Cryopreservation is used to stabilize cultures at specific points in time with specific genetic characteristics. Without the ability to cryopreserve our cell lines, we are forced to continuously subculture them, during which time the cells may accumulate genetic changes and become heterogeneous. Cryopreservation allows us to produce a bank of stock vials at specific passages with specific genetic characteristics. Using validated stock vials to initiate new experiments maximizes the long-term usefulness of a cell line and minimizes experimental variation.
During cryopreservation, most of the water is removed from the interior of cells and is converted to ice. This stops metabolism and allows cells to be stored at low temperatures for long periods of time. However, recovery of hESCs from cryopreservation is sometimes very poor, and because of the slow growth rate of hESCs, the time from thawing of the vial to having cultures suitable for experimentation can be weeks to months.
OVERVIEW
The methods outlined here work well in some laboratories but not others, for reasons that are not clear. This is an area of active research, to maximize the viability and maintain pluripotency of hESCs after cryopreservation. The most important issue is to make sure that the cells used for cryopreservation are in excellent condition, actively proliferating, and that the cultures have very little differentiation. Freezing the cells at high density appears to improve their viability after thawing, but since hESCs do not survive well after being dissociated into single cells, the exact densities cannot be easily quantified.
We provide two different methods in this chapter. The main method is a modification of a standard slow freezing protocol that works well for at least some hESC lines. For this protocol, we recommend that the entire population of a culture vessel be placed into 1–3 vials, and when thawed, the cells should be placed in the same size vessel. The second method, vitrification, is offered as an alternative procedure. Vitrification is a rapid freezing technique that minimizes formation of damaging ice crystals. While vitrification requires considerable skill, for some hESC lines it gives consistent results, and for researchers who master this method it is a recommended technique for cryopreserving small numbers of cells from newly derived hESC lines.
PROCEDURES
Slow freezing of cells
1. Prepare actively proliferating, high-density cells as you would for passaging. Change the culture medium just before harvesting the cells.
2. Label 1.8 mL cryovials with cell line name, date, and passage number.
3. Mix 2x stock cryopreservation medium (see Recipes) and keep on ice.
4. Dislodge the colonies from the plate mechanically using a sterile pipette tip. Alternatively, treat with 200 U/mL of collagenase IV for 5–10 min at 37°C. Remove collagenase and replace with ESC medium (3 mL for each well of a six-well dish).
5. For each well of a six-well dish, collect the cells in 3 mL of ESC medium and transfer to a 15 mL conical tube.
6. Centrifuge 5 min at 0.2 rcf (usually about 1000 rpm). Aspirate supernatant, leaving a small amount of medium.
7. Gently resuspend the pellet in protein-containing ESC medium (usually 1.5mL for each well of a six-well dish or one half of the final freezing volume). Use a 5 mL pipette to gently triturate the cells.
8. Drop wise, add an equivalent volume of ice-cold 2 stock cryopreservation medium, mixing constantly by tapping the tube.
9. Place 1.0 mL of cell mixture in each cryovial.
10. Rapidly transfer the vials to a precooled (4°C) Nalgene freezing container (containing isopropanol), and place immediately in a freezer at –70 to –80°C.
NOTE: Do not leave the cells in DMSO at room temperature for long periods of time. 11. Transfer cells to liquid nitrogen for long-term storage.
Thawing cells
There is a growth lag upon thawing the cells and it may take several days in order to be able to visualize the colonies. It is advisable to observe the cultures under 4 magnification 24 h after thawing, but not exchange the medium for at least 48 h. There will be a lot of floating debris and dead cells upon thawing the cells – this is normal.
1. Gently thaw the vial of cells by shaking it gently in a 37°C water bath and remove while a sliver of ice still remains.
2. Spray the tube with 70% EtOH and dry with a Kimwipe.
3. In the biosafety hood, aseptically remove the vial contents and place into a 15 mL conical tube.
4. Slowly, with gentle tapping, add 10 mL of room temperature culture medium.
5. Spin very gently at 0.2 rcf (1000 rpm) for 5 min.
6. Remove the supernatant.
7. Tap the tube to dislodge the pellet.
8. Add 3 mL of ESC medium to the tube and transfer to one well of a six-well dish that has been prepared with an inactivated feeder layer or extracellular matrix.
9. Place plate into the incubator and allow the cells to attach to the plate.
10. Allow 3–7 days for the cells to attach. During this time replace half of the medium every other day.
11. The medium should be replaced daily starting 4–7 days after thawing the cells, or when the cells appear to be attached.
PITFALLS AND ADVICE
Centrifugation
Centrifugation can damage sensitive cells. The major factors that can be important are: relative centrifugal force (200xg, 400xg, 800xg), time (5, 10, and 15 min), length of the column of liquid (3 mL, 6 mL, and 12 mL in 15 mL tubes) and the type of centrifuge (fixed angle or swinging bucket).
Cryoprotectants
DMSO could contribute to hESC death and differentiation. First, addition and removal of DMSO causes osmotic stress that may affect the survival of delicate cells. Second, DMSO itself has been shown to be a potent inducer of apoptosis and differentiation. Alternate cryoprotectants that are being investigated are permeable agents such as ethylene glycol, propylene glycol, glycerol and erythritol and non-permeable sugars and sugar-alcohols such as D-glucose and fructose, sucrose, trehalose, mannose, raffinose, adonitol, glucitol, and sorbitol.
ALTERNATIVE PROCEDURES
Freezing cells by open pulled straw vitrification
Open pulled straw (OPS) vitrification is not a trivial technique. It requires preparation of three different media and careful work under a dissecting scope. In this method, hESC colonies are dissected, and 10–12 individual undifferentiated pieces of colonies are carefully collected and placed into sequential vitrification media with increasing concentrations and combinations of cryoprotectants. The cells are then placed into straws and frozen by plunging into liquid nitrogen. In spite of the extra time spent preparing and the great care to freeze only undifferentiated clumps of cells, this method results in a very high (x90%) percentage recovery of the frozen cellular aggregates, with very low percentage of differentiation in the cultures following recovery from cryopreservation. It is possible that since there is low percentage of cell death and rapid recovery following OPS vitrification, the selective pressures that may be at play during more traditional cryopreservation methods may not be as much of an issue with this method. NOTE: This method was adapted from methods used at the Monash Institute of Medical Research Laboratory of Embryonic Stem Cell Biology.
Freezing cells by vitrification
For this method, hESC colonies are dissociated using a combination of dispase (10 mg/mL in serum-containing medium) and mechanical dissection into clumps of x100–200 cells each.
The sequential incubations are performed on a 37°C heated stage of a dissecting microscope. This procedure should not take more than 3min from the time the cells are placed into vitrification solution 1 (VS1) until they are placed into liquid nitrogen. Work quickly.
Set-up for vitrification
Label 4.5 mL cryovials with cell line, passage number, and date. Puncture vials with an 18G needle through the top and on the side so that liquid nitrogen can fill the vial. Use a four-well plate with three wells containing 1 mL each of holding medium (HM), VS1, VS2 vitrification solutions on a heating stage of a dissecting microscope (Figure 4.2). Transfer of the cells will be done in droplets of VS2 on the inside of the lid of the four-well dish.
