Name: From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs (Video) | JoVE Journal
Uploaded: 2008-06-04T23:27:00.000+00:00
Duration: 6 min 29 s
Description: Learn to culture human embryonic stem cells on mouse embryonic fibroblast feeder cells and maintain pluripotency using feeder-free matrigel conditions. This protocol guides researchers through essential stem cell biology techniques.

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5 minutes

Human embryonic stem cell studies in the Teitell lab are supported by a California Institute for Regenerative Medicine (CIRM) seed grant RS1-00313. We thank members of the Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, especially Dr. Amander Clark, Dr. Jerome Zack, and members of the UCLA Broad Institute Stem Cell Core Facility for their support of our studies.

Splitting human embryonic stem cells (hESCs) plated on mouse embryonic fibroblasts (MEFs)
Usually a confluent hESC plate can be split 1:6 to 1:10, depending on the particular hESC line. The split plate will become confluent again 5-7 days after splitting.
<ol>
<li>Two days before splitting, gelatinize plates using a 0.1% gelatin solution. For a 6-well plate, add 2ml into each well and incubate plate in a 37&deg;C, 5% CO2 tissue culture incubator overnight.</li>
<li>Plate MEFs on gelatinized 6-well plates the day before you plan on splitting the hESCs. (NOTE: MEFs can be plated as many as 3 days earlier if needed. After ~ 3 days, MEFs become flatter and more spread-out and cannot sustain hESCs as well as fresher MEFs can.) Take a vial of g-irradiated or mitomycin C treated CF1 MEFs, containing 5-6 x 106 cells, from liquid nitrogen and thaw for 2 min in a 37&deg;C water bath. Wash cells once with warm MEF Culture Media in a 50ml falcon tube and resuspend in the final volume of warm MEF Culture Media. At 5-6 x 106 cells, this is enough to plate two or three 6-well plates.</li>
<li>While the MEFs are being washed, take out the gelatinized plates, remove all solution from the wells and add 2.5 ml of MEF Culture Media per well. </li>
<li>Add 0.5 ml of MEF suspension per well to achieve 3ml as a final volume in each well. Make sure MEFs are evenly dispersed and place plates back in the incubator overnight to settle.</li>
<li>On the day of hESC splitting, prepare fresh or use &lt; 2 week-old sterile collagenase IV solution at 1mg/ml. Remove all the media from the hESC wells you want to split, wash once with 2ml per well of warm 1&times;PBS, pH 7.4, and add 1ml of collagenase IV solution. Incubate at 37&deg;C for 5-10 min. Take the 6-well hESC plate out of the incubator and add 1ml of ES media (without bFGF) to each well. Using the solution in each well, with a 1ml pipette suck up the media and blow the stem cells off of the plate. For each well this will take about 5 to 10 repetitions. Then, transfer the suspended hESCs into a 50ml falcon tube. Do this for all the wells being split and combine in one 50ml falcon tube. Pellet the hESCs at room temperature at 200g for 5 min and wash one time with ES media lacking bFGF.</li>
<li>While the hESCs are being washed, take out the MEF plates, remove all media from the wells and wash once with sterile, warm 1&times;PBS, pH 7.4 then add 2.5 ml of ES media (now supplemented with 10 ng/ml bFGF) per well.</li>
<li>Resuspend the pelleted hESCs in an appropriate volume of ES media, supplemented with 10ng/ml bFGF. (NOTE: When the hESCs are resuspended, they should be of roughly uniform colony size and shape. The resuspension volume depends on the splitting ratio.) Carefully pipette the hESC suspension up and down a few times to make the colonies smaller and more uniform, but not so much that single cells or very small colonies are generated. Add 0.5 ml of hESC suspension per well to the MEF plates to achieve 3 ml as a final volume in each well. Visually check to make sure that the hESCs are distributed evenly before placing the plates back in the incubator overnight to settle. After plating, it will usually take a few days for colonies to take on their characteristic shape and border appearance.</li>
</ol>

<ol><li>Thomson, J. amesA., Itskovitz-Eldor, J. oseph, Shapiro, S. anderS., Waknitz, M. ichelleA., Swiergiel, J. enniferJ., Marshall, V. ivienneS., Jones, J. effreyM. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts%5BTitle%5D)+AND+%22Science+%22%5BJournal%5D)">Embryonic Stem Cell Lines Derived from Human Blastocysts.</a> Science . 282, 1145-1147 (1998).</li>
<li>Xu, C. hunhui, Inokuma, M. argaretS., Denham, J. errod, Golds, K. athaleen, Kundu, P. ratima, Gold, J. osephD., Carpenter, M. elissaK. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feeder-free+growth+of+undifferentiated+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Nature+Biotechnology+%22%5BJournal%5D)">Feeder-free growth of undifferentiated human embryonic stem cells.</a> Nature Biotechnology . 19, 971-974 (2001).</li>
</ol>

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. In the last step before plating, when the hESCs are resuspended, they should be of roughly uniform colony size and shape. Carefully pipette the hESC suspension up and down a few times to make the colonies smaller and more uniform, but not so much that single cells or very small colonies are generated.Immunofluorescence staining and microscopy or flow cytometry for hESC pluripotency markers, such as Oct...

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from mefs to matrigel i: passaging hescs in the presence of mefs

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

Due to their significant potential in regenerative medicine, increasing attention is being focused on the biology and manipulation of human embryonic stem cells. In this video, we show you how to grow and maintain human embryonic stem cells in a pluripotent state using mouse embryonic fibroblast feeder cells, and feeder free conditions on matrigel. Hi, I am Jinjiang from a laboratory micro title in the Department of Pathology and Laboratory Medicine at University of California Los Angeles.Today we'll show you a procedure for culturing human embryonic stem cells on mouth embryonic fiber, blood feeder cells, and in feeder free conditions on metri gel. This procedure involves preparing mouth embryonic fibroblast cells generating meth condition media for growth on metri gel preparation of Magel place and masses for feeding and growing human Embry stem cells over time. So let's get started.To begin the culture procedure, human emry like stem cells are plated on mouse embryonic, fibroblasts, or mes to split human embryonic stem cells plated on mes. We start with confluent human embryonic stem cells and plate at a dilution of one to six, to one to 10, depending on the particular embryonic stem cell line. This will become confluent again in five to seven days after splitting before actually splitting the cells Prepared gelatinized plates two days in advance by using a 0.1%gelatin solution from checon.If you're using a six well plate add two mills into each well and let it sit in a 37 degree 5%CO2 tissue culture incubator overnight. On the next day plate maths onto Gelatinized. Six well plates masks can be plated as many as three days earlier if needed.However, it is not recommended to use cells older than three days. Take a stock vial of irradiated or mitomycin C treated CF OneFS containing five to six times 10 to the six cells from liquid nitrogen and thaw for two minutes in a 37 degree Celsius water bath. After the cells have thawed wash them once with warm meth media in a 15 milliliter Falcon tube with a total number of five to six times 10 to the six cells.One can see two or three six well plates with three mils of media in each. Well make sure the mes are evenly dispersed afterwards. You can place the plate back in the incubator overnight.On the day you are going to split the human embryonic stem cells. Prepare fresh sterile collagenase four solution at one mg per mil concentration. Note, the collagenase solution can be stored at four degrees Celsius and used for no longer than two weeks.Remove all the media from the human embryonic stem cell wells. You want to split and watch each well once with two mills of warm one XPDS pH 7.4. Next, add one milliliter of collagenase four solution and incubate the cells at 37 degrees Celsius for five to 10 minutes.After incubation, add one milliliter of stem cell media without basic fibroblast growth factor or BFGF to each well then stem cells can be lifted from the bottom of the wells by simply using a one milliliter pipette to pipet the medium already in the well up and down for each well. This will take about five to 10 repetitions. Afterwards, transfer the suspended human embryonic stem cells into a 15 milliliter falcon tube.After you've collected the cells, pellet the stem cells by centrifugation at room temperature at 200 times G for five minutes. Then wash one at a time with stem cell media lacking BFGF while the human embryonic stem cells are being washed. Take out the meth plates.Remove all the media from the wells and wash once with sterile warm one XPBS pH 7.4. Then add 2.5 milliliters of embryonic stem cell medium. Now supplemented with 10 nanograms per mil BFGF per well.Next resuspend the pelleted human embryonic stem cells in an appropriate volume of stem cell medium, supplemented with 10 nanograms per mil BFGF. The resuspended volume depends on the splitting ratio. Carefully pipette the suspension up and down a few times to make smaller clumps, but not so much that you have single cells or very small colonies.Add 0.5 milliliters of human embryonic stem cell suspension per well to the meth plated wells to achieve a three milliliter final volume in each well. You can visually check to make sure that stem cells are distributed evenly before placing the plates back in the incubator overnight to settle after plating. It will usually take a few days for colonies to take on their characteristic shape and border appearance.

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From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

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