Integrated system for delivering macromolecules into single cells using precise motion control and automation

Abstract

A system for delivering macromolecules or other materials into cells may include microneedles formed on microneedle chips, cell trapping chips, and systems for aligning microneedle chips with cell trapping chips. According to various embodiments, systems can include apparatuses for handling microneedle chips, a gantry, an alignment module, a microscope module, and a fluidics module. Methods for aligning the cell trapping chips and the microneedle chips and methods for performing cell engineering processes using the systems are also described.

Description

INTEGRATED SYSTEM FOR DELIVERING MACROMOLECULES INTO SINGLE CELLS USING PRECISE MOTION CONTROL AND AUTOMATIONCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 63 / 509,431 filed June 21, 2023 and U.S. Provisional Application No. 63 / 516,618 filed July 31, 2023, which are currently co-pending herewith and which are incorporated by reference in their entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to systems, apparatuses, processes, and methods for delivering macromolecules or other materials into cells. Particular implementations leverage precise motion control and faithful integration and interfacing of system components to create a system for precise intracellular cargo delivery.BACKGROUND

[0003] Cell engineering is a promising area for scientific research and medical treatment development with numerous potential applications in medicine, biology, pharmacology, plant science, and other fields. In many cell engineering applications, a goal may be changing a cell’s functionality, internal chemistry, or genetic material by introducing new matter into the cell. Mammalian cells are particularly difficult to transfect with high efficiency, viability, and recovery due to their fragility and aseptic handling requirements. Current cell engineering solutions include chemical methods (e.g., lipofection, calcium phosphate precipitation), viral methods (e.g., retroviral, lentiviral, adenoviral vectors), and other physical methods (e.g., electroporation, microinjection, gene gun, or impalef ection). These methods have several respective limitations and shortcomings. For example, chemical methods can cause cytotoxicity, have limited efficiency in certain cell types, and may induce immune responses. Viral methods may have potential for insertional mutagenesis, immunogenicity, oncogenicity, and ethical concerns. And other physical methods can cause cellular damage, may have varying efficiencies depending on cell type, mayrequire specialized equipment and expertise, and may be population based (e.g., they do not exhibit control at single cell resolution). Furthermore, conventional methods are challenging to scale in a bio safe and automated system for cell transfection at high resolution.

[0004] In some cell engineering applications, open arrays (e.g., an array containing nanoneedles and an array containing a porous membrane) can be aligned and / or planarized within a certain level of precision, accuracy, and reproducibility. Such alignment and / or planarization is difficult to achieve at the tolerances necessary at a nanometer scale. An added difficulty is operating the arrays in a system that optimizes footprint while integrating access to liquid handling, sensors, and biosafety controls. In view of all these issues in the public domain, there is a general need for a cell engineering system with precise, accurate, and repeatable motion control, planarization, and alignmentBRIEF DESCRIPTION OF THE DRAWINGS

[0005] For a more complete understanding of the present disclosure, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0006] FIG. 1 illustrates a perspective view of an example system according to aspects of the present disclosure;

[0007] FIG. 2 illustrates a perspective view of components and modules of an example system according to aspects of the present disclosure;

[0008] FIGs. 3 A and 3B illustrate a perspective exploded view of an example needle chip handling module according to aspects of the present disclosure;

[0009] FIG. 4 illustrates a perspective view of an example gantry stage module according to aspects of the present disclosure;

[0010] FIG. 5 illustrates a perspective view of an example tip-tilt platform module according to aspects of the present disclosure;

[0011] FIGs. 6A and 6B illustrate perspective views of an example microscope module according to aspects of the present disclosure;

[0012] FIG. 7 illustrates a perspective view of an example fluidics module according to aspects of the present disclosure;

[0013] FIG. 8 illustrates a flow diagram of an example process for performing a cell engineering workflow according to aspects of the present disclosure;

[0014] FIGs. 9A through 9D illustrate flow diagrams of an example process for performing a cell engineering workflow according to aspects of the present disclosure;

[0015] FIGs. 10A through 10G illustrate flow diagrams of an example process for performing a cell engineering workflow according to aspects of the present disclosure.

[0016] FIGs. 11A-11D illustrate a diagram of aspects of an example needle chip and cell loading process according to aspects of the present disclosure;

[0017] FIGs. 12A-12C illustrate a diagram of an example alignment process according to aspects of the present disclosure; and

[0018] FIG. 13 illustrates an example of a computing device that is operable to support cell engineering workflows and methods according to aspects of the present disclosure.

[0019] It should be understood that the drawings are not necessarily to scale and that the disclosed aspects are sometimes illustrated diagrammatically and in partial views. In certain instances, details which are not necessary for an understanding of the disclosed methods and apparatuses or which render other details difficult to perceive may have been omitted. It should be understood, of course, that this disclosure is not limited to the particular aspects illustrated herein.DETAILED DESCRIPTION

[0020] This disclosure describes systems, apparatuses, and methods for performing cell therapies and / or cell engineering with high precision. It is desirable in some cell engineering workflows to be able to insert a cargo (e.g., a chemical, mechanical, or genetic material) preciselyinto a single cell using microneedles. References made in this disclosure to microneedles, microneedle structures, or microneedle chips are intended to also refer respectively to, for example, nanoneedles, nanoneedle structures, and nanoneedle chips. References made in this disclosure to nanoneedles, nanoneedle structures, and nanoneedle chips are intended to also refer respectively to, for example, microneedles, microneedle structures, or microneedle chips. For the sake of clarity and conciseness, only one of the terms may be listed in aspects of this disclosure, but unless explicitly specified differently, a reference to one of these terms is intended to include the others. References simply to “needle chips” can include, for example, microneedle chips and / or nanoneedle chips.

[0021] In some cell engineering applications, single cells may be trapped at target locations on a cell trapping chip and then microneedles on a microneedle chip may be brought precisely into contact with the cells at the target locations. An example of trapping cells at a target location may include trapping cells at target locations in a porous membrane. Another example of trapping cells at a target location may include trapping cells in pores in a cell trapping chip. Another example may include both a porous membrane and a cell trapping chip used in combination. Other examples of trapping cells at target locations may also be possible.

[0022] Unaligned contact between the target locations and needles can cause damage or failure of the needles, the cell trapping chip, or both. However, alignment in this context is very difficult. The dimensions of target locations (e.g., pores on a cell trapping chip) and of the microneedles on a microneedle chip can be made in such small scales that there is little room for error in aligning the target locations with the microneedles. This can be especially true because of the high aspect ratio to many microneedle structures (e.g., microneedles can be several times taller than they are wide) — if there is even a slight a discrepancy in planar alignment between cell trapping chips and microneedle chips, the needles may miss the cell target locations. In many cases, the needles are fragile and cannot withstand contact with non-cellular materials (e.g., with other structures). Cell trapping chips may also be fragile. This fragility means that contact alignment is typically not a viable option for aligning the chips.

[0023] To combat misalignment between the chips, in various implementations discussed herein, the microneedle chips and the cell trapping chips are optically aligned such that the chips are co-planarized, or brought into a substantially parallel plane, to within 1 pm of errorin four edges. Systems and modules used to handle and bring the chips into alignment may require similar precision tolerances. In various aspects of this disclosure, a system may execute cell engineering workflows by bringing cells onto a cell trapping chip having a plurality of cell traps, aligning the cell trapping chip with a microneedle or nanoneedle chip having a plurality of microneedles or nanoneedles such that each cell trap of the cell trapping chip is aligned with a single microneedle on the microneedle chip, and causing the chips to come into contact such that materials can be transferred into the cells from the microneedles. These systems are discussed in greater detail below. Methods for performing cell engineering workflows are also discussed. Examples of how those methods may be applied to the disclosed systems are also provided.System description

[0024] Reference is now made to FIG. 1, which illustrates an example of a system 100 according to aspects of the present disclosure. System 100 may include an enclosure 110 for housing components and modules of the system. In various implementations, the enclosure 110 may include an air filter 112 (e.g., a HEPA air filtration system) configured to maintain a clean environment within the enclosure 110 suitable for working with microscopic materials and / or for facilitating a sterile environment. The system 100 may include vibration damping feet 114. Vibration damping feet 114 may be made of a hard rubber (e.g., a rubber having Shore rubber hardness of 90D) to reduce the effect of external vibrations on the system 100. In various implementations the vibration damping feet 114 may include integrated casters (not shown) so that the system 100 may be moved if needed. The enclosure 110 may also include brackets 116 so that the system 100 may be secured in place. In various implementations, brackets 116 may prevent the system 100 from tipping over (such as, for example, accidental tip over or tip over as a result of seismic activity).

[0025] The enclosure 110 may include a first clean air cavity 120 configured to house elements and / or modules of the of the system 100, including a module assembly 130. The individual components and modules of module assembly 130 will be described in greater detail below with respect to FIGs. 2-7. In various aspects, the module assembly 130 of the integrated system 100 may be configured to perform at least a portion of intracellular cargo delivery workflows. In various implementations, a work light 122 may be included in the first clean air cavity for illuminating module assembly 130. The work light 122 may facilitate optical alignmentprocesses performed by the module assembly 130 or improve user visibility during operation of the system 100. The first clean air cavity 120 may be enclosed by a first door 124. First door 124 may include vents 126.

[0026] Enclosure 110 may include a second cavity 140 for housing a fluid handling system 150. The second cavity 140 may be enclosed by a second door 144. Fluid handling system 150 may include a plurality of reagent containers 152, a liquid waste container 154, a pump 155, a valve 156, and a tubing network 158. In various implementations, the plurality of reagent containers 152 may store reagents, including as non-limiting examples, payload materials such as mRNA, DNA, RNP, and proteins, alcohols such as IPA or ethanol, buffers such as phosphate- buffered saline (PBS), and / or diluted bleach. In various non-limiting examples at least one of the plurality of reagent containers 152 may contain cell culture media. A cell culture media may be a media in which cells may be able to live and grow. In various instances the media may include cells (e.g., living cells) cultured within a solution. In other instances, the media may be a solution containing elements useful for cell survival and growth but into which cells have not been added. For example, the media may include a solution of PBS, Dulbecco's Modified Eagle Medium, Roswell Park Memorial Institute (RPMI) medium, and / or Fetal Bovine Serum.

[0027] In various instances, reagents stored in the plurality of reagent containers may be considered precious reagents. Precious reagents may include, for example, pay load materials and / or a cell culture media. In various instances, reagents stored in the plurality of reagent containers may include nonprecious reagents. Non-precious reagents may include, for example, alcohols, buffers, and / or sterilizing materials.

[0028] Liquid waste container 154 stores liquid waste from the system, including liquid waste from the module assembly 130. In various implementations, more than one liquid waste container 154 may be used. More than one liquid waste container 154 may, for example, prevent hazardous reactions between chemicals and / or reagents used during one or more processes using the system 100.

[0029] The fluid handling system 150 may include fluid transport devices for directing the flow of fluids within the system, such as pump 155, valve 156, and tubing network 158. Pump 155 may include more than one pump. Nonlimiting examples of the pump 155 may include asyringe pump, a peristaltic pump, a diaphragm pump, a vacuum pump, a pressure-based pump, and electroosmotic pump, a displacement pump and / or a piston pump. In various implementations, a pump 155 may include, for example, a syringe integrated with a rotary valve. Valve 156 may include more than one valve (e.g., rotary valves). Tubing network 158 may be made of flexible tubing materials such as Teflon and / or silicone. The tubing network 158 may be in fluid communication with the module assembly 130 and the other elements of the fluid handling system 150.

[0030] Reference is now made to FIG. 2, in which a perspective view of components and modules of an example integrated system according to aspects of the present disclosure is shown as module assembly 200. Module assembly 200 presents an example of the module assembly 130 in FIG. 1. Module assembly 200 may include a needle chip handling module 210 (e.g. a needle chip handler), a gantry module 220, a tip-tilt platform module 230 (e.g., an alignment module), a microscope module 240, a fluidics module 250, and vibration damping feet 260. Needle chip handling module 210 stores needle chips (not shown) disposed in needle chip holders 212 and supports the needle chips during system processes and workflows. Gantry module 220 moves needle chips, including during transport and alignment. Tip-tilt platform module 230 enables the movement and angular alignment of a cell trapping chip in a cassette 252 (e.g., a cartridge that holds and / or carries a cell-trapping chip). Microscope module 240 may facilitate coplanarization and optical alignment of the needle chips and cell trapping chips. Fluidics module 250 may include a cell trapping chip (not shown) disposed in a cassette 252. Fluidics module also may include devices and channels for facilitating fluid transport into and out of the module assembly 200. These modules will be described in greater detail below.

[0031] In various implementations, module assembly 200 may include vibration damping feet 260. Vibration damping feet 260 may mechanically isolate the module assembly 200 from at least a substantial amount of vibration. For example, vibration damping feet may dampen vibrations from the system 100 with respect to the module assembly 200 (e.g., module assembly 130). Vibration damping feet 260 may be distinct from and / or in addition to vibration damping feet 114. In various implementations, vibration damping feet 260 may be made of a hard rubber or some other suitable vibration damping material.

[0032] For the modules of module assembly 200 to enable the precise automation of workflows contemplated by this disclosure, the modules will preferably be integrated with and / or interface with other modules in a highly precise and faithful manner. For example, the modules may be integrated together within build tolerances of 1 pm to 500 pm. In various implementations, build tolerances for the module assembly 200 may be at least within industry standards for build tolerances. Build tolerances may also exceed the industry standard tolerance ranges. As non-limiting examples, the build tolerances may include machining tolerance ranges and / or registering tolerance ranges. An exemplary machining tolerance range may be between a tolerance of 150 pm and 200 pm. An exemplary range of registering tolerance may be within a tolerance of 25 pm to 200 pm.

[0033] Precisely integrated modules such as those discussed above may enable more precise motion control during cell engineering processes discussed more fully below with reference to FIGs. 8-10. The individual modules of the module assembly 200 will now be discussed in greater detail.Needle Chip Handling Module

[0034] Referring now to FIGs. 3A and 3B, a perspective exploded view of an example needle chip handler (e.g. a needle chip handling module) according to aspects of the present disclosure is shown as needle chip handling module 300. Needle chip handling module 300 presents an example of the needle chip handling module 210 of FIG. 2. Needle chip handling module 300 may include a plurality of needle chip holders 310, a hopper 320, a hopper rest 330, and a robotic gripper 340.

[0035] Each of the needle chip holders 310 may hold a needle chip (not shown). The needle chip may include an array of microneedles or nanoneedles. Because microneedles and nanoneedles can be fragile, handling needle chips directly can cause the needles to break. The needle chip holders 310 enable the chips to be handled and manipulated within the system with a significantly lowered chance of breaking the needles. Indirect handling of the needle chips may also help to preserve sterility of the needle chip. The needle chip holders 310 are examples the needle chip holders 212 of FIG. 2. As illustrated In FIG. 3B, for example, the needle chip holders 310 may include a grip section 312, a stop section 314, and a needle chip holding section 316.Grip section 312 enables a robotic gripper 340 to grip the needle chip holder 310. Stop section 314 may prevent the robotic gripper 340 from gripping portions of the chip holder 310 where the needle chip is stored. Stop section 314 may also protrude such that the needle chip holder 310 may rest by the protruding portion of stop section 314 in a well 322 of the hopper 320. The needle chip holding section 316 may grip, be adhered to, or otherwise hold the needle chip for use in the system. Needle chip holding section 316 may be at least partially hollow and may be open on an end to expose the needle chip. In other implementations, needle chip holding section 316 may be formed as a unitary structure with the needle chip disposed on an end of the needle chip holding section 316.

[0036] The hopper 320 may hold the plurality of needle chip holders 310 in wells 322. A hopper may be any suitable container for housing needle chip holders 310. In this example, the hopper 320 includes three wells 322, but a person of ordinary skill would recognize that configurations with either more or fewer than three wells could be made from this disclosure. Having a plurality of wells 322 may enable greater throughput and efficiency for the system 100. According to various aspects of the present disclosure, the hopper 320 may be disposed in a hopper rest 330. The hopper rest 330 may include a plurality of sensors 332. The sensors 332 may be able to detect the presence and / or the position of a needle chip in a corresponding needle chip holder 310. In various implementations, the plurality of sensors 332 may provide a well-defined position of the needle chip holders to the system 100, and / or to controllers for the system 100. For example, sensing the position of needle chip holders with precision may help automate cell engineering workflows.

[0037] Needle chip handler 300 may include a robotic gripper 340 for securely gripping the needle chip holder 310, as shown in FIG. 3B. The robotic gripper 340 may include, for example, gripper forceps 342, a limit sensor 344, pneumatic connections 346, and a mounting bracket 348. The gripper forceps 342 grip a needle chip holder 310 and hold it securely during system processes and workflows. Limit sensor 344 may detect the force applied by the gripper forceps to the needle chip holder, and may provide feedback to a system controller (not shown) to limit or increase the amount of force applied by the gripper forceps. Alternately or additionally, limit sensor 344 may measure pneumatic pressure and / or some other indicator of the force applied by the gripper forceps 342. The gripper forceps 342 may be activated by pneumatic pressure toachieve precision-controlled motion. To enable pneumatic control of the gripper forceps 342, the robotic gripper may include pneumatic connections 346. The robotic gripper may be mounted onto a vertical linear stage 416 of gantry module 400 by a mounting bracket 348, as illustrated in FIG. 4. While robotic gripper 340 is illustrated herein in FIG. 3B as a gripper that mechanically grasps needle chip holder (e.g., through pneumatic activation), robotic gripper 340 may be configured in other ways that would be known to those of ordinary skill in the art. For example, robotic gripper 340 may configured to lock into place with the chip holders 310 based on the shape of the robotic gripper, the chip holder, or a combination thereof.Gantry module

[0038] Referring now to FIG. 4, a perspective view of an example gantry module according to aspects of the present disclosure is shown as gantry module 400. Gantry module 400 can represent an example of the gantry module 220 of FIG. 2. In various implementations, the gantry module 400 may be used to move robotic gripper 340 to perform cell engineering workflows. For example, gantry module 400 may enable highly precise motion of the robotic gripper to pick up, move, and / or adjust the position of needle chips. For example, the precision motion of the gantry module 400 may bring needle chips into alignment for cell engineering workflows. Gantry module 400 may include linear stages, including a first horizontal linear stage 410, a second horizontal linear stage 412, a third horizontal linear stage 414, and a vertical linear stage 416.

[0039] Gantry module 400 may be a 4-degree of freedom (4DOF) gantry module. For example, the gantry module 400 may be configured to move gantry positions in each of the X, Y, and Z dimensions indicated by the reference coordinate system shown in FIG. 4. In the view of FIG. 4, the X dimension roughly corresponds to a direction parallel to first horizontal linear stage 410 (or roughly left-right in the view of FIG. 4), the Y dimension roughly corresponds to a direction parallel to the second horizontal linear stage 412 and the third horizontal linear stage 414, and the Z dimension roughly corresponds to a direction parallel to the vertical linear stage 416. The first horizontal linear stage 410 may also be termed an X linear stage 410, and the second horizontal linear stage 412 and the third horizontal linear stage 414 may be termed a first Y linear stage 412 and a second Y linear stage 416, respectively.

[0040] The linear stages 410, 412, 414, and 416 may include linear actuators 420, 422, 424, and 426 (e.g., ball screw linear actuators) to enable precise motion control of the linear stages.

[0041] Gantry module 400 may also rotate in a substantially horizontal plane about an axis substantially parallel to the Z dimension (e.g., yaw rotation). The rotational motion of the gantry module 400 may be facilitated by rotary compliance stages 432 and 434 coupled between the X linear stage and the respective Y linear stages. Rotational motion of the gantry module 400 in the horizontal plane may be achieved, for example, by extending one of the Y linear stages a different distance or at a different rate from the other Y linear stage.

[0042] In various implementations, the gantry module 400 may include sensors. Such sensors may be coupled to the linear stages and may precisely locate the position of the linear stages and / or determine the coordinates of the gantry module 400 within a coordinate frame of reference. For example, the gantry module 400 may include a first sensor 440, a second sensor 442, a third sensor 444, and a fourth sensor 446. The sensors 440, 442, 444, and 446 may include, as non-limiting examples, encoders, optical sensors, mechanical sensors, and / or electromagnetic sensors. In various implementations, the motions of the linear stages 410, 412, 414, and 416 may be controlled by a controller or plurality of controllers (not shown). In various implementations the controller or controllers may control the gantry module 400’ s motions based on feedback or other information obtained from the sensors 440, 442, 444, and 446.Tip-Tilt Platform Module

[0043] Reference is now made to FIG. 5, which illustrates a perspective view of an example of a tip-tilt platform module 500 (e.g. an alignment module) according to aspects of the present disclosure. The tip-tilt platform module 500 presents an example of the tip-tilt platform module 230 of FIG. 2. The tip-tilt platform module 500 may include a platform 510. The platform 510 may be a custom platform (e.g., it may be custom machined, milled, molded, or otherwise shaped).

[0044] Tip-tilt platform module 500 may be configured to adjust the pitch angle and / or roll angle of the platform 510 relative to the horizontal. This may allow the alignment module (e.g tip-tilt platform module) to adjust the planar angle of a chip (e.g., a cell trapping chip, or a needle chip) to align with the planar angle another chip. The pitch and / or roll angle of the alignmentmodule may be adjusted with a high degree of precision to enable planar alignment of microneedle structures. The platform 510 may be supported by a first positional linear stage 520 (e.g., a first pusher) coupled to the platform 510 via a first ball bearing 522, a second positional linear stage 530 (e.g., a second pusher) coupled to the platform 510 via a second ball bearing 532, and a third positional linear stage 540 (e.g., a third pusher) coupled to the platform 510 via a third ball bearing 542. Each of the pushers, including first positional linear stage 520, second positional linear stage 530 and third positional linear stage 540 may include a corresponding motor that extends or retracts the pusher. Examples of the motors are shown in FIG. 5 as first motor 524, second motor 534, and third motor 544. In various implementations, the motor positions may range between -12.5 and 12.5 mm relative to a resting position. In various implementations, when each of the motors 524, 534, and 544 are set to the same value, the pushers points of contact at the ball bearings 522, 532, and 542 will be the same height across the platform 510 and the platform will be level.

[0045] In various implementations, when viewed from above, (e.g., without the platform 510), the positional linear stages may form a triangle with the ball bearings located at the vertices. In various implementations, the triangle may be an equilateral (or substantially equilateral) triangle. In various implementations in which the triangle is an equilateral triangle, the base length of such an equilateral triangle may range between 285 mm and 365 mm. In various implementations, the base of the equilateral triangle may be 337.75 mm and the height of the equilateral triangle may be 292.5 mm. In various implementations, the platform 510 may be tilted up to 5 degrees above or below the horizontal by extending and / or retracting at least one of the positional linear stages.

[0046] The platform 510 may include a first aperture 512, a cassette socket 514, a second aperture 516, a securing member 518, and a fastener 519. First aperture 512 may accommodate a needle chip holder 310. First aperture 512 may allow a microscope module (e.g., microscope module 240 of FIG. 2 or microscope module 600 of FIG. 6A) to determine the relative position of a needle chip (not shown) in the needle chip holder 310. The first aperture 512 may have a diameter larger than the diameter of the needle chip holder 310 to provide sufficient clearance between the needle chip holder 310 and the walls of the first aperture 512. Such clearance may enable alignment adjustments to be made to the needle chip holder 310. Cassette socket 514 may be formed in platform 510. In various instances, cassette socket 514 may be formed in a faceof the platform 510 opposite from the positional linear stages (pushers) 520, 530, and 540. Cassette socket 514 may be configured to accept a cassette (not shown), such as the cassettes described in greater detail with respect to FIG. 7. The cassette may be secured in place in the cassette socket 514 via securing member 518. In various implementations, securing member 518 may be able to be fastened in place with fastener 519 (e.g., a hand-driven screw, a bolt, a spring fastener, a tabular fastener, a latch, a locking mechanism, or the like). The second aperture 516 may allow for a microscope module (such as microscope module 240 or 600) to determine the relative position of a cell trapping chip disposed in a cassette on the platform 510. In various implementations, determining the relative position of a cell trapping chip may allow the pitch and / or roll angle of the cell trapping chip to be aligned with the pitch and roll angle of the needle chip. The needle trapping chip may be formed with the same pitch of features and the same alignment fiducials as the needle chip to facilitate this alignment.

[0047] As such, in combination and precise coordination with the gantry stage module 400 described above with reference to FIG. 4, the tip-tilt platform module 500 can create an alignment system with six degrees of freedom for aligning the nanoneedle chip and the cell trapping chip. As discussed above, the gantry stage module 400 can control the motion of the needle chip in the X, Y, and Z directions, and its rotational (yaw) angle. A cell trapping chip can be mounted on the tip-tilt platform of the tip-tilt platform module 500, and the pushers (e.g., positional linear stages) 520, 530, and 540 can be engaged to adjust the platform 510 so that the pitch and roll of the cell-trapping chip matches the pitch and roll of the needle chip. Positional linear stages 520, 530, and 540 may also be configured to adjust the Z position of the cell trapping chip.

[0048] Each of the pushers 520, 530, and 540 may include a respective interface connection 526, 536, and 546 such that the respective motors 524, 534, and 544 may be powered and / or controlled by a controller (not shown). The interface connections 526, 536, and 546 may communicate pusher position information as feedback to the controller. In various implementations, the controller may be configured using a motor to world (MTOW) transformation. The MTOW transformation may translate a desired pitch and / or roll angle for the platform 510 into the required linear motion of the pushers 520, 530, and 540.Microscope Module

[0049] Referring now to FIGs. 6 A and 6B, a perspective view of an example microscope module according to aspects of the present disclosure is shown as microscope module 600. Microscope module 600 presents an example of the microscope module 240 of FIG. 2. Microscope module 600 may include a plurality of linear stages (collectively referred to as an XYZ linear stage). For example, Microscope module 600 may include a first horizontal linear stage 610 (e.g., an actuator in the X dimension) driven by a first motor 612, a second horizontal linear stage 620 (e.g., an actuator in the Y dimension) driven by a second motor 622, and a linear stage actuator 630 ((e.g., an actuator in the Z. dimension) driven by a third motor 632, a low magnification microscope 640, a first camera 642, a high magnification microscope 650, a second camera 652, and a mirror assembly 654. Although not identified in FIGs. 6A and 6B, microscope module 600 may also include one or more sources of illumination.

[0050] The microscope module will be preferably integrated precisely and faithfully into the module assembly 200 along with the other modules discussed above. In various implementations, the two microscopes may be mounted on the linear stage actuator 630. The motors 612, 622, and 632 may be configured to move the microscopes 640 and 650 along the axes of the XYZ linear stage into a position to view and / or adjust focus on the needle chips and cell trapping chips (e.g., during alignment and / or during cell engineering workflows) and to adjust the focus plane of each of the two microscopes.

[0051] A perspective view of aspects of the microscope module 600 including low magnification microscope 640 and high magnification microscope 650 is shown at FIG. 6B. The low magnification microscope 640 may be optically coupled to a first camera 642. The high magnification microscope 650 may be optically coupled to a second camera 652. In various implementations, the high magnification microscope 650 may be optically coupled to the second camera 652 via a mirror assembly 654. Mirror assembly 654 may enable the second camera 652 to detect images through the high magnification microscope 650 without needing to be in the straight-line optical path of light entering the high magnification microscope 650 through its lens 656. In various implementations, mirror assemblies may be likewise applied to the low magnification microscope 640, or may be included inside the low magnification microscope 640, such that the path light takes from lens 646 may be directed to the first camera 642 coupled to thelow magnification microscope 640. Configuring microscopes to use mirror assemblies like mirror assembly 654 may allow for a lower profile microscope module 600 to be used in the system 100.

[0052] The low magnification microscope 640 may be configured to perform rough alignment of the needle chip and the cell trapping chip. In various implementations, the low magnification microscope may be configured to evaluate cell trapping efficiency after a cell trapping process is performed. The high magnification microscope 650 may be configured to accurately determine and focus on the plane of the needle chip and cell trapping chip. Accurately determining the plane of the chips may enable precise alignment of the nanoneedles on the needle chip to the previously discussed target locations (e.g., pores) of the trapped cells on the celltrapping chip. Precise planar alignment thus enables the system 100 to perform cell membrane penetration and delivery of biomaterials into cells. The microscope module 600 may be controlled with an off-the-shelf control circuit (not shown) and programmed to focus on the respective fiduciary marks of the nanoneedle chip and cell trapping chip using a focus algorithm, such as the predictive focus algorithm discussed in greater detail below.Fluidics module

[0053] Referring now to FIG. 7, a perspective view of an example fluidics module according to aspects of the present disclosure is shown as fluidics module 700. Fluidics module 700 presents an example of the fluidics module 250 of FIG. 2.

[0054] Referring back to FIG. 7, fluidics module 700 is shown as integrated with other modules and elements of the module assembly 200, including needle chip handling module 300 and tip-tilt platform module 500 (e.g., alignment module). Fluidics module 700 may include a cassette 710, an opening 716, a cell trapping chip 718, a first syringe 720, a second syringe 722, a first syringe actuator 724, a second syringe actuator 726, a fluid handling manifold 730, fluid connections 732, and a flow rate sensor 734.

[0055] Cassette 710 may be disposed in a cassette socket 514 formed in the surface of the platform 510. Cassette 710 may also be referred to as a cartridge (e.g., a fluidic cartridge), a housing, or as a carrier for a cell trapping chip 718. The cassette 710 may include a cavity or chamber for fluid transport, storage, separation, etc. The cassette 710 may be secured in place in the cassette socket 514 via securing member 518. In various implementations, securing member518 may be able to be fastened in place with fastener 519. The cassette 710 may contain an opening 716. Opening 716 may expose cell trapping chip 718. Opening 716 may facilitate chip alignment or other cell engineering processes, as a microscope could view the cell trapping chip 718 through the opening.

[0056] The cell trapping chip 718 may include a porous membrane. The porous membrane can include target positions for cells, which may include pores. Those pores, for example, can be smaller than a dimension of the cells to be trapped on the cell trapping chip. These pores can be dimensioned such that when cells are present on the cell trapping chip 718 and an air pressure difference is applied to the pores, single cells become trapped at the pores. When a needle chip is properly aligned with the cell trapping chip 718, microneedles on the needle chip align with the previously discussed target locations and can impact or poke the cells to deliver biomaterials into the cells.

[0057] In various implementations, cell trapping chip 718 may be transparent or translucent. A transparent chip may enable better alignment between a needle chip and the cell trapping chip, as alignment marks on the needle chip could be seen through the cell trapping chip and aligned with alignment marks on the cell trapping chip. Transparent chips may also enable better determinations of cell trapping effectiveness. For example, if cells were to be trapped at target locations on the cell trapping chip, after the cell trapping, a microscope could be used to see through the cell trapping chip and determine the percentage of target locations that effectively trapped cells at their respective locations.

[0058] The fluidics module 700 may include syringes, such as first syringe 720 and second syringe 722, configured to deliver and / or retrieve fluids into the cassette 710 for cell engineering workflows. For example, the syringes 720 and / or 722 may deliver solutions containing cells (e.g., a blood sample, a plasma sample, or a cell culture media) onto the cell trapping chip 718. Additionally or alternatively, pumps having a fluid connection or connections with the cassette 710 may be used in place of syringes to transport material onto or off of the cell trapping chip 718. For example, displacement pumps and / or pressure control systems (such as a regulator corrected to a reservoir) may be used to transport fluids into the cassette 710. Other nonlimiting examples of fluidic pumps that could be employed in fluid transport in place of and / or in addition to syringes 720 / 722 may include a syringe pump, a peristaltic pump, a diaphragm pump,a vacuum pump, a pressure-based pump, and electroosmotic pump, a displacement pump and / or a piston pump. Other devices typical to fluid handling systems may be included, such as flow sensors, leak monitors, valves, flow regulators, and the like.

[0059] First syringe 720 and / or second syringe 722 may retrieve cells during a cell engineering workflow or after a cell engineering workflow has completed. Additionally or alternatively, the first syringe 720 and / or second syringe 722 may deliver and / or retrieve other fluids (e.g., buffers, reagents, cell culture media, and so on) to / from the cassette 710 for use in cell engineering workflows. First syringe 720 may be actuated by a first syringe actuator 724, and second syringe 722 may be actuated by a second syringe actuator 726. Fluids may also be delivered to and / or retrieved from the cassette 710 via a fluid handling manifold 730. Fluid handling manifold may include fluid connections 732. Fluid flow rate in the fluidics module 700 may be monitored by a flow rate sensor 734. In various implementations, information from the flow rate sensor 734 may be provided as feedback to a fluid flow rate controller (not shown). The fluid manifold 730 may be in fluid communication with the fluid handling system 150 of FIG. 1. Transport of fluids between the fluid handling system 150 and the fluidics module 700 may be accomplished using pumping mechanisms and other fluid transport devices, such as those discussed above.

[0060] Although embodiments of the system have been described in which a cell trapping chip may be housed in a cassette or cartridge, such that a needle chip may be moved into contact with the cell trapping chip, it is anticipated that other embodiments are possible. For example, various implementations may include having the needle chip housed in a fluidic cartridge (e.g., a cassette) and the cell trapping chip may be configured to move to the needle chip’s location for cell engineering procedures. In such implementations, corresponding adjustments may need to be made to the other modules to achieve equivalent functionality.System methods description

[0061] Reference is now made with respect to FIG. 8, in which FIG. 8 illustrates a flow diagram of an example process for performing a cell engineering workflow according to various aspects of the present disclosure. FIG. 8 illustrates a process 800 that may include a series of processes for performing cell engineering operations. According to various aspects, process 800may include a series of operations. At operation 801, a needle chip and a cell trapping chip are provided. The needle chip and the cell trapping chip may correspond to, but not limited to, various needle chips and cell trapping chips as described herein.

[0062] Operation 802 may include the step of functionalizing the needle chip. According to various implementations, functionalizing the needle chips may include performing steps to prepare the needles on the needle chips to hold cargo during the workflow. For example, in various implementations, functionalizing the needle chips may include applying a chemical treatment to a surface of the chips. In various implementations, functionalizing the needle chips may include applying a chemical treatment to a region of a needle chip including the needles. In various implementations, functionalizing the needle chip may include performing a secondary treatment, non-limiting examples of which include exposing the needle chip to UV light, applying a second chemical treatment to the needle chip, and / or flowing a gas over the needle chip.

[0063] In various implementations, functionalizing the needle chip may include adding a surface chemistry to the needle chip, where the surface chemistry may enable a payload to be carried by the needles. For example, the surface chemistry may enable and / or facilitate a pay load’s adhesion to the needle chip. Additionally or alternatively, the surface chemistry may facilitate a pay load’s release into a cell. In various implementations, functionalizing the needle chip may include storing the needle chip in a chemical solution. In various implementations, functionalizing the needle chip may include allowing a chemical treatment to dry and / or to cure on the needle chip. In various implementations, functionalized chips may be stored for later use. Depending on the chemical treatment applied, functionalized chips may be stored dry or wet to maintain their functionalization. In various implementations, functionalization of the needle chip or chips may include or correspond to functionalization operations discussed below in the examples of FIGs. 9 A and 10A.

[0064] Operation 803 may include the step of functionalizing the cell trapping chip(s). Examples of functionalizing the cell trapping chip(s) may include or correspond to various operations as described below with respect to FIGs. 9B and 10B. For example, functionalizing the cell trapping chip may include providing cell material, payload materials, buffers, or other materials to a portion or portions of the cell trapping chip. Functionalizing the cell trapping chip(s) may include preparing a housing for the cell trapping chip(s) (e.g., cassette 710 as shown in FIG.7) to receive or include cell material, payload materials, buffers, or other materials useful in performing cellular engineering applications.

[0065] Operation 804 may include the step of disposing the needle chip on an alignment module. For example, disposing the needle chip on an alignment module may include or correspond to operations as discussed below with respect to FIGs. 9A (e.g., with respect to operation 813) and 10A (e.g., with respect to operation 1018). According to various implementations, the alignment module may include or correspond to a portion or portions of the module assembly 200, such as, for example, the gantry stage module 220, the tip-tilt platform module 230, and / or the microscope module 240.

[0066] Operation 805 includes the step of aligning, by the alignment module, the needle chip with the cell trapping chip. Examples of the alignment process may include or correspond to operations discussed below with respect to at least FIG. 9A (e.g., operation 819) and / or FIG. 10D. For example, according to various implementations, alignment of the needle chip and cell trapping chip may be performed according to one or more methods as described below with respect to FIGs. 11 and 12A-12C.

[0067] Operation 806 may include the step of trapping a cell at a target location of the cell trapping chip by applying a pressure differential to the cell trapping chip. For example, trapping a cell at a target location of the cell trapping chip may include or correspond to functionality discussed above with respect to the fluidics module of FIG. 7. Additionally or alternatively, according to various implementations, trapping a cell at a target location of the cell trapping chip may include or correspond to operations discussed below with respect to FIG. 9C (e.g., operation 834) and / or FIG. 10E (e.g., operation 1052).

[0068] Operation 807 may include the step of impacting the cell with a needle on the needle chip by moving the needle chip toward the cell trapping chip. According to various implementations, impacting the cell with a needle on the needle chip may include or correspond to operations discussed below with respect to FIG. 9C (e.g., operation 836) and / or FIG. 10E (e.g., operation 1061).

[0069] Operation 808 may include the step of removing the needle chip from contacting the cell trapping chip. According to various implementations, removing the needle chip fromcontacting the cell trapping chip may include or correspond to, for example, operations discussed below with respect to FIG. 9C (e.g., operation 838) and / or FIG. 10E (e.g., process 1060 and / or process 1070).

[0070] Operation 809 may include the step of retrieving the cell from the cell trapping chip. For example, retrieving the cell from the cell trapping chip may include or correspond to functionality discussed above with respect to the fluidics module of FIG. 7. Additionally or alternatively, according to various implementations, retrieving the cell from the cell trapping chip may include or correspond to operations discussed below with respect to FIG. 9C (e.g., operation 838), FIG. 9D (e.g., operations 843, 848, and / or 851), FIG. 10E (e.g., process 1070), and / or FIG. 10F (e.g., process 1080).

[0071] Reference is now made to FIGs. 9A-9D, in which a flow diagram of an example process for performing a cell engineering workflow according to various aspects of the present disclosure is shown as a series of processes, including a process for loading needle chips 810, a process for loading cells 820, a process for poking cells 830, and a process for unloading cells 840. FIGs. 9A-9D may include or correspond to portions of process 800 of FIG. 8. According to various aspects, FIGs. 9A-9D may illustrate an example of the processes described above with respect to FIG. 8.

[0072] In various implementations, the needle chip loading process 810 shown in FIG. 9 A may include a series of operations. Operation 811 may include the step of functionalizing needle chips for use in the workflow. This functionalizing step at operation 811 may include or correspond to the functionalizing of needle chips as described in operation 802 above. Functionalizing the needle chips may include performing steps to prepare the needles on the needle chips to hold cargo during the workflow. Functionalizing the needle chips may include applying a chemical treatment to a surface of the chips. In various implementations, functionalizing the needle chips may include applying a chemical treatment to a region of a needle chip including the needles. In various implementations, functionalizing the needle chip may include performing a secondary treatment, non-limiting examples of which include exposing the needle chip to UV light, applying a second chemical treatment to the needle chip, and / or flowing a gas over the needle chip.

[0073] In various implementations, functionalizing the needle chip may include adding a surface chemistry to the needle chip, where the surface chemistry may enable a payload to be carried by the needles. For example, the surface chemistry may enable and / or facilitate a pay load’s adhesion to the needle chip. Additionally or alternatively, the surface chemistry may facilitate a pay load’s release into a cell. In various implementations, functionalizing the needle chip may include storing the needle chip in a chemical solution. In various implementations, functionalizing the needle chip may include allowing a chemical treatment to dry and / or to cure on the needle chip. In various implementations, functionalized chips may be stored for later use. Depending on the chemical treatment applied, functionalized chips may be stored dry or wet to maintain their functionalization .

[0074] Operation 813 may include the step of installing needle chips in a component of cell engineering platform, such as, for example, needle chip holders (e.g. needle chip holder 310).

[0075] Optionally, operation 815 may include the step of consolidating the needle chip holders to an organizational component on the platform, such as, for example, a hopper (e.g., hopper 320 discussed previously), a receptacle, a rest, a mount, a nest, a holder, a jacket, a chamber, a storage unit, or some other such device that can organize, receive, store, and / or hold needle chips and / or needle chip holders.

[0076] Operation 817 may include the step of selecting a needle chip for the workflow. For example, a needle chip may be selected based on its position in a needle chip holder such as have been described above with reference to FIG. 3.

[0077] Operation 819 may include the step of positioning the needle chip use in the work flow. Operation 819 may include securing the needle chip in its initial position in the component of the cell engineering platform. Alternatively, operation 819 may include moving the needle chip into an aligning position so that the needle chip can be co-planarized with a cell trapping chip. In various implementations, once the needle chip has been co-planarized, it may be moved into a position where it can be directly aligned with the cell trapping chip. When the needle chip is in position, the needle positioning process may be complete. Once the cell loading process 820 described below is also complete, the cell poking process 830 may be started.

[0078] Referring now to FIG. 9B, FIG. 9B illustrates a flow diagram of a cell loading process 820, in accordance with various implementations. The cell loading process 820 may include a series of operations. According to various implementations, the operations illustrated in FIG. 9B may include or correspond to operations to functionalize the cell trapping chip. For example, functionalizing the cell trapping chip may include preparing the cell trapping chip to receive cell material. Operation 821 may include the step of collecting cells in a first syringe. Operation 822 may include the step of collecting a payload in a second syringe.

[0079] Operation 823 may include the step of installing precious materials into a cassette (e.g., a fluidics cartridge, such as cassette 710). A cassette may be configured to hold precious materials and / or nonprecious materials. Precious materials may include cells and / or a culture of cells in a cell culture media. Precious materials may also include a genetic payload material that will be delivered to the cells. In some cases genetic payload materials (e.g. mRNA, RNP) can be expensive. Precious materials may be introduced to the cassette by syringes or wells directly attached to the cassette itself in order to minimize dead volume. In some instances, precious materials may include materials with a significant cost, limited supply, and / or significant effort needed to make the material. A cassette may be configured to hold a cell trapping chip. Operation 823 may include the step of installing a cell trapping chip within the cassette.

[0080] At operation 824, the cassette can be installed onto an alignment module. This operation may include securing the cassette in place. For example, the cassette may be installed at a predetermined location.

[0081] At operation 825, the cassette and the cell trapping chip may be primed with non-precious materials. This may include flowing a sequence of different solutions into the cassette. In various implementations, the solutions may be transferred using fluid transportation devices, such as tubing, pumps and valves. The flow rate during this step may be monitored by a flow rate sensor.

[0082] Operation 826 may include the step of priming the cassette with precious materials, including cell samples. Operation 826 may include flowing a cell or cells into the cassette from a syringe by applying positive pressure.

[0083] In various implementations, at least a portion of the cell loading process 820 may be performed, for example, before at least a portion of the needle chip loading process 810. For example, operation 821 through at least operation 824 may be performed prior to the needle chip loading process 810 to ensure the cassette is installed before the needle chip is positioned. This may also prevent unnecessary vibrations or displacement of the needle chip after it is held in position. At least a portion of the cell loading process 820 may be performed in parallel or approximately at a concurrent time as a portion of the needle chip loading process 810 (e.g., at least operations 825 and 826 may be performed concurrently with operations 811, 813, and 815).

[0084] Reference is now made to FIG. 9C, which shows an example a cell poking process 830 according to aspects of the disclosure.

[0085] Operation 832 may include the step of loading precious cargo onto the needles of the needle chip. The precious cargo may include materials to be inserted into a cell (e.g., payload materials, genetic materials, pharmaceutical materials, and / or chemical materials).

[0086] At operation 834, a vacuum may be applied to the cell trapping chip. For example, a vacuum may be applied to attract cells toward target locations as discussed previously. These target positions can be pores in the cell trapping chip. In various implementations, pores can be dimensioned with a smaller diameter than the cells. Thus, the cells may become trapped at the pores. In various implementations, applying a vacuum to the cell trapping chip may form a microfluidic channel between the microneedle chip and the cell trapping chip. In various implementations, the vacuum may be applied when the needle chip reaches a preset height above the pores on the cell trapping chip. In various implementations, the vacuum may be a hydraulic pressure difference in the fluid between one side of the cell trapping chip and the other side.

[0087] In various implementations, a low magnification microscope may scan over the cell trapping chip to determine the cell trapping efficiency. In various implementations, once the cell trapping chip is loaded with cells, the user will input the desired poking parameters (e.g., approaching velocity, insertion distance, and / or needle in-cytoplasm duration). Inputting the desired parameters may cause a controller to adjust the vacuum level on the chips, add fluid to the system, introduce secondary stage materials to the microfluidic interface, and / or adjust a timer fora system process. Alternatively, in various embodiments the relevant parameters may be preprogrammed into the system controller based on a particular cell engineering workflow.

[0088] Operation 836 may include the step of poking the cells with the needles on the needle chip. This operation may include causing the microneedles to contact, impact, or poke into the cells.

[0089] The cell poking may be performed according to the cell poking parameters. In various implementations, a video feed may be configured to record data during the process. In various implementations, sensors may be configured to record data during the process. In such implementations, data from the video feed and / or sensor may be saved in storage (e.g., a computer memory). In other such implementations, data may be transmitted remotely to a storage device (e.g., a cloud server).

[0090] Operation 838 may include the steps of releasing the vacuum and recapturing the cells. In various implementations, releasing the cell trapping chip from the vacuum may include applying positive pressure to the cell trapping chip for a preset time duration using a pump. In various such implementations, the pump may include a flow rate sensor configured to provide flow rate feedback to the system. Recapturing the cells may include collecting the cells to a retrieval chamber in the cassette. For example, a syringe may be used to provide the necessary pressure to move the processed cells into the retrieval chamber.

[0091] Reference is now made to FIG. 9D, which shows an example cell unloading process 840. Cell unloading process 840 may include a series of determinations and operations, examples of which are outlined below. At determination 841 of the cell unloading process 840, a determination may be made as to whether a cycle of a cell engineering process is complete. If the cycle of the cell engineering process is complete, then at operation 843 cells may be collected. Collecting cells may include flowing the cells out of the cassette. This may be done, for example, using a syringe. Collecting cells may be alternatively performed by a pumping system. The cells may then be collected into a container. In various implementations, poked and unpoked cells may be flowed into respective dedicated output reservoirs (e.g., chambers or cavities) in the fluidics cartridge such that at the end of the poking operation, users can retrieve cells from those reservoirs. In various situations, a fluidics cartridge may be used for multiple operations (e.g., multiplebatches of the same cell engineering process). It may be desirable in such circumstances to have cell samples stored in a reservoir so they may all be retrieved at one time.

[0092] A determination 844 may include determining whether a new cycle is to be performed. If a new cycle is to be performed, then the cell unloading process 840 may end and the new cycle may be started from operation 823 of FIG. 9B. In that case, in various instances, time to install a new cassette may be provided. If, at determination 841 of FIG. 9D, a new cycle is not to be performed, then at operation 845 the cell engineering process may completed, and a shutdown procedure may be initiated.

[0093] If, at determination 841 the cycle is not complete, a determination 842 may be made as to whether to reuse the cassette. If the cassette is not to be reused, the process may go to operation 843 and follow the same flow as if the cycle were complete, as described above.

[0094] If at determination 842 the cassette is to be reused, a determination 846 may be made as to whether to reuse the needle chip. If the needle chip is to be reused, then a determination 847 may be made as to whether to collect cells. In various implementations it may be inefficient to collect all the cells each time the process is run, and so cell collection may be preferably delayed. If cells are to be collected, then at operation 848 the cells may be collected. Operation 848 may be functionally equivalent to operation 843 described above. After the cells are collected at operation 848, or if the determination 847 cells were not to be collected, then at operation 849 the old needle chip may be returned to the location it was retrieved from. The cell unloading process 840 would then end, and needle chip loading process 810 of FIG. 9A may be rejoined at operation 817, where a new needle chip would be selected.

[0095] Returning to FIG. 9D, if, at determination 846, the needle chip is not to be reused, then a determination 850 may be made as to whether to collect cells. If cells are to be collected, then at operation 851 the cells may be collected. Operation 851 may be functionally equivalent to operation 843 described above.

[0096] After the cells are collected at operation 851, or if at the determination 850 cells were not to be collected, a determination 852 may be made as to whether the needle chip is to be cleaned. If the needle chip is to be cleaned, then the process flows to operation 853. Operation 853 may include the step of cleaning the needle chip. In various implementations cleaning theneedle chip may include washing the needle chip with ethanol, and then with phosphate buffered saline (PBS) or some other solvent compatible with the needle cargo. After the needle chip is cleaned, or if at determination 852 the needle chip was not to be cleaned, then the cell collection process may end and the overall process may proceed to operation 832 of FIG. 9C.

[0097] Another implementation of the process described in FIGs. 9A through 9D is shown in FIGs. 10A through 10G, which illustrate flow diagrams of an example process for performing a cell engineering workflow according to aspects of the present disclosure. Portions of FIGs. 10A to 10G may correspond to and / or overlap with portions of FIGs. 9 A through 9D. Cell engineering processes, steps, and subprocesses illustrated in FIGs. 10A to 10G may include the processes and steps as presented in FIGs. 10A to 10G, or a subset of the processes and steps as presented in FIGs. 10A to 10G. In various implementations, the processes and steps may be performed in a different order to the extent that such reordering is reasonable. Some of the processes, routines, subroutines, and steps may be optional and may not need to be performed in every instance.

[0098] Reference is now made to FIGs. 10A through 10G, which illustrate flow diagrams of an example process for performing a cell engineering workflow according to various aspects of the present disclosure. According to various aspects of the disclosure, the flow diagrams of FIGs. 10A through 10G may include or correspond to methods or portions of methods discussed herein, including with respect to FIG. 8 and FIGs. 9A-9D.

[0099] According to various aspects of the disclosure, the flow diagrams of FIGs. 10A through 10G may include several processes and / or subprocesses, including, for example, 1) a needle chip preparation process 1010, 2) a cassette assembly process 1020, 3) a needle chip and cartridge loading process 1030, 4) a chip planes synchronization and final XY alignment process 1040, 5) a cell capture and payload loading process 1050, 6) a cell poking process 1060, 7) a cell release and collection process 1070, 8) a post poke cell handling process 1080, and 9) a system shutdown process 1090.

[0100] Referring now to FIG. 10A, a needle chip preparation process 1010 may include various subprocesses and / or operations. According to various operations, the needle chip preparation process 1010 may include operations to functionalize the needle chips. For example,at operation 1011, the system may be configured to accept delivery of a needle chip or a plurality of needle chips. According to various implementations, the needle chips may be formed in a large batch and / or attached to each other, such as may be the case, for example, when needle chips are formed on and / or from a silicon substrate (e.g., a silicon wafer), on or from other semiconductor materials such as gallium arsenide, or on and / or from other substrates or materials suitable for microfabrication of needle chips. According to various implementations, at operation 1012 the needle chip(s) may be broken into individual dice of a single chip or multiple needle chips. For example, needle chip wafers may be scribed and broken, mechanically sawn (e.g., with a dicing saw), or cut with a laser cutter. This process may also be referred to as die singulation.

[0101] At operation 1013, the surface of the needle chip(s) may be cleaned. In various implementations, cleaning the surface may include dipping and / or spraying operations to remove impurities. For example, dipping may include dipping the wafer, dice, and / or individual needle chip(s) into a chemical solution or ultra-pure deionized water. In various implementations, spraying may include spraying a chemical in liquid or gas form onto a spinning wafer or onto individual chips. At operation 1014, surface plasma activation may be performed. For example, surface plasma activation may include sputtering with an inert gas and / or etching with chemically reactive plasma.

[0102] At operation 1015, the chip(s) may be immersed in a thiol solution. The immersion . At operation 1016, the chip(s) may be removed from the thiol solution and rinsed in an appropriate rinsing material. At operation 1017, the chip(s) may be dried and stored. At operation 1018, the needle chip(s) may be mounted into needle chip holders, such as, for example needle chip holders 310 as have been described herein.

[0103] Referring now to FIG. 10B, a flow diagram of a cassette assembly process 1020 is illustrated. According to various operations, the cassette assembly process 1010 may include operations to functionalize cell trapping chips and / or cassettes. In various implementations, at operation 1021, the cell trapping chip(s) (e.g., vacuum traps) may be singulated (e.g., separated into dice of individual chips or multiple chips). For example, the cell trapping chips may be separated from each other by being scribed and broken, mechanically sawn (e.g., with a dicing saw), or cut with a laser cutter. Operation 1021 may also include plasma treating the cell trapping 1chip(s). For example, the cell trapping chip(s) may be treated by sputtering the cell trapping chip(s) with an inert gas and / or etching with chemically reactive plasma.

[0104] At operation 1022, the cell trapping chip(s) (e.g., vacuum trap(s)) may be mounted into or onto a cassette (e.g., such as the cassette 710 of FIG. 7). At operation 1023, the process may include a step for loading priming syringes into the cassette and priming the syringe connection. At operation 1024, cells (e.g., cells in a cell solution) may be prepared and loaded into cell syringes. Cell syringes may be distinct from priming syringes. At operation 1025, the priming syringes may be swapped for (e.g., removed and replaced by) cell syringe(s) and payload syringe(s). Payload syringes may be prepared to include payload materials (e.g., genetic payloads), such as have been discussed herein.

[0105] Reference is now made to FIG. 10C, in which a flow diagram of a needle chip and cartridge loading process 1030 is illustrated. At operation 1031, a system check may be performed. At operation 1032 a system initialization may be performed. At operation 1033, needle chip(s) may be mounted on to the system. The needle chips may be functionalized and provided to operation 1033 from the output of operation 1018. The needle chip(s) may be mounted in needle chip holders. In various implementations, mounting needle chip(s) on to the system may include mounting a chip holder or needle chip holders in wells in a needle chip hopper or other organizational component as has been described herein. Additionally or alternatively, mounting needle chip(s) on to the system may include mounting the organizational component to the system.

[0106] At operation 1034, the cassette may be loaded onto the system. The cassette may be prepared for mounting from the output of operation 1025. The cassette may be secured in place by clamps or other securing mechanisms (e.g., securing member 518). At operation 1035, the cassette may be primed. Operation 1036 may include moving (e.g., picking up) a needle chip holder with a robotic gripper (e.g., robotic gripper 340 described above).

[0107] Reference is now made to FIG. 10D, in which a flow diagram of a chip planes synchronization and final XY alignment process 1040 is illustrated. In various implementations, the synchronization and final XY alignment process 1040 may be configured to align the needle chip with the cell trapping chip (or, more precisely, to align a needle on the needle trapping chip with target locations at which cells may become trapped in the cell trapping chip). At operation1041, the location of the vacuum chip (e.g., cell trapping chip) and the location of the needle chip may be determined. For example, the locations of the cell trapping chip and needle chip may be precisely determined according to methods described herein (e.g., with reference to the functionality described relative to the sensors 332 in FIG. 3A, the microscope module 600 of FIG. 6, and / or the methods described with reference to the alignment processes of FIGs. 11 and 12). At operation 1042, initial angular correction may be performed. At operation 1043, the chips may be translated to a pre-insertion XY position. According to various implementations, the pre-insertion XY position may include or correspond to an aperture in the cassette corresponding to the cell trapping chip. For example, the needle holder may be configured to be insertable into the aperture in the cassette. At operation 1044, the needle chip (and the needle chip holder) may be inserted into the cassette. At operation 1045 a diaphragm seal may be engaged. The diaphragm seal may be engaged until it is within a specified value at determination 1046. When the diaphragm seal is within a specified value, at operation 1047 the needle chip may be translated in a Z direction to its prepoke distance. At operation 1048, a final XY translation may be performed to complete the alignment process.

[0108] Reference is now made to FIG. 10E, in which flow diagrams of a cell capture and payload loading process 1050, a poking process 1060, and a release and collection process 1070 are illustrated. At operation 1051 of the cell capture and payload loading process 1050, a payload may be loaded into or onto the needles of the needle chip. At operation 1052, cells may be flowed into the cell trapping chip (e.g., from the cassette) and trapped at target locations in the cell trapping chip (e.g., by applying a pressure differential to the cell trapping chip). At operation 1061 of poking process 1060, the system may cause the needles of the needle chips to poke the cells trapped at the target locations of the cell trapping chip. The system may be configured to perform the poking process automatically. The poking process may include process steps to retract needles from the cell trapping chip after the poking has been accomplished. During the poking process 1060, parameters may be input and / or captured during the process. For example, the poking process may be configured based on parameters such as a descent velocity, a starting position, a poking distance (e.g., a distance into a cell that a needle may be poked), a residence time (e.g. how long the needle remains in the cell), an ascent velocity, and / or a number of times the cell is to be poked. Other parameters may be factored into the poking process, and not all parameters need to be measured and / or input for the process to be performed.

[0109] At operation 1071 of the release and collection process 1070, the flow on the bottom of the cassette may be reversed. This may cause cells to flow into an output reservoir in the cassette. At operation 1072, cells may be collected on or in a cassette well (e.g., the output reservoir). At operation 1073, the needle chip may be retracted. At operation 1074, the cells may be removed from the cassette well (e.g., the output reservoir). For example, the cells may be removed using a pipette, or the cells may be removed through some other method of transporting cell materials and / or cell solutions. The cells may be provided for operation 1081 as described below. At operation 1075, the cell trapping chip (e.g., vacuum trap) may be imaged.

[0110] Reference is now made to FIG. 10F, in which a flow diagram of a post poke cell handling process 1080 is illustrated. At operation 1081, the cells may be moved (e.g., using a pipette) into a plate. For example, the cells may be moved into a well or wells of a well plate, such as a 6-well plate, a 24-well plate, a 48-well plate, a 96-well plate, a 384 well plate, a 1536-well plate, a 3456-well plate, or a 9600-well plate. A well plate is described here only by example, as the cells may be moved into another appropriate location at this step. At operation 1082, the cell media may be modified (e.g., to facilitate analysis of the cells). At operation 1083, the cell or cells may be plated into an appropriate well plate. This may include a different well of the well plate described above, or a different well plate. At operation 1084, the cell health may be analyzed. For example, a cell count may be performed. Additionally or alternatively, cell viability may be determined. For example, cell viability may be calculated using a ratio of the total living cells to the total number of cells in the sample. At operation 1085, analysis may be performed on the cells and on cargo delivered to the cells. For example, changes in the cell viability may be analyzed. This analysis may take place over an extended period of time, such as over a day or a number of days.

[0111] Reference is now made to FIG. 10G, in which a flow diagram of a system shutdown process 1090 is illustrated. At operation 1091, the needle chip may be removed and mounted on a microscope fixture. The microscope fixture may include or correspond to the microscope module 600 described herein, or it may include some other microscope fixture. Optionally, at operation 1095, the needle chip may be imaged off system. At operation 1093, the cartridge may be disassembled and disposed of. At operation 1094 the system may enter a systemshut-down routine. At operation 1095 the system, or individual system components may be cleaned and / or decontaminated as needed.

[0112] In various implementations, some of the processes of FIGs. 10A-10G can be run in parallel. Processes that may run in parallel may start at the same time, or may be configured to start at different times. For example, the needle chip preparation process 1010 of FIG. 10A, the cassette assembly process 1020 of FIG. 10B, and the needle chip and cartridge loading process 1030 of FIG. 10C may be configured to run at least partially in parallel and provide inputs and / or outputs to each other, such as is shown in FIGs. 10A-10C.

[0113] In various implementations, processes may end at different times, disregarding which of the processes finish first. For example, the post poke cell handling process and the system shutdown process may finish at different times, as shown in FIGs. 10F and 10G.

[0114] In various instances some of the steps and processes of FIGs. 10A-10G may be performed by users or by automated systems. Any indication of whether a process is performed by a user or an automated system is included as an example and is not intended to limit the manner in which a process may be performed.

[0115] FIGs. 10F and 10G highlight examples of cell handling and system shut down processes that may be optional to the method. For example, analyzing cell health or analyzing cargo as in FIG. 10E may not need to be performed in every instance, or as part of the same process flow. Likewise, the timing indicated for the analysis in the last step of post poke cell handling process 8 in FIG. 10E may be performed within a few days, but is not limited to this time frame. In various instances the analysis may be performed momentarily after the cell poking process, and in other instances more time such as days later may be needed for proper analysis. In any case, the time frame is given as an example and not as a limitation. In FIG. 10G, shutting down the system after each time the process is performed may not be necessary, such as, for example, in instances when multiple processes may be performed concurrently using the same needle chip and / or cell trapping chip, or with new needle chips and / or cell trapping chips without shutting down the system completely.Application of method to system

[0116] FIGs. 11A-11D illustrate an example of aspects of the needle chip loading process 810 and the cell loading process 820 as applied to a system in accordance with various implementations, including, for example, system 100. As shown in FIG. 11 A, a user may load a cassette 710 into the cassette socket 514 on the platform 510. The cassette 710 may be secured in place with securing member 518 and fastened in place with fastener 519. FIG. 11A may include or correspond to operation 824 of cell loading process 820. The cassette 710 may be primed with fluids, including, for example, any number of a cell sample culture, a buffer, and / or a reagent. This may occur before the cassette 710 is loaded onto the platform 510, or after the cassette is loaded onto the platform 510.

[0117] In FIG. 1 IB, the hopper 320 loaded with needle chip holders 310 may be loaded onto the hopper rest 330 on platform 510. This may include or correspond to operation 815 of needle chip loading process 810 described above with reference to FIG. 9A. In various implementations, the hopper 320 may be installed such that the needle chip holders 310 are located at predefined known positions. In various implementations, the system may detect the number of needle chips being loaded. The hopper 320 may be configured to allow the system 100 to determine the position of all the needle chips beforehand, for example by using the sensors 332 in the hopper rest 330. When the positions of the needle chips have been determined, the robotic gripper 340 may move to the needle chip position and picks it up as shown in FIG. 11B. This process may include or correspond to operation 817 of needle chip loading process 810.

[0118] FIGs. 11C-11D may include or correspond to operation 819 of needle chip loading process 810. At FIG. 11C, the robotic gripper 340 gripping the needle chip holder 310 may be moved using the gantry stage module 400 (can also be referred to as a gantry module) to a first aperture 512 in the platform 510. The system may estimate the rough location of the needle chip using the low magnification microscope (e.g., low magnification microscope 640 of FIG. 6A) and a focus algorithm, such as a predictive focus algorithm. The system may also determine the precise focus plane of the needle chip and the cell trapping chip 718 using the high magnification microscope (e.g., high magnification microscope 650 of FIG. 6A). Additionally or alternatively, an image processing routine may be used to determine the precise location of the needle chip and cell trapping chip 718. The gantry stage module 400 and the tip-tilt platform module 500 may adjust the positions of the needle chip and the cell trapping chip 718 to bring them into the sameor a substantially parallel plane. At FIG. 11D, based on the planarization and the precisely determined needle chip location, the system may move the needle chip into the cassette 710 through opening 716 and into contact with the cell trapping chip 718.

[0119] In various instances some of the steps and processes of FIGs. 11A-11D may be performed by users or by automated systems. Any indication of whether a process is performed by a user or an automated system is included as an example and is not intended to limit the manner in which a process may be performed.Method of performing alignment

[0120] An alignment process for a cell engineering workflow may include estimating the coordinates of features of a needle chip, estimating the coordinates of features of a cell trapping chip, and based on the estimated coordinates of the features of the needle chip and the estimated coordinates of the features of the cell trapping chip, moving at least one of the needle chip and the cell trapping chip into alignment with the other chip. After the alignment process is complete, the needle chip and the cell trapping chip will have surfaces aligned into the same plane.

[0121] For example, the chips to be aligned may be a microneedle chip and a cell trapping chip. The needle trapping chip may be formed with the same pitch of features and the same alignment fiducials as the needle chip. Chips formed in this manner can enable the use of optical alignment systems and robotics to align every nanoneedle to every cell trap. In various implementations, the chips may be co-planarized to within 1 pm of error in four edges. In order to ensure every nanoneedle on the needle chip pokes into a single cell at a target location (e.g in pores) in the cell trapping chip, careful alignment is required. However, the nanoneedles on the chips may be fragile, and thus cannot afford to perform contact alignment. Cell trapping chips may also be too fragile for contact alignment. Chips designed for contact alignment would need a more complex mechanical design to protect the nanoneedles. Instead, alignments may be made using a microscope to locate key features of the chips in three-dimensional space and a tip-tilt platform to move one of the chips into alignment with the other chips as described below. Alignments made using a tip-tilt platform may be confirmed optically before cell engineering processes on the chips are begun. In various implementations, at least one of the cell trapping chipand the needle chip may be formed of a transparent material. This may facilitate effective alignment between the chips.

[0122] Reference is now made to FIGs. 12A to 12C, which show a diagram of an example alignment process according to aspects of the present disclosure. FIGs. 12A-12C include illustrations of stages of an alignment process 1200.

[0123] At the stage of FIG. 12A a high magnification microscope 1220 estimates the coordinates of key features of the needle chip 1210 using a sample of data points. The key features may include, for example, a first edge 1212 of the needle chip 1210, a second edge 1214 of the needle chip 1210, corners of the needle chip 1210, and / or fiducial marks located on the needle chip 1210. In various implementations, the coordinates of key features may first be roughly estimated by a low magnification microscope (not shown). A high magnification microscope 1220 can provide a narrow field of view. Using a low magnification microscope in a first stage can provide a wider field of view for an initial coordinate estimate. In various implementations, in which the key feature is an edge of the needle chip, the coordinate estimate may correspond to the position of three of the edges of the needle chip. Identifying, estimating, and / or locating the unique coordinates of three key features of the needle chip 1210 may define a plane in which the needle chip 1210 is located. In various implementations, the coordinate estimate may correspond to the location of fiducial marks. Such fiducial marks may be located at the edge of the needle chip 1210. Again, the coordinates of at least 3 fiducial marks may be needed to define the plane of the needle chip 1210.

[0124] In various implementations the coordinates may correspond to a coordinate vector in three dimensional space. In various implementations, coordinates may be estimated with reference to a cartesian coordinate system (e.g., X, Y, and Z coordinates). Additionally or alternatively, the coordinates may be estimated with reference to angles of rotation. For example, in FIG. 12A the coordinates of an edge 1212 of the needle chip 1210 are suggested by reference to an offset Az, an x plane, and an angle 9 between the needle chip 1210 and the x plane. Regardless of the particular coordinate system used, after the coordinates of key features are determined, the coordinate information may be stored, for example in a computer memory, for use in alignment algorithms as described with respect to FIG. 12C below.

[0125] In various implementations, the alignment process may use a microscope to accurately focus on fiducial marks that may be located at the corners of the needle chip and cell trapping chip. A predictive focus algorithm may be used to help locate a focus plane of the chip. Examples of a predictive focus algorithm will be discussed in further detail below.

[0126] Identifying, estimating, and / or locating the unique coordinates of three key features of the needle chip 1210 may define a plane in which the needle chip 1210 is located. Thus, once the coordinates of one of the key features of the needle chip 1210 are estimated, the coordinates of at least two other separate key features of the needle chip 1210 may be estimated in a similar manner. As an example, in various implementations, the coordinates of a first fiducial on a first corner (e.g., the top-left comer) of the needle chip 1210 may be determined and subsequently recorded. Next in this example, the coordinates of a second fiducial on a second comer (e.g., the top-right comer) of the needle chip 1210 may be determined and subsequently recorded. Finally, the coordinates of a third fiducial on a third corner (e.g., the bottom-right corner) of the needle chip 1210 may be determined and subsequently recorded. This example is intended to be illustrative and not limiting, and it is anticipated that any of several alternate key features or locations on a needle chip may be used to identify the a plane in which the needle chip may be located, just as any of several alternative orders in which a needle chip’s key features are located may be used.

[0127] At the stage of FIG. 12B, the microscope 1220 may be used to estimate the coordinates of the key features of the cell trapping chip 1240. This may be done in a similar manner to estimating the coordinates of key features of the needle chip 1210. As in stage a), the coordinates of key features of the cell trapping chip 1240 may be estimated using a high magnification microscope 1220 or multiple microscopes (e.g. a low magnification microscope and high magnification microscope 1220). The coordinates may be defined using the same coordinate system as used for key features of the needle chip 1210 for ease of comparing the coordinates of the cell trapping chip 1240 to the coordinates of the needle chip 1210.

[0128] Furthermore, the estimated coordinates of three separate key features of the cell trapping chip 1240 may be estimated in a similar manner to the three separate key features of the needle chip 1210. For ease of comparison and alignment between the two sets of coordinates, thethree key features of the cell trapping chip 1240 may correspond to the three key features of the needle chip.

[0129] FIG. 12C illustrates an example of aligning the cell trapping chip 1240 into planar alignment with needle chip 1210 by adjusting the angle of cell trapping chip 1240. As discussed above, estimating and / or locating the unique coordinates of three key features of the needle chip 1210 may define a plane in which the needle chip 1210 is located. Estimating and / or locating the unique coordinates of three key features of the cell trapping chip 1240 may define a plane in which the cell trapping chip 1240 is located. Alignment processes as discussed here in reference to stage c) may function to bring the cell trapping chip 1240 into the same plane as the needle chip 1210.

[0130] At the stage shown in FIG. 12C, an alignment algorithm may be used to compute the necessary movement parameters to align the chips based on the estimated coordinates of the needle chip 1210 and the cell trapping chip 1240. For example, the computed movement parameters may correspond to an offset between the coordinate vectors of the needle chip 1210 and the corresponding coordinate vectors of the cell trapping chip 1240. In various implementations, the coordinate vectors from the key features on each of the chips may form a pair of coordinate vectors. In various implementations, from the 3 pairs of coordinates, the correction angles needed may be computed. These corrections may then be converted to the position of motorized stages of a tip-tilt platform. An exemplary alignment algorithm is discussed in further detail below.

[0131] In various implementations, based on the computed movement parameters, a tiptilt platform may be used to move at least one of the cell trapping chip 1240 and the needle chip 1210 into a calculated orientation such that the chips are aligned into the same plane. The goal of the alignment process is to manipulate the needle chip so that the separation distance between at least three corners of the cell trapping chip 1240 and at least three corresponding corners of the needle chip 1210 is the same. If the separation distance is the same between the corresponding comers of the chips is the same for each corner, that implies that the chips are planarized.Alignment Algorithm

[0132] In various implementations of an alignment process, needle chips and cell trapping chips may be aligned using an alignment algorithm. The alignment algorithm may include steps of identifying the coordinates of key features of a needle chip, identifying the coordinates of key features of a cell trapping chip, determining the plane in which the needle chip is positioned, determining the plane in which the cell trapping chip is positioned, determining a misalignment between the plane of the cell trapping chip and the plane of the needle chip, calculating the movement of one of the chips to bring the chip into the plane of the other chip, and moving the chip into alignment.

[0133] A needle chip or a cell trapping chip may be disposed on a tip-tilt platform (e.g., an alignment module). The chip that is not disposed on the tip tilt-platform may be held in a fixed position relative to the other chip. A tip-tilt platform may include pushers (e.g., linear stages). In various instances, the pushers may be arranged at comers of the tip-tilt platform. In various implementations, the pushers may include motors. The motors may be connected to a controller. The controller may be configured to run the motors and extend and / or retract the pushers. Extending and / or retracting the pushers may move the tip-tilt platform and / or alter its pitch and / or roll angle relative to the fixed position of the other chip. In various instances, the controller may be configured using a transformation. For example, the transformation may translate a calculated plane of alignment into a calculated set of pusher positions. Moving the pushers to the calculated set of pusher positions may bring the chip on the tip-tilt platform into the calculated plane of alignment.Predictive focus algorithm

[0134] To achieve the planarization of less than 1 urn error in four edges discussed above, a microscope with a high magnification objective lens (e.g., 60x - lOOx) may be used to focus on fiducial markings on both the microneedle chip and the cell-trapping chip. A high magnification microscope can desirably create a shallow depth of field to determine the accurate focus plane. A high magnification microscope gives a narrow field of view. But the narrow field of view can be mitigated by the following exemplary predictive focus algorithm. The predictive focus algorithm may have 4 stages: image acquisition, image analysis, optimal focus determination, and focus adjustment. The stages are described below.

[0135] (1) Image acquisition. At a first stage, the high magnification microscope captures a series of images at different focal planes (or Z-positions) within a predefined range above and below an initial focal plane. Ideally, the range of focal planes will contain the focal plane at which the chip (either the microneedle chip or the cell-trapping chip) is located.

[0136] (2) Image analysis. At a second stage, the images may be analyzed to determine the sharpness and / or contrast within each image. Several methods can be used to analyze image sharpness, such as, for example, calculating the variance, sum-modified Laplacian, or Tenengrad function of the image.

[0137] (3) Optimal focus determination. At a third stage the image with the highest sharpness or contrast value may be identified. This image corresponds to the optimal focal plane for the specimen.

[0138] (4) Focus adjustment. At a fourth stage, the microscope's hardware components, such as, for example a motorized stage or a focusing mechanism, moves the objective lens or the stage to the optimal focal plane as determined by the algorithm.

[0139] As an example of how this predictive focus algorithm may be used in connection with the alignment process shown in FIGs. 12A-12C, the predictive focus algorithm may be configured to focus its image analysis on the key features of the needle chip 1210 or the cell trapping chip 1240.Advantages of precision alignment and automation

[0140] Alignment processes such as those described herein may enable precision automation of cell engineering workflows. Precision automation may provide several benefits to cell engineering workflows and applications. For example, at least the following benefits may be achieved.

[0141] Consistency and reproducibility: Automated systems can precisely follow predefined protocols, ensuring consistent results with minimal variability. This leads to improved reproducibility.

[0142] Increased throughput: Automation allows for the simultaneous handling of multiple samples, increasing the overall throughput and efficiency of the process. This may be particularly valuable when working with large sample numbers or high-throughput screening applications.

[0143] Reduced human error: Automated systems minimize the risk of human error, such as pipetting mistakes or inconsistencies in handling, which can compromise the quality of the transfection process and the subsequent data.

[0144] Enhanced safety: Some transfection methods involve the use of hazardous materials or biohazardous samples. Automated systems can reduce the risk of exposure to these materials, ensuring a safer working environment.Implementation of Methods and Workflows on a Computing Environment

[0145] Referring to FIG. 13, an example of a computing device that is operable to support cell engineering workflows and methods according to one or more aspects of the present disclosure is shown as a computing environment 1300 that includes a computing device 1310. The computing device 1310 may be operable to initiate or control cell engineering workflows including chip alignment processes or other cell engineering workflows of the stages of any of the processes described with reference to, for example, FIGs. 8-12.

[0146] The computing device 1310 includes at least one processor 1320 and system memory 1330. Depending on the configuration and type of computing device, the system memory 1330 may be volatile (such as random access memory or “RAM”), non-volatile (such as read-only memory or “ROM,” flash memory, and similar memory devices that maintain stored data even when power is not provided) or some combination of the two. The system memory 1330 typically includes instructions 1332 and one or more applications. The at least one processor 1320 may be operable to execute the instructions 1332 to perform one or more operations described herein, including, for example, operations of the method 810 of FIG. 9A, the method 820 of FIG. 9B, the method 830 of FIG. 9C, the method 840 of FIG. 9D, the method 900 of FIG. 11, or the method 1200 of FIGs. 12A-12C. Alternatively, the instructions 1332, the applications, or both, may be located at multiple computing devices, where the multiple computing devices are part of adistributed computing system. In this case, one or more of the multiple computing devices of the distributed system may comprise the representative computing device 1310.

[0147] The computing device 1310 may also have additional features or functionality. For example, the computing device 1310 may also include removable and / or non-removable data storage devices such as magnetic disks, optical disks, tape, and standard- sized or miniature flash memory cards. Such additional storage is illustrated in FIG. 13 by storage 1340. Computer storage media may include volatile and / or non-volatile storage and removable and / or non-removable media implemented in any method or technology for storage of information such as computer- readable instructions, data structures, program components or other data. The system memory 1330 and the storage 1340 are examples of computer storage media. The computer storage media may include, but is not limited to, RAM, ROM, electrically erasable programmable read-only memory (EEPROM), flash memory or other memory technology, compact disks (CD), digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store information and that can be accessed by computing device 1310. Any such computer storage media may be part of the computing device 1310. The computing device 1310 may also have input / output (VO) device(s) 1350, which may include input devices, such as a keyboard, mouse, pen, voice input device, touch input device, etc., output device(s), such as a display, speakers, a printer, etc., or a combination thereof.

[0148] The computing device 1310 also contains one or more communication interface(s) 1360 that allow the computing device 1310 to communicate with a cell engineering system 1380 via a wired or a wireless network 1370. The cell engineering system 1380 may include one or more cell engineering process modules, one or more computing devices, cell engineering tools or devices, or a combination thereof. In an illustrative embodiment, the cell engineering system 1380 may initiate or facilitate, for example, any of the stages or operations of the processes described with reference to FIGs. 9A-9D, FIGs. 10A-10G, FIGs. 11A-1 ID, or FIGs. 12A-12C.

[0149] The communication interface(s) 1360 are an example of communication media. By way of example, and not limitation, communication media may include wired media such as a wired network or direct-wired connection, and wireless media, such as acoustic, radio frequency(RF), infrared and other wireless media. It will be appreciated, however, that not all of the components or devices illustrated in FIG. 13 or otherwise described in the previous paragraphs are necessary to support embodiments as herein described. For example, the VO device(s) 1350 may be optional.

[0150] Those of skill in the art would understand that information and signals may be represented using any of a variety of different technologies and techniques. For example, data, instructions, commands, information, signals, bits, symbols, and chips that may be referenced throughout the above description may be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields or particles, or any combination thereof.

[0151] Components, the functional blocks, and the modules described herein include processors, electronics devices, hardware devices, electronics components, logical circuits, memories, software codes, firmware codes, among other examples, or any combination thereof. In addition, features discussed herein may be implemented via specialized processor circuitry, via executable instructions, or combinations thereof.

[0152] Those of skill would further appreciate that the various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the disclosure herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present disclosure. Skilled artisans will also readily recognize that the order or combination of components, methods, or interactions that are described herein are merely examples and that the components, methods, or interactions of the various aspects of the present disclosure may be combined or performed in ways other than those illustrated and described herein.

[0153] The various illustrative logics, logical blocks, modules, circuits, and algorithm processes described in connection with the implementations disclosed herein may be implementedas electronic hardware, computer software, or combinations of both. The interchangeability of hardware and software has been described generally, in terms of functionality, and illustrated in the various illustrative components, blocks, modules, circuits and processes described above. Whether such functionality is implemented in hardware or software depends upon the particular application and design constraints imposed on the overall system.

[0154] The hardware and data processing apparatus used to implement the various illustrative logics, logical blocks, modules, and circuits described in connection with the aspects disclosed herein may be implemented or performed with a general purpose single- or multi-chip processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, or any conventional processor, controller, microcontroller, or state machine. In various implementations, a processor may also be implemented as a combination of computing devices, such as a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration. In various implementations, particular processes and methods may be performed by circuitry that is specific to a given function.

[0155] In one or more aspects, the functions described may be implemented in hardware, digital electronic circuitry, computer software, firmware, including the structures disclosed in this specification and their structural equivalents thereof, or any combination thereof. Implementations of the subject matter described in this specification also may be implemented as one or more computer programs, that is one or more modules of computer program instructions, encoded on a computer storage media for execution by, or to control the operation of, data processing apparatus.

[0156] If implemented in software, the functions may be stored on or transmitted over as one or more instructions or code on a computer-readable medium. The processes of a method or algorithm disclosed herein may be implemented in a processor-executable software module which may reside on a computer-readable medium. Computer-readable media includes both computer storage media and communication media including any medium that may be enabled totransfer a computer program from one place to another. A storage media may be any available media that may be accessed by a computer. By way of example, and not limitation, such computer- readable media can include random-access memory (RAM), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM), CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that may be used to store desired program code in the form of instructions or data structures and that may be accessed by a computer. Also, any connection may be properly termed a computer-readable medium. Disk and disc, as used herein, includes compact disc (CD), laser disc, optical disc, digital versatile disc (DVD), floppy disk, hard disk, solid state disk, and Blu-ray disc where disks usually reproduce data magnetically, while discs reproduce data optically with lasers. Combinations of the above should also be included within the scope of computer-readable media. Additionally, the operations of a method or algorithm may reside as one or any combination or set of codes and instructions on a machine readable medium and computer-readable medium, which may be incorporated into a computer program product.

[0157] Various modifications to the implementations described in this disclosure may be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to various other implementations without departing from the spirit or scope of this disclosure. Thus, the claims are not intended to be limited to the implementations shown herein, but are to be accorded the widest scope consistent with this disclosure, the principles and the novel features disclosed herein.

[0158] Additionally, a person having ordinary skill in the art will readily appreciate, terms such as “left,” “right,” “above,” “below,” “upper,” and “lower” are sometimes used for ease of describing the figures, and indicate relative positions corresponding to the orientation of the figure on a properly oriented page, and may not reflect the proper orientation of any device as implemented.

[0159] Certain features that are described in this specification in the context of separate implementations also may be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation also may be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and eveninitially claimed as such, one or more features from a claimed combination may in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.

[0160] Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. Further, the drawings may schematically depict one more example processes in the form of a flow diagram. However, other operations that are not depicted may be incorporated in the example processes that are schematically illustrated. For example, one or more additional operations may be performed before, after, simultaneously, or between any of the illustrated operations. In certain circumstances, multitasking and parallel processing may be advantageous. Moreover, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described program components and systems may generally be integrated together in a single software product or packaged into multiple software products. Additionally, various other implementations are within the scope of the following claims. In some cases, the actions recited in the claims may be performed in a different order and still achieve desirable results.

[0161] As used herein, including in the claims, various terminology is for the purpose of describing particular implementations only and is not intended to be limiting of implementations. For example, as used herein, an ordinal term (e.g., “first,” “second,” “third,” etc.) used to modify an element, such as a structure, a component, an operation, etc., does not by itself indicate any priority or order of the element with respect to another element, but rather merely distinguishes the element from another element having a same name (but for use of the ordinal term).

[0162] The term “coupled” is defined as connected, although not necessarily directly, and not necessarily mechanically; two items that are “coupled” may be unitary with each other.

[0163] The term “or,” when used in a list of two or more items, means that any one of the listed items may be employed by itself, or any combination of two or more of the listed itemsmay be employed. For example, if a composition is described as containing components A, B, or C, the composition may contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination. Also, as used herein, including in the claims, “or” as used in a list of items prefaced by “at least one of’ indicates a disjunctive list such that, for example, a list of “at least one of A, B, or C” means A or B or C or AB or AC or BC or ABC (that is A and B and C) or any of these in any combination thereof.

[0164] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive “or.”

[0165] The term “substantially” is defined as largely but not necessarily wholly what is specified - and includes what is specified; e.g., substantially 90 degrees includes 90 degrees and substantially parallel includes parallel - as understood by a person of ordinary skill in the art. In any disclosed aspect, the term “substantially” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1, 1, 5, and 10 percent; and the term “approximately” may be substituted with “within 10 percent of’ what is specified.

[0166] The terms “a” and “an” are defined as one or more unless this disclosure explicitly requires otherwise.

[0167] The terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), and “include” (and any form of include, such as “includes” and “including”) are open-ended linking verbs. As a result, an apparatus or system that “comprises,” “has,” or “includes” one or more elements possesses those one or more elements, but is not limited to possessing only those elements. Likewise, a method that “comprises,” “has,” or “includes,” one or more steps possesses those one or more steps, but is not limited to possessing only those one or more steps.

[0168] Although the aspects of the present disclosure and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit of the disclosure as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular implementations of the process, machine, manufacture, composition of matter, means, methodsand processes described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure, processes, machines, manufacture, compositions of matter, means, methods, or operations, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding aspects described herein may be utilized according to the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or operations.

Claims

CLAIMSWhat is claimed is:

1. A system for performing cell engineering procedures, the system comprising: a needle chip handler; a gantry module; an alignment module; a microscope module; and a fluidics module.

2. The system of claim 1, wherein the needle chip handler comprises: at least one needle chip holder, comprising a needle chip; and a receptacle configured to house the at least one needle chip holder.

3. The system of any one of claims 1 and 2, further comprising a gripper coupled to the gantry module and configured to grasp a needle chip holder.

4. The system of claim 3, wherein the gripper is configured to retrieve needle chip holders from the receptacle.

5. The system of any one of claims 2 to 4, wherein the organizational device is configured to store a plurality of needle chip holders.

6. The system of any one of claims 1 to 5, wherein the gantry module comprises a plurality of linear stages.

7. The system of claim 6, wherein the gantry module further comprises at least one rotary compliance stage coupled between a first linear stage of the plurality of linear stages and a second linear stage of the plurality of linear stages.

8. The system of any of claims 1 to 7, wherein the alignment module comprises:a platform, a plurality of positional linear stages coupled to the platform.

9. The system of claim 8 wherein the platform is configured to accommodate a cell trapping chip.

10. The system of any of claims 8 or 9, wherein the plurality of positional linear stages is configured to modify an angle of the platform by changing a position of at least a first positional linear stage of the plurality of positional linear stages .

11. The system of any of claims 1 to 10, wherein the microscope module comprises:An XYZ linear stage, and a high magnification microscope coupled to the XYZ linear stage.

12. The system of claim 11, wherein the microscope module further comprises a low magnification microscope coupled to the XYZ linear stage.

13. The system of any of claims 11 to 12, wherein the microscope module further comprises a camera optically coupled to the high magnification microscope.

14. The system of any of claims 1 to 13, wherein the fluidics module comprises a cell trapping chip disposed in a fluidic cartridge; and at least one syringe.

15. The system of any of claims 1 to 15, further comprising an enclosure.

16. The system of claim 15, wherein the enclosure comprises: an air filtration system; and a fluid handling system in fluid communication with the fluidics module.

17. The system of claim 16, wherein the fluid handling system further comprises at least one of the following: a reagent container, a liquid waste container, a fluidic pump, or a valve.

18. A method for performing cell engineering procedures comprising: providing a needle chip, and providing a cell trapping chip; functionalizing the needle chip; functionalizing the cell trapping chip; disposing the needle chip on an alignment module; aligning, by the alignment module, the needle chip with the cell trapping chip; trapping a cell at a target location of the cell trapping chip by applying a pressure differential to the cell trapping chip; impacting the cell with a needle on the needle chip by moving the needle chip toward the cell trapping chip; removing the needle chip from contacting the cell trapping chip; and retrieving the cell from the cell trapping chip.

19. The method of claim 18, wherein the needle chip comprises a plurality of needles; wherein the cell trapping chip comprises a plurality of target locations corresponding to the plurality of needles.

20. The method of any one of claims 18 and 19 wherein functionalizing the needle chip includes at least one of the following: applying a surface chemistry to the needle chip, applying a cargo to the needle chip, treating the needle chip with a plasma, exposing the needle chip to ultraviolet light, or flowing a gas over the needle chip.

21. The method of any one of claims 18 to 20, wherein functionalizing the cell trapping chip includes at least one of the following: treating the cell trapping chip with a plasma, primingthe cell trapping chip, or installing a buffer, a reagent, a cell sample, or a cell culture onto the cell trapping chip.

22. The method of any one of claims 18 to 21, wherein aligning the needle chip with the cell trapping chip further comprises: moving the needle chip to a first position at which the location of the needle chip can be determined; and / or determining the location of the needle chip; and / or determining the location of the cell trapping chip; and / or determining an alignment error between the needle chip and the cell trapping chip; and / or moving the needle chip into rotational alignment with the cell trapping chip; and / or modifying, by the alignment module, the planar location of the cell trapping chip to a planar location substantially parallel to the planar location of the needle chip.

23. The method of claim 22, wherein determining the location of the needle chip includes determining the plane in which the needle chip is positioned; and wherein determining the location of the cell trapping chip includes determining the plane in which the cell trapping chip is positioned.

24. The method of claim 23, wherein determining the plane in which the needle chip is positioned comprises identifying coordinates of key features of the needle chip in three dimensional space; and wherein determining the plane in which the cell trapping chip is positioned comprises identifying coordinates of key features of the cell trapping chip in three dimensional space.

25. The method of any of claims 22 to 24 wherein the location of the needle chip and the location of the cell trapping chip are respectively determined by a microscope module.

26. The method of any of claims 22 to 25, wherein the location of the needle chip and the location of the cell trapping chip are respectively determined using a predictive focus algorithm.

27. The method of any of claims 22 to 26, wherein determining the alignment error between the needle chip and the cell trapping chip comprises comparing the planar positions of the needle chip and the cell trapping chip.

28. The method of any one of claims 18 to 27, wherein the alignment module comprises a platform and a plurality of positional linear stages coupled to the platform.

29. The method of claim 28, wherein aligning the needle chip with the cell trapping chip further comprises adjusting an angle of the platform by adjusting the positions of the plurality of positional linear stages.

30. The method of any one of claims 28 to 29, wherein the cell trapping chip is provided in a fluidic cartridge, and wherein disposing the cell trapping chip on the alignment module comprises securing the fluidic cartridge to the platform.

31. The method of claim 30, wherein the fluidic cartridge comprises an opening for exposing the cell trapping chip.

32. The method of any one of claims 18 to 31 wherein aligning the needle chip with the cell trapping chip comprises bringing the needle chip into a parallel plane as the cell trapping chip to within 1 micrometer of alignment error.

33. The method of any one of claims 18 to 32, further comprising a needle chip handler, wherein the needle chip handler comprises a needle chip holder and / or a gantry, and / or a gripper; and, optionally, a receptacle configured to house the needle chip holder.

34. The method of claim 33, wherein the gripper is configured to grasp the needle chip holder.

35. The method of any one of claims 33 to 34, wherein the receptacle is configured to house a plurality of needle chip holders.

Images