WO2023139272A1

WO2023139272A1 - Gelation device with piston

Info

Publication number: WO2023139272A1
Application number: PCT/EP2023/051607
Authority: WO
Inventors: Nicolas Fernandez; Pierre Chaumat
Original assignee: Scipio Bioscience
Priority date: 2022-01-24
Filing date: 2023-01-24
Publication date: 2023-07-27

Abstract

The present invention relates to a device for creating a cylindrical hydrogel.

Description

GELATION DEVICE WITH PISTON

FIELD OF INVENTION

[0001] The present invention relates to a device for creating a cylindrical hydrogel.

BACKGROUND OF INVENTION

[0002] To derive next generation sequencing (NGS) analysis, three tasks must occur: 1) sample preparation, 2) sequencing and 3) bioinformatics. Microfluidics have been exploited to improve the first of the three requirements, specifically by enabling high throughput (HT) parallelization of reactions and efficiencies of scale. One application that has an acute need for HT microfluidic sample preparation is single-cell gene expression analysis by RNA sequencing (single-cell RNAseq). The reason for this is that the number of cells to be analyzed can range from hundreds to hundreds of thousands and each workflow starts by first isolating single cells in individual reaction chambers. Thus, the HT parallelization reaction capacity of any microfluidic platform needs to match these cell number requirements.

[0003] Several single-cell RNA-seq technologies exist, among which microfluidics, especially water-in-oil droplet emulsions platforms. Those platforms all present technical drawbacks that were solved by the Applicant, who developed a method for single-cell gene expression analysis, that neither does require microfluidics chips nor does require droplets, while preserving the key benefit of droplet platforms in being able to process greater than thousands of cells (International patent publication WO 2018/203141).

[0004] This technology is based on the use of a hydrogel, preferably cylindrical, which upon solidification, is capable of trapping hundreds if not thousands of cells in a discrete manner. Individual cells can therefore be processed and analyzed in the hydrogel, by diffusion of biochemistry and molecular biology reagents. In particular, the use of a hydrogel solves three key problems commonly associated with previous technologies. First, any detergent level is supported by the hydrogel, creating the possibility of lysing any biological unit, whether a cell, a nucleus, a bacterium, etc., as well as supporting key biochemistry and molecular biology reactions. Second, multistep reactions can be performed with ease since small-sized soluble reagents can easily access and leave the reactor space through the hydrogel: subsequent reactions are performed by simply exchanging the solution in contact with the hydrogel. Third, there is no need for oils, chips and/or droplet generation instruments. For automation, an instrument may be used to manage the hydrogel reactor platform but is not required.

[0005] It is nonetheless a requirement that any diffusion of biochemistry or molecular biology reagents within the hydrogel be the most homogeneous possible at any time, in order to process and analyze all the cells in a comparable manner. There is therefore a need for an efficient, safe and easy way to create hydrogels offering this advantage.

SUMMARY

[0006] The present invention aims at solving this problem and thus relates to a gelation device configured for creating a cylindrical hydrogel, the gelation device comprising: a vial extending along a longitudinal axis, the vial displaying a first extremity, a middle section and a second extremity: o the first extremity being open and configured to cooperate with a removable cap, o the middle section displaying a general cylindrical shape, o the second extremity being closed and presenting a general convex support area, a piston configured to be removably insertable inside the vial, the piston also displaying a first extremity, a middle section and a second extremity, the first extremity being open, the middle section displaying a generally cylindrical shape, the second extremity comprising an aperture, wherein the device comprises at least one radial lamella extending along the longitudinal axis, the at least one radial lamella being situated on the vial or on the piston, wherein, when the piston is inserted inside the vial, the at least one radial lamella of the vial or piston is configured to cooperate with a corresponding surface of the vial or piston, said cooperation generating a space between the internal surface of the vial and the external surface of the piston all along the middle section of the vial, wherein the space has a width ranging from about 100 pm and about 3000 pm.

[0007] Thus, this solution achieves the above objective. In particular, it allows easy forming of a thin layer of hydrogel into which any small size reagents can diffuse efficiently. The present invention allows this hydrogel to be homogenous in thickness in order to increase spatial homogeneity of the diffused reagent concentration. The gel casting process to be carried out by the present invention further prevents intense shear and thus biological unit dislocation inside the hydrogel or hydrogel damaging. The specific design also leads to a gelation kit that is fully compatible with standard lab equipment.

[0008] The device according to the invention may include one or more of the following characteristics, taken in isolation from one another or in combination with one another: the device may comprise at least two radial lamellas, preferably four, distributed around the longitudinal axis over at least 180° of the device, the device may comprise at least three radial lamellas, preferably four, distributed around the longitudinal axis over at least 180° of the device, the space may present a constant width over the middle section of the vial, the vial may comprise, at its second extremity at least one internal radial lamella extending longitudinally along the longitudinal axis, the piston may comprise, at its first extremity, at least one external radial lamella extending longitudinally along the longitudinal axis, and when the piston is inserted inside the vial, the at least one internal radial lamella of the vial may be configured to cooperate with the external surface of the piston and the at least one external radial lamella of the piston may be configured to cooperate with the internal surface of the vial, said cooperation generating a space between the internal surface of the vial and the external surface of the piston all along the middle section of the vial, the vial may comprise at least three internal radial lamellas, preferably four, distributed around the longitudinal axis, the piston may comprise at least three external radial lamellas, preferably four, distributed around the longitudinal axis, each internal lamella of the vial may comprise a first extremity and a second extremity, the first extremity of the internal lamella being situated on the middle section of the vial and the second extremity of the internal lamella being situated on the second extremity of the vial, the second extremity of the vial may display a generally conical shape, the second extremity of the vial may display a concave apothem, the second extremity of the vial may comprise, on its external surface, at least one centrifugation blade, the support area of the second extremity of the vial may comprise at least three centrifugation blades, preferably six, equidistantly distributed around the longitudinal axis, the aperture of the piston may comprise a one-way valve which is configured: o to block any fluid circulation between the vial and the piston when the piston is inserted inside the vial, and o to let fluid circulate between the vial and the piston when the piston is removed from the vial, the vial may display a maximal volume of 0.5 L.

[0009] Another object of the present invention relates to a gelation kit comprising a gelation device according to any one of the precedent technical features, and a cap, said cap being configured to be removably secured to the first extremity of the vial.

[0010] A further object of the present invention is a gelation method for creating a cylindrical hydrogel by means of the gelation device and/or the gelation kit according to any one of the precedingly listed technical features, the gelation method comprising following steps: inserting a gellable solution, optionally comprising a biological sample, inside the vial, inserting the piston inside the vial and pushing the piston along the longitudinal axis as far as possible in order to push the gellable solution up along the inner surface along the internal surface of the middle section of the vial, letting the device rest with the piston inserted inside the vial at its maximal capacity, removing the piston once the gellable solution has solidified, thereby obtaining a cylindrical hydrogel optionally comprising a biological sample trapped therein.

[0011] A further object of the present invention is a method of analyzing discrete biological units, comprising the steps of: a) providing the gelation device and/or the gelation kit according to any one of the precedingly listed technical features; b) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units; c) diluting the biological unit/barcode unit complexes in a gellable solution; d) if steps b) and c) were performed outside the vial, pouring the gellable solution after step c) in the vial, then inserting the piston inside the vial and pushing the piston along the longitudinal axis as far as possible, in order to push the gellable solution along the inner surface up along the internal surface of the middle section of the vial; e) letting the device rest with the piston inserted inside the vial at its maximal capacity; f) removing the piston once the gellable solution has solidified, thereby obtaining a cylindrical hydrogel comprising discrete biological unit/barcode unit complexes trapped therein; and g) barcoding the biological unit’s nucleic acids within each of said biological unit/barcode unit complexes with the unique barcode. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention will be better understood, and other aims, details, characteristics and advantages thereof will emerge more clearly on reading the detailed explanatory description which follows, of embodiments of the invention given by way of illustration, purely illustrative and non-limiting examples, with reference to the accompanying drawings:

Figure 1 is a perspective view of a cap according to the present invention,

Figure 2 is a perspective view from a vial according to the present invention, Figure 3 is a perspective view of a piston according to the present invention, Figure 4a is a perspective view of the vial cooperating with the cap,

Figure 4b is a longitudinal cut of the vial cooperating with the cup,

Figure 5 is a longitudinal cut of the piston,

Figure 6a is a perspective view of the vial cooperating with the piston,

Figure 6b is a longitudinal cut of the vial cooperating with the piston,

Figure 7 is a schematical diagram illustrating a process for creating a cylindrical hydrogel by means of the gelation device according to the invention, and for analyzing discrete biological units.

DETAILED DESCRIPTION

Gelation device

[0013] As can be seen on Figure 6a, this invention relates to a gelation device 10 configured for creating a cylindrical hydrogel. The device 10 according to the present invention basically functions as a manual cylindrical hydrogel mold.

[0014] The term “hydrogel” refers to a high water-content 3D network of hydrophilic polymers. These polymers in water are typically found in two states, depending among others on the extent of the interaction between individual polymer molecules: a solution (or liquid) state, and a solidified (or gel or polymerized) state. In the first state, the gellable solution behaves as a viscous liquid, while in the solidified state, the hydrogel exhibits a finite yield stress. The transition from the liquid state to the solidified state typically occurs in response to certain physical stimuli (such as resting time, changes in temperature, electric fields, magnetic fields, solvent composition, light intensity, and/or pressure) and/or chemical stimuli (such as changes in pH, ions, crosslinker addition, catalyst addition, enzyme activation, and/or specific chemical compositions).

[0015] Hydrogels can be classified into physical and chemical hydrogels based on their cross-linking mechanism. In one embodiment, hydrogels are prepared from at least one natural polymer. In one embodiment, hydrogels are prepared from at least one synthetic polymer. In one embodiment, hydrogels are prepared from at least one natural polymer and at least one synthetic polymer. Examples of physical hydrogel crosslinks include, but are not limited to, entangled chains, hydrogen bonding, hydrophobic interaction, ion capture, ion chelation and crystallite formation. Physical hydrogel can be synthesized by ionic interaction, crystallization, stereocomplex formation, hydrophobic interaction, protein interaction and hydrogen bond. Such physical hydrogels may be permanent or transient, reversible or irreversible. Examples of chemical hydrogels crosslinks include, but are not limited to, covalent bounds. Chemical hydrogels can be synthesized by chain growth polymerization, addition and condensation polymerization and gamma and electron beam polymerization. They can be formed by polymerization of end-functionalized macromers. Such chemical hydrogels can be permanent or transient, reversible or irreversible.

[0016] Some specific examples of hydrogels are polysaccharide hydrogels. These include, but are not limited to, alginate, agarose, K-carrageenan, r-carrageenan, chitosan, dextran, dialdehyde starch, heparin, gellan, native gellan gum, rhamsan, deacetylated rhamsan, S-657, xanthan gum and welan. Polysaccharide hydrogels can be formed by covalent crosslinking, ionic crosslinking, chemical conjugation, esterification and/or polymerization. By way of example, when the polysaccharide hydrogel is alginate, it can be crosslinked by ionic crosslinking in presence of a multivalent cation, such as calcium.

[0017] Some other specific examples of hydrogels are protein-based hydrogels. These include, but are not limited to, collagen, fibrin, albumin, gelatin, and laminin. Some protein-based hydrogels can be solidified by cooling or heating; they can also be solidified by crosslinking using a crosslinker, such as cyanamide, diisocyanate, dimethyl adipimidate, epoxy compounds, ethylaldehyde, formaldehyde, glutaraldehyde, glyceraldehyde, hexamethylenediamine, terephthalaldehyde and mixture thereof, optionally in the presence of a crosslinking activator, such as carbodiimide.

[0018] Some other specific examples of hydrogels are polysaccharide hydrogels combined with proteins as described here above.

[0019] Hydrogels can also be non-biodegradable or/and synthetic hydrogels, including, but not limited to, compounds from vinylated monomers and/or vinylated macromers polymerization, in particular, 2 -hydroxy ethyl methacrylate, 2-hydroxypropyl methacrylate, acrylamide, acrylic acid, N-isopropylacrylamide, and poly A sopropylacrylamide.

[0020] Some hydrogels may be able to transition back from their solidified state to a solution state: this phenomenon is classically referred as “hydrogel depolymerization” or “hydrogel melting”, which liquefaction can occur in response to certain chemical stimuli (e.g., by change of ionic concentrations in the case of alginate; or by addition of reducing agent including, without limitation, phosphines [e.g., tris(2-carboxyethyl)phosphine, i.e., TCEP] or dithiothreitol, i.e., DTT, such as in the case of albumin), thermal stimuli (e.g., by temperature increase, such as in the case of thermosensitive or thermoreversible hydrogels if the temperature is raised above their melting point), or enzymatic activity (e.g., by enzymatic degradation, such as in the case of agarose using agarase).

[0021] Hydrogels, once the gellable solution has solidified, exhibit diffusion properties that depend on the size of the diffusing objects. Small diffusing molecules diffuse like in a viscous media at rest while largest molecules interact with the 3D polymer network and diffuse slower. The typical size threshold between viscous-like diffusion and polymer- network interacting diffusion is usually called pore size and depends on numerous parameters such as polymer concentration, crosslinking point concentration, polymer persistence length and/or polymer hydration properties. In any of the embodiments described herein, the hydrogel should typically have a pore size sufficiently small to trap a biological unit, a barcode unit and/or an analyte extracted or derived from a biological unit (z.e., to slow down their diffusion), while having a pore size sufficiently large to allow diffusion of biochemistry and molecular biology reagents. Such biochemistry and molecular biology reagents are well-known to the skilled artisan, and encompass all reagents known to perform biochemistry and molecular biology assays, such as solutions (buffer solutions, wash solutions, and the like), detergents, some enzymes, nucleic acid primers, and the like. These reagents can migrate in the hydrogel by diffusion, by convection or by the action of a field gradient (e.g., by electrophoresis). Typical pore sizes for hydrogels range between about 1 nm and 1 pm, preferably from about 2 nm to about 500 nm, more preferably from about 5 nm to about 100 nm.

[0022] As can be seen on Figures 2, 3 and 5, the gelation device 10 according to the present invention comprises: a vial 12 extending along a longitudinal axis X, a piston 14 configured to be removably insertable inside the vial 12.

[0023] The vial 12 and the piston 14 are configured to cooperate together by sliding, the piston 14 being configured to be removably insertable inside the vial 12 (see Figures 6a and 6b).

[0024] The piston 14 and the vial 12 are both made from biocompatible materials such as glass or plastic e.g. polystyrene, polypropylene, polyethylene, PFTE. In order to limit the hydrogel adhesion, the external surface of the piston 14 exterior surface is designed to display a very low roughness. More particularly, the roughness of the external wall of the piston 14 is such that the arithmetical mean deviation of the assessed profile (Ra) is smaller than 3 pm, preferably smaller than 0.5pm. The treatment of the external wall of the piston 14 can be physical (controlled roughness) or/and chemical (e.g. coating). The roughness of the internal wall of the vial 12 is higher than the roughness of the external wall of the piston 14 in order to improve the adherence of the hydrogel to the internal wall of the vial 12 while retrieving the piston 14.

[0025] As can be seen on Figure 2, the vial 12 displays a first extremity 121, a middle section 120 and a second extremity 122: the first extremity 121 is open (enabling air to enter the vial 12) and configured to cooperate with a removable cap 16 (see Figures 1, 4a and 4b), the middle section 120 displays a general cylindrical shape, the second extremity 122 is closed and displays a general convex support area 18.

[0026] The cap 16 is part of a gelation kit comprising a gelation device 10 according to the present invention, and a cap 16. The cap 16 is configured to be removably secured to the first extremity 121 of the vial 12. The first extremity 121 and the cap 16 may, for example, cooperate by means of screwing means (see Figures 4a and 4b).

[0027] The length of the vial 12 ranges from 15 mm to 300 mm, preferably is around 115 mm. The diameter of the middle section 120 and the first extremity 121 of the vial 12 ranges from 10 to 200 mm, preferably is around 28 mm. Generally speaking, the vial 12 displays a maximal volume of 0.5 L, preferably a volume around 40mL.

[0028] In the present specification, the wording “general cylindrical shape” refers to any shape that appears cylindrical when looked at it. It might be slightly conical (for example with a diameter reduction or draft angle of 1° along the longitudinal axis X), but has to appear cylindrical to the eye.

[0029] The convex support area 18 of the second extremity 122 is configured to cooperate with any sort of carrying device, like for example a laboratory tube rack or a centrifugation device. In some embodiments, the convex support area 18 of the second extremity 122 of the vial 12 displays a generally conical shape. More particularly regarding the embodiment depicted on Figure 2, the second extremity 122 of the vial 12 comprises, on its external surface, at least one centrifugation blade 24. In this case each centrifugation blade 24 is part of the support area 18 and the external profile of each centrifugation blade 24 contributes to the general convex shape of the convex support area 18. The internal profile of each centrifugation blade 24 follows the shape of the walls of the vial 12. In some embodiments, said vial 12 may present a conical shape with concave apothems. Two separate inflexion points are thus to be found. This enables the vial 12 to display a small reservoir 25 of about 1 to 5 mL, preferably 4mL at the tip of the vial 12. This small reservoir 25 enables to concentrate the biological sample to be poured inside the vial 12 and integrated to the hydrogel. The small reservoir 25 also enables to concentrate biological samples during centrifugation, for example. Regarding the embodiment depicted on Figure 2, the support area 18 of the second extremity 122 of the vial 12 comprises at least three centrifugation blades 24, preferably six, distributed around the longitudinal axis X. The blades are preferably equidistantly distributed.

[0030] The piston 14 also displays a first extremity 141, a middle section 140 and a second extremity 142. Like for the vial 12, the first extremity 141 of the piston 14 is open in order to let some air enter the piston 14.

[0031] The length of the piston 14 ranges from 15 to 300 mm, preferably is around 105 mm, in order to comfortably fit inside the vial 12. The diameter of the middle section 140 and the first extremity 141 of the piston 14 ranges from 10 to 200mm, preferably is around 26 mm.

[0032] As can be seen on Figure 3, the first extremity 141 of the piston 14 comprises a manipulation ring 15 at the first extremity 141 of the piston 14. The manipulation ring 15 may be crenellated in order to secure and ease the manipulation of the piston 14.

[0033] In some embodiments, the manipulation ring is an abutment ring 15 against which the first extremity 121 of the vial 12 abuts when the piston 14 has been inserted to the maximal capacity of the vial 12 (see Figures 6a and 6b). This way, the user cannot push the piston 14 too far inside the vial 12, hence cannot induce a non-homogeneous repartition of the hydrogel along the internal wall of the vial 12.

[0034] In some other embodiments, the maximal insertion capacity of the piston 14 inside the vial 12 is defined by the thickness of at least one radial lamella 22 situated either on the vial 12 or the piston 14. More precisely, it is the cooperation by friction or abutment of the piston 14 or the vial 12 with the at least one radial lamella 22 which defines the maximal insertion capacity of the piston 14 inside the vial 12. Further details will be given further below.

[0035] The middle section 140 displays a generally cylindrical shape, the second extremity 142 comprises an aperture 20 (see Figure 5). In a preferred embodiment of the device 10, the aperture 20 of the piston 14 comprises a one-way valve 21 which is configured: to block any fluid circulation between the vial 12 and the piston 14 when the piston 14 is inserted inside the vial 12, and to let fluid, more particularly air, circulate between the piston 14 and the vial 12 when the piston 14 is removed from the vial 12.

[0036] This one-way valve 21 might for example be an umbrella valve, a ball valve or a duckbill valve. The possibility to let air circulate between the piston 14 and the vial 12 while the piston 14 is removed, enables the piston 14 to be removed without damaging the solidified cylindrical hydrogel formed inside the vial 12. On the other hand, blocking all fluidic transfer from the vial 12 to the piston 14 when the piston 14 is inserted inside the vial 12 ensures that no sample can enter the piston 14 and is thus safely pushed up between the piston and the wall of the vial 12 while the piston 14 is inserted inside the vial 12.

[0037] In the embodiment depicted on Figure 3, the second extremity 142 of the piston 14 displays a conical shape with a concave apothem. This enables the piston 14 to fit the vial 12 in order to improve their cooperation.

[0038] As already mentioned here-above, the device 10 according to the present invention further comprises at least one radial lamella 22 extending along the longitudinal axis X. The device 10 according to the present invention preferably comprises at least two radial lamellas 22. Each lamella 22 fulfills the technical function of a spacer between the piston 14 and the vial 12.

[0039] As the radial lamellas 22 of the present invention extend along the longitudinal axis X, it allows the gellable solution to flow along the length of each radial lamellas 22, between the radial lamellas 22, in order to be homogeneously distributed between the radial lamellas 22. In some preferred embodiment, the radial lamellas 22 and the gellable solution (and later the hydrogel) extend in the same place, enabling to control the width w of the hydrogel. [0040] Each radial lamella 22 is thus situated on the vial 12 or on the piston 14. In case the device 10 comprises several radial lamellas, each of them is situated either on the vial 12 or on the piston 14. In some preferred embodiments, all the lamellas 22 of the device 10 have the same thickness. Thus, in this embodiment, the presence of at least two lamellas 22 ensures a homogeneous and constant space between the piston 14 and the vial 12. As can for example be seen on Figure 3, the device 10 comprises at least three radial lamellas 22, preferably four. All the radial lamellas 22 are distributed around the longitudinal axis X. They are distributed over at least 180° of the circumference of the gelation device 10. In a preferred embodiment, the radial lamellas 22 are equidistantly distributed around the longitudinal axis X. In the embodiments in which all the lamellas 22 are identical, this leads to the creation of a space 26 with a constant width w (or thickness w) all along the circumferences of the piston 14 and the vial 12. The space 26 thus presents a constant width w over the middle section 120 of the vial 12.

[0041] In the present application, the wording “constant width” refers to a globally or mainly constant width w. More precisely, this means that the majority of the space 26 presents a given constant width w and that a small fraction of this space 26 presents a larger width, due to the presence of the lamellas 22.

[0042] Each radial lamella 22 extends along the longitudinal axis X and display a general pyramidal shape. More precisely, each radial lamella 22 comprises a first extremity 221 and a second extremity 222. The first extremity 221 is directed towards the middle of the device 10, more particularly, of either the vial 12 or the piston 14 of the device 10. The second extremity 222 is directed towards one extremity of the device 10. It is directed either at the second extremity 122 of the vial 12 or at the first extremity 141 of the piston 14, depending where the radial lamella 22 is situated. The first extremity 221 of each lamella 22 is thinner and slimmer than the second extremity 222 of each lamella 22. This enables each lamella 22 to ensure a convenient clearance angle that facilitates the retrieval of firstly the piston 14 and then the cylindrical hydrogel from the vial 12 once the cylindrical hydrogel is created. This further enables a cooperation, by friction between each lamella 22 and the corresponding surface of the vial 12 or the piston 14, said cooperation increasing or diminishing along the axial direction depending on the relative movement between the piston 14 and the vial 12. This cooperation by friction enables the definition of a maximal insertion capacity of the piston 14 inside the vial 12, as already mentioned above. To further avoid damages to the hydrogel, the radial lamellas 22 present a longitudinal length that does not exceed half of the length of the device 10 (the vial 12 or the piston 14, depending on which element the radial lamella 22 is situated on). Too long radial lamellas 22 could prevent the gellable solution to be homogeneously distributed over the external surface of the piston 14 and the internal face of the vial 12 and generate some sorts of tunnels or grooves inside the cylindrical hydrogel.

[0043] The longitudinal length of each lamella 22 ranges from 10 to 80% of the total length of the vial 12, preferably 40% of the total length of the vial 12. In other words, regarding the embodiments, the longitudinal length of each lamella 22 therefore ranges from 10 to 200 mm, more preferably around 50 mm.

[0044] In some embodiments, the device 10 comprises a set of radial lamellas 22 on the internal side of the vial 12. In some other embodiments, the device 10 comprises a set of radial lamellas 22 on the external side of the piston 14. More particularly, in the embodiment depicted on Figures 2 to 6b, the vial 12 comprises, at its second extremity 122, a first set of at least one internal radial lamella 22 extending longitudinally along the longitudinal axis X and the piston 14 comprises, at its first extremity 141, a second set of at least one external radial lamella 22 also extending longitudinally along the longitudinal axis X.

[0045] As the piston 14 and the vial 12 can be turned around the longitudinally axis X independently from each other, the first and second set of radial lamellas 22 can be longitudinally aligned with each other, or not. The user can decide whether the radial lamellas 22 of the first and second set should be longitudinally aligned or not. Preferably, they should not. This further lower the risks of generating tunnels or grooves. The longitudinal gap between the two sets of radial lamellas 22 further improves the gellable solution repartition along the internal wall of the vial 12 while the piston 14 is inserted inside the vial 12: as the gellable solution is quite viscous, if the insertion of the piston 14 inside the vial 12 does not happen in alignment with the longitudinal axis X, the result might be some inhomogeneous repartition of the gellable solution along the internal wall of the vial 12, one side of the vial 12 being faster covered than the other, which could lead to some overflow of the gellable solution outside the vial 12. To avoid this, the circulation of the gellable solution is facilitated over the whole internal circumference of the vial 12 by the gap between the two sets of lamellas. This provides space for the gellable solution to circulate along the internal wall of the vial 12 and improve its homogeneous repartition while the piston 14 is inserted inside the vial 12. A non- alignment between the two set of lamellas 22 can also increase this homogeneous spreading effect, leading to a homogeneous gel to be cast.

[0046] In the present application, the word “homogeneous” designates objects that have at least a constant thickness or width. In some alternative embodiments, it can further refer to homogeneity of other physical, chemical or mechanical features, such as, for example, a homogeneous volumetric repartition of molecules or beads or a homogeneous elasticity, optical index etc.

[0047] According to this embodiment with two sets of radial lamellas 22, the vial 12 comprises at least three internal radial lamellas 22, preferably four, distributed around the longitudinal axis X. They preferably are equidistantly distributed around the longitudinal axis X. Regarding the embodiment depicted on Figure 4b, the first extremity 221 of the internal radial lamella 22 is situated on the middle section 120 of the vial 12 and the second extremity 222 of the internal radial lamella 22 is situated on the second extremity 122 of the vial 12 (see Figure 4b). In this embodiment, the maximal insertion capacity of the piston 14 inside the vial 12 is defined by the thickness of the second extremity 222 of the internal radial lamella 22 situated on the second extremity 122 of the vial 12. The piston 14, when inserted at its maximal capacity abuts against said second extremity 222 of the internal radial lamella 22.

[0048] In this very embodiment, the piston 14 comprises at least three external radial lamellas 22, preferably four, also distributed around the longitudinal axis X. They also are preferably equidistantly distributed around the longitudinal axis X, in order to improve the homogeneity of the created space 26. This leads, in some embodiments, to an improved constancy of the width w of the space 26 all around the circumference of the piston 14.

[0049] When the piston 14 is inserted inside the vial 12, each radial lamella 22 of the vial 12 or piston 14 is configured to cooperate with a corresponding surface of the vial 12 or piston 14, said cooperation generating a space 26 between the internal surface of the vial 12 and the external surface of the piston 14 all along the middle section 120 of the vial 12 (see Figure 6b). More precisely, regarding the embodiments in which the gelation device 10 comprises two sets of lamellas 22, one in the vial 12 and one on the piston 14, when the piston 14 is inserted inside the vial 12, each internal radial lamella 22 of the vial 12 is configured to cooperate with the external surface of the piston 14 and each external radial lamella 22 of the piston 14 is configured to cooperate with the internal surface of the vial 12. In this case also, said cooperation generates a space 26 between the internal surface of the vial 12 and the external surface of the piston 14 all along the middle section 120 of the vial 12.

[0050] Regardless of the embodiment, the space 26 has a width w (or thickness w) ranging from about 100 pm to about 3000 pm. Preferably, the width w (or thickness w) of the space 26 is about 500 pm in order to generate a cylindrical hydrogel of 500 pm thickness.

[0051] In some preferred embodiments, the space 26 has a constant width w (or thickness w) ranging from about 100 pm to about 3000 pm. Preferably, the width w (or thickness w) of the space 26 is constant and about 500 pm in order to generate a cylindrical hydrogel of 500 pm thickness.

Gelation method

[0052] The gelation device 10 and the corresponding cap 16 according to the present invention enable the implementation of a gelation method for creating a cylindrical hydrogel as illustrated on Figure 7. Preferably, the gelation device 10 and the corresponding cap 16 according to the present invention enable the implementation of a gelation method for creating a homogeneous cylindrical hydrogel with a constant thickness. [0053] The gelation method comprises following steps: inserting a gellable solution inside the vial 12, and more particularly inside the small reservoir 25 of the vial 12, inserting the piston 14 inside the vial 12 and pushing the piston 14 along the longitudinal axis X as far as possible, in order to push the gellable solution up along the internal surface of the middle section 120 of the vial 12, letting the device 10 rest with the piston 14 inserted inside the vial 12 at its maximal capacity while the gellable solution solidifies and becomes a hydrogel, removing the piston 14 once the gellable solution has solidified and has become a hydrogel with, depending on the embodiments, a constant thickness determined by the constant width w of the space 26 generated by the lamellas 22, thereby obtaining a preferably homogeneous cylindrical hydrogel.

[0054] While the piston 14 is inserted, the closed one-way valve 21 avoids any liquid to enter the piston 14 and forces the gellable solution to be distributed around the longitudinal axis X. When the piston 14 is removed, the one-way valve 21 is open and air can enter from the first extremity 141 of the piston 14 inside the piston 14 and enter the vial 12 through the aperture 20 in order to ease the retrieval of the piston 14 by preventing pressure difference with atmospheric pressure and subsequent damage on the cylindrical hydrogel.

[0055] In one embodiment, the gellable solution inserted inside the vial 12 comprises a biological sample. This way, the biological sample can be trapped inside the cylindrical hydrogel once it has solidified.

[0056] In one embodiment, the biological sample comprises a plurality of biological units. By “biological unit”, it is referred to biological structures, or to portions, components or combinations of biological structures. Examples of biological units include, but are not limited to, a cell or a group of cells, a virus, an organelle (such as a nucleus, a mitochondrion or a chloroplast), a macromolecular complex (such as an exosome), a biological macromolecule (such as a chromosome, a nucleic acid fragment, a contiguity preserved transposition DNA (CPT-DNA) fragment, a protein or a peptide). [0057] In one embodiment, the biological sample further comprises a plurality of barcode units. By “barcode unit”, it is referred to a substrate or support, which may be rigid, solid or semi-solid, bearing at least one “barcode” and preferably a plurality of barcodes. Barcodes are molecular patterns which can be used as unique identifiers, e.g., to uniquely identify a discrete biological unit. The term barcode further refers to the molecular pattern which is used to identify the source or origin of an analyte within a biological sample.

[0058] The composition, shape, form, and modifications of the barcode unit can be selected from a range of options depending on the application. Exemplary materials that can be used as a barcode unit include, but are not limited to, acrylics, carbon (e.g., graphite, carbon-fiber), cellulose (e.g., cellulose acetate), ceramics, controlled-pore glass, cross-linked polysaccharides (e.g., agarose, SEPHAROSE™ or alginate), gels, glass (e.g., modified or functionalized glass), gold (e.g., atomically smooth Au(l 11)), graphite, inorganic glasses, inorganic polymers, latex, metal oxides (e.g., SiO2, TiO2, stainless steel), metalloids, metals (e.g., atomically smooth Au(l l l)), mica, molybdenum sulfides, nanomaterials (e.g., highly oriented pyrolitic graphite (HOPG) nanosheets), nitrocellulose, NYLON™, optical fiber bundles, organic polymers, paper, plastics, polacryloylmorpholide, poly(4-methylbutene), polyethylene terephthalate), poly(vinyl butyrate), polybutylene, poly dimethylsiloxane (PDMS), polyethylene, polyformaldehyde, polymethacrylate, polypropylene, polysaccharides, polystyrene, polyurethanes, polyvinylidene difluoride (PVDF), quartz, rayon, resins, rubbers, semiconductor material, silica, silicon (e.g., surface-oxidized silicon), sulfide, and TEFLON™. Barcode units can be composed of a single material or of a mixture of several different materials. Barcode units can be simple square grids, checkerboard grids, hexagonal arrays and the like. Suitable barcode units also include, but are not limited to, beads, slides, chips, particles, strands, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, culture dishes, microtiter plates such as 768-well, 384-well, 96-well, 48-well, 24-well, 12-well, 8-well, 6-well, 4-well, 1-well and the like. In one embodiment, the barcode unit is a bead. In one embodiment, a single barcode unit in a plurality of barcode units may be a minimal, indivisible part of said plurality of barcode units. A single barcode unit in a plurality of barcode units may be, e.g., a single square on a grid, a single bead in a population of beads, a single well in a microtiter plate, etc. Alternatively, a single barcode unit in a plurality of barcode units may be a minimal part of said plurality of barcode units, wherein a single binding event between a biological unit and a barcode unit occurs at the molecular level. Alternatively, a single barcode unit in a plurality of barcode units may be a part of said plurality of barcode units ranging from about 1 pm² to about 1 mm², preferably from about 1 pm² to about 100 pm², more preferably from about 1 pm² to about 50 pm². In one embodiment, this size range is chosen for manufacturability. In one embodiment, this size range is chosen to ensure the formation of biological unit/barcode unit complexes with a 1:1 ratio.

[0059] In one embodiment, each single barcode unit in a plurality of barcode units comprises a unique identifier or “barcode”. In one embodiment, each single barcode unit in a plurality of barcode units comprises clonal copies of a unique barcode. Barcodes may be of a variety of different formats, including labels, tags, probes, and the like. For example, the barcode unit may be optically and/or non-optically barcoded. Optical barcodes include, but are not limited to, chromophores, fluorophores, quantum dots, styrene monomers, and combination thereof, which can be identified, e.g., by their spectrum such as Raman spectrum or electromagnetic spectrum; and/or by their intensity of color. Non-optical barcodes include, but are not limited to, biomolecular sequences such as nucleic acid sequence (z.e., DNA and/or RNA sequence) and/or protein sequences, which can be identified, e.g., by sequencing.

[0060] In one embodiment, the number of clonal copies of each unique barcode comprised in each single barcode unit in a plurality of barcode units ranges from 2 to about 10¹².

[0061] In a specific embodiment, the barcode unit comprises nucleic acid barcodes. Nucleic acid barcodes are preferably single- stranded but could be double- stranded. Nucleic acid barcodes can be DNA barcodes, RNA barcodes, or a mixture thereof. Nucleic acid barcodes may comprise from 5 to 20 nucleotides, preferably from 8 to 16 nucleotides. Nucleic acid barcodes can comprise degenerate sequences. [0062] It follows that the term “barcoding” refers to the covalent or non-covalent attachment of a barcode to a biological unit or to an analyte of said biological unit.

[0063] Barcoding of a biological unit’s nucleic acid can be achieved by primer template annealing of a nucleic acid barcode to the biological unit’s nucleic acid. It can also be achieved by primer-directed extension, or by ligation.

[0064] In order to allow immobilization of nucleic acid sequences of or from the biological units, it may be desirable that the nucleic acid barcode further comprises at least one nucleic acid sequence primer, in order to replicate, extend and/or amplify genetic information of or from the biological units after barcoding. Nucleic acid sequence primers may be a degenerate (z.e., random) nucleic acid sequence primer, or may be specific to a nucleic acid sequence of interest. Nucleic acid sequence primers may prime at multiple locations of the nucleic acid sequences of or from the biological units. Nucleic acid sequence primers may comprise from 5 to 50 nucleotides, preferably from 5 to 30 nucleotides.

[0065] Nucleic acid sequence primers may comprise a poly-dT sequence or a poly-dU sequence, in order to be specific for a poly-A sequence. Poly-A sequences may be found, e.g., on the 3’ end of mRNAs, within the poly-A tail.

[0066] Nucleic acid sequence primers may alternatively comprise a (dT)_nVN or (dU)nVN sequence, wherein n ranges from 5 to 50, V represents any nucleotide but T/U (z.e., A, C or G), and N represents any nucleotide (z.e., A, T/U, C or G), in order to be specific for a (A)_nBN sequence, wherein n ranges from 5 to 50, B represents any nucleotide but A (z.e., T/U, C or G), and N represents any nucleotide (z.e., A, T/U, C or G). (A)nBN sequences may be found, e.g., on the 3’ end of mRNAs, overlapping between the poly-A tail and the 3’ UTR or CDS.

[0067] Nucleic acid sequence primers may alternatively comprise a poly-I sequence. Accordingly, the nucleic acid sequence primer is non-specific and can prime to any nucleic acid sequence of or from the biological units. [0068] In one embodiment, a barcode unit comprises at least one oligonucleotide and preferably a plurality of oligonucleotides, each comprising a nucleic acid barcode and a nucleic acid sequence primer.

[0069] In order to allow replication, extension and/or amplification of nucleic acid sequences of or from the biological units, it may be desirable that the oligonucleotide comprising the nucleic acid barcode and optionally the nucleic acid sequence primer further comprises at least one PCR handle sequence. PCR handle sequences are preferably identical across all oligonucleotides and barcodes units. PCR handle sequences may comprise from 10 to 30 nucleotides, preferably from 15 to 25 nucleotides.

[0070] In one embodiment, each barcode unit further comprises at least one means for binding a biological unit, in order to form biological unit/barcode unit complexes.

[0071] The means for binding can provide an aspecific binding, or alternatively a specific binding of a biological unit. Such means for binding a biological unit comprise, but are not limited to, a protein or a fragment thereof, a peptide, an antibody or a fragment thereof, a nucleic acid (such as single-stranded or double-stranded DNA or RNA), a carbohydrate (such as a monosaccharide, disaccharide or polysaccharide), a lipid, a vitamin or a derivative thereof, a coenzyme or a derivative thereof, a receptor ligand or derivative thereof, a fatty acid, and a hydrophobic group (such as an alkyl group having from 2 to 8 carbon atoms or more, an aryl group, an acyl group, and the like).

Method of analyzing discrete biological units

[0072] By way of example, the gelation method for creating a cylindrical hydrogel according to the present invention can be implemented more broadly in a method of analyzing discrete biological units as illustrated on Figure 7.

[0073] In particular, analyzing discrete biological units can include: analyzing gene expression in discrete biological units; analyzing the genotype in discrete biological units; analyzing the haplotype of discrete biological units; and/or analyzing the epigenome in discrete biological units. [0074] All these methods have been described in details in WO 2018/203141, the content of which is incorporated herein by reference.

[0075] In more details, Figure 7 shows the subsequent steps of an exemplary method of analyzing gene expression in discrete biological units (e.g., cells at the single-cell level), as follows: a biological sample comprising biological unit is inserted in the small reservoir 25 of the vial 12; a gellable solution is poured in the vial 12 and mixed with the biological sample; the piston 14 is inserted inside the vial 12; the piston 14 is pushed along the longitudinal axis X as far as possible inside the vial 12, in order to push the gellable solution up along the inner surface along the internal surface of the vial 12; the gellable solution solidifies inside the device 10, creating a cylindrical hydrogel embedding the biological units; the piston 14 is removed from the vial 12; the cylindrical hydrogel is contacted with a lysis buffer inside the vial 12, thereby lysing the biological units; during this step, the biological units’ nucleic acids are released but remain in close proximity with the barcode unit(s) from their biological unit/barcode unit complexes with which they interact to be barcoded; the hydrogel is liquified, e.g., by addition of a degellation buffer; the barcoded nucleic acids can be retrieved and transferred to a 1.5 mL microtube, together with molecular biology reagents, e.g., reagents for nucleic acid reverse transcription and/or nucleic acid amplification. the amplified nucleic acids are separated from the barcode units for further processing.

[0076] The method according to the present invention can therefore comprise steps of: contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, diluting the biological unit/barcode unit complexes in a gellable solution; inserting the piston 14 inside the vial 12 comprising the gellable solution, and pushing the piston 14 along the longitudinal axis X as far as possible, meaning until the piston 14 reaches its maximal inserted position inside the vial 12, in order to push the gellable solution up along the internal surface of the middle section 120 of the vial 12; letting the device 10 rest with the piston 14 inserted inside the vial 12 at its maximal capacity while the gellable solution solidifies; removing the piston 14 once the gellable solution has solidified, thereby obtaining a cylindrical hydrogel comprising discrete biological unit/barcode unit complexes trapped therein; and barcoding the biological unit’ s analytes within each of said biological unit/barcode unit complexes with a unique barcode.

[0077] Contacting the plurality of biological units with the plurality of barcode units may be carried out outside the vial 12, or inside the vial 12. When carried out inside the vial 12, it is preferably carried out inside the small reservoir 25 of the vial 12. If carried out outside the vial 12, then the gellable solution comprising the biological unit/barcode unit complexes diluted is poured in the vial 12, preferably in the small reservoir 25 of the vial 12, prior to inserting the piston 14 inside the vial 12.

[0078] When the biological units are cells, vesicles, capsules or membrane-compartments, the method can further comprise a step of lysis of the biological units. This step allows the release of the biological units’ nucleic acids. Preferably, this step is carried out in the hydrogel, in order for these released nucleic acids to be barcoded with a unique barcode within each discrete biological unit/barcode unit complex trapped in the hydrogel.

[0079] Once the biological units or their analytes such as their nucleic acids have been barcoded in the hydrogel, biochemistry and molecular biology assays can be performed either on these biological units or their analytes trapped in the hydrogel; or alternatively, the hydrogel can be liquified and these assays can be carried out in solution and/or in bulk. Examples of such biochemistry and molecular biology assays include, but are not limited to, amplification (such as, e.g., by polymerase chain reaction [PCR]), reverse-transcription, purification, nucleic acid hydrolyzing, decapping, sequencing, transcriptome profiling, genotyping, epigenome profiling, phasing, haplotyping and the like.

[0080] In one embodiment, the biological units are cells, and the method is for analyzing discrete cells,

at the single-cell level.

Claims

CLAIMS Gelation device (10) configured for creating a cylindrical hydrogel, the gelation device (10) comprising: a vial (12) extending along a longitudinal axis (X), the vial (12) displaying a first extremity (121), a middle section (120) and a second extremity (122): o the first extremity (121) being open and configured to cooperate with a removable cap (16), o the middle section (120) displaying a general cylindrical shape, o the second extremity (122) being closed and presenting a general convex support area (18), a piston (14) configured to be removably insertable inside the vial (12), the piston (14) also displaying a first extremity (141), a middle section (140) and a second extremity (142), the first extremity (141) being open, the middle section (140) displaying a generally cylindrical shape, the second extremity (142) comprising an aperture (20), wherein the device (10) comprises at least one radial lamella (22) extending along the longitudinal axis (X), the at least one radial lamella (22) being situated on the vial (12) or on the piston (14), wherein, when the piston (14) is inserted inside the vial (12), the at least one radial lamella (22) of the vial (12) or piston (14) is configured to cooperate with a corresponding surface of the vial (12) or piston (14), said cooperation generating a space (26) between the internal surface of the vial (12) and the external surface of the piston (14) all along the middle section (120) of the vial (12), wherein the space (26) has a width (w) ranging from about 100 pm and about 3000 pm. Gelation device (10) according to the preceding claim, wherein the device (10) comprises at least two radial lamellas (22), preferably four, distributed around the longitudinal axis (X) over at least 180° of the device (10). Gelation device (10) according to the preceding claim, wherein the device (10) comprises at least three radial lamellas (22), preferably four, distributed around the longitudinal axis (X) over at least 180° of the device (10). Gelation device (10) according to any one of the preceding claims, wherein the space (26) presents a constant width (w) over the middle section (120) of the vial (12). Gelation device (10) according to any one of the preceding claims, wherein the vial (12) comprises, at its second extremity (122), at least one internal radial lamella (22) extending longitudinally along the longitudinal axis (X), wherein the piston (14) comprises, at its first extremity (141), at least one external radial lamella (22) extending longitudinally along the longitudinal axis (X), and wherein, when the piston (14) is inserted inside the vial (12), the at least one internal radial lamella (22) of the vial (12) is configured to cooperate with the external surface of the piston (14) and the at least one external radial lamella (22) of the piston (14) is configured to cooperate with the internal surface of the vial (12), said cooperation generating a space (26) between the internal surface of the vial (12) and the external surface of the piston (14) all along the middle section (120) of the vial (12). Gelation device (10) according to claim 4, wherein the vial (12) comprises at least three internal radial lamellas (22), preferably four, distributed around the longitudinal axis (X). Gelation device (10) according to claim 4, wherein the piston (14) comprises at least three external radial lamellas (22), preferably four, distributed around the longitudinal axis (X). Gelation device (10) according to any one of claims 4 to 6, wherein each internal lamella (22) of the vial (12) comprises a first extremity and a second extremity, the first extremity of the internal lamella (22) being situated on the middle section of the vial (12) and the second extremity of the internal lamella (22) being situated on the second extremity of the vial (12).

9. Gelation device (10) according to any one of the preceding claims, wherein the second extremity (122) of the vial (12) displays a generally conical shape.

10. Gelation device (10) according to the preceding claim, wherein the second extremity (122) of the vial (12) displays a concave apothem.

11. Gelation device (10) according to any one of the preceding claims, wherein the second extremity (122) of the vial (12) comprises, on its external surface, at least one centrifugation blade (24).

12. Gelation device (10) according to any one of the preceding claims, wherein the aperture (20) of the piston (14) comprises a one-way valve (21) which is configured: to block any fluid circulation between the vial (12) and the piston (14) when the piston (14) is inserted inside the vial (12), and to let fluid circulate between the vial (12) and the piston (14) when the piston (14) is removed from the vial (12).

13. Gelation device (10) according to any one of the preceding claims, wherein the vial (12) displays a maximal volume of 0.5 L.

14. Gelation method for creating a cylindrical hydrogel by means of the gelation device (10) and the gelation kit according to any one of claims 1 to 12 and claim 13, the gelation method comprising following steps: inserting a gellable solution, optionally comprising a biological sample, inside the vial (12), inserting the piston (14) inside the vial (12) and pushing the piston (14) along the longitudinal axis (X) as far as possible in order to push the gellable solution up along the inner surface along the internal surface of the middle section (120) of the vial (12), letting the device (10) rest with the piston (14) inserted inside the vial (12) at its maximal capacity, removing the piston (14) once the gellable solution has solidified, thereby obtaining a cylindrical hydrogel optionally comprising a biological sample trapped therein. A method of analyzing discrete biological units, comprising the steps of: a) providing a gelation device (10) according to any one of claims 1 to 12; b) contacting a plurality of biological units with a plurality of barcode units to form biological unit/barcode unit complexes, wherein each barcode unit comprises a unique barcode, and wherein said barcode units comprise at least one means involved with binding said biological units; c) diluting the biological unit/barcode unit complexes in a gellable solution; d) if steps b) and c) were performed outside the vial (12), pouring the gellable solution after step c) in the vial (12), then inserting the piston (14) inside the vial (12) and pushing the piston (14) along the longitudinal axis (X) as far as possible, in order to push the gellable solution along the inner surface up along the internal surface of the middle section (120) of the vial (12); e) letting the device (10) rest with the piston (14) inserted inside the vial (12) at its maximal capacity; f) removing the piston (14) once the gellable solution has solidified, thereby obtaining a cylindrical hydrogel comprising discrete biological unit/barcode unit complexes trapped therein; and g) barcoding the biological unit’s nucleic acids within each of said biological unit/barcode unit complexes with the unique barcode.