DE10150269B4 - Micro chamber for non-invasive measurement on biological material - Google Patents

Abstract

Microcentric for cultivation, manipulation and / or non-invasive measurement on biological material, comprising a housing (1, 2) comprising an upper part (1) and a lower part (2), with a container-shaped recess (4, 5) in the lower part (2 ) and a protuberance (3) in the upper part (1), with at least one respective liquid- or gas-conducting inlet and outlet (6, 7) into the container-shaped recess (5), the upper part (1) being opposite the lower part (2). with a hinge (8) is designed hinged, wherein the protuberance of the upper part (3) and the Behärmförmige recess (4, 5) of the lower part are arranged and configured so that in a closed position of lower part (2) and upper part (1) the container-like recess (5) of the lower part, forming a first chamber (5), is lid-like sealed by the protuberance of the upper part (3), wherein a closure means (9, 10) is provided for securing the closed position, and wherein at least one d it is provided in the container-shaped recess (4, 5) past and / or on the protuberance (3), respectively the supply and discharge line (6, 7) connecting liquid or gas leading line (18a, b) is provided, wherein the line (18a , b) has means for measuring zero checks.

Description

The invention relates to a micro-chamber for non-invasive measurement of biological material and their use.

There are already a number of Bioreactors to carry out of measurements on biological material, in particular for examination of metabolic processes or to carry out toxicological examinations, e.g. on water or air samples.

As examples can be cited miniPERM the company IN VITRO Systems & Services, a mini fixed-Biroeaktor the company Meredos, a fluidized bed reactor of the Research Center Jülich, BIACORE X from BIACORE, cellferm-per company DASGIP mbH, Cytosensor ^® from Molecular Devices , FLEXERCELL ^® from Flexcell, Suprafusion from Brandet, Vertical Schuler Organ Bath from HSE Harvard, the Rotary Cell culture system from Synthecon, the Wave Bioreactor Cellbase from WaveBiotech AG, and chambers from cell-lining. Most of these reactors or systems are only suitable for large volumes in the ml to I scale. DE 44 43 902 discloses a microscope chamber with specially arranged seals for optical investigation of metabolic processes on cells, and the company Minucells and Minutissue Vertriebs GmbH offers corresponding gradient culture chambers

The said chambers or reactors have Disadvantages to the effect that in each case large quantities of samples or large quantities required on expensive reagents such as hormones and membrane effectors are. An object of the invention was therefore one for work suitable in microliter scale Chamber provide, whose volume can be selected additional variable should. In addition, there was a need for a flexibly usable chamber, into the biological material at different positions can be introduced, in addition, as needed, as a 1-chamber or 2-chamber system can be operated to investigate with Kulturmedien- or Perform reagent gradient to be able to and in the most diverse measuring sensor system at different positions can be introduced i.e. for a versatile and easy-to-use flexible chamber.

The object has been solved by the in claim 1 in connection with 1 and 2 defined micro-chamber. Preferred embodiments are defined in claims 2 to 11. The invention further relates to the use of the micro-chamber as defined in claim 12.

1 and 2 represent two chambers that do not correspond to the invention. 3 shows an embodiment of the invention according to claim 1 in the unfolded state. 4 shows a chamber according to the invention according to claim 3. 5 is a plan view of the upper and lower part of the chamber according to the invention in the unfolded state, in which the lines for the measurement of zero controls are clearly visible. The connection modules for sensors according to claim 7 are shown in elevation. The following reference numerals are indicated in the figures:

• 1 upper part
• 2 lower part
• 3 protuberance
• 4 container-shaped recess
• 5 container-shaped recess or first chamber
• 6 liquid or gas-carrying Inlet or outlet
• 7 liquid or gas-carrying Inlet or outlet
• 8 joint
• 9 closure means
• 10 closure means
• 11 essay
• 12a, b sealing ring
• 13a, b sealing ring
• 14 membrane
• 15 disc or biosensor carrier
• 16 view opening
• 17 Second Chamber
• 18a, b line for measuring zero controls
• 19 connection module for sensors

Description of the preferred embodiments

The micro chamber is designed as a perfusion chamber and allows non-invasive Measurements on biological material on a miniaturized scale.

As a biological material, bacteria, algae, adherent growing plant or animal cells, plant or animal Cell suspensions, plant or animal tissue and plant or small animal organisms.

In the chamber according to the invention, the first and / or the second chamber ( 5 ) respectively. ( 17 ) Volumes in the microliter range. Typically, the volumes of the chamber (s) are each about 100 to 300 μl, preferably 120 to 270 μl, more preferably 150 to 250 μl. The miniaturization of the measuring system enables the installation of parallel operated perfusion chambers for industrial use with high sample throughput. The microchamber is used as a module of an overall measuring device, which has a sensor module in addition to the micro chamber.

The microchamber (flow cell) consists of a lower ( 2 ) and an upper part ( 1 ) (Cell body; housing ( 1 . 2 )), which have a joint ( 8th ) are connected together and by a closure means, preferably a quick release ( 9 . 10 ) are locked. Both the lower ( 2 ) as well as the upper cell body ( 1 ) have at least one inlet and at least one outlet ( 6 ). The hydrostatic or barometric pressure in at least one inlet ( 6 ) and at least one process ( 6 ) can be monitored and regulated within physiological limits. In the lower cell body ( 2 ), at least one feed ( 6 ) and at least one process ( 6 ) in a relatively small recess ( 4 . 5 ) down (to the outside of the housing ( 1 . 2 ) is preferably sealed over a transparent surface or alternatively via a planar microsensor chip (biochip).

The biochip can be made from the known ones Biosensors selected and includes e.g. tissue sections applied to a suitable disc-shaped carrier, Brain slices, antibodies, DNA, RNA, antigens, molecules of obese size and chemical Composition, cells, bacteria, algae, viruses, single cell layers etc. The biochip can also handle interdigital electrode structures (IDES sensors) and / or electrically stimulable by ring electrodes be designed.

Preferably, at least one of the feed ( 6 ) and at least the process ( 6 ) connecting line at the sample compartment (container-shaped recess ( 4 . 5 ) and serves to measure zero checks.

In the upper cell body ( 1 ) at least one feed ( 6 ) and at least one process ( 6 ) in a protuberance ( 3 ), which preferably consists of transparent material, over which preferably either a membrane insert ( 11 ) with a hydrophilic, hydrophobic or semipermeable membrane or insert ( 11 ) is inserted without membrane. That when using a membrane insert with membrane ( 14 ) resulting compartment between membrane and protuberance ( 3 ) is preferably via a sealing ring ( 12a . 12b ) sealed. Preferably, at least one lead ( 6 ) and at least one derivative ( 6 ) connecting cable passes the compartment and serves to measure zero checks.

When using a membrane insert ( 14 ) with membrane arise by merging the, via a joint ( 8th ), upper ( 1 ) and lower ( 2 ) Housing between the protuberance ( 3 ) of the upper part ( 1 ) and the recess ( 4 . 5 ) of the lower cell body two compartments (chambers 5 . 17 ), which through the preferred semi-permeable membrane of the membrane insert ( 14 ) are separated.

The inlets and outlets ( 6 ) open in the upper part ( 1 ) and in the lower part ( 2 ) preferably in the middle of the respective chamber, in order to ensure a uniform mixing of the contents. The volume of the upper compartment (second chamber ( 17 )), between protuberances ( 3 ) and membrane, is determined by the height of the protuberance ( 3 ) and, depending on the application, by replacing the protuberance ( 3 ) can be varied. The volume of the lower compartment (first compartment ( 5 )) is determined by the depth of the recess ( 4 ) of the lower part ( 2 ) and can be varied depending on the application by replacing the recess.

Because both the top ( 1 ) as well as the lower part ( 2 ) via in each case at least one inlet and at least one outlet ( 6 ), both parts ( 1 . 2 ) are charged with different aqueous and 1 or gaseous media and f or mixtures. The hydrostatic or barometric pressure can be adjusted and regulated in both chambers by conventional measures.

When using an insert ( 14 ) without a membrane is created by the merging of, via a joint ( 8th ), upper and lower housings ( 1 . 2 ) between the protuberance ( 3 ) of the upper part ( 1 ) and the recess ( 4 ) of the lower part ( 2 ) a central chamber (reaction space) ( 5 ), whose volume is determined by the height of the protuberance, and can be varied depending on the application.

Preferably, the protuberance ( 3 ) of the upper part ( 1 ) made of transparent material and the recess ( 4 ) of the lower part ( 4 ) down through a transparent material. Therefore, both chambers have ( 5 . 17 ) via an optical or the central chamber ( 5 ) via two optical windows. These windows allow exposure of the chamber (s) and / or visualization and / or process control via all common micro-optic and / or spectroscopic techniques.

In particular, the measurement can by Light microscopy or confocal laser microscopy done. As proof can if necessary, fluorescence detection methods are also used.

The transparent material can be arbitrary selected but it should not be in contact with the media used toxic products such as e.g. Emit plasticizer. For example, are Plexiglas, transparent plastics, quartz or glass suitable.

The sealing materials will be out the common ones selected and include e.g. Rubber, silicone or teflon.

Through holes in the upper and / or lower part ( 1 . 2 ) may be preferred in both chambers ( 5 . 17 ) or the central reaction space ( 5 ) additional sensors (eg optodes, pressure sensors) or electrodes (eg for deriving electrical signals or for electrical stimulation) are introduced. For example, measurements of pH, pO ₂ , impedance, fluorescence, temperature etc. can be carried out.

In both chambers ( 5 . 17 ) or the central Chamber ( 5 ), a non-contact driven magnetic stirrer can be introduced.

The membrane of the insert ( 14 ) may consist of conventional material used for membranes. It can be hydrophilic, hydrophobic or semipermeable and is suitably pretreated with suitable processes for the particular intended use. The membrane may also contain sandwich-embedded biological material, eg in an alginate matrix. The membrane may be coated with monolayers of cells or similar biological material. By different choice of hydrostatic or barometric pressure in the two chambers ( 5 . 17 ), a deformation of the cells applied to the membrane can be effected.

By merging the upper part ( 1 ) and the lower part ( 2 ) the protuberance ( 3 ) obliquely into the recess ( 4 ) of the lower part ( 2 ) and thereby prevents the inclusion of air bubbles when working with aqueous media. Usually the first chamber ( 4 ), then possibly the insert ( 14 ) on the protuberance ( 3 ), the second chamber ( 17 ) and then the protuberance by merging upper and lower part ( 1 . 2 ) obliquely into the first chamber ( 4 ) introduced.

The housing ( 1 . 2 ) can be tempered (heated or cooled) either via a connected water bath or electrically via integrated Peltier elements or by applying to a conventional hot plate.

The entire micro chamber is sterilizable.

At least one inlet ( 6 ) and / or at least one process ( 6 ) at least one sensor module is connected, in which at least one microelectrode for measuring the medium flowing through is integrated. On the side facing the microchamber, a sensor module has in each case a cannula which is pricked into a drain of the microchamber which is closed in each case with a sterile septum and permits a sterile connection between the microchamber and the sensor module.

The measurements can be for short or any length of time periods carried out are and are independent of species. It can For example, metabolic processes of individual species or toxicological Investigations of liquid or gaseous Samples can be performed by using certain species as a "sensor".

By the inventive chamber became for the first time a micro chamber for very small volumes in the microliter range provided, which is very easy to use and despite the low Size of the reaction chamber (s) robust and easy to handle. The volume of the chamber (s) is light variable. The structure allows an extremely versatile Application of various materials and methods of measurement.

Claims (12)

Microculture for cultivation, manipulation and / or non-invasive measurement of biological material, comprising a housing ( 1 . 2 ) from an upper part ( 1 ) and a lower part ( 2 ), with a container-shaped recess ( 4 . 5 ) in the lower part ( 2 ) and a protuberance ( 3 ) in the upper part ( 1 ), with at least one respective liquid- or gas-conveying inlet and outlet ( 6 . 7 ) in the container-shaped recess ( 5 ), the top ( 1 ) opposite the lower part ( 2 ) with a joint ( 8th ) is designed foldable, wherein the protuberance of the upper part ( 3 ) and the container-shaped recess ( 4 . 5 ) of the lower part are arranged and configured such that in a closed position of the lower part ( 2 ) and upper part ( 1 ) the container-shaped recess ( 5 ) of the lower part, a first chamber ( 5 ), lid-like by the protuberance of the upper part ( 3 ), a closure means ( 9 . 10 ) is provided for securing the closed position, and wherein at least one of the gerförmigenförmigen recess ( 4 . 5 ) passing and / or on the protuberance ( 3 ) passing, respectively the supply and discharge ( 6 . 7 ) connecting liquid or gas line ( 18a, b ), the line ( 18a, b ) Has means for measuring zero checks. Microchamber according to one of the preceding claims with an attachment ( 11 ), which on the protuberance ( 3 ) and in the container-shaped recess ( 4 . 5 ) can be used. Microchamber according to one of the preceding claims, wherein the protuberance ( 3 ) a second chamber ( 17 ), with at least one respective liquid- or gas-carrying inlet and outlet into the second chamber ( 17 ), the second chamber ( 17 ) a membrane ( 14 ) to the side, with the in the closed position, the Behinderungförmige recess ( 4 . 5 ), forming a first chamber, is closed like a lid. Microchamber according to one of the preceding claims, wherein holes in the upper and / or lower part ( 1 . 2 ) through which in both chambers ( 5 . 17 ) or the central reaction space ( 5 ) Biosensors and / or physical sensors. can be introduced. A microchamber according to any one of the preceding claims, wherein the first ( 5 ) and / or the second chamber ( 17 ) by means of sealing rings ( 12a . 12b . 13a . 13b ) is sealed or are. Microchamber according to one of the preceding claims, wherein the first and / or second chamber ( 5 . 17 ) for a micro-optical or spectroscopic observation at least partially are designed casual. Microchamber according to one of the preceding claims, wherein this at least partially made of thermally conductive material is made for heating. Microchamber according to one of the preceding claims one or more Peltier elements for heating or cooling. A microchamber according to any one of claims 3 to 9, wherein the membrane ( 14 ) is configured as a semipermeable membrane. Microchamber according to one of the preceding claims, wherein the supply or discharge lines ( 6 . 7 ) in the first and / or 2, chamber ( 5 . 17 ) at least one sensor module is connected, which may be hydrophilic, hydrophobic, coated with biological material on one or two sides or may contain embedded biological material sandwiched. A microchamber according to any one of claims 3 to 10, wherein the membrane ( 10 ) is configured as a non-permeable membrane which may be hydrophilic, hydrophobic, one or both-sided coated with biological material, or sandwiched with biological material. Use of a microchamber according to one of the preceding claims Cultivation, manipulation and / or non-invasive measurement on biological material in miniaturized scale.