Mouse embryonic stem cell culture

From Biowiki
Jump to: navigation, search

When performed correctly under the optimal conditions, mouse embryonic stem cells can be passaged indefinitely. Stem cell culturing is similar in theory to differentiated cell culturing, but with nuanced differences. The differences stem from the specialized environment, called a niche, which is inhabited by stem cells in vivo. Stem cell niches provide signals required to inhibit cellular differentiation. [1] However, similar to normal cell maintenance there are variations in culture techniques due to personal preferences.

History

In 1970 two articles were published, one from a lab lead by Gordon H. Sato and another by Boris Ephrussi. These two articles showed for the first time how mouse cells from a teratoma could be maintained and produce clonal cells in culture, in vitro. [2] [3] However, these groundbreaking pluripotency studies were undercut by the decrease in differentiation when injected into mice, which accompanied in vitro passaging. Martin Evans solved the problem with differentiation in 1972 by adding an irradiated chick fibroblast feeder layer. [4] Then, it wasn’t until 1981 that Evans and Kaufman were able to demonstrate culturing of pluripotent cells derived from from the inner cell mass of a mouse blastocyst. [5] [6]

Maintenance

Most stem cell research is conducted with mouse embryonic stem (mES) cells due to the controversy regarding human stem cells, which means the process of maintaining a mES cell line is relatively well documented. Cells are almost always maintained in an incubator at 37oC and 5% CO2. To minimize contamination, antibiotics can be added to media, but even with the addition of antibiotics to media, all tissue culture work should be performed under sterile conditions. [7] To prevent differentiation most media is supplemented with some combination of fetal bovine serum (FBS), leukemia inhibitory factor (LIF) and/or a two-inhibitor solution (2i). [8] LIF serves as substitute for feeder layers that can be a potential source of contamination. [9] FBS is used to provide necessary growth factors and should be certified to be ES cell quality, which indicates that it was screened for optimal ES cell conditions. [10] 2i was discovered in 2008 to be useful in mES cell culture as a substitute for FBS. The two drugs inhibit mitogen-activated protein kinase and glycogen synthase kinase-3. [11] There is currently a debate over whether 2i + LIF or FBS + LIF is superior in terms of maintenance of pluripotency, proliferation, cost and other experimental factors.[12] [13]

Passaging

Passaging of mouse embryonic stem (mES) cells requires greater attention because ES cells have a condensed cell cycle compared to differentiated cells and if the confluency gets too high then the pluripotent cells will start to differentiate. [14] When working with suspension cultures, passaging generally requires removing a predetermined quantity of cells and adding the volume to fresh media. [15] If not using a suspension culture, prior to passaging the plates or flasks should be covered in 0.1% gelatin that is necessary to help the mES cells adhere and should be removed after a period of time. The spent medium is aspirated or dumped off and the cells are washed with phosphate-buffered saline (PBS). Then, in order to get the cells into suspension, an enzyme called accutase or trypsin is typically used to break down the extracellular matrix while the cells are in an incubator at 37oC. However, timing is critical because if cell surface receptors are cleaved then mES cells could begin to differentiate from a lack of signals. [16] Gentle tapping can be used as necessary to help cells de-adhere. The dissociation agent is inactivated by the addition of FBS containing media, which means that if using serum-free media, then the ES cells do not need to be washed first. All trypsin or accutase containing solution should be removed and a fraction of resuspended cells should be added to complete the passaging process. [17] A hemocytometer may be used to count cells, which allows for approximate production of a desired concentration of cells per milliliter. [18]

Differentiation

Mouse embryonic stem cells have the potential to become any cell type from the three germ layers, mesoderm, endoderm and ectoderm. Even with media containing LIF, 2i and FBS, some small fraction of cells will start to spontaneously differentiate, especially in overly confluent cultures. [19] Functional differentiation can be achieved by using media without 2i or LIF and specific cell types can be obtained by altering the maintenance conditions, such as by forming embryoid bodies. [20]

References

  1. PMID 18295578 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  2. PMID 4936569 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  3. PMID 5514468 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  4. PMID 4672577 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  5. PMID 7242681 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  6. PMID 25890180 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  7. PMID 12480926 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  8. PMID 24339907 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  9. PMID 3143916 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  10. PMID 15110706 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  11. PMID 18497825 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  12. PMID 25720369 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  13. PMID 20658998 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  14. PMID 16972248 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  15. PMID 21527925 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  16. PMID 15110706 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  17. PMID 12480926 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  18. PMID 21527925 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  19. PMID 12480926 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand
  20. PMID 19444239 (PubMed)
    Citation will be completed automatically in a few minutes. Jump the queue or expand by hand