Download as a pdf Protocol Aspirate media and wash with PBS Incubate with PBS+Tryple for 1min30sec at RT Aspirate and wash with PBS Add RPMI(+)+Polybrene Add particles dropwise (aim to 20x final concentration Expectations Takes a long time in CMs to get expression compared to HEK cells, around 4 days. Non CMs have higher expression of the exogenous protein. Material PBS+Tryple mix 1to1 RPMI(+) with Polybrene (7.5uL polybrene/10mL of media); 1.2mL per 12well well (add cat num) Viral particles (ideally 30-45x concentrated) Sanity check Test particles at 1x concentrated on Cal51 or HEK cells 📄 Download this post as PDF

Download as a pdf There are several major protocols to follow when freezing cells: Stable cell lines (HEK, NHDFs etc.) using DMEM+FBS+DMSO Pluripotent stem cells and early differentiation (KOSR + DMSO) Differentiated cardiomyocytes (Bam banker-not working yet) Material FBS KOSR DMSO Tryple PBS Filter 0.45μm Freezing Vials Freezing Chamber Preparation Label freezing vials. Make sure freezing chamber is equlibrated to Room Temp Prepare freezing solution: For stable cell lines(10% DMSO; 90% Culture medium with FBS; 1.5mL per vial) For hPSCs and early derivated (10%DMSO; 90% KOSR) Filter freezing medium Put freezing medium on ice, use cold Process Aspirate medium from the cells. Wash with PBS. Add tryple (2mL per 10cm dish). Incubate up to 5 min at 37◦C and check for detachment of cells. Harvest cells in 10mL of media transfer to falcon tube. Centrifuge, discard pellet. Resuspend in 1mL of freezing medium and keep on ice. Count cells. Dilute to 1-2x10E6 cells/mL of freezing media. Pipette 1-1.5mL in the tubes (depends on the type). Put vials in the freezing chamber. Label the freezing chamber with name, hour, and date. Put in -80◦C (>2h). Move to liquid nitrogen.

Download as a pdf Materials DMEM high glucose Peniciline/streptomycine (100x) FBS Tryple 2% Gelatine PBS Media preparation DMEM high glucose 1% peniciline/streptomycine 10% FBS Dish coating Dilute 2% stock of Gelatine to 0.1% in PBS Sterile filter Incubate 30 min. at 37◦C Aspirate gelatine from the plates Cells Thawing Transfer cells from liquid nitrogen storage on dry ice into cell culture room Spray with ethanol, immerse in water bath Let thawing for 1-2 min. (check visually, remove when there is only a little piece of ice left inside the vial) Transfer content of the vial into a 15mL tube Dropwise add cold culture medium, shake gently between the media addition (first 5 ml) Add 5 more mL to reach 10-12 ml Colllect cells by centrifugation at 300g for 5 minutes Discard the supernatant into waste Flick the tube to release the cell pellet Cells counting Resuspend in 1mL of media take 10uL of susupension, mix with 10uL of trypan blue, inject into the counting slide count cells Cells seeding recomended seeding density is 2x104 cells/ cm2 dilute cells to proper concentration, gently pipette onto the plate Cell Passaging Prepare fresh dishes with gelatine coating (-30 min.) Aspirate the media Wash with PBS Add Tryple (1mL per 20cm2) Incubate for 5 min. at 37◦C Visually check the cells detachment Gently collect using P1000 pipette tip Collect to 10mL of medium Colllect cells by centrifugation at 300g for 5 minutes Discard the supernatant into waste Flick the tube to release the cell pellet Resuspend in 1 ml of medium Count cells Resuspend in fresh media to desired concentration • Seed onto the dishes Recomended cell densities Sparse cells 5 000 -20 000 cells/cm2 ...

Download pdf version IPS cells passage on 6cm dish Aspirate medium Wash with PBS Put RT TRYPLE 1mL, incubate 2-4min at 37C until cells appear isolated (but not floating) Carefully aspirate TRYPLE, add 3mL of E8 and gently dissociate cells using 1000uL pipette tip, take 10uL of suspension for countess measurment (confluent 6cm dish has 9-11million cells) Spin 300g/3min Prepare E8 complete with 1:2000 RI (4mL for 6cm dish, 2mL 6well, 1mL 12 well, 0.5mL 24well) Discard supernatant and resuspend to concentration 2million cells/mL in E8+RI Put E8+RI in a new matrigel coated (1:33 MG + 1:100 Geltrex) dish, add cell suspension (200uL for 6cm dish, 100uL for 6well, 50uL for 12well, 25uL for 24well) to seed 20 000cells/cm2 Rock to distribute cells evenly Change media daily (for first 3 days, change half of medium, spin the dish to congregate debris in the center of the dish to be aspirated) Passage when confluent (without borders between cells visible in monolayer, only cell nucleui should be visible) Cardiac differentiation from monolayer Note: The number of days before cells reach monolayer are variable, wait till you get full confluency with a good morphology ...

CM selection: Once CMs are contracting, i.e., from day 7 to day 11, cells are kept in RPMI supplemented with 1XB27(+insulin). To increase cardiomyocyte purity, cells are cultured in RPMI without glucose for three days (day 11 to day 14). (5mM sodium lactate supplement is recommended as an alternative energy source for CMs). Change the media to RPMI supplemented with 1XB27 (day 14 to day 16) On day 16, the iPSC-CMs are dissociated with TrypLE 10x and seeded in 6-well Matrigel-coated plates at a density of 2-3x106 per well in RPMI with 1XB27 containing 10% KOSR and ROCK inhibitor Y-27632 (replating media). After 2 days, cells are cultured in RPMI with 1XB27 without glucose for 3 days. Post-starvation, maintain them on RPMI B27+ for 2 days before switching to 3 ml of a metabolism-based maturation medium. Protocol cartoon ...

Time required: 45min - 1h30min Materials: PBS Fibronectin: Stem Cell technologies 1mg/ml Cat#07159 (old one) Sigma Aldrich Cat#1141 1mg/ml link Procedure: Optional: sterilize surfaces by UV in the cell culture hood for 30min Dilute Fibronectin in sterile PBS 1:100 to get 10ug/mL Cover cultivation surface Incubate at Room Temperature for 1h or 30 min at 37C Aspirate before cell seeding Notes: For hydrophobic surfaces (PDMS, Ibidi soft plates) use 0.5mL for 12well, 1mL for Ibidi plate For ibidi slides, use 200uL per well

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Download as a pdf Time Allow take 3-3.5 hours, add one more hour when seeding on ibidi dishes in the drop Materials Fibronectin (Sigma; Cat num. F1141-5mg; lot …) Pluronic Acid (Cat bum. 59005, lot P40614-101164) ddH20 RPMI (+) KOSR Rock Inhibitor Enzyme T Enzyme T diluent FBS PBS Counting Slide DIshes/slides Ibidi dish with the beads coated surface (dry); 3.14cm2 6well with 2cm stencil; 9.5cm2 Preparation steps Solutions Fibronectin Dilute 1mg/mL fibronectin to 50ug/mL in PBS Amount: 0.75mL per dish 2mL per 1w6 Pluroinc Acid Dilute to 1% from 10% stock in ddH20 Amount: 3mL per dish 3mL per 1e6 NOTE: filter through 22um filter Stop solution dilute FBS to 20% in PBS Amount: 2mL per 12well 1mL per 24 well Replating medium 10% KOSR in RPMI(+), add 1to2000 Rock Inhibitor Amount: 3mL per ibidi dish 3mL for 6well dish 1mL for counting of cells Enzyme T dissolve 33uL/mL of buffer X Amount: 400uL per 1w12 well 200uL per 1w24 well Process Coat and sterilze the plates Ibidi: Add 750uL drop on the ibidi dish, spread to edges using pipette (is very hydrophobic) 1w6: Add 2mL swirl to make sure all surface is coated Incubate 1hr at RT Aspirate FN, Wash with 3mL PBS 3x Add pluronic 3mL (both ibidi or 1w6) Incubate 30min at RT (Ideally start harvesting cells at this point) wash with PBS 3mL 3x Collect cells wash with PBS add diluted enzyme T (200uL/24 well, 400uL/12 well) incubate 8-10min Add Stop solution (300ul/24well, 600ul/12 well) Collect by pipetting, disrupt to single cells Add (500ul; 1mL) to wash down Spin at 300g for 3-5min Resuspend in 0.5mL replating media (for 24 well, for the rest, 1mL is enough) count Seed cells create a solution Ibidi: approx 100k cells/mL in replating solution 1w6: approx 30k cells/mL Deposit: Ibidi: as a drop approx 600uL, spread to the edges to fill the whole surface of insert 1w6: 3mL in the dish Add media (Ibidi only): Let the cells to sit down for 1 hour (check after 30 min if its okey), but 1hr is better Fill the rest of the vessel with the replating medium (2mL) Change media change media day after afterwards cheange media to RPMI (+) change every second day Measure from the day 3 onwards

Download this as a pdf link Time expected 4 h Device sterilization (can be done before) 1 h Matrigel prep (can be done before) 1.5-2 h Seeding Device sterilization Put 0.2mL ethanol in the device well, in the device lid, enclose all in a container and place for 4h-overnight at 80 C to let the ethanol evaporate Coating *It is important to coat just the diamond, so place a small enough drop on the top, which does not spill ...