PimCell. Cell Culture Starter Kit ADVANCED CELL CULTURE PIMBIO CATALOGUE & APPLICATION NOTES

Transcription

1 2018 PimCell Cell Culture Starter Kit ADVANCED CELL CULTURE PIMBIO CATALOGUE & APPLICATION NOTES

2 PimCell Microfluidic Cell Culture Starter Kit PimCell line is a group of related cell culture microfluidic devices developed and manufactured by PimBio B.V. (The Netherlands). With our plug-and-play microfluidic technology you will significantly accelerate the development of your own 3D cell culture products which comply with microscopic slide and SBS microtiter plate standards. Advantages of PimCell plug & play microfluidic technology highly reproducible adapted to existing lab equipment and automation comply with microscopic slide standards adapted to 96 and 1536 SBS microtiter plate standards economical access to the fields of organ-on-a-chip technology adapted to every desired cell culture scaffolding short learning curve static and dynamic cell culture excellent imaging simple maintenance One PimCell Microfluidic Cell Culture Starter Kit contains: ten PimCell microfluidic miniplates M5 with 3 independent cell culture units per miniplate one handling Frame for the spacing of a 96 well SBS microtiter plate five medium Reservoirs with caps and Adapters one pump Fluid Connector About PimBio B.V. PimBio B.V. is a privately held company founded in 2014 to develop new and promising microfluidic devices for bioengineering of physiologically relevant living tissue for different applications. The development of a platform technology producing 3D physiologically relevant in vitro models will enable the generation of fully differentiated living tissue types and whole organs which can be used in scientific research, regenerative medicine and industry. PimBio has the vision to operate as a market leader in the manufacturing of microfluidic devices, by investing in quality, innovation and capacity. PimBio supports the 3Rs, the guiding principles of reduction, refinement and replacement of animal experiments. Having a basis in The Netherlands with our partner network extending into West Europe we can provide existing processes and manufacturing methods for in vitro applications. All our products are manufactured and packaged in clean rooms by our partners certified according to ISO 9001 and ISO The essence for PimBio is to work together with customers on innovative and reliable solutions in the complete trajectory from idea to manufacturing. The mission of PimBio is to provide research and industrial community a unique combination of micro technologies, materials, microfluidics knowledge and customer application know-how, which enables PimBio to offer customers innovative and sustainable solutions with 3Rs potential to improve people s lives. PIMBIO B.V. 1

3 PimCell microfluidic miniplate M5 M5 is our first PimCell Microfluidic Plate s model. This model is a microscopy-slide-sized and microfluidics based cell culture miniplate that enables culturing and screening of an unlimited number of physiologically relevant organ and tissue models. All PimCell microfluidic devices are based on the SBS microtiter 96 & 1536 well plate standards, robot- and pipette operatable and support a wide range of assays. The miniplates are made from a thermoplastic medical grade polymer which is completely nonpolar and amorphous. It has a very low autofluorescence, a very low permeability for water vapour and a low capacity for the absorption of water. PimCell miniplates are compatible with all microscopy and readout equipment, including confocal microscopy and luminescent readers. The PimCell microfluidic miniplate M5 contains three independent cell culture units (chips) on a slide of standard microscopy format. Each unit has one open access cell culture chamber and two separated flow channels with high fluidic resistance central parts which can allow to perform gravity driven perfusion of microfluidic channels (please see our Application Notes for more information). Each flow channel has one inlet and one outlet connected to own inlet/outlet port. A part of wall of the high fluidic resistance part of each channel is open into the open access cell culture chamber. The main utility of the high resistance part is to reduce the flow rate of cell culture media during long-term experiments with unidirectional flow in the microfluidic channels. The microfluidic channels are designed for culturing blood endothelial cells or perfusion of cell culture chamber. Technical Features: - application: one-use R&D only - available: sterile - material: olefin - cell culture chamber 1.43mmX1.43mmX0.4mm with 0.86 µl volume - short microfluidic channel has 140 µm depth, 1.5 mm width and 2.87 µl volume - long microfluidic channel has 140 µm depth, 1.5 mm width and 4.58 µl volume - high fluidic resistance part has 25 µm depth, 250 µm width, 1.43 mm length and about 9nL volume - one well has 59 µl volume - oil sensitive - can be used with Silicone immersion oil only! Fig. 1: Top view of PimCell microfluidic miniplate M5 with three cell culture units (chips) per miniplate. One cell culture unit has one open access cell culture chamber, one short microfluidic channel and one long microfluidic channel PIMBIO B.V. 2

4 Handling Frame for 96 well microtiter plate spacing To interface PimCell microscopy-slide-sized microfluidic miniplates we offer a stackable handling Frame which complies with the SBS microtiter 96-well plate standard. The handling Frame can therefore be handled with standard laboratory automation equipment and support the integration of PimCell microfluidic miniplates into your lab workflow. This handling Frame places PimCell microscopy-slide-sized microfluidic miniplates on the positions of the wells of a 96 well microtiter plate. Four PimCell microscopy-slide-sized microfluidic miniplates can be securely fixed in this handling Frame. Technical Features: - application: for R&D only - available: unsterile Fig. 2: Handling Frame for microscopy slide format miniplates to comply with spacing of a 96 well microtiter plate PIMBIO B.V. 3

5 Medium Reservoir with cap and Luer interfaces This reusable media Reservoir with a volume of 500 µl has a standard Male Luer interface to connect to an incorporated Female Luer port of a PimCell miniplate. The advanced design of the cap with a small open access can eliminate problems associated with media evaporation from a closed reservoir. A sterile filter connected to the open access of the cap through a Male Luer Adapter or covering with breathable adhesive film can reduce also contamination risk. The cap can stay open and the Reservoir can be also covered with a breathable adhesive film (not included) to provide better microscopic view when Reservoir should be connected to an open access cell culture chamber. Technical Features: - application: R&D cell culture only - depth 2 cm and volume 500 µl (25 µl per 1 mm depth) - available: unsterile - can be autoclavable C 20 min - suitable for PimCell miniplates with female Luer adapters - material: polypropylene (opaque) Fig. 3: Medium Reservoir with cap has an additional Male Luer Adapter on its cap PIMBIO B.V. 4

6 Pump Fluid Connector Our pump Fluid Connector allows to couple Female Luer interfaces on the microfluidic platforms with tubing deriving from pumps, reservoirs or other microfluidic channels. A Luer Slip Elbow Connector made from a clear transparent plastic allows to ensure an easy visualisation of cell culture under dynamic conditions. Technical Features: - application: R&D cell culture only - available: not sterile - can be autoclavable C 20 min - suitable for PimCell miniplates with Female Luer adapters - material: opaque (polypropylene), clear transparent Luer Slip Elbow (olefin) and tubing (silicone) Fig. 6: A transparent Luer Slip Elbow Connector allows an easy visualisation of cell culture chamber with an inverted microscope under flow conditions PIMBIO B.V. 5

7 Application notes With our plug-and-play PimCell Microfluidic Cell Culture Starter Kit you can open new ways to significantly accelerate development of your own OOC products which comply with worldwide accepted microscopic slide and SBS microtiter plate standards. These application notes can help you to run you experiments with our 3D cell culture system and describe first basic steps to start with microfluidic PimCell miniplates. This part introduces how to operate PimCell miniplates for advanced cell culture. Using our Starter Kit you can create either static or dynamic cell culture. Option 1 Static cell culture (without flow inside microfluidic system) Fig. 8: A bottom view of a microfluidic cell culture unit of PimCell microfluidic miniplate M5. A short microfluidic channel (AB) has one inlet port (A) and one outlet port (B). A long microfluidic channel (DE) has also one inlet port (D) and one outlet port (E). These two microfluidic channels direct communicate through an open access cell culture chamber (C) Fig. 9: A scheme of cell culture without flow between a cell culture chamber (C) and microfluidic channels (AB and DE) PIMBIO B.V. 6

8 Option 2 Dynamic cell culture with flow from the cell culture chamber into the microfluidic channels Fig. 10: A scheme of 3D cell culture with flow (white arrows) from the cell culture chamber (C) into the microfluidic channels (AB and DE) Option 3 Gravity driven cell culture with flow along the cell culture chamber Fig. 11: A scheme of 3D cell culture with flow (white arrows) inside microfluidic channels (AB and DE) along the cell culture chamber (C) PIMBIO B.V. 7

9 Fig. 12: A bottom view of high fluidic resistance central parts (FG and HI) of microfluidic channels. These two microfluidic channels allow long-term gravity driven unidirectional perfusion without any additional equipment like a perfusion rocker platform (for more information please see Application Notes). PIMBIO B.V. 8

10 Static cell culture without flow between the cell culture chamber and the microfluidic channels (Option 1) The most simple way to use PimCell miniplates for 2D cell culture is to introduce cell culture media and cells into the cell culture chamber of one microfluidic unit of PimCell miniplate. This Application Note can be easy adapted for 3D cell culture just by introducing a 3D cell culture scaffold like hydrogel into an open access cell culture chamber. 1. Preparation steps 1.1 PimCell coating PimCell microfluidic miniplate M5 is made from a thermoplastic medical grade polymer which is biocompatible and permits direct cell growth but it is very hydrophobic and should be coated for efficient adhesion of most cells. All coating solutions can be calculated for a specific amount of coating material per area (µg/cm²) recommended by the manufacturer s reference. Dilute a coating solution to the desired concentration according to the manufacturer s specifications or references. For the coating setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 coating solution one sterile plastic 1.0 ml Insulin syringe one sheet of sterile (para)film Don t use glass or metal syringes to avoid miniplate damaging Work under sterile conditions Open the sterile packaging of the miniplate and gently connect an Insulin syringe loaded with 100 µl coating solution to the well of the open access cell culture chamber Gently introduce the coating solution into microfluidic system by pushing the syringe s plunger The solution will appear inside inlet/outlet wells of microfluidic channels Fig. 12: Male Luer Slip of an Insulin syringe connected to Female Luer Slip of the well of the open access cell culture chamber Cover the wells with sterile parafilm or breathable adhesive film to avoid evaporation and incubate according to the manufacturer s specifications or references Fig. 13: A well of one microfluidic channel filled with a coating solution PIMBIO B.V. 9

11 1.1.3 After incubation, remove the (para)film from the wells and gently aspirate the microfluidic system through inlets or outlets ports completely using the connected to the cell culture chamber syringe by pulling its plunger. Avoid aspiration through the open access cell culture chamber to protect integrity of its coating Add 50 µl ultra-pure water or PBS per each well of the microfluidic unit and gently aspirate the microfluidic system through inlets or outlets ports completely a syringe by pulling its plunger Repeat Step three times to rinse the microfluidic system. Aspirate all microfluidic system completely and let it dry at room temperature. PimCell microfluidic system is ready to use. Store under sterile conditions and use as soon as possible Fig. 14: Insulin syringe with a standard Male Luer Slip can be easy connected to incorporated Female Luers of PimCell miniplate 1.2 Sterilization Our reusable cell culture materials which direct contact with cell culture like Reservoirs and Connectors are produced in cleanroom facilities and clean areas to prevent physical impurities and contamination. The materials are not sterilized when delivered. Our reusable materials are compatible with gamma, autoclave and ethanol (EtOH) sterilization. Sterilise unsterile Reservoirs using a steam sterilizer C 20 min or 70% EtOH. It is highly recommended to rinse medium Reservoirs thoroughly with sterile water, buffer or medium before cell culturing to remove EtOH residues from the Reservoirs. 1.3 Degasification Place PimCell microfluidic miniplate M5 at 37 0 C for 24h before PimCell miniplate loading (e.g. into a cell culture incubator) At a higher temperature plastic material can take up less gas than at a lower temperature. If plastic materials like olefin have been stored at room temperature, the gas (air) that is solved in plastic will be released from plastic material when heated up in the incubator. Air bubbles will then emerge PIMBIO B.V. 10

12 inside microfluidic channels or tubing and close the flow pathway. Therefore, you have to prevent this effect by degassing all the parts involved beforehand. Each time you take the system out of the incubator, this process will start over. Take care that you work as fast as possible at room temperature to constant keep the temperature of the system at plus 37 0 C. Never leave the microfluidic system outside the incubator for more than 15 minutes. Always warm media and PBS in a 37 0 C water bath before opening. This will avoid the potential contamination from air being drawn into a cold bottle of media/pbs upon opening. 2. 2D Cell Culture for Option 1 For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 one sterile plastic 1.0 ml Insulin syringe five Reservoirs with caps transparent sterile breathable adhesive film cells and cell culture media 2.1 Calculate number of cells per ml cell suspension Prepare cell suspension and cell culture media according to the manufacturer s specifications or reference 2.2 Place one degassed and coated PimCell miniplate, five sterile Reservoirs, one syringe and one sheet of sterile adhesive film into a sterile laminar flow cabinet and unpack all materials 2.3 Gently but tightly connect Reservoirs to the inlet/outlets wells of the microfluidic channels and cover it with the sterile breathable adhesive film (see fig. 4) 2.4 Connect a syringe loaded with 1 ml of cell culture media to the cell culture chamber and gently introduce the cell culture media into the microfluidic system by pushing the syringe s plunger Fig. 16: Cell culture medium introduction into microfluidic system PIMBIO B.V. 11

13 2.5 Replace the connected to the cell culture chamber syringe with a Reservoir and gently add 20 µl cell suspension to the cell culture chamber media Reservoir via the wall above the cell culture medium Cover the cell culture chamber media Reservoir with the breathable adhesive film Incubate at 37 0 C and 5% CO 2 for half an hour for cell attachment Fig. 17: Cell suspension introduction 2.6 After cell attachment, remove the breathable adhesive film from the cell culture chamber media Reservoir and gently add 200 µl cell-free medium via the wall above the cell suspension Don t trap air bubbles Avoid pointing the pipet tip directly onto the channel s inlet 2.7 After that, tightly cover the cell culture chamber Reservoir with the sterile breathable adhesive film and incubate at 37 0 C and 5% CO 2 If you want to cultivate the cells for more than one day under static conditions, a complete media exchange has to be made every 24 hours 3. Medium exchange 3.1 For medium changing remove the breathable adhesive film and aspirate the old media from all Reservoirs Avoid pointing the pipet tip directly onto microfluidic channel s inlets to prevent aspiration of media from microfluidic channels 3.2 After that, gently add 240 µl of fresh media into the cell culture chamber Reservoir via the wall above microfluidic channel s openings Don t trap air bubbles Fig. 18: Medium aspiration Avoid pointing the pipet tip directly onto the channel s openings Fig. 19: Medium introduction into an empty Reservoir PIMBIO B.V. 12

14 3.3 Add 190 µl of fresh media into the Reservoirs of the microfluidic channels via the wall above microfluidic channel s openings. Gravity driven flow from the Reservoir of the cell culture chamber into the Reservoirs of the microfluidic channels will refresh all media inside microfluidic channels during media level equilibration Cover the Reservoirs with the sterile breathable adhesive film and incubate at 37 0 C and 5% CO D Cell Culture for Option 1 The most simple way to adapt Option 1 to 3D cell culture is to introduce desired 3D cell culture scaffold (e.g. hydrogel) direct into a cell culture chamber. Suggestion: Since two microfluidic channels of one cell culture unit can be easy divided by 3D cell culture scaffold we have different options for coating of microfluidic channels: 1) coating before scaffold introduction; 2) coating after scaffold introduction; 3) no coating at all since 3D scaffold offers excellent cell attachment surface. It is possible to use different coatings in different microfluidic channels of one cell culture unit. For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 two sterile plastic 1.0 ml Insulin syringes five Reservoirs with caps sterile breathable adhesive film 3D cell culture scaffold (e.g. Matrigel) cells and cell culture medium 4.1 Prepare desired hydrogel/cell mix and cell culture medium according to the manufacturer s specifications or references 4.2 Place one degassed PimCell miniplate, five sterile Reservoirs, two sterile syringes and a sterile breathable adhesive film into a sterile laminar flow cabinet and unpack all materials 4.3 Gently introduce 1 µl of hydrogel or hydrogel/cell mix into the cell culture chamber of warm PimCell miniplate direct above the central part of the cell culture chamber to fill it with the hydrogel Fig. 20: Hydrogel introduction PIMBIO B.V. 13

15 Tip! Let op, use only warm miniplates to stimulate direct polymerisation of a temperature sensitive hydrogel like Matrigel. The polymerisation should occur direct on the miniplate before liquid hydrogel can escape from the cell culture chamber into the microfluidic channels and close it Fig. 21: Polymerized hydrogel inside the cell culture chamber 4.4 Gently but tightly connect a Reservoir to the well of the cell culture chamber After that, gently add 240 µl of cell culture media into the cell culture chamber s Reservoir via the wall of the Reservoir above the polymerized hydrogel or hydrogel/cell mix and cover this Reservoir with the breathable adhesive film (see Fig. 4) Don t trap air bubbles Avoid pointing the pipet tip directly onto the hydrogel Fig. 22: Medium introduction via the wall of a Reservoir above the polymerized hydrogel 4.5 Add 50 µl cell culture media to the inlet well of the short microfluidic channel. Connect an empty syringe to the outlet port of the short microfluidic channel and very gently aspirate media from the outlet well through the microfluidic channel by pulling the plunger of the syringe until media appears inside the outlet well. Let op, push the syringe s plunder very gently and slowly to avoid the hydrogel disconnection from the bottom of the cell culture chamber by hard flow inside the high resistance central part of each microfluidic channel Tip! In most cases hydrogel/plastic interactions are non-covalent and this means that hydrogels have very fragile connection with plastic surfaces of microfluidic miniplates. Be very gently with medium introduction into microfluidic system loaded with a hydrogel to avoid the hydrogel dissociation from the microfluidic pathway. Never push media inside a microfluidic channel and use aspiration method only! 4.6 Disconnect the syringe from the short microfluidic channel 4.7 Repeat Step 4.5 with the long microfluidic channel (see DE of Fig.8) 4.8 Disconnect the syringe from the long microfluidic channel and connect the wells of both microfluidic channels to the media Reservoirs 4.9 Add 190 µl of the cell culture media per one media Reservoir of the microfluidic channels via the wall above microfluidic channel s openings and close its caps Cover the caps with the breathable adhesive film to prevent contamination and reduce media evaporation. Incubate at 37 0 C and 5% CO 2 PIMBIO B.V. 14

16 Dynamic cell culture with flow from the cell culture chamber into the microfluidic channels (Option 2) The most simple way to use PimCell miniplates for Option 2 is cell culture media pumping direct through a cell culture chamber using our transparent Pump Flow Connector. Fig. 23: Pump Fluid Connector direct attached to a cell culture chamber 1. Preparation steps are general for 2D and 3D cell culture 1.1 PimCell coating See Steps of Option Sterilisation Sterilise the unsterile Reservoirs and the Pump Fluid Connector using a steam sterilizer C 20 min or 70% EtOH. It is highly recommended to rinse the accessories thoroughly with sterile water, buffer or medium before cell culturing to remove EtOH residues from the Reservoirs and the Pump Fluid Connector. 1.3 Degasification Place PimCell microfluidic miniplate M5 and the Pump Fluid Connector at 37 0 C for 24h before PimCell chip loading (e.g. into a cell culture incubator) 2. 2D cell culture for Option 2 For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 one sterile syringe for a syringe pump when you use a syringe pump one sterile plastic 1.0 ml Insulin syringe five Reservoirs with caps one pump Fluid Connector one sheet of breathable adhesive film one handling Frame one (syringe) Pump cells and cell culture media PIMBIO B.V. 15

17 For 2D cell seeding see Steps of Option After 2D cell attachment, load the pump syringe with desired volume of cell culture media and connect it to the pump Fluid Connector 2.7 Load the pump Fluid Connector with the cell culture media by pushing the syringe s plunger and remove air bubbles from the system by gently pushing down on the plunger of the syringe 2.8 Disconnect the cell culture chamber media Reservoir from the cell culture chamber and connect the loaded pump Fluid Connector to the cell culture chamber 2.9 Insert the chip into the handling Frame for stability, connect the syringe to the pump and start pumping. Incubate at 37 0 C Tip! You can adapt our (syringe) pump Fluid Connector to a pressure driven pump by interfacing e.g. PTFE tube with a small piece of silicone tube of our pump Fluid Connector Cut the silicone tube of the pump Fluid Connector off and connect it to the PTFE tube Fig. 24: Connecting PTFE tube to silicone tube 3. 3D cell culture for Option 2 Pumping through 3D scaffold populated with 3D cells is the most advanced 3D cell culture technology to mimic interstitial flow through living tissue. The most simple way to use PimCell miniplate for a combination of 3D cell culture with Option 2 is direct pumping cell culture medium into microfluidic channels through an open access cell culture chamber loaded with desired 3D cell culture scaffold. For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 one sterile syringe for a syringe pump when you plan to use a syringe pump two sterile plastic 1.0 ml Insulin syringe four Reservoirs with caps one pump Fluid Connector one sheet of our breathable adhesive film one handling frame one (syringe) Pump cells, cell culture media and hydrogel (e.g. Matrigel) PIMBIO B.V. 16

18 3.1 Prepare desired hydrogel/cell mix and cell culture media according to the manufacturer s specifications or references 3.2 Place degassed PimCell miniplate, one pump Fluid Connector, four sterile Reservoirs, three sterile syringes and one sheet of our sterile breathable adhesive film into a sterile laminar flow cabinet and unpack all materials 3.3 Gently introduce 1 µl of hydrogel/cell mix into the cell culture chamber of warm PimCell miniplate direct above the central part of the cell culture chamber to fill it with the hydrogel Please, use only warm miniplates to stimulate direct polymerisation of a temperature sensitive hydrogel like Matrigel 3.4 After polymerisation, gently add 7 µl of cell culture media into the cell culture chamber via the wall above the polymerized hydrogel Don t trap air bubbles Avoid pointing the pipet tip directly onto the hydrogel 3.5 Load the pump syringe with the cell culture media and tightly connect the loaded syringe to the pump Fluid Connector 3.6 Load the pump Fluid Connector with cell culture media and remove air bubbles from the system by gently pushing down on the plunger of the syringe 3.7 Connect the loaded pump Fluid Connector to the cell culture chamber If a little bit of the cell culture media leaks during the connecting just gently clean it up with a sterile napkin Fig. 25: Cell culture chamber connected to a syringe for pumping with a syringe pump 3.8 Add 50 µl cell culture media to the inlet well of the short microfluidic channel. Connect an empty syringe to the outlet port of the short microfluidic channel and very gently aspirate media from the outlet well through the microfluidic channel by pulling the plunger of the syringe until media appears inside the outlet well. 3.9 Disconnect the syringe from the short microfluidic channel Repeat the Step 3.8 with the long microfluidic channel (see DE of Fig.8) Disconnect the syringe from the long microfluidic channel and connect all wells of both microfluidic channels with medium Reservoirs PIMBIO B.V. 17

19 3.9.2 Add 150 µl of the cell culture media per one media Reservoir via the wall above microfluidic channel s openings Close the caps of all media Reservoirs and cover all caps with the breathable adhesive film to prevent media evaporation from the microfluidic channels during incubation Insert PimCell miniplate into the handling Frame for stability (optional) and start pumping Incubate at 37 0 C Let op, use very low flow rates only (about 0,1-1 µl/hour) to emulate interstitial flow between 3D cells inside cell culture scaffolding and to avoid pressure accumulation inside the cell culture chamber! Fig. 26: Assembling microfluidic system for Option 2 PIMBIO B.V. 18

20 Gravity-driven cell culture with flow along the cell culture chamber (Option 3) Gravity is the driving force that accomplishes the transfer of fluids. Gravity-driven flow is movement of fluids from place to place (e.g. from one media Reservoir to another) due to hydrostatic pressure exerted by a difference in height (relative to gravitational field) between these two places. The most simple way to use PimCell miniplates for Option 3 is creation a hydrostatic pressure difference ( P) between an inlet and an outlet Reservoir of a microfluidic channel to establish gravity-driven flow of cell culture media through the high resistance central part of the microfluidic channel. Fig. 27: A schema of gravity-driven flow inside a microfluidic channel due to hydrostatic pressure difference between two media Reservoirs Considering a pressure driven Poiseuille flow in a long, straight and rigid channel, the volume flow rate Q is determined by the ratio of the driving pressure difference ΔP and the hydraulic resistance R (of the high resistance part of PimCell microfluidic channel in our case): Q = P/R With a given driving pressure difference ΔP, the volume flow rate Q is based on the hydraulic resistance R. The higher the hydraulic resistance R, the lower the volume flow rate Q. The flow rate and fluidic shear stress of the high resistance central part of the microfluidic channel next to the open access cell culture chamber can be controlled by modifying the hydraulic resistance R. The hydraulic resistance of a straight channel (the high resistance flow channel) with rectangular Rrec cross-sections is: where w, h and L are the width, height, and length of the straight channel, and η is the fluidic viscosity. When the fluidic viscosity is 0,01 dynes/cm 2 s (like cell suspension) the hydraulic resistance of the high resistance flow channel will be about 4,68 x10 6 (please see Table 1 with estimated flow rates). PIMBIO B.V. 19

21 P [cm] Q [µl/hour] Q [µl/day] Days of stable perfusion per mm of P drop 1 0,07 1,84 13,5 0,8 0,06 1,47 16,9 0,6 0,04 1,10 22,6 0,4 0,03 0,73 33,9 0,2 0,01 0,36 67,8 0,1 0,007 0,18 135,6 Table 1: Estimated flow rates of gravity-driven perfusion by hydrostatic pressure drop of media with the fluidic viscosity of 0,01 dynes/cm 2 s. Low flow rates inside the high resistance flow channel allow sufficient media refreshment inside this channel since the volume of the high resistance flow channel is only about 9 nl. 1. Preparation steps are general for 2D and 3D cell culture 1.1 PimCell coating See Steps of Option Sterilisation Sterilise the unsterile Reservoirs and the Pump Fluid Connector using a steam sterilizer C 20 min or 70% EtOH. It is highly recommended to rinse the accessories thoroughly with sterile water, buffer or medium before cell culturing to remove EtOH residues from the Reservoirs and the Pump Fluid Connector. 1.3 Degasification Place PimCell microfluidic miniplate M5 and the Pump Fluid Connector at 37 0 C for 24h before PimCell chip loading (e.g. into a cell culture incubator) 2. Cell culture for Option 3 For the setup per one cell culture unit you need the following materials: one PimCell microfluidic miniplate M5 sterile plastic 1.0 ml Insulin syringe(s) five media Reservoirs with caps one sheet of breathable adhesive film one handling Frame (optional) cells, cell culture media and hydrogel (e.g. Matrigel) 1 Prepare desired hydrogel/cell mix and cell culture media according to the manufacturer s specifications or references 1.1 Place degassed PimCell miniplate, sterile Reservoirs, sterile syringe(s) and sterile breathable adhesive film into a sterile laminar flow cabinet and unpack all materials 1.2 For 3D cell culture preparation see Steps of Option Add appropriate amount of cell culture media to each media Reservoir to create desired hydrostatic pressure difference and gravity-driven flow between Reservoirs PIMBIO B.V. 20

22 1.4 Close the caps of all media Reservoirs and cover all caps with the breathable adhesive film to prevent media evaporation from the microfluidic channels during incubation. FAQ 1 Q: Can I use PimCell miniplate to create a body-on-a-chip system? A: Yes, it is possible to interconnect different cell culture units using our Pump Fluid Connector. 12 interconnected cell culture units of four PimCell miniplates M5 inserted in a Handling Frame could generate coculture of 12 different in vitro models. Interconnected Handling Frames could generate more complex systems. 2 Q: How can I use PimCell miniplate for anaerobic cell culture? A: PimCell miniplate connected to a pump is a closed and gas impermeable system when you replace our silicone tubing with a gas impermeable tubing. There are different options known e.g.: 1) completely fill the flow pathway with an anaerobic cell culture media under anaerobic conditions and pump the anaerobic media through the microfluidic system; 2) cover anaerobic cell culture media in all reservoirs with a special oil to create an anaerobic barrier between air and media. 3 Q: How can I use PimCell miniplate for explants cultures? A: The most simple way to use PimCell miniplates for explant cultures is to use an open access cell culture chamber as a perfusion chamber: 1) place explant into a cell culture chamber; 2) connect the chamber to a pump via our Pump Fluid Connector; and 3) start perfusion of a culture media through the chamber into microfluidic channels. Tip! - Cut a small piece of an explant out using a sterile biopsy punch with 1.5 mm hole - When you have a permanent moving explant like intestinal mucosa you can cut a 4.0 mm diameter out from a highly porous membrane (using a punch with 4.0 mm hole) to cover & fix the moving explant inside the cell culture chamber by fixing the membrane with an Elbow Connector 4 Q: I need a system for live visualization of epithelial-endothelial interactions (2D/2D). How can I use PimCell miniplate to create such in vitro model? A: You can create a 2D membrane between microfluidic channels and a cell culture chamber using coating technology e.g.: 1) coat an open access cell culture chamber with a hydrogel like Collagen type I which could create a very thin membrane between microfluidic channels and the cell culture chamber; 2) seed e.g. blood endothelia cells into the microfluidic channels and epithelial cells e.g. respiratory epithelial cells into the cell culture chamber. Additionally, the open cell culture chamber allows to create an air-liquid interface. PIMBIO B.V. 21

23 Disclaimer Whilst we endeavour to ensure that the information in this catalogue is correct, PimBio B.V. reserves the right not to be responsible for the correctness, completeness or quality of the information provided. Liability claims regarding damage caused by the use of any information provided, including any kind of information which is incomplete or incorrect, will therefore be rejected. All offers are not-binding and without obligation. Parts of the pages or the complete publication including all offers and information might be extended, changed or partly or completely deleted by PimBio B.V. without separate announcement. All products and services offered within this catalogue are intended for R&D use only. PimBio B.V. Kloosterstraat AB Oss The Netherlands info@pimbio.com PIMBIO B.V. 22