Bioreactor

The 3-D hollow-fiber bioreactor system (Stem Cell Systems, Berlin, Germany) used in this study consists of three independent but interwoven capillary bundles (Figure 2): two of them serve for countercurrent medium perfusion (media I and II) and the third for internal oxygenation (gas). Cells are cultured in the extracapillary space, which has a volume of 0.5 mL. They are allowed to adhere to the hollow-fiber scaffold and supplied with nutrients and oxygen through the capillary membranes (Figure 3A and C).

3-D bioreactor.

Note: The photograph shows the bioreactor with tube connectors for two medium capillary bundles, one gas capillary bundle, and cell inoculation, a tube connected to the space between the capillaries (cell compartment).

Culture of primary human hepatocytes in 3-D bioreactors and 2-D cultures.

Notes: (A) Cell aggregates being cultured between the hollow-fiber capillaries of the bioreactor, providing the cells with gas and nutrients. (B) 2-D monolayer culture on collagen-coated culture vessels. Exemplary microscopy of (C) paraffin-embedded bioreactor culture and (D) 2-D monolayer culture. Hollow-fiber capillaries are marked with an asterisk.

The capillary bed is incorporated into a polyurethane housing equipped with connectors for mounting the perfusion tubing and for cell inoculation. The structure of the perfusion circuit is shown in Figure 4. Culture medium is constantly recirculated through the bioreactor by means of a peristaltic pump. At the same time, fresh culture medium is fed into the perfusion circuit via a second pump, while the corresponding excess medium is rinsed out into the waste vessel. A port integrated into the perfusion circuit enables substance injection and sampling from the recirculating medium. An electronic gas-mixing unit enables supply of air–CO₂ at a defined mixture and flow rate.

Schematic illustration of the bioreactor perfusion circuit.

Notes: The tubing system contains two independent pumps for medium recirculation through the bioreactor and fresh medium feed, respectively, while rinsing out of used medium is driven by hydrostatic pressure increase (red, bioreactor-inflow tubing; blue, bioreactor-outflow tubing). The bioreactor disposes of two medium-perfusion capillary systems, which are countercurrently perfused to enhance mass exchange. Each capillary layer consists of alternating medium and oxygen capillaries, with cells being cultured in the space between the capillaries. Cells are inoculated via a separate tube line. An electronically controlled gas-mixing unit provides defined flow rates of air and CO₂ and allows for regulation of gas concentrations in the supplied gas mixture (gray lines). The temperature in the bioreactor chamber is constantly kept at 37°C via an electronically controlled heating unit.