DE102016215269B4 - Device and method for separating biological cells - Google Patents

Abstract

Device (10) for separating biological cells (28) on a microdosing device (20) via individual liquid drops (26) of a suspension of the cells (28) released from the dosing channel (22) of the microdosing device (20) onto a plurality of target vessels (60), containing: at least one detector unit (30) assigned to the dosing channel (22) and arranged directly on it, containing several light sources (32) and several detectors (36) for detecting light-induced fluorescence, the detector unit (30) being arranged so that a Individual drops of liquid (26) dispensed from the dosing channel (22) can be illuminated by the light sources (32) and any fluorescent light emitted therefrom can be registered by the detectors (36) in at least two spatial directions; actuators (64) for step-by-step switching of the target vessels (60) relative to the metering channel (22); andprogrammed evaluation unit (40), connected via a signal connection (42) to the detectors (36) for the detector signal-related generation of control signals at a signal connection (44) to an actuator (24) for the recurring triggering of the dispensing of liquid drops (26) at the metering channel ( 22) of this microdosing device (20) and of control signals at a signal connection (46) to the actuator system (64) for triggering a step-by-step switching of the target vessels.

Description

The invention relates to an integral device and a method for the automatic isolation of biological cells from a suspension of cells and their distribution to several target vessels.

The filing, i.e. the isolation of individual biological cells, is an essential process step within the automatable cultivation of cells and in biological assays. The so-called “single cell technology” for cultivating and analyzing individual cells is becoming increasingly important. It allows a new approach to the study and control of the development of cells and cell lines. First of all, the cells are free in a suspension and are then to be distributed individually on substrates or target vessels for further process steps, that is to say deposited, for example on several similar wells of a multiwell or microtiter plate.

Known methods for cell isolation are difficult to automate. The cells are usually separated out under manual control, for example under the microscope; known automatic cell sorting processes often subject the cells to high physical stress. Such processes are not suitable for fully automatic high throughput processes for the rapid and uncomplicated provision of a large number of individual cells of uniform quality from a cell suspension provided. Another problem is the long dwell time of the cells in the process of cell isolation, which can already lead to significant differences in the quality of the isolated cells from one another. In addition, a large proportion of cells are often destroyed or significantly damaged during the process. For certain analyzes, however, it is necessary to obtain as much as possible all cells of the suspension presented and to be able to examine them further.

It is therefore desirable to have a method that can be carried out in a fully automated manner, in particular, for the rapid and safe isolation and distribution of cells from a cell suspension provided to a number of target vessels, preferably on wells of a microtiter plate, the cell isolation being carried out fully automatically and reliably standardized according to defined criteria and the time for isolation and the physical stress for each cell is significantly reduced.

Out US 2015/0152497 A1 a device for the isolation of biological cells at a microdosing device emerges via liquid droplets of a suspension of the cells discharged from the dosing channel of the microdosing device onto a plurality of target vessels.

DE 10 2009 032 428 B1 discloses an arrangement for depositing cells on a substrate.

US 2007/0065808 A1 discloses a method and apparatus for sorting out particles from a particle stream.

Microdosing devices for dosing biological fluids, including cell suspensions, are known. These single or multi-channel microdosing devices have recently become suitable for dosing small or very small amounts of liquid, in portions or drops from 1 nl to 500 nl and at a high repetition rate (approx. 10 to 600 Hz). The 1-DOT nanodosing technology (Fraunhofer Institute for Manufacturing Engineering and Automation IPA) is a high-throughput method for the automatic processing of volumes in the nanoliter range. Drops of approx. 1 nl can be triggered individually and are dosed repeatedly via pressure pulses (shock waves) from a template with a nozzle onto the target vessels below. The present invention is based on the technology of such microdosing devices.

The technical problem on which the present invention is based consists in the provision of improved methods and means for carrying out a simple and safe isolation of cells from a cell suspension which has been submitted, suitable for automated high-throughput methods. The technical problem is completely solved by a device according to claim 1 for separating biological cells by means of liquid drops of a suspension of these cells in one or more target vessels that are individually dispensed from the dosing channel of a microdosing device.

The device contains in particular at least one, preferably exactly one, detector unit which is assigned to the dosing channel of a microdosing device and preferably arranged directly on it. This contains several light sources, especially UV light sources, suitable for exciting fluorescence in an especially fluorescence-marked and / or autofluorescent biological cell, and several detectors or detector fields associated with these light sources, suitable and specifically designed for the detection of light-induced fluorescence. The detector unit is arranged on a microdosing device and designed in such a way that individual drops of liquid dispensed from the dosing channel can be individually illuminated by this light source and any fluorescent light emitted by this can be registered on the detector or detector field.

In the context of the disclosure of this invention, the term “UV light source” is also used understood any light source emitting fluorescence. As a rule, this is short-wave, high-energy light in the UV range, but blue light or light of greater wavelength can also be suitable, depending on the fluorescent dye or fluorescent structure. It is preferred that the wavelength of the light source matches the excitation spectrum of the fluorescent dye. This can ultimately also be yellow or red light if the cells are colored accordingly. A preferred variant emits light with a wavelength of 460 nm.

The device also contains a programmed evaluation unit. This is in contact with the at least one detector of the detector unit via a first signal connection. It is programmed to generate at least one control signal from the detector signal with automatic application of specific evaluation criteria, which can be routed via a signal connection to an actuator of the microdosing device for the purpose of the recurring triggering of the delivery of individual liquid drops from the respective dosing channel of the microdosing device that can be connected to it.

The device also has an actuator: servomotor, robot arm or the like, for step-by-step switching of the target vessels on the respective metering channel. According to the invention, this is in contact with the programmed evaluation unit via a further signal connection, with a control signal being able to be generated in the programmed evaluation unit according to predetermined criteria, which can trigger the step-by-step switching of the target vessels to this actuator.

The actuator system for step-by-step switching of the target vessels preferably has a carrier or rack for receiving a plurality of target vessels under the dosing device, for example individual vials. In an alternative and preferred embodiment, multiwell plates, that is to say microtiter plates, are accommodated. There the individual target vessels are arranged in a regular form (mostly 96-fold, also 384, 1536 or 3456-fold) as an addressable two-dimensional array. The actuator system for switching the target vessels (wells) is preferably designed in such a way that the microtiter plates can be pushed in rows and rows under the dosing device in the X and / or Y direction. Preferably, the individual target vessels (wells) can be freely addressed.

In an alternative variant, the actuator system is designed in such a way that the arranged target vessels remain at rest relative to the overall apparatus during separation, but the dosing device, in particular via a robotic arm, can be moved step-by-step in order to select a different target vessel in response to the change signal from the programmed evaluation unit to approach the arranged target vessels.

The invention makes use of the knowledge that the fluorescence of specifically fluorescence-labeled cells or the autofluorescence of specific structures of the biological, in particular intact, vital cells in a single liquid drop of a suspension of such cells allows the presence of a cell in this liquid drop to be detected on the basis of this fluorescence signal .

An essential aspect is that the suspension can be reliably dosed in individual drops. According to the invention, the concentration of the cells of the cell suspension is set such that each individual dosed liquid drop or dispensed liquid portion either contains a maximum of one cell or no cell on average. It can be optimized in such a way that the majority of the dosed drops of liquid or portions of liquid do not contain any cells and, if at all, only contain exactly one cell. The proportion of fluid portions that contain two or more cells can thus be minimized. The high repetition frequency of the serial delivery of these small portions of liquid in the microdosing devices mentioned allows the serial separation, which can be automated and standardized quickly and easily, with a simultaneously low proportion of incorrect dosages, in which two or more cells are dosed into a single target vessel.

The invention provides an algorithm according to which such individual liquid drops or liquid portions are repeatedly dosed into a first target vessel until a living, intact cell has been detected in a single one of the serially dosed liquid drops or liquid portions by means of its fluorescence signal by means of the detector unit. In this way, the liquid metering into this first target vessel can be completed and a step-by-step switching of the actuators that can be transmitted via the programmed evaluation unit, the further metering can be directed to another target vessel. The repetitive serial dosing process of the small liquid drops or portions of the cell suspension can then be carried out into the further target vessel until a cell is detected again on the basis of its fluorescence in one of the dispensed liquid portions.

One element of the device according to the invention is the detector unit, which is positioned as close as possible to the metering channel where the liquid droplet emerges in order to register the fluorescence of a cell contained in the liquid droplet there. For this purpose, the detector unit has several UV light sources, which preferably have a light wavelength which corresponds to the excitation wavelength of the fluorescent dye or autofluorescent structures of the biological cell used. The fluorescence signal generated by a single fluorescently labeled or autofluorescent cell is inherently weak. At the same time, due to the light refraction conditions within the individual dispensed liquid drop, there is the problem that fluorescent light emitted by a cell contained there is refracted in the liquid drop in such a way that no or too little light falls into the detector of the detector unit. To avoid this, provision is made for two or more detectors or detector fields to be provided in the same detector unit at the same time, which detectors can detect a fluorescence signal in at least two different spatial directions. In a preferred variant, a single ring-shaped detector or a detector with a ring-shaped detector field is provided, the ring being arranged around the liquid drop passing there, so that a fluorescence signal can be detected in several spatial directions. Additional improvements to the fluorescence detection provide a detector which is specifically designed for the temporal integration of the fluorescent light of the droplet flying past the detector field, with fluorescent light from further spatial directions advantageously being able to be registered on the same detector field.

The detector is preferably a photomultiplier known per se or another light amplifier unit which is suitable for detecting a fluorescence signal of an individual cell in a liquid drop with sufficient external signal spacing. The detector is preferably selected from: avalanche photodiode-based detectors (APD), silicon photomultiplier-based detectors (SiPM) and photoelectron multiplier tubes (PMT).

In a particularly preferred variant of the invention, the detector itself, which is highly sensitive on the one hand, and takes up space on the other, is arranged outside the measuring field of the metering channel. It is provided that the fluorescent light emitted by the dispensed liquid drop on the metering channel is received via one or more suitable light guides (glass fiber or the like) and guided to this external detector. Accordingly, in one variant, the device has at least one external detector with at least one light guide with a detector field, which is arranged on the metering channel in such a way that fluorescent light from the metered liquid drops falls on this field. In a specific variant of this, several light guides of a preferably individual or single external detector form detector fields at different positions in order to detect fluorescent light from several spatial directions. In an alternative variant of this, the light guide of an external detector ends in a ring-shaped detector field (ring-shaped light collector) in order to detect light from several spatial directions.

In an alternative or additional embodiment, the light source is also placed outside, and its light is guided to the metering channel via at least one light guide (glass fiber or the like). Accordingly, in these variants the device has an external light source with at least one light guide which is arranged on the metering channel in such a way that fluorescence-triggering light from the light guide falls onto the liquid droplets. In a variant of this, the light guide of the UV light source ends in a ring-shaped illumination on the metering channel.

The device according to the invention is specifically suitable to be upgraded to a known microdosing device, or multichannel microdosing device for the recurring serial delivery of small drops of liquid or portions of liquid with high repetition frequency, in order to functionalize this microdosing device for the automatic isolation of cells from a cell suspension. Accordingly, another object of the invention is a correspondingly upgraded microdosing device for the isolation of biological cells via individual liquid drops of a suspension of these cells released from the dosing channel of the microdosing device onto one or more target vessels, which contains the inventive device described herein.

• In einem ersten Schritt (1) wird die Abgabe jeweils eines einzelnen Flüssigkeitstropfens oder einer einzelnen Flüssigkeitsportion aus dem jeweiligen Dosierkanal der Mikrodosiereinrichtung in ein erstes Zielgefäß ausgelöst.

The device according to the invention is specifically suitable for carrying out an automatable or automatic method for cell isolation by means of a microdosing machine. Accordingly, another object of the invention is a method for the in particular fully automatic isolation of biological cells which are presented in a cell suspension by means of a microdosing device, the suspension of the cells being dispensed via at least one metering channel of the microdosing device in the form of individual liquid drops. The method according to the invention contains at least the following steps, which in a preferred embodiment are run through several times in one cycle:

• In a first step (1), the delivery of a single drop of liquid or a single portion of liquid from the respective dosing channel of the microdosing device into a first target vessel is triggered.

In a subsequent or simultaneously occurring second step (2), individual drops of liquid which have been released may be converted from this when irradiated Drops of liquid with light of a suitable wavelength, especially fluorescent light emitted by UV light, which according to the teaching of the invention should originate from a fluorescent cell, is detected. In a preferred embodiment, the cells of the cell suspension provided are each marked with a suitable fluorescent dye in a preceding step and the fluorescence of this fluorescent dye is excited and recorded in step (2). In an alternative variant, the wavelength of the light which stimulates the fluorescence is chosen such that autofluorescent structures of the cell are stimulated to fluoresce and this fluorescence is registered in step (2); a previous staining of the cells can then be omitted.

In a third step (3), one or more control signals are generated from the signal from the fluorescence detector by automatic evaluation. The control signal is suitable for triggering the delivery of a further drop of liquid or a further portion of liquid from this dosing channel or to replace the first target vessel on this dosing channel with a second or further target vessel, whereby the further dosing of liquid into the second or further target vessel will take place.

According to the invention, the criterion for generating the control signals is the sensor signal of the fluorescence detection registered with the detector when the liquid drop is irradiated with light, whereby in alternative (a) a sensor signal that is absent or is subliminal when the liquid drop is dispensed indicates the absence of a cell in this dispensed liquid drop, which leads to the generation of a control signal which triggers a further delivery of another drop of liquid from the metering channel. In alternative (b), a supra-threshold sensor signal registered when the liquid drop is dispensed is interpreted as that a cell is present in this liquid drop. As a result, a control signal is then generated, which triggers switching to a second or further target vessel before a further drop of liquid is released from the dosing channel.

In particular, it is provided that the threshold value of the amplitude of the sensor signal is determined in absolute terms on the basis of predetermined values or in a relatively self-adaptive manner using a drift-compensating band pass or high pass. An absolute or relative lower threshold value of a minimum fluorescence intensity and preferably also an absolute or relative upper threshold value of a maximum acceptable fluorescence intensity are provided. If the lower threshold value is not reached, this is assessed in such a way that no or no living cells are contained in the registered individual liquid drop or jet.

In a special variant, a further query is included, according to which, if a maximum threshold value is exceeded, which, depending on the specification, should lead to the signaling of incorrect dosing, which possibly triggers a further switching of the target vessel.

Carrying out the method with a microdosing device using a recurring dispensing of individual portions of liquid or drops of liquid, with each individual drop of liquid containing exactly one single cell on average or none, allows the entire cell suspension of the template to be delivered in small quantizations, each with a maximum of one cell on average contained, can be tested serially, whereby if a cell is present in a quantum, measures for cell isolation can be carried out. Since the number of cells in a dose corresponds to a distribution function, there is a high probability that a liquid drop will contain a maximum of one cell. No matter how low the cell concentration in the template is, there is always the theoretical possibility of having two or more cells in one dosed drop. The invention provides for the cell concentration in the suspension provided to be set in such a way that - with a given individual dosing amount and dosing frequency - within a permitted time window for the dosing and isolation process as a whole, the desired quality of the isolation, i.e. the permissible proportion of incorrect dosages with more than a cell in a target vessel is achieved. For a better quality of the separation, the aim is to minimize the probability that a metered drop contains more than one cell. A preferred variant of the method therefore provides that the concentration of the cells in the suspension of the template is set in such a way that the probability that a metered drop contains more than one cell is minimized. An optimized variant of the method provides that the concentration of the cells in the suspension of the template is set in such a way that each individual drop of liquid released from the metering channel contains on average 0.1 cells, preferably 0.01 cells, especially 0.001 cells. It is preferred that on average only every 10th, preferably only every 50th, only every 100th or only every 1000th dosed drop contains a cell. It is preferred that the ratio of the number of dosed drops containing exactly one cell and the number of drops containing two or at least two cells is 5: 1, preferably 10: 1, more preferably 100: 1 or greater. An alternative, preferred, optimized variant of the method provides that the concentration of the cells in the suspension of the template is set in such a way that each individual drop of liquid dispensed from the metering channel contains exactly one cell on average.

By using known microdosing devices for the recurring dosing of the smallest amounts of liquid at high frequency, the cell suspension can be serially and completely checked for the presence of cells in a very short time and these can then be sorted out and distributed. In a preferred embodiment, the sequence of steps (1), (2) and (3a) is therefore carried out at a high frequency of 1 Hz up to 600 Hz, preferably more than 10 Hz, more than 50 Hz, 100 Hz or more , especially from 100 to 400 Hz, is repeated, in which case up to about 600 individual liquid drops or portions are then dosed per second and each time examined for the presence of a cell.

• 1 zeigt schematisch den Prinzipaufbau der erfindungsgemäßen Zellvereinzelungsvorrichtung (10) an einer Mikrodosiereinrichtung (20) mit Dosierkanal (22). Die Vorrichtung weist eine Detektoreinheit (30), bestehend aus einer Lichtquelle (32) und einem Detektor oder Detektorfeld (36) auf. Detektor (36) ist über eine Signalverbindung (42) mit einer Auswerteeinheit (40) verbunden. Die Auswerteeinheit (40) ist wiederum über eine erste Signalleitung (44) mit einem Aktor (24) einer Mikrodosiereinrichtung (20) verbindbar. Gleichzeitig ist die Auswerteeinheit (40) über eine zweite Signalverbindung (46) mit einer Aktorik (64) verbunden, welche, im dargestellten Fall eine Mikrotiterplatte (62) mit einer Vielzahl von Zielgefäßen (60) trägt. Diese Platte (62) ist relativ gegenüber dem Dosierkanal (22) verschiebbar. Ein aus Dosierkanal (22) abgegebener Flüssigkeitstropfen (26) ist schematisch dargestellt zum Zeitpunkt des Passierens der Detektoreinheit (30). Der Flüssigkeitstropfen (26) wird in diesem Augenblick vom Licht (34) der UV-Lichtquelle (32) beleuchtet. Eine in dem Flüssigkeitstropfen (26) enthaltene Zelle (28) wird dadurch zur Fluoreszenz angeregt. Das Fluoreszenzlicht (35) kann mittels Detektor (36) registriert werden. Wird eine überschwellige Fluoreszenz detektiert, schaltet die Auswerteeinheit (40) das Zielgefäß (60) über die Aktorik (64) weiter, so dass jede weitere Dosierung aus Dosierkanal (22) in ein weiteres Zielgefäß (60) erfolgt. Wird bei Dosierung von Flüssigkeitstropfen (26) aus dem Dosierkanal (22) kein oder ein unterschwelliges Fluoreszenzsignal registriert, wird über Auswerteeinheit (40) und Signalverbindung (44) ein Aktor (24) der Mikrodosiereinrichtung (20) veranlasst, einen weiteren Flüssigkeitstropfen (26) aus dem Dosierkanal (22) abzugeben. Zellfreie Flüssigkeit abgegebener Flüssigkeitstropfen (26) wird von dem darunter liegenden Zielgefäß (60) aufgenommen, so lange bis eine erste und einzige Zelle (28) in dem Flüssigkeitstropfen (26) registriert wird, welche dann ebenfalls in dieses Zielgefäß (60) abgegeben wird. Danach wird das Zielgefäß (60) erneut weitergeschaltet.
• 2A und 2B zeigen Draufsichten auf die Detektoreinheit (30) der erfindungsgemäßen Vorrichtung in Blickrichtung der Dosierrichtung des Flüssigkeitstropfens (26). Anregungslicht einer externen UV-Lampe fällt hier über einen Lichtleiter (33) an den Messort. Von einer in den Flüssigkeitstropfen (26) vorhandenen Zelle (28) abgegebenes Fluoreszenzlicht fällt auf einen Lichtleiter (37), der mit einem externen Fluoreszenzdetektor verbunden ist. 2A zeigt eine einfache Variante mit einem einzigen Anregungslichtleiter (33) und einem einzigen Detektorlichtleiter (37). 2B zeigt eine Variante mit mehreren Anregungslichtleitern (33) zur optimalen Anregung der Fluoreszenz und mehreren Detektorlichtleitern (37) zur Registrierung von Fluoreszenzlicht in mehr als einer Raumrichtung.

The invention is explained in more detail by the following figures and examples:

• 1 shows schematically the basic structure of the cell separation device according to the invention ( 10 ) on a microdosing device ( 20th ) with dosing channel ( 22nd ). The device has a detector unit ( 30th ), consisting of a light source ( 32 ) and a detector or detector field ( 36 ) on. Detector ( 36 ) is via a signal connection ( 42 ) with an evaluation unit ( 40 ) connected. The evaluation unit ( 40 ) is in turn via a first signal line ( 44 ) with an actuator ( 24 ) a microdosing device ( 20th ) connectable. At the same time the evaluation unit ( 40 ) via a second signal connection ( 46 ) with an actuator ( 64 ), which, in the case shown, is a microtiter plate ( 62 ) with a variety of target vessels ( 60 ) wearing. This record ( 62 ) is relative to the dosing channel ( 22nd ) movable. On off dosing channel ( 22nd ) dispensed liquid drop ( 26th ) is shown schematically at the time of passing the detector unit ( 30th ). The drop of liquid ( 26th ) is at this moment from the light ( 34 ) the UV light source ( 32 ) illuminated. One in the drop of liquid ( 26th ) contained cell ( 28 ) is stimulated to fluorescence. The fluorescent light ( 35 ) can by means of a detector ( 36 ) can be registered. If fluorescence above the threshold is detected, the evaluation unit ( 40 ) the target vessel ( 60 ) via the actuators ( 64 ) so that every further dosing from dosing channel ( 22nd ) into another target vessel ( 60 ) he follows. Is used when dosing liquid drops ( 26th ) from the dosing channel ( 22nd ) no fluorescence signal or a subliminal fluorescence signal is registered, the evaluation unit ( 40 ) and signal connection ( 44 ) an actuator ( 24 ) the microdosing device ( 20th ) causes another drop of liquid ( 26th ) from the dosing channel ( 22nd ) to submit. Cell-free liquid released liquid droplets ( 26th ) is taken from the target vessel below ( 60 ) until a first and only cell ( 28 ) in the drop of liquid ( 26th ) is registered, which is then also transferred to this target vessel ( 60 ) is delivered. Then the target vessel ( 60 ) advanced again.
• 2A and 2 B show top views of the detector unit ( 30th ) of the device according to the invention in the viewing direction of the dosing direction of the liquid drop ( 26th ). Excitation light from an external UV lamp falls through a light guide ( 33 ) to the measurement location. From one in the liquid drop ( 26th ) existing cell ( 28 ) emitted fluorescent light falls on a light guide ( 37 ) connected to an external fluorescence detector. 2A shows a simple variant with a single excitation light guide ( 33 ) and a single detector light guide ( 37 ). 2 B shows a variant with several excitation light guides ( 33 ) for optimal excitation of the fluorescence and several detector light guides ( 37 ) for the registration of fluorescent light in more than one spatial direction.

Claims (9)

Device (10) for separating biological cells (28) on a microdosing device (20) via individual liquid drops (26) of a suspension of the cells (28) released from the dosing channel (22) of the microdosing device (20) onto a plurality of target vessels (60), containing: at least one detector unit (30) assigned to the metering channel (22) and arranged directly on it, containing several light sources (32) and several detectors (36) for detecting light-induced fluorescence, the detector unit (30) being arranged so that one from the Dosing channel (22) of dispensed individual liquid drops (26) can be illuminated by the light sources (32) and any fluorescent light emitted therefrom can be registered in at least two spatial directions on the detectors (36); Actuators (64) for step-by-step switching of the target vessels (60) relative to the metering channel (22); and programmed evaluation unit (40), connected via a signal connection (42) to the detectors (36) for the detector signal-related generation of control signals at a signal connection (44) to an actuator (24) for the recurring triggering of the dispensing of liquid drops (26) at the metering channel ( 22) of this microdosing device (20) and of control signals at a signal connection (46) to the actuator system (64) for triggering a step-by-step switching of the target vessels. Device according to Claim 1 , wherein an annular detector field of a detector (36) surrounding the liquid droplets (26) is assigned to the metering channel (22), so that from the Liquid droplets (26) emitted fluorescent light can be detected in several spatial directions. Device according to one of the preceding claims, wherein the detector (36) has several light guides (37) which are arranged on the metering channel (22) in such a way that fluorescent light (34) emitted by the liquid drop (26) is incident on these light guides (37) . Device according to one of the preceding claims, wherein the light source (32) has several light guides (33) which are arranged on the metering channel (22) in such a way that fluorescence-triggering light from the light guides (33) falls onto the liquid drop (26). Microdosing device (20) for the automatic isolation of biological cells (28) via individual liquid drops (26) of a suspension of the cells (28) released from a dosing channel (22) of this microdosing device (20) onto a plurality of target vessels (60) containing the device (10) ) according to one of the preceding claims. Method for the isolation of biological cells by means of a microdosing device with a dosing channel for the recurring delivery of individual liquid drops of a suspension of these cells to several target vessels, comprising the steps: (1) Triggering a delivery of a single drop of liquid from the dosing channel into a first target vessel, (2) irradiating the liquid drop with fluorescence-triggering light and detecting light-excited fluorescent light from the individual liquid drop in the detector unit (30) characterized in one of the preceding claims, (3) automatic generation of control signals from the signal of the detector unit (30) with the proviso, (a) that a missing or subliminal fluorescence signal indicates the absence of a cell in this liquid drop, and a further delivery of a single liquid drop from the metering channel is then triggered, or (b) that a supra-threshold fluorescence signal indicates the presence of a cell in this drop of liquid and then switching to a second or further target vessel is triggered before a further delivery of a single drop of liquid is triggered from the dosing channel. Procedure according to Claim 6 , the concentration of the cells in the suspension of the template being set so that on average only a maximum of every 10te of liquid drop dispensed from the metering channel contains one cell. Procedure according to Claim 6 or 7th , wherein the cycle from steps (1), (2) and (3a) of the repeated delivery of the liquid drop is run through at a repetition frequency of more than 50 Hz. Method according to one of the Claims 6 to 8th , wherein the cells are marked with a fluorescent dye and in step (2) the fluorescence of the fluorescent dye is excited and registered.

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