The essenTials of life science ReseaRch. Globally DeliveReD™. ATCC® STem Cell CulTure Guide tips and techniques for culturing stem cells. Growing cells in the laboratory is known as cell culture. Human embryonic stem cells (hESCs) are generated by transferring cells from a preimplantation-stage. Embryonic stem cells have the ability to differentiate into more cell types than adult stem cells. Differentiation is triggered by various factors in vivo, some of which can be replicated in in vitro stem cell cultures. The nature of stem cells necessitates the use of special stem cell culture media and reagents.Stem Cell Culture Learning · Pluripotent Stem Cell Culture · EBiSC.
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The culture confluence determines when hPSC colonies should be passaged: Stem cell culture of the method used, it is prudent to set aside one well of cells from the same culture used for passaging for an additional 1—2 days in case of errors or contamination during passaging.
Calculate how stem cell culture medium and solution are needed for this process For harvesting and plating hPSCs on iMEFs, the following are required: Return the plates to the incubator if there is no visible contamination.
Take the approved iMEF plates and place them in the biosafety cabinet. Label the plates with the cell line name, new passage number, the date and initials.
Completely aspirate the medium from the iMEF plate s with a Pasteur pipette.
Cell Culture Media and Supplements Overview
Stem cell culture swirl the stem cell culture to rinse the well walls. Return the plate s to the incubator. Take the hPSC plate s from the incubator and evaluate under a microscope. If using a colony marker, mark the differentiated colonies on the bottom of the plate and place in the biosafety cabinet.
Using a Pasteur pipette, aspirate all marked differentiated cells in the plate by touching the colonies with the tip of the pipette. The differentiated areas will be sucked away leaving the undifferentiated colonies in the stem cell culture.
Aspirate the remaining medium along with the floating differentiated cells or cell debris using a straight Pasteur pipette.
Set the timer at 3 minutes and 10 minutes, respectively. Start the timer After 3 minutes stem cell culture incubation, check the plate s under a microscope to determine whether the cell colonies are ready to be harvested.
If stem cell culture are ready, the perimeter of the colony should appear folded back, separating from the iMEFs. Colonies before a and after collagenase treatment b. Colonies are ready to be harvested if edges of colonies are clearly curled and lifted off feeder layer arrow, b.
Incubation length depends on the freshness of the collagenase solution. The stem cell culture it is, the longer the incubation time will be. However, do not exceed 15 minutes of incubation time.
When stem cell culture incubation is complete, aspirate collagenase from the wells in the biosafety cabinet. Add 2 ml culture medium to each well in the plate s.
Using a sterile 5 ml glass pipette, take up most of the medium from one well. Pipette up and down gently to minimize bubbles.
Stem Cell Basics III.
Leave the contents in the well until cells in all of the wells are detached. Rinse all the wells in the plate with another 3 mls of CM. If there are more plates, harvest remaining cells into the same tube. These stem cells can become any stem cell culture in the body, excluding a placenta.
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