gastric fundic glands isolation: a method to isolate fundic glands from human stomach tissue

Gastric Fundic Glands Isolation: A Method to Isolate Fundic Glands from Human Stomach Tissue

To begin, collect the prepared tissue in a large plastic container filled with calcium/magnesium-free DPBS. Leave the plastic container on ice until use. Next, with the help of forceps wash the tissue vigorously in a sterile beaker, containing 100 milliliters of calcium/magnesium-free DPBS, for about 30 seconds. Then, use a sterile gauze to wipe away the mucus layer from the epithelium.
Next, use large forceps to hold the muscle layer of the tissue firmly. Then, use large curved hemostatic forceps and scrape the epithelium with force. Store the scraped epithelium in a sterile polystyrene Petri dish. To optimize the tissue digestion, mince the epithelial tissue into fragments. Then, wash the tissue with calcium/magnesium-free DPBS supplemented with antibiotics.
Wash multiple times to remove the blood. Discard the wash in a waste beaker. Then, collect the washed epithelial fragments in a 125-milliliter glass round bottom flask with a 25-milliliter stir bar and pre-warmed incubation medium. After collecting the tissue fragments, seal the round bottom flask with a rubber septa. Then, insert a 20G spinal needle in the septa. Connect the septa to an oxygen tank with rubber hosing.
Insert 10 to 15 outflow needles into the septa to prevent its rupture and then use clamps to secure the setup to a ring stand. Then, switch on the oxygen outflow on low. After this, calibrate the water bath at 37 degrees Celsius on a stir plate for 30 to 45 minutes. Once calibrated, place the setup in the water bath. After 15 minutes of incubation, check for dissociated glands.
After the desired incubation time, add 50 milliliters of preheated DMEM/F-12 to the incubation medium using a serological pipette. Next, filter the gland mixture obtained through a sterile gauze in four 50-milliliter conical tubes. Then, incubate the filtrate on ice for 15 minutes. This will allow the gland to settle to the bottom of the conical tube. Use a serological pipette to discard 40 milliliters of the supernatant from the top of the gland suspension.
Resuspend the remaining 10 milliliters of the gland mixture in 10 milliliters of DPBS supplemented with antibiotics. Then, distribute the entire mixture into 5-milliliter cell culture test tubes. Centrifuge the tubes at 65 x g for 5 minutes at 4 degrees Celsius. After centrifugation, carefully remove the supernatant.

gastric-fundic-glands-isolation-method-to-isolate-fundic-glands-from

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Assays and techniques

EoE Sub Articles

eoe sub articles

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Establishing Human-derived 3D Gastric Fundic Organoids
NOTE: To avoid contamination, perform protocol in its entirety within a sterile tissue culture hood. Human fundic tissue was collected from patients undergoing sleeve gastrectomy.

<ol>
	<li>Collect the tissue from patients undergoing sleeve gastrectomy and place it in the ice-cold Ca2+/Mg2+-free Dulbecco&#39;s Phosphate Buffered Saline (DPBS) in a large plastic container.</li>
	<li>Using forceps, wash the tissue vigorously in a sterile beaker containing 100 mL Ca2+/Mg2+-free DPBS. Wash for approximately 30 s or until the debris and the blood has been removed. Next, using sterile gauze, wipe away the mucous layer from the epithelium.</li>
	<li>Using large forceps, firmly grasp the muscle layer of the tissue and, with force, scrape away the epithelium using the large curved hemostatic forceps. Collect the scraped epithelial fragments into a sterile, polystyrene Petri dish.</li>
	<li>Mince the epithelial tissue into approximately 2.0 x 2.0 mm2 sized fragments using razors to optimize tissue digestion. Wash the tissue fragments with Ca2+/Mg2+ -free DBPS supplemented with antibiotics (Ca2+/Mg2+ -free DPBS, 1% Penicillin/Streptomycin, 0.25 mg/mL Amphotericin B /10 mg/mL Gentamicin, 50 mg/mL Kanamycin), until the wash is free of blood. Perform multiple washes if needed. Discard off the wash by carefully filtering the wash through a sterile gauze into a waste beaker.</li>
	<li>Collect washed fragments into a 125 mL glass round bottom flask with a 25 mm stir bar and pre-warmed incubation media (basal media supplemented with 2 mM L-glutamine, 1% Penicillin/Streptomycin, 10 mM HEPES Buffer, 1 mg/mL Collagenase Type 1, 2 mg/mL cell culture grade Bovine Serum Albumin). Seal the round bottom flask with rubber septa.</li>
	<li>Insert a 20G spinal needle into the septa and connect it to an oxygen tank with rubber hosing. To filter out any contaminants, insert tissue paper into the tubing to create a filter. Turn on the oxygen outflow on low. Insert 10&#8211;15 outflow needles into the septa to avoid the rupture of septa.</li>
	<li>Secure the set up to a ring stand using clamps and place it in a water bath calibrated to 37 &#176;C with a stir plate for 30&#8211;45 min. After the tissue has been incubating for 15 min, remove 50 &#181;L of incubation media and check visually for dissociated glands. If glands are not dense or have not separated from the tissue, leave for an additional 5&#8211;10 min.</li>
	<li>Immediately following incubation, add 50 mL pre-heated DMEM/F-12 to incubation media using a sterile serological pipette or by pouring directly into the incubation mixture.</li>
	<li>Filter the gland mixture through a sterile gauze into four 50 mL conical tubes. Keep the filtrate on ice for 15 min to allow extracted glands to settle to the bottom of the conical tube. Be careful not to disturb the settled glands; remove and discard off the top 40 mL of supernatant using a serological pipette. Resuspend the remaining 10 mL in 10 mL of DPBS supplemented with antibiotics.</li>
	<li>Distribute the mixture evenly into 5 mL cell culture test tubes. Centrifuge for 5 min at 65 x g at 4 &#176;C and remove the supernatant carefully using a pipette. 
	NOTE: It is crucial to perform this step on the ice and to avoid polymerization of the basement membrane matrix while mixing. Additionally, it is important to visually inspect the gland density by sampling for 50 &#181;L. The optimal density is ~70% confluency.</li>
	<li>Next, plate the glands in 50 &#181;L of the basement membrane matrix using a wide-tipped pipette. Avoid producing any bubbles while plating.</li>
	<li>To allow basement membrane matrix to polymerize, incubate for 10 to 15 min at 37 &#176;C. If plating in a 12-well culture plate, add 1 mL of hFGO media (Advanced Dulbecco&#39;s modified Eagle medium/F-12 medium supplemented with 2mM L-glutamine, 1% Penicillin/Streptomycin, 0.25 mg/mL Amphotericin B /10 mg/mL Gentamicin, 50 mg/mL Kanamycin, 10 mM HEPES Buffer, 1 mM n-Acteylcystine, 1 x N2, 1 x B27, 50% Wnt-conditioned medium, 20% R-spondin-conditioned medium supplemented with 100 ng/mL bone morphogenetic protein inhibitor, 1 nM gastrin, 50 ng/mL Epidermal Growth Factor, 200 ng/mL Fibroblast Growth Factor 10, 10 mM Nicotinamide, and 10 &#181;M Y-27632 ROCK inhibitor) per well. If not using a 12-well culture plate, add enough media to submerge the basement membrane matrix bubble.</li>
	<li>Culture the cells at 37 &#176;C in a 5% CO2&#160;humidified cell culture incubator. Change media every 4-5 days.</li>
</ol>

<table><tbody><tr><td>Ca2+/Mg2+ -free DPBS</td><td>Fisher&amp;nbsp;</td><td>21-031CV</td><td></td></tr><tr><td>Sterile gauze</td><td></td><td></td><td></td></tr><tr><td>Curved hemostatic forceps</td><td></td><td></td><td></td></tr><tr><td>Sterile Petri dish</td><td></td><td></td><td></td></tr><tr><td>Sterile razor blades</td><td></td><td></td><td></td></tr><tr><td>Advanced Dulbecco&#039;s Modified Eagle/F12 medium (basal media)</td><td>Life Technologies&amp;nbsp;</td><td>12634-010</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Scientific</td><td>SV30010</td><td></td></tr><tr><td>Amphotericin B/Gentamicin</td><td>Thermo Scientific</td><td>R01510</td><td></td></tr><tr><td>GlutaMAX (L-glutamine)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Kanamycin</td><td>Thermo Fisher&amp;nbsp;</td><td>RO1510</td><td></td></tr><tr><td>Collagenase Type 1&amp;nbsp;</td><td>Worthington</td><td>LS004214</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma Aldrich</td><td>A9418</td><td></td></tr><tr><td>HEPES buffer&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>H0887</td><td></td></tr><tr><td>125 mL Round bottom flask</td><td></td><td></td><td></td></tr><tr><td>1 in (25 mm) Stir bar</td><td></td><td></td><td></td></tr><tr><td>Rubber Septa</td><td></td><td></td><td></td></tr><tr><td>20G Spinal needles of 3.5 in length&amp;nbsp;</td><td>Thermo Fisher&amp;nbsp;</td><td>405182</td><td></td></tr><tr><td>Kimwipes (tissue paper)</td><td>Fischer Scientific&amp;nbsp;</td><td>06-666C</td><td></td></tr><tr><td>Oxygen Tank with rubber hosing</td><td></td><td></td><td></td></tr><tr><td>Ring Stand Clamps</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C Water bath</td><td></td><td></td><td></td></tr><tr><td>5 mL Cell culture test tubes&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>14-956-3C</td><td></td></tr></tbody></table>

Source: Teal, E. et al. <a href="https://www.jove.com/video/57435" target="_blank">Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration.</a> J. Vis. Exp. (2018)
This video describes a method to isolate gastric epithelial cells from the human fundic tissue. The isolated epithelial cells can be used as an in vitro model to study their regulatory roles in gastric physiology and pathophysiology.

Source: Teal, E. et al. Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration. J. Vis. Exp. (2018)
This ...

In humans, the fundus is the uppermost part of the stomach. Fundic epithelium consists of numerous gastric glands that secrete gastric juice, which aids in digestion.
To isolate the gastric fundic glands, begin by taking human fundic tissue in a beaker. Add a suitable buffer to the beaker and wash the fundic tissue vigorously. Washing removes the contaminating debris and blood from the fundic tissue.
Transfer the clean tissue to a Petri plate. Using sterile gauze, wipe away the protective mucous layer, thus exposing the underlying epithelial layer. Using forceps, forcefully scrape the uppermost epithelial layer from the tissue.
Transfer the scraped epithelium to another Petri plate and mince it into smaller pieces. Add the minced pieces to a flask containing a buffer supplemented with collagenase enzymes. Incubate at the desired temperature with constant agitation to prevent clumping.
Collagenase digests the extracellular matrix proteins present in the epithelial layer, releasing the dissociated fundic glands into suspension. Filter the suspension into a fresh tube to remove any undissociated clumps.
Keep the glandular filtrate undisturbed on ice to sediment the glands. Discard the supernatant containing cell debris and resuspend the glandular sediment in a fresh buffer. Centrifuge and store the glandular pellet until further use.

1.1K ビュー. 胃底腺の分離:ヒトの胃組織から眼底腺を分離する方法

Gastric Fundic Glands Isolation: A Method to Isolate Fundic Glands from Human Stomach Tissue

記録

Gastric Fundic Glands Isolation: A Method to Isolate Fundic Glands from Human Stomach Tissue