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.
