WO2018081298A1

WO2018081298A1 - Systems and methods for injection of biological specimens

Info

Publication number: WO2018081298A1
Application number: PCT/US2017/058337
Authority: WO
Inventors: Richard Novak; Susan CLAUSON; Vishal KESHARI; Donald E. Ingber
Original assignee: President And Fellows Of Harvard College
Priority date: 2016-10-26
Filing date: 2017-10-25
Publication date: 2018-05-03

Abstract

A device for injecting biological specimens includes (i) a top casing; (ii) a hopper disposed in the top casing; (iii) a bottom casing disposed below the top casing, the bottom casing defining a chamber and an outlet; (iv) a slidable carriage at least partially disposed within the chamber, the slidable carriage defining a cavity and a needle guide; and (v) a needle disposed within the needle guide. The cavity is configured, upon linear translation of the slidable carriage within the chamber toward the needle, to align with and receive a biological specimen from the hopper that is penetrated by the needle upon further linear translation of the slidable carriage toward the needle. The cavity is further configured to align with the outlet upon a reverse linear translation of the slidable carriage away from the needle after the biological specimen is penetrated.

Description

SYSTEMS AND METHODS FOR INJECTION OF BIOLOGICAL SPECIMENS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/413,244, filed October 26, 2016, and U.S. Provisional Patent Application No. 62/543,007, filed August 9, 2017, both of which are hereby incorporated by reference in their entireties.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant no. W911NF-16-C- 0050 awarded by U.S. Department of Defense, Advanced Research Projects Agency. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present invention relates to injection of biological specimens. More specifically, the invention relates to a systems and methods for an automated injector for high through-put injection of biological specimens.

BACKGROUND

[0004] Injection in biological experiments includes manual injection of a biological specimen under a microscope and the Roboinject platform that injects oocytes, embryos, larvae, or cells of similar size in well plates. Both approaches have similar limited throughput and involve handling individual biological samples. Furthermore, Roboinject systems are robotic systems that are complex and can be cost prohibitive.

SUMMARY

[0005] According to one aspect of the present invention, a device for injecting biological specimens includes (i) a top casing; (ii) a hopper disposed in the top casing configured to receive and contain one or more biological specimens; (iii) a bottom casing disposed below the top casing, the top casing defining a chamber and an outlet; (iv) a slidable carriage at least partially disposed within the chamber and between the top casing and the bottom casing, the slidable carriage defining a cavity and a needle guide; and (v) a needle disposed within the needle guide and between the top casing and the bottom casing. The cavity is configured, upon linear translation of the slidable carriage within the chamber toward the needle, to align with and receive a biological specimen from the hopper that is penetrated by the needle upon further linear translation of the slidable carriage toward the needle. The cavity is further configured to align with the outlet upon a reverse linear translation of the slidable carriage away from the needle after the biological specimen is penetrated to allow an injected biological specimen to exit the cavity and enter the outlet.

[0006] According to another aspect of the present invention, a method for injecting biological specimens includes (i) loading a plurality of biological specimens in a solution into a hopper; (ii) allowing a first of the plurality of biological samples to settle into a cavity of a carriage that is in fluid communication with the solution in the hopper; (iii) linearly translating the carriage toward a needle until the first biological specimen is penetrated, the needle traversing a needle guide space defined by the carriage; (iv) allowing the needle to remain within the first biological specimen for a predetermined amount of time based on the flow rate of an injection solution within the needle; (v) linearly translating the carriage away from the needle immediately after the lapse of the predetermined amount of time past the hopper to an outlet; (vi) allowing the first biological specimen to exit the cavity and enter the outlet; and (vii) after the first biological specimen exits the cavity, linearly translating the carriage toward the hopper to allow a second of the plurality of biological specimens to settle into the cavity.

[0007] Additional aspects of the invention will be apparent to those of ordinary skill in the art in view of the detailed description of various embodiments, which is made with reference to the drawings, a brief description of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 illustrates an exploded view of an exemplary injection device for a biological specimen, according to aspects of the present disclosure.

[0009] FIG. 2 illustrates a top view of an exemplary carriage of an injection device for a biological specimen, according to aspects of the present disclosure.

[0010] FIG. 3 illustrates a perspective view of the exemplary injection device of FIG. 1, according to aspects of the present disclosure.

[0011] FIG. 4 illustrates a cross-section through an exemplary injection device with biological specimens loaded into a hopper but prior to a specimen falling into a carriage, according to aspects of the present disclosure. [0012] FIG. 5 illustrates an exemplary cross-section through the injection device of FIG. 4 after a specimen has fallen into the carriage, according to aspects of the present disclosure.

[0013] FIG. 6 illustrates an exemplary cross-section through the injection device of FIG. 4 of the biological specimen being injected, according to aspects of the present disclosure.

[0014] FIG. 7 illustrates an exemplary cross-section through the injection device of FIG. 4 after the biological specimen has been injected but prior to the specimen entering an outlet of the injection device, according to aspects of the present disclosure.

[0015] FIG. 8 illustrates an exemplary cross-section through the injection device of FIG. 4 of the biological specimen entering the outlet as the specimen exits the injection device, according to aspects of the present disclosure.

[0016] FIG. 9 illustrates an exemplary top perspective view for a bottom casing of an injection device for a biological specimen, according to aspects of the present disclosure.

[0017] FIG. 10 illustrates an exemplary bottom perspective view for a carriage of an injection device for a biological specimen, according to aspects of the present disclosure.

[0018] FIG. 11 illustrates an exemplary bottom perspective view for a top casing of an injection device for a biological specimen, according to aspects of the present disclosure.

[0019] FIG. 12A-12B illustrate top and cross-sectional views of an exemplary injection device as a biological specimen has been loaded into a chamber, according to aspects of the present disclosure.

[0020] FIG. 13A-13B illustrate top and cross-sectional views of the exemplary injection device of FIG. 11 after a biological specimen has been loaded into a chamber and before injection, according to aspects of the present disclosure.

[0021] FIG. 14A-14B illustrate top and cross-sectional views of the exemplary injection device of FIG. 11 during an ejection step for the biological specimen, according to aspects of the present disclosure.

[0022] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the invention is not intended to be limited to the particular forms disclosed. Rather, the invention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. DETAILED DESCRIPTION

[0023] While this invention is susceptible of embodiment in many different forms, there is shown in the drawings and will herein be described in detail preferred aspects of the invention with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the broad aspect of the invention to the aspects illustrated. For purposes of the present detailed description, the singular includes the plural and vice versa (unless specifically disclaimed); the word "or" shall be both conjunctive and disjunctive; the word "all" means "any and all"; the word "any" means "any and all"; and the word "including" means "including without limitation."

[0024] It should be understood that the inventions described herein are not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of various embodiments described herein, which is defined solely by the claims.

[0025] As used herein and in the claims, the singular forms "a", "an" and "the" include the plural reference and vice versa unless the context clearly indicates otherwise. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

[0026] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g. " is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g. " is synonymous with the term "for example."

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.

[0028] An injection system and method, as described in this disclosure, addresses a need to automate many biological experiments for improved accuracy, increased throughput, and reduced risk when working with pathogens or other laboratory hazards. The systems and methods that are described generally include providing for transferring of an embryo, or a similarly sized biological specimen, to a needle, injecting a known and controlled dose of pathogen or other hazard into the embryo, and transferring the embryo to a test/observation site.

[0029] A non-limiting exemplary aspect of an automated injector system for high throughput embryo experimentation is now described. The automated injector system comprises: (i) a hopper that holds one or more embryos; (ii) a needle to inject a pathogen into an embryo; (iii) a carriage that transports a single embryo to the injection site and guides the needle to embryo, the carriage having a cavity shape that holds the embryo in place during injection; (iv) a pump that supplies steady flow of pathogen during injection where the flow rate of the pathogen times residence time of needle in embryo determines injected dose which minimizes having to calibrate each needle as is commonly required for pressure-based injections; (v) an automation of the carriage movement to control the dose of pathogen as a function of time the needle penetrates the embryo, and/or the depth and/or region of the injection; and/or (vi) an outlet that releases the injected embryo before picking up a next embryo.

[0030] A non-limiting exemplary aspect of a method of using an automated injector system comprises an embryo batch being loaded into the hopper, and the embryos settling down to the bottom of the hopper. One embryo falls into the carriage. The carriage slides toward the needle until the embryo is impaled or otherwise penetrated. Pressure relief grooves are included in the base to enable fluid to flow around the carriage during carriage motion. The time of the needle in the embryo times the flow rate of the injection solution determines the injected dose. After injection, the carriage slides back, past the input hopper, to the outlet. The embryo falls into the outlet with gravity. The carriage slides back to the input hopper port to repeat the process.

[0031] It is contemplated that the above-described single-axis operation can be further automated using a linear actuator (e.g., servo, stepper motor, etc .). In some aspects, multiple systems can be operated by a single actuator, or multiple injection systems can be within a single device, such as through parallel embryo slots in a single carriage.

[0032] Referring now to FIGS. 1-8, exemplary aspects of the present systems and methods are illustrated for an injection system for biological samples that allows for high throughput flow-through injection of a pathogen into the sample. The injector system can include a single-axis motion system with a gravity feed for the biological specimens. Furthermore, the injector system can provide a high-degree of injection control— including volume, rate, and depth of injection - through the application of a motorized control. [0033] It is contemplated that an injection system for biological specimens can be built from a variety of materials including materials that can be machined. Properties of a material can include sealing capabilities and being non-porous. Examples of contemplated materials include acrylics, polyetherimide, polycarbonate, other thermoplastics or resins, and metals. Materials exhibiting heat resistance, solvent resistance, and flame resistance, such as ULTEM™, can be desirable in medical and chemical applications where durability and tighter tolerances are desirable.

[0034] Referring now to FIG. 1, an exploded view of an exemplary injection device 100 for a biological specimen is illustrated. The injection device 100 includes top casing 110 that sits on top of a bottom casing 120. The bottom casing 120 includes a chamber 130 into which a carriage 140 can be disposed. A front side of the bottom casing 120 can include a needle mount 150 and the back side of the bottom casing 120 can include a shaft mount 160. The injector system can further include a shaft 170 extending through the shaft mount 160 and extending into the bottom casing 120 for moving the carriage 140 within the chamber 130. One or more O-rings 180, 190 can be fitted around the shaft 170 or at the interfaces of the needle mount 150 and shaft mount 160 to seal any fluids in the chamber 130 within the bottom casing 120.

[0035] FIG. 2 illustrates a top view of an exemplary carriage 240 of an injection device for a biological specimen. As illustrated in FIG. 1, the carriage may be removal and is positioned in a chamber of a bottom casing. Furthermore, the carriage 240 can translate during operation in a direction parallel to the generally horizontal plane that is created by the interface between the top casing 110 and the bottom casing 120 and in a direction that aligns with a needle extending into the chamber 130 from the needle mount 150. A needle guide 242 is positioned toward the needle-mount side of the injector system for receiving the needle. The needle guide 242 extends into a cavity 245 that includes a biological specimen to be injected as part of the injection process.

[0036] The needle guide 245 acts as an opening into the carriage 240 that allows the needle to inject a biological specimen in the cavity 245. In some aspects, the needle for an injection system comprises fragile materials (e.g., Borosilicate glass) where it would be desirable for the needle to not come into contact with the walls forming the needle guide 242. In some aspects it is contemplated that a needle may be electroplated or otherwise reinforced, such as a composite or plastic coating, to provide additional robustness properties where the needle is more resistant to fracture than a standard glass needle. In some aspects, needles made with silicon or silicon nitride are contemplated. [0037] FIG. 3 illustrates a perspective view of the exemplary injection device of FIG. 1. The carriage 340 is illustrated in an operational position within the chamber 330. A needle mount 350 is attached to the bottom casing 320 and includes a needle port 355. A shaft mount 360 is attached to the bottom casing 320. A shaft 370 extends through the shaft mount 360 and into the bottom casing 320 and is operationally connected to the carriage 340 to provide linear translational force(s) to the carriage 340 (e.g., a single axis motion). The shaft 370 may be moved manually, connected to a motor, connected to an actuator, or otherwise moved such that the shaft translates in a way that linearly progresses and retracts the carriage 340 toward and away from an injection needle.

[0038] FIGS. 4-8 illustrate cross-sections through an exemplary injection device where biological specimens are loaded into a hopper. Similar to the embodiments illustrated in FIGS. 1-3, the injection device includes a top casing 410, a bottom casing 420, a chamber 430, a carriage 440, a needle mount 450 with a needle port 455, a shaft mount 460 and shaft 470, a needle guide 442, a cavity 445, and O-rings 480, 490. The cross-sections further illustrate a hopper 442 for receiving biological specimen(s) 403, 405, an outlet 435, and the injection needle 457.

[0039] In some aspects, the injection system is contemplated to be a gravity-driven system for handling the biological specimens, such as embryos. A biological specimen, such as biological specimens 403, 405, in FIG. 4 may be stored within a fluid in a vial prior to being introduced into the injection system. The biological specimens are then introduced into the hopper 442, which also contains a fluid (e.g., water, salt solution) where the biological specimens sink into the hopper within the fluid under the forces of gravity. The bottom of the hopper 442 is connected to a hopper outlet 443 that is configured to connect directly with the carriage 440. The injection system is configured such that one biological specimen at a time falls into the carriage 440. It is contemplated that the cavity 445 of the carriage 440 is sized to only allow one biological specimen at a time to enter the cavity from the hopper outlet 443.

Thus, the hopper can hold a large batch of biological specimens yet inject only one specimen at a time. The illustrated configuration is particularly desirable because handling by the injection system user is limited where the specimens are always immersed in a fluid (e.g., from vial to hopper) and a large number of biological specimens can be placed in the hopper rather than just one at a time. The system provides a generally vertical gravity-feed passage that minimizes disruptions to the biological specimen by allowing the biological specimen to travel into and out of the carriage based on differences in densities between the biological specimen and its surrounding fluid. In some aspects, it is contemplated that the fluid may be denser than the biological specimens where the specimens actually float from an opening in the bottom casing up into a cavity in the carriage and out through an opening in the top casing. Referring specifically to FIG. 4, a cross-section through an exemplary injection device is illustrated with biological specimen(s) 403, 405 loaded into a hopper 442 but prior to specimen 403 falling into the cavity 445 of carriage 440.

[0040] Injection systems are also contemplated that use perfusion to force embryos from a hopper, such as hopper 442, and into a carriage, such as carriage 440, and from the carriage to an outlet, such as outlet 443. It is further contemplated that an injection system can use vibration to move embryos from the carriage and into an outlet. For example, vibration can be used in conjunction with gravity or pump-driven flow to maintain embryo motion and prevent adhesion to walls or edges or other embryos in the system.

[0041] FIG. 5 illustrates an exemplary cross-section through the injection device of FIG. 4 after a specimen 403 has fallen into a well or cavity 430 of the carriage 440. Between FIGS.

4 and 5, the shaft 470 is linearly translating the carriage 440 toward the needle 457. The biological specimen 403 is being moved within the cavity to a position just prior to the specimen 430 being impaled or otherwise penetrated by the needle 457.

[0042] Referring now to FIG. 6, an exemplary cross-section through the injection device of

FIG. 4 is illustrated with the biological specimen 403 being penetrated. Proceeding from

FIG. 5, the shaft 470 further translates the carriage 440 linearly toward the needle 457 such that the biological specimen 403 has now moved within the cavity 430 to a position where the specimen 403 has been impaled or otherwise penetrated by the needle 457.

[0043] Referring now to FIGS. 7, an exemplary cross-section through the injection device of

FIG. 4 after the biological specimen has been injected but prior to the specimen entering an outlet of the injection device. Proceeding from FIG. 6, the shaft 470 has now begun translating the carriage 440 linearly away from the needle 457 such that the biological specimen 403 has now moved within the cavity 430 to a position where the specimen 403 has moved beyond the hopper outlet 443 (i.e., the cavity inlet) and right above the outlet 435 where the specimen can move through the fluid via gravity, or via density differences between the fluid and biological specimen, and exit the cavity through the outlet 435. In some aspects, a device can be operated upside down, such as in the exemplary case of biological specimens being less dense than the surrounding fluid, where the specimens are allowed to move in an upwardly direction from the hopper and into the cavity of the carriage and out of the carriage into an outlet. In some aspects, a flushing mechanism may also be used to assist the exit of the biological specimen 403 from the cavity 430. [0044] FIG. 8 illustrates an exemplary cross-section through the injection device of FIG. 4 of the biological specimen 403 entering the outlet 435 as the specimen 403 exits the injection device.

[0045] In some aspects, it is contemplated that a hopper, hopper outlet, or a combination of both can hold a plurality of biological specimens, such as embryos, in a stacked arrangement. In some aspects, this will include up to ten specimens and in other aspects it will include more than ten specimens. The specimen size will also be determinative of the number of stacked specimens that can be contained in the hopper, hopper outlet, or combination. To minimize the specimens from being stressed as the bottommost specimen (e.g., as biological specimen 403moves between the stages of FIGS. 4 and 5) drops in the cavity of the carriage and the carriage starts translating toward the needle, the machining or shaping of the top casing, bottom casing, chamber, and the carriage are done to tight tolerance (e.g., 50 to 100 microns) to minimize gaps between the various components of the injection system that could trap the biological specimens. In addition, the biological system is essentially only acted upon by gravity as the specimen is suspended in a fluid, further minimizing any stress experienced by the biological specimen and its protective membrane.

[0046] It is contemplated that different sized biological specimens can be injected using the described injection systems of the present disclosure. In some aspects, the carriage size is adjusted including the thickness of the carriage, the height of the cavity, the lateral dimensions of the cavity, or combinations thereof. For example, the same primary injection system components can be used to inject biological specimens varying as much as an order of magnitude or more in size (e.g., frog eggs vs. mosquito eggs) by using carriages of different sizes where the cavity of the different carriages is sized to accommodate the different sized biological specimens. In is also contemplated that the hopper outlet (e.g., element 443) and the outlet 435 in the bottom casing would need to be similarly adjusted to accommodate the size of the specimen being injected. In some aspects, it is contemplated that the tolerances between the carriage and the adjacent top casing or bottom basing are approximately ten percent or less of the diameter of the biological specimen. For example for a biological specimen that is one millimeter in diameter, the gap between the carriage and the top casing and the gap between the carriage and the bottom casing will be approximately 100 microns or less. In some aspects, the tolerance for total vertical movement of the carriage between the top and bottom casings will be approximately ten percent or less than the diameter of the biological specimen. [0047] Various alternatives or optional aspects of the above described automated injector system and method are contemplated.

[0048] In some aspects, an injector system can be used for injecting various reagents, compounds, cells, microbiota, fungi, etc., into any number of receiving structures or organisms. In some aspects, the injector is configured for injecting amphibian, fish, insect, and other embryos. In yet further aspects, the injector is configured for organoid, biopsy, tissue, spheroid, or egg injection. In some aspects, the injector is configured for inducing damage into the above organisms/tissues.

[0049] In some aspects, a needle diameter and composition can be modified for the specific application (e.g., large diameter for injury vs. small diameter for injecting small eggs).

[0050] In some aspects, a motorized control can be added to better control injection parameters and reduce damage. For example, a drive screw, belt, magnetic, pneumatic, hydraulic, or other actuation method can be used.

[0051] In some aspects, an injector system can be parallelized for greater throughput, such as by having multiple carriages, multiple embryo cavities, etc.

[0052] In some aspects, an injector system can be integrated with imaging or other manipulation systems.

[0053] In some aspects, an injector system can be integrated with upstream or downstream analysis such as cytometry.

[0054] In some aspects, an injector system can include a nonlinear actuation system such as a lever or rotor.

[0055] In some aspects, the size of all features of an injector system can be adjusted to fit specific embryos or tissues.

[0056] In some aspects, materials for fabricating an injector system can be selected to suit specific purpose(s) (e.g., low drug absorbing, optically clear, opaque for light sensitive operations, etc.). In some aspects, acrylic, Ultem®, and/or other transparent materials are contemplated.

[0057] In some aspects, an injection in an injector system can be implemented using continuous perfusion, pressure driven perfusion, and/or done continually or in pulses.

[0058] In some aspects, a pressure relief in an injector system can be implemented using fluidic channels, grooves, tubing, and/or external ports.

[0059] In some aspects, an injector system can include flushing of non-injected material(s) from the chamber. [0060] An automated embryo (or biological specimen injector) injector, such as the injector described herein provides for high throughput flow-through injection of a pathogen to meet the analytical needs of embryo experimentation. The injector can include a single-axis motion system with gravity feed of embryos. The single-axis motion system allows for a potential for a high degree of injection control (e.g., volume, rate and depth of injection, etc.) with the addition of motorized control.

[0061] Desirable aspects of the injector systems and methods of the present disclosure include the ability to minimize moving components within the injector system and having a configuration that allows for gravity to position the embryos (or biological specimens) throughout the injection operation. These benefits provide the scalability to handle single embryos without damage while further providing high throughput injection and minimizing the need to manipulate single biological specimens by hand. A further benefit includes that the continuous perfusion of the needle avoids having to calibrate each needle to assure a precise dose of injected material in contrast to standard pressure-driven systems that are controlled by the needle geometry and variations in geometry.

[0062] Other desirable aspects of an injection device for biological specimens can include both gravity-driven, pump-driven, or combined systems that at least partially use perfusion- style mechanisms to force embryos through the injection device.

[0063] Referring now to FIGS. 9-14, exemplary aspects for additional injection devices and related components for biological specimens are described, including aspects applicable for perfusion-style systems. Gravity-driven systems may be desirable where less throughput is needed for embryo injection operations. In some aspects, active types of perfusion-style systems downstream from the injection device can be beneficial for pulling embryos through the device. Pumping-type systems, such as ones using a peristaltic pump, can be particularly beneficial for increasing throughout of embryo injections by actively driving embryos through the systems faster than gravity feed alone, especially where the combination of the pumping-type system and injection device address pressure buildup, carriage motion, and embryo movement through the various ports or channels of the injection device. For example, consideration given to minimizing pressure buildup in the loading port and embryo cavity can have beneficial results in minimizing embryo viability losses. Furthermore, systems addressing any piston-like motion of the carriage that can drive fluid backwards into the hopper region would also be beneficial to minimize any piston-like action from pushing embryos out of the desired loading zone and further minimize the need to wait for the embryos to settle back down again. Additional consideration given to the loading into and ejection from the carriage due to constricted ports that minimizes multiple embryos from entering/exiting at the same time can also be beneficial.

[0064] It is contemplated that in some aspect gravity feed is desirable where gentle handling of embryos and other injected objects is preferred and throughput is of less or minimal concern. In some aspects, adding pump systems and other improvements to an injection device leads to increased operations with greater robustness than what could be accomplished with gravity alone, while still maintaining high levels of embryo viability.

[0065] Turning to FIG. 9, an exemplary top perspective view of a bottom casing 900 is illustrated for an injection device for a biological specimen. The casing 900 includes pressure-relief channel (s) 910 that relieve pressure relief during continuous peristaltic fluid flow through the injection device. In some aspects, the pressure-relief channel 910 can assist with priming of the pumping system by allowing for the removal of bubbles and providing improved fluid flow to remove debris or injected material that may be found inside the injection device. The illustrated channel 910 is discontinuous to allow for valving action during the sliding motion of the carriage (e.g., element 1000 in FIG. 10) during loading- injection-ejection steps further for an injection device that are described in more detail in FIGS. 11-14. The pressure-relief channel 910 can be used when embryos are not being loaded or ejected from the injection system.

[0066] Turning now to FIG. 10, an exemplary bottom perspective view of a carriage 1000 is illustrated for an injection device for a biological specimen. Drainage can be provided through channel systems that allow for fluid equalization as the carriage moved forward and back within a chamber (e.g., element 940 in FIG. 9) of a bottom casing, such as casing 910. In some aspects, carriage 1000 can include chamfered corner(s) 1010, 1015 to provide a fluid path to the ceiling of a chamber defined by the bottom casing 910 and a top casing (e.g., casing 1100 in FIG. 11). Furthermore, the bottom casing 910 can also include drainage channels 920, 925 that run parallel to the carriage as the carriage is sliding back and forth within the chamber as a result of piston motion for the injection device. In some aspects, fluid equalization is improved by connecting side fluidic port(s) 930 with the drainage channel(s) 920, 925. The fluidic port can also serve as the source of fluid for perfusing and flushing the system to take in fluid from the top of the hopper of the injection system. One or more of the injector device improvements allow for changes in volume due to carriage motion to minimize pressure buildup on the embryos, whether positive and/or negative pressure. Thus, the likelihood of embryos getting pushed out of a hopper port or damaged by getting forced through too rapidly through the injector device is minimized during injection operations.

[0067] It is contemplated that the placement of fluidic channels in an injection device also improves embryo loading and ejection by modulating fluid flow based on the position of the carriage 1000. For example, a carriage 1000 may include a short microfluidic channel 1020 opposite a needle guide 1030.

[0068] Turning to FIG. 11, an exemplary bottom perspective view of a top casing 1100 is illustrated for an injection device for a biological specimen. The top casing 1100 can define the upper surface or ceiling 1110 of a chamber (e.g., defined by the top and bottom casing) where the carriage 1000 is disposed in the chamber and translates horizontally. A microchannel 1120 may be disposed in the top casing 1100 such that the microchannel is a formed in the ceiling or upper surface of the chamber. An embryo can then move through the microchannel to an area 1130 or end of the microchannel where upon the carriage translating to align with an outlet of the injector device, the embryo is pushed into the outlet. Thus, the combined injector device with a top casing similar to top casing 1100 drives embryos from the chamber efficiently to the outlet via the microchannel in the top casing when the carriage has translated to an ejection position, see FIG. 14. This has the beneficial effect of routing fluid flow to the top of the embryo and pushing it down into the outlet port of the injection device.

[0069] Turning to FIGS. 12A-14B, top and cross-sectional views of an exemplary injection device is illustrated at various stages of the injection process, including as a biological specimen is being loaded into a chamber (FIGS. 12A-12B), as the specimen is ready to be injected (FIGS. 13A-13B), and during the post-injection ejection step (FIGS 14A-14B). Arrows are used in FIGS. 12B, 13 A, 14 A, and 14B to illustrate the fluid flow through the injection device based on the valving system created through the connections between channels and valves of the carriage and casing(s) as the carriage translated horizontally from loading, to injection, and to the ejection stage.

[0070] FIGS. 12A-12B illustrate an exemplary embryo loading step where a pressure-relief channel 1210 in the bottom casing is blocked (see also FIG. 9). Normally in the loading position, fluid can flow easily from a chamber 1240 directly to an outlet 1250, thereby driving embryos into the chamber 1240 for loading. To increase the rate of loading, such as when a pump system is connected to an injector device at an outlet port, and to minimize pressure buildup due to high perfusion rates, a valving system including carriage valves, such as valves 1220, 1230, can be applied across the pressure relief channel 1110. The pressure- relief channel 1110 is normally open to allow pressure relief but closed only during the embryo loading stage, as illustrated in FIGS. 12A-B. Closure directs most fluid flow from the chamber to the outlet without having a fluid bypass to reduce that flow. As illustrated in FIG. 12B, the fluid can flow down from the hopper (A) and through chamber via the needle guide (B) through an embryo loading suction channel (C) and into an outlet port (D¹).

[0071] Turning now to FIGS. 13A, 13B, 14A, and 14B, the carriage at the next stages in the process is translated forward for injection (FIGS. 13A-B) or backward for ejection (FIGS. 14A-B) which opens the valving system and provides pressure relief by allowing flow between via the pressure-relief channel and the carriage valve(s), along with other channels defined by the carriage or casing(s). For example, in FIG. 13 A during injection, fluid that is pumped through the system flows into the side inlet port (Α'), then flows along the perfusion channel connected with the side inlet port (Β'), then flows along the chamber near the piston (C), followed by flow along another perfusion channel (D'), and then into the pressure-relief channel (Ε') before exiting via the outlet port. As another example, in FIGS. 14A-B during ejection, fluid that is pumped through the system can flow into the side inlet port (A"), then flow along the perfusion channel connected with the side inlet port (B"), followed by flowing along the chamber behind the carriage along microchannel 1120 (C") - see FIG 11 - before flowing into the outlet port (D"). Fluid can also flow into the outlet through the needle guide (E"). The fluid flows illustrated in FIGS. 13A, 14A, and 14B are also beneficial for flushing the injector device.

[0072] Each of the above described aspects and obvious variations thereof are contemplated as falling within the spirit and scope of the claimed invention, which is set forth in the following claims. Moreover, the present concepts expressly include any and all combinations and subcombinations of the preceding elements and aspects. For example, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated may be further modified to incorporate features shown in any of the other embodiments disclosed herein.

Claims

CLAIMS What is claimed is:

1. A device for injecting biological specimens, the device comprising:

a top casing;

a hopper disposed in the top casing configured to receive and contain one or more biological specimens;

a bottom casing disposed below the top casing, the bottom casing defining a chamber and an outlet;

a slidable carriage at least partially disposed within the chamber and between the top casing and the bottom casing, the slidable carriage defining a cavity and a needle guide; and

a needle disposed within the needle guide and between the top casing and the bottom casing,

wherein the cavity is configured, upon linear translation of the slidable carriage within the chamber toward the needle, to align with and receive a biological specimen from the hopper that is penetrated by the needle upon further linear translation of the slidable carriage toward the needle, and

wherein the cavity is further configured, upon a reverse linear translation of the slidable carriage away from the needle after the biological specimen is penetrated, to align with the outlet to allow an injected biological specimen to exit the cavity and enter the outlet.

2. The device of claim 1, further comprising a shaft connected to the slidable carriage, the shaft configured to apply linear translation forces to the carriage.

3. The device of claim 2, further comprising an actuator connected to the shaft, the actuator imparting a force to the shaft that is converted into the linear translation forces applied to the carriage.

4. The device of claim 1, wherein the hopper is configured to contain a plurality of specimens in a stacked configuration.

5. The device of claim 1, wherein the one or more biological specimens are suspended in a fluid within the hopper, cavity, and outlet.

6. The device of claim 5, wherein the density of the biological specimen is greater than the density of the fluid.

7. The device of claim 5, wherein the density of the biological specimen is less than the density of the fluid.

8. The device of claim 5, wherein the bottom casing includes pressure relief grooves to allow fluid to flow around the carriage during linear translation of the carriage within the chamber.

9. The device of claim 8, wherein the bottom casing further includes an inlet, the inlet and outlet being hydraulically connected to a pumping system such that fluid continuously flows via the inlet into the chamber before exiting at the outlet during the linear translation of the carriage within the chamber.

10. The device of claim 1, wherein the received one or more biological specimens include embryos.

11. The device of claim 1, wherein the top casing, hopper, bottom casing, and slidable carriage comprise inert transparent materials.

12. The device of claim 1, wherein the needle comprises a composite material.

13. A method for injecting biological specimens, the method comprising:

loading a plurality of biological specimens in a solution into a hopper;

allowing a first of the plurality of biological samples to settle into a cavity of a carriage that is in fluid communication with the solution in the hopper; linearly translating the carriage toward a needle until the first biological specimen is penetrated, the needle traversing a needle guide space defined by the carriage;

allowing the needle to remain within the first biological specimen for a predetermined amount of time based on the flow rate of an injection solution within the needle; linearly translating the carriage away from the needle immediately after the lapse of the predetermined amount of time past the hopper to an outlet; allowing the first biological specimen to exit the cavity and enter the outlet; and

after the first biological specimen exits the cavity, linearly translating the carriage toward the hopper to allow a second of the plurality of biological specimens to settle into the cavity.

14. The method of claim 13, further comprising a linear actuator and shaft for linearly translating the carriage to and from the needle.

15. The method of claim 13, wherein the hopper is configured to contain the loaded plurality of biological specimens in a stacked configuration.

16. The method of claim 13, wherein the plurality of biological specimens are suspended in a fluid within the hopper, cavity, and outlet.

17. The method of claim 16, wherein the density of the plurality of biological specimens is greater than the density of the fluid.

18. The method of claim 16, wherein the density of the plurality of biological specimens is less than the density of the fluid, the plurality of biological specimens moving upwardly from the hopper into the cavity of the carriage.

19. The method of 16, wherein the fluid is allowed to flow around the carriage during linear translation of the carriage.

20. The method of claim 13, wherein the loaded plurality of biological specimens includes embryos.