Culturing human embryonic stem cells (hESCs) requires a significant commitment of time and resources. It takes weeks to establish a culture, and the cultures will require daily attention. Once hESC cultures are established, they can, with skill and the methods described below, be kept in continuous culture for years.
A word of caution for those with experience culturing mouse embryonic stem cells: they are not the same! Both mouse and human ESCs are diploid, they are pluripotent, and they are relatively stable in culture. However, the stability of mouse ESC lines is regularly measured because the objective of almost all genetic manipulation is to make new lines of mice. If an ESC line can generate a mouse, as we term it, “go germline,” we know that it is clearly pluripotent. This has given us an operational definition for pluripotence and stability in culture for mouse ESCs.
hESC lines were originally derived using very similar culture medium and conditions as those developed for the derivation and culture of mouse ESC lines. However, these methods were suboptimal for hESCs, and have evolved considerably in the years since hESC lines were derived. Compared with mouse ESCs, hESCs are very difficult to culture – they grow slowly, and most importantly, since we have no equivalent assays for germline competence, we cannot assume that the cells that we have in our culture dishes are either stable or pluripotent. This makes it far more critical to assay the cells frequently, using characterization methods such as the karyotyping, immunocytochemistry, gene expression analysis, and fluorescence activated cell sorting (FACS) methods provided in this manual.
In this chapter we outline protocols for the culture of hESCs, starting as one would usually do, by being handed a culture by an experienced colleague. Other chapters focus on cryopreservation and establishing hESC cultures from frozen stocks, and on the variety of culture conditions, including the preparation of various types of feeder layers, conditioned medium, and extracellular matrix substrata.
The methods we recommend are those that are the most straightforward and have worked well in our hands; these are offered as the recommended methods and reagents. We also offer alternative methods and reagents that work but are not routinely used in most laboratories. The key variables that we outline in this chapter are:
- Culture medium
– Basal medium
– Serum or serum substitute
- Passaging cells
– Manual passage
– Non-enzymatic dissociation
– Enzymatic dissociation.
While optimizing and standardizing conditions in your lab, it is important to keep in mind that changing one thing in a system may have unexpected impact on the entire system.
PROCEDURES
Tips for successfully culturing hESCs
- Feed cells every day, except for 1 or 2 days following passage.
- Examine the cultures every day under 4 and 10 phase contrast. This will allow you to become familiar with the morphologies of undifferentiated and differentiated cells and colonies.
- When they are cultured on feeder layers some hESC lines tend to undergo spontaneous differentiation in the centers of the colonies. When passaging, take care to avoid passaging these differentiated “centers” to the new culture.
- Most hESC lines double every 31–35 h.
- Store medium at 4°C and discard any unused medium after 10 days. Best results are achieved when medium is prepared in small batches once a week.
The single most important skill in successful culturing of hESCs may be the ability to recognize the morphology of undifferentiated cells under a variety of conditions.
Phase contrast micrographs from the same culture, 4 days after it was passaged onto a feeder layer (human foreskin fibroblasts, ATCC HS27). (A) Typical colonies with smooth, phase-bright edges, with the fibroblast feeder layer forming whorls around the colonies (10x magnification). In contrast, in the same culture there are colonies with obvious differentiation at the edges (B – 4x magnification) and in the center. (C – 10 magnification). In selecting colonies for passage and expansion, only the ones shown in (A) would be acceptable. The others should not be passaged to the next culture dish.
For routine expansion of hESCs, we recommend that the cells be cultured at a relatively low density so that individual colonies can be easily monitored and selected against differentiation. hESCs can be cultured to high density (Figure 1.2), but a higher proportion of differentiated cells must be expected.
Passaging hESCs
hESCs, unlike mouse ESCs, do not survive well when dissociated to single cells. Therefore, the most reliable method for passaging undifferentiated hESC cultures is manual dissection of the colonies. This method may seem tedious, but it is virtually foolproof and we recommend that novices use this method until they have familiarity with the cells and can easily recognize differentiation in the culture. We also recommend manual passaging for producing cell banks of low-passage hESCs. Enzymatic dissociation methods are provided in Alternative Procedures.
NOTE: Using the number of passages as a measure of the age of an hESC line is an unfortunate historical accident. Because of the inconsistencies in hESC culture procedures in different labs, cells are passaged at different time intervals, ranging from 4 to 7 days. Therefore the number of passages for one line might be twice that of another, even though the cells have been in culture for exactly the same amount of time. For example, in a year of continuous culture, a cell line could be passaged as few as 52 and as many as 90 times. A better measure would be the number of doublings, but to count the number of cells in a culture is difficult since the cells form tight clusters and are not passaged as single cells.
General guidelines
- The cells should be passaged at about 1:3 every 5–7 days.
- Prepare the feeder layer or extracellular matrix (ECM) substrata the day before passaging.
- Depending on the cell line, passaging on Friday may be a good routine. The cells can usually be left undisturbed for 2 days following passaging, which allows them to settle down on the substrata, attach and begin dividing before the medium is changed.
- There will be considerable variation in the size of colonies in a single dish. Compared with their mouse counterparts, hESCs do not substantially pile up on each other, and their colonies can grow to a large diameter while remaining undifferentiated. Culture conditions affect the flatness of the colonies, but as an approximation, they are ready to split when the diameter fills the 10x field when observed under the microscope. As shown in Figure 1.3, a colony about half the diameter of the 10x field contains about 4400 cells. A colony filling the field would contain about 15 000 cells.
- For routine passaging by any method, do not make a single-cell suspension; dissociate the colonies into smaller colonies of a few hundred cells.
- Examine the culture daily for colony morphology under the phase contrast or dissecting microscope.
- With experience, one can get a good overview of colony morphology by holding the dish up to a light and looking at the bottom of the dish. The differentiated colonies will have ragged edges and hollow centers.
- On the bottom of the dish, mark colonies that are badly differentiated or parts of the colony that you do not wish to transfer to a new culture dish.
- To be certain that the colonies selected are undifferentiated, it is advisable to dissect the colonies while viewing the dish under a dissecting microscope with illumination from the base. But this is not absolutely necessary, and some prefer to passage the cells without magnification.
Mechanical dissociation
1. Evaluate the culture under 4x or 10x phase contrast optics.
2. The cells can be split among 3–6 dishes of the same size as the original culture, depending on the density of the original culture. If you wish to put the cells in different-sized dishes, calculate the amount of volume to add based on surface area of each type of dish.
3. Mark (or remove) overtly differentiated colonies so as not to disturb these during the dissociation process.
4. Remove the medium from the dish and replace with fresh hESC medium.
5. Dissect the colonies by hand, either under a low-power dissecting microscope (in a horizontal flow hood) or without a microscope, in the tissue culture hood.
NOTE: Several implements can be used to slice up or break up the colonies. Because they are inexpensive and sterile, we recommend either a 20 ?L pipettor which has a sterile filter tip attached, or a sterile 23G needle.
6. Figure 1.4 shows the method used for slicing the colonies into about 100 pieces. The colony is cut into strips, and then into squares. Each piece of the colony has a few hundred cells.
7. Break up each colony by moving the tip around and across each colony in a crosshatch or a spiral motion.
NOTE: Since the colonies are large at the time of passage, it is relatively easy to see individual colonies on the plate and, with practise, one can quickly dissociate an entire plate in less than 20 min.
8. After all of the colonies are dissected, use a 5 mL pipette to transfer the culture medium containing the dissected colonies to a 15 mL conical tube. Rinse the plate with hESC medium and add this to the same 15 mL tube.
9. Bring up the final volume in the tube to 8–10 mL with hESC medium.
10. Gently triturate the cell clumps using a sterile 10 mL pipette and divide the suspension into the prepared culture dishes on feeder layer or ECM-coated plates. Do not make a single-cell suspension; triturate gently, trying to achieve a relatively uniform suspension of cell clumps containing a few hundred cells each.
ALTERNATIVE PROCEDURES
Enzymatic dissociation
Enzymatic dissociation methods vary widely, and the exact conditions need to be developed for each laboratory. Most importantly, cultures that have been maintained by manual passaging cannot be passaged by enzymatic dissociation unless exceptional care is taken to adapt and monitor the cells.
The type of enzyme used for dissociation is critical. For example, passaging with trypsin appears to put more selective pressure on the cultures than other methods, resulting in a higher incidence of drift of hESC lines toward aneuploidy. But some hESC lines have been derived using trypsin from the outset; these lines can be rountinely passaged using whatever enzymatic technique is provided by the supplier.
Microbial collagenase is preferred by many laboratories, perhaps because of the way in which it is used. Collagenase is used to loosen the hESC colonies from the dishes, not to dissociate them to single cells, and the cell clumps have to be further dissociated by trituration.
NOTE: Keep in mind that enzymes are not highly purified recombinant products, and they may contain animal products. Trypsin is prepared from porcine (pig) tissue, and collagenase is a crude microbial product.
Collagenase dissociation
1. Remove the culture medium.
2. Rinse culture with Dulbecco’s PBS (D-PBS).
3. Treat the culture with 200 U/mL of collagenase IV for 5–10 min at 37°C until the edges of the colonies start to curl up – observe the culture under the microscope.
4. Remove the collagenase and replace with 2 mL of hESC medium (if using a sixwell or 35 mm dish).
5. Using a 5 mL pipette, gently dislodge the “good” colonies from the plate and place them in a 15 mL conical tube. Alternatively, one could remove the differentiated colonies prior to treating the culture dish with collagenase.
6. Gently triturate the cell clumps using a sterile 10 mL pipette and plate on feeder layer- or ECM-prepared dishes. Try to achieve a relatively uniform suspension of cell clumps containing several hundred cells each.
7. The cells can be split among 3–6 dishes of the same size as the original culture, depending on density of the original culture. If you wish to put the cells in different sized dishes, calculate the dilution based on surface area of each type of dish.
Non-enzymatic cell dissociation
Ca2x- and Mg2-free saline solutions containing EDTA or EGTA have not been as widely used for hESC dissociation as the methods described above, but they should offer advantages for assays that require intact cell surface proteins such as flow cytometry and immunocytochemistry. Commercial formulations are available, such as Cell Dissociation Buffer (Invitrogen catalog no. 13150016), which contains glycerol as well as a proprietary mixture of salts and chelators.
If you decide to try this method, remove all of the protein-containing medium and rinse the cells briefly with the dissociation buffer. Add enough buffer to cover the cells and monitor them under the microscope until the edges of the colonies begin to lift, then triturate the cells gently to dissociate. If the cells are to be recultured, don’t dissociate them into single cells, and be certain to check the karyotype of the cells after 10 passages; until you prove otherwise, you should assume that any untested passaging method is selecting for chromosomal abnormalities.
Accutase and Accumax (Millipore/Chemicon catalog no. SCR005 and SCR006)
These products are proprietary mixtures of proteolytic and collagenolytic enzymes in EDTA that the manufacturer states is free of mammalian- or bacterial-derived products. Accumax also contains DNAse. If you test this method, start with a 5-minute room temperature incubation and monitor the cells under the microscope. While the manufacturer indicates that inactivation of the enzymes with protein is not necessary, we recommend that protein-containing medium be used to dilute out the enzyme after the cells are dissociated, to prevent clumping and sticking of the cells to the pipettes.
Trypsin-like Enzyme (TrypLE Select, Invitrogen catalog no. 12563-029)
This is a single enzyme, a recombinant fungal serine protease with trypsin-like activity. Anecdotal reports suggest that hESC line that have been mechanically passaged can be successfully transitioned to single-cell enzymatic passaging using TrypLE Select. If you decide to try this method, we recommend a saline rinse, then a 5-minute incubation in the 1x enzyme solution as provided by the manufacturer. Monitor the cells under the microscope and add protein-containing medium to the culture before triturating.
HyQTase (HyClone catalog no. SV30030.01)
This is a cell detachment solution in D-PBS with EDTA. The composition is proprietary. According to the manufacturer, HyQTase is composed of a naturally derived complex of proteolytic and collagenolytic enzymes in D-PBS containing EDTA. According to the manufacturer it can be used for either serum-containing or serumfree cultures. The manufacturer states that it does not contain mammalian or bacterial derived products and is non-recombinant.
PITFALLS AND ADVICE
Monitoring drift in hESC cultures
Since hESC cultures are often kept in continuous culture for months, even years, it is very important to monitor for drift in the cultures. The best way to avoid drift is to generate a large bank of frozen cells as soon as possible after the cultures are first expanded. The importance of this cannot be overemphasized; the value of discoveries based on hESC cells depends on the reproducibility of results. See Chapter 26 for methods for setting up an hESC lab.
Genetic drift
We know that hESCs acquire chromosomal abnormalities over long periods of culture, so karyotyping or other genetic analysis methods must be performed on a regular basis. For detailed information about how to monitor genetic drifts, see Chapters 5–7 and 26. Keep in mind that changes during the time the cells are cultured in your lab can only be detected if you analyze the cells very soon after you obtain them.
Developmental drift
hESCs can also drift toward a more differentiated state over periods of extended culture. Since there is no assay for pluripotence equivalent to germline transmission of mouse ESCs, surrogate markers, such as antibody markers, should be routinely checked, especially if the morphology of the cells seems to be different from the earlier cultures. The gold standard for measuring the pluripotency of an hESC line is to transplant it to an immune-deficient mouse to form a teratoma tumor (Chapters 12 and 13). Keep in mind that it will require histological expertise to identify cell types and tissues in the tumors. In vitro, differentiation of hESCs using embryoid body culture will allow at least a cursory analysis of hESC differentiation potential. However, embryoid bodies never achieve the maturity of cells that develop in teratomas, and since the methods used to assess differentiation in vitro usually involve a small number of markers assayed by PCR (Chapter 10) or immunocytochemistry (Chapter 9), it is more difficult to judge the full range of pluripotence.
The best approach to monitoring developmental drift is to pick a particular method and differentiated cell type to check periodically (see Chapters 14 on embryoid body and neuroepithelial differentiation, as well as the specific chapters on neuronal, cardiac, and hematopoietic cells, Chapters 15–18).
Contamination of cultures
hESCs are usually cultured without antibiotics; with good culture technique, bacterial contamination should not be a problem. However, we recommend that antibiotics be used while new investigators are being trained in the techniques. Antibiotics such as penicillin and streptomycin do not have any effect on mycoplasma. Mycoplasma is a serious problem in laboratories that culture multiple cell lines or have inadequately trained personnel. Cultures must be monitored for mycoplasma on a regular basis, and contaminated cultures destroyed. Methods for mycoplasma detection are provided in the quality control section of this chapter, and in Chapter 26.
EQU I PMENT
- Tissue culture hood: Class II A/B3
- Tissue culture incubator, 37°C, 5% CO2, in humidified air
- Inverted phase contrast microscope with 4x, 10x, and 20x objectives
- Centrifuge, low speed 300–1000 rpm
- Water bath, 37°C
- Pipettors, such as Eppendorf p-2, p-20, p-200, p-1000
- Pipette aid, automatic pipettor for use in measuring and dispensing media
- Aspirator in the hood, with flask
- Refrigerator, 4°C
- Freezers: –20°C, –80°C, and –140°C.
Supplies
- 5 mL, 10 mL, 25 mL sterile disposable pipettes
- Six-well culture dishes
- 15 mL sterile conical tubes
- 50 mL sterile conical tubes
- Sterile 9x Pasteur pipettes
- Pipette tips for Eppendorf or similar pipettor.
KnockOut™ serum replacement (Invitrogen catalog no. 108280-028)
This product has a short shelf-life and should be aliquoted into 50 mL tubes and stored at –20°C. Thaw at 37°C just prior to use.
Additional information
KnockOut serum replacement (KSR) is a brand name for an Invitrogen product that is composed of BSA, transferrin, insulin, and other protein and non-protein components. The exact formulation is proprietary, but its composition was published (July 16, 1998) in an International Application Published under the Patent Cooperation Treaty (PCT), designated WO98/20679. See Epoline (ofi.epolin.org) to view the entire patent application.
L-Glutamine (200mM)
L-Glutamine (Invitrogen catalog no. 25030-081) is unstable and must be stored frozen at –20°C. Thaw the bottle completely just prior to use and aliquot in 10 mL tubes. Do not refreeze tubes, store at –4°C and discard unused glutamine after two weeks.
2-Mercaptoethanol
2-Mercaptoethanol (2-ME) has been used in ESC culture media since the first derivation of mouse ESCs in 1981. Originally included as a reducing agent because of concern about oxidation of culture components, it continues to be used in hESC media. Since the final concentration is 0.1 mM, and the pure solutions of 2-ME are 14.3 M, it is necessary to start with a stock solution.
Several companies sell diluted solutions of 2-ME; the 55 mM solution in PBS (Invitrogen catalog no. 21985-023) is a convenient concentration for a stock. If you wish to make your own stock, we suggest that you make a 1000x stock from the generally available concentrated solution (14.3 M).
For 1000x stock: dilute 35 ?L of 14.3 M 2-ME (Sigma catalog no. M7522) into 5 mL of PBS to make a 0.1 M stock solution. Filter before use.
QUALITY CONTROL METHODS
Lot-to-lot variability of reagents
It is important to keep in mind the actual source of the materials and reagents used in the culture and maintenance of hESCs. Since many are derived from animal sources, there is inherent lot-to-lot variability of the product. While vendors make every effort to control the variability by setting production specifications, these are usually ranges and as long as the product falls within the approved range, the product passes inspection and is distributed.
Ideally, you should have your own quality control methods to test new lots of products. At the very least, record the lot numbers of reagents used; if an experimental result cannot be replicated, or a cell line fails to thrive, you will save considerable time if the problem is traceable to a bad reagent.
Monitoring for mycoplasma contamination
Mycoplasma are the smallest forms of bacteria (0.2–0.3 ?m in diameter) and they can pass through the typical microbiology 0.2 ?m filter used in cell culture and do not produce the characteristic turbid growth shown by other bacteria. Because they lack cell walls, they are unaffected by the standard antibiotics used in culture media (penicillin and streptomycin that act on bacterial cell walls). Serious infections can be detected in cultures by DAPI or Hoescht staining for DNA; stained cell nuclei will be surrounded by fluorescing structures in the cytoplasm.
Mycoplasma infections drastically affect cell metabolism, gene expression and antigenicity, and can be devastating to a hESC laboratory. Infections are difficult to get rid of once they take hold, and some tissue culture collections recommend that contaminated cells be destroyed as soon as mycoplasma are detected.
Mycoplasma are highly infectious and cross-contamination commonly occurs when new cells are introduced into laboratories. The ATCC (American Type Culture Collection) estimates that 16% of cell cultures are contaminated by mycoplasma. The bacteria can also come from tissue culture reagents such as serum and media supplements and from laboratory staff.
The best defense against mycoplasma contamination is good aseptic technique, and the laboratory should not allow inexperienced or careless workers to share cell lines, solutions, or equipment. As a precaution, the cell lines should be tested at least four times a year. Testing for mycoplasma can be done by enzymatic, polymerase chain reaction (PCR), fluorescent staining, or culture methods (see list below).
