Device for manipulating cells, particles and / or liquids through magnetic fields and by suction

Abstract

Device for manipulating cells, particles and / or liquids by means of two modes of operation, a magnetic mode and a suction mode, comprising: two actuating structures (5 and 5'), one for each mode, capable of sliding independently through a corresponding channel (25, 25') of the main tubular element (23), two rods (4, 4'), each rod corresponding to one mode of operation and integral with one of the drive structures (5, 5'), coupling pieces (9, 10), one for the magnetic mode and one for the suction mode, each provided with a channel (25, 25') through which the corresponding rod (4, 4') runs, where one coupling piece (9) is provided with means (18, 19) for housing a disposable cover (16) for the magnetic mode and another coupling piece (10) is provided with means (21) for housing a capillary (15) for the suction mode, springs, levers and slots that cooperate to activate one or the other mode of operation.

Description

[0001] DEVICE FOR MANIPULATING CELLS, PARTICLES AND / OR LIQUIDS THROUGH MAGNETIC FIELDS AND BY ASPIRATION

[0002] DESCRIPTION

[0003] Technology sector

[0004] The invention falls within the field of biotechnology, nanotechnology and microstructures, more specifically within laboratory devices intended for cell cultures and used in assisted reproduction.

[0005] Background of the invention

[0006] Cell manipulation, both individually and in groups, is a constant and routine procedure present in every process and technique performed in laboratories, from cell culture labs to assisted reproduction labs, at all stages of in vitro embryo production. This constant manipulation means that the human factor poses a significant risk of damage and loss of valuable biological material, as well as slowing down workflows. Working with cells requires a high level of training and qualification on the part of laboratory personnel.Thus, the risk of human error translates, on the one hand, into a considerable probability of cell loss during handling, especially important in assisted reproduction laboratories where each cell (oocyte and / or embryo) acquires enormous individual value due to its limited availability. On the other hand, the time required for cell mobilization tasks is limited by the skill required for such handling, particularly when working with small cell groups (e.g., spheroids or embryos) or individual cells such as oocytes. This situation creates a complex environment for working with all types of cells and is of particular interest when handling times must be especially short, such as during the use of cryoprotectants in vitrification and cryopreservation techniques.The use of magnetic nanoparticles or microparticles that adhere to cells now has a wide range of applications. Virtually any type of cultureable cell can now be functionalized with magnetic nanoparticles or microparticles capable of being attracted by an external magnetic field. This has led to advances and improvements in the field of cell cultures, enabling the creation of three-dimensional cultures through the application of a magnetic force. This technology has provided cell cultures with advantages such as three-dimensional growth (spheroids / organoids) that enhances their metabolic capabilities and greatly benefits the objectives of the study and its functionality. Culture in spheroids / organoids simulates the three-dimensional structures of cell growth, providing a better model for studying cell progression in vitro.Cell culture systems are becoming increasingly complex and are better able to mimic culture conditions to closely resemble in vivo conditions. The ability to direct cells, cell groups, or spheroids / organoids would be advantageous in manipulating this material, and the ability to move them across surfaces adapted to the needs of the assays—for example, microchannels coated with other cell types—would be of great interest.

[0007] The type of individual manipulation required for oocytes and embryos is even more complex than when working with groups of cells. Within the field of assisted reproductive technologies, this difficulty is compounded by the high value of each of these cells, which often represents the only available specimen of a genetically relevant trait. As mentioned, the manipulation of oocytes and embryos must be performed individually, and during an in vitro fertilization program, the operator's skill becomes paramount. Through the continuous changes in the media that these cells undergo, the risk of cell loss or stress increases, which can mean the loss of irreplaceable material.

[0008] On the other hand, one of the main tools widely used in biotechnology, and especially in assisted reproduction techniques, is the freezing or vitrification of cells, particularly for the vitrification of oocytes and embryos. Vitrification has replaced slow or traditional freezing in this field, as the former technique reduces the damage caused by the formation of ice crystals. Vitrification has proven to be a fundamental methodology in both human and animal assisted reproduction. The ability to cryopreserve female gametes without compromising their quality has been a key advance for preserving fertility in women undergoing cancer treatments. Furthermore, its application in the cryopreservation of gametes and embryos produced in vitro or obtained in vivo in animals has been vital for the livestock sector, as well as for the recovery of endangered animal species.

[0009] There are different techniques and systems that can be used for vitrification. Initially, a manual vitrification method was used, employing different cryoprotectants with gradually increasing concentrations. Advances in this field have led to the development of new vitrification systems, mostly intended for the field of human reproduction, which involve the use of easily stored vitrification devices with pre-established vitrification protocols. Currently available vitrification devices, which allow for the manipulation of oocytes or embryos, can be either closed or open. In closed systems, the material to be vitrified does not come into contact with the liquid nitrogen thanks to a cap that protects the device. However, open systems do not have this cap.Even with improvements in technique, this process remains highly dependent on the operator's skill, since it involves working with cryoprotectants at very high concentrations and they should not remain in them for longer than the established time.

[0010] The devices developed to date rely on the surface tension created on a small circular surface and the working liquid reagent, providing the adhesion surface for the oocytes or embryos. Loss of biological material during the handling steps involved in vitrification is very common, even with the new commercial devices used, where the oocyte and / or embryo must be carefully placed on a tab using a stereoscopic magnifying glass as a guide.

[0011] Regarding the current state of the art, there are known patents and articles related to the manipulation of cells using magnetic fields, the most relevant of which are listed below:

[0012] Document WO9940444A1 describes a device for manipulating magnetic particles, consisting of a tube with a disposable cover at its rear end and a movable stem inside, with a magnet at its tip, that attracts the magnetic particles to be captured. Among the applications mentioned are the manipulation or separation of cells in biochemical analyses.

[0013] Document W02013019212A1 refers to a device for facilitating the manipulation of magnetized cells. The device consists of a tubular structure containing a guide that moves a magnet fixed at its end, bringing it close to the magnetized cells and trapping them by magnetic attraction onto a disposable cover located at the end of the tubular structure and in contact with the cell culture. Manual operation of the magnet's guiding mechanism is provided.

[0014] US patent 5647994A describes a magnetic particle separation device for a solution. In one embodiment, the device consists of a pipette tube attached to a handle at the top and a movable ring magnet located externally to the tube at the bottom and mounted on a movable element. The magnet can be manually moved vertically, attracting the magnetic particles to be separated as it approaches. The device is intended for the separation of biomaterials, particularly cells, using magnetic particles.

[0015] Patent application US8118754B1 discloses a magnetic biopsy device for extracting tumor cells labeled with superparamagnetic nanoparticles. The device consists of a cannula containing a stem with a magnet at one end that moves toward the biopsy site, exerting magnetic attraction on the magnetically labeled cells, which adhere to a disposable cover.

[0016] Document W00189705A2 describes a robot for manipulating magnetic particles attached to RNA or DNA molecules in solution, comprising a series of movable magnets for retaining and releasing said molecules from a sample located in a receiving tube that receives inside the magnet movable between two positions by the action of a guiding mechanism.

[0017] However, none of the devices described so far are capable of operating both with magnetic fields and by means of the aspiration technique.

[0018] Summary of the invention

[0019] The present invention offers a solution to the problems of handling particles, cells, and / or liquids arising from the various techniques used in biotechnology laboratories, thanks to the integration of two functionalities into a single device: an aspiration mode and a magnetic mode. The device allows for a simple, easy, and quick switch between magnetic and aspiration modes. This dual capability is of great importance in laboratory work because, at certain times, for example, when cells (oocytes, embryos, or spheroids / organoids) are not bound with nanoparticles, manipulation via aspiration systems becomes necessary. Specifically, the invention provides a device for manipulating cells, particles, and / or liquids using two operating modes: a magnetic mode and an aspiration mode.comprising a main tubular element provided with two internal channels, two actuating structures, one for each mode, capable of sliding independently along a corresponding channel of the main tubular element, two rods, each rod corresponding to an operating mode and attached to one of the actuating structures, two coupling pieces, one for the magnetic mode and the other for the aspiration mode, each provided with a channel through which the corresponding rod runs, wherein one coupling piece is provided with means for housing a disposable cover for the magnetic mode and the other coupling piece is provided with means for housing a capillary for the aspiration mode, wherein the rod of the magnetic mode supports a magnet and the rod of the aspiration mode runs inside the capillary for the aspiration mode, four springs arranged in two pairs, one pair for each operating mode,Each pair consists of an internal spring and a concentric external spring, two actuating levers, each associated with and integral to its corresponding coupling piece, and in turn associated with a longitudinally extending groove along part of the main tubular element. This groove is provided with a recess, such that when the lever slides into the groove and engages in the recess, the coupling piece corresponding to that mode is displaced towards the rear of the device. This causes the springs to cooperate, allowing the corresponding actuating structure to slide and move the corresponding stem, thus activating one or the other operating mode. Optional implementations are described in the dependent claims.

[0020] In this way, a multi-function device is obtained according to the needs in the laboratory, being able to work through a system of magnetic fields or by aspiration of cells, groups of these, particles and / or liquids in a practical, safe and simple way.

[0021] Description of the figures

[0022] In order to aid a better understanding of the characteristics of the invention, according to a preferred embodiment thereof, a series of drawings are provided as an integral part of said description, where, for illustrative and non-limiting purposes, the following has been presented:

[0023] Figs. 1A and 1B: show a side elevation view of the device (top image; FIG 1A) and a sagittal section (bottom image; FIG 1B).

[0024] Fig. 2: shows a side elevation view of the internal structures of the device.

[0025] Figs. 3A and 3B: show a side elevation view of the complete driven device (top image; FIG 3A) and a sagittal cross-section (bottom image; FIG 3B) for use through magnetic fields.

[0026] Fig. 4: shows a view of the disposable cover with detail of the protrusion at its front end. Figs. 5A, 5B and 5C: show a view of the cylindrical piece (FIG. 5A) which has a grooved piece at its end (FIG. 5B), as well as a sagittal section of the same (FIG. 5C), which is used to attach a disposable cover for magnetic mode.

[0027] Fig. 6: shows a view of a cylindrical capillary coupling piece for aspiration mode.

[0028] Fig. 7: shows a view for use in magnetic mode with the cap arranged at its rear end for the protection of the material attached to the disposable cover when the magnetic mode has been activated.

[0029] Figs. 8A and 8B: show a side elevation view of the complete device (top image; FIG. 8A) and a side elevation view of the device in sagittal section (bottom image; FIG. 8B) for use in aspiration mode.

[0030] Figs. 9A and 9B: show a side elevation view of the device actuated for use in aspiration mode (image above; FIG. 9A), to subsequently show a detail of the concentric arrangement of the springs when they are actuated (aspiration mode, at the top of the device in this case) and when they are not (magnetic mode, at the bottom of the device in this case) (FIG. 9B).

[0031] Figs. 10A, 10B and 10C: show a side elevation view of the device actuated for use in aspiration mode (FIG. 10A) with a detail of the main tubular element of the device (FIG. 10B) and the arrangement of the springs when the device is (at the top of the device in this case) or not (at the bottom of the device in this case) activated (FIG. 10C).

[0032] Detailed description of the invention

[0033] With reference to Figures 1A and 1B, the cell, cell group, particle, and / or liquid handling device (1) of the invention comprises a tubular main element (23), a manual actuation portion (3) at the front end of the main element (end closest to the user) consisting of two independent actuation structures (5 and 5') that are elongated and narrower than the tubular main element (23). These structures are inserted inside the tubular main element (23) and slide independently of each other within two channels (25, 25', FIG. 10B). The actuation structures (5, 5') are attached at their rear end to a stem (4, 4'). The stem thus runs independently within the internal channels (25, 25') for each actuation mode of the device (aspiration or magnetic) (FIGS. 2 and 3).The drive structures (5, 5') have two sections, an outer and an inner section that never protrudes from the main tubular element (23), the inner section being narrower. Surrounding the inner section of structures 5 and 5' are coupling pieces (9, 10) independent of the internal drive structure (5, 5'), corresponding to each operating mode. In a preferred example, the coupling pieces are cylindrical and screw-on (9, 10). The coupling piece serves to attach a disposable cover (16) in magnetic mode (9) or a capillary (15) in suction mode (10) (FIGS. 5 and 6). The front end of the coupling piece (9, 10) is connected (for example, by means of a thread) to an actuating lever (13, 13'). At the rear end, the coupling pieces (9, 10) have a channel (20, 20') for the stem (4, 4') to run through.In the case of the coupling piece for the aspiration mode (10), there is another capillary fitting hole (21) with a larger diameter than the hole for the stem (20) (FIG. 6). The coupling piece for the magnetic mode (9) is provided, in one implementation, with a cavity to house the cylindrical piece (11) together with the magnet (12) when the magnetic device is not actuated, in addition to serving as a fitting for the disposable cover (16) (FIG. 4) thanks to the stud (17) that it has, which is inserted through a notch (18) provided in the coupling piece (9) and which, when this coupling piece is rotated in the internal guide (19), serves to hold the disposable piece (16) in place (FIG. 5). On the other hand, both suction and magnetic mechanisms have associated springs (7, 7'; 8, 8') for their activation (FIGS. 9 and 10).Thus, there is a first spring (8, 8') that is fixed to the rear end of the inner section of the drive structure (5, 5'), while the rear end of the first spring (8, 8') is attached to the inner part of the coupling piece of the disposable cover (16) for the magnetic mode (9) or of the capillary (15) for the suction mode (10) (FIGS. 9 and 10). Therefore, when the drive lever (13, 13') is slid through a slot (24, 24') and coupled to a recess in said slot (14, 14'; FIGS. 2, 3, 8 and 9), each coupling piece (9, 10) moves axially along the main tubular element (23), causing the concentric spring (8, 8') associated with the coupling piece to extend. All of this means that the coupling piece (9 or 10) is housed at the rear of the main tubular element (23).Furthermore, in the inner channels (25, 25') of the main tubular element (23) there are two second concentric springs (7, 7') to the first springs 8 and 8' and of larger diameter which are associated in their front part with the actuating lever (13, 13') and in their rear part are optionally housed in the inner flange (26) of the main tubular element (23) designed for this purpose.

[0034] To use the device in either mode (aspiration or magnetic), the operating lever (13 or 13') must be moved along the corresponding slot (24 or 24') and into the recess (14 or 14'). This causes the initially compressed internal concentric spring (8, 8') to extend towards the rear of the main tubular element (23), in turn displacing the coupling piece (9 or 10) used to attach the capillary (15) and the disposable cover (16), respectively. Furthermore, moving the lever (13 or 13') also compresses the second external concentric springs (7, 7'), which were initially extended, moving them towards the rear of the main tubular element (23) in each internal channel (25, 25').Once the lever (13 or 13') is in the recess (14 or 14') and therefore the coupling pieces (9, 10) have moved towards the rear of the main tubular element (23), the device can be used in its suction mode if lever 13 has been moved, or in its magnetic mode if lever 13' has been moved. In a second step, to activate each mode, the front part of the actuation mechanism (5, 5') is pressed, which in turn moves the internal stem (4, 4), which runs axially inside the corresponding coupling piece (9, 10) through the holes (20, 20') to exit at its rear end.

[0035] The internal stem 4' serves as a support for a magnet (12), preferably via a cylindrical piece (11). For this purpose, the cylindrical piece (11) has a cavity where the magnet is placed and an opening on the other side for press-fitting onto the stem. In aspiration mode, the stem 4 runs inside the capillary (15), allowing the aspirated volume to be increased or decreased proportionally to the distance traveled. Because the coupling elements (9) and (10) are provided with an opening (21) for press-fitting the capillary (15) or with means for attaching the disposable cover (16), both the capillary and the disposable cover can be easily changed. This makes them consumable, offering the advantage of being able to be replaced between procedures when sterile elements are required.

[0036] In magnetic mode, the magnetic particles or cells capable of being attracted to a magnetic field adhere to the outer part of the rear end of the disposable cover (16), as the magnet is located near this end when the device is activated. Figure 3B shows how, when the device (1) is activated, the lever (13') is first placed in the recess (14'), then the tubular element (5') is pressed (for example, by means of an actuation button (6')), causing the magnet (12) attached to the internal stem (4') to slide inside the disposable cover (16) until it reaches its inner end at the rear. When the action on the manual actuation structure (5') is released, the spring 7' expands, retracting the stem (4') to a forward position, and thus the magnet (12) retracts to the front inner end of the disposable cover (16).Therefore, when the device is not powered, the magnet (12) remains inside the disposable cover (16) at its front.

[0037] Figure 7 shows how a cap (22) can be attached to the disposable cover (16), allowing the material adhered to the surface of the distal end of said disposable cover (16) to be stored in liquid nitrogen without contamination problems during vitrification processes, or any other procedure that requires it. This cap can be provided with a zone for inserting heavy material, allowing the disposable cover, along with the attached cap, to be immersed in a liquid medium for cell storage.

[0038] The disposable cover (16) may have a finish on its distal part a flat surface, a flat surface with a flange, a porous lattice or any other element.

[0039] To use the device (1) by aspiration (FIGS. 2, 8, 9), the lever (13) is slid as previously described, which in turn slides the coupling piece (10) and allows the capillary (15) to be attached. The stem (4) runs axially through the capillary, with the stem inside the capillary and the capillary held by pressure in the orifice (21) of the coupling element (10). Figures 8A and 8B show how, when the device (1) is activated, the internal stem (4) runs inside the capillary (15). By eliminating the need to operate the manual actuation mechanism (5), it becomes possible to aspirate volumes with or without structures located near the rear end of the capillary (15). The system in aspiration mode has a working volume of a few microliters, so it does not require a large air chamber and does not need any other modifications.

[0040] In view of this description and figures, a person skilled in the art may understand that the invention has been described according to some preferred embodiments thereof, but that multiple variations may be introduced in said preferred embodiments, without exceeding the object of the invention as claimed.

Claims

CLAIMS 1. Device for the manipulation of cells, particles and / or liquids by means of two operating modes, a magnetic mode and an aspiration mode, comprising: - a main tubular element (23) provided with two internal channels (25, 25'), - two manual drive structures (5 and 5'), one for each mode, capable of sliding independently along a corresponding channel (25, 25') of the main tubular element (23), - two stems (4, 4'), each stem corresponding to a mode of operation and attached to one of the drive structures (5, 5'), - two coupling pieces (9, 10), one for the magnetic mode and the other for the aspiration mode, each provided with a channel (25, 25') through which the corresponding stem (4, 4') runs, wherein one coupling piece (9) is provided with means (18, 19) for housing a disposable cover (16) for the magnetic mode and another coupling piece (10) is provided with means (21) for housing a capillary (15) for the aspiration mode, wherein the stem (4') of the magnetic mode supports a magnet (12) and the stem (4) of the aspiration mode runs inside the capillary (15) for the aspiration mode, - four springs distributed in two pairs, one for each operating mode, each pair consisting of a first internal concentric spring (8, 8') and a second external concentric spring of larger diameter (7, 7'), - two actuating levers (13, 13'), each associated with and integral to the corresponding coupling piece (9,10), and in turn associated with a groove (24, 24') extending longitudinally along part of the main tubular element (23), where the groove is provided with a cavity (14, 14'), so that when the lever (13, 13') is slid in one mode through the corresponding groove (24, 24') and lodged in the cavity (14, 14'), the coupling piece corresponding to one mode (9, 10) is displaced towards the rear end of the device (1), causing the springs to cooperate and allowing the corresponding actuating structure (5, 5') to slide the corresponding stem (4, 4') and activate one or the other mode of operation.

2. Device for the manipulation of cells, particles and / or liquids according to claim 1, characterized in that the internal stem 4' of the magnetic mode serves as a support for the magnet (12), through a cylindrical piece (11) which is provided with a cavity for the magnet (12) and an opening on the other side to insert the stem by pressure.

3. Device for the manipulation of cells, particles and / or liquids according to claim 2, characterized in that the coupling piece corresponding to the magnetic mode (9) is provided with a cavity to house the cylindrical piece (11) together with the magnet (12) when the magnetic device is not actuated, in addition to serving as a fitting for the disposable cover (16).

4. Device for the manipulation of cells, particles and / or liquids according to any of claims 1 to 3, characterized in that the disposable cover (16) has a finish on its distal part of a flat, flat with a flange or porous mesh.

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