For preparation of a 0.5 M EDTA (ethylenediamine tetraacetic acid) stock solution, 3.84 g EDTA were dissolved in 50 ml deionised water. The pH-value was adjusted to 8.0 by titration of HCl. The prepared solution was sterilized by filtration (0.2 µm) and thereby storable at ambient conditions.

The lysis buffer was used to disintegrate the CellBeads^© prior to the vitality staining with trypan blue. The principle of the lysis buffer is the depolymerisation of the alginate layer through the formation of a complex with the bivalent cations. The break up of the alginate causes the release of the immobilized cells. For the preparation of this buffer, PBS (phosphate buffered saline, Biowest, Nuaille, France) was supplemented with 10 mM EDTA (2 ml EDTA stock solution per 98 ml PBS buffer) and 0.1% BSA.

Tris buffer was used for the preparation of SYBR Green solution. 3.14 g Tris were dissolved in 250 ml deionised water. The pH-value was adjusted to 8.0 using HCl. The prepared solution was autoclaved and stored at room temperature.

The fluorescence dyes SYBR Green and propidium iodide were used for visualization of the vitality of the encapsulated cells. A 20-fold concentrated stock solution of SYBR Green was prepared using the original 10.000 fold SYBR Green + DMSO solution. The stock solution is stable at -20°C for up to one year. The working solution was prepared by adding 500 µl SYBR Green stock solution and 500 µl EDTA solution to 1500 µl Tris-HCl buffer. For preparation of the propidium iodide solution, 5 ml PBS were added to 25 mg propidium iodide (Sigma-Aldrich). The solution is stable in an opaque bottle at 4°C for up to 6 months.

Nile red is a fluorescent lipid staining dye and used to verify the adipogenic differentiation status of the cells [12]. A 1 mg/ml stock solution of nile red in ethanol was diluted in PBS to a final concentration of 1 µg/ml and clarified by filtration (0.22 µm) prior to use.

The CellBeads^© were cultured in EMEM (minimal essential medium with Earle’s salts, Biochrom AG, Berlin, Germany), supplemented with 10% BGS (Bovine Growth Serum, Thermo Fisher Scientific, Schwerte, Germany), 100 U/ml penicillin and 100 µg/ml streptomycin. The medium was used for cultivation without adipogenic differentiation.

The induction medium was used to induce the adipogenic differentiation of the encapsulated cells.

The induction medium consisted of DMEM-HG (Dulbecco’s modified Eagle medium - high glucose, Biowest, Nuaille, France), which was supplemented with 10% BGS, 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich), 1 μM dexamethason (Sigma-Aldrich), 0.2 mM indomethacin (Sigma-Aldrich), 0.01 mg/ml insulin (Sigma-Aldrich) and 0.5 mM 3-isobutyl-1-methyl-xanthin (Sigma-Aldrich).

DMEM-HG supplemented with 10% BGS, 10 mg/l insulin, 100 U/ml penicillin and 100 µg/ml streptomycin were used as a culture medium between the induction phases of adipogenic differentiation.

The CellBeads^© were supplied in a frozen cryo vial. Prior to use they were thawed by placing the cryo vial in a 37°C water bath for 1-2 minutes. Afterwards, the CellBeads^© were transferred to a 25-cm² tissue culture flask containing 20 ml conditioned culture medium, cultured for 1 hour at 37°C in a humidified 5% CO₂ incubator and subsequently introduced into the fixed bed bioreactor system.

The core of the fixed bed cultivation system is the reactor which consists of a commercially available single-use plastic syringe and a special lathed piston, which enables the perfusion of the reactor and the embedding of the package (Fig. 2 and 3). The funnel-shaped inflow area forces a uniform velocity profile upward at the inflow boundary of the fixed bed. The prototype of the piston is made of biocompatible polyetheretherketone (PEEK), covered at the top with a stainless steel mesh with an aperture size of 100 µm to retain the CellBeads^©. Two O-rings made of the autoclavable Viton ^© serve as seals between syringe and piston.

Fig. (1). Light-microscopical images of CellBeads© at a magnification of 40x (a) and 200x (b).

Fig. (2). Syringe and piston drawn in the assembled condition.

Fig. (3). Handcrafted oxygen measurement chamber with integrated oxygen mini sensor for the measuring of the oxygen concentration in the medium outflow.

Fig. (4). Schematic of the fixed bed reactor system and the corresponding periphery.

Fig. (5). Vitality of the CellBeads©, cultured in the fixed bed reactor and tissue culture flasks. The vitalities were determined after lysis of the alginate capsules by the trypan blue exclusion method. The data (fixed bed) represents the mean ± standard deviation of two cultivations.

Fig. (6). SYBR Green and propidium iodide stained CellBeads© after 100 hours cultivation in the fixed bed reactor (a) and tissue culture flask (b). Independent of the cultivation method, many necrotic cells, indicated by red luminescence, are visible.

Fig. (7). SYBR Green and propidium iodide staining after 500 h cultivation in the fixed bed reactor (a) and tissue culture flask (b). No red luminescent necrotic cells are observable.

Fig. (8). Nile red staining of CellBeads© cultured under adipogenic (a) and unstimulated non-adipogenic (b) conditions in the fixed bed reactor.

Fig. (9). Nile red staining of CellBeads© cultured under adipogenic (a) and unstimulated non-adipogenic (b) conditions in shaken tissue culture flasks.

Fig. (10). Oxygen saturation concentration at the medium outlet during the adipogenic differentiation cultivation. The peaks are due to the medium changes.

A schematic of the experimental setup, including the small-scale fixed bed reactor (volume: 3 ml, diameter: 9 mm) and the periphery, is illustrated in Fig. (4). The system was perfused using a precision peristaltic pump (IPC-8, Ismatec, Glattbrugg, Switzerland), which enabled small volume flows with reduced pulsation. Two 250 ml flasks (Duran flask, Schott AG, Mainz, Germany), equipped with sterile filters (Midisart 0.22 µm, Sartorius AG, Goettingen, Germany) to maintain pressure equilibrium, were used as conditioning and waste vessels, respectively. A self-made measurement chamber, consisting of an oxygen mini sensor (PreSens, Regensburg, Germany) inserted into a glass tube fitted with PEEK hose connectors, enabled the noninvasive monitoring of the dissolved oxygen in the medium outflow (Fig. 3). The mini sensor consists of a glass disc coated by a luminescent dye. Molecular oxygen caused a quenching of the luminescence and the oxygen dependant signal was measured via optical fibre (Fibox3, Presens) [13, 14].

After autoclaving (121°C, 20 minutes), the conditioning flask was filled with medium (EMEM + 10% BGS), which was circulated by pumping to fill the tubings and to remove air bubbles. The system was placed in a humidified incubator (37°C, 5% CO₂; Galaxy B, RS Biotech, Alloa, UK) for 1 hour to allow conditioning of the medium. Afterwards, the reactor chamber was opened by removing the piston to insert the CellBeads^© (1 ml package volume containing about 4500 CellBeads^©, 2100 cells per CellBead^©) and then closed by inserting the piston back into the syringe. During this procedure, the influx and efflux tubings were pinched to avoid a back flow of medium. All steps after autoclaving were carried out under sterile conditions.

The whole system except the peristaltic pump was placed in the incubator during cultivation. The medium flow was adjusted to 0,5 ml/min, leading to a dissolved oxygen concentration in the outflow of 78-86% of air saturation. The inflow was saturated with oxygen. The medium was completely changed every 3-4 days. The CellBeads^© were cultured for 200 and 500 hours, respectively. In each case, two cultivation runs were performed. Additionally, cultivations of 100 µl CellBeads^© in 25-cm² tissue culture flasks with the same medium (20 ml) and medium changing intervals were executed as a reference. To ensure a homogeneous nutrient and oxygen concentration profile in the CellBead-medium suspension, the tissue culture flasks were positioned on an orbital shaker (30 rpm) and placed in the incubator. In addition, orbital shaking increased the oxygen transfer into the medium and thus supported optimized culture conditions for the reference CellBeads^© [15]. After cultivation the CellBeads^© were analysed for vitality.

SYBR Green and propidium iodide are fluorescence dyes which intercalate between the double helix of nucleic acids. SYBR Green can pass through the membrane of viable cells whereas propidium iodide is only able to enter necrotic cells with disintegrated membranes [16].

In each case, 100 µl CellBeads^© were transferred into 6-well cell culture plates. After adding 200 µl PBS, 10 µl propidium iodide and lastly 20 µl SYBR Green working solutions, the samples were cultured for 5 minutes in the dark. Pictures were taken for evaluation using a fluorescence microscope (Eclipse 80i, Nikon, Tokyo, Japan) at an excitation wavelength of 488 nm.

For a quantitative evaluation of the vitality of the CellBeads^© the alginate capsule and matrix was dissolved using the EDTA containing lysis buffer.

500 µl CellBeads^© were transferred into a 50 ml plastic tube and washed with PBS twice to remove traces of medium. Afterwards, 20 ml lysis buffer and 3 ml trypsine (Biowest, Nuaille, France) were added. After incubation for 20 minutes in a humidified incubator (37°C, 5% CO2), the suspension was resuspended 20-30 times to induce a certain sheer stress that causes the complete disintegration of the CellBeads^©. Subsequently, 100 µl cell suspension were mixed with 100 µl trypan blue and incubated at ambient conditions for about 5 minutes. The number of vital cells and number of blue-stained dead cells were determined using a phase contrast microscope (DMIL, Leica AG, Wetzlar, Germany) by means of a Neubauer counting chamber.

Additional adipogenic cultivations were performed to investigate the differentiation ability of the CellBeads^© during the cultivation process in the disposable syringe fixed bed reactor.

The CellBeads^© were cultured using the setup described above but instead of normal cultivation, medium induction and maintanance medium were used. Induction medium was applied for 3 days, followed by 4 days cultivation with maintenance medium. This cycle was repeated 3 times. After each medium change, the first 20 ml were discarded by means of perfusion of the system to avoid a mixture of the two media in the tubes. Because of the higher sodium bicarbonate content in DMEM, the CO₂ concentration in the incubator was increased to 10 %. Additional reference cultivations in 25-cm² tissue culture flasks were executed.

The nile red staining was performed after the adipogenic differentiation cultivation to verify the adipogenic differentiation of the CellBeads^©.

To verify adipogenic differentiation about 50 CellBeads^© were transferred into a 6-well cell culture plate, rinsed three times with PBS after medium removal, fixed by the addition of 1ml methanal and incubated for one hour. Afterwards, the methanal was removed, followed by three washing steps with PBS. The subsequent incubation with 400 µl nile red solution for one hour was carried out in the dark. Pictures were taken using a fluorescence microscope (Eclipse 80i ) at an excitation wavelength of 550 nm.