DE202004006024U1 - Flow chamber as an insert in the well of a micro-titration dish, for the perfusion of adherent cell/tissue cultures, comprises that it forms a flow channel with the base for observation by a microscope or spectroscope - Google Patents

Abstract

A flow chamber with a flow channel for microscopy, e.g. for the perfusion of adherent cell and tissue cultures, comprises a chamber body and a sealing ring to fit exactly as a plastics insert within the well of a micro-titration dish, and form a flow channel with the base of the micro-titration plate (5). The chamber with a flow channel for microscopy, e.g. for the perfusion of adherent cell and tissue cultures, has a chamber body and a sealing ring to fit exactly as a plastics insert within the well of a micro-titration dish, and form a flow channel with the base of the micro-titration plate (5). The channel walls are contained by the sealing ring and the chamber body. The chamber body is pressed home by a clamping clip (7) and a frame (8), and a support plate (9) prevents any bending at the dish base. The circular chamber body has two coaxial drillings (10,10') with an adapter at one end for a medium feed, and with an oval or round shape with a chamfer at the other end.

Description

Field of the Invention

Flow chambers are used to represent both moving and immovable molecules, liquids or cells and their mutual effect on each other using different light microscopic or spectroscopic techniques such as high resolution microscopy, Fluorescence microscopy, phase contrast microscopy or confocal Laser microscopy, etc.). Observations of adherent or suspended objects are conceivable.

State of the art

There is a biological medical laboratory rely on a variety of different investigation methods. You can methodological problems in feeding of the material to be examined for the various examination methods. It is in the economic interest of the user if possible to have few different transport containers ready.

Carrying out flow chamber experiments has so far mostly used a transfer of the Examination material on a carrier material ahead, which can be introduced into the flow chamber or this forms together with other parts. This requires an additional one Work step for implementation of trial and may be inconvenient Influences on have the living, biological specimen. Further processing for example in the sense of coloring of the specimen for Different microscopic techniques mostly take place in standardized containers in the form of so-called microtiter plates. Between 6 and up to well over 100 Sample chambers are arranged on a standardized (ANSI / SBS) base. The previously known river chambers can not be used with the above. Microtiter plates agree on what the methodological already mentioned Disadvantages leads.

Working with contaminating biological Materials such as bacteria, viruses, fungi or others are used Part unresolved Flow chamber requirements. Contamination is of great importance the hose connections and the chamber contents, causing problems cleaning and sterilization of the flow chamber.

Numerous chambers in which the Cultivation of living biological materials and perfusion of one liquid are known. Different types are described, whereby a basic one Building a flow between two parallel plates and thus the exercise more defined shear for example adherent Cells.

In DE 10148210 , the chamber space is formed in a microscope slide and liquid reservoirs at the inlet and outlet create a communicating system through the channel, in which the examination of predominantly suspended examination objects is possible. In this case, however, the flow channel is closed, which makes it difficult or impossible to introduce adhesive test material into the chamber.

In contrast, in DE 10007398 described a chamber with a microscopic flow channel, which is formed by a sealing washer, a chamber body and a plastic washer, the cells being cultivated beforehand on the latter, which thus makes it possible to observe adherent examination objects. However, in order to introduce the material to be examined, its implementation is necessary. The design with a heated chamber body and attachment of an electrical sensor complicates the use as a contamination-uncritical disposable item.

The design of US 4974952 Made of stainless steel with attaching the channels that feed and discharge the flow medium within the steel frame makes cleaning in the event of contamination more difficult.

description the invention

It is the task of the present Invention to develop a flow chamber that is fast and simple experiment setup guaranteed, with the usual microscopic methods applicable, not critical to microbial Contamination, compatible with standardized (ANSI / SBS) microtiter dishes as well as being easy to manufacture.

These problems are solved with the in Protection claim (1-8) mentioned features solved.

Accordingly, the chamber body ( 1 ) (see 1 u. 2 ) is formed by a cylindrical plastic insert flattened on two sides, which fits exactly into the 6-world microtiter dish ( 2 ) enables. There is a groove at the bottom of the chamber body ( 3 ' ) to accommodate a biocompatible O-ring made of MVQ ( 3 ). The sealing ring in the groove surrounds the ceiling, which is recessed by 250 μm compared to the rest of the chamber body floor ( 4 ) of the flow channel. The flow channel is formed from the bottom in the form of the well bottom of the microtiter dish ( 5 ), the walls in the shape of the deformed ( 6 ) sealing ring in the groove ( 3 ) and the ceiling ( 4 ).

Using a tension bracket ( 7 ) that ensures light penetration, and two screws ( 6 ) the chamber body on the in the frame ( 8th ) inserted microplate is clamped. The support plate ( 9 ) prevents possible damage and bending of the microtiter plate. The frame and support plate can be heated if necessary.

Two prepared chambers can be quickly inserted into the microtiter dish and two screws tightened with each one Manufacture SI / SBS standardized microtiter dishes.

The working distance between the observed Cell culture and microscope objective is only slightly enlarged. to The entire flow channel is available under microscopic assessment.

The chamber according to the invention enables according to computational Fluid Dynamics (CFD) defines the exercise of flow-induced shear forces adherent Cell and tissue cultures in standard 6-well microtiter dishes. It are additional through the use of these standardized cell and tissue culture carriers and functionality impairing Test steps opposite comparable chambers saved. Use in automated Systems are possible.

The chamber body has two bores each in the form of a Luer cone ( 10 . 10 ' ). Here, the medium is fed in and out via a hose connection adapter (Luer / Luer lock male or female). In the ceiling of the flow channel ( 4 ) the channel in the body opens to a longitudinal oval inflow ( 11 ) and a round drain ( 11 ' ). Inflow and outflow are provided with a chamfer 0.5 mm × 45 ° to avoid peak shear forces. The other holes ( 12 ) are optional and are used to attach the chamber body to the clamping bracket. Twisting the chamber in the well is prevented by fitting the side flats of the chamber body into a groove ( 13 ) guaranteed in the bottom of the clamp.

Due to the construction described above is a simple manufacture of the chamber body possible, which the training of Flow chamber made possible as a disposable item. Thus, by the advantageous design The handling of contaminating substances is not critical to the invention.

The height of the flow channel is not determined by influences the sealing ring, but is only responsible for the manufacturing tolerances the 250 μm deepening in the chamber body, which shows a high reproducibility by CFD objectified flow conditions guaranteed.

The features of the invention go except out the claims also from the description, with the individual features for themselves alone or in several advantageous protective versions in the form of combinations represent for protection is requested with this document. The combination exists from known (river chamber for cultivation, local perfusion, microscopic observation) and new elements (formation of the chamber in the well of an ANSI / SBS Multititer bowl, possible Use of the chamber to use adherent cell and tissue cultures as a disposable item and thus use with contaminating substances, simplest production e.g. as an injection molded part), mutually influence and in their new overall effect a use advantage and result in the desired success, which is that the easy one automatable construction of a flow channel and by CDF objectified shear forces allows becomes.

embodiment

The invention is explained below of the figures on an embodiment explained in more detail without limited to this example to be. Show it:

1 a perspective view of the fully assembled chamber with hose connection adapter in a 6-well microtiter dish

2 a bottom view of the clamping bracket with two Einhetten the assembled and sealed chamber body

The generally in the figures with ( 1 ) flow chamber essentially consists of a cylindrical body ( 1 ) made of a suitable translucent plastic. The body ( 1 ) is constructed in one piece and has two coaxial, directly opposite Luer cone bores ( 10 . 10 ' ), which are used for media supply and discharge via adapters. The holes end at a certain depth on the longitudinal oval inlet ( 11 ) or round drain ( 11 ' ). Inflow and outflow end in the ceiling of the flow channel formed by the body bottom ( 4 ). The side walls of the flow channel are made from the deformed sealing ring ( 3 ) educated. The channel is closed by inserting the construction according to the invention into the microtiter dish through the bottom of the cell culture vessel, the necessary contact pressure being achieved by screwing the clamping bracket ( 7 ) using the screws ( 7 ' ) on the frame ( 8th ) is reached. By supporting the bottom of the microtiter dish using a support plate ( 9 ) sufficient thickness prevents deformation and a secure seal of the flow channel is achieved.

Claims (5)

Chamber with a microscopic flow channel, e.g. for the perfusion of adherent cell and tissue cultures under defined flow conditions, consisting of a chamber body ( 1 ) and a sealing ring ( 3 ), characterized in that the chamber body is inserted into a microtiter bowl (ANSI / SBS) and with the bottom of the bowl a flow channel is built up, the walls of which continue through the sealing ring ( 3 ) and the body ( 1 ) are closed, with the chamber body on the microtiter dish using a clamp ( 7 ) and a frame ( 8th ) and a bending of the bottom of the microtiter tray via a support plate ( 9 ) is prevented. Chamber according to claim 1, characterized in that the round chamber body ( 1 ) two coaxial holes ( 10 . 10 ' ), which verse at one end with an adapter for the media feed can be hen, and are provided on the other end in a longitudinal oval or round version with a chamfer. Chamber according to claim 1 to 3, characterized in that the surfaces of the flow channel from the bottom of a microtiter plate ( 5 ), the bottom of the chamber body ( 4 ) and the sealing ring ( 3 ) are formed. Chamber according to claim 1 to 3, characterized in that a rotation of the chamber by a groove in the clamping bracket ( 13 ) is prevented. Chamber according to claim 1 to 4, characterized in that the height of the flow channel only is defined by the recess in the bottom of the chamber body.