WO2025109542A1

WO2025109542A1 - Dispensing device

Info

Publication number: WO2025109542A1
Application number: PCT/IB2024/061725
Authority: WO
Inventors: Elena Bianchi; Paola DE STEFANO; Gabriele Angelo DUBINI; Monica PIERGIOVANNI; Giovanni Tonon; Oronza Antonietta BOTRUGNO
Original assignee: Ospedale San Raffaele SRL; Politecnico di Milano
Current assignee: Ospedale San Raffaele SRL; Politecnico di Milano
Priority date: 2023-11-23
Filing date: 2024-11-22
Publication date: 2025-05-30
Anticipated expiration: 2026-05-23

Abstract

Object of the present invention is a method for the controlled delivery of delivery volumes up to 15 ml, fractionated into multiple deliveries of volumes comprised between 4 microlitres and 1 ml, of thermo-sensitive fluid comprising cellular aggregates, comprising: -a delivery device (1); -a volumetric pump (21); wherein said delivery device (1) comprises: -a conduit (3) made of biocompatible material with an inner diameter comprised between 50 micrometres and 2.5 mm, said conduit (3) having an inlet port (10) and an outlet port (11); -a cooling element (2); -a thermally insulating coating (6); where said conduit (3) runs in a path that, for most of its length, is almost perpendicular to the direction of the force of gravity; wherein said conduit (3) is connected to said volumetric pump (21) by means of a connector (9).

Description

DISPENSING DEVICE

STATE OF THE ART

The process of drug discovery and development is notoriously long and expensive. Among the potential candidates to become new drugs, only 2.5% reach the pre-clinical study phase and, of this 2.5%, only 5-10% reach the clinical trial.

The main reason for this high failure rate is the lack of adequate biological models capable of replicating in vitro the required pathology. Thanks to recent advances in cell biology, it has been possible to develop a wide range of 3D cell culture methods. In this context, organoids are emerging as promising models as they reproduce on a small scale and in 3D the tissue from which they are derived, mimicking ex vivo physiology, pathology, and response to therapy. Therefore, the international scientific community is showing a growing interest in methodologies that use organoids to reproducibly replicate complex systems in the laboratory. The possibility of using organoids to develop personalised treatments offers an interesting and unique alternative in the current drug development process, which often makes use of animal models that are difficult to develop, expensive and often pose ethical problems. However, the strategies currently in use for the formation of 3D constructs are difficult to integrate into large-scale systems as the instrumentation present in High-Throughput (HTS) systems is unable to adequately manage the automatic deposition of the biological matrices required for optimal organoid growth.

Matrigel® (Coming) is the biological matrix commonly used for organoid culture. However, Matrigel® is a thermo-sensitive material characterized by a complex rheology that prevents easy management thereof with the common automated pressure control systems. The Matribot® instrumentation (Corning) enables the printing of thermosensitive hydrogels with a "syringe-based extrusion" method coupled with a temperature control system. However, it cannot handle the dispensing of the biological matrix containing cells or organoids in a single step, as it is affected by the phenomenon of sample sedimentation. Maintaining solution homogeneity is, in fact, a critical step for the optimal success of the experiment.

Jeong Mi-Hyeon et al., in Int J Mol Sci 2023, 24: 1006. https://doi.org/10.3390/ijms24021006 describes a fully automated system for dispensing matrices having the characteristics of Matrigel®, wherein said system is capable of delivery volumes of up to 4 microlitres of single cell suspensions in diluted matrices. EP4067051 describes a dispensing system based on the progressive mixing of the desired biological matrix and cell suspension.

WO2015/017579 describes a dispensing system wherein the cell suspension is injected into a container containing the desired biological matrix. Said container is placed in rotation so as to make the solution uniform and then deposit it through multiple capillary structures.

There is a strong need for an accessible, robust system that allows reproducibility in the large-scale delivery of biological matrices containing cell suspensions or organoids for the performance of biological assays.

DESCRIPTION

Object of the present invention is a method for the controlled delivery of delivery volumes up to 15 ml, fractionated into multiple deliveries of volumes comprised between 4 microlitres and 1 ml, where said fluids are thermo-sensitive fluids comprising cellular aggregates, such as for example organoids, spheroids, aggregates.

Said thermo-sensitive fluids are liquids at low temperatures (between 1 and 4 °C) and gels at room temperature and therefore they need to be handled at temperatures close to 4 °C. The viscosity of said fluids, as well as the temperature, also depends on the flow velocity, specifically on the shear stress that characterizes the flow itself.

A further object of the present invention is a method for the delivery of suspensions in thermo-sensitive fluid comprising biological material. In one embodiment, said biological material consists of cell suspensions or organoids.

DESCRIPTION OF THE FIGURES

Figure 1: (A) device for the delivery of thermo-sensitive fluid, perspective view. (B) kit comprising a volumetric pump and a device for the delivery of thermo-sensitive fluid comprising cellular aggregates.

Figure 2: device for the delivery of thermo-sensitive fluid comprising cellular aggregates, in two embodiments, (A, C) exploded. (B, D) perspective view.

Figure 3: (A) kit comprising a support. (B, C_z D) three embodiments of the top of the pins. (E) multiplicity of supports.

Figure 4: example of drops seeded with the method according to the present invention on a multiplicity of supports. (A, B) photographs representative of supports with drop. (C) 4X microscope image of a drop, arrows indicate the organoids.

Figure 5: 3D cell viability and proliferation assay CellTiter-Glo® (CTG, CellTiter- Glo® 3D Cell Viability Assay - Promega) in cultures of patient-derived organoids (A) on organoids plated according to the method of the present invention; (B) on organoids plated according to the prior art. NT = untreated, FOX1, FOX2, FIRI1 and FIRI2 = exposures to the combinations of actives indicated in Example 2.

Figure 6: 3D cell viability and proliferation assessment assay CellTiter-Glo® in patient-derived organoid cultures. For organoids #6 (A) and #17 (B), the counts carried out at 0 and 96h are compared, working in 4 microlitres and 8 microlitres of structures in Matrigel® 1:1 (5 mg/ ml protein concentration).

Figure 7: 3D cell viability and proliferation assay CellTiter-Glo® in patient-derived organoid cultures. For organoids #17, the counts carried out at 0 and 96h are compared, working in volumes of 8 microlitres of structures in BME (Basal Membrane Extract) 70% (about 7 mg/ ml protein concentration) in PBS.

Figure 8: 3D cell viability assay CellTiter-Glo® in patient-derived organoid cultures. Evaluation of the seed homogeneity of 8 microlitre boluses, delivered in sequence (cartridge with 1 mm inner diameter tube, 375,000 aggregates/ml - lx) at different flow rates. For organoids #17, the counts carried out at Oh are compared, working in volumes of 8 microlitres of structures in Matrigel® 1:1 (5 mg/ml protein concentration) in PBS obtained with different flow rates.

Figure 9: Data shown in Figure 8, aggregated and processed. For organoids #17, the counts carried out at Oh are compared, working in volumes of 8 microlitres of structures in Matrigel® 1:1 (5 mg/ml protein concentration) in PBS obtained with different flow rates. The region highlighted in green refers to the expected cell viability values. The coefficient of variation CV is reported.

Figure 10: 3D cell viability assessment assay CellTiter-Glo® in patient-derived organoid cultures. For organoids #17, the counts carried out at Oh are compared, working in volumes of 8 microlitres of structures in Matrigel® 1:1 (5 mg/ ml protein concentration) in PBS obtained with the cartridge in a vertical and horizontal position. Data (A) are reported point by point and (B) aggregated. The coefficient of variation CV is reported. The highlighted regions refer to the expected cell viability values.

Figure 11: 3D cell viability assessment assay CellTiter-Glo® in patient-derived organoid cultures. For organoids #17, the counts carried out at Oh are compared, working in 8 microlitres volumes of structures in Matrigel® 1:1 (5 mg/ ml protein concentration) and Matrigel® 1:4 (1.25 mg/ ml protein concentration) in PBS. Data (A) are reported point by point and (B) aggregated. The highlighted regions refer to the expected cell viability values.

Figure 12: 3D cell viability assessment assay CellTiter-Glo® in patient-derived organoid cultures. For organoids #17, the counts carried out at Oh are compared, working in volumes of 4, 6 and 8 microlitres of structures in Matrigel® 1:1 in PBS (5 mg/ ml protein concentration) obtained with cell density 375,000 aggregates/ml (lx) and 750,000 aggregates/ ml (2x). Data (A) are reported point by point and (B) merged. The highlighted regions refer to the expected cell viability values.

DETAILED DESCRIPTION

Object of the present invention is a method for the delivery of controlled volumes of a thermo-sensitive fluid comprising cellular aggregates.

In one embodiment, said method comprises:

- Making available a delivery kit 20 that comprises, with reference to Figure 1A, B:

■ a delivery device 1;

■ a volumetric pump 21; wherein said delivery device 1 comprises: o A conduit 3 of biocompatible material of inner diameter comprised between 50 micrometres and 5 mm, or between 70 micrometres and 2.5 mm, or between 500 micrometres and 2 mm and length between 5 cm and 10 m, having an inlet port 10 and an outlet port 11; o A cooling element 2; o A thermally insulating coating 6; where said conduit runs along a path that, for most of its length, is almost perpendicular to the direction of the force of gravity; wherein said volumetric pump is in fluid connection with said conduit through a connector 9;

- Loading said conduit comprised in said delivery device, which conduit is initially empty and dry, with a thermo-sensitive fluid comprising cellular aggregates; by way of example, said loading can take place using a syringe;

- Connecting said conduit to the volumetric pump, through said connector;

- Dispensing the desired volume of thermo-sensitive fluid comprising cellular aggregates on one or more media.

In one embodiment, said thermo-sensitive fluid comprises a diluted matrix with protein concentration between 2 and 12 mg/ml, or between 3 and 6 mg/ ml, preferably about 5 mg/ ml.

In one embodiment, said thermo-sensitive fluid is Matrigel, preferably diluted.

Preferably, said matrix is diluted in PBS.

In one embodiment, said aggregates formed by said cells have a characteristic size comprised between 10 and 70 micrometres.

In one embodiment, said thermo-sensitive fluid comprises from 200 thousand to 1 million aggregates/ ml, preferably between 375,000 and 750,000 aggregates/ ml.

In one embodiment, said volumetric pump is functional for the delivery of the thermo-sensitive fluid comprising homogeneously distributed cellular aggregates when loaded in said conduit and determines the magnitude of the individual volumes delivered consecutively by means of the movement of a buffer fluid. Said pump allows volumetric control to be maintained regardless of the rheology of the fluid to be delivered. In particular, said volumetric pump delivers a buffer fluid, which is for example PBS, Phosphate Buffer Solution, which is kept separate from the thermosensitive fluid to be delivered, for example by means of an air bubble of reduced size, in relation to the diameter of the tube. A lower volume bubble corresponds to the diameter of the lower tube. By way of example, said two fronts, the buffer front and the buffer front, are spaced by about 10 mm, where said 10 mm are occupied for example by said air bubble. Said separator prevents the buffer fluid from mixing with the thermo-sensitive fluid, ensuring a correct transfer of the movement of the buffer fluid to the thermosensitive fluid, thus allowing the extrusion thereof.

In one embodiment, said conduit 3 is a spiral-wound tube coil of semi-rigid material (e.g. PTFE).

In one embodiment, said conduit is a spiral tube coil of rigid material.

In one embodiment, said conduit is a path obtained in a support of rigid material. In one embodiment, said cooling element 2 is accommodated in the central volume of said spiral tube coil.

Alternatively, said cooling element is adjacent to the conduit outside said tube coil and inside the thermally insulating coating, positioned in such a way that the cooling is constant and homogeneous (not depicted).

In one embodiment, said cooling element 2 is ice. Alternatively, it is a Peltier cell with a thermally conductive element, or a system with recirculation with a coolant.

Said volumetric pump is, by way of example, a syringe pump, roller or membrane pump.

Said thermally insulating coating is preferably partly or totally transparent, thus allowing to see the flow inside, wherein also said conduit 3 is made of transparent material.

In one embodiment, said thermally insulating coating is a sleeve of thermally insulating material, in which said conduit is housed.

In one embodiment, with reference to Figure 2, said thermally insulating coating is a container 7 having an upper base 12 and a lower base 13 comprising a hole 8 in a position proximal to said lower base 13. In this embodiment, said conduit 3 is conveniently housed in said container 7, said inlet port 10 of said conduit 3 is proximal to said upper base 12 and the distal portion of said conduit 3, terminating with said outlet port 11, protrudes through said hole 8 from said container 7.

By way of example, said container 7 is a test tube, with a conical or concave bottom. Said thermally insulating coating is, in this embodiment, a container functional for collecting, on the lower base thereof, dripping due to condensation.

In one embodiment, said hole 8 is positioned at the bottom of said container 7 (Figure 2 A, B). In one embodiment, it is positioned on one of the side walls of said container (Figure 2C, D).

In one embodiment, said delivery device 1 also comprises a support 5 to the end portion of said conduit 3, wherein said support is useful when said conduit is made of semi-rigid material being functional to direct the delivery direction.

Said volume to be delivered is comprised between 50 microlitres and 15 ml, or between 100 microlitres to 5 ml, fractionated into multiple deliveries of volumes comprised between 4 microlitres and 1 ml, or between 4 and 400 microlitres, or between 4 and 10 microlitres of fluids, wherein said fluids are thermo-sensitive fluids comprising cellular aggregates.

Advantageously, in said conduit the filling volume (priming) is equal to or greater than the total volume of the biological sample to be delivered. That is, the conduit contains at least the volume of fluid to be delivered in the multiplicity of wells/ supports that it is desired to seed homogeneously.

With reference to Figure 3A, the kit 20 for delivering, seeding and maintaining 3D cell cultures in culture also comprises a support 22 on which the suspension of thermosensitive fluid delivered by said device is deposited and gelled.

In one embodiment, said kit also comprises at least one culture plate for static and/ or dynamic cell cultures.

In one embodiment, said support 22 is a pin, the top of which is surrounded by a crown structure, as schematized in Figure 3B. Alternatively, said support 22 is a pin whose top is cup-shaped (Figure 3C). Alternatively, said support 22 is a pin whose top is cup-shaped with a central pin (Figure 3D). Each of these embodiments of the top of said pin is adapted to facilitate the deposition and maintenance in situ of the thermo-sensitive fluid delivered thereon, while allowing an easy exchange with the medium in which it will be subsequently immersed.

In a preferred embodiment, the diameter of said pins is about 2 mm.

In a preferred embodiment, said supports are made of polymeric material, for example, worked with stereolithographic 3D printing (SLA) technology, certified for biomedical applications. Said supports are sterilisable. Said supports are conveniently tested for compatibility with the culture required by the experimental setting.

Figure 3E shows a multiplicity of supports, which are conveniently loaded with the kit and the method according to the present invention.

Said arrangement in the space of the conduit guarantees a zero or minimal slope over long sections of said conduit, and this strongly limits the undesired effects of the sedimentation of the cellular aggregates in a direction parallel to the flow. In one embodiment, said slope is less than 45°, or less than 40°, or less than 15°. In one embodiment, it is about 10°.

In one embodiment, said volumetric pump dispenses with flow rates comprised between 15 microlitres/ min and 800 microlitres/ min, in one embodiment of about 200 microlitres/ min, in volume control a buffer fluid (e.g. PBS), with an accuracy > 95%, defining the volumes to be delivered greater than 1 microlitre. The displacement of the buffer fluid induces the corresponding delivery of said thermo-sensitive fluid.

The combination of the diameter of the tube, the size of the cellular aggregates, the viscosity of the thermo-sensitive fluid and the flow rate set on the volumetric pump makes it possible to identify a working point useful for delivery samples of equal volumes, avoiding the sedimentation of the cellular aggregates and keeping the suspension homogeneous.

Alternatively, said device is already connected to the volumetric pump and to the syringe containing the sample to be delivered by means of a three-way connector. In this embodiment, the filling of the conduit and the insertion of the air bubble take place through the connector, simplifying the start of delivery that can take place after rotation of the pin and selection of the syringe - conduit connection in conjunction with the start of the volumetric pump.

Preferably, said conduit is cold when it is loaded, so as not to expose the mixture to temperature rises during loading, wherein even minor temperature changes would vary the physical characteristics of said thermo-sensitive fluid, affecting the correct seeding.

In one embodiment, said method is implemented in a 1 mm diameter conduit, in which a matrix diluted to 5 mg/ml protein concentration, comprising about 375,000 aggregates/ ml, is flowed at a flow rate of 200 microlitres/ minute, wherein said aggregates are of a characteristic size comprised between 10 and 70 micrometres.

In one embodiment, said method is implemented in a 1.6 mm diameter conduit, in which a matrix diluted to 5 mg/ml protein concentration, comprising about 375,000 aggregates/ ml, is flowed at a flow rate of 200 microlitres/ minute, wherein said aggregates are of a characteristic size comprised between 10 and 70 micrometres.

Advantageously, the method according to the present invention allows to generate a high number of 3D constructs for the culture of cells or organoids for drug screening assays.

The 3D constructs, deposited on special supports, are then kept in culture and, thanks to the specific configuration of the supports, they can also be recovered individually, in a non-destructive way, for subsequent analyses (e.g. genomic and transcriptomic analyses).

The following examples are merely intended to illustrate some embodiments of the invention, they are not intended to be limiting thereof, the scope of protection of which is defined by the claims that follow.

Example 1: deposition of thermo-sensitive fluid comprising organoids

A kit comprising a delivery device has been made available, wherein said spiral tube coil is made of PTFE and has a length of 1 m and an inner diameter of 1 mm.

Said tube coil was loaded with a syringe with 800 microlitres of Matrigel® diluted 1:1 with PBS, containing a suspension of tumour organoids derived from patient with liver metastases of colorectal cancer, after which it was connected to a volumetric pump which is a Harvard Apparatus PHD2000 syringe pump, loaded with a Hamilton Gas Tight TLL glass syringe, loaded with sterile PBS which is the buffer fluid, set the flow rate 200 microlitres/ min, set the volume for each delivery of 8 microlitres.

Supports were also made available to receive the delivered material, in this case supports had cup-shaped tops.

Delivery was started which led to the deposition of 8 microlitres of mixture comprising organoids on each support. Representative photographs of some loaded supports are reported in Figure 4 A and 4B. Figure 4C shows a 4X magnification microscope image of a drop on the support. The white arrows indicate the presence of organoids.

Example 2: drug dose-response assay performed on organoids seeded according to the method of the present invention and control organoids

Following the method referred to in Example 1, organoids obtained from patient surgical specimens, defined herein as organoids #63, suspended in a volume of 8 microlitres of thermo-sensitive fluid, were seeded on the supports. Said supports are overturned and immersed in 60 microlitres of culture medium in wells of a standard 384- well plate. The same was repeated by seeding organoids obtained from a second patient, referred to as organoids #61. For comparison purpose, the same organoids #61 and #63 were seeded in accordance with a method commonly used in laboratory practice, described in Vlachogiannis G et al. 2018 Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359: 920-926.

Said 384-well multi-well plate for cell cultures where said supports are immersed was previously prepared so that:

- a first group of wells, control wells, were loaded with standard culture medium (NT);

- a second group of wells were loaded with two combinations of chemotherapy drugs FOLFOX (FOX) and FOLFIRI (FIRI), which mimic the chemotherapy regimens most commonly used in the clinical management of liver metastases of colorectal cancer, combinations tested at two different concentrations, indicated as follows:

FOX1: 5-Fluorouracil (5-FU) 5 microg/ ml, oxaliplatin (OXA) 0.5 microg/ ml.

FOX2: 5-FU 50 microg/ ml, OXA 5 microg/ ml.

FIRI1: 5-FU 5 microg/ml, 10 nM 7-ethyl-10-hydroxy-camptothecin (SN38).

FIRI2: 5-FU 50 microg/ml, SN38100 nM.

In parallel, in the 96-well multi-well plate for the comparative experiment, organoids #63 and #61 were seeded. 48 microlitres of a 1:1 suspension of Matrigel® (5 mg/ml protein concentration) in PBS and organoids were used for each well. After solidification, treatment was started with the actives in the combinations FOX1, FOX2, FIRI1, FIRI2 as used in the experiment according to the invention.

After a drug exposure time of 96 hours, the cell viability of the organoids was assessed in the wells according to the invention and in the comparative wells by measuring ATP levels in the cells by means of the CellTiter-Glo® assay (CTG, CellTiter- Glo® 3D Cell Viability Assay - Promega).

The results obtained are reported in Figure 5. The results of three (Figure 5A) and two (Figure 5B) independent experiments generated using GraphPad Prism are reported. The results obtained with the method according to the invention are reported in Figure 5A and in Figure 5B the comparison with the method of the prior art. The results are presented as mean percentage of viable cells in the treated samples, compared to the untreated control cells calculated over six (Figure 5A) and three (Figure 5B) technical replicates for each condition. The lower standard deviation observed in the graphs of Figure 5A, method according to the invention, makes it evident that delivery drops by means of the system object of the invention allows to obtain repeatable results. The homogeneity of the delivered samples is evaluated by comparing the values obtained with the CellTiter-Glo® assay: the compared values indirectly correspond to the amount of cellular material present after a growth of 96h. The comparison between the values obtained therefore highlights both the cell distribution homogeneity and the homogeneity of the growth in the different conditions, normalized on the values obtained with the untreated NT samples. The organoids seeded according to the prior art show a higher standard deviation (Figure 5B) index of a lower homogeneity.

Example 3: proliferation assays performed on organoids seeded according to the method of the present invention

Following the method referred to in Example 1, using a device comprising a tube coil of length 25 cm and diameter 1.6 mm, said coil was loaded with a syringe with 800 microlitres of Matrigel® diluted 1:1 with PBS (5 mg/ ml protein concentration), containing a suspension of patient tumour organoids, organoids #6 and organoids #17. The volumetric syringe pump, filled with sterile PBS, was set at a flow rate of 400 microlitres/ min, with delivery volumes of 4 microlitres and 8 microlitres.

With CTG assay, the viability, homogeneity and proliferation of the organoids were verified, by verifying products of the system of deposition of the cell suspension in thermo-sensitive fluid, in the delivery of volumes of different entities (4 and 8 microlitres), on two populations of different organoids (#6 and #17) at time 0, of seeding, and after 96 hours of culture. The data are reported in Figure 6A and 6B. The homogeneity of the delivered samples is evaluated by comparing the values obtained with the CTG assay: the compared values indirectly correspond to the amount of cellular material present at the time of delivery, 10 h, and after a growth of 96 h. The comparison between the values obtained therefore highlights both the homogeneity of the cell distribution in the delivery of different volumes (4 and 8 microlitres), and the growth at 96h.

The same test was repeated using a delivery device comprising a spiral tube coil of length 1 m and inner diameter 1 mm.

Said coil was loaded with a syringe with a volume of 800 microlitres of BME® diluted to 70% (approximately 7 mg/ml protein concentration) with PBS, containing a suspension of patient-derived tumour organoids, organoids #17. The volumetric syringe pump, filled with sterile PBS, was set at a flow rate of 200 microlitres/ min, with a delivery volume of 8 microlitres.

With CTG assay, the viability, homogeneity and proliferation of the organoids in the constructs/ structures deposited on the supports were verified, at time 0 of seeding, and after 96 hours of culture. The data obtained are reported in Figure 7. The compared values indirectly correspond to the amount of cellular material present at the time of delivery, t Oh, and after a growth of 96h. The comparison between the values obtained therefore highlights both the homogeneity of the cell distribution at the time of delivery, and the growth at 96h.

Example 4: by varying the flow rate, the sedimentation of the aggregates varies

Following the method referred to in Example 1, using a device comprising a tube coil of length 50 cm with an inner diameter of 1 mm, a solution of Matrigel® diluted in PBS 1:1 (5mg/ml protein concentration), comprising 375,000 aggregates/ ml, was loaded.

Six identical experiments were performed, varying the flow rate comprised between 25 and 400 microlitres/minute. For each experiment, 30 samples were seeded on crown supports (as per Figure 3B) and their viability was measured with CTG® assay (RLU). As highlighted by the data in Figure 8, optimal results are obtained when the flow rate is 200 microlitres/ minute. In the graph obtained with the flow rate at 200 microlitres/ minute, the circles and squares indicate two experiments performed under the same conditions (technical replicates)

The collected and aggregated data are reported in Figure 9: the value of the coefficient of variation CV of the individual experiments shows that optimal results are obtained when the flow rate is 200 microlitres/minute.

Example 5: impact of the position of the delivery device

Following the method referred to in Example 1, using a device comprising a tube coil of length 50 cm with an inner diameter of 1 mm, a solution of Matrigel® diluted in PBS 1:1 (5 mg/ml protein concentration), comprising 375,000 aggregates/ ml, was loaded. The seeding method was started, with a flow rate of 200 microlitres/minute.

Figure 10A reports the measurement of the cell viability of the samples obtained by means of Cell-Titer Gio®. The squares are the samples seeded with the device held vertically, and thus with the horizontal conduit. The circles are the samples seeded with the device held horizontally, and thus with the vertical conduit. The reference area is highlighted in the centre of the graph, where the samples would be expected to fall (expected values). The results obtained by keeping the conduit parallel to the plane are considerably better.

In confirmation of the above, the same data, aggregated and processed, are reported in Figure 10B, explaining the values of the coefficient of variation. Example 6: seeding homogeneity of the samples, impact of the matrix protein concentration.

Following the method referred to in Example 1, using a device comprising a tube coil of length 25 cm with an inner diameter of 1.6 mm, a solution of Matrigel® diluted in PBS 1:1 (5 mg/ ml protein concentration), comprising 375,000 aggregates/ ml was loaded. The seeding method was started, with a flow rate of 50 microlitres/ minute. The same experiment was conducted with 1.25 mg/ml protein concentration solution.

Figure 11A reports the samples seeded under the two conditions, protein concentration 1.25 mg/ml (squares) and 5 mg/ml (circles). The highlighted area shows the reference area of the expected values. The data clearly show how the low concentration of the matrix makes the samples inadequate due to sedimentation.

The same data, aggregated and processed, are reported in Figure 11B, explaining the values of the coefficient of variation.

Example 7: seeding homogeneity of the samples, impact of cellular aggregate concentration and volume of biological matrix dispensed.

Following the method referred to in Example 1, using a device comprising a tube coil of length 50 cm with an inner diameter of 1 mm, a solution of Matrigel® diluted 1:1 in PBS (5mg/ml protein concentration), comprising 375,000 aggregates/ ml, or 750,000 aggregates/ ml, was loaded.

The seeding method was started, with a flow rate of 200 microlitres/ minute and drops of 4, 6 and 8 microlitres were seeded, for both conditions.

Figure 12 reports the data obtained, aggregated and processed, with the values of the coefficients of variation.

References:

1. Device for the delivery of thermo-sensitive fluid.

2. Cooling element.

3. Spiral tube coil

5. End section support

6. Coating

7. Container

8. Hole

9. Inlet connector

10. Inlet port

Claims

1. A method for the delivery of controlled volumes of a thermo-sensitive fluid comprising cellular aggregates wherein said method comprises:

- Making available a delivery kit (20) that comprises:

• a delivery device (1);

• a volumetric pump (21); wherein said delivery device (1) comprises: o A conduit (3) of biocompatible material of diameter comprised between 50 micrometres and 5 mm, or between 70 micrometres and 2.5 mm, or between 500 micrometres and 2 mm and length between 5 cm and 10 m, having an inlet port (10) and an outlet port (11); o A cooling element (2); o A thermally insulating coating (6); where said conduit runs along a path that, for most of its length, is almost perpendicular to the direction of the force of gravity;

- Loading said conduit (3) comprised in said delivery device, which conduit is initially empty and dry, with a thermo-sensitive fluid comprising cellular aggregates;

- Dispensing the desired volume of fluid on one or more supports.

2. The method according to claim 1, wherein said volumetric pump delivers a buffer fluid kept separate from the thermo-sensitive fluid comprising cellular aggregates to be delivered, for example by means of an air bubble.

3. The method according to claim 1, wherein said thermally insulating coating (6) is a container (7) having an upper base (12) and a lower base (13) comprising a hole (8) in a position proximal to said lower base (13), wherein said conduit (3) is housed in said container (7), said inlet port (10) is proximal to said upper base (12) and the distal portion of said conduit (3), terminating with said outlet port (11), protrudes through said hole (8) from said container (7).

4. The method according to claim 1, wherein said conduit (3) is a spiral-wound tube coil of semi-rigid material.

5. The method according to claim 3, wherein said hole (8) is positioned on the side walls of said container (7).

6. The method according to one of claims 1 to 5, wherein said kit also comprises a support (22) on which the suspension of thermo-sensitive fluid delivered by said device is deposited and gelled.

7. The method according to claim 6, wherein said support (22) is a pin of about 2 mm diameter, the top of which is preferably surrounded by a structure that is crownshaped, or cup-shaped, or cup-shaped with a central pin.

8. The method according to claim 1, wherein said thermo-sensitive fluid is a diluted matrix, with protein concentration comprised between 2 and 12 mg/ ml, preferably between 3 and 6 mg/ ml, even more preferably about 5 mg/ ml.

9. The method according to claim 1, wherein said aggregates formed by said cells have a characteristic size comprised between 10 and 70 micrometres.

10. The method according to claim 1, wherein said thermo-sensitive fluid comprises from 200 thousand to 1 million aggregates/ ml, preferably between 375,000 and 750,000 aggregates/ ml.

11. The method according to one of claims 1 to 10, which is implemented in a conduit of 1 mm inner diameter, in which a matrix diluted to 5 mg/ml protein concentration, comprising about 375,000 aggregates/ ml, is flowed at a flow rate of 200 microlitres/ minute, wherein said aggregates are of a characteristic size comprised between 10 and 70 micrometres.

12. The method according to one of claims 1 to 10, which is implemented in a 1.6 mm inner diameter conduit, in which a matrix diluted to 5 mg/ ml protein concentration comprising about 375,000 aggregates/ ml is flowed at a flow rate of 200 microlitres/ minute, wherein said aggregates are of a characteristic size comprised between 10 and 70 micrometres.