Apparatus and method for harvesting cells

Abstract

A device for lifting and collecting cells and methods of using the same are disclosed. The device includes a cell collection head; an arm connected to the cell collection head and extending proximally from the cell collection head to form a device body; and at least two flow channels, wherein a first inlet of a first of the at least two flow channels is disposed proximally of the arm and a first outlet of the first of the at least two flow channels is disposed distally of the cell collection head, and an outlet of a second of the at least two flow channels is disposed proximally of the arm and an inlet of the second of the at least two flow channels is disposed distally of the cell collection head.

Description

[0001] Cross-referencing This application claims priority to Israeli Patent Application No. 310822, filed on February 13, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention generally relates to cell harvesting apparatus and methods. More specifically, this invention relates to apparatus and methods for separating and collecting cells from the surface of cell cultures. Background Technology

[0003] Many laboratory procedures require culturing tissue for subsequent analysis and diagnostic testing. Tissues are cultured in tissue culture containers, such as flasks or petri dishes. Tissue culture containers are used by depositing a controlled amount of liquid growth medium into the container. A small sample of the tissue to be cultured is then placed into the container. The container is closed by placing the lid on the tubular neck or by placing the top wall on top of the opening defined by the side walls. The container is then stored in an environment conducive to tissue growth. The growing tissue in the container must be removed and analyzed periodically. In some cell types, the growing tissue may adhere to the bottom wall of the container, so it must be scraped off for analysis.

[0004] Cell scrapers are used to remove tissue from the bottom of tissue culture containers. A typical cell scraper consists of an elongated handle integrally molded from a rigid plastic material, which connects to a scraper blade integrally molded from plastic, comprising a flat scraper edge. The blade size will vary depending on the intended application, and particularly on the size of the tissue culture container.

[0005] Harvesting cell cultures from the inner surface of a flask using a cell scraper requires access through the typically narrow opening of the flask. Furthermore, cells must be removed from the surface in a manner that causes minimal damage to the cells. Therefore, this process is time-consuming and usually requires the expertise of a skilled technician.

[0006] Cell dissociation is typically achieved using proteolytic enzymes such as trypsin. However, incubating cells with excessively high trypsin concentrations for too long can damage the cell membrane and potentially kill the cells. Furthermore, the use of trypsin and other proteolytic enzymes can leave residues in the harvested cells, and therefore may be prohibited in some cases. Therefore, there is a need for an apparatus and method for harvesting cells from the surface of cell cultures without using proteolytic enzymes or damaging the culture.

[0007] The above examples of related technologies and their related limitations are intended to be illustrative rather than exclusive. Other limitations of the related technologies will become apparent to those skilled in the art after reading the specification and studying the accompanying drawings. Summary of the Invention

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods that are intended to be exemplary and illustrative, and not limiting.

[0009] One embodiment provides an apparatus comprising: an apparatus body including: a cell collection head; and at least two flow channels, wherein a first inlet of a first of the at least two flow channels is connectable to a fluid source, and a first outlet of the first of the at least two flow channels is disposed distal to the cell collection head, wherein an outlet of a second of the at least two flow channels is disposed proximal to a fluid collection reservoir, and an inlet of the second of the at least two flow channels is disposed distal to the cell collection head, and wherein the fluid source is at least one of an internal fluid reservoir and an external fluid reservoir.

[0010] According to some embodiments, the main body of the device is shaped like an arm connected to the cell collection head, extending proximally from the cell collection head to form a single unit.

[0011] According to some implementations, at least two flow channels can be arranged inside the main body of the device.

[0012] According to some embodiments, the apparatus may further include at least one pump fluidly connected to at least one of the first inlet of the first flow channel and the second outlet of the second flow channel.

[0013] The device may also include a control system to control the movement of the cell collection head.

[0014] According to some embodiments, at least one pump has at least a first operating state and at least a second operating state, wherein in the first operating state, the at least one pump sprays fluid from a fluid source onto the surface of a cell culture via a collection head, and in the at least second operating state, the at least one pump draws fluid from the surface of a cell culture into a fluid collection reservoir via a cell collection head.

[0015] The device may also include a control system to control the operating status of at least one pump.

[0016] According to some embodiments, the device may also include a filter configured to separate the aspirated fluid from the cells in the aspirated fluid.

[0017] Some aspects of the present invention include a method for harvesting cells, the method comprising spraying fluid from a first fluid source onto a cell culture surface via a first fluid channel to separate cells from the cell culture surface; and aspirating the sprayed fluid and at least a portion of the separated cells via a second fluid channel to harvest cells from the cell culture surface.

[0018] The method according to some implementations may also include controlling the operation of one or more pumps according to at least one of the following: a predetermined scheme and a standard for real-time measurement.

[0019] According to some implementations, the predetermined scheme includes one or more of a predetermined flow rate, a predetermined duration, and a fluid type.

[0020] According to some implementation methods, the criteria for real-time measurement are selected from the volume of the ejected fluid, the volume of the aspirated fluid, the number of collected cells, and the mass of collected cells.

[0021] In some implementations, the predetermined flow rate is between 0.3 ml / sec and 50 ml / sec.

[0022] In some embodiments, the fluid is a liquid, and the predetermined flow rate is selected from 0.3 ml / sec, 0.6 ml / sec, and 1 ml / sec. In other embodiments, the fluid is air, and the predetermined flow rate is selected from 2.51 ml / sec, 8 ml / sec, and 11.41 ml / sec.

[0023] According to some embodiments, the method may also include scraping the surface of the cell culture with the cell collection head of the cell harvesting device to further separate the cells from the surface of the cell culture.

[0024] In addition to the exemplary aspects and implementations described above, other aspects and implementations will become apparent from the accompanying drawings and by studying the following detailed description. Attached Figure Description

[0025] Exemplary embodiments are illustrated in the figures. The dimensions of the components and features shown in the figures are generally chosen for convenience and clarity and are not necessarily shown to scale. The figures are as follows.

[0026] Figure 1 This is a schematic diagram of a device for harvesting cells according to an embodiment; and Figure 2A and Figure 2B This is a flowchart of a method for harvesting cells according to some implementation methods. Detailed Implementation

[0027] This document discloses an apparatus and method for harvesting cells. The apparatus and method described herein can be used to lift, retrieve, and, in some embodiments, collect cells and / or fluids (e.g., liquids) from a cell culture surface. Advantageously, the apparatus and method can facilitate cell lifting by applying appropriate shear force to maintain acceptable cell viability. Acceptable cell viability can be considered as at least 70%. According to some embodiments, the apparatus and method can facilitate cell lifting without contact between the apparatus and the cell culture surface by applying a fluid (e.g., air, water, etc.) at a predetermined flow rate to and collecting cells / fluid therefrom on the cell culture surface.

[0028] As used in this article, the cell is defined as the basic structural and functional unit of all life forms.

[0029] The apparatus and method disclosed herein offer significant advantages over existing cell harvesting technologies. By utilizing controlled fluid dynamics and mechanical action, this method enables efficient cell separation and collection without relying on chemical reagents such as trypsin or other proteolytic enzymes. Omitting the use of such chemicals eliminates the potential risks associated with residual enzyme activity, which could damage cell membranes, alter cell properties, or harm the application tissue. This chemical-free method preserves cell viability and the natural characteristics of the harvested cells, which is particularly important for sensitive cell types or downstream applications requiring unchanged cell states. Furthermore, the absence of chemical reagents simplifies regulatory compliance and reduces costs associated with specialized reagents. Compared to traditional enzymatic methods, the precise control of fluid flow rate and mechanical force enables gentler cell harvesting, potentially increasing yield and reducing cell stress. This method is particularly beneficial for applications in regenerative medicine, cell therapy, and other fields where maintaining cell integrity and function is crucial.

[0030] As used herein, the term "cell culture surface" refers to any type of surface on which cells are at least partially attached or adhered (i.e., cells bind / attach to the surface) while maintaining the viability of adherent and / or growing cells. Suitable cell culture surfaces allow for the proliferation and growth of adherent cells. Non-limiting examples of suitable cell culture surfaces include plastics or coated plastics (e.g., coated with collagen), methylcellulose, organic and inorganic polymers, and nanoparticle surfaces such as glass slides, cell culture plates, multi-well plates (e.g., 2-well, 6-well, 24-well, 48-well, 96-well, etc.), culture dishes, and flasks.

[0031] The term "adherent cells" refers to cells, such as prokaryotic or eukaryotic cells, that are attached to, fixed to, or in contact with the surface of a cell culture medium. This type of cell can withstand or survive washing and culture medium exchange processes after culture, which are conditions for many cell-based cultures and / or assays.

[0032] The device of the present invention can be advantageously used in automated systems, such as components (integral or detachable) within cell culture systems / bioreactors.

[0033] Now for reference Figure 1 The illustration shows a device 100 according to one embodiment for lifting and collecting cells and / or fluids from the surface of a cell culture. Cells / fluids can be collected from flat surfaces, such as tissue culture flasks, chambers, or trays, or from the surface of three-dimensional particles, such as beads and scaffolds. In a non-limiting example, the scaffold can be printed, chemically designed, etc.

[0034] For ease of description of device 100, Figure 1 The diagram shows three orthogonal axes. The axis labeled "Longitudinal Axis" refers to the central axis extending along the length of the arm 102 of device 100 from the proximal end 104 to the distal end 106 of the cell collection head 108. The axis labeled "Vertical Axis" extends along the width of the cell collection head 108 from the first side 110 to the second side 111. The axis labeled "Transverse Axis" represents the "thickness" (also called "depth") of device 100 and is perpendicular to the vertical and longitudinal axes.

[0035] The device 100 may include at least two flow channels 112 and a cell collection head 108 (e.g., a blade). In some embodiments, the device 100 may further include an arm 102 connected to and extending proximally from the cell collection head 108 to form a device body 101, such that movement of the arm 102 translates into corresponding movement of the cell collection head 108. In some embodiments, the arm 102 is rigidly connected to and extends proximally from the cell collection head 108. In some embodiments, the connection is detachable. In some embodiments, the arm 102 is equipped with a connector (not shown) to allow flexibility of the arm 102, for example, flexibility relative to the collection head 108. In some embodiments, the arm 102 is flexible, and the orientation of the distal portion of the arm 102 may be adjusted along longitudinal and / or vertical and / or transverse axes relative to the orientation of the proximal portion of the arm 102, thereby affecting the orientation of the distally positioned cell collection head 108 relative to the proximal portion of the arm 102.

[0036] In some embodiments, at least one of the cell collection head 108 and arm 102 accommodates at least a portion of at least two flow channels 112. In some embodiments, at least a portion of at least two flow channels 112 is disposed within the device body 101. In some embodiments, at least two flow channels 112 are disposed within the device body 101.

[0037] In some embodiments, at least two flow channels 112 may include a first flow channel 112a and a second flow channel 112b. Figure 1 In the non-limiting example shown, a first flow channel 112a is used to supply fluid to the cell culture, and a second flow channel 112b is used to collect fluid / cells from the cell culture. A first inlet 114 of the first flow channel 112a of the at least two flow channels 112 may be located proximally to the arm 102, and a first outlet 116 of the first flow channel 112 may be located distally to the cell collection head 108. A second outlet 118 of the second flow channel 112b of the at least two flow channels 112 may be located proximally to the arm 102, and a second inlet 120 of the second flow channel 112b may be located distally to the cell collection head 108.

[0038] In some embodiments, the dimensions (e.g., orifices) of the first outlet 116 and the second inlet 120 can be between 15G and 34G, such as 15G, 20G, 21G, 24G, 25G, 28G, 30G, 33G, 34G, and any values ​​or ranges therebetween. In some embodiments, at least the second inlet 120 can be at least 20% larger than the cell size, for example, at least 20% larger than 25 μm. In non-limiting examples, the dimensions (e.g., inner diameter) can be between 30 μm and 200 μm.

[0039] In some embodiments, the outlet 116 of the first flow channel 112a and / or the inlet 120 of the second flow channel 112b may be located on the distal collection surface 122 of the cell collection head 108. The distal collection surface 122 may be designed to elevate cells from the cell culture surface. In some embodiments, the distal collection surface 122 may be configured to scrape the culture surface. Thus, the cell collection head 108 may be a scraper configured to physically separate cells from the cell culture surface with or without the use of fluids such as air, saline, etc.

[0040] According to some embodiments, the head 108 may not need to actually contact the cell culture surface so that cells can be separated from the surface and collected simply by applying fluid at a predetermined flow rate, such as applying liquid (e.g., saline) at a flow rate of 0.3 ml / sec, 0.6 ml / sec, 1 ml / sec, 2 ml / sec, 4 ml / sec or even up to 10 ml / sec, or applying air at a flow rate of 2 ml / sec, 2.51 ml / sec, 8 ml / sec, 11.41 ml / sec, 13 ml / sec, 15 ml / sec, 41 ml / sec and up to 50 ml / sec, for a predetermined duration, such as 15 seconds, 30 seconds, 45 seconds, 60 seconds, 90 seconds, 120 seconds, etc.

[0041] In a first non-limiting example, the fluid is a liquid (e.g., an aqueous solution, such as saline solution (e.g., phosphate-buffered saline (PBS), serum-free culture medium, etc.)) with a predetermined flow rate between 0.3 ml / sec and 4 ml / sec, and any value or range therebetween. In a second non-limiting example, the fluid is air with a predetermined flow rate between 1 ml / sec and 50 ml / sec, and any value or range therebetween.

[0042] According to some embodiments, fluid may be applied from one or more first outlets 116 and collected from one or more second inlets 120. According to some embodiments, fluid may be applied from the center of the collection head 108 and / or from one or more sides of the collection head 108 (e.g., from outlets 116 disposed on the sides of the collection head 108). According to some embodiments, a controller may control from which outlet 116 fluid is applied and from which inlet 120 fluid and cells are collected. According to some embodiments, this may be done based on cell type, desired flow rate, etc.

[0043] In some embodiments, two or more first outlets 116 and two or more second inlets 120 may be arranged alternately. This configuration allows for more efficient and uniform fluid distribution and cell collection on the cell culture surface. By alternating the use of the first outlets 116 and the second inlets 120 for fluid injection and collection, the device can produce a more balanced and efficient harvesting process. The alternating arrangement helps minimize inaccessible areas or areas of uneven fluid flow, potentially improving the overall efficiency of cell separation and collection. Furthermore, this configuration allows for more precise control of the hydrodynamics on the cell culture surface, thereby optimizing shear forces and fluid exchange rates.

[0044] In some embodiments, the device 100 may further include one or more fluid reservoirs 124 for storing fluid collected via a first flow channel 112a and / or fluid dispensed via a second flow channel 112b. In some embodiments, the one or more fluid reservoirs 124 include a first fluid reservoir 124a for storing fluid (e.g., wash fluid and feed fluid) intended to be dispensed onto the surface of a cell culture via the first flow channel 112a; and a second fluid reservoir 124b for storing fluid collected via the second flow channel 112b. The one or more fluid reservoirs 124 are fluidly connected to at least one of the at least two flow channels 112. In some embodiments, the one or more fluid reservoirs 124 are fluidly connected to a proximal inlet 114 and / or a proximal outlet 118. Fluid may enter and / or exit the one or more fluid reservoirs 124 sequentially and / or simultaneously. In some embodiments, the fluid is selected from liquids, gases, and combinations thereof. In some embodiments, the fluid also includes particles. In some embodiments, the fluid also includes cells and / or components thereof.

[0045] In some embodiments, the device 100 may further include at least one pump 130 fluidly connected to at least one of a proximal inlet 114 and a proximal outlet 118 for pumping fluid into and / or out of the device 100, respectively. In a non-limiting example, fluid pumped from the first fluid reservoir 124a is distributed onto the cell culture surface via a distal outlet 116 of the first fluid channel 112a. In another non-limiting example, fluid pumped from the cell culture surface via a distal inlet 118 of the second fluid channel 112b is collected into the second fluid reservoir 124b. In some embodiments, the second reservoir 124b may be equipped with a filter (not shown) to separate the collected cells from the aspirated fluid, for example, to prevent cells from entering the second reservoir 124b. Those skilled in the art will understand that cells separated from the fluid by the filter can be returned to a new cell culture surface to grow new cell lines. It should be further understood that the fluid separated from the cells can be analyzed and / or reused for further recycling of cells harvested from the same or another cell culture surface.

[0046] In some embodiments, the device 100 may further include a control system 140. The control system may adjust the movement of the arm 102 to cause a corresponding movement of the cell collection head 108. In some embodiments, the control system may control a pump to regulate the pumping of fluid into and / or out of one or more fluid reservoirs 124 via one or more flow channels 112.

[0047] In some embodiments, at least one pump 130 may have at least a first operating state and at least a second operating state, wherein in the first operating state, at least one pump 130 sprays fluid from a fluid source (e.g., a first fluid reservoir 124a) into the cell culture through a collection head 108, and in at least the second operating state, at least one pump 130 draws fluid from the cell culture into a fluid collection reservoir (e.g., a second fluid reservoir 124b) through a cell collection head. In some embodiments, the pump may operate in one or more operating cycles. Each operating cycle may include a first operating state and a second operating state. In some embodiments, the controller 140 may control the number of operating cycles. For example, at each harvest period, pump 130 may perform 2, 3, 4, 5, 6 or more operating cycles.

[0048] In some embodiments, controller 140 can control the operating state of at least one pump 130. For example, controller 140 can control the duration of operation of at least a second operating state. In some embodiments, the duration can be between 5 and 30 seconds, and any value or range therebetween, such as 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds or longer, as described in the Embodiments section below.

[0049] According to some embodiments, the control system 140 may include a controller or processor (not shown) that actively communicates with one or more pumps 130 and can control the operation of one or more pumps 130 according to a predetermined scheme (e.g., during a predetermined time period and / or at a predetermined flow rate), or control the operation of one or more pumps according to real-time measured criteria, such as the volume of fluid discharged, the volume of fluid collected, the number of cells collected, the mass of cells collected, etc.

[0050] Now for reference Figure 2A , Figure 2A This is the use of the embodiments of the present invention. Figure 1 The flowchart shows a method for lifting and collecting cells from the surface of a cell culture using a device (also known as cell harvesting).

[0051] In step 2010, a fluid, such as air, saline, or any other suitable liquid or gas, can be sprayed from a fluid source onto the surface of the cell culture from which cells are to be harvested. The fluid source can be a fluid reservoir inside the cell harvesting device, or it can be an external fluid source or reservoir. According to some embodiments, the fluid source can be connected to or can be connected to a fluid channel within the cell harvesting device (e.g., ...). Figure 1 The cell collection head of the device 100 in the middle ( Figure 1 There is an exit at point 108.

[0052] According to some implementations, spraying fluid from a fluid source onto the surface of a cell culture may include controlling one or more pumps via a controller. Figure 1 (130 in the text). According to some implementations, the control of one or more pumps can be based on at least one of a predetermined scheme and a real-time measurement standard.

[0053] According to some embodiments, the predetermined flow rate may include one or more of a predetermined flow rate, a predetermined duration, and a fluid type. According to some embodiments, the predetermined flow rate is between 0.3 ml / sec and 50 ml / sec. According to other embodiments, the fluid is a liquid, and the predetermined flow rate is between 0.3 ml / sec and 10 ml / sec. For example, it is selected from 0.3 ml / sec, 0.6 ml / sec, and 1 ml / sec. In still other embodiments, the fluid is air, and the predetermined flow rate is between 2 ml / sec and 50 ml / sec, for example, it is selected from 2 ml / sec, 2.51 ml / sec, 8 ml / sec, 11.41 ml / sec, and 41 ml / sec.

[0054] According to some implementation methods, the criteria for real-time measurement can be selected from: the volume of the ejected fluid, the volume of the aspirated fluid, the number of collected cells, and the mass of collected cells.

[0055] According to some embodiments, the scheme may also include scraping the surface of the cell culture with the cell collection head of the cell harvesting device to further separate the cells from the surface of the cell culture.

[0056] In step 2020, the ejected fluid can be drawn back, for example through the fluid inlet at the cell collection head and via the fluid channel. The fluid drawn back, including the fluid ejected and / or the cells separated from the cell culture surface by the cell collection head scraping the surface, can be collected into a reservoir, such as reservoir 124b.

[0057] According to some implementations, aspirating fluid from the surface of a cell culture may include controlling one or more pumps via a controller. Figure 1 (130 in the text). According to some embodiments, the control of one or more pumps can be based on at least one of a predetermined scheme and a real-time measured standard, as described above for jet fluids.

[0058] For example, a predetermined scheme for aspirating fluid from the surface of a cell culture may include starting aspiration at a predetermined time after the fluid jet has begun. According to some embodiments, aspiration may be initiated after a predetermined volume of fluid has been jetted, or it may be started and operated in parallel with the fluid jet. Aspiration of fluid and cells may be stopped when stopping conditions are met, such as when a predetermined volume of fluid and cells has been aspirated, the fluid reservoir (124b) is full, or a predetermined mass of cells has been collected.

[0059] Now for reference Figure 2B It illustrates a method according to some implementations. For example... Figure 2B As shown, the distal portion of the cell collection head 108, disposed distally on the device body 101, is placed on the surface of the cell culture, allowing the distal collection surface 122 to contact the cell culture surface (250), or to approach the surface 122 without actual contact. Fluid is introduced into the first inlet 114 (252) of the first fluid channel 112a disposed together with the proximal arm 102 of the device body 101. In some embodiments, fluid is introduced from the first fluid reservoir 124a. Fluid is ejected from the first fluid channel 112a via the first outlet 116 disposed on the distal cell collection head 108 of the device body 101 (254). In some embodiments, the ejected fluid may induce shear forces suitable for maintaining cell viability and loosening cell adhesion. In some embodiments, the ejected fluid may loosen cells adhering to the surface of the cell culture. The proximal arm 102, rigidly connected to the cell collection head 108, may be moved in a direction parallel to the plane of the cell culture surface (256). The connection between arm 102 and cell collection head 108 is used to convert movement of arm 102 into corresponding movement of cell collection head on or near the cell culture surface to elevate cells from the cell culture surface (258). In some embodiments, the movement of cell collection head 108 is adapted to elevate cells while maintaining cell viability. In some embodiments, this movement facilitates elevation of cells from the cell culture surface via distal collection surface 122. In some embodiments, the elevated cells are assimilated in fluid. In some embodiments, this movement spans a transverse axis, a longitudinal axis, and / or rotational motion. Fluid is aspirated into a second inlet 120 (260) of a second fluid channel 112b disposed together with the cell collection head 108 disposed distally to the device body 101. The aspirated fluid is discharged from the second fluid channel 112b via a second outlet 118 disposed proximal to arm 102 (262). In some embodiments, the aspirated fluid is collected in a second reservoir 124b (262).

[0060] In some embodiments, steps 254 and 260 may be performed sequentially and / or at least partially simultaneously. In some embodiments, simultaneous application of steps 254 and 260 may result in circulatory movement of fluid, which may promote cell release from the cell culture surface. In some embodiments, each of steps 254, 258, and 260 may be performed independently. In some embodiments, steps 254, 258, and 260 may be performed simultaneously or in any order. In some embodiments, steps 258 and 260 may be performed sequentially and / or at least partially simultaneously. In some embodiments, each of steps 254 and 260 may be performed once or more in any order prior to step 258.

[0061] In some implementations, the control system can adjust the performance of each step. In some implementations, the control unit can adjust the injection and aspiration of fluids and the movement of the cell collection head to maintain cell viability.

[0062] In some embodiments, the control unit can operate the first and second flow channels to apply an optimal shear force suitable for maintaining cell viability. Those skilled in the art will understand that the optimal shear force depends on the cell type. Furthermore, the optimal shear force applied may depend on the type of biological function desired (e.g., differentiation, migration, etc.).

[0063] The present invention can be a system, method, and / or computer program product. A computer program product may include a computer-readable storage medium (or media) having computer-readable program instructions thereon for causing a processor to execute aspects of the present invention.

[0064] Computer-readable storage media can be tangible means capable of retaining and storing instructions for use by an instruction execution device. Computer-readable storage media can be, for example, but not limited to, electronic storage devices, magnetic storage devices, optical storage devices, electromagnetic storage devices, semiconductor storage devices, or any suitable combination of the foregoing. A non-exhaustive list of more specific examples of computer-readable storage media includes the following: portable computer disks, hard disks, random access memory (RAM), read-only memory (ROM), erasable programmable read-only memory (EPROM or flash memory), static random access memory (SRAM), portable optical disc read-only memory (CD-ROM), digital multifunction disc (DVD), memory sticks, floppy disks, mechanical encoding devices such as punched cards or raised structures in recesses on which instructions are recorded, and any suitable combination of the foregoing. The computer-readable storage media used herein should not be construed as transient signals, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through waveguides or other transmission media (e.g., light pulses through fiber optic cables), or electrical signals transmitted through wires.

[0065] The computer-readable program instructions described herein can be downloaded from a computer-readable storage medium to various computing / processing devices, or downloaded via a network to an external computer or external storage device, such as the Internet, a local area network (LAN), a wide area network (WAN), and / or a wireless network. This network may include copper cables, optical fibers, wireless transmission, routers, firewalls, switches, gateway computers, and / or edge servers. A network adapter card or network interface in each computing / processing device receives the computer-readable program instructions from the network and forwards these instructions to a computer-readable storage medium within the respective computing / processing device.

[0066] Computer-readable program instructions used to perform the operations of this invention may be assembly instructions, instruction set architecture (ISA) instructions, machine instructions, machine-dependent instructions, microcode, firmware instructions, state setting data, or source code or object code written in any combination of one or more programming languages, including object-oriented programming languages ​​such as Java, Smalltalk, C++, etc., and traditional procedural programming languages ​​such as the "C" programming language or similar programming languages. The computer-readable program instructions may execute entirely on the user's computer, partially on the user's computer, as a standalone software package, partially on the user's computer, partially on a remote computer, or entirely on a remote computer or server. In the latter case, the remote computer may be connected to the user's computer via any type of network, including a local area network (LAN) or a wide area network (WAN), or may be connected to an external computer (e.g., via the Internet through an Internet service provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGAs), or programmable logic arrays (PLAs) may be personalized using state information from the computer-readable program instructions to perform aspects of the invention.

[0067] This document describes aspects of the invention with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the invention. It should be understood that each block in the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer-readable program instructions.

[0068] These computer-readable program instructions can be provided to the processor of a general-purpose computer, special-purpose computer, or other programmable data processing device to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing device, create a flowchart and / or block diagram for implementation. Figure 1The computer-readable program instructions may also be stored in a computer-readable storage medium that can instruct a computer, a programmable data processing device, and / or other means to operate in a particular manner, such that the computer-readable storage medium in which the instructions are stored includes an article of art comprising implementation flowcharts and / or blocks. Figure 1 Instructions for various aspects of the functions / actions specified in one or more boxes.

[0069] Computer-readable program instructions may also be loaded onto a computer, other programmable data processing equipment, or other apparatus to perform a series of operational steps on the computer, other programmable equipment, or other apparatus, thereby producing a computer-implemented process, such that the instructions, which execute in the computer, other programmable equipment, or other apparatus, implement a flowchart and / or block diagram. Figure 1 The function / action specified in one or more boxes.

[0070] The flowcharts and block diagrams in the figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various embodiments of the present invention. In this regard, each block in a flowchart or block diagram may represent a module, segment, or portion of instructions comprising one or more executable instructions for implementing a specified logical function. In some alternative embodiments, the functions described in the blocks may appear in the order shown in the figures. For example, depending on the functions involved, two blocks shown consecutively may actually be executed substantially simultaneously, or these blocks may sometimes be executed in reverse order. It will also be noted that each block in the block diagrams and / or flowcharts, and combinations of blocks in the block diagrams and / or flowcharts, can be implemented by a dedicated hardware-based system that performs a specified function or action or executes a combination of dedicated hardware and computer instructions.

[0071] Example Several cell harvesting experiments were conducted using the apparatus (such as apparatus 100) and the methods disclosed above. In all cell harvesting experiments, the control included harvesting by scraping only, i.e., mechanically scraping the surface of the cultured cells. Two parameters were tested: yield (%) and viable cells (%) compared to the control.

[0072] In all experiments, the cell culture surface included microglial mesenchymal stem cell line, DMEM medium, Pen-strep (wt. 1%), and fetal bovine serum (10 wt%). Harvest assays were performed using two types of containers (6-well plates and T75 flasks), as is known in the art. Several needle gauges ranging from 21 G to 30 G were used. Each test procedure was performed by applying repetitive short pulses, with each pulse lasting 5, 10, 15, and 20 seconds, for a total of 5, 10, 30, 45, 60, and 120 seconds. For liquids, the short pulses were 5, 10, 15, and 20 seconds, for a total of 15, 30, 45, 60, 120, and 240 seconds.

[0073] Cells cultured in 6-well plates were collected into 15 ml conical tubes, and cells cultured in T75 flasks were collected into 50 ml conical tubes.

[0074] Example 1 - Liquid-Assisted Harvesting Three settings were tested: A) control – scraping for different durations, B) injecting PBS liquid and aspirating the sprayed liquid, and C) combining scraping with liquid injection and aspiration.

[0075] Table 1 summarizes the results of Example 1: Where N represents the number of tests performed.

[0076] Three yield categories were defined compared to the control-based approach: 1) below 75% was defined as insufficient yield, 2) between 75% and 125% was defined as comparable to the control, and 3) above 125% was defined as superior to the control.

[0077] Regarding vitality, a value of 80% is defined as the critical value, where anything below this critical value is considered insufficient, while anything above 80% is considered sufficient.

[0078] As shown, optimal yield results were obtained by simultaneously performing liquid injection and scraping in both G21 and G25 needles, and by performing liquid injection only in the 25G needle. In some experiments, the yield was higher than the control yield, exceeding the control by 125%. Regarding the viability of the collected cells, all assay methods provided adequate results (e.g., between 80% and 100%).

[0079] When testing the duration of each harvesting action, at least 30 seconds of liquid injection and aspiration were required when harvesting using only liquid. In 6-well plates and T75 flasks, yields increased when injection and aspiration lasted 45 and 50 seconds, respectively. When combined with mechanical scraping, the optimal duration was found to be between 15 and 45 seconds.

[0080] Example 2 - Air-assisted harvesting Three scenarios were tested: A) control – scraping for different durations, B) air injection and cell aspiration, and C) a combination of scraping and air injection with aspiration.

[0081] Table 2 summarizes the results of Example 2. Where N represents the number of tests performed.

[0082] Three yield categories were defined compared to the control: 1) below 75% was defined as insufficient yield, 2) between 75% and 125% was defined as substantially the same as the control, and 3) above 125% was defined as superior to the control.

[0083] Regarding viability, a value of 80% is defined as the critical value; values ​​below this critical value are considered insufficient, while values ​​above 80% are considered sufficient. When using air, optimal yield results were obtained when harvesting cells from 6 wells using only air and a 25G syringe. A combination of air and scraping using 25G and 30G needles also yielded acceptable yields and viability.

[0084] When testing the duration of air injection, the optimal duration was found to be between 15 and 45 seconds. Applying air for 120 seconds or longer using a 25G needle resulted in reduced yield and viability.

[0085] The various embodiments of the present invention are described for illustrative purposes only and are not intended to be exhaustive or limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles of the embodiments, their practical application, or technical improvements to technologies found in the market, or to enable those skilled in the art to understand the embodiments disclosed herein.

Claims

1. An apparatus for harvesting cells from the surface of a cell culture, comprising: The main body of the device includes: Cell collection head; and At least two flow channels, The first inlet of the first flow channel in the at least two flow channels is connectable to a fluid source, and the first outlet of the first flow channel is located distal to the cell collection head. The outlet of the second flow channel of the at least two flow channels is located proximal to the fluid collection reservoir, and the second inlet of the second channel is located distal to the cell collection head. The fluid source is at least one of an internal fluid reservoir and an external fluid reservoir.

2. The device of claim 1, wherein the body is shaped as an arm connected to the cell collection head, extending proximally from the cell collection head to form a single unit.

3. The device according to any one of claims 1 or 2, wherein the at least two flow channels are disposed inside the device body.

4. The apparatus according to any one of the preceding claims, comprising two or more first outlets and two or more second inlets arranged alternately.

5. The apparatus according to any one of the preceding claims, wherein the size of the second inlet is between 15G and 34G.

6. The apparatus according to any one of the preceding claims, wherein the fluid is selected from air and aqueous solution.

7. The apparatus according to any one of the preceding claims further comprises at least one pump fluidly connected to at least one of the first inlet of the first flow channel and the second outlet of the second flow channel.

8. The apparatus according to any one of claims 2 to 7, further comprising a control system for controlling the movement of the cell collection head.

9. The apparatus of claim 7, wherein the at least one pump has at least a first operating state and at least a second operating state, wherein in the first operating state, the at least one pump sprays fluid from the fluid source onto the surface of the cell culture via the collection head, and in the at least second operating state, the at least one pump draws fluid from the surface of the cell culture into the fluid collection reservoir via the cell collection head.

10. The apparatus of claim 9 further includes a control system for controlling the operating state of the at least one pump.

11. The apparatus of claim 10, wherein the control system controls the duration of operation and the number of operation cycles of the at least second operating state.

12. The apparatus of claim 11, wherein the duration is between 5 and 30 seconds.

13. The apparatus according to any one of the preceding claims, wherein the fluid is a liquid and the predetermined flow rate is selected from 0.3 ml / sec, 0.6 ml / sec and 1 ml / sec.

14. The apparatus according to any one of claims 1 to 12, wherein the fluid is air, and the predetermined flow rate is selected from 2.51 ml / sec, 8 ml / sec, and 11.41 ml / sec.

15. The apparatus according to any one of the preceding claims further includes a filter configured to separate the aspirated fluid from the cells in the aspirated fluid.

16. A method for harvesting cells, comprising: Fluid is sprayed from a first fluid source onto the surface of a cell culture via a first fluid channel to separate cells from the surface of the cell culture. and Cells are harvested from the surface of the cell culture by aspirating the ejected fluid and at least a portion of the separated cells via a second fluid channel.

17. The method of claim 16, wherein injecting the fluid comprises controlling the operation of one or more pumps according to at least one of the following: a predetermined scheme and one or more standards measured in real time.

18. The method of claim 17, wherein the predetermined scheme includes one or more of a predetermined flow rate, a predetermined duration, and a fluid type.

19. The method of claim 17, wherein one or more criteria for real-time measurement are selected from the jet volume of fluid, the aspirated volume of fluid, the collected cell count, and the collected cell mass.

20. The method of claim 18, wherein the predetermined flow rate is between 0.3 ml / sec and 50 ml / sec.

21. The method of claim 18, wherein the fluid is a liquid, and the predetermined flow rate is selected from 0.3 ml / sec, 0.6 ml / sec, and 1 ml / sec.

22. The method of claim 18, wherein the fluid is air, and the predetermined flow rate is selected from 2.51 ml / sec, 8 ml / sec, and 11.41 ml / sec.

23. The method according to any one of claims 16-22, further comprising scraping the surface of the cell culture with the cell collection head of the cell harvesting device to further separate the cells from the surface of the cell culture.

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