DE102017000228A1 - Method and device for gentle, automatic parallel dispensing and aspiration of liquids and biological suspensions - Google Patents

Abstract

Dispensing comb for simultaneously distributing a liquid in a plurality of arrays arranged in arrays, comprising a linear supply channel (2, 3) connected to a horizontal straight distribution channel (4) connected along its length, at regular intervals, with at least 2, usually 8 or 16, in parallel to each other lying Dispensierkanäle (6), wherein: - form mutually perpendicular longitudinal sections of the supply channel (2, 3) Symmetriepläne (SP1, SP2), - the distribution channel (4), plane symmetrical to SP1, d. H. in the middle longest, connected to the supply channel (2, 3), - the capillary dispensing channels (6) are connected plane symmetrical to SP1, characterized in that capillary dispensing channels (6) open at an angle of 0 to 90 degrees to the symmetry plan SP2 ,

Description

The invention relates to a method and apparatus for the gentle, automatic parallel dispensing and aspiration of liquids and in particular cell-containing or other shear-sensitive liquids such as suspensions, dispersions or solutions.

From the US4417610 For example, a system for dispensing viscous fluids is known. The US4995432 describes a dosing system for liquids during the US5105859 discloses a reagent dispenser for multiwell plates and it in the US5865224 goes to an automatic Dispensiergerät.

The reproducible and gentle dispensing of shear-sensitive components such as proteins or stem cells in microtiter plates, in particular simultaneous dispensing into a plurality of containers arranged in arrays, is currently not possible under ideal conditions due to the shear stress that commercial systems exert on the shear-sensitive components. Shear stress-induced differentiation or even damage poses a hazard to stem cell handling. Protecting proteins may also damage the proteins. This is often noticed only during storage.

Commercially available systems also offer only limited possibility to exchange components with cell contact.

It was therefore an object to provide a solution which enables a reproducible and gentle dispensing of a liquid containing shear-sensitive components in containers, in particular microtiter plates.

Other tasks were to design this solution so that all components that come in contact with the liquid can be easily replaced. Ideally, a compact integrated design should also be provided, allowing for sterile operation of the device under a microbial safety cabinet (cleanbench).

• – zu einander senkrechte Längsschnitte des Versorgungskanals (2, 3) Symmetriepläne (SP1, SP2) bilden,
• – der Verteilungskanal (4), plansymmetrisch zu SP1, d. h. mittig längst, verbunden mit dem Versorgungskanal (2, 3) ist,
• – die Kapillar-Dispensierkanäle (6) plansymmetrisch zu SP1 verbunden sind.

dadurch gekennzeichnet, dass Kapillar-Dispensierkanäle (6) mit einem Winkel von 0 bis 90 Grad zum Symmetrieplan SP2 münden.The first object has been achieved by a dispensing comb for the simultaneous distribution of a liquid in a plurality of containers arranged in arrays, comprising a linear supply channel ( 2 . 3 ) connected to a horizontal straight distribution channel ( 4 ) connected along its length, at regular intervals, with at least 2, usually 8 or 16, parallel to each other Dispensierkanäle ( 6 ), in which:

• - to vertical longitudinal sections of the supply channel ( 2 . 3 ) Form symmetry plans (SP1, SP2),
• - the distribution channel ( 4 ), plane symmetrical to SP1, ie longest in the middle, connected to the supply channel ( 2 . 3 ),
• The capillary dispensing channels ( 6 ) are connected symmetrically to SP1.

characterized in that capillary dispensing channels ( 6 ) at an angle of 0 to 90 degrees to the symmetry plan SP2.

This dispensing comb allows a controlled, precise delivery of a liquid to be dispensed with high throughput of filled containers per unit time.

For the purposes of the application, liquids to be dispensed are in particular biological suspensions containing shear-sensitive components, in particular cell-containing or protein-containing suspensions, for example stem cell suspensions.

Dispensing in the sense of the application means the delivery of a controlled amount of liquid.

For the purposes of the application, a symmetry plan is a symmetry line which divides the dispensing comb into 2 congruent parts.

For the purposes of the application, the term "plan symmetric" refers to the fact that a feature, eg. B. the capillary channels, at equal intervals over a defined length z. B. the length of the distribution channel is distributed and at the same time the individual characteristics of the feature symmetrical on both sides of a symmetry line - for example, the supply channel - are distributed.

For the purposes of the application, a capillary channel - also referred to as capillary dispensing channel - the last, finest channel through which the liquid to be dispensed is performed. It is usually round and has a diameter of 0.2 to 1 mm. With a small diameter, the shear stress for the liquid increases. With a large diameter, the risk of dripping losses of the liquid to be dispensed increases. Drip losses should be avoided as far as possible, as this will cause the amount of dispensed liquid to fluctuate, meaning that the dispensing volumes are not uniform. In addition, the inner diameter of the capillary channels must not be too large in relation to the flow cross-section of the distribution channel. Among other things, this depends on the uniform distribution of the liquid over all channels.

For the purposes of the application is a supply channel, the central first channel of the Dispensierkamms, in which the liquid to be dispensed is introduced.

For the purposes of the application, a distribution channel is the channel which receives the liquid to be dispensed from the supply channel and forwards it to the capillary channels.

Preferably, the capillary dispensing channels open at an angle of 40 to 50 degrees to the symmetry plan SP2. This has the effect that the liquid to be dispensed is directed to the container wall and not directly into the liquid, which is located in the container. Thus, the risk of cross-contamination by liquid drops, which arrive at the introduction of the liquid to be dispensed in adjacent wells minimize. Moreover, an introduction to the container wall also protects the components in the liquid to be dispensed and any components already present in the container. This is the case, for example, if already sedimented or grown cells are located at the bottom of the containers (wells), since they could be detached from the ground again by a direct irradiation.

In a particular embodiment, all channels are introduced in a comb body.

Preferably, the comb body is made of a biocompatible material, for example of a biocompatible plastic such as polyoxymethylene (POM) or other known biocompatible materials such as glass, stainless steel or coated materials.

In the comb body, the channels can be introduced with the usual methods of manufacturing technology so z. B. be milled or the entire body be made by injection molding.

Preferably, the comb body comprises the distribution channel, particularly preferably the supply channel.

Preferably, the distribution channel is arranged vertically in relation to the supply channel and thus runs parallel to the SP1.

Furthermore, no capillary channel is preferably accommodated in the extension of the supply channel. Instead, the supply channel opens in an extended area of the distribution channel to achieve a uniform flow distribution.

In a preferred embodiment of the dispensing comb, the capillary dispensing channels are formed by separate capillary dispensing nozzles / tubes.

For the purposes of the application, separate capillary dispensing nozzles / tubes are capillary channels which are not included in the comb body.

These capillary dispensing nozzles / tubes are preferably made of a biocompatible material.

In this embodiment of the dispensing comb, the distribution channel has openings for receiving the capillary dispensing nozzles. The capillary dispensing nozzles / tubes are typically pressed into these openings or possibly glued.

These openings for receiving the capillary dispensing nozzles / tube are preferably channel-shaped and have the appropriate angle, so that the capillary dispensing nozzles / tube are inserted in the openings with the correct angle. The angle can be between 0-90 ° and preferably between 40-50 °. Particularly preferred is an angle of 45 °.

This embodiment has the advantage that the Dispensierkamm can be produced inexpensively and the complete Dispensierkamm including the already inserted capillary Dispensierdüsen / tube good and stable steam sterilizable z. B. is autoclavable.

In addition, it is conceivable to design the entire Dispensier comb as a single-use article.

For example, the distribution channel ( 4 ) has a rectangular cross-section of 1.5 × 2.5 mm ² over a length L of 68 mm. In the inflow region of the flow cross-section of the distribution channel is extended over a length of 10 mm to 1.5 × 5 mm ² .

The duct and connection dimensions are optimized for the smallest possible dead space volume.

For the purposes of the application, a dead volume is an amount of the liquid to be dispensed which has been introduced into the dispensing comb, but does not leave it during the normal dispensing operation and thus reduces the efficiency of the dispensing comb.

The dimensions of the distribution channel are adapted to the microplate or array of containers.

For the purposes of the application, an array is a spatially fixed arrangement of containers wherein the containers are arranged side by side on the same plane. Typically, the planes of the arrays are rectangular.

On rectangular array planes there are thus containers arranged side by side along the long side of the rectangle. For the purposes of this application, they are in line.

In addition, there are containers which are arranged side by side along the shorter side of the rectangle - colloquially over or under each other. For the purposes of this application, these containers are in a column.

Preferred arrays are microtiter plates, in particular 6-, 12-, 24-, 48-, 96- and 384-1536 well plates.

Thus, the 3 wells of a 6-well plate are in line, while each 2 wells of the 6-well plate are in a column.

Usually, the distribution channel ( 4 ) so long that all containers in a column can be addressed. Its cross-section can round or square in particular square with a diameter or in the angular embodiment, cross-sectional height and width of 1 mm to 5 mm, preferably 1.5 mm to 2.5 mm, wherein width and height can be selected independently. The dimensions of the distribution channel are limited by the acceptable volume loss.

However, it is equally conceivable that the distribution channel ( 4 ) is so long that all lying on a line container can be addressed

Usually, the cross-sectional area of the distribution channel is ≦ cross-sectional area of the output of the supply channel connected thereto.

With the distribution channel, the capillary dispensing channels are diagonally inclined to the o. G plan symmetrical to SP1. Angle connected via their inputs, d. H. distributed at equal intervals over the length of the distribution channel and symmetrically on both sides of the supply channel.

In addition, the Dispensierkamm 4, 8 or 16, preferably 8 or 16 capillary dispensing nozzles / tubes or capillary channels. In other words, the dispensing comb is typically designed for 96- or 348-well plates. However, any other number of capillary dispensing nozzles / tubes or capillary channels is also conceivable.

Preferably, the distance between the capillary dispensing channels usually corresponds to the geometry of the microtiter plate to be filled or array of containers used. Said geometry of the microtiter plates is standardized by the standard of the Society for Biomolecular Science (SBS).

In the case of 384-well plates, that is a dispensing comb with 16 capillary dispensing channels, the dispensing comb is spaced 4.5 mm between the capillary dispensing nozzles / capillary channels (measured from the center of the capillary dispensing nozzles / capillary channels) ).

In a first embodiment, the inputs are ( 7 ) of the dispensing channels ( 6 ) in the symmetry diagram SP2 of the dispensing comb opposite to the connection to the supply channel. In this embodiment, the distribution channel is typically ( 4 ), not only plane symmetrical to SP1 but also to SP2, ie also in the middle of the output of the supply channel ( 2 . 3 ) connected.

Alternatively and preferably, however, are the inputs ( 7 ) of the dispensing channels ( 6 ) arranged in a straight line parallel to the symmetry diagram SP2 in the width of the distribution channel, opposite to the connection to the supply channel ( 2A and 2 B ). Usually, the distance of this line to SP2 is 2 mm to 4 mm. In this embodiment, the cross section of the distribution channel is preferably angular, more preferably quadrangular.

Preferably, then the supply channel is not connected in the middle but laterally of the distribution channel. The distribution channel is best connected in the extension of the supply channel, as in 2A presented with the advantage of reducing dead volume. Optionally, the transition may be rounded in the angles formed between the walls of the channels.

In a preferred embodiment, the distribution channel is closed with a transparent film, preferably a biocompatible adhesive tape, so that the distribution channel ( 4 ) has an optical window over its entire length. For example, ViewSEAL ^™ (greiner bio-one) adhesive tape was used to seal the distribution channel. This example of a film suitable for sealing the distribution channel has the advantage that only areas stick in which the adhesive contained in microcapsules has been activated by pressing. Thus, it is possible to glue the film so that the wall of the distribution channel formed by the film does not stick. Alternatively, it would also be possible to weld on a material closing off the distribution channel, for example by laser welding.

Through this window, faults in the distribution channel (air bubbles or blockages) can be checked if necessary. The dispensing comb is also rinsed during the priming process with a few mL of dispensed material (together with the other components of the tubing). The optical window allows the visual detection of any existing air bubbles to check the quality of the priming. Typically, this visual inspection is done by the user. An automation z. B. by image capture and analysis would also be possible.

For the purposes of the application, a priming or a priming process is the first filling of the dispensing comb or dispensing unit.

Usually, the length of the dispensing channels ( 6 ) are selected in this embodiment so that they do not overhang the body ( 1B ). Thus, the Dispensierkämme are compact and stackable well without the risk of damaging the capillary dispensing nozzles / tube.

Preferably, the comb is tool-clipped into the receptacle provided in the Dispensierunit. This results in a play-free and robust coupling in the device.

The described dispensing comb makes it possible, for example, to minimize unwanted damage caused by shear stress to proteins or stem cells. In the case of stem cells, moreover, an undesired differentiation triggered by shear stress is minimized.

• – mindestens ein Mittel zur Aufnahme der zu dispensierenden Flüssigkeit aus einer drucklosen Flüssigkeitsvorlage, verbunden mit
• – mindestens einem Transportsystem zur Beförderung der zu dispensierenden Flüssigkeit, verbunden mit
• – dem oben beschriebenen Dispensierkamm,

wobei das Transportsystem mindestens einen intermediären Druckbehälter umfasst, in dem abwechselnd Überdruck/Unterdruck erzeugt werden kann.A further subject of the application is a Dispensierunit for a device for dispensing one or more liquids, comprising the following elements:

• - At least one means for receiving the liquid to be dispensed from a pressureless liquid reservoir, associated with
• - At least one transport system for transporting the liquid to be dispensed, associated with
• The above-described dispensing comb,

wherein the transport system comprises at least one intermediate pressure vessel, in which alternately positive pressure / negative pressure can be generated.

For the purposes of the application, a dispensing unit is a device that can independently pick up a liquid to be dispensed and dispense it into the containers of an array. Preferably, the Dispensierunit also prepare this process independently, z. By performing a priming operation. Moreover, the dispensing unit is preferably designed so that different columns of an array can be filled with different components and / or with different volumes.

An example of a pressureless fluid reservoir is an open vessel in which the liquid to be dispensed is located.

An example of an intermediate pressure vessel in which alternating positive pressure / negative pressure can be generated is the body of a syringe. The use of a syringe without a piston as an intermediate pressure container has the advantage that the syringe is sterile and disposable or designed for single use.

Preferably, the positive pressure / negative pressure is externally applied by means of two pumps, for example, as in 3 shown with the help of the vacuum pump (P1) and the compressor (P2).

Preferably, the dispensing unit is used to dispense biological fluids. For the purposes of this application, biological fluids are solutions of biopolymers or biopolymers such. As proteins or nucleic acids or cell-containing suspensions or dispersions.

The gentle dispensing of the biological suspension, z. B. a cell suspension is prepared by using an intermediate pressure vessel (eg., As in 3 shown) achieved in the transport system. This is filled by a previously applied vacuum from an external vacuum reservoir and then acted upon with a finely adjustable pressure from an external pressure reservoir. Both external reservoirs are adjusted to their respective desired pressures by electronic pressure regulators. This has the advantage that the pressure in the Dispensierunit remains stable and a non-pressurized liquid template can be used or the liquid template can be connected without pressure.

Preferably, the intermediate pressure vessel is monitored by a level sensor and the pump is controlled by the corresponding signal. This solution has the particular advantage that the changeover from underpressure when taking up the liquid to overpressure during delivery and dispensing of the liquid can be carried out variably and with particular care depending on the sensitivity of the liquid.

The transport of the liquid preferably takes place via hose lines and is controlled by precise switching pinch valves.

Typically, commercially available disposable components are used to transport the liquid. The following components were used in a test facility. Silicone Platinum Tube, ID = 1.6mm, OD = 3.2mm, WS = 0.8mm about 5 m 50 mL syringe, sterile, Luer-Lock (BD Plastiplak Product Number: 309653 or equivalent) 2 pieces Male Luer Lock to Barb Connector, 3/32 inch ID Tubing, z. Qosina 11535 or equivalent 7 pcs T Connector, All Female Luer Locks, z. Qosina 88214 or equivalent 1 piece Syringe filter PTFE 25 mm 0.2 μm, Luer-Lock 2 pieces

The biological suspension is passed into the dispensing comb described above, whose central distribution channel opens into dispensing capillaries, from which the dispensed material is dispensed. The shear stress exerted on the cells in this way is comparable to manual pipetting.

The dispensing unit preferably comprises a device with which liquid can be aspirated from the containers of an array, for example a suction comb.

Furthermore, the dispensing unit preferably comprises further components, for example a controller, drivers, motors, silencers, pressure regulating valves, ventilation.

In one example, the position of the dispensing comb within the dispensing unit is fixed. In this example, a suction comb and the microtiter plate to be filled move. Usually, the movement of the Absaugkamms and the Dispension of the Dispensierkamms with the movement of the microtiter plate clocked. Support and transport for the plates are commercially available. For example, the elements of Igus were used in a test unit. The transport system was self-developed.

The cycle, the pump and switching Pinquet valves are computer controlled by means of a correspondingly configured central control unit connected to a user interface.

By using a touch screen-based control, the device is also very comfortable to use and results in reproducible dispensing results. The entire process is automated and user-controlled. The dispensing is initially designed for the microtiter plates listed below, but can be adapted to other vessels to be filled by adjusting the device or the software. Preferably, the dispenser is completely operated by means of a built-in touch screen device.

The high hygienic requirements are achieved through an integrated cleaning concept. All components with cell contact are user-replaceable or steam sterilizable. In addition, the device has a corresponding wash program for short-term cleaning requirements, which leads a disinfectant solution through the fluid paths. Finally, all tubings and adapters with liquid contact are interchangeable (disposable).

In addition to carrying out the respective dispensing protocol, the user must ensure that a calibration adapted to the medium takes place. Furthermore, a priming procedure should be carried out before each dispensing and the wash and emptying protocol should be carried out after the activities have been completed. To operate the wash and emptying protocol, the dispensing unit preferably additionally has an external liquid waste container, including a tubing, which is equipped with a vacuum.

Characters:

The figures show particular embodiments of the Dispensierkamms and Dispensierunit invention, without being limited thereto.

1A and 1B show a dispensing comb for 384-Wellmikrotiterplatten with 16 capillary-Dispensierkanälen in side view and from above.

2A shows the cross-section of the dispensing comb 1 along the symmetry plan SP1

2 B shows the cross-section of the dispensing comb 1 parallel to the SP2 symmetry plan on a capillary dispensing channel.

3 shows a schematic representation of a Dispensierunit comprising the Dispensierkamm 1 - here referred to as Dosierkamm.

LIST OF REFERENCE NUMBERS

SP1, SP2

Symmetry plans of the Dispensierkamms, which are formed by mutually perpendicular longitudinal sections of the supply channel.

1

comb body

2

Supply channel shell

3

Supply channel lower part

4

distribution channel

5

Window closed (eg with a foil, which is not shown)

6

Capillary dispensing channel

7

Entrance of the capillary dispensing channel

8th

Output of the capillary dispensing channel = dispensing nozzle

4 shows the precision of dispensing cell culture medium in a 96-well microtiter plate. The extinction values at 570 nm in each well are given after dispensing a Brillant Black solution (Aldrich, Cat # 21184-2, 10 ppm in DMEM: F-12, gibco Cat # 21041-025) with a valve opening time of 298 msec at overpressure of 0.5 bar, corresponding to 150 μL / well.

5 : Comparison of Precision Dispensing of Brilliant Black Solution (Aldrich, Cat # 21184-2, 10 ppm in DMEM: F-12, gibco Cat # 21041-025) into two independent 96 well microtiter plates. The deviations (coefficient of variation, CV) of the extinction values at 570 nm after dispensing at different pressures corresponding to approximately 150 μL / well (96-well MTP) or 50 μL (384-well MTP) are indicated. Both the averaged CVs within an MTP (intra-plate CV) and those between two different MTPs (inter-plate CV) are indicated.

6 Cell viability of dispensed MC3T3 cells in 96-well (left) or 384-well format (right)

Example:

A dispensing comb for 384 well microtiter plates with 16 capillary dispensing channels and a dispensing comb for 96 well microtiter plates with 8 capillary dispensing channels were tested. The design of the combs resulted in a uniform dispensing of Dispensierguts with minimized dead volume (see 4 ). The precision of the dispensing result within a microtiter plate (MTP) is very high with a coefficient of variation (CV) of 1.6%. As the overpressure increases, the dispensing rate of the dispensing product increases, but in this case the CV also increases to values of, for example, 10. B. 3.1% at 0.7 bar (see 5 ). The CV between two different plates (inter-plate CV) is also very good with values between 0.1 and 1.9% (see 5 ). CV values increase slightly when using the 16-needle comb and the 384-well MTP but remain below 5% CV in all cases observed. The low shear stress experienced by the dispensing material can be determined, for example, by determining the cell vitality of a dispensed cell line ( 6 ). In this example murine MC3T3 cells were used. At harvest, the adherent cell cultures were in 70% confluency. Two 96-well MTP were each filled with 100 μL / well and two 384-well MTP with 40 μL / well each. This happened at the three system pressures 0.3; 0.5 and 0.7 bar. In addition, two plates were pipetted manually (using a 200 μL pipette). The panels were then by pipette with 10 ul / well (96-well MTP) and 4 .mu.l / well (384-well MTP) alamarBlue ^® (Thermo Fisher) was added and incubated overnight at 37 ° C and 5% CO 2 atmosphere incubated. The fluorescence measurement was carried out at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. From the vitality data, it can be seen that the automatic dispensing leads to comparable, sometimes even better results than manual pipetting.

The work that led to this application was made in accordance with the Grant Agreement "Bio.NRW: StemCellFactory, Grant Number: 005-1007-0026, Project Number: z0911bt027f" NRW Program: Bio.NRW Ziel2 Competition (2009) Funded under the European Regional Development Fund (ERDF).

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 4417610 [0002]
• US 4995432 [0002]
• US 5105859 [0002]
• US 5865224 [0002]

Cited non-patent literature

• Grant Agreement "Bio.NRW: StemCellFactory, Grant Number: 005-1007-0026, Project Number: z0911bt027f" NRW Program: Bio.NRW Ziel2 Competition (2009) [0082]

Claims (2)

Dispensing comb for the simultaneous distribution of a liquid in a plurality of arrays arranged in arrays, comprising a linear supply channel ( 2 . 3 ) connected to a horizontal straight distribution channel ( 4 ) connected along its length, at regular intervals, with at least 2, usually 8 or 16, parallel to each other Dispensierkanäle ( 6 ), wherein: - longitudinal sections of the supply channel perpendicular to each other ( 2 . 3 ) Symmetry plans (SP1, SP2) form, - the distribution channel ( 4 ), plane symmetrical to SP1, ie longest in the middle, connected to the supply channel ( 2 . 3 ), - the capillary dispensing channels ( 6 ) are connected symmetrically to SP1, characterized in that capillary-dispensing channels ( 6 ) at an angle of 0 to 90 degrees to the symmetry plan SP2. Dispensing unit for a device for dispensing one or more liquids, comprising the following elements: - At least one means for receiving the liquid to be dispensed from a pressureless liquid reservoir, associated with - At least one transport system for transporting the liquid to be dispensed, associated with The above-described dispensing comb, wherein the transport system comprises at least one intermediate pressure vessel, in which alternately positive pressure / negative pressure can be generated.

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