TITLE: Maintenance of hPSC
AUTHORS: Lyn Healy, Valeria Fernandez Vallone, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
This protocol describes maintenance of established hPSC lines in expansion media on matrix coated culture vessels.

[GUIDELINES]
Depending on the growth characteristics of specific hPSC line, cells must be passaged every 5-7 days in
order to maintain log phase of growth and avoid induction of differentiation.
In general hPSC lines are maintained in a 6 well format, however information on other tissue culture vessel formats are also included in this protocol as a guidance. 

[STEPS]
SECTION: Culture Monitoring
2. Inspect hPSC cultures daily using a microsope to monitor morphology, the presence of spontaneous differentiated cells, and confluence. Based on these observations, determine if the cultures require further action (e.g. removal of differentiated cells, passaging). Use reference images in the protocol: Reference pictures of hiPSC cultured in defined conditions to guide your assessment of the cultures. Cell lines may vary in their growth rate and morphology in different media/matrix combinations.
SECTION: Culture Monitoring
3. If the cells require passaging, refer to the protocol Non-enzymatic passaging of hPSC or Single cell passaging of hPSC. Otherwise performe a complete media change (refer to Culture <70% confluent tab).

 

SECTION: Culture Monitoring
4. Use a stereo microscope in a biological safety cabinet to visually identify regions of differentiation.

 

SECTION: Culture Monitoring
5. Remove differentiated regions by scraping the cells with a scraping tool.
 

SECTION: Culture Monitoring
6. Tap the side of the plate to resuspend losen cell aggregates and remove the medium from the wells using an aspirator pipette. 
 

SECTION: Culture Monitoring
8. Add fresh medium by gently adding it to the side of the well or flask using a 5 ml or 10 ml pipette. 
SECTION: Culture Monitoring
3.1. The volume of media is tissue culture vessel dependent. Recommended volumes of maintenance media per tissue culture vessel are shown in the table below.

     Culture Vessel             Surface Area [cm2]*      Total   volume[ml]        6 well plate       1 well      ~9,6      2        6 wells        12        12 well plate      1 well      ~4      1        12 wells        12        24 well plate      1 well      ~2      0,5        24 wells        12        96 well plate      1 well      ~0,35      150 µl        96 wells        14,4        T25 flask      -      25      5        T75 flask      -      75      25   

SECTION: Culture Monitoring
9. Incubate the vessels at 37 °C  and at 5 % volume CO2.


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3. Prepare required amount of media for washing and for further culture, see Materials and Table 2. 
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4. Pre-warm the medium37 °Cin a water bath for 15 min or equilibrate the medium to room temperature ( 22 °C ± Temperature  2 °C ) by placing outside the fridge for  30 min



SECTION: Culture Monitoring
3. Prepare required amount of media for further culture, see Materials and Table 3. 


 

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5. Aspirate and discard the medium from the wells. 
 

SECTION: Culture Monitoring
6. Add gently fresh medium through the side of the well or flask using a 5 mL or 10 mL pipette. Refer to Table 3 for recommended volumes. 
SECTION: Culture Monitoring
7. Incubate the vessels at 37 °C  and at 5 % volume CO2.






SECTION: Culture Monitoring
7. Wash gently once with media. Refer to Table 2 for recommended volumes.
SECTION: Culture Monitoring
3.1. The volume of media is tissue culture vessel dependent. Recommended volumes of maintenance media per tissue culture vessel are shown in the Table 3.


 

SECTION: Selection of culture conditions
1. The following points should be considered when choosing hPSC culture conditions:

A number of media will work with an array of  different matrices. 
The rate of growth of cell lines will differ between different matrix/media combinations and cell lines may exhibit a  transient change in morphology until adapted to the new culture conditions. 
When transitioning cell lines between different media/matrix combinations  follow the manufacturers guidance. Usual recommendations: do not switch the media and matrix at the same time. Make a gradual change of media including proportions until full transition.
hPSC often require a couple of passages to adapt to the new culture conditions. 
Following transition to new culture conditions, perform quality control testing as appropriate for the intended use of the cells.
For commercial media, prepare, store and use as directed by the manufacturer. For  media made in house, use and store under the same conditions established for the validation of the media.