FR3161221A1 - Tissue dissociation process and associated kit - Google Patents

Abstract

Tissue dissociation method and associated kit The invention relates to a kit and its method (4) for tissue dissociation comprising: arranging a biological tissue (3) in a closed container (2) comprising at least one bead (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 container (2), at least one cycle (42) of tissue dissociation, in a bead stirring apparatus (1), in which the closed container (2) is stirred so as to induce, by the at least one bead (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) of the biological tissue (3). Figure for abstract: Fig. 3B

Description

Tissue dissociation procedure and associated kit

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, biopharmaceutical development, elucidation of pathologies and tissue engineering.

STATE OF THE ART

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

Effective tissue dissociation allows, for example, the characterization of cellular composition in 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 predicting response to therapy and selecting a treatment.

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.

Chemical dissociation works on the principle that cations participate in maintaining intercellular bonds and the intracellular matrix. By introducing EDTA or EGTA, which binds these cations, the intercellular bonds are broken.

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

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

Choosing the right method or combination of methods remains complex. Numerous conflicting opinions exist in the scientific literature. Therefore, there is a need for a standardized protocol for tissue dissociation, for example, based on tissue type.

A tissue dissociation method based on cell separation by a rotating system within a closed container is known. The plastic tubes containing the sample include a rotor assembly which, driven by the machine's motor, rotates and disrupts the tissue by compressing it against protrusions arranged on a stator. Through lamination along a shear plane between the rotor and the stator, the tissue is disintegrated, allowing the recovery of live cells. For several years, the leading automated technique has been the GentleMACS™ system from Miltenyi Biotec™, for example, described in document EP2540394 A1. Due to this lamination process, however, this solution can induce significant heating in the shear plane between the rotor and the stator. Furthermore, this method requires the acquisition of equipment specifically designed for tissue dissociation applications.

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

The other objects, features, and advantages of the present invention will become apparent from an examination of the following description and accompanying drawings. It is understood that other advantages may be incorporated.

SUMMARY

• une disposition d’un tissu biologique dans un récipient clos comprenant au moins une bille,
• un ajout d’un milieu liquide comprenant au moins une enzyme de dissociation tissulaire apte à être active pour la dissociation du tissu biologique dans le récipient clos,
• au moins un cycle de dissociation tissulaire, dans un appareil d’agitation par bille, dans lequel le récipient clos est agité de façon à induire, par l’au moins une bille, un effet de cisaillement dans le volume du milieu liquide, de façon à obtenir une suspension de cellules comprenant au moins 20 %, et de préférence au moins 50 %, de cellules viables et dissociées du tissu biologique.

To achieve this objective, the first aspect involves a tissue dissociation process comprising:

• an arrangement of biological tissue in a closed container comprising at least one bead,
• the addition of a liquid medium comprising at least one tissue-dissociating enzyme capable of being active for the dissociation of biological tissue in the closed container,
• at least one cycle of tissue dissociation, in a ball-stirring apparatus, in which the closed container is agitated so as to induce, by 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%, and preferably at least 50%, of viable cells dissociated from the biological tissue.

Thus, the process uses a ball mill or, equivalently, a ball mill, also known as a ball agitation device, conventionally used for the preparation of chemical or biological samples by grinding and destroying cells. Unlike existing solutions, the device used is not specifically dedicated to and optimized for tissue dissociation. This avoids a considerable additional investment for laboratories and companies wishing to perform tissue dissociation. The invention allows the use of a single device for both grinding/homogenizing biological tissues and tissue dissociation, while maintaining the performance expected with reference instruments.

In general, homogenizers that use the principle of bead-beating to grind biological samples are used at too high a force to dissociate living cells. Unlike conventional tissue dissociation equipment, bead-beating homogenizers are designed to generate a bead impact force on the samples powerful enough to fragment cells and isolate intracellular contents. Therefore, using a bead-beating homogenizer for tissue dissociation is entirely counterintuitive.

Here, the bead-shearing device is used according to a bead-shearing principle. Performing tissue dissociation with a bead shaker, or equivalently a bead homogenizer, is a technical challenge that offers several advantages over conventional techniques: time savings due to parallel processing, improved reproducibility because the process is more homogeneous, and faster execution because the process can be more efficient thanks to volume processing (and therefore a potentially larger "apparent" surface area).

Furthermore, compared to conventional tissue dissociation solutions, dissociation here occurs within the volume (and therefore in three dimensions) of the liquid medium in the closed container, through volumetric shear induced by the movement of the ball(s). The dissociation thus results from a random and homogeneous process throughout the volume of the liquid medium. This differs from two-dimensional dissociation by lamination, in a lamination plane between a rotor and a stator. Tissue dissociation by volumetric shear allows for a more homogeneous medium, facilitating the dissipation of heat generated locally by shear forces. Moreover, the duration of mechanical agitation and the level of stress on the cells are reduced. The amount of debris in the resulting cell suspension can also be reduced.

• le récipient clos comprenant l’au moins une bille,
• l’au moins une enzyme de dissociation tissulaire.

Another aspect concerns a tissue dissociation kit for implementing the process according to any one of the preceding claims, comprising:

• the closed container containing at least one marble,
• at least one tissue dissociation enzyme.

BRIEF DESCRIPTION OF THE FIGURES

The aims, objects, features and advantages of the invention will become clearer from the detailed description of an embodiment thereof, which is illustrated by the following accompanying drawings in which:

FIG. 1 There FIG. 1 represents a schematic overview of a ball homogenizer according to an example embodiment.

FIG. 2 There FIG. 2 represents a closed container comprising beads, biological tissue and liquid medium comprising a cell dissociation enzyme, according to an example embodiment.

FIG. 3 FIG. 3 Figures 3A and 3B schematically illustrate the tissue dissociation process, according to an example of implementation.

FIG. 4 FIG. 4 Figures 4A and 4B represent 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.

FIG. 5 FIG. 5 Figures 5A and 5B represent schematic diagrams illustrating the shear effects in a case of bead-shearing.

FIG. 6 FIG. 6 Figures 6A and 6B represent schematic diagrams illustrating the shear effects in a case of bead-beating.

FIG. 7 There FIG. 7 illustrates the dissociation kit according to an example of implementation.

The drawings are provided by way of example and are not intended to limit the scope of the invention. They are schematic representations of the principle intended to facilitate understanding of the invention and are not necessarily to scale with practical applications. In particular, the relative dimensions of the illustrated elements, and especially of the cells, beads, and container, are not representative of reality.

DETAILED DESCRIPTION

Before beginning a detailed review of embodiments of the invention, optional features that may be used in combination or alternatively are stated below.

According to one example, during at least one cycle of tissue dissociation, the closed container is agitated according to agitation parameters configured to be within a dissociation window, said window being delimited by a first threshold of 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 a cell viability of less than 50% of the dissociated cells.

Thus, the operating parameters of the bead homogenizer are chosen to limit cell destruction by defining an operating range that allows for the production of a cell suspension containing at least 50% viable cells dissociated from the biological tissue. More specifically, the agitation frequency, agitation speed, and, where applicable, the agitation duration can be selected to fall within the dissociation window.

• une fréquence d’agitation F comprise entre 4 Hz et 45 Hz, de préférence entre 20 et 42 Hz, et
• une vitesse moyenne d’agitation v comprise entre 0,2 m/s et 2,5 m/s.

As an example, during at least one cycle of tissue dissociation, the closed container is agitated according to:

• an agitation frequency F between 4 Hz and 45 Hz, preferably between 20 and 42 Hz, and
• an average agitation speed v between 0.2 m/s and 2.5 m/s.

The container is thus agitated with a repeated motion and at an average speed that optimizes tissue dissociation and cell viability within the dissociation window. Below this speed, cell dissociation from the tissue will be less satisfactory, and above it, the risk of significant cell death is increased.

As an example, for each tissue dissociation cycle, the closed container is agitated for a duration _t1 of between 3 and 30 seconds , preferably between 5 and 10 seconds. This agitation time range optimizes tissue dissociation and cell viability within the dissociation window, particularly in synergy with the agitation frequency and average speed mentioned above.

In one example, at least one bead occupies a volume proportion between 0.1% and 10% of the volume of the closed container. This volume proportion minimizes the probability of significant impact between the bead(s) and the container wall, while generating sufficient shear force to allow cell dissociation.

In one example, the container contains between 1 and 15 marbles.

In one example, at least one bead has a density between 0.5 g/ ^cm³ and 8 g/ ^cm³ . This density range allows for movement within the fluid that is appropriate for generating sufficient shear force to dissociate cells. In another example, at least one bead is based on or made of a material chosen from the group consisting of latex, glass, ceramic, metal, and stainless steel. These materials are particularly suitable for contact with biological material in solution.

For example, at least one bead 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 beads impacts their movement in the liquid medium during agitation, and in particular their speed of movement and the resulting impacts.

As an example, the closed container has a volume between 2 and 50 mL, preferably between 2 and 15 mL. The tube's volume affects its dimensions, and therefore the potential for collisions between the bead(s) and the container wall. These volumes are advantageous for optimizing tissue dissociation and cell viability, while remaining compatible with the container volumes commonly used in bead homogenizers.

In one example, the liquid medium occupies a volume fraction of the closed container between 0.3 and 0.7. The proportion of liquid medium in the container affects the dissociation efficiency. Less liquid promotes homogenization.

For example, during at least one cycle of tissue dissociation, the closed container is agitated by a precessional motion. The container holding the biological tissue, typically on a support tray in the homogenizer, is thus agitated around a central axis, the axis describing a cone during this motion. A precessional motion induces a displacement of the container(s) in a direction perpendicular to this movement. Some ball homogenizers employ an oscillating translational or rotational motion to move the containers or tubes holding the sample. The precessional motion offers an advantage in terms of the direction of movement of the ball(s). Their movement can be more random within the volume and not simply unidirectional as with other homogenizers.

As an example, the process involves, prior to each tissue dissociation cycle, incubating the biological tissue in the presence of at least one tissue dissociation enzyme, preferably within the closed container. This enhances the enzyme's action prior to the mechanical action of the bead(s). This facilitates tissue dissociation through the synergistic interaction of enzymatic and mechanical action.

As an example, the process comprises several cycles of tissue dissociation , these cycles preferably being separated by periods of no agitation. This allows for the temporal distribution of the mechanical action on the sample. This promotes the dissipation of heat generated by localized heating caused by collisions between the bead(s) and the container wall. Cell viability is therefore enhanced. Furthermore, this facilitates enzymatic action on the biological tissue by allowing at least one enzyme to act between several phases of mechanical action, and thus between several stages of biological tissue degradation.

According to one example, the process further comprises, prior to adding at least one tissue dissociation enzyme to the closed container, selecting 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.

For example, the kit includes several different tissue dissociation enzymes, allowing the user to select at least one enzyme to add to the tissue dissociation process. Selecting the enzyme or enzyme mixture allows the process to be tailored to the specific biological tissue being dissociated. The process and kit can therefore be applied to a wide variety of different biological tissues.

According to one example, at least one tissue dissociation enzyme is chosen from the group consisting of dispase, collagenase, DNase, trypsin, papain, hyaluronidase, elastase.

According to one example, the kit includes the stirring device or, equivalently, the ball homogenizing device.

In the following description, the term "on" does not necessarily mean "directly on." Thus, when it is stated that a part or component A rests "on" a part or component B, this does not mean that parts or components A and B are necessarily in direct contact with each other. These parts or components A and B may be either in direct contact or supported by one or more other parts. The same applies to other expressions such as, for example, "A acts on B," which can mean "A acts directly on B" or "A acts on B through one or more other parts."

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

A parameter that is "approximately equal to/greater than/less than" a given value means that the parameter is equal to/greater than/less than the given value, within ±10% of that value. A parameter that is "approximately between" two given values means that the parameter is at least equal to the smaller of the two given values, within ±10% of that value, and at most equal to the larger of the two given values, within ±10% of that value.

Tissue dissociation process 4 and associated kit 5 are now described according to specific examples of implementation.

The term "sample grinder", or equivalently "homogenizer" or "stirring device", broadly refers to any type of translational, rotary or precession device that processes samples, including not only high-power shaker-type grinders as described in this document, but also other laboratory equipment such as vortices, shakers and agitators.

As illustrated by the FIG. 1 For example, the homogenizer 1 conventionally comprises a device designed to hold one or more closed containers 2, or equivalently one or more tubes 2, containing samples. The tubes 2 are generally arranged on a support 10 driven in rotation, and more specifically in precession, by a rotating shaft 11. The support 10 is mounted on the homogenizer 1 via the shaft 11, which is itself mounted on a frame 13. The device 1 and the tubes 2 can be arranged under a cover 12 that pivots between an open and a closed position. During the precession movement, the support 10 is driven in rotation, and more specifically in precession, about a central axis A, the axis describing a cone of angle α during this movement, inducing a displacement of the tubes in a direction perpendicular to this movement. The samples are thus subjected to a precession movement.

In tissue dissociation process 4, and as illustrated for example by the FIG. 2 A biological tissue 3 is placed in a liquid medium 22 in a tube 2. The tube 2 is sealed and therefore fluid-tight. The tube 2 is preferably sterile. The tube 2 may include a lower portion 20 having one or more walls and closed by a stopper 21. The tube 2 includes at least one bead 24 configured to exert shear forces in the liquid medium 22 to induce tissue dissociation. For the purposes of this text, it is assumed, for the sake of completeness, that the tube 2 contains several beads 24. Note that, unless otherwise specified, the following characteristics may apply to the example in which the tube contains a single bead 24.

As illustrated, for example, by Figures 3A and 3B, efficient tissue dissociation yields a suspension of viable and functional cells. Typically, biological tissue comprises undissociated cells surrounded by an extracellular matrix. Biological tissue can contain cells of various types, depending on the nature of the tissue.

• une concentration de cellules dissociées suffisante ; en-deçà la dissociation est partielle et insuffisante. Par exemple, une concentration suffisante peut être sensiblement supérieure ou égale à la valeur seuil permettant l’efficacité de l’analyse, selon l’application donnée. Cette valeur seuil peut être comprise entre 10⁴et 10⁶cellules par mL,
• un pourcentage de cellules dissociées vivantes suffisant : au-delà on considère que le tissu est homogénéisé (ou broyé de façon équivalente). Par exemple, un pourcentage de viabilité peut être sensiblement supérieur ou égal au seuil nécessaire pour garantir l’efficacité de l’analyse, selon l’application donnée. Ce seuil est de préférence supérieur ou égal à 50 % des cellules dissociées du tissu 3 (ce pourcentage ne comptant donc pas les éventuelles cellules 30 restant dans le tissu 3 à l’issue de la dissociation).

Effective tissue dissociation corresponds to:

• A sufficient concentration of dissociated cells is required; below this concentration, dissociation is partial and insufficient. For example, a sufficient concentration may be significantly greater than or equal to the threshold value required for effective analysis, depending on the application. This threshold value may be between ^10⁴ and ^10⁶ cells per mL.
• A sufficient percentage of dissociated cells must remain viable; beyond this, the tissue is considered homogenized (or equivalently ground). For example, a viability percentage can be significantly greater than or equal to the threshold required to ensure the effectiveness of the analysis, depending on the specific application. This threshold is preferably greater than or equal to 50% of the dissociated cells from tissue 3 (this percentage therefore does not include any cells 30 remaining in tissue 3 after dissociation).

The aim, therefore, is to generate a fluid shear force through the synergy between the beads 24 inside the tube 2 and the agitation motion, preferably precession, with a high but not excessively high velocity gradient, to create a suspension 33 of viable and functional cells 31 via bead-shearing and not bead-beating. If the agitation conditions are too gentle, then the number of dissociated cells 31 is too low, and the cell suspension 34 is therefore inadequate. If the agitation conditions are too strong, then the number of dissociated cells 31 is high, but the cell death rate is too high, with too many lysed cells 32. The cell suspension 35 is then also inadequate.

For this reason, and as illustrated for example in FIG. 3 The process 4 involves adding the biological tissue 3 to be dissociated to the tube 2 containing the beads 24. A liquid medium 22 is added, comprising one or more tissue dissociation enzymes 23. In the process 4, a mechanical action by the beads 24 is thus combined with an enzymatic action through the addition of one or more enzymes 23 to 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 from, a cell culture medium. The appropriate enzyme(s) and culture medium 22 are preferably used to facilitate dissociation and preserve the integrity of cells 31. The appropriate culture medium for a given tissue can be easily identified by a person skilled in the art, for example, from the literature or from established protocols.

Dissociation enzymes may include enzymes configured to facilitate the dissociation of cells 30 from the extracellular matrix. These enzymes may include proteases, collagenases, dispases, DNases, trypsins, papains, hyaluronidases, and elastases. These enzymes may be tailored to the type of biological tissue 3. To this end, the process 4 may include a selection step 40 of the enzyme or enzyme mixture to be used, depending on the biological tissue 3. The enzymes 23 may be added to the liquid medium 22 prior to or after the addition of the biological tissue 3 to the liquid medium 22. For this purpose, and as illustrated in FIG. 7 For example, kit 5 may include tube 2 containing beads 24, and several enzymes 23. Kit 5 can thus be adapted to many tissues 3.

Prior to at least one, and preferably each, tissue dissociation cycle 42, the process 4 may include an incubation 41 of the tube 2 to promote enzymatic action. For this purpose, the tube 2 may be placed in an incubator at a defined temperature T (for example, approximately 37 °C) for a duration _t2 . The temperature T and the duration _t2 may be adjusted according to the biological tissue 3.

During a tissue dissociation cycle 42, the tube 2 is agitated, preferably with a precessional motion, for a duration _t1 , at an agitation frequency F and an average agitation speed v. The process 4 may comprise several tissue dissociation cycles 42, preferably separated temporally by periods 43 of non-agitation. By non-agitation, it is understood that the tube 2 is not agitated in the ball homogenizer 1. Another form of agitation, for example manual agitation, is possible during these periods 43. Equivalently, successive dissociation cycles 42 can be said to be temporally separated. Successive dissociation cycles 42 may be separated by incubations 41, as illustrated by way of non-limiting example.

Process 4 may then include a step 44 or a set of steps 44 for the recovery and/or analysis of the resulting cell suspension. For example, the beads 24 may be removed from the cell suspension 33 by filtration. This initial filtration step will also allow the removal of large pieces of residual (undissociated) tissue.

Process 4 may further include, preferably following filtration, centrifugation and/or rinsing steps to recover isolated and live cells 31. The process may further include an assessment of cell viability, for example by counting isolated and live cells 31 against the number of dead cells 32. Any method of assessing cell viability may be considered, for example the Trypan blue method or by flow cytometry.

The parameters of process 4 can be defined to enable efficient tissue dissociation, and therefore the suspension of viable and functional cells. Defining and optimizing a set of parameters (e.g., bead composition, size and quantity, tube volume, enzyme solution composition, and agitation programs – speed, frequency, and duration) can optimize tissue dissociation and cell viability.

Tissue dissociation can be governed by a set of agitation and geometric parameters defining a dissociation window 422, allowing for the production of a suitable cell suspension 33 for the intended application. As illustrated, for example, by the FIG. 4 The dissociation window 422 can be delimited, on the one hand, by a first dissociation threshold 420, below which the tissue is not dissociated 36 or the number of dissociated cells in suspension 34 is insufficient, as described previously. On the other hand, the dissociation window 422 can be delimited by a second cell death threshold 421, above which excessive cell death 32 occurs in a suspension 35: homogenization then takes place.

Inappropriate agitation and geometric parameters can lead to a situation where dissociation is insufficient even though the percentage of live cells is already below the second threshold 421, as illustrated for example in FIG. 4 There is then no tissue dissociation.

Before detailing more specific parameter examples, the phenomena in solutions on either side of the second threshold 421 are described as an 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.

As illustrated in Figures 5A and 5B, the agitation of 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 F _cis can be:

• δ la distance entre billes,
• µ₁la viscosité du milieu liquide 22
• v_bla vitesse de la bille pouvant être donnée par la relation suivante sur la force de trainée :

with :

• δ the distance between balls,
• µ ₁ the viscosity of the liquid medium 22
• v_{_b,} the speed of the ball, can be given by the following relationship on the drag force:

• m_bla masse de la bille,
• ρ_f: masse volumique du fluide,
• S_bla section de la bille,
• C_xle coefficient de trainée de la bille.

with :

• m _b the mass of the ball,
• _ρf : density of the fluid,
• Let S _be the cross-section of the ball.
• C _x is the drag coefficient of the ball.

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 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 agitation parameters (and in particular agitation 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.

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

During homogenization, the agitation of tube 2 and the resulting movement of beads 24 in the liquid medium 22 also induce collisions between beads 24 and biological tissue 3, as illustrated for example by the FIG. 6 The force of a mechanical impact F_{_col} occurring over a time dt can be:

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, and 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 by the FIG. 6 This threshold 421 is the cell death limit. This threshold can be determined to a first order by the stirring speed and the characteristics of the biological sample, and to a second order by the characteristics of the tube 2, the liquid medium 22 and the beads 24.

From this threshold 421, the homogenization efficiency can be defined as a homogenization rate per cycle. Increasing the stirring frequency and/or speed and/or duration further reduces the percentage of live cells. The homogenization rate can be determined, to a first approximation, by the characteristics of the tube 2, the liquid medium 22, and the beads 24, and to a second approximation by the stirring speed.

The agitation of the tube 2 and the resulting movement of the balls 24 in the liquid medium 22 also induce collisions between 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 frequency, speed and duration of agitation and can be dependent on the characteristics of the tube 2, the liquid medium 22 and the balls 24.

When the temperature exceeds a certain threshold, cells can degrade and die. This threshold is primarily determined by the characteristics of the biological sample. An increase in the agitation time and/or the temperature of the medium 22 can therefore reduce the percentage of live and dissociated cells 31.

Based on the above and standard fluid mechanics equations, the following parameters can optimize tissue dissociation and cell viability. These parameters are presented in a table and can be used individually or in combination, in whole or in part.

Parameter type Size Designation Unit Lower limit Upper limit Container Diameter (D2) D _t mm 5 25 Height (H2) H _t mm 30 50 Volume V _t mL 2 15 Fluid Volume V _f mL 1 10 Dynamic viscosity µ_f (corresponding to µ₁previously) mPa.s 0.25 1.5 Density ρ _f g/ ^cm³ 0.9 1.1 Marble(s) Volume proportion in tube 2 p _b % 0.1 10 Density ρ _b g/ ^cm³ 0.5 8 Diameter (D1) D _b mm 1 7, preferably 5 Number N _b - 1 15 Section S _b mm² 0.5 20 Mass m _b mg 1 600

Parameter type Size Designation Unit Lower limit Upper limit Homogenizer Frequency of agitation F Hz 4.0 45, preferably 42 Stirring speed v m/s 0.2 2.5 Duration t ₁ s 3 30

The stirring frequency is defined as the number of movements per second. These movements can be rotation, translation, precession, a combination of these movements, or any other cyclically repeating motion. It is expressed here in Hz to generically correspond to different types of ball homogenizers. For example, the stirring frequency ranges from 1000 rpm to 5500 rpm (rotations per minute).

The agitation speed, or average agitation speed, is defined as the average speed of tube 2 during its movement. Since the movement is cyclical, it may accelerate and decelerate cyclically, and therefore its instantaneous speed may not fall within the described range. This is why an average speed, taken over the entire cycle of the movement, is used.

The agitation time considered corresponds to the time over all the movements of the tube, in a 42 cycle of tissue dissociation.

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.

Specific examples of tissue dissociation are now described.

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

Identification of ball parameters

The initial tests below were carried out on whole pinkie worms.

• Les billes CK28 (3 ou 7 billes d’oxyde de zirconium de 2,8 mm diamètre) dans 2.5 mL de PBS et un protocole de 1,4 m/s à 24,9 Hz x 10 secondes,
• Les billes CK50 (1 ou 3 billes d’oxyde de zirconium de 5,0 mm diamètre) dans 2.5 mL de PBS et un protocole de 1,4 m/s à 24,9 Hz x 5 secondes.

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

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

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

For CK28 pellets, tests with a quantity of pellets greater than 15 showed a mortality rate of more than 90%.

CK14 beads (zirconium oxide, 1.4 mm diameter) were unable to grind the sample. We believe that a longer homogenization cycle would lead to an increase in cell mortality. This type of bead is therefore excluded from further testing.

Test 1: CK 28 beads with 5 mL of PBS Number of marbles Sample mass (mg) quality of the ground material for a stirring time of 3 seconds quality of the ground material for a stirring time of 5 seconds Count result (microscope) 67 43.4 AVERAGE OK > 90% of dead cells 30 44 AVERAGE OK > 90% of dead cells 15 37.5 slightly crushed OK 7 40 very little crushed slightly crushed 3 35 Intact intact 1 48 Intact intact 2nd test - CK14, CK28 and CK50 with 2.5 mL PBS Type of marbles Number of marbles Sample mass (mg) quality of the ground material for a stirring time of 10 seconds Count result (microscope) CK14 7 51.2 intact 15 48.4 very little crushed CK28 3 35.7 OK < 80% dead cells 7 41 OK < 80% dead cells CK50 1 28 OK < 80% dead cells 3 44.8 OK < 80% dead cells 3rd test - CK50 with 2.5 mL PBS Type of marbles Number of marbles Sample mass (mg) quality of the ground material for a stirring time of 5 seconds Count result (microscope) CK50 1 39 OK < 80% dead cells 3 50 OK < 80% dead cells

• les billes CK50 (1 bille) dans 2.5 mL de PBS et un protocole de 1,4 m/s à 24,9 Hz x 5 sec (25%)
• les billes CK50 (3 billes) dans 2.5 mL de PBS et un protocole de 1,4 m/s à 24,9 Hz x 5 sec (22%)

The best combination of beads was tested at several agitation speeds. Based on quantitative visual assessment, the tests identified two bead combinations that appear most promising for retaining more than 20% of live 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 (25%)
• 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%)

CK 28 and CK 50 tests with 2.5 mL PBS Type of marbles Number of marbles Sample mass (mg) Count result (microscope) Number of living cells Number of dead cells % Viability Quality/debris Live cells per mL CK28 3 37 5 40 11 AVERAGE 50,000 7 34 3 20 13 GOOD 30,000 CK50 1 34.1 5 15 25 Alright 50,000 3 42.8 7 25 22 Alright 70000 GentleMACS™ comparative test with 2.5 mL PBS Program Sample mass (mg) Counting result (microscope) Number of living cells Number of dead cells % Viability Quality/debris Live cells per mL 0.2 m/s at 4.08 Hz – 10s 38.8 4 20 16 AVERAGE 40,000 1.1 m/s at 20.75 Hz – 10s 28 5 17 30 Alright 50,000 0.6 m/s at 12.42 Hz – 10s 43 4 14 22 GOOD 40,000 1.1 m/s at 20.75 Hz – 10s 29 3 22 12 AVERAGE 30,000

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 significantly more debris.

CK50 test with 5 ml of HEPES buffer + Enzymes Type of marbles Number of marbles Sample mass (mg) Counting result (microscope) Number of living cells Number of dead cells % Viability Quality/debris Live cells per mL CK50 1 40.8 3 6 33.33 Alright 3.10 ⁶ 3 41.3 5 6 45.45 Alright 5.10 ⁶ GentleMACS™ comparative test with 2.5 mL PBS Program Sample mass (mg) Counting result (microscope) Number of living cells Number of dead cells % Viability Quality/debris Live cells per mL mlung_01 and 02 41.6 5 7 41.66 AVERAGE 5.10 ⁶

Lung dissociation protocol and liver

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

Sample Animal identification Sex Weight (g) Fabric Tissue weight (mg) Precellys® (P) or GentleMACS™ (G) test 1 SPN 006008-23 #45 Male 25.64 ½ liver 760.21 G 2 ½ liver 717.21 P 3 ½ lung 70.67 G 4 ½ lung 61.95 P 5 SPN 006007-23 #44 Male 26.48 ½ liver 657.46 G 6 ½ liver 811.27 P 7 ½ lung 80.15 G 8 ½ lung 75.78 P

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

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 seconds to 3 seconds for both tissue types.

Fabric gentleMACS™ Program Precellys® Program Lung 37C_Multi_C ^First agitation: 3 seconds at 1.4 m/s at 24.9 Hz
Incubation: 30 min at 37 °C
2nd post-incubation agitation: 3 seconds at 1.4 m/s at 24.9 Hz Liver 37C_Multi_C

At the end of each of the dissociation programs, the GentleMACS™ and Precellys® samples were treated in the same way.

After mechanical/enzymatic dissociation of the samples, cell counting (using Trypan blue-stained cells) was performed under a microscope to determine cell concentration. Cytometric analysis requires a cell concentration below 10⁶ 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.

The liver samples were very dense and contained a lot of debris. This phenomenon was observed for both GentleMACS™ and Precellys®. Microscopic counting of the liver samples was not performed due to poor visibility under the microscope. The lung samples, however, produced lysates of very good visual quality (clear and free of debris). Counting was performed on only one of the mice. The results are shown in the table below.

Cell counting Lung (Mouse 1) Lung (Mouse 1) – correction per mg GentleMACS™ Precellys® GentleMACS™ Precellys® Living cells 155 174 0.20 0.24 Dead cells 12 18 0.02 0.02 Total average cells 41.75 48 0.05 0.06 Total cell concentration 83,500 cells per mL 960,000 cells per mL 1098 cells per mg 1263 cells per mg Cells/mL or mg 8.3 ^{x 10⁵} cells per mL 9.6 ^{x 10⁵} cells per mL 1.09.10³ cells per mg 1.2 ^{x 10³} cells per mg

The total average number of cells corresponds to 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.

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

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

Sample Debris (%) Total cells (%) Dead cells (%) Live cells (%) GentleMACS™ 3 48.8 22.1 6.6 93.3 4 63.7 12.8 6.54 93.4 Average 56.25 17.45 6.57 93.35 Standard deviation 10.54 6.58 0.04 0.07 Precellys® 7 51.3 33.7 14.3 85.6 8 53.2 30.8 25.2 74.6 Average 52.25 32.25 19.75 80.10 Standard deviation 1.34 2.05 7.71 7.78

Flow cytometry results show that the percentage of viable cells is higher with GentleMACS™, despite a slightly higher amount of debris. However, the total number of cells is higher with Precellys®.

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

The protocol identified on the Precellys can be retained as is. An optimization test could be considered, either by reducing the incubation time to 37°C or by eliminating the initial stirring before incubation.

Spleen dissociation protocol And heart

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

Sample Animal identification Sex Weight (g) Fabric Tissue weight (mg) Precellys® (P) or GentleMACS™ (G) test 1 SPN 005240-23 No. 32 L7-IRES Female 21.39 ½ spleen 35.95 G 2 ½ spleen 35.95 P 3 ½ heart 43.18 G 4 ½ heart 52.6 P 5 SPN 005722-23 No. 36 L7-IRES Female 18.66 ½ spleen 35.74 G 6 ½ spleen 34.22 P 7 ½ heart 44 G 8 ½ heart 38.66 P 9 SPN 005241-23 No. 33 L7-IRES Female 20.53 ½ spleen 31.19 G 10 ½ spleen 26.02 P 11 ½ heart 37.02 G 12 ½ heart 47.71 P

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

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 agitation.

Fabric GentleMACS™ Program Precellys® Program Missed 37C_m_SDK_1 Incubation: 15 min at 37 °C
Post-incubation agitation: 2 x 5 seconds at 1.4 m/s at 24.9 Hz

All spleen samples were initially processed identically on the Precellys®: 5 seconds of post-incubation agitation. However, after agitation, a visual inspection of the samples revealed that the spleens were still intact. Therefore, a second 5-second agitation was deemed necessary for the spleen samples.

At the end of each of the dissociation programs, the GentleMACS™ and Precellys® samples were treated in the same way.

After mechanical/enzymatic dissociation of the samples, cell counting (using Trypan blue-stained cells) was performed under a microscope to determine cell concentration. Cytometric analysis requires a cell concentration below 10⁶ 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.

Regarding the spleen samples, the lysates were of very good visual quality (clear and free of debris). The count was performed on only one of the mice. The results are shown in the table below.

Cell counting Rate (Mouse 1) Rate (Mouse 1) – correction per mg GentleMACS™ Precellys® GentleMACS™ Precellys® Living cells 1596 2051 44.39 57.05 Dead cells 16 19 0.45 0.53 Total average cells 403 517.5 11.21 14.39 Total cell concentration 8,060,000 cells per mL 10,350,000 cells per mL 224,200 cells per mg 287,900 cells per mg Cells/mL or mg 8.06 ^{x 10⁶} cells per mL 1.03 ^{x 10⁷} cells per mL 2.2 ^{x 10⁵} cells per mg 2.8 ^{x 10⁵} cells per mg

The cell concentration obtained for spleen samples with Precellys® was higher than that obtained with GentleMACS™, with a very high viability rate (>95%) for both processes.

The results for the spleen samples were excellent for both machines (>90%), with a slightly higher viability percentage for the Precellys®. Furthermore, the amount of debris was less than 20% for both the GentleMACS™ and the Precellys®.

Sample Debris (%) Total cells (%) Dead cells (%) Live cells (%) GentleMACS™ 1 21.4 73.1 6.74 93.3 5 16.4 72.8 9.49 90.5 9 21.5 63.1 12.40 87.6 Average 19.77 69.67 9.54 90.47 Standard deviation 2.92 5.69 2.83 2.95 Precellys® 2 15.7 74.1 5.02 95 6 25.8 58.5 4.79 95.3 10 12.9 72.4 5.02 95 Average 18.13 68.33 4.94 95.10 Standard deviation 6.79 8.56 0.13 0.17

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 show that red blood cell lysis significantly increases cell mortality, either due to the duration of the manipulation or the toxicity of the lysis buffer. The mortality rate increased to over 40% with Precellys® and over 60% with GentleMACS™, as shown in the table below.

Sample Debris (%) Total cells (%) Dead cells (%) Live cells (%) GentleMACS™ 1 22.7 72.5 77.60 22.2 5 15 80.5 84 15.9 9 29.5 59.4 48 51.9 Average 22.40 70.80 69.87 30 Standard deviation 7.25 10.65 19.21 19.23 Precellys® 2 16.7 75 32.6 67.4 6 14.1 80.6 60.5 39.5 10 22.6 65.1 36 64 Average 17.80 73.57 43.03 56.97 Standard deviation 4.36 7.85 15.22 15.22

Regarding the spleen samples, the results are excellent, with a viability rate of 95% for Precellys® and 90% for GentleMACS™. Furthermore, the amount of debris observed is very low (<20%).

Synthesis

The results obtained over 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 for the spleen, for which the percentage of debris was higher with Precellys (see table below).

GentleMACS™ Precellys® Debris (%) Live cells (%) Concentration per mL Debris (%) Live cells (%) Concentration per mL Lung 56.25 ± 10.54 93.35±0.07 8.3.10 ⁵ 52.25±1.34 80.10±7.78 9.6.10 ⁵ Missed 19.77±2.92 90.47±2.85 8.06.10 ⁶ 18.13±9.79 95.10±0.17 1.03.10 ⁷ Liver 49.75±3.75 90.20±3.11 1.45.10 ⁷ 62.20±3.68 87.35±9.12 1.04.10 ⁷ Heart 59.45±21.43 69.10±2.40 41.45±4.88 87.35±0.49

In general, samples treated with Precellys® showed less debris and a higher total cell count than GentleMACS™. Sometimes this difference in debris count was visible to the naked eye, especially in heart samples. However, inter-sample variability in the concentration of dissociated cells appeared to be greater with Precellys®. It is possible that the random movement of the beads inside the tubes impacts 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).

For comparison, it is also possible to note certain 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, 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 induce tissue disaggregation through light pressure. In an Application Note written by Cytiva for the VIA Extractor™ tissue disaggregator, the cell concentration observed by hemocytometer and the viability percentages obtained on liver samples are similar to or lower than those obtained in this study, as illustrated in the table below.

Sample Method Viability (%) Haemocytometer (cells/mL) 1 GentleMACS™ 56 2.2.10 ⁶ 2 GentleMACS™ 49 2.9.10 ⁶ 3 GentleMACS™ 51 2.3.10 ⁶ 4 VIA Extractor™ 77 4.9.10 ⁶ 5 VIA Extractor™ 75 5.1.10 ⁶ 6 VIA Extractor™ 64 5.9.10 ⁶ 7 Precellys® 94 1.04.10 ⁷

The gating strategy (selecting an area on the point cloud generated during flow cytometry to determine which cells to continue analyzing; this is a method for discriminating between cell populations) used to identify cells of interest in flow cytometry analysis is also similar to that used for the reference method. The percentage of lymphocytes isolated from lung samples appears comparable (~30%) between GentleMACS™ and Precellys®.

The invention is not limited to the embodiments described above and extends to all embodiments covered by the invention. The present invention is not limited to the examples described above. Many other embodiments are possible, for example, by combining features described above, without departing from the scope of the invention. Furthermore, the features described with respect to one aspect of the invention can be combined with another aspect of the invention.

Claims (16)

Tissue dissociation method (4) comprising:

• an arrangement of biological tissue (3) in a closed container (2) comprising at least one bead (24),

• the addition of a liquid medium (22) comprising at least one tissue dissociation enzyme (23) capable of being active for the dissociation of biological tissue (3) in the closed container (2),

• at least one cycle (42) of tissue dissociation, in a ball-stirring apparatus (1), in which the closed container (2) is agitated so as to induce, by 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 cells and dissociated (31) from the biological tissue (3).

A tissue dissociation method (4) according to the preceding claim, wherein, during at least one tissue dissociation cycle (42), the closed container (2) is agitated according to agitation parameters configured to be within 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 cell viability of less than 50% of the dissociated cells. A tissue dissociation method (4) according to any one of the preceding claims, wherein, during at least one tissue dissociation cycle (42), the closed container (2) is agitated according to:

• an agitation frequency F between 4 Hz and 45 Hz, and
• an average agitation speed v between 0.2 m/s and 2.5 m/s.

A tissue dissociation method (4) according to any one of the preceding claims, wherein, for each tissue dissociation cycle (42), the closed container (2) is agitated for a duration t₁ between 3 and 30 seconds. Method (4) of tissue dissociation according to any one of the preceding claims, wherein at least one bead (24) occupies a volume proportion of between 0.1% and 10% of the volume of the closed container (2). Method (4) of tissue dissociation according to any one of the preceding claims, wherein at least one bead (24) has a density between 0.5 g/ ^cm3 and 8 g/ ^cm3 . Method (4) of tissue dissociation according to any one of the preceding claims, wherein at least one bead (24) has at least one dimension (D1), for example a diameter, of between 0.1 mm and 5 mm, preferably between 1 mm and 5 mm. Method (4) of tissue dissociation according to any one of the preceding claims, wherein the closed container (2) has a volume between 2 and 50 mL. Method (4) of tissue dissociation according to any one of the preceding claims, wherein the liquid medium (22) occupies a volume fraction of the closed container (2) of between 0.3 and 0.7. Method (4) of tissue dissociation according to any one of the preceding claims, wherein, during at least one cycle (42) of tissue dissociation, the closed container (2) is agitated in a precessional motion. 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). Method (4) of tissue dissociation according to any one of the preceding claims, comprising several cycles (42) of tissue dissociation. Tissue dissociation kit (5) for carrying out the method (4) according to any one of the preceding claims, comprising:

• the closed container (2) comprising at least one ball (24),
• at least one tissue dissociation enzyme (23).

Kit (5) according to the preceding claim, comprising several different tissue dissociation enzymes (23), so as to be able to select at least one tissue dissociation enzyme (23) to be added in the tissue dissociation process (4). Kit (5) according to any one of the two preceding claims, wherein at least one tissue dissociation enzyme (23) is selected from the group consisting of dispase, collagenase, DNase, trypsin, papain, hyaluronidase, elastase. Kit (5) according to any one of the three preceding claims, the kit comprising the ball-type stirring device (1).