Crypt culture and human enteroid and colonoid generation from whole tissue. (A) Jejunal crypts plated in basement membrane matrix after isolation. The crypts are closing up after 3 to 4 hr and start to balloon up to form enterospheres beyond this time. At 7 days, the jejunal enteroids are formed. (B) After isolation and culture, ileal crypts behave like the jejunal crypts and form ileal enteroids. (C) Colonic crypts are plated in basement membrane matrix after isolation. The crypts close and form colonoids after 7 days (Scale bars: 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52483/52483fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52483/52483fig4highres.jpg" /> 
Figure 4.&#160;Crypt culture and human enteroid and colonoid generation from biopsy. (A) Duodenal crypts plated in basement membrane matrix after isolation. The crypts are closing up after 3 to 4 hr and start to balloon up beyond this time to form enterospheres. At 7 days, the enteroids are formed. (B) After isolation and culture, colonic crypts are plated in basement membrane matrix. The crypts close up to form colonospheres then colonoids after 7 days (Scale bars: 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52483/52483fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52483/52483fig5highres.jpg" /> 
Figure 5.&#160;Intestinal lineages of the human enteroids. (A) Human enteroid after 6 days in culture. (B) Hematoxylin-eosin sections of the enteroids in (A) demonstrate the epithelial lining (Scale bars: 100 &#181;m). (C-D) Confocal imaging of enteroids after EdU staining (magenta) shows the presence of proliferative cells. (E) Confocal imaging of enteroids demonstrates the presence of: E-cadherin for epithelial cells (ECAD, green) (Scale bar: 50 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52483/52483fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52483/52483fig6highres.jpg" /> 
Figure 6.&#160;Crypt culture and human enteroid generation from diseased tissue. (A) Enteroids established from a cystic fibrosis specimen. (B) Enteroids established from a congenital tufting enteropathy specimen (Scale bars: 100 &#181;m). <a href="https://www.jove.com/files/ftp_upload/52483/52483fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Movie 1.&#160;Enterosphere forming human enteroid in culture. 32 hr time-lapse movie shows an enterosphere established from human small intestine retracting to form an enteroid in culture. <a href="https://www.jove.com/files/ftp_upload/52483/Movie1_v2.mp4" target="_blank">Please click here to view this video.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Solvent</td>
			<td>Stock Concentration</td>
			<td>Final Concentration</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate buffered saline Ca2+, Mg2+ free (DPBS)</td>
			<td>Life technologies; Gibco</td>
			<td>14190-144</td>
			<td>-</td>
			<td>-</td>
			<td>1x</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine 
			tetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>431788</td>
			<td>Ultrapure dH2O</td>
			<td>0.5 M</td>
			<td>2 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Fischer Scientific</td>
			<td>BP439-500</td>
			<td>DPBS</td>
			<td>Powder</td>
			<td>2%</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fischer Scientific</td>
			<td>BP220-1</td>
			<td>DPBS</td>
			<td>Powder</td>
			<td>1%</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA) Fraction V</td>
			<td>Fischer Scientific</td>
			<td>BP1600-100</td>
			<td>DPBS</td>
			<td>Powder</td>
			<td>1%</td>
			<td></td>
		</tr>
		<tr>
			<td>Gzntamycin/Amphotericin B solution</td>
			<td>Life technologies; Gibco</td>
			<td>R-015-10</td>
			<td>-</td>
			<td>500x</td>
			<td>1x</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt-3A conditionned medium</td>
			<td>in house</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life technologies; Gibco</td>
			<td>12634-028</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Life technologies; Gibco</td>
			<td>15630-080</td>
			<td>-</td>
			<td>1 M</td>
			<td>10 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX (glutamine)</td>
			<td>Life technologies; Gibco</td>
			<td>35050-061</td>
			<td>-</td>
			<td>100X</td>
			<td>1X</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies; Gibco</td>
			<td>15140-148</td>
			<td>-</td>
			<td>100X</td>
			<td>1X</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Life technologies; Gibco</td>
			<td>17502-048</td>
			<td>-</td>
			<td>100X</td>
			<td>1X</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Life technologies; Gibco</td>
			<td>17504-044</td>
			<td>-</td>
			<td>50X</td>
			<td>1X</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td>DPBS</td>
			<td>1 M</td>
			<td>1 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotidamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td>DPBS</td>
			<td>1 M</td>
			<td>10 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, GFR, Phenol free (basement membrane matrix)</td>
			<td>Corning</td>
			<td>356231</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>REQUIRED</td>
		</tr>
		<tr>
			<td>human recombinant Noggin</td>
			<td>R&#38;D</td>
			<td>6057-NG/CF</td>
			<td>DPBS</td>
			<td>100 &#956;g /ml</td>
			<td>100 ng/ml</td>
			<td rowspan="3">Other suppliers: R&#38;D; Anaspec and Preprotech</td>
		</tr>
		<tr>
			<td>human recombinant R-Spondin</td>
			<td>Preprotech</td>
			<td>120-38</td>
			<td>DPBS</td>
			<td>1 mg/ml</td>
			<td>1 &#956;g/ml</td>
		</tr>
		<tr>
			<td>human recombinant EGF</td>
			<td>Sigma-Aldrich</td>
			<td>E9644-.2MG</td>
			<td>DBPS</td>
			<td>500 &#956;g/ml</td>
			<td>50 ng/ml</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503-1MG</td>
			<td>DPBS</td>
			<td>10 mM</td>
			<td>10 &#956;M</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>DMSO</td>
			<td>500 &#956;M</td>
			<td>500 nM</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067-5MG</td>
			<td>DMSO</td>
			<td>30 mM</td>
			<td>10 &#956;M</td>
			<td></td>
		</tr>
		<tr>
			<td>human [Leu]15-Gastrin 1</td>
			<td>Sigma-Aldrich</td>
			<td>G9145-.1MG</td>
			<td>DPBS</td>
			<td>100 &#956;M</td>
			<td>10 nM</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>DMSO</td>
			<td>10 mM</td>
			<td>2.5 &#956;M</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiazovivin</td>
			<td>Stemgent</td>
			<td>04-0017</td>
			<td>DMSO</td>
			<td>10 mM</td>
			<td>2.5 &#956;M</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1X), phenol red (cell dissociation enzyme)</td>
			<td>Life technologies; Gibco</td>
			<td>12605-010</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>For passaging</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life technologies; Gibco</td>
			<td>10082-147</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>For passaging</td>
		</tr>
		<tr>
			<td>CTS Synth-a-Freeze Medium (freezing medium)</td>
			<td>Life technologies; Gibco</td>
			<td>A13713-01</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>For freezing</td>
		</tr>
		<tr>
			<td>L Wnt-3A cell line</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>Wnt-3A conditionned media production</td>
		</tr>
		<tr>
			<td>Renilla luciferase assay</td>
			<td>Promega</td>
			<td>E2710</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>Wnt-3A conditionned media activity</td>
		</tr>
		<tr>
			<td>human recombinant Wnt-3A</td>
			<td>R&#38;D</td>
			<td>5036-WN/CF</td>
			<td>DPBS</td>
			<td>100 &#956;g /ml</td>
			<td>100 ng/ml</td>
			<td>Wnt-3A conditionned media activity</td>
		</tr>
		<tr>
			<td>HEK293 TOPflash cell line</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>Wnt-3A conditionned media activity; Gift from Hans Clevers laboratory</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Detailed reagent list with preferred manufacturer and catalog number.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td>Consumable</td>
			<td>Tools</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>15- and 50 ml conical tubes</td>
			<td>Dumont #5 standard forceps (F.S.T.; #11251-20)</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Microfuge tubes</td>
			<td>Dumont #7, curved fine forceps (F.S.T.; #11274-20)</td>
		</tr>
		<tr>
			<td>Stereo-microscope</td>
			<td>24-well plates</td>
			<td>Fine scissors (F.S.T.; #14060-09)</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>0.22 &#956;m filters (Sartorius)</td>
			<td>Vannas spring scissors (F.S.T.; #15018-10)</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Serological pipettes</td>
			<td>0.2 mm diameter minutien pins (F.S.T.; #26002-20)</td>
		</tr>
		<tr>
			<td>Freezing container (Nalgene)</td>
			<td>Micropipette tips</td>
			<td>0.1 mm diameter minutien pins (F.S.T.; #26002-10)</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone (Dow Corning)</td>
			<td>150 &#956;m mesh openings, nylon screening (Dynamic Aqua-supply)</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dish</td>
			<td>5 ml round-bottom polypropylene tubes (Falcon)</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettor</td>
			<td>18G blunt fill needle (BD)</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>3 ml lsyringes with Luer-Lock tips (BD)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Cryovials</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Detailed consumables, tools, and equipment needed for the crypt isolation and culture.

Watch this Scientific Journal Video about Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy at JoVE.com

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

establishment-human-epithelial-enteroids-colonoids-from-whole-tissue

Research

JoVE Journal

Medicine

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

3D Whole-heart Myocardial Tissue Analysis

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other biologicals that enhance angio- or vasculogenesis into the infarct border zone (IBZ), tissue perfusion is improved, and the myocardium can be protected from further damage. For maximum therapeutic effect, it is hypothesized that the regenerative substance is best delivered to the IBZ. This requires accurate injections and has led to the development of new injection techniques. To validate these new techniques, we have designed a validation protocol based on myocardial tissue analysis. This protocol includes whole-heart myocardial tissue processing that enables detailed two-dimensional (2D) and three-dimensional (3D) analysis of the cardiac anatomy and intramyocardial injections. In a pig, myocardial infarction was created by a 90-min occlusion of the left anterior descending coronary artery. Four weeks later, a mixture of a hydrogel with superparamagnetic iron oxide particles (SPIOs) and fluorescent beads was injected in the IBZ using a minimally-invasive endocardial approach. 1 h after the injection procedure, the pig was euthanized, and the heart was excised and embedded in agarose (agar). After the solidification of the agar, magnetic resonance imaging (MRI), slicing of the heart, and fluorescence imaging were performed. After image post-processing, 3D analysis was performed to assess the IBZ targeting accuracy. This protocol provides a structured and reproducible method for the assessment of the targeting accuracy of intramyocardial injections into the IBZ. The protocol can be easily used when the processing of scar tissue and/or validation of the injection accuracy of the whole heart is desired.

This protocol describes a novel method for the 3D comparison of whole-heart myocardial tissue with MRI. This is designed for the accurate assessment of intramyocardial injections in the infarct border zone of a chronic porcine model of myocardial infarction.

Cardiac regenerative therapies aim to protect and repair the injured heart in patients with ischemic heart disease. By injecting stem cells or other ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.