Search Thermo Fisher Scientific
Search Thermo Fisher Scientific
Pluripotent Stem Cells (PSCs) can divide indefinitely, self-renew, differentiate and functionally develop into almost any cell in the body, given the right conditions. There are several kinds of pluripotent stem cells:
PSCs are generally maintained on a layer of feeder cells for many passages without any compromise to proliferation, pluripotency or differentiation potential. Feeder cells are usually murine embryonic fibroblasts (MEF), which must be irradiated or chemically treated to inactive them (noted as iMEF) prior to culturing with PSCs. Alternatively, PSCs can be maintained in feeder-free conditions using specialized media systems on a matrix-coated tissue culture surface. This introduction will discuss the feeder-dependent and feeder-free culture of human ESCs and iPSCs (noted as hESCs and hiPSCs), which will be referred to as PSCs.
Feeder-based medium is comprised of basal medium supplemented with 15–20% KnockOut™ Serum Replacement (KSR) and other additives. Feeder-free medium is either Conditioned Medium (CM), which is comprised of feeder-based medium that has been conditioned on iMEFs and therefore can be used to grow PSCs in the absence of feeders, or commercially available media that support feeder-independent growth of PSCs.
Optional: If cells cannot be fed both weekend days, you may skip a single day and just feed your cultures an additional 1-2 mL of media the day before.
Figure 1. A microscope with a 5X Objective and Phase contrast or DIC is ideal to observe a PSC colony morphology (panel A). Traditionally cultured hESCs or hiPSCs appear as compact colonies surrounded by fibroblast shaped feeder cells (iMEF) (panel B).
Figure 2. H9 ESC cultured on iMEF. PSCs grow in a compact colony formation with very well defined borders. PSCs have a high nucleus-to-cytoplasm ratio and the colonies grow in a 3-dimensional radial pattern.
Figure 3. PSC colonies that are beginning to differentiate show loss of defined edges and the emergence of large differentiated cells. The central core remains compact and it is possible to rescue this colony by scrapping out the differentiated cells at the edges.
Figure 4. Differentiating colonies show loss of defined edges, possess large differentiated cells and a heterogeneous central core that is not typical of a PSC.
In general, split cultures when the first of the following occurs:
Enzymatic passaging of PSCs will vary from cell line to cell line. Some hESC cell lines or hiPSCs may not react in the same manner to enzymatic passaging, and consequently the enzyme’s type, concentration, and exposure time must be empirically determined for the particular cell line to be passaged. If the hESC or hiPSC line being cultured is not optimally passaged enzymatically, manual or mechanical passaging must be performed.
Figure 5. PSC colonies can be harvested in bulk using enzymatic methods such as treatment with Collagenase Type IV for 30–60 minutes. During this time, attached PSC colonies (panel A) curl up and detach from the dish (panels B–E), leaving behind iMEFs (panel F). When the colonies begin to curl up (panels B-C), cell clusters can be gently dislodged with a 5-mL pipette. Care should be taken to not over expose the PSCs to the enzyme as it may affect the efficiency of recovery.
During the culture of PSCs, it may be necessary to manually dissect PSC colonies to either remove undesired portions of a colony or whole colonies, or to break up and passage individual colonies. Traditional tools include a drawn-out glass Pasteur pipette and needles that can be used to dissect individual colonies. In particular, this method is used for maintenance of PSC colonies by removing unwanted differentiated colonies and for manually passaging colonies of PSCs that cannot be passaged enzymatically. Mechanical passaging is also an important tool for hiPSC selection and maintenance.
Figure 6. PSC colonies (panel A) may be manually dissected using a needle and syringe. Straight cuts in horizontal and vertical directions create a checker board pattern of small cell clusters that can be re-seeded for expansion and passaging (panel B). Uneven cuts (panel C) and microscale manipulations make this procedure labor-intensive.
Figure 7. Colonies can be mechanically scored using the StemPro® EZPassage™ tool. The entire dish is scored with two quick movements and the broken-down parts of the colony are transferred to a fresh iMEF coated dish.
A typical passage schedule for PSC is as follows:
Figure 8. Cells 24 hours post-passage (Day 2). Examples of areas containing colonies with good (top panels versus bad morphologies (bottom panels).
Figure 9. 48 to 72 hours post-passage (Days 3–4). Well-separated, seeded-down colonies continue to expand and grow in size while maintaining their morphology.
Figure 10. hPSC colonies are ready for passage 96 hours post-passage (Day 4). Most colonies are large (at 5X magnification they fit the entire visual field) and the neighboring colonies begin to grow into each other.
MAN0006676 03-Mar-2012
For research use only. Not intended for any animal or human therapeutic or diagnostic use.
For Research Use Only. Not for use in diagnostic procedures.