DE102010037923A1 - Bioreactor arrangement for cells, comprises closed bioreactor, cell pellet carrier, device for supplying nutrient solution into cell pellet, optical oxygen probes, laser, detector and control and signal transmitting device - Google Patents

Abstract

Bioreactor arrangement for cells, comprises (a) a closed bioreactor (10), (b) a cell pellet carrier (12) for receiving a cell pellet, (c) a device for supplying nutrient solution into the cell pellet, (d) optical oxygen probes for mixing the cells in the cell pellet, (e) a laser (14) directed on the oxygen probe, (f) a detector (20) for receiving phosphorescence signals emitted from the oxygen probes, and (g) a control- and signal transmitting device (18) for generating a value representing the oxygen content in the cell pellet.

Description

Technical area

• (a) einen abgeschlossenen Bioreaktor;
• (b) ein Zellpelletträger zur Aufnahme eines Zellpellets; und
• (c) Mittel zum Zuführen von Nährlösung in das Zellpellet.

The invention relates to a bioreactor arrangement for cells comprising:

• (a) a sealed bioreactor;
• (b) a cell pellet carrier for receiving a cell pellet; and
• (c) Means for supplying nutrient solution into the cell pellet.

Such bioreactors serve the cultivation of cells, in particular the cultivation of stem cells. The cells form a cell pellet and are located on a cell pellet carrier with a nutrient solution. The cell pellet carrier is cup-shaped and disposed within a bottle-like bioreactor. Depending on the cell type, it is important that the environmental conditions of the cell type are adjusted. Thus, knee cartilage cells are best cultured when increased mechanical pressure is exerted on the cells.

Cell development, especially stem cell development in multi-dimensional tissue, depends on the oxygen content in the immediate environment. The oxygen content in the cell pellet carrier differs from the oxygen content in the nutrient medium. It is therefore desirable to measure the oxygen content in the region of the cells.

State of the art

Bioreactors are known which direct a laminar flow through a bioreactor and thus produce an increased mechanical pressure.

Arrangements for measuring the oxygen content by means of optical oxygen probes are known. Laser light is introduced with a sensor with a light guide in a material. In the material phosphorescent microspheres are arranged whose phosphorescence properties depend on the oxygen content. A disadvantage of this technique is that the sensors damage the cells and influence the flow conditions.

Disclosure of the invention

It is an object of the invention to provide a bioreactor arrangement of the type mentioned, in which the oxygen content in the immediate vicinity of the cells can be monitored without damaging the cells and Zellpelletträger and without affecting the flow conditions.

• (d) optische, den Zellen in dem Zellpellet beigemischte, Sauerstoffsonden;
• (e) einen auf die Sauerstoffsonden gerichteten Laser;
• (f) einen Detektor zur Aufnahme eines von den Sauerstoffsonden emittierten Phosphoreszenzsignals; und
• (g) eine Steuer- und Signalauswerteelektronik zur Erzeugung eines den Sauerstoffgehalt in dem Zellpellet repräsentierenden Wertes.

According to the invention the problem is solved by:

• (d) optical oxygen probes admixed to the cells in the cell pellet;
• (e) a laser directed to the oxygen probes;
• (f) a detector for receiving a phosphorescence signal emitted by the oxygen probes; and
• (g) control and signal evaluation electronics for generating a value representing the oxygen content in the cell pellet.

The arrangement allows the contactless measurement of the oxygen content. The cells and cell carriers are not damaged and the flow conditions remain unchanged. With the laser, the oxygen probes are excited to phosphorescence. The emitted phosphorescence signal is recorded with the detector and passed to an evaluation. From the phosphorescence signal, the oxygen content can be determined.

Preferably, the bioreactor has translucent windows and the laser and detector are located outside the bioreactor. The culture then remains completely undisturbed. The windows preferably have a low absorption in the range of the wavelengths of the laser light and the phosphorescence signal.

The optical oxygen probes are based in particular on phosphorescent dyes. When excited with, for example, violet or blue light, they emit an intense, longer-wave, in particular red or orange phosphorescence. This is quenched by molecular oxygen (O ₂ ) in the environment. The process is also called "quenching". As the oxygen content increases, both the intensity and the decay time of the phosphorescence signal decrease. Under cooldown here is the Duration of afterglow referred to. In practice, it is advantageous to determine the decay time as a measured variable, since intensities are subject to a variety of disturbing influences. Such disturbances are, for example, absorption and scattering by the sample itself. To measure the cooldown, the sample can be excited with an example rectangular light pulse. The afterglow is detected with a suitable detector, for example a photodetector. The signal is then evaluated with suitable evaluation electronics with a computer and suitable plug-in card or oscilloscope. The cooldown is often in the range of microseconds. From this, the oxygen content can be determined via a calibration curve. These calibration curves are temperature-dependent, which is taken into account in the evaluation.

Commercial optical oxygen meters with probes usually use a measuring method in which the optical sensor is excited with sinusoidally modulated light. The cooldown is determined by measuring the phase shift. The short-wave blue excitation light is sinusoidally intensity-modulated. The phosphorescent probe emits longer-wave, red, also sinusoidally modulated light. Depending on the oxygen content, the emission of the phosphorescence signal takes place more or less time-delayed, ie with a phase shift. From the phase shift, it is known to calculate a mean decay time and thus determine the oxygen content via a calibration curve.

In a particularly preferred embodiment of the invention, the radiation emitted by the laser is simultaneously modulated with two different frequencies. The modulation frequencies can be above 1 kHz.

Unlike known arrangements, in the present invention the laser beam is modulated at two different frequencies simultaneously. This ensures that noise and background signals can be corrected. If, in addition to the phosphorescence signal, fluorescence also exists in the same wavelength range, the signals overlap with a new, smaller phase shift. Interference signals with small intensities are sufficient to cause significant measurement errors. Such interference signals are caused for example by optical components in the beam path, colored nutrient solutions or other cells. The modulation with two frequencies allows the calculation and correction of the background signals. This is based on the finding that fluorescence signals are emitted in phase with virtually no time delay. The modulation with two frequencies is independent of the amplitude in many areas.

In the present invention, the modulation of the exciting laser light with two frequencies allows the measurement independent of the geometric position of the laser and detector. This has the further advantage that the signal components and signal strengths may change during the growth process of the cells without the measurement results being falsified. Also, parts of the reactor for controlling the flow can be mechanically moved without affecting the measurement result.

Preferably, means for generating mechanical pressure on the cell mass in the bioreactor are provided in the bioreactor arrangement. In such bioreactors, non-contact oxygen measurement is of particular advantage.

In a preferred embodiment of the invention, the oxygen probes are formed by microspheres with a diameter of 10 to 150 micrometers, which are stained with Pt propyrin, ruthenium phenanthroline or their derivatives.

In a particularly simple bioreactor arrangement, the laser is formed by a diode laser.

Embodiments of the invention are the subject of the dependent claims. An embodiment is explained below with reference to the accompanying drawings.

Brief description of the drawings

1 is a schematic representation of a bioreactor with oxygen measurement.

2 shows the dye PT (II) -tetrapentafluorophenylporphyrin

3 illustrates the energy states of the dye 2

4 is a typical course of the cooldown depending on the oxygen content of the dye 2 in a solid matrix

5 shows decay curves at different oxygen content.

6 illustrates the determination of the decay time by means of phase modulation

7 illustrates the interference by fluorescence

8th illustrates the masking of the fluorescence signal by two-frequency phase modulation with measurement at frequency ω ₁ and measurement at frequency ω ₂ , showing a background signal (no phase shift, τ ₂ << τ ₁ ⇒ τ ₂ ≈ 0), sensor signal decay time τ ₁ and total signal (Background + sensor) with occurring phase shifts. The measurements are carried out at Φ _App and φ _App at ω ₁ and ω ₂ , evaluation of the unchanged τ ₁ . It applies

Description of the embodiment

1 shows a general with 10 designated bioreactor. The bioreactor 10 is about bottle-shaped. Inside the bioreactor 10 a laminar flow and thus an increased mechanical pressure is generated. In the middle area of the bioreactor 10 is a cell pellet 12 arranged with cell cultures. The cell cultures are mixed with phosphorescent sensor particles. The sensor particles are in the form of microspheres with a diameter in the range of 100 microns. The microspheres are colored with phosphorescent dyes, for example Pt-tetrapenta-fluorophenylporphyrin. The structural formula of this dye is in 2 shown.

On the upper part of the bioreactor, a holding device is installed. In the fixture is a diode laser 14 held at a wavelength of 450 nm. In an alternative embodiment, a blu-ray laser with a wavelength of 405 nm is used. The diode laser 14 is on the cell pellet 12 directed. For this purpose, the inlet of the bioreactor is provided with a window made of quartz glass. It can be seen that the laser beam emitted by the diode laser 16 walking freely through the room. The diode laser 14 is controlled by a laser control. In particular, the control is such that the laser light with two different known frequencies of 63 kHz and 33 kHz are modulated, as in 7 and 8th is shown. The order of magnitude depends on the cooldown of the dye used.

Based on 3 is shown schematically how the dye is excited to phosphorescence. The laser light displaces the dye of the microspheres from the ground state 26 in an excited singlet state 28 , This is by an arrow 30 represents. From the excited state 28 The dye particles go back to the ground state with emission of radiation. The radiation is emitted in all directions. Part of the radiation is generated by fluorescence. This is by an arrow 32 represents. The fluorescence radiation sounds very fast. Another part of the radiation is produced by dye atoms, which phosphoresce over a longer period of time. For this purpose, a radiationless transition into the excited triplet state takes place first 34 , This has a longer life than the singlet state. Accordingly, the atoms get out of the triplet state more slowly 34 under emission of phosphorescent radiation 36 back to the ground state. Only the phosphorescent radiation 36 is being measured.

The through a window 22 Radiation exiting the bioreactor is collimated with a lens, separated by an optical filter from scattered excitation light, and a detector 20 detected. The detector signal is directed to a signal evaluation. That's an arrow 24 represents. An example of the measured signals is in 4 and 5 shown. 5 shows the intensity of a phosphorescence signal as a function of time (decay curve) for different samples with different oxygen content. 4 shows the cooldown depending on the oxygen content.

The signal evaluation measures the phase shifts 40 at both modulation frequencies ω, as in 6 is shown. Subsequently, a background correction is performed, the decay time is determined and the oxygen content is determined by comparison with stored calibration curves. The phase shift φ is related to the decay time τ after τ = (tan φ) / ω.

The background correction is in 8th illustrated. Assuming that the decay time τ _{2 of} the background signal is much smaller than the decay time τ _{1 of} the phosphorescence and thus that the phase shift of the background and sensor signal φ _{App is} smaller than the phase shift φ by the phosphorescence, one obtains from the measured Signal an entire phase shift φ. Together with the modulation frequencies ω ₁ and ω ₂ , the background-corrected phase shift can be determined according to the in 8th determine the formula shown.

The signals thus evaluated provide a measure of the oxygen content in the immediate vicinity of the cells without their being affected during their development.

Further alternative embodiments use lasers having a longer wavelength in the range of 532 nm. These scatter less and therefore penetrate deeper into the cell carrier. In addition, longer-wave lasers produce less disturbing, photochemical reactions, as the rivoflavin contained in nutrient media absorbs in the short-wave blue or violet wavelength range. This activates activated oxygen. This distorts the result and can damage the cells. An excitation light source at 532 nm is outside the absorption band of riboflavin and is gentle to the detector.

Claims (7)

Bioreactor arrangement for cells comprising: (a) a sealed bioreactor ( 10 ); (b) a cell pellet carrier ( 12 ) for receiving a cell pellet; and (c) means for delivering nutrient solution into the cell pellet; characterized by (d) optical oxygen probes admixed to the cells in the cell pellet; (e) a laser directed to the oxygen probes ( 14 ); (f) a detector ( 20 ) for receiving a phosphorescence signal emitted by the oxygen probes; and (g) control and signal evaluation electronics ( 18 ) for generating a value representing the oxygen content in the cell pellet. Bioreactor arrangement according to claim 1, characterized in that the bioreactor translucent window ( 22 ) and the laser ( 14 ) and the detector ( 20 ) outside the bioreactor ( 10 ) are arranged. Bioreactor arrangement according to one of the preceding claims, characterized in that that of the laser ( 14 ) emitted radiation is simultaneously modulated with two different frequencies. Bioreactor arrangement according to claim 3, characterized in that the modulation frequencies are above 1 kHz. Bioreactor arrangement according to one of the preceding claims, characterized in that means for generating mechanical pressure on the cells in the bioreactor ( 10 ) are provided. Bioreactor arrangement according to one of the preceding claims, characterized in that the oxygen probes are formed by microspheres with a diameter of 10 to 150 micrometers, which are stained with Pt-propyrin, ruthenium-phenanthroline or derivatives thereof. Bioreactor arrangement according to one of the preceding claims, characterized in that the laser ( 14 ) is formed by a diode laser.

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