WO2018065349A1 - Procédé d'analyse de particules sécrétées et/ou endocytosées par des cellules en culture cellulaire - Google Patents

Procédé d'analyse de particules sécrétées et/ou endocytosées par des cellules en culture cellulaire Download PDF

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Publication number
WO2018065349A1
WO2018065349A1 PCT/EP2017/074950 EP2017074950W WO2018065349A1 WO 2018065349 A1 WO2018065349 A1 WO 2018065349A1 EP 2017074950 W EP2017074950 W EP 2017074950W WO 2018065349 A1 WO2018065349 A1 WO 2018065349A1
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Prior art keywords
cuvette
particles
culture medium
culture
block
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PCT/EP2017/074950
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English (en)
Inventor
Alice GALBIATI
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Alma Mater Studiorum - Università di Bologna
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Publication of WO2018065349A1 publication Critical patent/WO2018065349A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
    • G01N2021/513—Cuvettes for scattering measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6482—Sample cells, cuvettes

Definitions

  • the present invention relates to a method and to the relative cuvette for real-time analysis of particles secreted or endocytosed by cells in cell culture, such as for example exosomes or other cellular products.
  • the studied particles have size in general terms smaller than micron and are for example exosomes or other kind of vesicles, mammospheres, liposomes, protein complexes, colloids or viruses.
  • particle is in fact any physical or biological object composed by an atomic or molecular aggregate, naturally occurring or produced by means of nanotechnologies, characterized by particular chemical and physical properties and by sizes that range from nanometers to tens of micrometers.
  • particle is univocally assigned the above-mentioned meaning.
  • culturing cells in the laboratory envisages keeping the cells immersed in suitable liquid culture media (also called cell growth media) which contain the appropriate amount of necessary substances dissolved in them, in conveniently treated plastic containers (culture flasks and plates), in incubators that are able to maintain temperature, carbon dioxide partial pressure and humidity controlled.
  • suitable liquid culture media also called cell growth media
  • the cells interact with the surrounding environment and expel or absorb a number of particles in or from the culture medium.
  • Said particles produced or absorbed by the cells in culture are usually analyzed using ordinary laboratory techniques which permit to measure physical and chemical features related to the particles suspended in a liquid: for example size, Z-potential and fluorescence.
  • the Z-potential which is the potential at the level of the solvation layer of the particle, represents the main interaction force between the particles one with each other and the particles and the cells.
  • the measurement of the Z-potential depending on the pH permits to determine their isoelectric point.
  • a sample of the culture medium is generally collected from the cell culture and placed within particular cuvettes suitable to be inserted in a specific housing of the measuring system.
  • These cuvettes are usually disposable and made of transparent plastic material, such as polystyrene.
  • Size measurement is usually performed using DLS technique (Dynamic Light Scattering, diffusione dinamica della luce).
  • DLS technique Dynamic Light Scattering, diffusione dinamica della luce
  • a laser beam is used to illuminate the sample and intensity fluctuations of scattered light as a function of time are measured.
  • the particles move faster or slower depending on their size, giving rise to more or less rapid intensity fluctuations of scattered light. From the measurement of the rate of intensity fluctuation it is possible to calculate the diffusion coefficient of the particles which is converted through the Stokes-Einstein equation into the hydrodynamic diameter.
  • the DLS technique envisages the use of a cuvette of standard size and parallelepiped shape, having an open upper part for inserting the sample.
  • the sample is usually placed within a particular cuvette characterized by a U-shaped channel suitable to contain the sample and having electrodes at its extremities.
  • the sample is illuminated by a laser beam and at the same time an electric field is applied between the electrodes of the cuvette.
  • the charged particles move toward the electrode of opposite sign, causing a variation of the frequency of light scattered by the sample which is directly proportional to the to the electrophoretic mobility.
  • the Z-potential is in turn proportional to the electrophoretic mobility.
  • the DLS technique for the measurement of the hydrodynamic diameter and the technique to measure the Z-potential use both a laser beam which hits a sample and a detector to detect scattered light. These two measurements can be performed using the same machine (e. g. Z-sizer, Malvern Instruments Ltd). For this purpose, it is to highlight that the cuvette with U-shaped channel for the measurement of the Z-potential can be used in this kind of machines for the measurement of the hydrodynamic diameter too.
  • fluorescence measurement such measurement is typically used to identify particular fluorescent compounds (fluorochromes) which can be bound through specific antibodies to proteins of interest or used to label the cells.
  • the fluorochromes become excited upon absorption of light emitting fluorescence signal.
  • Flow cytofluorimetry systems employed in cell fluorescence analysis can also be used for the analysis of micro- and nanoparticles such as those produced by cells in culture.
  • the sample with the suspended cells is inserted in a fluidic system which, letting the sample flow inside a nozzle, causes the cells to pass one at a time through the measurement point.
  • the cell which passes through the measurement point is hit by a light beam which excites the fluorochromes. Once the fluorochromes are excited, they emit a fluorescence signal which is detected by sensors which measure its intensity.
  • the flow cytometry system with tradename FACSaliburTM also envisages an optional device for the biomedical research which permits to sort and isolate the cells of interest once they are identified by fluorescence measurements.
  • This device electrically charges non- fluorescent cells positively and fluorescent cells negatively and conveys them in separated collector ducts by applying an appropriate magnetic field.
  • the technical problem underlying the present invention is therefore to provide a method for the analysis of particles in a culture medium of a cell culture that overcomes the drawbacks observed in the prior art, permitting more flexibility in researching and monitoring the cell life phenomena.
  • a method for the analysis of particles secreted and/or endocytosed by cells in culture comprising the following steps: seeding a cell sample within a cuvette in the presence of a culture medium; placing the cuvette in controlled atmosphere within a thermostated block so as to keep the cell sample alive and promote the production and/ or absorption of particles towards and/ or from the culture medium; measuring in real-time by means of a signal measurement block at least one feature of the particles immersed in said culture medium; interpreting and analyzing in real-time by means of a computer system connected to the signal measurement block the measurements of said at least one feature of the particles.
  • the term "cuvette” is conferred a functional meaning, which generically comprises every container adapted to permit to perform a spectrophotometric and/ or spectrofluorimetric analysis.
  • the term “cuvette” is therefore not to be understood in any way limited to standard shapes and/or sizes currently employed in the field.
  • the method outlined above can be performed using a cell culture flask, even of large size, which is to be considered to all intents and purposes a cuvette in the above defined sense.
  • the method according to the present invention envisages for example the measurement of size, Z- potential and/ or fluorescence.
  • the method envisages for example real-time measurement of at least two features selected from the group consisting of size, Z-potential and fluorescence.
  • the method envisaged real-time measurement of at least size or Z-potential.
  • an analysis system is provided, comprising at least the thermostated block, the signal measurement block and the computer system. Said analysis system and/or said cuvette are preferably set up to permit real-time measurement of Z-potential of the particles immersed in the culture medium.
  • the method also comprises a cell sorting step on the basis of said signal.
  • the measurement of size and Z-potential can be performed using the technique of dynamic light scattering.
  • While a fluorescence analysis is preferably performed labelling with fluorochromes the particles immersed in the culture medium and then measuring the intensity of the signal emitted by the fluorochromes when they are excited by a light beam.
  • Particles of particular interest which are suitable to be analyzed with the method according to the present invention are, for example, exosomes and/ or spheroids. This does not exclude the possibility to identify, analyze and / or sort other micro / nano particles which play an important role in cell life phenomena.
  • the method can comprise a cell sorting step on the basis of the value of the measurement of said at least one feature using a sorting block.
  • exosomes and/ or spheroids for example, it is possible to sort them on the basis of their size or charge, permitting specific analysis on defined and purified samples (lipid, protein and nucleic acid composition of the produced exosomes).
  • the sorted particles can then be conveyed in respective containers maintained at controlled temperature in a fractioning block.
  • the fractioning block can be placed out of, integrated or partially integrated within the sorting block.
  • the sorted particles are preferably extracted from the cuvette together with part of the culture medium which contains them.
  • the volume of the culture medium contained in the cuvette can be kept constant by means of a constant volume maintainer block, which tops up within the cuvette an amount of culture medium equal to the amount of the extracted culture medium.
  • a cuvette for a method for the analysis of particles secreted and/ or endocytosed by cells in culture as described above, said cuvette internally defines at least one culture chamber adapted to contain a cell sample in the presence of a culture medium, in which at least one inner surface of the culture chamber is treated with a substance suitable to promote cell adhesion; the cuvette further comprises a seeding opening for inserting the cell sample within said culture chamber.
  • the above-mentioned seeding opening can correspond with the upper opening of the cuvette or it can be obtained ad hoc on a side wall, in any case it has to be such that it is possible to seed the cell sample from the outside ensuring access to the treated inner surface of the culture chamber.
  • Said cuvette can be constituted by a simple transparent container, i. e. of the type traditionally used in particle size measurements.
  • the noteworthy modification compared to the prior art is the treatment of the inner surface for cell adhesion.
  • the cuvette can be of the type traditionally used for Z-potential measurement, i. e. it can comprise a U-shaped channel adapted to accommodate at least part of the culture medium contained within the cuvette and a pair of electrodes for the application of an electric field within the U-shaped channel.
  • the culture chamber - provided with at least one inner surface conveniently treated for cell adhesion - can be realized in communication with the U-shaped channel, preferably under it.
  • the cuvette can also advantageously comprise isolation means for the reversible isolation of the culture chamber with respect to the U-shaped channel thereabove; the isolation means are operated during the Z-potential measurement so as to hermetically isolate the cell sample from the electric field applied to the U- shaped channel.
  • the treated inner surface of the cuvette can advantageously be realized so as to be removed from the rest of the cuvette in order to recover the cell sample at the end of the analysis of the particles. Since it is necessary, for treating the cells in culture, to avoid external contamination, the cuvette is preferably of the disposable type.
  • the cuvette ready for use is kept sterile inside a case; before the use it is extracted from the case and, when the analysis is finished, it is disposed and not re-used for another sample in order to avoid contamination.
  • the cuvette preferably comprises at least one lid to achieve, in use, sterile closure of the cuvette itself.
  • Figure 1 represents a diagram of the functional blocks integrated in a system for the realization of the method according to the invention
  • Figure 2 represents a perspective view of a cuvette, intended for size analysis, for the method according to the invention with lifted lid;
  • Figure 3A represents a front view of the cuvette of Figure 2 with lid in closure position;
  • Figure 3B represents an oblique axonometric projection at 45° of the cuvette of Figure 2;
  • Figure 4 represents a perspective view of the cuvette of Figure 2 with a first variant with holes of the lid;
  • Figure 5 represents a top view of the lid of the cuvette of Figure 4.
  • Figure 6 represents a side view of the lid of Figure 4 with suction/ replenishing ducts inserted in the holes;
  • Figure 7 represents a perspective view of the cuvette of Figure 2 with a second variant with holes of the lid;
  • Figure 8 represents a top view of the lid of the cuvette of Figure 7;
  • Figure 9 represents a side view of the lid of Figure 7 with suction/ replenishing ducts inserted in the holes;
  • Figure 10 represents a perspective view of a cuvette, intended for Z-potential analysis, according to the invention.
  • Figure 1 1 represents a front view of the cuvette of Figure 10;
  • Figure 12 represents a side view of the cuvette of Figure 10
  • Figure 13 represents a partial cut-away, side view of the cuvette of Figure 10;
  • Figure 14 represents a detail of the upper part of the cuvette of Figure 10 with lifted lid with inserted suction/ replenishing ducts
  • Figure 15 represents a perspective view of a lid of Figure 14 with inserted suction/ replenishing duct
  • Figure 16 represents a perspective view of a lid of the cuvette of Figure 10;
  • Figure 17 represents the detail of Figure 14 with lids realized in a variant
  • Figure 18 represents a front view of the cuvette of Figure 10 which shows isolation means built-in in the cuvette;
  • Figure 19A represents a front view of the lower part of the cuvette of Figure 10 which shows a detail of the isolation means of Figure 18 in an open configuration;
  • Figure 19B represents a front view of the lower part of the cuvette of Figure 10 which shows a detail of the isolation means of Figure 18 in a closed configuration;
  • Figure 20 represents a front view of a detail of the lower part of the cuvette of Figure 10 with a variant of the isolation means depicted in detail in open configuration
  • Figure 21 represents a front view of a detail of the lower part of the cuvette of Figure 10 with the isolation means depicted in detail in configuration closed by piston mechanism integrated in the instrument;
  • Figure 22 represents a front view of a detail of the lower part of the cuvette of Figure 10 with another variant of the isolation means depicted in detail, comprising an inflatable occluder with needle integrated in the instrument used to introduce air in the occluder;
  • Figure 23A represents a top view of the deflated inflatable occluder with needle inserted inside (open configuration);
  • Figure 23B represents a top view of the inflated inflatable occluder with needle inserted inside (closed configuration).
  • the term particle will be used to refer to any physical or biological object composed by an atomic or molecular aggregate, naturally occurring or produced by means of nanotechnologies, characterized by particular chemical and physical properties and by sizes that range from nanometers to tens of micrometers.
  • the term "particle” is univocally assigned the above- mentioned meaning.
  • the method can also be used to sort different particles in a formulation, i. e. for the massive purification of particles with defined size and charge.
  • reference numbers 1 and 1 ' identify cuvettes to be used in a method for the analysis of particles secreted and/ or endocytosed by cells in cell culture, according to the present invention.
  • Possible references to position employed in the description comprising references as anterior or posterior, in front of or behind, lower or upper, under or on, or similar expressions, will always be referred to the orientation of the previously said walls, corresponding to what depicted in Figures 2, 4, 7, 10 and 18.
  • Said cuvettes 1 , 1 ' are preferably sterilizable with gamma rays and made of transparent plastic material, such as polystyrene, which permits the light to pass through.
  • the side walls of the cuvette could also be made of quartz.
  • the cuvette 1 is specifically intended for measurements of particle size.
  • the cuvette 1 essentially consists of a box container, approximately parallelepiped-shaped and with square base, defined by walls that enclose a culture chamber 7. As showed in Figure 2, 3A and 3B, the cuvette has in particular an anterior wall 2, a posterior wall 3 opposed to the anterior wall 2, two side walls opposed to each other 4a and 4b, a bottom which in particular defines the seeding base 5, and an upper opening 6.
  • the cuvette 1 further comprises a removable lid 8, 8', 8" adapted to cover its upper opening 6 so as to prevent the passage of microorganisms that could contaminate the content of the culture chamber 7.
  • the seeding base 5 is preferably made of polystyrene, and has an inner surface 5a covered with polylysine to facilitate cell adhesion on it.
  • polylysine to facilitate cell adhesion on it.
  • Other materials and/ or substances known in the field can alternatively be used, alone or in combination to promote cell adhesion. Only by mean of example can be mentioned lysine, fibronectin and collagen.
  • the treatment of the inner surface 5a promotes cell adhesion on it, stimulating cell proliferation and viability. If the cell culture is kept in controlled atmosphere (37°C; 5% CO2) the cells continue to live and are able to carry on processes of secretion and absorption of particles from the culture medium.
  • the cuvette 1 can have a removable seeding base 5, for the recovery of the cell sample at the end of the analysis performed according to the method of the present invention.
  • the cell sample is inserted through a seeding opening 9 that corresponds with the upper opening 6; once the cells have been inserted together with the culture medium inside the culture chamber 7, the lid 8, 8', 8" is placed.
  • the cuvette 1 has a variant without holes of the lid 8.
  • the cuvette 1 has a first variant with holes of the lid 8', in which are done a suction hole 8a and a replenishing hole 8b.
  • a suction hole 8a and a replenishing hole 8b In the two holes are inserted by tight-fit respectively a suction duct 10a for the suction from the culture chamber 7 of the culture medium with the suspended cells and a replenishing duct 10b for the topping up of new culture medium within the culture chamber 7.
  • the suction holes 8a and the replenishing holes 8b are positioned on a diagonal of the square that defines the contour of the lid 8'.
  • the cuvette 1 has a second variant with holes of the lid 8", wherein suction holes 8a and replenishing holes 8b are positioned in an alternative configuration, on a median of the square.
  • the cuvette 1 ' is specifically intended for Z-potential measurements.
  • the cuvette 1 ' has a U-shaped channel 1 1 that causes a reduction of the capacity of the culture chamber 7 and an alteration of the upper part of the cuvette 1 ' compared to the parallelepiped shape, according to typical features of cuvettes related to Z-potential measurement.
  • the U-shaped channel 1 1 is circumscribed by the anterior wall 2, by the posterior wall 3, and by the two side walls 4a and 4b, and delimits at the top the culture chamber 7 with which it is in communication through a passage 12.
  • Each side wall 4a and 4b has a lateral protrusion 13, in correspondence of which an electrode 14 is positioned.
  • the cuvette 1 ' further comprises a closure upper surface 15 on which are done the suction hole 8a and the replenishing hole 8b of the culture medium.
  • the suction hole 8a and the replenishing hole 8b correspond with the two openings at the extremities of the U-shaped channel 1 1.
  • Figure 10 shows a pair of lids for hole 16 used for closing the suction hole 8a and the replenishing hole 8b.
  • FIG. 16 shows an alternative embodiment of the lid for suction hole 16a' and of the lid for replenishing hole 16b'.
  • the seeding opening 9 is obtained on one of the side walls 4a or 4b, in correspondence with the culture chamber 7, i. e. in proximity of the seeding base 5, and has a closable, small, inner lid 9a made of silicone (visible in Figure 1 1).
  • the cuvette 1 ' further comprises isolation means 17 of the culture chamber 7 adapted to occlude the passage 12 that connects the U-shaped channel 1 1 with the culture chamber 7.
  • some isolation means 17 can have the form of a rigid shaft preferably made of plastic and L-shaped.
  • Said rigid shaft comprises a vertical tract 17a which passes internally along the anterior wall 2 of the cuvette 1 ', a horizontal occluder 17b integral with the lower extremity of the vertical tract 17a and placed in correspondence with and insertable by tight-fit in the passage 12, and a horizontal extension 17c of the vertical tract 17a in correspondence with the upper extremity of the vertical tract 17a, parallel to the horizontal occluder 17b and coming out through an opening obtained in the anterior part 2 of the cuvette 1 '.
  • the rigid shaft comprising the vertical tract 17a, the extension 17c and the occluder 17b constitute the lever system that realizes the operating mechanism of these isolation means 17.
  • the extension 17c When the extension 17c is pushed toward the inside of the cuvette 1 ', it causes the horizontal tract 17b to advance and hermetically occlude the passage 12 ( Figure 19B). A movement of the extension 17c in the opposite direction causes instead the re-opening of the passage 12 ( Figure 19A).
  • the isolation means 17 as described above are integrated inside the cuvette and have an operating mechanism that is suited to be performed manually. It is useful to apply such a conformation of the cuvette when the instrument is not integrated in a thermostated block, i. e. it is accessible to the operator during the steps of the described method.
  • Figures 20-21 show an alternative embodiment of the isolation means 17'.
  • the isolation means 17' envisage a horizontal occluder 17b' that can be inserted from the outside of the cuvette 1 ' inside a channel 70 that emerges into the channel 12 and a rubber lid 70a hinged at the wall of the passage 12.
  • Figure 20 represents isolation means 17' in open configuration in which the rubber lid 70a occludes the access of the channel 70 to the passage 12
  • Figure 21 represents the isolation means 17' in closed configuration in order to isolate the cells during measurement in which the horizontal occluder 17b' is inserted in the channel 70, causing the rotation of the rubber lid 70a which then occludes the passage 12.
  • Figures 23A and 23B show a further embodiment of the isolation means 17" comprising an inflatable occluder 17b" having an inner cavity 18 circumscribed by an expandable material (e. g. rubber) that ensures the inflation of the occluder 17b" when air is injected under pressure in cavity 18.
  • an expandable material e. g. rubber
  • the cavity 18 is connected with the outside of the cuvette 1 ' through an inflation duct 18b inside of which there is a partition 18a. Inflation of the occluder 17b" is performed through insertion, from the outside, of a needle 19 inside said inflation duct 18b, through the partition 18a, till reaching the cavity 18.
  • the needle 19 is connected to a system with compressor, placed outside cuvette 1 ', which supplies air under pressure for the inflation of the occluder 17b".
  • Figure 23A shows the isolation means 17" in an open configuration in which the inflatable occluder is deflated and does not occlude the passage 12.
  • the needle 19 is inserted from the outside inside the inflation duct 18b and passing through the partition 18a it reaches the cavity 18; at this point the system with compressor is activated that, blowing up air in the cavity through the needle 19, inflates the occluder 17b" until the total occlusion of the passage 12.
  • Figure 23B shows instead the isolation means 17" in a closed configuration in which the inflatable occluder is inflated and the passage 12 is closed.
  • the last two embodiments of the isolation means 17' and 17" envisage an operating mechanism that needs an actualization system external to the test tube that interacts with it and is controlled by the computer system 22.
  • the method for the analysis of particles secreted and/ or endocytosed by cells in culture according to the present invention which employs the cuvettes 1 and 1 ' described above.
  • the method envisages in first place seeding a sample of cells suspended in a liquid culture medium within the culture chamber 7 of the cuvette 1 , 1 '. Once the cells have been introduced in the culture chamber 7, they will tend to settle adhering to the inner surface 5a of the seeding base 5.
  • the seeding procedure should be such as to fill only the culture chamber 7 (keeping the volume of the seeding medium under the passage 12) for seeding the cells, after about 2h from seeding the cells adhere to the polylysine layer 5a and the rest of culture medium can be inserted at a later time to completely fill the U-shaped channel (in this way the cells do not flow in the U-shaped channel).
  • the cuvette 1 , 1 ' in then inserted inside an incubation thermostated block 20.
  • Said thermostated block 20 consists in an incubation chamber which accommodates the cuvette 1 , 1 ' within which a controlled atmosphere in terms of temperature, CO2 partial pressure and humidity is maintained by means of a conditioning system which is typical of known incubators for cell culture.
  • a temperature of 37°C and a CO2 partial pressure equal to 5% are preferably maintained such as to keep the cells alive.
  • the cells in culture within the cuvette 1 , 1 ' held in controlled atmosphere are in an optimal condition to proliferate and interact with the surrounding culture medium producing and/o absorbing particles.
  • the method according to the present invention further envisages the measurement of physical and chemical features of the particles suspended in the culture medium within the cuvette 1 , 1 '.
  • the measurements are performed in a signal measurement block 21 that relates with the thermostated block 20 so as to perform the measurements without modifying the conditions of the controlled atmosphere in the thermostated block 20.
  • the signal measurement block 21 can be integrated or partially integrated within the thermostated block 20.
  • a computer system 22 such as for example a computer or a tablet, on which the specific software for real-time analysis and processing of measurement data are installed, is connected to a signal measurement block 21.
  • the computer system 22 further permits to implement the measurements according to the needs of the operator.
  • the performed measurements are relative to the size and Z-potential and fluorescence of the particles suspended in the culture medium within the cuvette 1 , 1 '.
  • the signal measurement block 21 comprises the measuring systems described in the prior art for measurement of hydrodynamic diameter and Z-potential of particles suspended in a liquid.
  • the cuvette 1 ' contrary to cuvette 1 that cannot be used for Z-potential measurement, can be used for both size and potential measurement.
  • the culture medium which is in the U-shaped channel 1 1 of cuvette 1 ' is isolated from the culture chamber 7.
  • the isolation is performed upon activation of the isolation means 17, 17' and 17" that causes the hermetical closure of the passage 12 between the U-shaped channel 1 1 and the culture chamber 7.
  • said isolation is fundamental to protect the cells attached to the seeding base 5 of the culture chamber 7, maintaining the electric field used for the Z-potential measurement within the U-shape channel 1 1.
  • the method further envisages the possibility to perform a fluorescence analysis of the particles suspended in the culture medium.
  • the signal measurement block 21 comprises also a flow cytofluorimetry system.
  • the particles suspended in the culture medium need to be previously labelled with specific fluorochromes.
  • the culture medium is collected from the cuvette 1 , 1 ' through the suction duct 10a and conveyed to the fluidic system of the flow cytofluorimetry instrument. After the analysis is completed the particles can be sorted on the basis of their signal by the sorting block.
  • the method further comprises sorting the analyzed particles on the basis of at least one of their measured physical and chemical features.
  • Said sorting step is in any case to be considered optional, even if it is strongly preferable in order to obtain sorted samples, each containing a single particle species (defined size or defined Z-potential).
  • Particle sorting is performed using a sorting block 23 that sorts the particles on the basis of the value of the measurement of size, Z-potential or fluorescence.
  • the criteria for sorting the particles are set by the user through the computer 22 which is also connected to the sorting block 23.
  • Particle sorting performed in the sorting block 23 preferably envisages the capture of the particles inside a capillary tube that slowly suctions the culture medium funneling the particles single-file, the specific measurement on the funneled single particle and the subsequent sorting of the particle on the basis of the measured value.
  • the particles sorted by the sorting block 23 are then stocked in a fractioning block 24 that keeps the particles separated within different containers maintained at controlled temperature.
  • the sorting block 23 can integrate a device as the one used by the FACScaliburTM machine according to the prior art that permits to select particles on the basis of fluorescence measurement; i. e. as soon as the signal measurement block 21 measures the fluorescence of the single particle, it is conveyed in separated containers on the basis of the measured signal.
  • the particles are extracted from the cuvette 1 , 1 ' through the suction duct 10a, along which they are funneled.
  • the signal measurement block 21 has also a system for measuring the size and/ or Z-potential of each particle that passes through the duct. After the measurement has been performed, the sorting block 23 directs the particle directly into a specific container of the fractioning block 24 on the basis of the result of the measurement of the size or Z-potential.
  • the sorting block 23 can advantageously make use of a system of the type disclosed by CellPly which permits particle transport within ducts using the electric field generated by electrodes placed on the outer wall of the duct.
  • the sorted particles are extracted from the cuvette 1 , 1 ' together with part of the culture medium contained in the culture chamber 7.
  • the extracted culture medium is then topped up by a constant volume maintainer block 25 that pumps new culture medium within the culture chamber 7 of the cuvette 1 , 1 ' through the replenishing duct 10b ensuring the maintenance of a constant volume of the culture medium within the culture chamber 7 for the entire duration of the analysis.
  • a method for the analysis of particles secreted and/ or endocytosed by cells in cell culture solves the technical problem and gains several advantages, among which first of all to foresee the cell culture directly within the cuvette used for measuring the particle features.
  • the cuvettes currently used are not configured to permit cell growth.
  • the method according to the invention permits the sorting of the particles contained in the culture medium on the basis of the physical and chemical features such as the size, the Z-potential and the fluorescence. Said characterization of the particles over the time can be used to study the variation over the time of the number of specific particles contained in the culture medium.
  • the latter type of analysis is useful to build the so-called uptake curves relative to the absorption over the time of specific particles by the cells in culture or to perform a titration of the amount of virus produced by infected cells over the time monitoring the increase of the number of particles produced in the culture medium that correspond to the virus size, but also the variation over the time of the number and the composition (diameter and potential) of the particles secreted/ esocytosed by the cells.
  • the method according to the present invention can also envisage the use of the technique of flow cytofluorimetry to characterize and/ or sort the particles by real-time identification of specific structures labelled with fluorochromes comprised in the particles, such as for example proteins or nucleic acids contained in exosomes produced by the cells.
  • the advantageous applications of the method according to the present invention in the field of the research on exosomes or on spheroids are discussed more in detail hereinafter.
  • the method permits to monitor and to sort from the total pool of exosomes/ spheroids, produced by cells in culture, specific subgroups of exosomes/ spheroids depending on the needs of the study; that have identical features on one, two or three of the proposed parameters (fluorescence, size and surface charge).
  • This aspect of the method permits to conduct specific analysis only on the subclass of exosomes/ spheroids of interest for the researcher (examples of subsequent analysis on purified samples: quantitative and qualitative assays of RNA, DNA, miRNA and proteins).
  • the method permits to analyze in real-time the features of the exosomes/ spheroids produced, so that it is possible to develop kinetics of exosomes release and growth curves of the spheroids.
  • the method permits to follow in real time the decrease/ increase of the number of exosomes produced by cells in culture, proving the role of the protein in the exocytosis process from a qualitative and a quantitative point of view.
  • the method can also be used to test a molecule known to inhibit/ promote exosome production, permitting to analyze the effect of the molecule in real time (activity kinetics of the molecule), possibly also on a specific subpopulation of the produced exosomes.
  • the effect of the molecule on the cell can be: inhibiting/ promoting only the production of the exosomes of a specific size or surface potential; varying the amount of exosomes belonging to two populations which are specific for size or surface potential, letting unchanged the total number of produced exosomes; etc.
  • the method uses two monitoring or sorting parameters (fluorescence and size or fluorescence and surface potential) with the method to determine: - if the protein of interest is expressed at specific points of the formation of the spheroid in real-time; if the protein is comprised only in spheroids of specific size or potential or in all spheroids so it is essential for the formation of the spheroid itself.
  • the possibility to measure the fluorescence intensity and not only the presence of fluorescence permits to use the method to determine the variation of concentration of the protein of interest in the spheroid population, for example the protein is much more abundant in spheroids of small size but it is comprised also in the spheroids of larger size.
  • the method can also be employed in other research and diagnostic fields.
  • virology for example with the method it is possible to calculate in realtime the viral titer and the kinetics of the exiting of the virus from the cell from the instant of the infection examining only the size, while the size related to the surface potential can be used to value the homogeneity of the viral stock.

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Abstract

La présente invention concerne également un procédé d'analyse de particules sécrétées et/ou endocytosées par des cellules dans une culture cellulaire, comprenant les étapes suivantes consistant : à ensemencer un échantillon cellulaire à l'intérieur d'une cuvette (1, 1') en présence d'un milieu de culture; à placer ladite cuvette (1, 1') dans une atmosphère contrôlée à l'intérieur d'un bloc thermostaté (20) de manière à maintenir l'échantillon cellulaire en vie et à favoriser la production et/ou l'absorption de particules vers et/ou à partir du milieu de culture; à mesurer en temps réel au moyen d'un bloc de mesure de signal (21) au moins une caractéristique desdites particules immergées dans ledit milieu de culture; à interpréter et à analyser en temps réel au moyen d'un système informatique (22) connecté audit bloc de mesure de signal (21) les mesures de ladite caractéristique au moins desdites particules.
PCT/EP2017/074950 2016-10-03 2017-10-02 Procédé d'analyse de particules sécrétées et/ou endocytosées par des cellules en culture cellulaire WO2018065349A1 (fr)

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