To sterilize PimCell^® silicone inserts wet and dry autoclaves are recommended.

PimCell^® adaptors should be sterilized by washing in 70% ethanol.

Reagents

• Deionized (DI) water
• Ethanol: 70% in sterile DI water

Equipment & Materials

• Biosafety cabinet
• Pipette tip box
• Gloves
• Sonicator
• Tweezers
• Autoclave
• Washing reservoir
• Drying Oven
• Glass beaker
• Glass cover slips
• Aluminum foil

PimCell^® silicone inserts sterilization procedure

• In the biosafety cabinet, using clean gloves and tweezers, transfer the PimCell^® silicone inserts from the delivery container to a washing reservoir.
• Rinse inserts in 70% ethanol for 15 minutes followed by 3 times washing with a large volume of deionized water.
• Use tweezers to transfer the PimCell^® silicone inserts from the washing reservoir to a glass beaker with deionized water. For effective sterilization check that the inserts not stuck to each other. Cover the glass beaker with aluminum foil. Sterilize the inserts at 120°C for 20 min in an autoclave (a wet autoclave).
• In the biosafety cabinet, using clean gloves and tweezers, transfer the PimCell^® silicone inserts from the glass beaker to a clean pipette tip box for dry autoclave sterilization. Place the inserts with the channel side up. Cloze the box and sterilize again at 120°C for 20 min (a dry autoclave).
• Place the box containing sterile devices in an over at 80°C for at least 4 hours.

The sterilized devices can be stored at room temperature 23°C for up to a month before bonding to desired cover.

PimCell^® adaptors sterilization procedure

• Use surgical scissors to cut PimCell^® adaptors at a location that allows closing a desired plate with its lid when adaptors are attached to PimCell^® inserts .
• PimCell^® adaptors should be sterilized by washing in 70% ethanol for 15 minutes followed by 3 times washing with a large volume of deionized water.

How do I clean glass coverslips to seal the PimCell^® silicone inserts? 

Cleaning glass coverslips procedure

• Sonicate the glass slides in a water bath sonicator for 30 minutes.
• In the biosafety cabinet, using clean gloves and tweezers, rinse the glass slides with 70% ethanol.
• Wash slides three times with deionized water.
• Place the glass in a clean in a clean pipette tip box using a pair of sterilized tweezers and autoclave at 120°C for 20 min (a dry autoclave).
• Dry the autoclaved glass in an oven at 80°C for more than 4 hours.

How can PimCell^® silicone inserts be sealed?

Sealing methods can be classified into irreversible and reversible bonding^1-4. Irreversible sealing is the preferred option for working with high-pressure fluid without leakage problems. The PimCell^® silicone inserts are designed for gravity driven perfusion. Membrane free microfluidic technology is not adapted to high-pressure which can very easy destroy a membrane free 3D cell culture. Reversible sealing is recommended for assembly of the PimCell^® silicone inserts.  

Reagents

• Deionized (DI) water
• Ethanol: 70% in sterile deionized water
• Poly-L-lysine (PLL), MW 70,000–150,000, 1 g (cat. no. P1274, Sigma)
• Sodium tetraborate, 99%, 100 g (cat. no. 221732, Sigma)

Equipment & Materials

Biosafety cabinet

0.2 mm filter

Gloves

Digital balance

Tweezers

Expanded plasma cleaner

Pipette with tips

Drying Oven

Sterile dish

Assembly procedure

• Prepare 100μg/ml solution of PLL. Dissolve 400 mg of PLL (1 mg ml^-1) in borate buffer solution by stirring for 30 min. Sterilize the solution by filtration with 0.2 mm filter. PLL solution can be stored at -20°C for future use.
• In the biosafety cabinet, using clean gloves and tweezers, place clean glass coverslips in PLL solution for 12 h.
• Immerse in DI water for at least 2 h. Wash twice with DI water.
• Dry glass coverslips overnight in the biosafety cabinet under sterile conditions.
• Store PLL-coated glass coverslips in a 4°C refrigerator until use. PLL-coated glass coverslips can be used within 2 weeks.
• Place a sterilized PimCell^® silicone insert on a PPL-coated glass coverslip with its channel side down and lightly touch the PimCell^® silicone insert until it is sealed to the glass.

The assembled PimCell^® silicone insert must be used within 1–2 h. Fill the open top chambers and channels with DI water for later use on the same day.  

NB! The PimCell^® silicone insert can be sealed directly to the glass of 24 well microtiter plate (product code 2031240202011) using PLL coating. 

What is a general guideline for 3D cell culture using PimCell^® silicone insert?

The direct advantage of PimCell^® silicone inserts is a 3D cell culture without any dead volume always presented inside inlets or outlets of (hydro)gel filled microfluidic channels. Using an open top cell culture chamber we introduce desired 3D cell culture support directly into the open top cell culture chamber (i.e. gel or gel mixture with desired cells) for gelation. The polymerized gel separates content of microfluidic channels from the space above the cell culture chamber allowing seeding different cell types on the different part of a common 3D cell culture support. The direct advantage is an opportunity to create a novel or to modify existing in vitro 3D models for producing more valuable and reliable data. This protocol covers a general usage of PimCell^® inserts such as filling an open top cell culture chamber with a 3D cell culture support like gel and hydrating media channels. Seeding monolayer cells on the media channel, staining etc. are beyond of the scope of this general illustration but could be easily adapted from different publications⁵. 

• Harvest desired cells according to their dissociation protocol and prepare a cell suspension with the optimal cell density for seeding.
• In the biosafety cabinet, using clean gloves and tweezers, take assembled PimCell^® inserts from the sterile dish, connect its microfluidic channels to PimCell^® adaptors and place in a sterile 24 well plate (one insert per one well). Keep these at room temperature (RT, ~20–25 °C) at least 1 hour.
• Prepare the required amount of gel mixture. The PimCell^® Voice has one open top chamber per unit and needs about 3 µL gel mixture per one chamber + 40% extra. The PimCell^® Tube has two chambers per one unit and needs about 5 µL per one chamber + 40% extra . Keep on ice and use gel immediately within 10 minutes after preparation.
• Place your pipette tip slightly above the bottom coverslip and gently dispense the gel mixture into the open top cell culture chamber of PimCell^® Repeat this step to entirely fill all PimCell^® insert.

N.B. Correct positioning of the pipette tip with gel mixture above the open top chamber allows capillary forces to pull the gel mixture under a CPB along the adjusted microfluidic channel without the gel overflows into the channel. Increase the loading volume (i.e. to 3,5 µL) in case of incomplete filling.

PimCell^® inserts with unpolymerized gel must be handled with care. Excessive impact or agitation may cause leaking unpolymerized gel mixture into the adjusted microfluidic channel.

• Gently pipet 50 µL of sterile PBS into each well in the free space between the PimCell^® insert and the wall of the well to prevent gel dehydration during gelation.
• Cover the plate with its lid and place in the a humidified incubator at 37 °C and 5% CO₂ for 20 min to allow gelation.
• After gel polymerization, open the lid of the plate and gently dispense 50 µL of desired cell culture medium above each open top chamber with the polymerized gel.
• Gently add 100 µL of desired medium to each perfusion medium inlets and wait till the medium appears in each perfusion outlet.
• Gently add 50 µL of medium to the perfusion medium outlet after cell attaching on the bottom of the perfusion channels.
• Close the lid of the plate and place in the a humidified incubator at 37 °C and 5% CO₂.
• Refresh medium every 2–3 days on each open top cell culture chamber, inlet and outlet.

N.B. Then you need more volume of cell culture medium (i.e. for differentiation of iPSCs or organoids ) you can remove the sterile PBS from the space around inserts and add more medium to each well.

• Take beautiful pictures and send to our customers gallery. Publish your data using PimCell^® devices, mention our company and get 15 devices for free in your next order.

In this way you can modify different protocols for gravity driven 3D cell culture incl. vascularization of different tissues.

References

1. Park, J.W. et al. Microfluidic culture platform for neuroscience research.

    Nature Protocols 1, 2128-36 (2006)   

2. Harris, J. et al. Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices.

    J. Vis. Exp. 9, e410 (2007) 

3. Shin, Y. et al. Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.

    Nature  Protocols 7, 1247-1259 (2012)   

4. Hajal, C. et al. Engineered human blood–brain barrier microfluidic model for vascular permeability analyses.

    Nature Protocols 17, 95-128 (2022) 

5. Bonanini, F. et al. In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed.

    Angiogenesis 25, 455–470 (2022)