Expansion of hPS Cell Derived Pancreatic Progenitors

A. Initiating de novo expansion from pancreatic progenitor (PP) cells

1. Estimate cell yield from differentiation (~6 million per well of a 6-well plate (6wpw) and coat the appropriate number of wells with Matrigel diluted in DMEM/F12 (1:50) or Vitronectin-N (20 ug/ml) diluted in DMEM/F12 at least one hour before proceeding, considering that each 6wpw should be seeded with 3 million cells for a successful expansion.

2. Wash the wells of PP cells with 2 ml of DPBS and incubate with 1 ml TryPLE, warmed at 25° C, at 37 °C for 2-3 min until you see the cells rounding up.

3. Further break up cells by gently triturating 3 x with a P1000 tip. Stop enzyme activity by immediately adding 1 ml of 2% BSA in high glucose DMEM containing20 µM of ROCKi. Process in parallel only up to three wells.

4. Once done will all wells, gently triturate the cells a further 3X using a P1000 in allthe wells before passing through a 40um strainer (Corning, FALC352340) into anempty 6wpw.

5. Transfer the cell suspension to a 15 ml falcon, wash each well with 1 ml 2% BSA in high glucose DMEM 10 uM ROCKi and add it to the corresponding falcon before spinning down for 4 min at 300xg.

6. Aspirate the supernatant and resuspend the pellet in 1 ml of expansion media containing 10 µM of ROCKi. Take 25 ul of the cell solution and mix with 25 ul ofTrypan blue solution. Add 10 ul of the well-mixed cell/trypan blue solution to a chamber of the Cell CountessTM counting chambers and proceed to count using the Cell CountessTM II (see note 1).

7. Seed 3 million (see note 2) live cells per 6wpw, coated as above, in a total volume of 2 ml (cell suspension + expansion media) media containing 10 uM ROCKi.
8. Next day wash with 2 ml DPBS, add 2 ml of fresh 37 C prewarmed expansion media. Do daily washes and media changes.

9. For subsequent passages (see note 3) seed 2 million (see note 2) cells per 6wpw, coated as above, in a total of 2 ml of expansion media containing 10 uM ROCKi.

    

   Note 1 To count cells, place 10ul of trypan blue / cell solution into each of the counting chambers of the slide for Cell Countess II. Take both counts and then the average for better accuracy. If values differ by 10% or more, mix cell suspension again as above, retake both measurements and discard the outlier before calculating the average number.

   Note 2 When seeding cells, it is essential that the seeding density be adjusted considering the cell death rate. For instance, if the total cell count is 3 million cells, with 2.7 million live cells and 0.3 million dead cells (10% death rate), theseeding should be done with 2.2 million live cells (2 million + 2 million x % ofdead cells).

   Note 3 Passaging is typically done every four days but, after P5 and on rare occasions, cells might need splitting earlier. Never leave cells for more than four days before passaging because if cells become overconfluent they start losing PP identity. TryPLE digestion of a good culture should not exceed 3-4 minutes.

B. Cryopreservation of expanding PP cells:

1. Warm TryPLE to 25°C.
2. Aspirate medium from the well and wash once with 2 mL of DPBS.
3. Add 1 mL of TryPLE per 6wpw and incubate at 37°C for 2 minutes and check to see if the cells are rounded up. Increase incubation time as necessary until cellsare rounded up.
4. Gently mix by pipetting 3x with P1000 to break up cells and remove from the well and immediately add 2% BSA in high glucose DMEM to each well to stop the reaction. Pipette further to obtain a single-cell suspension. Transfer it to a 15 mL conical tube. Rinse the well with an additional 1 mL of DMEM to collect anyremaining cells.
5. Add 25 ul of cell solution to 25 ul of 1x trypan blue. Mix well and place 10ul of solution into the counting chamber for Cell Countess II, count cells (see note 1) while the falcon is spinning down.
6. Centrifuge the falcon at 300xg for 4 minutes during the counting step.
7. Centrifuge the cells, gently resuspend them in mFreSR at a concentration of 10 x106 cells/ml (see note 2) and transfer the cell suspension in 1.2 ml labelled cryovials. Cell concentration is important ! If not enough cells for 1 ml, adjust the volume accordingly. Add ROCKi at a final concentration of 20 uM.
8. Freeze vials using a standard slow rate-controlled cooling protocol that reduces temperatures at approximately 1°C/min. Place vials in the Thermo Mr Frosty filled with fresh absolute isopropanol up to the filling limit at room temperature andthen store at -80 C, followed by long-term storage at liquid nitrogen the next day.

C. Thawing Cryopreserved PP Cells

Note The thawing procedure should be carried out as quickly as possible.

1. Add 5mL of 37 C prewarmed high glucose DMEM to a 15 mL tube.
2. Quickly thaw the cells in a 37°C water bath by gently and continuously shaking the cryovial until a very small frozen cell pellet remains.
3. Transfer the contents of the cryovial to the 15 mL conical tube and centrifuge cells at 300xg for 4 minutes at room temperature.
4. Aspirate the supernatant with the vacuum pump until <200ul are left and the remaining with the P200 as not to disturb the pellet. Resuspend the pellet in 1 ml of PP Expansion medium. Mix by pipetting and count the cells by mixing 25 ul of the cells with 25 ul Trypan blue (0.4 %). Add 10 ul of the well-mixed cell/trypan blue solution to a chamber of the Cell CountessTM counting chambers and proceed to count using the Cell CountessTM II (see note 1).
5. Seed 3x106 cells/well (see note 2) in hES qualified Corning Matrigel (1:50) pre- coated 6-well plate as above containing 2 ml of PP Expansion medium + ROCKi at 10uM.
6. Place the plate in a 37°C incubator. Change media as soon as possible the next morning.
7. Perform daily medium changes using 2 ml/well of PP Expansion medium.

D. Differentiation of expanded PP cells

1. Resuspend cells in 1 ml of Expansion medium + ROCKi 20 µM and count them with Cell Countess II (please see note 1). Adjust the cell concentration at 0.6 millioncells/ml (for 2000 cells / microwell of Aggrewell 800) with expansion medium.
2. Add 1 mL of the cell suspension in each well previously prepared with 1ml of full expansion medium.
3. After the first day incubation with expanding medium, completely replace 2ml expansion medium with PEP medium + 1XPEP factors + 0.25X each of EGF, bFGF and FGF18 used in expansion.
4. After the end of the second day continue differentiation with PEP medium as for directly differentiated PP cells.

E. Storage and preparation of various reagents

TrypLE Express (Life Technologies Cat. No. 12604013)

Store at 4C, warm the required amount to 25 C before use

Trypan Blue Solution, 0.4% (Life Technologies T10282)

Pass through a 22um filter and store at room temperature

ROCKi (Miltenyi Cat. No. 130-104-169)

20 mM in H2O, long term storage at -20 °C, working aliquot at 4 C

Matrigel stock preparation and use

Dilute 1:5 with DMEM/F12 and store as 600 ul aliquots in -20 °C Always keep on ice when thawing and handling to prevent gelling

After thawing, dilute stock a further 1:10 with DMEM/F12 (1:50 final) before using

Vitronectin stock preparation and use

Dilute 1:5 with DMEM/F12 and store as 500 ul aliquots in -20 °C

Always keep on ice when thawing and handling to prevent gelling

After thawing, dilute stock a further 1:5 with DMEM/F12 (1:25 final) before using

Final concentration: 20ug/ml

Working stocks at 4 C or -20 C are good for 2-3 weeks Stocks at -80 C are good for a year

ALL SUPPLEMENT STOCKS AND WORKING STOCKS IN ULTRA-LOW BINDING TUBES !

AVOID REPEATED FREEZE THAW CYCLES !

*** Use tight cap tubes