Tissue dissociation method and associated kit

Abstract

The invention relates to a kit and to an associated tissue dissociation method (4), comprising: • arranging a biological tissue (3) in a closed container (2) containing at least one ball (24); • adding a liquid medium (22) containing at least one tissue dissociation enzyme (23) that is capable of being active in dissociating the biological tissue (3) in the closed container (2); • carrying out at least one tissue dissociation cycle (42), in a ball stirring device (1), wherein the closed container (2) is stirred so that the at least one ball (24) induces a shearing effect in the volume of the liquid medium (22), so as to obtain a suspension (33) of cells comprising at least 20% viable dissociated cells (31) from the biological tissue (3).

Description

[0001] “Tissue dissociation process and associated kit”

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of tissue dissociation. It finds particularly advantageous application in biopharmaceutical research, the fields of cell therapy, regenerative medicine, development of biomedicines, elucidation of pathologies and tissue engineering.

[0004] STATE OF THE ART

[0005] Tissue dissociation aims to isolate primary cells for numerous biomedical and therapeutic applications. To obtain suspensions of living individual cells, tissues are treated in a gentle enough manner to successfully dissociate the cells while preserving their viability (ability to be cultured and preservation of their physiological activity).

[0006] Efficient tissue dissociation, for example, allows the characterization of cellular composition for heterogeneous tissues and the study of the behavior of individual cell populations in healthy and / or diseased tissues. This preparation step (transition from tissue to isolated cells) can be used for disease diagnosis based on single-cell sequencing, as well as for the study of predicting response to therapy and selecting a treatment.

[0007] There are three main types of methods for tissue dissociation, which can be combined. These include: enzymatic dissociation, chemical dissociation, and mechanical dissociation. Enzymatic dissociation is the process of using tissue dissociating enzymes configured to digest cut pieces of tissue, thereby releasing the cells from the tissue. Many different types of enzymes are used in this process and they can also be used in combination (Trypsin, Collagenase, etc.). Some enzymes are more effective with certain tissues, so it is best to identify the enzyme or combination of enzymes for each specific tissue type.

[0008] Chemical dissociation works on the principle that cations help maintain intercellular bonds and the intracellular matrix. By introducing EDTA or EGTA, which binds these cations, the intercellular bonds are broken.

[0009] Finally, mechanical dissociation allows the tissue to be cut into small pieces and, coupled with gentle agitation, completes tissue disintegration.

[0010] The selection of a dissociation method can be based on the tissue type, tissue origin, and the methods that have been shown to be most effective in the literature. The goal of most tissue dissociation processes is to obtain the largest quantity of cells and the most viable cells possible, so that the dissociation process or system does not negatively impact cell viability and / or function.

[0011] Choosing the right method or combination of methods remains complex. There are many conflicting opinions in the scientific literature. Therefore, there is a need for a standardized protocol for tissue dissociation, for example, according to tissue type.

[0012] A tissue dissociation process is known based on the separation of cells by a rotation system inside a closed container. The plastic tubes containing the sample include a rotor assembly which, driven by the machine's motor, rotates and participates in the disruption of the tissue by compressing the tissue against protrusions arranged on a stator. By lamination along a shear plane between the rotor and the stator, the tissue is disintegrated, allowing the recovery of living cells. For several years, the automated technique that has been used has been the GentleMACS™ system from Miltenyi Biotec™, for example described in document EP2540394 A1. Due to this lamination process, this solution can however induce significant heating in the shear plane between the rotor and the stator. In addition, this process requires the acquisition of equipment specific to the tissue dissociation application.

[0013] An object of the present invention is therefore to propose a tissue dissociation solution which is improved compared to existing solutions, and in particular which makes it possible to overcome at least one of the aforementioned drawbacks.

[0014] Other objects, features, and advantages of the present invention will become apparent from the following description and accompanying drawings. It is understood that other advantages may be incorporated.

[0015] SUMMARY

[0016] To achieve this objective, according to a first aspect, a tissue dissociation method is provided comprising:

[0017] - an arrangement of a biological tissue in a closed container comprising at least one bead, - an addition of a liquid medium comprising at least one tissue dissociation enzyme capable of being active for the dissociation of the biological tissue in the closed container,

[0018] - at least one tissue dissociation cycle, in a bead stirring apparatus, in which the closed container is stirred so as to induce, by the at least one bead, a shearing effect in the volume of the liquid medium, so as to obtain a cell suspension comprising at least 20%, and preferably at least 50%, of viable cells dissociated from the biological tissue.

[0019] Thus, the method uses a ball mill or equivalently a ball homogenization device, also referred to as a ball mill, conventionally used for the preparation of chemical or biological samples by grinding and destroying cells. The device used is thus not dedicated and optimized specifically for tissue dissociation, unlike existing solutions. This therefore avoids a considerable additional investment for laboratories and companies wishing to perform tissue dissociation. The invention makes it possible to use a single device to perform both grinding / homogenization of biological tissues and tissue dissociation, while preserving the performance expected with reference instruments.

[0020] Generally speaking, homogenizers that use the bead-beating principle to grind biological samples are used too powerfully to be able to dissociate living cells. Unlike conventional tissue dissociation equipment, bead-beating homogenizers were designed to generate a bead-beating force on the samples, powerful enough to fragment cells and isolate intracellular contents. Using a bead-beating homogenizer for tissue dissociation is therefore completely counterintuitive.

[0021] Here, the bead-shearing apparatus is used according to a bead-shearing principle. Performing tissue dissociation with a bead-shearing apparatus or, equivalently, a bead-shearing apparatus is a technical challenge that has the following advantages over conventional techniques: time savings due to parallelization, improved reproducibility because the process is more homogeneous, and speed of execution because the process can be more efficient thanks to the volume work (and therefore a potentially larger "apparent" surface area).

[0022] Furthermore, compared to conventional tissue dissociation solutions, the dissociation is carried out here in the volume (and therefore in three dimensions) of the liquid medium of the closed container, by the volume shear induced by the movement of the bead(s). The dissociation therefore results from a random and homogeneous process in the volume of the liquid medium. This is therefore distinguished from a two-dimensional dissociation by lamination phenomenon, in a lamination plane between a rotor and a stator. Tissue dissociation by volume shear provides a more homogeneous medium facilitating the dispersion of heat created locally by the shear forces. Furthermore, the duration of mechanical agitation and the level of stress caused to the cells are thus reduced. The quantity of debris in the resulting cell suspension can also be reduced.Another aspect relates to a tissue dissociation kit for carrying out the method according to any one of the preceding claims, comprising:.

[0023] - the closed container comprising at least one ball,

[0024] - at least one tissue dissociating enzyme.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] The aims, objects, as well as the characteristics and advantages of the invention will emerge more clearly from the detailed description of an embodiment thereof which is illustrated by the following accompanying drawings in which:

[0027] Figure 1 represents a schematic overview of a ball homogenizer according to an exemplary embodiment.

[0028] Figure 2 represents a closed container comprising beads, the biological tissue and the liquid medium comprising a cell dissociation enzyme, according to an exemplary embodiment.

[0029] Figures 3A and 3B schematically illustrate the tissue dissociation process, according to an exemplary embodiment.

[0030] Figures 4A and 4B show schematic diagrams respectively for: an example in which the first dissociation threshold and the second cell death threshold delimit a dissociation window, and an example in which the first dissociation threshold and the second cell death threshold are such that the dissociation window does not exist.

[0031] Figures 5A and 5B represent schematic diagrams illustrating the shear effects in a bead-shearing case.

[0032] Figures 6A and 6B represent schematic diagrams illustrating the shear effects in a bead-beating case.

[0033] Figure 7 illustrates the dissociation kit according to an exemplary embodiment.

[0034] The drawings are given as examples and are not limiting of the invention. They constitute schematic representations of principle intended to facilitate the understanding of the invention and are not necessarily to the scale of practical applications. In particular, the relative dimensions of the elements illustrated, and in particular of the cells, the beads and the container are not representative of reality.

[0035] DETAILED DESCRIPTION

[0036] Before commencing a detailed review of embodiments of the invention, optional features which may possibly be used in combination or alternatively are set out below.

[0037] According to one example, during the at least one tissue dissociation cycle, the closed container is agitated according to agitation parameters configured to be within a dissociation window, said window being delimited by a first threshold for dissociation of the biological tissue into dissociated cells, the agitation parameters being insufficient below the first threshold to induce tissue dissociation, and by a second threshold called cell death, the agitation parameters being too intense beyond the second threshold and resulting in cell viability of less than 50% of the dissociated cells. Thus, the operating parameters of the bead homogenizer are chosen so as to limit cell destruction by defining an operating range allowing the obtaining of the cell suspension comprising at least 50% of viable cells dissociated from the biological tissue.More specifically, the stirring frequency, stirring speed, and where appropriate the stirring duration can be chosen to fall within the dissociation window.

[0038] According to one example, during the at least one tissue dissociation cycle, the closed container is agitated according to:

[0039] - a stirring frequency F between 4 Hz and 45 Hz, preferably between 20 and 42 Hz, and

[0040] - an average stirring speed v between 0.2 m / s and 2.5 m / s.

[0041] The container is then agitated with a repeated movement and at an average speed optimizing tissue dissociation and cell viability within the dissociation window. Below this, the dissociation of cells from the tissue will be less satisfactory, and beyond this the risk of significant cell mortality is increased.

[0042] According to one example, for each tissue dissociation cycle, the closed container is stirred for a time ti of between 3 and 30 seconds, preferably between 5 and 10 seconds. This range of stirring times makes it possible to optimize tissue dissociation and cell viability in the dissociation window, particularly in synergy with the stirring frequency and average speed above.

[0043] According to one example, the at least one bead occupies a volume proportion of between 0.1% and 10% of the volume of the closed container. This volume proportion makes it possible to minimize the probabilities of significant impact between the bead(s) and the wall of the container, while generating a shear force sufficient to allow cell dissociation.

[0044] In one example, the container contains between 1 marble and 15 marbles.

[0045] According to one example, the at least one ball has a density of between 0.5 g / cm3 and 8 g / cm 3 . This density range allows for movement in the fluid, suitable for generating sufficient shear force for cell dissociation. According to one example, the at least one bead is based on or made of a material selected from the group consisting of latex, glass, ceramic, metal, and stainless steel. These materials are particularly suitable for being in contact in solution with the biological material.

[0046] According to one example, the at least one ball has at least one dimension, for example a diameter, between 0.1 mm and 5 mm, preferably between 1 mm and 5 mm, and even more preferably between 2 and 5 mm. The size of the balls impacts their movement in the liquid medium during stirring, and in particular their speed of movement and the resulting impacts.

[0047] In one example, the closed container has a volume of between 2 and 50 mL, preferably between 2 mL and 15 mL. The volume of the tube impacts its dimensions, and therefore the collisions between the bead(s) and the container wall that can occur. These volumes are advantageous for optimizing tissue dissociation and cell viability, while being compatible with the container volumes commonly used in bead homogenizers. In one example, the liquid medium occupies a volume fraction of the closed container of between 0.3 and 0.7. The proportion of liquid medium in the container has an effect on the dissociation efficiency. Less liquid promotes homogenization.

[0048] According to one example, during the at least one tissue dissociation cycle, the closed container is agitated according to a precessional movement. The container containing the biological tissue, typically on a support tray in the homogenizer, is thus agitated according to a movement around a central axis, the axis describing a cone during this movement. A precessional movement induces a movement of the container(s) in a direction perpendicular to this movement. Some ball homogenizers implement an oscillating translational or rotational movement to drive the containers or tubes containing the sample. The precessional movement offers an advantage over the direction of movement of the ball(s). Their movement can be more random in the volume and not simply unidirectional as on other homogenizers.

[0049] According to one example, the method comprises, prior to each tissue dissociation cycle, an incubation of the biological tissue in the presence of the at least one tissue dissociation enzyme, preferably in the closed container. The action of the enzyme, prior to the mechanical action of the bead(s), is thus promoted. This facilitates tissue dissociation in synergy between an enzymatic action and a mechanical action.

[0050] In one example, biological tissue is disposed in a plurality of closed tubes comprising at least one bead. The biological tissue may be the same or different between the tubes. The liquid medium comprising at least one tissue dissociating enzyme may be added to each of the plurality of tubes. The liquid medium and / or the dissociating enzyme may be the same or different between the tubes.

[0051] According to one example, during the at least one tissue dissociation cycle, in the ball mill configured to receive the several tubes, the several closed tubes are agitated, preferably simultaneously, so as to induce, by the at least one ball, a shearing effect in the volume of the liquid medium, so as to obtain a cell suspension comprising at least 20% of viable cells dissociated from the biological tissue.

[0052] Cell dissociation can thus be parallelized, for one or more tissues for example. Screening of cell dissociation conditions can be carried out for example.

[0053] Viable and dissociated cell suspension can be obtained in the closed tube after at least one tissue dissociation cycle.

[0054] In one example, the closed tube is configured such that viable and dissociated cells remain in the closed tube until the completion of the at least one tissue dissociation cycle.

[0055] In one example, the liquid medium is contained in the closed tube until the cell suspension is obtained. In one example, there is no circulation of the liquid medium out of the closed tube.

[0056] In one example, the tube is closed with a cap, for example the cap is clipped or screwed onto the tube.

[0057] According to one example, the tube is not connected to a fluidic circuit for circulating the liquid medium. According to one example, the method comprises several tissue dissociation cycles, these cycles preferably being separated by non-agitation phases. This makes it possible to temporally distribute the mechanical action on the sample. This promotes the heat dissipation of local heating induced by collisions between the bead(s) and the wall of the container. Cell viability is therefore promoted. This also makes it possible to facilitate an enzymatic action on the biological tissue, by allowing the at least one enzyme to act between several phases of mechanical action, and therefore between several stages of degradation of the biological tissue.

[0058] According to one example, the method further comprises, prior to adding at least one tissue dissociation enzyme to the closed container, selecting the at least one tissue dissociation enzyme from a plurality of distinct tissue dissociation enzymes, and where appropriate preparing an enzyme mixture from the selected tissue dissociation enzymes.

[0059] In one example, the kit comprises several different tissue dissociation enzymes, so that the at least one tissue dissociation enzyme to be added to the tissue dissociation process can be selected. The selection of the enzyme or enzyme mixture allows the process to be adapted to the biological tissue to be dissociated. The process and kit can thus be applicable to a large number of different biological tissues.

[0060] According to one example, the at least one tissue dissociating enzyme is selected from the group consisting of dispase, collagenase, DNase, trypsin, papain, hyaluronidase, elastase.

[0061] In one example, the kit includes the stirring apparatus or equivalently the ball homogenization apparatus.

[0062] In the remainder of the description, the term "on" does not necessarily mean "directly on". Thus, when it is indicated that a part or member A is supported "on" a part or member B, this does not mean that the parts or members A and B are necessarily in direct contact with each other. These parts or members A and B may be either in direct contact or be supported on each other by means of one or more other parts. The same applies to other expressions such as, for example, the expression "A acts on B", which may mean "A acts directly on B" or "A acts on B by means of one or more other parts".

[0063] For the purposes of this disclosure, the expression "A and / or B" means (A), (B), or (A and B). For the purposes of this disclosure, the expression "A, B and / or C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A and B and C).

[0064] A parameter that is "substantially equal to / greater than / less than" a given value means that this parameter is equal to / greater than / less than the given value, within plus or minus 10% of this value. A parameter that is "substantially between" two given values ​​means that this parameter is at least equal to the smallest given value, within plus or minus 10% of this value, and at most equal to the largest given value, within plus or minus 10% of this value.

[0065] The tissue dissociation method 4 and the associated kit 5 are now described according to particular embodiments. The term "sample grinder", or equivalently "homogenizer" or "stirring device", broadly refers to any type of translational, rotary or precessional device that processes samples, including not only high-power shaker-type grinders as described herein, but also other laboratory equipment such as vortexers, shakers and agitators.

[0066] As illustrated by Figure 1 by way of example, the homogenizer 1 comprises in a completely conventional manner a device designed to carry one or more closed containers 2, or equivalently one or more tubes 2, containing samples. The holding support 10 can be configured to receive several tubes. The tubes 2 can be arranged on a holding support 10 driven in precession. The tubes 2 are generally arranged on a holding support 10 driven in rotation, and more particularly in precession, by a shaft

[0067] 11 itself rotating. The support 10 is mounted on the homogenizer 1 via the shaft 11 itself mounted on a frame 13. The device 1 and the tubes 2 can be arranged under a cover

[0068] 12 pivoting between an open position and a closed position. During the precession movement, the support 10 is driven in rotation, and more particularly in precession, around a central axis A, the axis describing a cone of angle a during this movement, inducing a movement of the tubes in a direction perpendicular to this movement. The samples are thus subjected to a precession movement.

[0069] In the tissue dissociation method 4, and as illustrated for example by FIG. 2, a biological tissue 3 is placed in a liquid medium 22 in a tube 2. The tube 2 is closed, and therefore the tube 2 is fluid-tight. The tube 2 is preferably sterile. The tube 2 may comprise a lower portion 20 having one or more walls, and closed by a stopper 21. The tube 2 comprises at least one ball 24 configured to exert shear forces in the liquid medium 22, to induce tissue dissociation. In the following, it is considered, without limitation, that the tube 2 contains several balls 24. Note that unless incompatibility exists, the following characteristics may apply to the example in which the tube comprises a single ball 24.

[0070] As illustrated for example by Figures 3A and 3B, efficient tissue dissociation makes it possible to obtain a suspension 33 of viable and functional cells 31. In a completely conventional manner, the biological tissue 3 comprises non-dissociated cells 30 surrounded by an extracellular matrix. The biological tissue 3 may comprise cells 30 of various types, depending on the nature of the biological tissue 3.

[0071] Effective tissue dissociation corresponds to:

[0072] - a sufficient concentration of dissociated cells; below this, dissociation is partial and insufficient. For example, a sufficient concentration may be significantly greater than or equal to the threshold value allowing the analysis to be effective, depending on the given application. This threshold value may be between 10 4 and 10 6 cells per mL,

[0073] - a sufficient percentage of live dissociated cells: beyond this, the tissue is considered to be homogenized (or ground in an equivalent manner). For example, a percentage of viability may be significantly greater than or equal to the threshold necessary to guarantee the effectiveness of the analysis, depending on the given application. This threshold is preferably greater than or equal to 50% of the cells dissociated from tissue 3 (this percentage therefore not counting any cells 30 remaining in tissue 3 after dissociation).

[0074] It is therefore a question, in synergy between beads 24 inside the tube 2 and the stirring movement, and preferably precession, of generating a fluid shear, with a high but not too high speed gradient, to create a suspension 33 of viable and functional cells 31 via bead-shearing and not bead-beating. If the stirring conditions are too gentle, then the quantity of dissociated cells 31 is too low, the cell suspension 34 is then not adequate. If the stirring conditions are too strong, then the quantity of dissociated cells 31 is high, but the mortality too high with too many lysed cells 32. The cell suspension 35 is then not adequate either.

[0075] For this, and as illustrated for example in Figure 3B, the method 4 comprises the addition of the biological tissue 3 to be dissociated into the tube 2 comprising the beads 24. A liquid medium 22 is added, comprising one or more tissue dissociation enzymes 23. In the method 4, a mechanical action by the beads 24 is thus associated with an enzymatic action thanks to the addition of one or more enzymes 23 into the liquid medium. Cell viability also depends on the liquid medium 22 in which they are located. The liquid medium 22 is therefore based on or made of a cell culture medium. The appropriate enzyme(s) and a culture medium 22 are preferably used to facilitate the dissociation and preserve the integrity of cells 31. The appropriate culture medium for a given tissue can be identified without difficulty by those skilled in the art, for example from the literature or pre-established protocols.

[0076] The dissociation enzymes may comprise enzymes configured to facilitate the dissociation of cells 30 from the extracellular matrix. These enzymes may comprise proteases, collagenases, dispases, DNases, trypsins, papains, hyaluronidases and elastases. These enzymes may be adapted to the type of biological tissue 3. For this, the method 4 may comprise a step 40 of selecting the enzyme(s) to be used, depending on the biological tissue 3. The enzymes 23 may be added to the liquid medium 22 prior to the biological tissue 3 or following its addition to the liquid medium 22. For this and as illustrated in FIG. 7 for example, the kit 5 may comprise the tube 2 comprising the beads 24, and several enzymes 23. The kit 5 may thus be adapted to numerous tissues 3.

[0077] Prior to at least one and preferably each tissue dissociation cycle 42, the method 4 may comprise an incubation 41 of the tube 2 to promote the enzymatic action. For this, the tube 2 may be placed in an incubator at a defined temperature T (for example substantially equal to 37°C) for a duration t2. The temperature T and the duration t2 may be adapted according to the biological tissue 3.

[0078] During a tissue dissociation cycle 42, the tube 2 is agitated, preferably with a precessional movement, for a duration ti, at an agitation frequency F and an average agitation speed v. The method 4 may comprise several tissue dissociation cycles 42, preferably separated in time by non-agitation phases 43. By non-agitation, it is meant that the tube 2 is not agitated in the ball homogenizer 1. Another form of agitation, for example manual, is possible in these phases 43. Equivalently, it can be said that the successive dissociation cycles 42 are temporally separated. Successive dissociation cycles 42 may be separated by incubations 41, as illustrated by way of non-limiting example.

[0079] As illustrated in Figure 1, several tubes 2 may each comprise a tissue 3 and at least one bead 24 and a liquid medium 22 for cell dissociation. These tubes may be agitated according to the methods described, and in particular simultaneously.

[0080] The method 4 may then comprise a step 44 or a set of steps 44 allowing the recovery and / or analysis of the cell suspension obtained. For example, the beads 24 may be removed from the cell suspension 33 by filtration. This initial filtration step will further allow the removal of large pieces of residual (undissociated) tissue.

[0081] The method 4 may further comprise, and preferably following filtration, centrifugation and / or rinsing steps to recover isolated and living cells 31. The method may further comprise an evaluation of cell viability, for example by counting isolated and living cells 31 compared to the number of dead cells 32. Any method for evaluating cell viability may be envisaged, for example the Trypan blue method or by flow cytometry.

[0082] The parameters of process 4 can be defined to enable efficient tissue dissociation, and thus a suspension of viable and functional cells. Defining and optimizing a set of parameters (e.g., composition, size and quantity of beads, tube volume, composition of the enzyme solution, and stirring programs - speed, frequency, and duration) can optimize tissue dissociation and cell viability.

[0083] Tissue dissociation may be governed by a set of stirring parameters and geometric parameters defining a dissociation window 422, making it possible to obtain a cell suspension 33 suitable for the intended application. As illustrated for example by FIG. 4A, the dissociation window 422 may be on the one hand delimited by a first dissociation threshold 420, below which the tissue is not dissociated 36 or the number of dissociated cells in suspension 34 is not sufficient, as described previously. The dissociation window 422 may on the other hand be delimited by a second cell mortality threshold 421, above which too high a mortality of the cells 32 is obtained, in a suspension 35: there is then homogenization.

[0084] Inappropriate agitation parameters and geometric parameters can lead to a situation where dissociation is insufficient while the percentage of living cells is already below the second threshold 421, as illustrated for example in Figure 4B. There is then no tissue dissociation.

[0085] Before detailing examples of more specific parameters, the phenomena in solutions on either side of the second threshold 421 are described by way of example, with reference to Figures 5A to 6B, to better illustrate the difference between a bead-shearing dissociation regime and a bead-beating homogenization regime.

[0086] As illustrated by Figures 5A and 5B, the agitation of the tube 2 induces a movement of the balls 24 in the liquid medium 22 which in turn creates a fluid shear force in the medium 22. The fluid shear force Fois can be: with :

[0087] - 5 the distance between balls,

[0088] - pi the viscosity of the liquid medium 22

[0089] - Vb the speed of the ball can be given by the following relation on the drag force: with :

[0090] - mb the mass of the ball,

[0091] - pf: density of the fluid,

[0092] - Sb the section of the ball,

[0093] - Cx the drag coefficient of the ball.

[0094] When the speed of the beads 24 exceeds a certain threshold, this force exceeds the shear strength of the bond between cell 30 and extracellular matrix in the tissue 3, the cell 30 then dissociates from the cellular tissue 3. This threshold 420 is the limit of first dissociation, which can be determined to the first order by the stirring parameters (and in particular stirring frequency, speed and duration), as well as the characteristics of the biological sample and the enzymatic cocktail, to the second order by the characteristics of the tube 2, the liquid medium 22 and the beads 24.

[0095] From this threshold, the dissociation efficiency can be defined by a dissociation rate per cycle. An increase in the agitation frequency and / or the agitation duration and / or the agitation speed increases the concentration of dissociated cells 31. The dissociation rate can more particularly be determined to the first order by the characteristics of the tube 2, the liquid medium 22 and the beads 24, to the second order by the agitation speed.

[0096] In homogenization mode, the agitation of the tube 2 and the movement of the balls 24 in the resulting liquid medium 22 also induce collisions between the balls 24 and the biological tissue 3, as illustrated for example in Figure 6A. The force of a mechanical impact Fcoi occurring over a time dt can be: v b Fcol K m b fa

[0097] When the speed of the balls 24 exceeds a certain threshold, the force of these collisions exceeds the breaking strength of the cell 30 or 31, the cell is then destroyed 32. The shear force described above can also exceed this strength if the distance between balls is sufficiently small, leading to a similar effect, as illustrated for example in Figure 6B. This threshold 421 is the limit of cell mortality. This threshold can be determined to the first order by the stirring speed and the characteristics of the biological sample, to the second order by the characteristics of the tube 2, the liquid medium 22 and the balls 24.

[0098] From this threshold 421, the homogenization efficiency can be defined by a homogenization rate per cycle. An increase in the stirring frequency and / or the stirring speed and / or the stirring duration further reduces the percentage of living cells. The homogenization rate can more particularly be determined to the first order by the characteristics of the tube 2, the liquid medium 22 and the beads 24, to the second order by the stirring speed.

[0099] The agitation of the tube 2 and the resulting movement of the balls 24 in the liquid medium 22 also induce collisions between the balls 24 and the ball and wall of the tube 2. These collisions release energy which increases the temperature of the liquid medium 22. The temperature of the liquid medium 22 can increase with the frequencies, speed and duration of agitation and can be dependent on the characteristics of the tube 2, the liquid medium 22 and the balls 24.

[0100] When the temperature exceeds a certain threshold, cells can degrade and die. This threshold is mainly determined by the characteristics of the biological sample. An increase in the stirring time and / or the temperature of the medium 22 can therefore reduce the percentage of living and dissociated cells 31 .

[0101] From the above and by the usual equations of fluid mechanics, the following parameters can optimize tissue dissociation and cell viability. These parameters are grouped in the form of a table and can be used separately or in combination with all or part of each other.

[0102] [Table 1]

[0103] [Table 2]

[0104] The agitation frequency is defined as the number of movements per second, the movement being a rotation, a translation, a precession, a combination of these movements or any other movement repeating cyclically. It is indicated here in Hz to correspond generically to different types of ball homogenizer. In one example, the agitation frequency is between 1000 rpm and 5500 rpm (rotations per minute). The agitation speed, or average agitation speed, is defined as the average speed of the tube 2 during its movement. Since the movement is cyclical, it may be accelerated and decelerated cyclically and therefore its instantaneous speed is not included in the range described. This is why an average speed is used, taken over the entire cycle of the movement.

[0105] The agitation duration considered corresponds to the duration over all the movements of the tube, in a cycle 42 of tissue dissociation.

[0106] These parameters can, quite advantageously, be used in combination together to optimize tissue dissociation and tissue viability for a large number of different biological tissues 3.

[0107] Specific examples of tissue dissociation are now described.

[0108] In the following examples, following the tissue dissociation process, filtration through a 70 μm porosity filter was performed. The filter was then washed with DMEM / F-12, HEPES at room temperature. The filtrate and filter wash solution were then centrifuged and resuspended in DMEM / F-12, HEPES + 2% FCS. Trypan blue was then added to count live and dissociated cells 31 and dead cells 32. Cell viability was further analyzed by flow cytometry.

[0109] Identification of ball parameters

[0110] The first tests below were carried out on whole pinkie worms.

[0111] Initial tests have identified, based on a qualitative visual assessment, four combinations of the most promising beads for maintaining a high relative percentage of living cells.

[0112] - CK28 beads (3 or 7 zirconium oxide beads of 2.8 mm diameter) in 2.5 mL of PBS and a protocol of 1.4 m / s at 24.9 Hz x 10 seconds,

[0113] - CK50 beads (1 or 3 zirconium oxide beads of 5.0 mm diameter) in 2.5 mL of PBS and a protocol of 1.4 m / s at 24.9 Hz x 5 seconds.

[0114] These combinations showed a considerable amount of live cells and little debris, particularly the CK50 beads.

[0115] For CK28 beads, tests with a quantity of beads greater than 15 showed a mortality rate of more than 90%.

[0116] CK14 beads (1.4 mm diameter zirconium oxide) failed to grind the sample. We consider that a longer homogenization cycle would lead to an increase in the cell mortality rate. This type of bead is therefore excluded for further testing.

[0117] [Table 3]

[0118] The best combination of beads was tested for several agitation speeds. The tests made it possible to identify, on the basis of a quantitative visual assessment, two combinations of beads that seem the most promising for maintaining a percentage greater than 20% of living cells: - CK50 beads (1 bead) in 2.5 mL of PBS and a protocol of 1.4 m / s at 24.9 Hz x 5 sec

[0119] (25%)

[0120] - CK50 beads (3 beads) in 2.5 mL of PBS and a protocol of 1.4 m / s at 24.9 Hz x 5 sec (22%)

[0121] [Table 4]

[0122] A new series of tests was therefore carried out to validate the protocol with another type of tissue (zebrafish muscle tissue). The tests demonstrated that the results obtained with the preparation of 3 CK50 beads on a precessional bead homogenizer (Precellys® Evolution, marketed by Bertin) are similar to those obtained with the GentleMACS™. In terms of cell viability, the results obtained with the fish samples are better (> 40% viability, see table below). Visually, under the microscope, the lysates obtained with the GentleMACS showed much more debris.

[0123] [Table 5]

[0124] Lung and liver dissociation protocol

[0125] Two mice (Black-6) were sacrificed and their organs were removed by a laboratory technician. The organs were divided into two halves and immediately placed in PBS and put on ice. [Table 6]

[0126] The enzyme mixture for Precellys® used in these tests consists of: 2437.5 pL of Enzyme A + 2437.5 pL of Enzyme B + 125 pL of Enzyme C for a preparation of 5 mL per sample. This mixture is a cocktail of collagenases, proteases and endonucleases.

[0127] Once the enzyme mixtures were dispensed into the corresponding tubes, the samples were transferred to the respective dissociation tubes. The dissociation programs used in each machine are detailed in the table below. The post-incubation agitation time on the Precellys was reduced from 10 sec to 3 sec, for both tissue types.

[0128] [Table 7]

[0129] At the end of each dissociation program, the GentleMACS™ and Precellys® samples were treated in the same way.

[0130] After mechanical / enzymatic dissociation of the samples, a cell count (labeled with Trypan blue) was performed under a microscope to determine the cell concentration. Cytometry analysis requires a cell concentration of less than 10 6 per mL. The average concentration of the samples was therefore calculated in order to dilute them, if necessary. All samples were diluted halfway with PBS.

[0131] The liver samples were very dense and contained a lot of debris. This time, this phenomenon was observed for both the GentleMACS™ and the Precellys®. Microscopic counting for the liver samples was not performed due to poor visibility under the microscope. For the lung samples, the lysates were of very good visual quality (clear and without debris). Counting was performed on only one of the mice.

[0132] The results are displayed in the table below.

[0133] [Table 8] The total cell average is the total number of cells observed, multiplied by the dilution factor (10,000 cells / mL) and divided by the number of squares analyzed in the counting chip.

[0134] The cell concentration obtained with Precellys® was higher than that obtained with GentleMACS™, with a very high viability rate (>90%) for both processes.

[0135] The viability results obtained with GentleMACS™ and Precellys® are similar and very encouraging.

[0136] [Table 9]

[0137] Cytometry results show that the percentage of viability is higher with the GentleMACS™, despite the slightly higher amount of debris. However, the total amount of cells is higher with the Precellys®.

[0138] For the lung, viability results are similar between Precellys® and GentleMACS™, with a viability rate greater than 80%. Cell viability is slightly higher with GentleMACS™. However, the number of cells observed with Precellys® is higher than that obtained with GentleMACS™.

[0139] The protocol identified on the Precellys can be retained as is. An optimization test would be possible with a reduction in the incubation time at 37°C or the elimination of the first agitation before incubation.

[0140] Spleen and Heart Dissociation Protocol

[0141] Three mice (L7-IRES) were sacrificed and their organs were removed by a laboratory technician. The organs were divided into two halves and immediately placed in PBS and put on ice. [Table 10]

[0142] The enzyme mixture used consists of: 2437.5 pL of Enzyme A + 2437.5 pL of Enzyme B + 125 pL of Enzyme C for a preparation of 5 mL per sample. This mixture is a cocktail of collagenases, proteases and endonucleases.

[0143] Once the enzyme mixtures were dispensed into the corresponding tubes, the samples were transferred to the respective dissociation tubes. The dissociation programs used in each machine are detailed in the table below. This time the samples were incubated at 37°C without prior shaking. [Table 11]

[0144] All spleen samples were initially processed in the same way on the Precellys®: 5 seconds of post-incubation agitation. However, after agitation, a visual check of the samples observed that the spleens were still intact. For this reason, a second 5-second agitation was considered necessary for the spleen samples.

[0145] At the end of each dissociation program, the GentleMACS™ and Precellys® samples were treated in the same way.

[0146] After mechanical / enzymatic dissociation of the samples, a cell count (labeled with Trypan blue) was performed under a microscope to determine the cell concentration. Cytometry analysis requires a cell concentration of less than 10 6 per mL. The average concentration of the samples was therefore calculated in order to dilute them, if necessary. All samples were diluted halfway with PBS.

[0147] Regarding the spleen samples, the lysates were of very good visual quality (clear and without debris). The count was performed on only one of the mice. The results are displayed in the table below.

[0148] [Table 12]

[0149] The cell concentration obtained for spleen samples with the Precellys® was higher than that obtained with the GentleMACS™, with a very high viability rate (>95%) for both processes. The results for spleen samples were excellent for both machines (>90%), with a slightly higher viability percentage for the Precellys®. In addition, the amount of debris was less than 20% for both the GentleMACS™ and the Precellys®.

[0150] [Table 13] To test the effect of red blood cell lysis on cell viability, a portion of the spleen sample was treated with RBC lysis buffer. The results obtained show that red blood cell lysis significantly increases cell mortality, either by the duration of handling time or by the toxicity of the lysis buffer. The mortality rate increased to more than 40% on the Precellys® and more than 60% for the GentleMACS™, as shown in the table below. [Table 14]

[0151] The results for the spleen samples are excellent. The viability rate for Precellys® is 95% and for GentleMACS™ is 90%. Furthermore, the amount of debris observed is very low (<20%). Summary

[0152] The results obtained during the several days of testing demonstrate that, with optimized protocols, the performance of Precellys® can be very similar to that of the reference method, GentleMACS™. In terms of yield (concentration) and cell viability, this observation was confirmed on all types of murine tissue analyzed except the spleen, for which the percentage of debris was higher on Precellys, see table below. [Table 15]

[0153] Generally, samples treated with Precellys® showed less debris and a higher total cell count than GentleMACS™. Sometimes this difference in debris count was visually observable, especially for heart samples. However, inter-sample variability in dissociated cell concentration appears to be higher with Precellys®. It is possible that random movement of beads within tubes may impact the reproducibility of results for the same tissue type. The magnitude of this variability has not yet been quantified, given the limited number of mice available for analysis. That said, the observed difference never exceeded 12% between samples for the same tissue type (e.g., liver).

[0154] For comparison, it is also possible to note some similarities with results published by other manufacturers on the same types of tissue. For example, the VIA Extractor™ tissue disaggregator consists of a device where biological samples are introduced into a sterile and hermetically sealed bag. The bag is then placed in an instrument where large paddles (similar to those of a defibrillator) gently “massage” the bag and exert tissue disaggregation by light pressure. In an Application Note written by Cytiva for the VIA Extractor™ tissue disaggregator, the concentration of cells observed with the haemocytometer and the viability percentages obtained on liver samples are similar or lower than those obtained in this study, as illustrated in the table below. [Table 16]

[0155] The gating strategy (selection of an area on the point cloud generated during flow analysis that decides which cells to continue analyzing. It is a method of discrimination of cell populations) used for the identification of cells of interest in flow cytometry analysis is also similar to that used for the reference method.

[0156] The percentage of lymphocytes isolated from lung samples appears comparable (-30%) between GentleMACS™ and Precellys®.

[0157] The invention is not limited to the embodiments previously described and extends to all embodiments covered by the invention. The present invention is not limited to the examples previously described. Many other variant embodiments are possible, for example by combining features previously described, without departing from the scope of the invention. In addition, the features described in relation to one aspect of the invention may be combined with another aspect of the invention.

Claims

Claims 1. Method (4) of tissue dissociation comprising: • an arrangement of a biological tissue (3) in a closed tube (2) comprising at least one ball (24), • an addition of a liquid medium (22) comprising at least one tissue dissociation enzyme (23) capable of being active for the dissociation of the biological tissue (3) in the closed tube (2), • at least one cycle (42) of tissue dissociation, in a ball mill (1) configured to receive several tubes (2), in which the closed tube (2) is agitated so as to induce, by the at least one ball (24), a shearing effect in the volume of the liquid medium (22), so as to obtain a suspension (33) of cells comprising at least 20% of viable and dissociated cells (31) from the biological tissue (3).

2. Method (4) of tissue dissociation according to the preceding claim, in which, during the at least one cycle (42) of tissue dissociation, the closed tube (2) is agitated according to agitation parameters configured to be located in a dissociation window (422), said window being delimited by a first threshold (420) of dissociation of the biological tissue (3) into dissociated cells (31), the agitation parameters being insufficient below the first threshold (420) to induce tissue dissociation, and by a second threshold (421) called cell death, the agitation parameters being too intense beyond the second threshold (421) and resulting in a cell viability of less than 50% of the dissociated cells.

3. Method (4) of tissue dissociation according to any one of the preceding claims, in which, during the at least one cycle (42) of tissue dissociation, the closed tube (2) is agitated according to: • a stirring frequency F between 4 Hz and 45 Hz, and • an average stirring speed v between 0.2 m / s and 2.5 m / s.

4. Method (4) of tissue dissociation according to any one of the preceding claims, in which, for each cycle (42) of tissue dissociation, the closed tube (2) is agitated for a duration ti of between 3 and 30 seconds.

5. Method (4) of tissue dissociation according to any one of the preceding claims, in which the at least one bead (24) occupies a volume proportion of between 0.1% and 10% of the volume of the closed tube (2).

6. Method (4) of tissue dissociation according to any one of the preceding claims, in which the at least one bead (24) has a density of between 0.5 g / cm 3 and 8 g / cm 3 .

7. Method (4) of tissue dissociation according to any one of the preceding claims, in which the at least one bead (24) has at least one dimension (D1), for example a diameter, between 0.1 mm and 5 mm, preferably between 1 mm and 5 mm.

8. Method (4) of tissue dissociation according to any one of the preceding claims, in which the closed tube (2) has a volume of between 2 and 50 mL.

9. Method (4) of tissue dissociation according to any one of the preceding claims, in which the liquid medium (22) occupies a volume fraction of the closed tube (2) of between 0.3 and 0.

7.

10. Method (4) of tissue dissociation according to any one of the preceding claims, in which, during the at least one cycle (42) of tissue dissociation, the closed tube (2) is agitated in a precessional movement.

11. Method (4) of tissue dissociation according to any one of the preceding claims, comprising, prior to each cycle (42) of tissue dissociation, an incubation (41) of the biological tissue (3) in the presence of at least one tissue dissociation enzyme (23).

12. Method (4) of tissue dissociation according to any one of the preceding claims, comprising several cycles (42) of tissue dissociation.

13. Tissue dissociation kit (5) for implementing the method (4) according to any one of the preceding claims, comprising: • the closed tube (2) comprising at least one ball (24), the closed tube (2) comprising a lower portion (20) having one or more walls, the tube being (2) closed by a stopper (21), • at least one tissue dissociation enzyme (23).

14. Kit (5) according to the preceding claim, comprising several different tissue dissociation enzymes (23), so as to be able to select the at least one tissue dissociation enzyme (23) to be added in the tissue dissociation method (4).

15. Kit (5) according to any one of the two preceding claims, in which the at least one tissue dissociation enzyme (23) is chosen from the group consisting of dispase, collagenase, DNase, trypsin, papain, hyaluronidase, elastase.

16. Kit (5) according to any one of the three preceding claims, the kit comprising the ball mill (1) configured to receive several tubes (2).

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