Production of microfluidics (µF) chips:
Design of the photomask using CAD or Illustrator Software. The file of the photomask we used for reprogramming experiments is available on request.
Print the photomask on a transparency film in high-resolution (>2400 dpi).
a. Silicon Wafer preparation:
i. Wash wafer with 2-Propanol.
ii. Wash wafer with distilled water.
iii. Dry wafer on a hot plate at 120°C for 30 minutes.
b. Photoresist Coating:
i. Place the wafer on the chuck of the spin coater and power the vacuum pump.
ii. Dispense 1 ml of photoresist for each inch of wafer diameter.
iii. Spin at 500 r.p.m. for 10 s with acceleration of 100 r.p.m/s.
iv. Spin at 500 r.p.m. for 30 s with acceleration of 1500 r.p.m/s.
c. Photoresist Soft Baking:
i. Bake the coated wafer on a hot plate at 65°C for 5 minutes and at 95°C for 30 minutes.
i. Exposure through a photomask to UV light at exposure energy of 310 mJ/cm2.
e. Post-Exposure Baking:
i. Bake the coated wafer on a hot plate at 65°C for 5 minutes and at 95°C for 10 minutes.
i. Place the wafer in a glass dish and put it on a shaker.
ii. Pour propylene glycol monomethyl ether acetate in the glass and shake for 15 minutes.
iii. Wash wafer with 2-Propanol.
iv. Dry the wafer with compressed air.
g. Photoresist Hard Baking:
i. Bake the coated wafer on a hot plate at 80°C for 1h.
- Replica Moulding:
a. Place the patterned wafer in a Petri dish and then in a desiccator, containing a small beaker with few drops of hexamethyldisilazane (HMDS).
b. Connect the desiccator to the vacuum line to reduce the pressure inside the desiccator; keep the wafer under vacuum for 30 minutes.
c. Pour Sylgard 184 PDMS in a plastic cup in a 10:1 base:curing-agent ratio, mix it thoroughly for at least 3 minutes.
d. Place the uncured PDMS in a desiccator and connect it to the vacuum line to evacuate the air; turn off the vacuum and keep the PDMS under vacuum in order to remove the bubbles. After 15 minutes vent the desiccator.
e. Place the wafer with PDMS into the vacuum desiccator for a 10 minutes and check for complete removal of bubbles.
f. Place the Petri dish containing both the wafer and PDMS in an oven, cure it at 70°C for 1.5 hour.
g. Separate the cured PDMS block from the wafer.
h. Cut the PDMS block into pieces according to the pattern of the wafer.
i. Use disposable 1-mm biopsy punches to produce inlet and outlet holes for each culture chamber.
j. Prepare the reservoirs as PDMS pieces of 5 x 0.5 cm and punch them with a 3-mm biopsy punch.
k. Clean thoroughly a microscopy glass slide with MICRO-90 cleaning solution (2% v/v in distilled water) and rinse with distilled water.
l. Place the PDMS replica, with the patterned surface facing up, and a glass slide in an air plasma cleaner for surface activation at 0.3 mbar of filtered air and 30 W for 2 minutes.
m. Place in conformal contact the activated surfaces of both PDMS and glass (block1).
n. Place the device, with the PDMS surface up, and the PDMS reservoir in an air plasma cleaner for surface activation at 0.3 mbar of filtered air and 30 W for 2 minutes.
o. Place in conformal contact the PDMS reservoir with the flat side of the PDMS (block1) in order to seal the reservoirs.
p. Place the device in an oven at 80°C for 1 hour to complete the bonding process.
q. Cool the bonded microfluidic device to room temperature.
r. Pipette 12 µL of 2-propanol inside each culture chamber to clean the chamber from production process by-products, and rinse by flowing 30 µL of distilled water into the chambers before 2-propanol evaporation.
s. Dry the chambers by aspirating the water with a 200 µL pipette and package the microfluidic device inside an autoclave bag.
t. Autoclave at 121˚C for 15 minutes, and let it dry at the end.
Resuspend each mmRNA (POU5F1, SOX2, MYC, KLF4, NANOG, with or without nGFP) and mix them following manufacturer datasheet, omitting LIN28 mmRNA. The molar ratio among different factors should be 3:1:1:1:1:1, with POU5F1 being the most abundant. Each mmRNA is resuspended at a concentration of 100 ng/µl and the following volumes of each factor are mixed together: POU5F1 520µl, KLF4 183µl, SOX2 155µl, cMYC 200µl, NANOG 136µl, nuclear eGFP 124µl. Aliquot mixed mmRNAs in small amount (5 to 10 µl) in 0.2 ml sterile microtubes. Avoid freezing-and-thawing more than 2 times. Alternatively, mmRNA can be produced in-house as described by Warren and colleagues (Warren et al., 2010).
Supplement DMEM with 10% FBS. Addition of antibiotics is optional. We normally perform reprogramming without antibiotics.
Complete Primed Reprogramming Medium (PRM):
Add to StemMACS ReproBREW XF Basal Medium, the 50X StemMACS ReproBREW XF Supplement and store at 4C. Add B18R (final concentration 0.2 µg/ml), RI (5 µM), CH (1 µM) only when reprogramming starts and do not store for more than 72 h.
RSeT for Naïve Reprogramming:
Add the 5x Supplement, 500x Supplement and 1000x Supplement to RSeT Basal Medium according to manufacturer’s instructions and store at 4C. Add B18R (final concentration 0.2 µg/ml) only when reprogramming starts and do not store for more than 72 h.
- Day -1 - Seeding of Human Fibroblasts in microfluidics (µF) chip:
a. Place the µF chip inside a 100 mm cell culture dish. Please see Supplementary Figure 1A of Giulitti et al., 2018 for a diagram showing the different parts of the microfluidics chip and how it is used.
b. Add 3 ml PBS-/- to the 100mm cell culture dish containing the µF chip and let it spread in the dish, without covering the µF chip. The dish works as a sterile humidified chamber. Throughout the reprogramming protocol make sure the dish is humidified by adding PBS-/- when needed.
c. Prepare a 25 μg/ml fibronectin solution in PBS-/- and inject 12 µl in each channel.
d. Incubate for 30 min at room temperature.
e. Calculate the number of fibroblasts needed for the reprogramming experiment. We want to plate fibroblasts at 25 cells/mm2 and the total surface of one channel is ~27 mm2, thus 675 cells per channel are needed. In fact, for each channel we have to use 1500 cells, because inlet and outlet volumes must be taken into account.
f. Detach fibroblasts by washing with PBS-/- twice, then add trypsin and incubate at 37C for 3-4 minutes. Add at least 3 volumes of Fibroblast Medium and transfer the cell suspension in a sterile 15 ml tube.
g. Count fibroblasts with chamber and transfer to sterile tube only the number of fibroblasts previously calculated in (e). Spin at 300 rcf for 4 minutes in a benchtop microcentrifuge.
h. Resuspend the cell pellet in fresh Fibroblast Medium. For each µF channel, 12 µl are needed. For example, for 20 channels 30,000 cells have to be resuspended in 240 µl of Fibroblast Medium.
i. Inject 12 µl of cell suspension per channel. Please note that the volume of the µF channel is 6 µl, but 12 µl are needed to fill also the inlet and outlet of each channel.
j. Remove excess of liquid from reservoirs (leave enough medium to form a meniscus).
k. Culture overnight in Low oxygen incubator (37°C, CO2 5%, O2 5%).
- Day 0, 9 a.m. - Preparation for the first transfection:
a. Calculate the total volume of PRM needed for the first 3 days of reprogramming experiment as follows: 12µl2 (number of daily media changes)number of channels3 days1.2 (to allow 20% excess of medium). For example, for 20 channels 1,728 µl. The same amount of complete PRM will be needed also at day 3.
b. Add RI (5 µM), CH (1 µM) and B18R (0.2 µg/ml) and do not store for more than 3 days.
c. Leave complete PRM medium at room temperature for at least 30 minutes and add 12 µl to each channel. Remove excess of exhausted medium from reservoirs.
d. Transfer µF chips back into the incubator for at least 1 hour before transfection. B18R is a component of the complete PRM that reduces the innate immune response and prepares cells for transfection of mmRNA. It must be present in all media used throughout the reprogramming protocol.
- Day 0, 10 a.m. - Prepare mmRNA Transfection mix:
a. All volumes indicated in this section refer to one µF channel, please multiply them by the number of channels used in the reprogramming experiment.
b. Thaw mmRNA aliquots on ice, use StemMACS mRNA Transfection Kit transfection reagent (TR) + transfection buffer (TB). In RNase-free sterile 0.2 ml tubes prepare the following mixes:
i. Mix A: 0.27 µl TB + 0.09 µl mmRNAs
ii. Mix B: 0.333 µl TB + 0.027 µl TR
c. Transfer Mix B to Mix A and mix gently 4-5 times. The total volume of transfection mix for one channel should be 0.72 µl.
d. Incubate for 20 min at RT.
e. Gently transfer 0.7 µl of Mix A+B to a tube containing 11.3 µl of complete PRM. Mix gently 4-5 times.
f. Dispense 12 µl of mix to each channel. Remove excess of exhausted medium from reservoirs.
g. Incubate in Low oxygen incubator (37°C, CO2 5%, O2 5%) for 8 hours.
- Days 0, 6 p.m.
a. warm at 37°C complete PRM and add 12 µl to each channel. Remove excess of exhausted medium from reservoirs.
b. Incubate in Low oxygen incubator (37°C, CO2 5%, O2 5%) overnight.
- Days 1-5 – MET induction:
a. 9 a.m., check for nuclear EGFP (after first transfection expect >50% nEGFP+ cells)
b. 9 a.m., proceed with transfection, as described in point 3.
c. Starting from day 2, the amount of mmRNA transfected should be gradually increased only if fibroblasts appear healthy and proliferative. If cell population is steady and/or stressed, do not increase the amount of mmRNA transfected.
d. The aim is to maximize mmRNA transfection efficiency, which can be estimated by looking at nuclear EGFP signal. At the same time excessive mmRNA amount could lead to cell stress and reduced proliferation.
e. To increase mmRNA amount multiply by 1.25X, 1.5X or 1.75x the volumes indicated in point 3 (see Table 1). Typically, mmRNA amount is increased every second day, reaching the 1.75x amount by day 5-6.
f. 6 p.m., add fresh complete PRM
- Days 6-12 – Naïve Reprogramming completion:
a. Over the first 6 days fibroblasts should undergo Mesenchymal to Epithelial Transition (MET) (see Figure 1e of Giulitti et al., 2018). If MET is not observed, iPSC colonies will very unlikely form, so we would suggest to restart the whole protocol.
b. If MET is observed, cells should be exposed to RSeT medium rather than PRM, from Day 6 onwards.
c. Calculate the amount of complete RSeT medium needed as done for PRM in point 2a. Add B18R (0.2 µg/ml) to RSeT and store complete RSeT at 4C for maximum 72 h.
d. 9 a.m., prepare transfection, see point 3, using RSeT instead of PRM.
e. Typically, the 1.75x dose of mmRNA is used from day 6 to the end of the reprogramming protocol (day 12).
f. Small compact colonies should start to emerge from day 9 and become mature around day 12-14.
g. In order to perform immunostaining of primary naïve iPSC colonies we use a mmRNA mix without nEGFP from day 8. This allows EGFP clearance by day 12.
h. 6 p.m., add 12 µl of warm B18R-supplemented RSeT medium to each channel.
- Days 12-14 – Expansion of naïve iPSC colonies out of the µF chip:
a. Wash twice with PBS-/- each channel containing naïve iPSC colonies. Incubate cells with 12 µl Accutase (or TrypLE) for 3-7 min at room temperature. The timing depends on the number of cells remaining in the channel. In some cases, several colonies will form on top of a dense layer of unreprogrammed fibroblasts.
b. When all cells are fully detached, add to each channel 12 µl of RSeT and collect cells from reservoir into a 1.5 ml microtube. Repeat this step until all cells have been collected.
c. Add 1 ml RSeT with 10 µM RI to the 1.5 ml microtube and plate on inactivated MEF in a well of a 24-well plate. If reprogramming efficiency is low we suggest to pool together colonies from several µF channels to maximise the success of expansion of naïve iPSC colonies.
d. Add 0.6 ml of fresh RSeT medium every day and passage colonies every 3-5 days according to the size of colonies and level of proliferation.
e. A split ratio between 1:1 and 1:4 should be used according to the number of colonies, their size and proliferation rate. The first two passages are critical to stabilise the naïve iPSC line.
f. We succeeded in generating a stable niPSC line starting from as little as 3 colonies, by passaging them every 5-6 days at a split ratio of 1:1. Conversely, starting from >50 colonies we obtained stable niPSC lines after 2 passages at a split ratio of 1:2 – 1:3 every 3 days.
g. Stable niPSC lines are routinely passaged every 3-4 days at a split ratio of 1:3 – 1:4, with daily medium change under low oxygen condition on feeder cells.
h. Naïve pluripotent identity should be checked during expansion by quantitative real-time RT PCR and Immunostaining as described in Figures 2 and S2 of Giulitti et al., 2018.