DE10150269A1 - microchamber - Google Patents

Abstract

A micro-chamber for carrying out non-invasive measurements, cultivation and / or manipulation of biological material is described with a housing (1, 2) consisting of an upper part (1) and a lower part (2), with a relative recess (4, 5) in the lower part (2) and a protuberance (3) in the upper part (1), each with at least one liquid or gas-carrying inlet and outlet (6, 7) into the relative recess (4, 5), the upper part (1) is designed to be foldable relative to the lower part (2) with a joint (8), the protuberance of the upper part (3) and the relative recess (4, 5) of the lower part being arranged and designed such that in a closed position of the lower part (2 ) and the upper part (1) the relative recess (4, 5) of the lower part, forming a first chamber (5), is sealed like a lid by the protuberance of the upper part (3), a closure means (9, 10) for securing the closed positionis provided, as well as the use of this micro-chamber for the cultivation, manipulation and / or implementation of non-invasive measurements on biological material.

Description

The invention relates to a micro-chamber and its use.

State of the art

There are already a number of bioreactors for performing Measurements on biological material, especially for the investigation of Metabolic processes or to carry out toxicological Investigations, e.g. B. on water or air samples.

Examples can be miniPERM from IN VITRO Systems & Services, a mini fixed bed biroeactor from Meredos, a fluid bed reactor from Forschungszentrum Jülich, BIACORE X from BIACORE, cellferm-pro from DASGIP mbH, Cytosensor® from Molecular Devices, FLEXERCELL® from Flexcell, Suprafusion from Brandel, Vertical Schuler Organ Bath from HSE Harvard, the Rotary Cell culture system from Synthecon, the Wave Bioreactor Cellbase from WaveBiotech AG, and Chambers from the company cell-lining. Most of these reactors or systems are only suitable for large volumes on the ml to l 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 chambers or reactors mentioned have disadvantages in that that large amounts of samples or large amounts of expensive reagents how hormones and membrane effectors are required.

An object of the invention was therefore to work for Microliter scale to provide suitable chamber, its volume should also be variably selectable. There was also a need for one flexibly usable chamber in which the biological material different positions can be introduced, which also depend on Can be operated as a 1-chamber or 2-chamber system Conduct investigations with culture media or reagent gradients can, and in different measuring sensor systems at different Positions can be introduced, d. H. on a versatile and simple flexible chamber.

The object was achieved by the micro-chamber defined in claim 1 in connection with the micro-chamber defined in FIGS. 1 to 4. 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.

Brief description of the figures

Fig. 1, 2, 3 and 4 illustrate two embodiments of the chamber according to the invention The figures show the following reference numerals are given. 1 top
2 lower part
3 protuberance
4 container-shaped recess
5 Container-shaped recess or first chamber
6 Liquid or gas supply or discharge
7 Liquid or gas-carrying supply or discharge
8 joint
9 closure means
10 closure means
11 essay
12a, b sealing ring
13a, b sealing ring
14 membrane
15 disk or biosensor carrier
16 viewing opening
17 Second chamber

Description of the preferred embodiments

The micro-chamber is designed as a perfusion chamber and allows cultivation, Manipulation and / or non-invasive measurements on biological material on a miniaturized scale.

Bacteria, algae, adherently growing can be used as biological material plant, animal or human cells, plant, animal or human Cell suspensions, plant, animal or human tissue and small plant or animal organisms are used.

In the chamber according to the invention, the first and / or the second chamber ( 5 ) or ( 17 ) have volumes in the microliter range. The volumes of the chamber (s) are typically approximately 10 to 1000 μl, preferably 100 to 300 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 micro-chamber is used as a module of an overall measuring device, which has a sensor module in addition to the micro-chamber.

The micro-chamber (flow cell) consists of a lower ( 2 ) and an upper part ( 1 ) (cell body; housing ( 1 , 2 )), which are connected to each other via a joint ( 8 ) and by a closure means, preferably a quick-release fastener ( 9 , 10 ) can be locked. Both the lower ( 2 ) and the upper cell body ( 1 ) have at least one inlet and at least one outlet ( 6 , 7 ). The hydrostatic or barometric pressure in at least one inlet ( 6 , 7 ) and at least one outlet ( 6 , 7 ) can be monitored and regulated within physiological limits. At least one inlet ( 6 , 7 ) and at least one outlet ( 6 , 7 ) opens into the lower cell body ( 2 ) in a relative recess ( 4 , 5 ), which preferably extends downwards (to the outside of the housing ( 1 , 2 )) a transparent surface or alternatively is sealed by a planar microsensor chip (biochip).

The biochip can be selected from the known biosensors and includes e.g. B. applied to a suitable disc-shaped carrier Tissue sections, brain sections, antibodies, DNA, RNA, antigens, molecules any size and chemical composition, cells, bacteria, algae, Viruses, single cell layers, etc. The biochip can also be interdigital Have electrode structures (IDES sensors) and / or through Ring electrodes can be designed to be electrically stimulable.

Preferably, at least one line connecting the inlet and at least the outlet leads past the sample compartment (container-shaped recess ( 4 , 5 )) and is used to measure zero controls.

At least one inlet ( 6 , 7 ) and at least one outlet ( 6 , 7 ) open into a protuberance ( 3 ) in the upper cell body ( 1 ), which preferably consists of transparent material, via which preferably either a membrane insert ( 11 ) with a hydrophilic one , hydrophobic or semipermeable membrane or an insert ( 11 ) without membrane is inserted. The compartment formed between membrane and protuberance ( 3 ) when using a membrane insert with membrane ( 14 ) is preferably sealed by means of a sealing ring ( 12 a, 12 b). Preferably, at least one feed line leads (6, 7) and at least one discharge (6, 7) connecting the line to the compartment over and serves for the measurement of zero controls.

When using a membrane insert ( 14 ) with a membrane, the upper ( 1 ) and lower ( 2 ) housing, which is connected via a joint ( 8 ), is brought together 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 are separated by the preferably semipermeable membrane of the membrane insert ( 14 ).

The feed and discharge lines ( 6 , 7 ) open in the upper part ( 1 ) and in the lower part ( 2 ), preferably approximately in the middle of the respective chamber, in order to ensure uniform mixing of the contents

The volume of the upper compartment (second chamber ( 17 )) between the protuberance ( 3 ) and the membrane is determined by the height of the protuberance ( 3 ) and can be varied depending on the application by changing the protuberance ( 3 ). The volume of the lower compartment (first chamber ( 5 )) is determined by the depth of the recess ( 4 ) of the lower part ( 2 ) and can be varied depending on the application by changing the recess.

Since both the upper part ( 1 ) and the lower part ( 2 ) each have at least one inlet ( 6 , 7 ) and at least one outlet ( 6 , 7 ), both parts ( 1 , 2 ) can have different aqueous and / or gaseous ones Media and / or mixtures are loaded. The hydrostatic or barometric pressure can be set and regulated in both chambers using conventional measures.

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

The protuberance ( 3 ) of the upper part ( 1 ) is preferably made of transparent material and the recess ( 4 ) of the lower part ( 4 ) is closed at the bottom by a transparent material. Therefore, both chambers ( 5 , 17 ) each have an optical window and the central chamber ( 5 ) has two optical windows. These windows allow exposure of the chamber (s) and / or visualization and / or process control using all common micro-optical and / or spectroscopic techniques. A corresponding embodiment is shown in FIGS. 1 and 2.

In particular, the measurement can be carried out by light microscopy or confocal Laser microscopy done. You can also use it as proof Fluorescence detection methods are used.

The transparent material can be selected as desired, but it should be in Contact with the media used no toxic products such as B. Dispense plasticizer. Examples are transparent plastics, quartz or glass suitable.

The sealing materials are selected from the usual ones include e.g. B. rubber, silicone or Teflon.

Through bores in the upper and / or lower part (1, 2), (5), additional sensors (B. optodes, pressure sensors z.) Or electrode (may preferably be in both chambers (5, 17) and the central reaction chamber z. B to derive electrical signals or for electrical stimulation). So z. B. measurements of pH, pO ₂ , impedance, fluorescence, temperature, etc. are carried out.

A contactlessly driven magnetic stirrer can be introduced into both chambers ( 5 , 17 ) and the central chamber ( 5 ).

The membrane of the insert ( 14 ) can consist of the usual material used for membranes. It can be hydrophilic, hydrophobic, semi-permeable or non-permeable and is suitably pretreated using suitable processes for the respective application. The membrane may also contain sandwiched biological material, e.g. B. in an alginate matrix. The membrane can be coated with monolayers of cells or similar biological material. Different selection of the hydrostatic or barometric pressure in the two chambers ( 5 , 17 ) can cause the cells applied to the membrane to deform.

By bringing the upper part ( 1 ) and the lower part ( 2 ) together, the protuberance ( 3 ) is inserted obliquely into the recess ( 4 ) of the lower part ( 2 ), thereby preventing the inclusion of air bubbles when working with aqueous media. Usually the first chamber ( 5 ) is filled, then the attachment ( 11 ) is placed on the protuberance ( 3 ), the protuberance is inserted obliquely into the first chamber ( 5 ) by bringing the top and bottom parts ( 1 , 2 ) together and then the second chamber ( 17 ) filled through the feed lines.

The housing ( 1 , 2 ) can be heated (heated or cooled) either via a connected water bath or electrically via integrated Peltier elements or by placing it on a conventional heating plate. In this case, the housing consists of heat-conducting material, e.g. B. aluminum or the like.

The entire micro-chamber can be sterilized.

At least one sensor module is connected to at least one inlet ( 6 , 7 ) and / or to at least one outlet ( 6 , 7 ), in which at least one microelectrode for measuring the medium flowing through is integrated. A sensor module has a cannula on the side facing the micro-chamber, which is inserted into a drain of the micro-chamber, which is closed with a sterile septum, and enables a sterile connection between the micro-chamber and the sensor module.

The measurements can be made over short or any length of time are carried out and are species-independent. For example Metabolic processes of individual species or toxicological studies of liquid or gaseous samples by using certain Species can be performed as a "sensor".

Through the chamber according to the invention, a microchamber for provided very small volumes in the microliter range that are very simple operable and despite the small size of the reaction chamber (s) robust and is easy to handle. The volume of the chamber (s) can be easily varied. The Construction enables an extremely versatile application of various Examination materials and measuring methods.

Claims (12)

1. micro chamber for the cultivation, manipulation and / or non-invasive measurement of biological material, with a housing ( 1 , 2 ) consisting of an upper part ( 1 ) and a lower part ( 2 ), with a relative recess ( 4 , 5 ) in the lower part ( 2 ) and a protuberance ( 3 ) in the upper part ( 1 ), each with at least one liquid or gas-carrying inlet and outlet ( 6 , 7 ) into the relative recess ( 5 ), the upper part ( 1 ) relative to the lower part ( 2 ) is designed to be foldable with a joint ( 8 ), the protuberance of the upper part ( 3 ) and the relative recess ( 4 , 5 ) of the lower part being arranged and designed such that in a closed position of the lower part ( 2 ) and the upper part ( 1 ) the relative recess ( 5 ) of the lower part, forming a first chamber ( 5 ), is sealed like a lid by the protuberance of the upper part ( 3 ), a closure means ( 9 , 10 ) for securing the closed position is provided. 2. Micro chamber according to claim 1 with a relative to the relative recess ( 4 , 5 ) and / or with a past the protuberance ( 3 ) leading, each of the inlet and outlet ( 6 , 7 ) connecting liquid or gas-carrying line. 3. Micro chamber according to one of the preceding claims with an attachment ( 11 ) which can be placed on the protuberance ( 3 ) and inserted into the relative recess ( 4 , 5 ). 4. Micro chamber according to one of the preceding claims, wherein the protuberance ( 3 ) has a second chamber ( 17 ), each with at least one liquid- or gas-carrying inlet and outlet in the second chamber ( 17 ), the second chamber ( 17 ) has a membrane ( 14 ) on the side with which in the closed position the relative recess ( 4 , 5 ), forming a first chamber ( 5 ), is closed like a lid. 5. Micro chamber according to one of the preceding claims with at least one in the first ( 5 ) and / or second chamber ( 17 ) introduced biosensor and / or physicochemical sensor. 6. Micro chamber according to one of the preceding claims, wherein the first ( 5 ) and / or the second chamber ( 17 ) with the help of sealing rings ( 12 a, 12 b, 13 a, 13 b) is or are sealed. 7. Micro chamber according to one of the preceding claims, wherein the supply and discharge lines ( 6 , 7 ) in the first and / or second chamber ( 5 , 17 ) are provided with interchangeable different connection modules. 8. Micro chamber according to one of the preceding claims, wherein the first and / or second chamber ( 5 , 17 ) are designed to be at least partially transparent for micro-optical or spectroscopic observation. 9. Micro chamber according to one of the preceding claims, wherein these are at least partially made of thermally conductive material Heating is made. 10. Micro chamber according to one of the preceding claims one or more Peltier elements for heating or Cooling. 11. Micro chamber according to one of claims 3 to 10, wherein the membrane ( 14 ) is designed as a hydrophobic, hydrophilic, semi-permeable or non-permeable, with biological material coated on one or two sides or containing biological material sandwiched embedded. 12. Use of a micro-chamber according to one of the preceding Claims for cultivation, manipulation and / or non-invasive Measurement of biological material on a miniaturized scale.