An Introduction to Stem Cell Maintenance
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:
- Embryonic Stem Cells: Isolated from the inner cell mass of the blastocyst stage of a developing embryo. These early cells were destined to create a fetus following implantation.
- Embryonic Germ Cells: Derived from aborted fetuses. These early cells were destined to become sperm and eggs.
- Embryonic Carcinoma Cells: Isolated from certain types of fetal tumors.
- Induced Pluripotent Stem Cells: Generated via ectopic expression of one or more genes to reprogram an adult somatic cell.
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.
- General maintenance of PSC cultures requires daily removal of spent media and replenishment with fresh PSC medium. It is crucial to add fresh bFGF, aseptically on a daily basis, to the pre-warmed media prior to adding to the cells. Daily visual inspection of cell morphology is highly recommended for proper growth and for the removal of any differentiating colonies via manual dissection.
As daily maintenance of the PSC cultures is required, it is helpful to develop an optimal working schedule. PSCs should be split every 3–4 days, based on colony size and distribution. Hands-on experience and a keen eye are most important in PSC culture. To avoid spontaneous differentiation of the colonies, do not:
- allow colonies to overgrow and touch each other
- over incubate the colonies in enzyme, when passaging them
- passage huge colonies
Generally, a manageable 7-day schedule for PSC culture is employed as follows:
- Monday: Feed existing PSC cultures and make iMEF plates
- Tuesday: Split PSC cultures onto iMEFs prepared Monday. A 1:3 or 1:4 split, meaning one dish passaged into 3 dishes or 4 dishes, respectively, is good for maintenance.
- Wednesday: Feed PSC cultures.
- Thursday: Feed existing PSC cultures and make iMEF plates (unless you have remaining iMEFs from Monday, which may be used at this time).
- Friday: Split PSC cultures onto iMEFs. A 1:4 or 1:5 split is good for maintenance over the weekend.
- Saturday/Sunday: Feed PSC cultures.
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.
In general, split cultures when the first of the following occurs:
- iMEF feeder layer is 10 days old; usually differentiation will occur more frequently if MEFs are too old
- PSC colonies are becoming too dense or too large
- Increased differentiation occurs
Enzymatic Passaging of hPSCs
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.
Mechanical Passaging of hPSCs
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.
Mechanical Passaging of hPSCs Using the StemPro® EZPassage™ Disposable Stem Cell Passaging Tool
A typical passage schedule for PSC is as follows:
- Day 0: Seed iMEFs on Attachment Factor–coated tissue culture plates or dishes.
- Day 1: Seed PSCs on attached iMEF feeder layer.
- Day 2–4: Change media and monitor for distribution and morphology of attached colonies.
- Day 5: Passage PSCs onto iMEF coated dishes.
- It is important to remove spent culture media and add fresh media every day. Most often, differentiated cells at the edges of each colony can be mechanically removed with a 27-gauge needle before passaging. It is critical to observe the distribution of attached colonies 24 hours post-passage.
- Colonies sometimes can all be clustered towards the middle, close to each other or on top of each other. An even distribution of colonies is critical for the maintenance of undifferentiated colonies during expansion. It is also important to monitor the attachment and morphology of the colonies. In all dishes there will always be some areas with unattached cells or cells that look differentiated. However, the majority of the colonies in the dish must be well-separated, with a pristine morphology.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.