EP1434851A2

EP1434851A2 - Microchamber

Info

Publication number: EP1434851A2
Application number: EP02772369A
Authority: EP
Inventors: Edgar Wagner; Christoph Beck
Original assignee: Individual
Current assignee: Individual
Priority date: 2001-10-11
Filing date: 2002-10-10
Publication date: 2004-07-07
Also published as: AU2002337150A1; WO2003033642A3; DE10150269A1; DE10150269B4; WO2003033642A2

Abstract

A microchamber for carrying out non-invasive measurements, cultivation and/or manipulation of biological materials is disclosed, comprising a housing (1, 2), made from an upper piece (1) and a lower piece (2) with a receptacle-shaped recess (4, 5) in the lower piece (2) and a protuberance (3) on the upper piece (1), with at least one liquid- and gas-supplying supply and drain line (6, 7) in the receptacle-shaped recess (4, 5). The upper piece (1) is embodied such as to pivot relative to the lower piece (2), by means of a joint (8), whereby the projection of the upper piece (3) and the receptacle-shaped recess (4, 5) in the lower piece are so arranged and embodied that, in a closed position of lower piece (2) and upper piece (1), the receptacle-shaped recess (4, 5) of the lower piece is sealed by the projection on the upper piece (3) in the manner of a lid to form a first chamber (5) and a locking means (9, 10) is provided to secure the closed position. The invention also relates to the use of said microchamber for the cultivation, manipulation and/or carrying out of non-invasive measurements on biological material.

Description

Ri rokαmmer

The invention relates to a micro chamber and the use thereof.

State of the art

There are already a number of bioreactors for performing measurements on biological material, in particular for examining metabolic processes or for carrying out toxicological studies, e.g. on water or air samples.

Examples include 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 cell-lining. Most of these reactors or systems are only suitable for large volumes in the ml to I scale. DE 44 43902 discloses a microscope chamber with specially arranged seals for the optical examination 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 large amounts of sample or large amounts of expensive reagents such as hormones and membrane effectors are required. It was therefore an object of the invention to provide a chamber suitable for work on a microliter scale, the volume of which should also be variable. There was also a need for a flexibly usable chamber into which the biological material can be introduced at different positions, which can also be operated as a 1-chamber or 2-chamber system as required, in order to carry out tests with culture media or reagent. To be able to carry out gradients, and in which a wide variety of measuring sensor systems can be introduced at different positions, ie in a versatile and easily flexible chamber.

The object was achieved by the micro chamber defined in FIGS. 1 to 4. Preferred embodiments are defined in claims 2 to 1 1. The invention further relates to the use of the micro-chamber as defined in claim 12.

Brief description of the figures

Figures 1, 2, 3 and 4 illustrate two embodiments of the chamber according to the invention. The following reference numerals are given in the figures:

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 supply or discharge

8 joint

9 closure means

10 closure means

1 1 essay 12α, b sealing ring

13α, 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 plant, animal or human cells, plant, animal or human cell suspensions, plant, animal or human tissue and plant or animal small organisms can be used as biological material.

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 one at least one process (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. tissue sections, brain sections, antibodies, DNA, RNA, antigens, molecules of any size and chemical composition, cells, bacteria, algae, viruses, single cell layers etc. applied to a suitable disc-shaped carrier. The biochip can also have interdigital electrode structures (IDES sensors) and / or be configured to be electrically stimulable by ring electrodes.

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 for measuring zero controls.

At least one inlet (6, 7) and at least one outlet (6, 7) open into an protuberance (3) in the upper cell body (1), which preferably consists of transparent material, via which either a membrane insert (11) with a hydrophilic, hydrophobic or semipermeable membrane or an insert (1 1) is inserted without membrane. 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 (12a, 12b). A line connecting at least one feed line (6, 7) and at least one discharge line (6, 7) preferably leads past the compartment and is used for measuring zero checks. When using a membrane insert (14) with a membrane, the upper (1) and lower (2) housing, which is connected via a joint (8), is created 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 one 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 Figures 1 and 2.

In particular, the measurement can be carried out by light microscopy or confocal laser microscopy. If necessary, fluorescence detection methods can also be used for detection.

The transparent material can be selected as desired, but should not be toxic products such as e.g. in contact with the media used. Dispense plasticizers. For example, transparent plastics, quartz or glass are suitable.

The sealing materials are selected from the common ones and include e.g. Rubber, silicone or teflon.

Through bores in the upper and / or lower part (1, 2), additional sensors (for example optodes, pressure sensors) or electrodes [for example for deriving electrical signals or. Can preferably be in both chambers (5, 17] or the central reaction chamber (5) for electrical stimulation). For example, measurements of pH, p0 ₂ , impedance, fluorescence, temperature etc. can be carried out.

A contactlessly driven magnetic stirrer can be introduced into both chambers (5, 17) or the central chamber (5). The membrane of the insert (1 4) 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 can also contain sandwiched biological material, for example 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 brought into the first chamber (5) at an angle by bringing the upper and lower part (1, 2) together introduced and then the second chamber (1 7) 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 is made of thermally conductive material, e.g. 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 one on the side facing the microchamber Cannula, which is inserted into a drain of the micro-chamber, each sealed with a sterile septum, and enables a sterile connection between the micro-chamber and the sensor module.

The measurements can be carried out over short or any length of time and are species-independent. For example, metabolic processes of individual species or toxicological analyzes of liquid or gaseous samples can be carried out by using certain species as a “sensor”.

The chamber according to the invention for the first time provided a micro-chamber for very small volumes in the microliter range, which is very easy to use and, despite the small size of the reaction chamber (s), is robust and easy to handle. The volume of the chamber (s) can be easily varied. The structure enables an extremely versatile application of various examination materials and measurement methods.

Claims

Patent claims:

1 . Micro-chamber for 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) in the relative recess (5), the upper part (1) facing the U 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 upper part (1), the container-shaped recess (5) of the lower part, forming a first chamber (5), is sealed like a lid by the protuberance of the upper part (3), with a closure means (9, 10) for securing the closed position is provided.

2. Mikrokam mer according to claim 1 with one at the relative recess (4, 5) leading, and / or with one at the protuberance (3) leading, each 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 (1 1) 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 (1 7), each with at least one liquid- or gas-carrying inlet and outlet in the second chamber (1 7), the second chamber (1 7) has a membrane (1 4) 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 (1 7) inserted biosensor and / or physicochemical sensor.

6. Micro chamber according to one of the preceding claims, wherein the first (5) and / or the second chamber (1 7) is or are sealed with the aid of sealing rings (1 2a, 1 2b, 1 3a, 1 3b).

7. Mikrokam mer according to any one of the preceding claims, wherein the supply and discharge lines (6, 7) in the first and / or second chamber (5, 1 7) are provided with interchangeable different connection modules.

8. Mikrokam mer according to any one of the preceding claims, wherein the first and / or second chamber (5, 1 7) for a micro-optical or spectroscopic observation are at least partially transparent.

9. Mikrokam mer according to any one of the preceding claims, which is at least partially made of thermally conductive material for heating.

0. Mikrokam mer according to any one of the preceding claims with one or more Peltier elements for heating or cooling.

1 . Micro chamber according to one of claims 3 to 1 0, wherein the membrane (1 4) is designed as a hydrophobic, hydrophilic, semipermeable or non-permeable membrane containing one or two sides coated with biological material or embedded in biological material in a sandwich-like manner. 2. 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.