WO2024046548A1

WO2024046548A1 - Automated well unit, computer implemented method of automated perfusing and washout of solutions, non-transitory computer-readable storage medium encoded with a computer program, and automated screening platform for screening biological samples

Info

Publication number: WO2024046548A1
Application number: PCT/EP2022/074126
Authority: WO
Inventors: Adriana NAGY- DABACAN; Vasile-vlad MOCA; Raul-Cristian Muresan; Harald BARZAN
Original assignee: Asociatia Transylvanian Institute of Neuroscience
Priority date: 2022-08-30
Filing date: 2022-08-30
Publication date: 2024-03-07

Abstract

This invention is directed to an automated well unit comprising rows; each row comprising wells; each well adapted for accommodating biological samples, provided with a row inlet and a row outlet; each well provided with a well inlet and a well outlet; one perfusing pump connected to the row inlets configured to pump in a new solution through the row inlets; a plurality of inlet tubes, each inlet tube connecting each row inlet with corresponding well inlets, a plurality of inlet filters, each inlet filter covering a corresponding well inlet; one washout pump connected to the row outlets, configured to pump out an existing solution from the row outlets; a plurality of outlet tubes, each outlet tube connecting each row outlet with corresponding well outlets; a plurality of outlet filters, each outlet filter covering a corresponding well outlet; a well control module, electronically and mechanically connected to the perfusing pump and to the washout pump, being configured to send specific perfusing instructions to the perfusing pump, and corresponding specific washout instructions the washout pump; means for electronically and mechanically connection to an observation unit of a screening platform, and means for electronically and mechanically connection to a global control unit of said screening platform. The automated well unit is configured to operate the automated perfusing of the new solution into the wells and the corresponding automated washout of the existing solution from said wells. This invention is further directed to a computer implemented method of automated perfusing and washout of solutions carried out by the automated well unit; to a non-transitory computer-readable storage medium encoded with a computer program and to an automated screening platform comprising the automated well unit, an observation unit, and a global control unit, configured to carry out screening of the biological samples.

Description

Automated well unit, computer implemented method of automated perfusing and washout of solutions, non-transitory computer-readable storage medium encoded with a computer program, and automated screening platform for screening biological samples

Field of the invention

The invention is related to automated systems and methods for screening biological samples. In particular, the invention is related to an automated well unit, a computer implemented method of automated perfusing and washout of solutions, a non-transitory computer-readable storage medium encoded with a computer program, and an automated screening platform for screening biological samples comprising the automated well unit.

The projects leading to this patent application have received funding from the European Research Council (ERC) and European Research Executive Agency (REA) under the European Union’s Horizon 2020 research and innovation programme (grant agreements No. 899262 and 952096).

Terms used in this invention

“Drugs”, alternatively called “candidate drugs” - are interchangeable terms used to designate candidate substances for use in human and animal medicine that need testing on biological samples before releasing them on the market, said testing being carried out by using a screening platform.

"Biological samples”, can be cellular cultures, organ slices or fragments, oocytes or small living animals in all stages of development that can be studied using well plates of various dimensions and characteristics.

“Screening of the biological samples”- an ensemble of processes carried out for the purpose of assessing the impact of the drugs on said biological samples.

“Zebrafish model larvae”, alternatively called zebrafish model of disease - a group of zebrafish larvae grouped by models of disease to be studied, e.g. neurological diseases. In the large majority of cases zebrafish larvae and not zebrafish adults are used for testing candidate drugs on them.

“Screening of the zebrafish model larvae” - an ensemble of processes carried out on the zebrafish model larvae, aiming at phenotyping features in said zebrafish model of disease, and assessing the impact of the drugs on said zebrafish model of disease.

“User” - the person using the screening platform carrying out the screening of the biological samples, typically a researcher or a team or researchers. “Fluids”, alternatively called “solutions”- solutions adapted for the life of the biological samples, with or without the drugs.

Background of the invention

In the past few years, the importance and need for accurate screening of the biological samples have increased considerably, therefor various screening platforms have been developed.

A typical screening platform has three units, with reference to Fig. 1 :

- A well unit A comprising multi-well plates filled with biological samples and a fluid of choice.

- An observation unit B including a motion tracking module, the motion tracking module including a motion tracking recording sub-module for recording the results of the tracking,

- A global control unit C comprising a control module of the ambient parameters - such as temperature, humidity and illumination; a stimulation controller including means for stimulation of the biological samples such as sound or vibration stimulation, light stimulation, electrical stimulation.

The delivery of the fluids in the state of the art is carried out via pipettes, either manually or via a robot acting on the pipettes. In order to be relevant, the experiments need a reference situation when there is no drug in the solution, thus the experiments typically start with delivering only the solution. The drugs are added at a later stage, reason for which the solution and the drugs are administered separately. The washout of the drugs in the state of the art is carried out by taking out the biological samples and, after that, by taking out the fluid via pipettes.

During an ongoing experiment, it is often needed to make changes related either to the drug or the parameters of the drug delivery or both.

For any changes related to the drug during the ongoing experiment, i.e. change of concentration level, change of drug, when using the screening platforms of the prior art, the major disadvantage is that the user has to interrupt the screening of the biological samples, to take out the multi-well plates and the biological samples, to replace the fluids, to put back the biological samples, to recalibrate manually the recordings sub-modules, and to resume the screening of the biological samples.

Disadvantages of prior art

The major disadvantage of interruption and resuming of the screening process leads to specific disadvantages such as but not limited to:

- the biological samples are disturbed by the operations of taking them out and putting them in again, the consequence of the stress being that their behavior is altered in respect to what is considered normal behavior, this is in particular relevant when the biological samples are living organisms, such as the zebrafish larvae,

- significant loss of time by multiple manipulation related to the taking out the multi-well plates, etc.; additionally, this loss of time cannot be estimated. Knowing that the lifetime of some biological samples such as the zebrafish larvae is short, the disadvantage of losing time is even bigger,

- loss of accuracy of the results due to multiple manipulations related to the taking out the multi-well plates etc., with each recalibration of the recordings sub-modules, the more frequently the recalibration is needed, the less accurate the results are,

- the recordings can be made only when the user is present during usual working hours, which leads to the impossibility to make remotely-controlled recordings and adjustments as well as the impossibility to make recordings round the clock,

- full automatization of the screening of the biological samples is not possible, because the delivery stage is not automated, requiring human intervention,

- the recordings can concern only the time between two successive changes, thus longterm screening and generalization of the screening results are not possible,

- the high potential of some types of short-lived biological samples, in particular of living organisms such as the zebrafish larvae is curtailed due to the impossibility to change fluids without removing and putting again the biological samples,

- high costs for at least two reasons: multiple manipulations require full-time employed personnel and, due to multiple manipulations, many experiments fail, thus the total number of experiments required to reach valid research conclusions is large.

Problem solved by the invention

The problem to be solved by the invention is to provide automated delivery of the solutions and automated washout of same for the screening platforms used to carry out screening biological samples, to avoid interrupting the screening of the biological samples for taking out the multi-well plates and the biological samples, replacing the fluids, putting back the biological samples, recalibrating manually the recording sub-modules after each solution change. Summary of the invention

In order to solve the problem, the inventors conceived in a first aspect of the invention, an automated well unit comprising:

- one or more rows, each row comprising wells,

- each well adapted for accommodating biological samples,

- each row provided with a row inlet and a row outlet,

- each well provided with a well inlet and a well outlet,

- one perfusing pump connected to the row inlets, the perfusing pump configured to pump in a new solution through the row inlets,

- a plurality of inlet tubes, each inlet tube connecting each row inlet with corresponding well inlets,

- a plurality of inlet filters, each inlet filter covering a corresponding well inlet,

- one washout pump, connected to the row outlets, the washout pump configured to pump out an existing solution from the row outlets,

- a plurality of outlet tubes, each outlet tube connecting each row outlet with corresponding well outlets,

- a plurality of outlet filters, each outlet filter covering a corresponding well outlet,

- a well control module, electronically and mechanically connected to the perfusing pump, and to the washout pump, the well control module being configured to send specific perfusing instructions to the perfusing pump, and corresponding specific washout instructions the washout pump,

- means for electronic and mechanic connection to an observation unit of a screening platform,

- means for electronic and mechanic connection to a global control unit of said screening platform.

The automated well unit is configured to operate the automated perfusing of the new solution into the wells and the corresponding automated washout of the existing solution from said wells, according to specific perfusing instructions and, respectively specific washout instructions from the well control module.

In a second aspect of the invention, it is provided a computer implemented method of automated perfusing and washout of solutions carried out by the automated well unit of any preferred embodiment, the method comprising the following steps:

- perfusing by the perfusing pump the new solution into the wells, upon receipt of specific perfusing instructions from the well control module, -washing out by the washout pump the existing solution from the wells, upon receipt of specific washout instructions from the well control module.

In a third aspect of the invention, it is provided non-transitory computer-readable storage medium encoded with a computer program, the computer program stored in the well control module, and the computer program comprising specific perfusing instructions and specific washout instructions executable by the automated well unit of any preferred embodiment, which, upon such execution by the automated well unit, causes the automated perfusing of the new solution into the wells and the corresponding automated washout of the existing solution from said wells.

In a fourth aspect of the invention, it is provided an automated screening platform comprising the automated well unit of any preferred embodiment, an observation unit, and a global control unit, wherein the automated screening platform is configured to carry out screening of the biological samples.

Advantages of the invention

The main advantage of the invention is to allow the user to make changes during the ongoing experiment without taking out the multi-well plates and the biological samples and without recalibrating manually the recordings sub-modules.

The main advantage leads to specific advantages such as but not limited to:

- when the biological samples are living organisms, they are not anymore stressed by the operations of taking them out and putting them in again,

- significant time is gained by the reduction of manipulation operations,

- an increase of accuracy of the results because there is no interruption of the experiment and multiple manual recalibrations of the recording sub-modules are no longer necessary,

- the possibility to view and record in real time the values of the relevant parameters, the possibility to work round the clock without the user near the screening platforms; the possibility to make remotely-controlled recordings and adjustments, to show statistics of the values of said relevant parameters during the entire time of the experiment,

- automatization of the entire experiment of screening of the biological samples is possible; allowing the user to intervene more often in the ongoing experiment and to broaden the type of decisions, for example allowing go/no go decisions depending on response thresholds,

- the recordings concern the entire duration of the experiment, making possible generalization of the screening results as the screening results correspond to a significantly larger uninterrupted period of time,

- does not curtail the potential of the short-lived biological samples such as the zebrafish at the larval stages as a model organism for high-throughput screening,

- less costs resulting from less need of personnel and less failed experiments,

- one significant step towards modularity of the screening platforms because the well unit of the invention can be adapted to function together with any observation units and/or control units.

Brief description of the drawings

Fig. 1 Schematic representation of the screening platform of the state of the art

Fig. 2a Schematic representation of the automated well unit according to the invention - view from above.

Fig. 2b Schematic representation of the automated well unit according to the invention - vertical cross-section

Fig. 3a Schematic representation of the first example - view from above

Fig. 3b Schematic representation of the first example - cross section

Fig. 3c Schematic representation of the first example - isometric projection

Fig. 4a Schematic representation of the second example - view from above

Fig. 4b Schematic representation of the second example - cross section

Fig. 4c Schematic representation of the second example - isometric projection

Fig. 5a Schematic representation of the third example - view from above

Fig. 5b Schematic representation of the third example - cross section

Fig. 5c Schematic representation of the third example - isometric projection

Fig. 6a Schematic representation of the fourth example - view from above

Fig. 6b Schematic representation of the fourth example - cross section

Fig. 6c Schematic representation of the fourth example - isometric projection

Fig. 6d Schematic representation of one well of the fourth example

Fig. 7a Schematic representation of the one-piece detachable filter - isometric projection

Fig. 7b Schematic representation of the one-piece detachable filter - side view

Fig. 7c Schematic representation of the one-piece detachable filter -view from above

Fig. 8 Schematic representation of the screening platform of the invention List of references in the drawings:

Screening platform from the state of the art

A Well unit

B Observation unit

C Global control unit

Automated screening platform of the invention

11 Automated well unit

11 1 rows of wells

11 11 row inlet

11 12 row outlet

11 13 wells

11131 well inlet

11132 well outlet

11133 well evaporation sensors

113 perfusing pump

114 washout pump

115 inlet tubes

116 outlet tubes

115-116 inlet-outlet tube

117 filters

1171 inlet filters

1172 outlet filters

1173 one-piece detachable filter

118 well control module

12 Observation unit

13 Global control unit

Detailed description

In a first aspect of the invention is it disclosed an automated well unit 11 adapted for perfusing a new solution and washing out an existing solution during an experiment, said experiment comprising various stages that require changes of the existing solution with the new solution. The components of the automated well unit 11 are illustrated schematically with reference to Fig. 2a and Fig. 2b, whereas Fig. 3 to Fig.7 illustrate various non-limiting alternative examples of realization of said automated well unit 11 or of its components.

With reference to Fig. 2, the automated well unit 11 comprises the following components:

- one or more rows 111 , each row 1 11 comprising wells 1113,

- one perfusing pump 113,

- a plurality of inlet tubes 115,

- a plurality of inlet filters 1171 ,

- one washout pump 1 14,

- a plurality of outlet tubes 116,

- a plurality of outlet filters 1172,

- a well control module 118.

The number of rows 111 of the automated well unit 1 1 depends on each experiment.

Each row 1 11 comprises the wells 1113, said wells 1 113 arranged such that the new solution and, respectively the existing solution be maintained at the same level in all wells 11 13 of the same row 111.

Each well 1113 is adapted for accommodating biological samples. The shape, the volume and the design of the automated well unit 11 respond to the requirements ensuring adequate vital conditions of the biological samples during the experiment and ensuring the laminar flow of the new and of the old solution.

The number, size and shape of the wells 1 113 of the same row 1 11 depend on each experiment. The preferred shape of the wells 1113 is rectangular cuboid for easy manufacturing and manipulating reasons. The wells 1 113, each well 1113 having a well bottom side, and a well upper side, the well upper side being open.

Each row 111 is provided with a row inlet 1111 and with a row outlet 1112.

Each well 11 13 is provided with a well inlet 11 131 and with a well outlet 1 1132.

The perfusing pump 1 13 is connected to the row inlets 1111 , being configured to pump in the new solution through the row inlets 11 11.

One row or more rows 111 can be connected to the same perfusing pump 113. In case the experiment requires the same changes of the solution for all rows 111 , then all rows 111 are connected to the same perfusing pump 113. On the contrary, if the experiment requires different solutions for each row 1 11 , each row 11 1 is connected to a different perfusing pump 113.

Each inlet tube 115 of the plurality of inlet tubes 115 connects each row inlet 1111 with corresponding well inlets 11131 .

One inlet tube 115 connects one or more well inlets 1 1131 to each row inlet 11 11. The row inlets 111 1 , the plurality of inlet tubes 1 15, and the corresponding well inlets 11131 , are configured for allowing the new solution to be perfused into the wells 1113.

The well outlets 11132, the plurality of outlet tubes 116 and the row outlets 11 12 are configured for allowing the existing solution to be washed out from said wells 11 13.

In some preferred embodiments, the neighboring wells 1113 communicate between themselves- as shown in Fig. 3a, Fig. 3b, Fig. 3c. There is a continuity between the inlet tubes 115 and the outlet tubes 116 between said neighboring wells 1113. One inlet tube 115 connects with one outlet tube 116 creating an inlet-outlet tube 115-116. The wells 1113 that are communicating have the advantage of simpler and cheaper design and shorter inletoutlet tubes 115-116. These embodiments are advantageous for example when the biological samples of one well 1113 are not perturbed by the traces of the biological samples from other wells 1113.

In other preferred embodiments, the neighboring wells 1 113 do not communicate between themselves, as shown in Figs. 4a to 6d. The inlet tubes 1 15 and the outlet tubes 116 are separated from one another. These embodiments are advantageous for example when the biological samples of one well 1113 are perturbed by the traces of the biological samples from other wells 11 13.

The shapes and the sizes of the cross-section, the total length of the inlet tubes 115, are designed to ensure the distribution of the new solution according to two basic principles:

- distribution of the new solution as a single-phase fluid flow into flow channels uniformly, the pressure of perfusing the new solution being equal through all well inlets 11131 that are connected by the same inlet tube 115 to said row inlet 1111 ,

- efficient and rapid replacement of the existing solution within the wells with the new solution, the new solution being perfused, at low speeds and pressures, that are compatible with biological samples.

The shapes and the sizes of the cross-section, as well as total length of the outlet tubes 116 are dimensioned corresponding to the shape and the size of the cross-section, as well as the total length of the corresponding inlet tubes 115 in order to ensure the reverse action of washout of the existing solution according to the same principles mentioned above.

The distribution of the new solution from the row inlets 1111 to the well inlets 11131 is accomplished through bifurcation of the inlet tubes 115 that symmetrically and uniformly split the upstream flow of the new solution from the row inlets 111 1 into one or more tiers of downstream flows until the flow of the new solution reaches the well inlets 11 131 .

The same mechanism of distribution applies to the washout of the existing solution, through bifurcation of the outlet tubes 116 that symmetrically and uniformly collect one or more tiers of downstream flows of the existing solution from the well outlets 1132 to the upstream flow of the existing solution until said existing solution reaches the row outlets 11 12.

The bifurcation of the outlet tubes 116 mirrors the bifurcation of the inlet tubes 115.

If needed, hydraulic distributors are used to symmetrically and uniformly split the upstream flow of the new solution into the tiers of downstream flows and, correspondingly symmetrically and uniformly collect the downstream flows to the upstream flow of the existing solution.

The positioning, the angle of the bifurcations and the form and size of the cross section of the inlet tubes 115 and of the corresponding outlet tubes 1 16 are designed considering the viscosity inertia, gravity, and surface tensions of the new solution and of the existing solution.

The examples depicted in Figs. 2a to 6c showing one inlet tube 115 connecting two well inlets 11131 shall not limit the invention to the content of the figures.

Given the small dimension of the biological samples, they could escape from the wells 113 through well inlets or outlets. The plurality of inlet filters 1171 is configured to prevent the biological samples to escape from the wells 1113. Each inlet filter 1171 covers a corresponding well inlet 11131 , the size and configuration of said inlet filter 1171 being adapted to the size of the biological samples.

The washout pump 114 is connected to the row outlets 1112. The washout pump 114 is configured to pump out the existing solution from the row outlets 1112.

Just like in the case of the perfusing pump 113, one row or more rows 1 11 can be connected to the same washout pump 114.

The number of washout pumps 114 is equal to the number of perfusing pumps 113.

Each outlet tube 116 of the plurality of outlet tubes 1 16 connects each row outlet 1112 with corresponding well outlets 1 1132.

One outlet tube 116 can connect one or more well outlets 11 132 to each row outlet 1112. The shape of the outlet tube 116 as well as the number of the well outlets 11132 are such that to ensure that the pressure of washing out the existing solution is equal through all well outlets 11 132 that are connected by the same outlet tube 116 to said row outlet 1 112. The examples depicted in Fig. 2a to 6c showing the outlet tube 116 connecting two well outlets 11 132 shall not limit the invention to the content of the figures.

The plurality of outlet filters 1172 is configured to prevent the biological samples to escape from the wells 11 13. Each outlet filter 1172 covers a corresponding well outlet 11 132, the size and configuration of said outlet filters 1172 being adapted to the size of the biological samples.

The well control module 118 is electronically and mechanically connected to the perfusing pump 113 and to the washout pump 114, for controlling the perfusing pump 113 and the washout pump 114. The well control module 118 is configured to send specific perfusing instructions to the perfusing pump 113 and corresponding specific washout instructions to the washout pump 1 14, said specific perfusing instructions and said corresponding specific washout instructions based on observation data received from the observation unit 12 and/or based on control data received from the global control unit 13.

The well control module 118 is configured to control either a single perfusing pump 113 with its corresponding washout pump 114 or more perfusing pumps 113 with their corresponding washout pumps 1 14.

The row outlets 1 112, and, respectively, the row inlets 1111 are holes whose respective cross-sections depend on the washout volume, and, respectively, on the perfusion volume. The shape of the cross-section of the row outlets 1 112 and row inlets 111 1 is, for example, circular or square.

The well inlets 11 131 and the well outlets 11 132 are holes whose respective crosssections, and inclination of the axes in respect to the vertical and their distance in respect to the wells bottom side or to the wells upper side are calculated depending depend on parameters such as: volume and pressure of the new, respectively existing solution. The cross-section of the well inlets 11131 well outlets 11132 is, for example, circular or square. For example, the diameter is chosen in order to have allow enough fluid volume change without inducing very high flow values which might disturb the biologic samples, the inclination is chosen in order to ensure balanced perfusion of wells by laminar flow and the inlet/outlet position is chosen such as to ensure optimal fluid exchange during the perfusion/washout operations.

The automated well unit 11 may comprise an illumination means from below, for example a LED strip surface, not represented in the figures.

The automated well unit 11 also comprises means for electronic and mechanical connection to an observation unit 12 and means for electronic and mechanical connection to a global control unit 13 of a screening platform, schematically represented in Fig. 8. The observation unit 12 and the global control unit 13 are connected via electrical connectors and cables with the automated well unit 11 in order to ensure appropriate electrical connectivity.

The means for electronically and mechanically connecting to the observation unit 12 and to the global control unit 13 include adaptations of the size and shape of the components of the automated well unit 11 to the mechanical and space constraints of the observation unit 12 and of the global control unit 13.

The automated well unit 11 is configured to operate the automated perfusing of the new solution into the wells 1113 and the corresponding automated washout of the existing solution from said wells 1113, according to specific perfusing instructions and, respectively specific washout instructions from the well control module 118.

One particular example of biological samples are zebrafish.

Zebrafish is emerging as one of the most versatile organisms for functional studies and high-throughput screening in translational studies as diverse as cancer, cardiovascular and neurological research.

The advantage of zebrafish lies in its high genetic homology to humans, comparable to traditional models² (zebrafish: 75%; mouse: 85%), combined with the flexibility of simple animal models: large number of progeny, rapid development, stereotyped behavior, simplified neuronal architecture and ease of genetic modification.

Due to their intrinsic advantages, zebrafish larvae are used for testing candidate drugs alone or combined.

In order to carry out said testing, the zebrafish larvae are grouped by models of disease to be studied, i.e. neurological diseases, creating zebrafish models of disease. For each zebrafish model of disease, there are two groups of activities to be carry out: i) phenotyping features in said zebrafish model of disease, and ii) assessing the impact of the drugs on said zebrafish model of disease.

The phenotyping of the features is carried out by analyzing one or more phenotypic parameters. One of the most frequently used phenotypic parameters is the motor phenotype.

Currently, a repertoire of transgenic reporter lines is available for the live monitoring of major cellular cascades, such as apoptosis³ and autophagy⁴, as well as specific signaling developmental pathways such as the TGF , Notch, Bmp, Wnt and Shh pathways⁵, to name just a few.

Apart from the general advantages of the invention, for the experiments where the biological samples are zebrafish larvae, the automated delivery and washout of the respective fish fluids has the advantage that it extends the actual time of the in vivo imaging of said cellular processes as compared with prior art, which enhances the reliability of the results of the in vivo imaging of said cellular processes.

In a preferred embodiment, the automated well unit 1 of the invention is adapted for accommodating zebrafish larvae. The specific adaptations are as follows:

- each well 11 13 is adapted for accommodating zebrafish model larvae,

- the plurality of inlet filters 1171 and the plurality of outlet filters 1172 are configured to prevent the zebrafish model larvae to escape from the wells 1 113,

- the new solution is a new fish solution and the existing solution is an existing fish solution.

For this purpose, the automated well unit 1 of this preferred embodiment comprises:

- the one or more rows 111 , each row 111 comprising the wells 1 113,

- each well 11 13 adapted for accommodating the zebrafish model larvae,

- each row 111 provided with the row inlet 1111 and the row outlet 1 112,

- each well 11 13 provided with the well inlet 11131 and the well outlet 11132,

- the perfusing pump 113 connected to the row inlets 1111 , the perfusing pump 113 configured to pump in the new fish solution through the row inlets 1111 ,

- the plurality of inlet tubes 115, each inlet tube 115 connecting each row inlet 1 111 with the corresponding well inlets 11131 ,

- the plurality of inlet filters 1171 configured to prevent the zebrafish model larvae to escape from the wells 1113, each inlet filter 1171 covering the corresponding well inlet 11 131 ,

- the washout pump 114, connected to the row outlets 1 112, the washout pump 114 configured to pump out the existing fish solution from the row outlets 1112,

- the plurality of outlet tubes 116, each outlet tube 116 connecting each row outlet 1 112 with the corresponding well outlets 11132,

- the plurality of outlet filters 1172 configured to prevent the zebrafish model larvae to escape from the wells 1113, each outlet filter 1172 covering the corresponding well outlet 11 132,

- the well control module 118, electronically and mechanically connected to the perfusing pump 113 and to the washout pump 114, the well control module 118 being configured to send the specific perfusing instructions to the perfusing pump 113 and the corresponding specific washout instructions the washout pump 114,

- the means for electronically and mechanically connection to an observation unit 12 of a screening platform, - the means for electronically and mechanically connection to a global control unit 13 of said screening platform.

In another preferred embodiment, used irrespective of the nature of the biological sample, the automated well unit 11 is configured for perfusing a volume of new solution equal to the volume of the existing solution washed out.

This is ensured by equaling the washout volume to the perfusion volume. In this case the specific perfusing instruction is sent simultaneously with the specific washout instruction.

This embodiment has the advantage of simplicity and efficiency, being used in ambiences where the evaporation of the existing solution does not exceed an evaporation threshold, thus neither the experiment nor biological samples are put in danger.

In an alternative preferred embodiment, advantageous for ambientes where the evaporation of the existing solution exceeds the evaporation threshold and the value of the evaporation can be estimated, the automated well unit 11 is configured for perfusing a volume of new solution equal to the volume of the existing solution washed out times a predetermined evaporation coefficient.

The predetermined evaporation coefficient is computed by the user knowing the temperature, humidity of the ambient and duration of the experiment on the one hand and knowing the volume of each well 1113 on the other hand, to avoid over-perfusing of said each well 1 113.

This embodiment has the advantage of allowing optimum conditions for the biological samples by avoiding evaporation of the existing solution.

In an alternative preferred embodiment, advantageous for ambientes where the evaporation of the existing solution exceeds the evaporation threshold, but either it is difficult to estimate the volume of the evaporation or said volume can vary a lot from one experiment to another, or from one ambient to another, the automated well unit 11 further comprises an evaporation sub-module (not represented graphically) of the well control module 118, and a plurality of evaporation sensors 1 1133. Said evaporation sub-module is in connection with the plurality of evaporation sensors 11133, each evaporation sensor 1 1133 placed in a corresponding well 11 13 configured to sense a level of the existing solution, such as depicted for example in Fig.7.

The plurality of evaporation sensors 11 133 is configured to send to the evaporation submodule 1181 a signal when the level of the existing solution is below the evaporation threshold.

The evaporation sub-modules are configured to calculate, upon receipt of the signal, an evaporation replacement new solution volume, and are configured to trigger a perfusion instruction to the perfusing pump 113 for said evaporation replacement new solution volume.

The perfusing pump is configured to perfuse upon receiving the perfusion instruction said evaporation replacement new solution volume.

This embodiment has the advantage of allowing optimum conditions for each individual experiment for the biological samples by avoiding evaporation of the existing solution.

In other preferred embodiments, the plurality of inlet filters 1171 of the same row 111 of the automated well unit 11 is connected to the corresponding plurality of outlet filters 1172 such that to form a one-piece detachable filter 1173.

In one of the preferred examples of realization depicted in Fig. 7, the plurality of inlet filters 1171 and, correspondingly the plurality of outlet filters 1172 have the shape of two connected opposed blades. Each blade covers one lateral side of the wells 1113 that include the respective filters: the inlet filters 1171 on one side of the wells 1 113, and the outlet filters 1172 on the opposed side of the wells 1113.

The connection of the plurality of inlet filters 1171 to the plurality of outlet filters 1172 is preferably done at the wells upper side.

The preferred embodiments when the automated well unit 11 is adapted for accommodating zebrafish larvae can be combined with any one or more from the preferred embodiments as follows:

- with the embodiments teaching the configurations for perfusing a volume of new solution equal to the volume of the existing solution washed out,

- with the embodiments teaching the configurations for perfusing the volume of new solution equal to the volume of the existing solution washed out times the predetermined evaporation coefficient,

- with the embodiments teaching the configurations to further comprise the evaporation sub-module of the well control module 1 18, said evaporation sub-module in connection with the plurality of evaporation sensors 11133,

- with the embodiments that use either communicating or non-communicating neighboring wells 1113 of the same row 111 ,

- with the embodiments that use the one-piece detachable filter 1 173.

Description of the examples of realization

All the examples of realization refer to the automated well unit 1 of the invention adapted for accommodating the zebrafish larvae. Example 1 of realization

With reference to Fig. 3a, Fig. 3b, Fig. 3c, in the first example of realization, the automated well unit 1 1 comprises a single row 11 1 , said row 111 comprising 4 wells 11 13.

The automated well unit 11 comprises 5 inlet-outlet tubes 115-116, out of which one inlet-outlet tube 115-116 connects the row inlet 11 11 with the first well 1 113, three inletoutlet tubes 115-116 connect the 4 wells 1113 two by two among themselves, and the last inlet-outlet tube 115-1 16 connects the row outlet 1 112 with the last well 1113. The fish solutions pass from one well 1113 to another well 1113.

Both the perfusing pump 113 and the washout pump 114 are placed at the wells upper side, being connected to the inlet-outlet tube 115-116 by means of the row inlet 111 1 and the row outlet 1112, respectively.

The well inlets 11131 and the well outlets 11132 are placed at the same height in respect to a horizontal surface.

The well inlets 11131 and the well outlets 11132 are cylindrical holes.

This example of realization has the advantages of simplicity, less tubing, easy access to the row inlet 1111 and the row outlet 1112. The first example of realization also provides the advantage to maintain the balance of the fish solution level in all the wells 11 13 by virtue of the principle of communicating vessels.

Examples 2 and 3 of realization

Example 2 - as depicted in Fig. 4a, Fig. 4b, Fig. 4c, and example 3- as depicted Fig. 5a, Fig. 5b, Fig. 5c have some common features.

Common features of examples 2 and 3

The automated well unit 11 has a single row 111 , said row 111 comprising 4 wells 1113.

The row inlet 1111 and the inlet tubes 115 are placed in diagonal in respect to the washout pump 1 14, the row outlet 1112 and the outlet tubes 1 16 with respect to the horizontal axis of symmetry of the row 1 11.

The neighboring wells 1113 do not communicate between themselves.

The balance of the fish solution level in the wells 11 13 is ensured by a bifurcated inlet tube 115 and a bifurcated outlet tube 116. The 4 wells 11 13 are grouped into 2 groups of 2 wells, the wells 1113 inside each group of wells 1113 connected by two inlet tubes 115 of a downstream tier. Another inlet tube 1 15 of an upstream tier, connects the two inlet tubes 115 of the downstream tier, creating the bifurcated inlet tube 115 connecting the 4 wells 11 13 of each group. The same configuration applies to the outlet tubes 116, namely 2 outlet tubes 1 16 of first tier, an outlet tube 116 of second tier, composing together the bifurcated outlet tube 116.

The second and third example of realization have the common advantages of simplicity, easy access to the row inlet 1111 and to the row outlet 1112 as well as not stressing the zebrafish larvae by sensing traces of other zebrafish larvae from other wells 1113.

Specific features of example 2

In the second example, the angle of the bifurcated inlet tube 115, respectively the bifurcated outlet tube is substantially 90°. The inlet tubes 115 and the outlet tubes 116 are square in cross-section.

The well inlets 11131 and the well outlets 11132 are placed at the same height in respect to said horizontal surface.

Specific features of example 3

In the third example, the angle of the bifurcated inlet tube 115, respectively the bifurcated outlet tube is >90°. The inlet tubes 115 and the outlet tubes 1 16 are circular in cross-section and the circular in cross-section, and the obtuse angle ensures laminar flow of the fish fluids by preventing the formation of air bubbles.

The well inlets 11131 are placed at a higher height than the well outlets 11132 in respect to said horizontal surface. The well inlets 11131 are closer to the wells upper side, whereas the well outlets 11132 are closer to the wells bottom side. The inlet tubes 115 and the outlet tubes 1 16 are circular in cross-section.

The well inlets 1 1131 and the well outlets 11132 are square holes, whose inclination in respect to the horizontal surface is an acute angle.

Example 4 of realization

With reference to Fig. 6a, Fig. 6b, Fig. 6c, in this example of realization, the automated well unit 11 comprises 4 rows 111 , each row 111 comprising 8 wells 1113.

Each row 111 is provided with its own perfusing pump 113 with its corresponding row inlet 111 1 and, correspondingly, each row 1 11 is provide with its own washout pump 114 with its respective row outlet 1 112.

The row inlets 1 11 1 are located at the wells upper side, and the row outlets 1112 located at the wells bottom side.

The neighboring wells 1113 do not communicate between themselves.

The balance of the fish solution level in the wells 11 13 is ensured by a bifurcated inlet tube 115 and a bifurcated outlet tube 116. The 8 wells 11 13 are grouped into 4 groups of 2 wells, the wells 1113 inside each group of wells 11 13 connected by 4 inlet tubes 115 of first downstream tier. Two inlet tubes 115 of second downstream tier connects the two neighboring groups of wells 1 113 of row inlet 1111 , whereas one inlet tube 115 of the upstream tier connects the two inlet tubes 115 of second tier, the inlet tubes 115 of first, second and third tier composing together the bifurcated inlet tube 115.

The same configuration applies to the outlet tubes 116, namely 4 outlet tubes 1 16 of first tier, 2 outlet tubes 116 of second tier, and one outlet tube 116 of first tier, all composing the bifurcated outlet tube 116.

The inlet tubes 115 and the outlet tubes 116 are circular in cross-section.

The angle of the bifurcated inlet tube 1 15, respectively the bifurcated outlet tube is substantially 90°.

The well inlets 11131 are placed at a higher height than the well outlets 11132 in respect to said horizontal surface. The well inlets 11131 are closer to the wells upper side, whereas the well outlets 1 1132 are closer to the wells bottom side.

The well inlets 1 1131 and the well outlets 11132 are cylindrical holes, whose inclination in respect to the horizontal surface is an acute angle.

As shown in Fig. 6d, the diagonal position of the well inlets 11 131 and the well outlets 11 132 on the opposite walls of the wells 1 113 is such that to ensure the longest trajectory possible for the fluids from the well inlets 1 1131 to the well outlets 11 132, for the purpose of ensuring the best conditions for the full replacement of the existing solution with the new solution.

The inlet filters 1171 and the outlet filters 1172 are represented only in one of the rows 11 1 for simplicity reasons. This example of realization uses the one-piece detachable filter 1173.

The fourth example of realization has the advantage of permitting more observation to be carried out in respect to the preceding examples of realization, while maintaining easy access to the row inlet 111 1 and to the row outlet 1112, as well as the advantage of not stressing the zebrafish larvae by sensing traces of other zebrafish larvae from other wells 11 13.

- Perfusing by the perfusing pump 113 the new solution into the wells 1113, upon receipt of specific perfusing instructions from the well control module 1 18,

- Washing out by the washout pump 114 the existing solution from the wells 1113, upon receipt of specific washout instructions from the well control module 118.

The perfusing steps can be either simultaneous or not simultaneous with the washout steps.

Before the experiment starts, the new solution is perfused into the automated well unit upon receipt of specific perfusing instructions from the well control module 118. As there is no existing solution at this stage, there is no specific washout instruction.

When the experiment ends, the existing solution is washed out from the wells 1113, upon receipt of specific washout instructions from the well control module 118. As there is no new solution to be pumped in, there is no specific perfusing instruction.

In a third aspect of the invention it is provided a non-transitory computer-readable storage medium encoded with a computer program, the computer program stored in the well control module 118, and the computer program comprising specific perfusing instructions and specific washout instructions executable by the automated well unit 1 1 of any preferred embodiment, which, upon such execution by the automated well unit 11 , causes the automated perfusing of the new solution into the wells 1113 and the corresponding automated washout of the existing solution from said wells 11 13 according to the method of automated perfusing and washout of solutions.

As the nature of the instructions does not require high processing power, the well control module 118 on which the computer program of the third aspect is stored can be either on a separate electronic control unit, controller or similar provided together with the automated well unit 11 or downloaded on a general-purpose hardware processing unit, having the advantage of reduced costs.

In a fourth aspect of the invention with reference to Fig. 8, it is provided an automated screening platform comprising the automated well unit 11 , an observation unit 12, and a global control unit 13. The automated screening platform is configured to carry out screening of the biological samples.

In a preferred embodiment, the automated screening platform of the invention is configured to carry out screening of the zebrafish model larvae.

Any feature in one aspect of the invention may be applied to other aspects of the invention, in any appropriate combination. In particular, method features may be applied to device features, and vice versa.

Wherever applicable, means - plus - function features may be expressed alternatively in terms of their corresponding structure. Particular combinations of the various features of the invention can be implemented and/or supplied and/or used independently.

While the description of the invention was disclosed in detail in connection to preferred embodiments and examples of realization, those skilled in the art will appreciate that modifications may be made to adapt a particular situation without departing from the essential scope to the teaching of the invention.

Reference numerals appearing in the claims are by way of illustration only and shall have no limiting effect on the scope of the claims.

Industrial applicability The invention shall be used in the field of research for the pharmaceutical sector, specifically for any type of assay using biological samples where drug administration is necessary. The invention is particularly useful for the screening platforms used in the assays aiming for phenotyping features in the zebrafish model of disease, and assessing the impact of the drugs on said zebrafish model of disease.

BIBLIOGRAPHICAL REFERENCES

1. Jordi J et al., Am J Physiol Regul Integr Comp Physiol. (2015) 309(4) :R345-57.

2. Barbazuk WB et al., Genome Res. (2000) 10(9): 1351 -8.

3. van Ham et al., FASEB J. (2010) 24(11 ):4336-42.

4. He C. et al., Autophagy (2009) 5(4):520-6.

5. Moro E. et al., Mol Genet Genomics (2013) 288(5-6): 231-242.

6. Knopfel T. Nat Rev Neurosci (2012) 13(10):687-700.

Claims

Patent Claims

1 . An automated well unit (11 ) comprising:

- one or more rows (111 ), each row (11 1 ) comprising wells (1113),

- each well (11 13) adapted for accommodating biological samples,

- each row (11 1) provided with a row inlet (11 11 ) and a row outlet (1112),

- each well (11 13) provided with a well inlet (11131 ) and a well outlet (11 132),

- one perfusing pump (113) connected to the row inlets (1111 ), the perfusing pump (113) configured to pump in a new solution through the row inlets (11 11 ),

- a plurality of inlet tubes (115), each inlet tube (115) connecting each row inlet (1111 ) with corresponding well inlets (1 1131 ),

- a plurality of inlet filters (1171 ), each inlet filter (1171 ) covering a corresponding well inlet 11131 ,

- one washout pump (1 14), connected to the row outlets (1112), the washout pump (114) configured to pump out an existing solution from the row outlets (1112),

- a plurality of outlet tubes (1 16), each outlet tube (116) connecting each row outlet (1112) with corresponding well outlets (11 132),

- a plurality of outlet filters (1172), each outlet filter (1172) covering a corresponding well outlet (11132),

- a well control module (118), electronically and mechanically connected to the perfusing pump (1 13) and to the washout pump (114), the well control module (118) being configured to send specific perfusing instructions to the perfusing pump (113), and corresponding specific washout instructions the washout pump (114),

- means for electronic and mechanic connection to an observation unit (12) of a screening platform,

- means for electronic and mechanic connection to a global control unit (13) of said screening platform, wherein the automated well unit (11 ) is configured to operate the automated perfusing of the new solution into the wells (11 13) and the corresponding automated washout of the existing solution from said wells (1113), according to specific perfusing instructions and, respectively specific washout instructions from the well control module (1 18).

2. The automated well unit (11 ) of claim 1 , wherein - each well (1113) is adapted for accommodating zebrafish model larvae,

- the plurality of inlet filters (1171 ) and the plurality of outlet filters (1172) are configured to prevent the zebrafish model larvae to escape from the wells (1 113),

3. The automated well unit (11 ) of claim 1 or 2, wherein the automated well unit (1 1) is configured for perfusing a volume of new solution equal to the volume of the existing solution washed out.

4. The automated well unit (11 ) of claim 1 or 2, wherein the automated well unit (1 1) is configured for perfusing a volume of new solution equal to the volume of the existing solution washed out times a predetermined evaporation coefficient.

5. The automated well unit (11 ) of claim 1 or 2, wherein

- the automated well unit (11 ) further comprises an evaporation sub-module of the well control module (118), said evaporation sub-module in connection with a plurality of evaporation sensors (11133), each evaporation sensor (11133) placed in a corresponding well (1113) and configured to sense a level of the existing solution,

- the plurality of evaporation sensors (11 133) is configured to send to the evaporation sub-module a signal when level of the existing solution is below an evaporation threshold,

- the evaporation sub-module is configured to calculate upon receipt of the signal an evaporation replacement new solution volume and configured to trigger a perfusion instruction to the perfusing pump (113) for said evaporation replacement new solution volume,

- the perfusing pump is configured to perfuse upon receiving the perfusion instruction said evaporation replacement new solution volume.

6. The automated well unit (11 ) of any of the claims 1 to 6, wherein the plurality of inlet filters (1171 ) of the same row (111 ) is connected to the corresponding plurality of outlet filters (1172) such that to form a one-piece detachable filter (1 173).

7. Computer implemented method of automated perfusing and washout of solutions carried out by the automated well unit (11 ) of any of the claims 1 to 6, the method comprising the following steps:

- Perfusing by the perfusing pump (113) the new solution into the wells (1 113), upon receipt of specific perfusing instructions from the well control module (118),

-Washing out by the washout pump (114) the existing solution from the wells (1113), upon receipt of specific washout instructions from the well control module (118).

8. A non-transitory computer-readable storage medium encoded with a computer program, the computer program stored in the well control module (118), and the computer program comprising specific perfusing instructions and specific washout instructions executable by the automated well unit (11 ) of any of the claims 1 to 6, which, upon such execution by the automated well unit (1 1 ), causes the automated perfusing of the new solution into the wells (1113), and the corresponding automated washout of the existing solution from said wells (1113).

9. Automated screening platform comprising:

- the automated well unit (11) of any of the claims 1 to 6,

- an observation unit (12), and

- a global control unit (13),

- wherein the automated screening platform is configured to carry out screening of the biological samples.

10. The automated screening platform of claim 9, wherein the automated screening platform is configured to carry out screening of the zebrafish model larvae.