Mouse embryonic stem cell culture

6.1.1 General preparations before cell culture

Cell culture grade sterile reagents, plastic ware and glassware was used throughout all experiments. Experiments were performed in a laminar air flow hood which was switched on and UV-sterilized with the glass front closed for at least 15 min prior to work. The “warm up phase” of 15 min ensured a stable air flow. The working surface, in addition to any working material that entered the flow hood, was sprayed with 70 % Ethanol and wiped clean prior to work. Sterile cell culture plasticware was opened and closed exclusively under the laminar air flow. According to good cell culture practice, bottles, containers and plates would be closed during the working process whenever possible. After finishing cell culture work, the laminar air hood was cleaned and wiped with 70 % Ethanol to disinfect the working surface. The germicidal UV-lamp was switched on, with the blower running, for at least 15 min after finishing the work.

Liquid culture materials such as D-PBS, H2O, gelatin, media or any other non-

commercial and as sterile declared solution was autoclaved or sterile filtrated using a 0,22 µm Stericup Filter Unit (Millipore, SCGPU05RE). Cell culture media was sterile filtrated, aliquoted in 500 mL bottles and stored at 4 °C. Media was pre-warmed to ~ 37 °C in a water bath prior to use.

Cells were cultured at 37 °C and 5 % CO2 in a humidified incubator. The humidity

chamber was filled with water on a weekly basis. The inside of the incubator and all its stainless steel shelfs were cleaned with 70 % Ethanol once a month. The incubator was used for cell culture only after humidity and CO2 levels were equilibrated.

6.1.2 Preparation of gelatin coated plates

0,1 % (w/v) gelatin solution was prepared as follows: 1 g pork gelatin (SIGMA; G1890) and 600 mL autoclaved water were added to a sterile 1 L beaker. The solution was swirled and microwaved till its boiling point (solution needs to boil to ensure proper solution of gelatin in water). The solution was topped up to a final volume of 1 L with H2O (RT).

After a short cooling period (till liquid reached approx. 50 °C), gelatin was sterile filtered in the laminar air hood using a 0,22 µm Stericup Filter Unit (Millipore; SCGPU05RE). Gelatin was aliquoted in 1 L bottles and stored at 4 °C.

Cell culture dishes were coated with 0.1 % gelatin (RT) latest 20 min before the experiment. The coating volume was chosen according to the culture volume of the respective dish. After checking that the entire dish surface was covered, gelatin coated plates were stored in the incubator till used. Prior to performing the experiment, gelatin was aspirated and the dish air dried before fresh media was added to the plate.

6.1.3 mES cell lines

All stable transgenic cell lines were derived from AB2.2 cell line (ATCC®SCRC- 1023™). The AB2.2 cell line was regularly karyotyped and tested for mycoplasma contamination.

6.1.4 mESC maintenance and propagation

Mouse embryonic stem cells (mESCs) were maintained without feeders under standard pluripotency conditions in ES complete media (LIF+Serum) (Table 3) on gelatin coated plates, and maintained at 37 °C with 5 % CO2 in a humidified incubator.

Table 3: ES complete - media compositio n.

Component Comment Supplier (Number) Concentration

DMEM high glucose,

w/o glutamine

Invitrogen (11960) 80 % (v/v)

Fetal Bovine Serum ES-qualified Millipore (ES-009-B) 15 % (v/v)

100x Non-Essential Amino Acids 10 mM glycine 10 mM L-alanine 10 mM L-asparagine 10 mM L-aspartic acid 10 mM L-glutamic acid 10 mM L-proline 10 mM L-serine Invitrogen (11140) 1x (100 µM) 100x L-Glutamine 200 mM Invitrogen (25030) 1x (2 mM)

100x Sodium Pyruvate 100 mM Invitrogen (11360) 1x (100 µM)

100x Penicillin- Streptomycin 10,000U mL-1 10,000U mL-1 Invitrogen (15140) 1x (100 U mL-1 Pen./Strep.) 2-Mercaptoethanol 55 mM in D-PBS Invitrogen (21985) 100 µM

Murine LIF Recombinant EMBL PEPCore 10 ng/mL

The media was sterile filtrated using a 0,22 µm Stericup Filter Unit (Millipore; SCGPU05RE) and aliquoted in 500 mL bottles with subsequent storage at 4 °C. ES complete media was changed daily, whereas mESCs were passaged every other day. Cell dilution for passaging was chosen as such that the culture reached ~ 80 % confluency on the day of passaging. Cell culture media was replaced 2-3 hours before splitting cells. Prior to passaging, cells were washed with D-PBS (2,7 mM KCL, 1,47 mM KH2PO4,

137 mM NaCl, 8,1 mM N2HPO4, pH 7,4) (RT). Subsequently, 0,05 % Trypsin-EDTA

solution (Invitrogen, 25300) was added in a dropwise manner (~ 20 µL/cm2) to cover the entire surface of the well. Trypsin was incubated for 5 min at 37 °C. Trypsin was inactivated by addition of ES complete media (volume was chosen according the respective plate volume). Single cell suspension was generated by pipetting the cell suspension 10-15 times, while flushing the surface of the well. 50 µL of the cell suspension was diluted in 50 µL of 0,4 % (w/v) Trypan blue (Invitrogen, 15250) and cells were counted using a hemocytometer (Neubauer improved chamber). Cell viability was expected to be ≥ 98 %. Pre-prepared gelatin coated plates were aspirated, and fresh ES

complete media (37 °C) was added to the dish before cells were seeded at a density of ~ 40,000 cells/cm2 and incubated at 37 °C with 5 % CO2 in a humidified incubator.

6.1.5 Freezing mouse embryonic stem cells

Cells to be frozen had reached ~ 80 % of confluency with a cell viability of ≥ 98 %. Culture media was changed 2-3 hours before freezing cells. Cells were washed with D- PBS (RT) before adding 0,05 % Trypsin-EDTA solution (Invitrogen, 25300) in a dropwise manner (~ 20 µL/cm2) to cover the entire surface of the well. Trypsin was incubated for 5 min at 37 °C and inactivated by addition of ES complete media (inactivation volume according to culture volume of the respective dish). Single cell suspension was achieved by pipetting the cell suspension 10-15 times while flushing the surface of the well. The entire cell suspension was transferred to a labeled and sterile 15 mL falcon. Cells were spun down (5 min, 500 rpm, RT), the supernatant was removed and the cell pellet resuspended in 1 mL (from a 6 well) of freezing media (10 % DMSO (v/v) (SIGMA, D8418) in ES-qualified EmbyroMax Fetal Calf Serum (Millipore, ES- 009-B)). The cell suspension was transferred to a cryo-vial (1 mL per vial) and cryo-vials were placed in a polystyrene sandwich at - 80 °C, overnight. Cryo-vials were transferred to liquid nitrogen for long-term storage.

6.1.6 Thawing mouse embryonic stem cells

Prior to thawing, 15 mL falcon tubes were labeled and filled with 10 mL of pre-warmed ES complete media (~ 37 °C). Pre-prepared gelatin coated plates were aspirated and air dried in the laminar air flow hood. Cryo-vials were transported on dry ice from liquid nitrogen storage to the tissue culture room. Cryo-vials were placed in 37 °C water bath untill a small core of frozen cell solution remained. 1 mL of pre-warmed ES complete media (~ 37 °C) was added to the vial and the cell suspension was transferred to a 15 mL falcon tube. The falcon was closed and inverted multiple times to dilute the DMSO. The tube was spun down (5 min, 500 rpm, RT), the supernatant was aspirated and the cell pellet resuspended in 2 mL of pre-warmed ES complete media. Cell suspension was plated in a 6 well plate which was incubated at 37 °C with 5 % CO2 in a humidified

6.1.7 Counting ES cells using the Neubauer chamber

Single cell suspension was generated as described previously. 50 µL of the cell suspension was diluted in 50 µL of 0,4 % (w/v) Trypan blue (Invitrogen, 15250). Neubauer improved chamber was mounted according to manufacturer recommendations and 10 µL of the cell dilution was loaded onto the chamber (Neubauer improved, depth: 0,1 mm, 0,0025 mm2). The chamber was placed on an inverted DM IL LED microscope from Leica with an HI PLAN 10x/0,22 PH1 objective. Both, live cells (transparent) and dead cells (blue) were counted according to manufacturer recommendations in all grey shaded groups squares (one group square consist of 16 single squares) (Figure 29).

Figure 29: Counting grid of a Neubauer improved chamber.

Cells alive (transparent) and dead (blue) were counted within the grey indicated areas of the grid. One group quadrat consists of 16 smaller quadrats. The sum of all cells was counted in all four group quadrats according to manufacturer recommendations.

The final cell concentration was calculated as follows:

𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 =∑ 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑥 10,000 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛Q.)(*+

𝑁_{R+*ST US.V+.(W} X

𝑐𝑒𝑙𝑙𝑠 𝑚𝐿 Z

Cell viability was calculated as follows:

𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = ∑ 𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠

∑ 𝑎𝑙𝑙 𝑐𝑒𝑙𝑙𝑠 (𝑙𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 + 𝑑𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠) 𝑥 100 [%]