InFlow Modular perfusion bioreactor

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2 InFlow is an incubator compatible, modular bioreactor for the controlled, bidirectional and interstitial perfusion of up to 9 cell seeded scaffolds with culture medium. InFlow is ideal for general purpose uses and is particularly suitable for the long-term culture of cylindrical fragments of BONE and CARTILAGE tissues. Main features - Three biologically and hydraulically independent perfusion lines - Bi-directional perfusion - Flow rate and direction set by user - Timings of perfusion set by the user for Timer-based and ACE controlled versions - Fast and safe priming function (manual or automated culture chamber by-pass) - Different size scaffold holders - Compatible with cylindrical porous scaffold - Different flow range available - Repeatability of conditions: no air couplings, pump acts directly on incompressible fluid. - Time course experiment, removing a single line each time 1

3 Single perfusion line: Each line presents a medium reservoir which ensures culture medium oxygenation through the presence of a hydrophobic filter and medium sampling thanks to a Luer lock septum. Additionally, the circuit is made of silicone platinum cured tubing for culture medium oxygenation, ph balancing and minimum priming volume. Single perfusion line components: - Anodized aluminium line support, with a design to hold and keep in position each component of the circuit - 25 [ml] autoclavable medium reservoir - Autoclavable caps with 4 ports (0.22 [µm] filter port, inlet port, outlet port, sample/filling port) - Autoclavable perfusion chamber with luer lock connections - Silicone scaffold adapters - 2 oxygenator coils for silicone tubing - PharMed tubing for medium reservoir and two-stops pump tubing - Platinum cured silicone tubing set - 2 Y shape barbed connectors for the by-pass circuit - Dual 3-way pinch valve to by-pass culture chamber and fast priming (for ACE controlled versions) - By-pass circuit through manual clamps systems (for basic and timer based versions) - Barbed and luer lock connectors to ensure process integrity 2

4 TECHNICAL DATA Culture chamber Autoclavable culture chamber in polycarbonate (FDA-approved) with luer lock connection and o-ring. Every chamber hosts a silicone adapter (scaffold holder) for cylindrical porous scaffold. Standard scaffold sizes: - 8 [mm] x h 2 [mm] Perfused diameter 6 [mm] - 8 [mm] x h 4 [mm] Perfused diameter 6 [mm] - 10 [mm] x h 2 [mm] Perfused diameter 8 [mm] - 10 [mm] x h 4 [mm] Perfused diameter 8 [mm] - 12 [mm] x h 2 [mm] Perfused diameter 10 [mm] - 12 [mm] x h 4 [mm] Perfused diameter 10 [mm] Other measures on request (change only the silicone adapters). Could be provided in disposable sterile bags. 3

5 Medium reservoir Autoclavable media bottle (25 ml) with special GL25 cap, with four accesses: - Inlet/outlet port (barbed fitting) connected to the circuit - Sampling/medium exchange port (luer lock fitting) - 0,22 [µm] filter port (luer lock fitting) Oxygenation Two oxygenation coil structures per line, hosts up to 3.6 meters (1.8 meters each one) of platinum cured silicone tubing with high permeability to O 2 and CO 2 minimizing the occupied volume. Made of anodized aluminium, to allow the coiling of oxygenator tubing, avoiding handling difficulties and guaranteeing easy operations under GLP conditions. 4

6 By-pass circuit For a fast priming of the circuit (at maximum flow rate) excluding the culture chamber in order to avoid cell damages due to too high pressure or perfusion velocity. Connected to perfusion circuit by means of 2 Y shape barbed connectors. Available in two version: - dual 3-way pinch valve to by-pass culture chamber and fast priming with connector (for ACE controlled versions) - by-pass circuit through manual clamps systems (for basic and timer based versions) Peristaltic pump Peristaltic pump, to set perfusion flow rate and direction (clockwise / anticlockwise). Available with different flow rate range and compatible with tubing size ID 1/32 and ID 1/16. Supplied with up to 9-channel head and up to 9 cartridges. Compatible with two-stop PharMed BPT tubes. Standard flow rate range: ml/min (1-100 rpm, 0.1 rpm step). Other options available. Rotational speed (rpm) vs. Flow rate (ml/min) vs. Perfusion velocity according to scaffold perfused diameter (µm/s) are presented in the following table (NB: a complete table is attached to user manual). 5

7 Rotational speed [rpm] Flow rate [ml/min] Speed [µm/s] perfused diameter = 6 [mm] Speed [µm/s] perfused diameter = 8 [mm] Speed [µm/s] perfused diameter = 10 [mm] 1 0, , , , , , , , , , , , , , , , , , , Table 1: Rotational speed [rpm], flow rate [ml/min] and speed of perfusion [µm/s] relation. 6

8 Sterilization The whole system could be autoclaved or sterilized in EtO. Control System InFlow system could be purchased without controller and used with external third parties controllers or just in combination with a peristaltic pump. The version without controller allows to set, directly on the peristaltic pump, flow rate and direction of perfusion. Otherwise InFlow can be equipped with 2 different controllers: - timer based version - ACE embedded controller version Timer based version Option that allows to control only perfusion patterns (timing and direction). Manual flow rate and priming set directly on the pump. Main features - Set time and direction of a perfusion pattern (symmetric or asymmetric) - Easy to use - Compact system, cost and space saving - Real-time control 7

9 ACE embedded version ACE embedded controller, InFlow version, with TFT display and keypad user interface to control: - Pump (flow-rate, start-stop, priming, direction) - Timing and perfusion patterns - Pinch valves (to bypass culture chambers) - Sensors Main features - Real-time control and monitor of culture parameters - Real-time plot of sensor values - Easy to use interface - Space saving - Data logging and saving on SD card support - Remote monitoring - IP65 enclosure (could be placed inside incubator) Optical sensors option InFlow ACE embedded version can be integrated with optical sensors to real-time on-line monitor culture parameters. Available upgrades: ph optical sensors to monitor one perfusion line (the central reservoir) in each module. ph and/or O 2 flow through optical sensors connected in series with the hydraulic circuit. 8

10 ph technical data Measuring range ph Response time < 120 sec. Resolution at ph = 7 ± 0.01 ph Accuracy at ph = 7 ± 0.05 sensor spot calibration ± 0.10 sensor batch calibration Drift at ph = 7 < ph per day Temperature range from 5 C to 50 C O2 technical data Measuring range 0-100% O 2 Limit of detection 0.03% Resolution ± 0.01% O 2 at 0.21% O 2 ± 0.1% O 2 at 20.9% O 2 Accuracy ± 0.4% O 2 at 20.9% O 2 ± 0.05% O 2 at 0.2% O 2 Response time < 6 sec. Temperature range from 5 C to 50 C 9

11 InFlow - Order information System InFlow Module (3 lines) + pump Timer-based controller ACE controller Sensors ph sensor O 2 sensor ph spot bag O 2 spot bag Order Code B B B Order Code On request On request On request On request Spare parts / replacements Order Code Platinum cured silicone tubing (3 lines) D-012 PharMed tubing (3 lines) D-014 Two-stop pump tubing set (3 lines) D-015 Tubing set and connectors (3 lines) D-013 Reservoir + multiport cap (q.ty 1) D-022 Perfusion chamber (q.ty 1) B-007 Silicone scaffold holders (1 size) B-008 Tubing pinch clamp D-019 Fittings and connectors D SKE Research Equipment C/O Leonardino Srl via Don Giovanni Minzoni, Milano Italy tel/fax: website: For information: info@ske.it Sales request: sales@ske.it 10