Freezing Human ES Cells

Summary

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines.

Abstract

Here we demonstrate how our lab freezes HuES human embryonic stem cell lines. A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction. Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature. During this time cells can be observed rounding, and colonies lifting off the plate surface. Gentle repeated pipetting will remove cells and colonies from the plate surface. Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun. Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media, a concentration such that one frozen aliquot is sufficient to resurrect a culture on a 10cm plate. After cells are adequately resuspended, ice cold freezing media is added at equal volume. Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours. Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Protocol

1. A healthy, exponentially expanding culture is washed with PBS to remove residual media that could otherwise quench the Trypsin reaction. 
2. Warmed 0.05% Trypsin-EDTA is then added to cover the cells, and the plate allowed to incubate for up to 5 mins at room temperature. During this time cells can be observed rounding, and colonies lifting off the plate surface. 
3. Gentle repeated pipetting will remove cells and colonies from the plate surface. 
4. Trypsinized cells are placed in a standard conical tube containing pre-warmed hES cell media to quench remaining trypsin, and then spun. 
5. Cells are resuspended growth media at a concentration of approximately one million cells in one mL of media.
6. After cells are adequately resuspended, ice cold Freezing Media is added at equal volume.
7. Cell suspensions are mixed thoroughly, aliquoted into freezing vials, and allowed to slowly freeze to -80C over 24 hours. 
8. Frozen cells can then moved to the vapor phase of liquid nitrogen for long term storage, or remain at -80 for approximately six months.

Note: such a frozen aliquot at a concentration of 106 cells/ml is sufficient to resurrect a culture on a 10cm plate.

Discussion

The technique we describe for freezing HuES cell lines works well for efficiently storing and resurrecting HuES cell lines and also any mammalian cell of interest. Freezing a small proportion of cells at each passage is an important consideration when working with hES cell lines, as karyotype abnormalities can arise and abnormal cells can quickly take over the line. Thus, having frozen splits at every passage in addition to large banks at certain passages is highly desirable.

Materials

Material Name	Type	Company	Catalogue Number	Comment
Name	Company	Catalog Number	Comments
HuES Medium	medium			For aq total of 651.5 ml, add: 500 ml KO-DMEM + 6.5 ml PenStrep + 6.5 ml L- Glut + 6.5 ml NEAA 6.5 ml + beta-mercaptoethanol 0.6ul (14.3 M stock) + 65 ml Serum Replacement + 65 ml Plasmanate + 0.65 ml bFGF2 (~10 ng/mL final)
Freezing Medium	medium			80% FCS (fetal calf serum) + 20% DMSO (dimethyl sulphoxide). Stored at 4C.