Density gradient liquid formation with sample particles

Abstract

A system for dispensing a density gradient liquid in a container is described. The system pumps a volume of sample particles and a density modifier into a mixing chamber in fluid communication with the proximal end of the probe. The mixing chamber mixes the sample particles and the density modifier together to at least partially form a density gradient liquid. The system dispenses a density gradient liquid into the container through the distal end of the probe. A density of the density gradient liquid varies between the first end and the second end, and at least a portion of the density gradient liquid between the first end and the second end includes a dispensed volume of sample particles.

Description

[0001] This application was filed as a PCT International Application on February 13, 2024, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 485,136, filed on February 15, 2023, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0002] Particles including viral vectors (e.g., adenovirus and adeno-associated virus (AAV)), extracellular vesicles (e.g., exosomes), and nucleic acids (e.g., plasmid DNA) can have a variety of cellular functions, structures, and mechanisms of action. For example, AAV can have different payloads, resulting in AAVs that differ not only in molecular weight but also in density. In some cases, density gradients can be used to separate these types of particles.

[0003] Typically, to separate particles using a density gradient, a sample of the particles is placed in a container containing a density-adjusting material to form a homogenous solution with uniform density. The container is then centrifuged to form the density gradient. Ultimately, the particles move through the density gradient until they reach a density equal to their own. The time typically required to form a density gradient by centrifugation can take several hours to complete, in addition to the time it takes for the particles to move through the density gradient to a point where their density matches theirs. Summary of the Invention

[0004] In general, the present disclosure relates to separating particles by using a density gradient. In one possible configuration, the density gradient is formed by automatically distributing a distribution volume of the density gradient containing the sample particles without centrifugation. In another possible configuration, the distribution volume of the sample particles is contained within a certain range of the density gradient. Various aspects including, but not limited to, the following aspects are described in the present disclosure.

[0005] One aspect relates to a system for dispensing a density gradient fluid in a container, the system comprising: a processing circuit system having a memory for storing instructions that, when executed by the processing circuit system, cause the processing circuit system to: pump sample particles and a density modifier into a mixing chamber in fluid communication with a proximal end of a probe, the mixing chamber mixing the sample particles and the density modifier together; and dispense a density gradient fluid into the container through a distal end of the probe, the density of the density gradient fluid varying between a first end and a second end, at least a portion of the density gradient fluid between the first end and the second end comprising a dispensed volume of sample particles.

[0006] Another aspect relates to a method for dispensing a density gradient fluid in a container, the method comprising: pumping sample particles and a density modifier into a mixing chamber fluidically connected to a proximal end of a probe, the mixing chamber mixing the sample particles and the density modifier together; and dispensing the density gradient fluid into the container through the distal end of the probe, the density of the density gradient fluid varying between a first end and a second end, at least a portion of the density gradient fluid between the first end and the second end comprising a dispensing volume of the sample particles.

[0007] Various additional aspects will be described in the following description. These aspects may relate to individual features as well as to combinations of features. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative and do not limit the broad inventive concepts on which the embodiments disclosed herein are based. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The following drawings, which form a part of this application, are illustrative of the described technology and are not intended to limit the scope of the disclosure in any way.

[0009] Figure 1 An example of a system for generating a density gradient for centrifugation is schematically shown.

[0010] Figure 2 Shown is housed in Figure 1 Example of a system with a manifold and mixer within the mixing chamber.

[0011] Figure 3 is an isometric view of a probe having a distal end inserted into a vessel and connected to a Figure 1 The proximal end of the system's manifold and mixing chamber.

[0012] Figure 4 Shows the connection to Figure 1 Example of the system's manifold and mixing chamber proximal to the probe.

[0013] Figure 5 Schematically shows that Figure 1 An example of a method performed by a system to generate a density gradient for separating particles.

[0014] Figure 6 Graphically shows the Figure 1 The system is based on Figure 5 An example of the method of distributing the radial length of the density gradient to distribute the volume of each component.

[0015] Figure 7 Schematically shows that Figure 1 Another example of a method for generating a density gradient for separating particles performed by a system.

[0016] Figure 8 Graphically shows the Figure 1 The system is based on Figure 7 Another example of the method of operation is to distribute the radial length of the density gradient liquid to distribute the volume of each component.

[0017] Figure 9 Schematically shows Figure 1 Examples of computing component hardware of a system. DETAILED DESCRIPTION

[0018] Figure 1 An example of a system 100 that can generate a density gradient for centrifugation is schematically shown. The system 100 is computer-controlled to precisely dispense any type, slope, or shape of gradient within a container 110. For example, the system 100 can generate a linear density gradient having a gradually increasing density from top to bottom, as well as a step density gradient having at least two discrete steps of different densities.

[0019] In some examples, system 100 includes features similar to features in systems described in U.S. Provisional Patent Application No. 63 / 369,306, filed on July 25, 2022, entitled “AUTOMATICDISPENSE OF DENSITY GRADIENTS,” U.S. Provisional Patent Application No. 63 / 369,299, filed on July 25, 2022, entitled “NON-DESTRUCTIVE MEASUREMENT OF DENSITY GRADIENTS,” and U.S. Provisional Patent Application No. 63 / 369,315, filed on July 25, 2022, entitled “REPLICATION OF DENSITYGRADIENTS,” the disclosures of which are incorporated herein by reference in their entirety.

[0020] The system 100 includes reservoirs 102, each containing a separate component for generating a density gradient within a container 110. Each reservoir 102 is connected to a pump 104 for pumping the components contained in the reservoir 102 into a manifold and mixing chamber 106. The pump 104 is programmed to pump the components from the reservoir 102 at predetermined volumes and rates for mixing within the manifold and mixing chamber 106.

[0021] exist Figure 1In the example shown, the system 100 includes four reservoirs, such as a first reservoir 102a connected to a first pump 104a for pumping a first component into a manifold and mixing chamber 106, a second reservoir 102b connected to a second pump 104b for pumping a second component into the manifold and mixing chamber 106, a third reservoir 102c connected to a third pump 104c for pumping a third component into the manifold and mixing chamber 106, and a fourth reservoir 102d connected to a fourth pump 104d for pumping a fourth component into the manifold and mixing chamber 106. The system 100 may include more than four reservoirs for holding more than four individual components for generating a density gradient, or may include fewer than four reservoirs for holding fewer than four individual components for generating a density gradient in the container 110.

[0022] The components contained in the reservoirs 102 are liquids that are pumped into the manifold and mixing chamber 106 to distribute the fluid stream into the container 110. As an illustrative example, the first reservoir 102a can contain deionized (DI) water, the second reservoir 102b can contain a density modifier, the third reservoir 102c can contain a buffer solution, and the fourth reservoir 102d can contain sample particles. As illustrative examples, the sample particles can include viral vectors (e.g., lentivirus, adenovirus, and adeno-associated virus (AAV)), lipid nanoparticles carrying mRNA, extracellular vesicles (e.g., exosomes), nucleic acids (e.g., plasmid DNA), and other types of biological or synthetic nanoparticles.

[0023] All four components contained in the reservoirs 102a through 102d can be introduced into a single stream through the manifold and mixing chamber 106. For example, DI water can be pumped from the first reservoir 102a into the manifold and mixing chamber 106 by the first pump 104a, the density adjuster can be pumped from the second reservoir 102b into the manifold and mixing chamber 106 by the second pump 104b, the buffer solution can be pumped from the third reservoir 102c into the manifold and mixing chamber 106 by the third pump 104c, and the sample particles can be pumped from the fourth reservoir 102d into the manifold and mixing chamber 106 by the fourth pump 104d. Thus, various combinations of the contents contained in the reservoirs 102a through 102d can be pumped into the manifold and mixing chamber 106.

[0024] In some examples, the pumps 104a-104d include peristaltic pumps for providing a smooth pumping flow. In some other examples, the pumps 104a-104d include syringe pumps, which can be used when more precise pumping is required.

[0025] Figure 2 An example of a mixer 200 housed within the manifold and mixing chamber 106 of the system 100 is shown. Figure 1 and Figure 2 Various combinations of DI water, density modifiers, buffer solutions, and sample particles are introduced into a single stream through the manifold and mixing chamber 106. Within the manifold and mixing chamber 106, a mixer 200 includes mixing elements 202a through 202f that mix the components as they pass through the mixing elements. The mixer 200 mixes the components to generate a homogenous flow of fluid for the probe 108 to dispense a density gradient fluid having a spatial variation in density across the radial length of the container 110 based on the relative concentrations of the components mixed by the mixer 200.

[0026] In some examples, the mixer 200 is a static mixer, and the mixing elements 202a-202f include alternating helical elements. In alternative examples, the manifold and mixing chamber 106 can include alternative types of mixers and mixing elements, including non-static mixers.

[0027] exist Figure 2 In the example provided in , each spiral element is set at 90° with the adjacent spiral element to provide thorough mixing of the components over the length L of the mixer 200 within the manifold and mixing chamber 106. The mixing elements 202a to 202f mix the components together to eliminate pockets of low density and / or high density material. The mixing elements 202a to 202f together cut and rotate the components multiple times to produce a substantially homogenous flow for the probe 108 to dispense the density gradient liquid into the container 110. As an illustrative example, the mixer 200 can include 12 mixing elements having an outer diameter OD of about 2.3 mm to about 2.4 mm, a total length L of about 27 mm to about 29 mm, and a single missing element length to diameter ratio of about 1.

[0028] The flow rate and mixing chamber parameters, such as the outer diameter OD, the total length L, or the number of mixing elements on the mixer 200, are selected to avoid shear damage to the sample particles. In some examples, different mixers can be used interchangeably within the manifold and mixing chamber 106 based on the type of sample particles being mixed by the mixer 200. For example, different mixers having different sizes and / or designs and / or materials can be used interchangeably within the manifold and mixing chamber 106 to maximize mixing while minimizing shear / sample damage to sensitive sample particles, such as lentivirus.

[0029] Figure 3is an isometric view of a probe 108 having a distal end 112 that is inserted into a container 110 and a proximal end 114 that is in fluid communication with the manifold and mixing chamber 106. In some examples, the proximal end 114 of the probe 108 is directly connected to the manifold and mixing chamber 106. Alternatively, the proximal end 114 of the probe 108 can be indirectly connected to the manifold and mixing chamber 106 via a tube.

[0030] like Figure 3 As shown, the distal end 112 is positioned toward the bottom of the interior volume 122 of the container 110, such that the probe 108 is ready to dispense the density gradient within the interior volume of the container. In examples where the system 100 dispenses the density gradient using an underlay process, the probe 108 remains fixed in the same position while the density of the homogenous stream dispensed by the probe 108 steadily increases. Alternatively, in examples where the system 100 dispenses the density gradient using an overlay process, the probe 108 can be moved upward while the density of the homogenous stream dispensed by the probe 108 steadily decreases.

[0031] like Figure 3 As shown, during the dispensing of the density gradient, the container 110 is fixedly positioned relative to the probe 108 by the holder 116. Figure 3 In the example shown, the retainer 116 includes a clamp for securely securing the container 110 to the frame 118 of the system 100 .

[0032] Figure 4 An example of the proximal end 114 of the probe 108 connected to the manifold and mixing chamber 106 is shown. The manifold and mixing chamber 106 includes a manifold portion 402 having inputs 404a to 404b, each of which receives a component pumped from a reservoir 102a to 102d by a pump 104a to 104d, respectively. The manifold and mixing chamber 106 also includes a mixing portion 406 that houses the mixer 200 for mixing the components pumped from the reservoirs before they reach the proximal end 114 of the probe 108.

[0033] The proximal end 114 of the probe 108 is secured by a set screw 408, which can be tightened or loosened around the proximal end 114 of the probe 108. The manifold and mixing chamber 106 are attached to a motor-driven mechanism that precisely moves the probe 108 up and down to a desired position within the container 110. In some examples, the probe 108 can be manually lowered to a desired position within the container 110.

[0034] In some examples, the probe 108 includes a coating of a non-stick material. In some examples, the coating includes and / or similar types of materials. In some examples, the coating is hydrophobic and / or non-wettable. The coating included on probe 108 prevents the density gradient solution dispensed in container 110 from adhering to or accumulating on probe 108. This allows probe 108 to be removed from container 110 without inadvertently mixing portions of the density gradient solution. In addition, the coating can prevent sample particles and other components dispensed through probe 108 from being adsorbed on probe 108.

[0035] Additionally, the manifold and mixing chamber 106 and probe 108 can be sterilized after each use of the system 100. Furthermore, the manifold and mixing chamber 106 and probe 108 are free of endotoxins to protect the integrity of sample particles and all components dispensed therethrough.

[0036] Reference Figure 1 The system 100 may include a control panel 130 for receiving input from a user to generate a desired density gradient. In some examples, the control panel 130 includes a user interface 132, such as a touch screen display, which can be used by the user to create a desired density gradient, perform measurements thereof, and store the distribution of the density gradient. In other examples, the user interface 132 may include additional input devices, such as one or more physical buttons that can be selected to control the operation of the system 100.

[0037] Separation sample particles can be performed by equilibrium zonal centrifugation, which usually includes layering the particle sample on the top of a density gradient liquid, and then using centrifugal force to make the particle move at different speeds according to its mass. When the particle moves downward through the density gradient liquid, because the faster sedimentation particles move in front of the slower particles, a zone is formed that contains particles of similar size. The zone of sample particle layering limits the sample volume that can be accommodated by the density gradient liquid. In addition, due to the required time for the particle of interest to precipitate in the density gradient liquid band and the additional time to form the density gradient liquid, the centrifugation time of this technology is usually up to several hours.

[0038] Alternative methods for separating sample particles may include isopycnic gradient ultracentrifugation (DGUC), which generally involves mixing sample particles with a density-forming material such as cesium chloride (CsCl), potassium bromide, iodixanol, to produce a homogeneous solution of defined density.

[0039] The homogenized solution is then placed in a container for centrifugation, where centrifugal force causes a density gradient to form. After the density gradient is formed, the sample particles in the sample migrate along the density gradient to a point where the buoyant density of the particles matches the density of the surrounding medium, allowing the sample particles to reach a stable equilibrium. As used herein, a stable equilibrium means that the sample particles have been sufficiently separated to be isolated for extraction, even though the sample particles may not be completely still.

[0040] The time it takes to develop a density gradient is largely dependent on the gravity applied during centrifugation, which becomes a key limiting factor for large-scale workflows because gravity decreases with increasing volume. For example, increasing the volume of density gradient used in a large-scale workflow can reduce throughput because more time is required to develop the density gradient due to the lower centrifugation speed.

[0041] When using DGUC techniques to separate sample particles, additional factors may also contribute to reduced throughput. For example, temperature can affect the time required to form a density gradient, as using lower temperatures to protect sample particle integrity may increase the time required to form a density gradient due to slower diffusion rates. Furthermore, the type of density modifier used, particularly its molecular weight and / or diffusion rate and viscosity, can affect the time required to form a density gradient.

[0042] Figure 5 An example of a method 500 for generating a density gradient for separating sample particles is schematically shown. System 100 can perform method 500 to significantly reduce the centrifugation time required to reach equilibrium and separate particles at high resolution. Method 500 is advantageous for workflows with large numbers of particles. Method 500 is also advantageous for small-scale workflows performed to separate particles that are unstable during high gravity, because centrifugation is performed at a lower speed without significantly increasing the time required to reach a stable equilibrium.

[0043] like Figure 5 As shown, the method 500 includes an operation 502 of lowering the distal end 112 of the probe 108 to near the bottom of the interior volume 122 of the container 110. An example of this arrangement is shown in FIG. Figure 1 and Figure 3 Shown in.

[0044] Method 500 includes an operation 504 of dispensing a density gradient solution into container 110. The density gradient solution is dispensed as a homogenous mixture of a density modifier pumped from the second reservoir 102b, a buffer solution pumped from the third reservoir 102c, and sample particles pumped from the fourth reservoir 102d, such that the sample particles are directly dispensed into the density gradient solution. This differs from the equilibrium zonal centrifugation and isopycnic DGUC described above.

[0045] In some examples, operation 504 includes performing a bottoming process, in which the distal end 112 of the probe 108 remains positioned near the bottom of the interior volume 122 of the container 110 while the density of the homogenous flow dispensed by the probe 108 steadily increases. Alternatively, operation 504 may include performing a covering process, in which the distal end 112 of the probe 108 moves upward in the container 110 while the density of the homogenous flow dispensed by the probe 108 steadily decreases.

[0046] In some examples, the density gradient dispensed in operation 504 is a continuous gradient of gradually decreasing density moving along the radial length of container 110. In this case, the continuous gradient can be linear or logarithmic. In alternative examples, the density gradient dispensed in operation 504 is a step gradient with defined interfaces between different layers of different densities.

[0047] Each position along the radial length of the density gradient fluid has a density based on the relative concentrations of the components pumped from the reservoir 102. For example, increasing the amount of density modifier mixed by the manifold and mixing chamber 106 increases the density of a particular portion of the density gradient fluid, while decreasing the amount of density modifier mixed by the manifold and mixing chamber 106 decreases the density of a particular portion of the density gradient fluid.

[0048] Next, method 500 includes determining whether the density gradient solution is complete in operation 506. When the density gradient solution is not complete (i.e., "No" in operation 506), method 500 continues to dispense the density gradient solution in operation 504. In at least some examples, when the density gradient solution is complete (i.e., "Yes" in operation 506), method 500 may proceed to operation 508, which dispenses a volume having a density lighter than the lightest density of the density gradient solution at the top of the density gradient solution.

[0049] In some examples, the volume dispensed on top of the density gradient consists primarily of DI water. Particularly when container 110 is a sealed tube, operation 508 can be performed to eliminate air pockets within the sealed tube left by removing probe 108, as air pockets can otherwise weaken the sealed tube, particularly under high gravity during centrifugation. Furthermore, by dispensing a top volume consisting primarily of DI water, probe 108 is cleaned and ready for dispensing a second density gradient in another container. In other examples, such as when container 110 is an open-top tube, operation 508 is optional.

[0050] Next, method 500 includes operation 510 of removing probe 108 from container 110. Operation 510 may include slowly removing probe 108 so as not to disturb the density gradient fluid. As discussed above, probe 108 may include a coating to prevent the density gradient fluid from adhering to probe 108 during removal of probe 108.

[0051] Next, method 500 includes placing container 110 in a centrifuge and performing centrifugation so that sample particles are separated in the density gradient solution generated by method 500. In some examples, operation 512 can include that a user manually places container 110 in a centrifuge, and causes the user to operate the centrifuge to perform centrifugation as needed. In other examples, operation 512 is automated. For example, a mechanical actuator such as a mechanical arm can be used to automatically place container 110 in a centrifuge, and thereafter, the centrifuge automatically performs centrifugation of container 110. In some cases, when sample particles are separated in the density gradient solution formed by method 500, ultracentrifugation is performed to analyze the sample particles.

[0052] In method 500, each of the pumps 104a to 104d is programmed to control the flow rate of each liquid component into the manifold and mixing chamber 106 to have a given dispense volume for generating a density gradient having a spatial variation in density over the radial length of the container 110. This enables the system 100 to precisely control the concentration of each liquid component in each portion of the density gradient dispensed by the probe 108.

[0053] Figure 6 An example of the dispensed volume of each component along the radial length (y-axis) of a density gradient fluid 600 dispensed by the system 100 according to operation of the method 500 is graphically illustrated. In this example, the density gradient fluid 600 is a continuous gradient fluid. Each portion along the radial length of the density gradient fluid 600 includes a combination of the density modifier pumped from the second reservoir 102b, the buffer solution pumped from the third reservoir 102c, and the sample particles pumped from the fourth reservoir 102d, such that the sample particles are dispensed directly into the density gradient fluid 600. Therefore, in this example, the density gradient fluid 600 includes a dispensed volume of sample particles along the entire radial length of the density gradient fluid 600.

[0054] like Figure 6 As shown, the top portion of the density gradient fluid 600 (i.e. Figure 6 The left side of the density gradient 600 includes the lowest dispensed volume of the density modifier, so that it has the lowest density in the density gradient 600, and the bottom portion of the density gradient 600 (i.e. Figure 6 The right side of the density gradient 600 includes the highest dispensed volume of the density modifier, so that it has the highest density in the density gradient 600, thereby gradually increasing the density of the density gradient 600. In some examples, the density gradient 600 has a density range between about 1.0 g / mL and 1.8 g / mL. This range can accommodate viral vectors, such as adenovirus and adeno-associated virus (AAV), whose density range can be between about 1.3 g / mL and 1.5 g / mL.

[0055] exist Figure 6 In the illustrative example shown, the density gradient 600 has a total volume of approximately 39 mL, which includes approximately 21 mL of sample particles. By reducing the slope of the distribution volume of the density modifier component, or by using a stock solution of a higher concentration of density modifier, even larger volumes of sample particles can be introduced into the density gradient 600.

[0056] Because the density gradient is formed during operation 504 of method 500, the centrifugation time typically required to form the density gradient is significantly reduced or even eliminated, which reduces the overall time required to separate the sample particles. For example, when starting with a homogenous solution of the density gradient material mixed with the sample particles (e.g., an isopycnic DGUC), two equilibria are reached, and at different times: a first equilibrium is reached when the density gradient stabilizes, and a second equilibrium is subsequently reached when the movement of the sample particles along the radial length of the density gradient stabilizes. When the density gradient is initially formed, sample equilibrium is not fully reached. Instead, reaching sample equilibrium may take several more hours.

[0057] Method 500 eliminates the first step of this two-step process because centrifugation is performed only for the second step (e.g., sample equilibration), and thus centrifugation for density gradient formation is significantly reduced or even eliminated. Method 500 enables sample particles to migrate to their respective equilibrium positions without first having to form a density gradient. Method 500 can reduce an isopycnic DGUC process for separating sample particles, which typically takes approximately 20 hours, to less than 5 hours.

[0058] Dispensing particles from a sample within a continuous density gradient is not easily accomplished using conventional density gradient formation techniques, which are primarily manual processes. This is because it is impossible to manually control sample introduction and density gradient formation with the necessary level of precision.

[0059] Figure 7 Another example of a method 700 for generating a density gradient for separating sample particles is schematically shown. Method 700 can be performed by system 100 to further reduce centrifugation time for separating particles. Method 700 is particularly advantageous for early development, analysis, and other low-volume workflows where fast turnaround time is critical for a variety of samples.

[0060] The method 700 includes an operation 702 of lowering the distal end 112 of the probe 108 proximate the bottom of the interior volume 122 of the container 110. Operation 702 is substantially similar to operation 502 in the method 500 described above.

[0061] Next, method 700 includes operation 704 of dispensing a first portion of the density gradient solution into container 110. In operation 704, the first portion includes a homogenous mixture of DI water pumped from first reservoir 102a, a density adjuster pumped from second reservoir 102b, and a buffer solution pumped from third reservoir 102c. Sample particles are not dispensed into the first portion of the density gradient solution.

[0062] In some examples, operation 704 includes performing a bottoming process in which the distal end 112 of the probe 108 remains positioned near the bottom of the interior volume 122 of the vessel 110 while the density of the homogenous flow dispensed by the probe 108 steadily increases. Alternatively, operation 704 may include performing a covering process in which the distal end 112 of the probe 108 moves upward in the vessel 110 while the density of the homogenous flow dispensed by the probe 108 steadily decreases.

[0063] Figure 8 An example of the dispensed volume of each component in each portion of the density gradient fluid 800 dispensed by the system 100 according to the operation of the method 700 is shown graphically. Figure 8 As shown in the example provided in FIG, the first portion 802 of the density gradient fluid 800 includes a combination of DI water, a density modifier, and a buffer solution. The first portion 802 does not include sample particles and its dispense volume is 0. In the first portion 802, the dispense volume of DI water gradually decreases while the dispense volume of the density modifier steadily increases, resulting in a gradual increase in the density of the first portion 802 of the density gradient fluid 800. This indicates bottom layer processing.

[0064] exist Figure 8 In the example provided in , the density gradient 800 is a continuous gradient having gradually decreasing density moving along the radial length of the container 110. In an alternative example, the density gradient 800 dispensed by the method 700 can be a step gradient having distinct interfaces between different portions having different densities.

[0065] Reference Figure 7 , method 700 next includes operation 706 of determining whether the first portion of the density gradient solution is complete. When the first portion of the density gradient solution is not complete (i.e., "No" in operation 706), method 700 continues to dispense the first portion of the density gradient solution in operation 704. When the first portion of the density gradient solution is complete (i.e., "Yes" in operation 706), method 700 proceeds to operation 708 of dispensing the second portion of the density gradient solution into container 110.

[0066] In operation 708, the second portion includes a homogenous mixture of sample particles pumped from the fourth reservoir 102d, the density modifier pumped from the second reservoir 102b, and the buffer solution pumped from the third reservoir 102c. In some examples, operation 708 includes performing a bottoming process, in which the distal end 112 of the probe 108 remains positioned near the bottom of the interior volume 122 of the container 110 while the density of the homogenous flow dispensed by the probe 108 steadily increases. Alternatively, operation 708 may include performing a covering process, in which the distal end 112 of the probe 108 moves upward in the container 110 while the density of the homogenous flow dispensed by the probe 108 steadily decreases.

[0067] like Figure 8 As shown in the example provided in FIG, the second portion 804 of the density gradient fluid 800 includes a combination of sample particles, a density modifier, and a buffer solution. In this example, the second portion 804 does not include DI water and its dispensed volume is 0. Instead, the dispensed volume of the sample particles in the second portion 804 replaces the dispensed volume of DI water. In an alternative example, the second portion 804 may include a dispensed volume of DI water, in which case the sample particles in the second portion 804 will have a larger volume than the dispensed volume of DI water. Figure 8 The dispense volume shown in the example of FIG. 8 is lower to accommodate the gradually increasing density of the second portion 804 .

[0068] In the second portion 804, the dispensed volume of the sample particles gradually decreases, while the dispensed volume of the density modifier steadily increases, causing the density of the second portion 804 to gradually increase. This indicates bottom layer processing. Figure 8 As shown, the sample particles are directly partitioned into the second portion of the density gradient, which is different from equilibrium zonal centrifugation and isopycnic DGUC.

[0069] Reference Figure 7 , method 700 next includes operation 710 of determining whether the second portion of the density gradient solution is complete. When the second portion is not complete (i.e., "No" in operation 710), method 700 continues with dispensing the second portion of the density gradient solution in operation 708. When the second portion of the density gradient solution is complete (i.e., "Yes" in operation 710), method 700 proceeds to operation 712 of dispensing the third portion of the density gradient solution into container 110.

[0070] In operation 712, the third portion includes a homogenous mixture of DI water pumped from the first reservoir 102a, density adjuster pumped from the second reservoir 102b, and buffer solution pumped from the third reservoir 102c. The sample particles are not dispensed into the third portion of the density gradient fluid.

[0071] In some examples, operation 712 includes performing a bottoming process in which the distal end 112 of the probe 108 remains positioned near the bottom of the interior volume 122 of the vessel 110 while the density of the homogenous flow dispensed by the probe 108 steadily increases. Alternatively, operation 712 may include performing a covering process in which the distal end 112 of the probe 108 moves upward in the vessel 110 while the density of the homogenous flow dispensed by the probe 108 steadily decreases.

[0072] like Figure 8 As shown, the third portion 806 of the density gradient fluid 800 includes a combination of DI water, a density modifier, and a buffer solution. The third portion 806 does not include sample particles and its dispense volume is 0. In the third portion 806, the dispense volume of DI water gradually decreases, while the dispense volume of the density modifier steadily increases, resulting in a gradual increase in the density of the third portion 806 of the density gradient fluid 800. This indicates bottom layer processing.

[0073] Method 700 includes an operation 714 to determine whether the third portion of the density gradient solution is complete. When the third portion is not complete (i.e., "No" in operation 714), method 700 continues to dispense the third portion of the density gradient solution in operation 712. When the third portion of the density gradient solution is complete (i.e., "Yes" in operation 714), method 700 can proceed to operation 716 to dispense the top volume, followed by operation 718 to remove the probe 108 from the container 110, and then to operation 720 to place the container 110 in a centrifuge for centrifugation to separate the sample particles in the density gradient solution by method 700. As described above, operations 716 to 720 can be substantially similar to operations 508 to 512 of method 500.

[0074] like Figure 8 As shown, the density gradient fluid 800 generated by method 700 includes a partitioned volume of sample particles within the density gradient fluid 800. For example, the density gradient fluid 800 includes a partitioned volume of sample particles only in the second portion 804 sandwiched between the first portion 802 and the third portion 806, while the first portion 802 and the third portion 806 do not include a partitioned volume of sample particles. In another example, the density gradient fluid 800 may include multiple portions of the partitioned volume including sample particles. The multiple portions of the partitioned volume including sample particles may be discontinuous relative to each other, such that a portion of the partitioned volume including sample particles is separated by a portion of the partitioned volume not including sample particles.

[0075] Dispensing sample particles into discrete locations within a continuous density gradient is not easily accomplished using conventional density gradient techniques, which are primarily manual processes, because it is impossible to manually control the sample particle dispensing and density gradient formation with the necessary level of precision. Advantageously, method 700 enables density gradient 800 to have even shorter centrifugation times for separating sample particles because sample particles can be dispensed closer to the locations along the radial length of density gradient 800 where their expected density lies. This reduces the distance that particles must travel to reach their equilibrium position, and thus further shortens centrifugation times.

[0076] In some examples, the system 100 uses a predictive model to automatically determine one or more desired parameters of the density gradient based on the type of sample particles to be separated, such as the relationship between the highest density and the lowest density in the gradient, such as the slope of the radial length and / or the distributed volume of components across the radial length, whether the gradient is linear, logarithmic, continuous, or stepped, and when an uneven distribution of sample particles in the density gradient is desired, determining the position for distributing sample particles in the density gradient based on the length of the container 110 or the estimated density of the sample particles.

[0077] In addition, in order to maximize the efficiency of the centrifuge, the density gradient liquid distributed according to the operation of method 500, 700 can be distributed to accurately match the known gradient liquid composition specified, or to match the experimental gradient liquid that is shown to be successful. For example, container size and / or geometry, centrifuge rotor geometry, temperature, centrifuge speed (rpm / rcf) and other parameters can determine the optimal density gradient liquid distribution for separating sample particles. When distributing density gradient liquid to match the optimal density gradient liquid distribution, the prediction model can also take these parameters into account.

[0078] The volume of sample particles introduced into the density gradient fluid 800 generated by method 700 is less than the amount of sample particles introduced into the density gradient fluid 600 generated by method 500. In this example, approximately 5.7 mL of sample is introduced into a total volume of 39 mL of density gradient fluid 800. Therefore, method 700 can be particularly advantageous for early development, analysis, and other small-batch workflows where fast turnaround times are critical for a variety of samples.

[0079] By selectively dispensing sample particles directly into these continuous density gradients to create density gradients 600, 800, the throughput of separating sample particles can be significantly increased by reducing the overall centrifugation time. This can maximize the throughput and efficiency of separating large sample amounts. In addition, this can minimize the time it takes to effectively separate relatively small sample amounts.

[0080] Additionally, with respect to density gradient formation, because centrifugation is eliminated or otherwise significantly reduced by methods 500, 700, these methods can utilize a wider variety of density modifier materials, such as sucrose, which would otherwise be impractical to use in equilibrium zonal centrifugation and isopycnic DGUC. For example, methods 500, 700 can generate continuous density gradients using density modifiers with lower densities (e.g., sucrose), which would require longer centrifugation times and / or greater centrifugal forces, which are impractical in equilibrium zonal centrifugation and isopycnic DGUC techniques.

[0081] As another example, in the traditional particle separation technology using density gradient liquid, using iodixanol as density regulating agent may be impractical sometimes.Different from cesium chloride (CsCl), iodixanol has relatively high molecular weight and is also viscous.At high speed, iodixanol forms very steep gradient liquid, and this may limit the resolution between sample types.At low speed, by using the density gradient liquid that iodixanol forms will form very slowly.Method 500,700 can overcome and use iodixanol as these challenges that are associated with density regulating agent, because the slope of the density gradient liquid formed by method 500,700 can be controlled when not sacrificing centrifugation time, this is owing to being used to form the centrifugal required usually of density gradient liquid and significantly reduce or even eliminate.

[0082] Figure 9 Schematically illustrates an example of computing hardware for system 100 for implementing aspects of the present disclosure. Figure 9 As shown, system 100 includes one or more processing devices 902, memory storage devices 904, and a system bus 906 that couples memory storage devices 904 to the one or more processing devices 902. The one or more processing devices 902 may include a central processing unit (CPU). In some cases, the one or more processing devices 902 are part of a processing circuit system having a memory for storing instructions that, when executed by the processing circuit system, cause the processing circuit system to perform various aspects, features, and functions described herein.

[0083] like Figure 9 As shown, memory storage 904 may include random access memory ("RAM") 908 and read only memory ("ROM") 910. Basic input and output logic, including the basic routines that help to transfer information between elements within system 100, such as during startup, may be stored in ROM 910.

[0084] The system 100 may also include a mass storage device 912, which may include an operating system 914 and store software instructions and data 916. The mass storage device 912 is connected to the processing device 902 via the system bus 906. The mass storage device 912 and associated computer-readable data storage media provide non-volatile, non-transitory storage for the system 100.

[0085] Although the descriptions of computer-readable data storage media contained herein refer to mass storage device 912, those skilled in the art will appreciate that a computer-readable data storage medium may be any available non-transitory physical device or article of manufacture from which system 100 can read data and / or instructions. Computer-readable storage media may include entirely non-transitory media. Mass storage device 912 is an example of a computer-readable storage device.

[0086] Computer-readable data storage media includes volatile and nonvolatile media, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable software instructions, data structures, program modules, or other data. Example types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technology, or any other medium that can be used to store information and can be accessed by a device.

[0087] The system 100 can operate in a networked environment using logical connections to other devices via a network 920. The system 100 is connected to the network 920 via a network interface unit 918 connected to the system bus 906. The network interface unit 918 can also connect to additional types of communication networks and devices, including via Bluetooth, Wi-Fi, and cellular telecommunication networks (including 4G and 5G networks). The network interface unit 918 can connect the system 100 to additional networks, systems, and devices. The system 100 also includes an input / output unit 922 for receiving and processing input and output from peripheral devices.

[0088] The mass storage device 912 and the RAM 908 may store software instructions and data. The software instructions may include an operating system 914 suitable for controlling the operation of the system 100. The mass storage device 912 and / or the RAM 908 may also store software instructions and data 916 that, when executed by the processing device 902, provide the functionality of the system 100 discussed herein.

[0089] The various embodiments described above are provided by way of illustration only and should not be construed as limiting in any way. Various modifications may be made to the above embodiments without departing from the true spirit and scope of the present disclosure.

Claims

1. A system for dispensing a density gradient in a container, the system comprising: processing circuitry having a memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to: pumping the sample particles and the density modifier into a mixing chamber in fluid communication with the proximal end of the probe, the mixing chamber mixing the sample particles and the density modifier together; and The density gradient fluid is dispensed into the container through the distal end of the probe, wherein the density of the density gradient fluid varies between a first end and a second end, and at least a portion of the density gradient fluid between the first end and the second end includes a dispensing volume of the sample particles.

2. The system according to claim 1, wherein: The density gradient fluid is a continuous gradient fluid with increasing density between the first end and the second end.

3. The system according to claim 1, wherein: The density gradient solution is a step gradient solution having interfaces of different densities between the first end and the second end.

4. The system according to claim 1, wherein: The instructions, when executed by the processing circuitry, further cause the processing circuitry to: The density gradient fluid is dispensed to cover the entire dispense volume of the density gradient fluid including the sample.

5. The system according to claim 1, wherein: The instructions, when executed by the processing circuitry, further cause the processing circuitry to: The density gradient fluid is dispensed to include the dispensed volume of the sample particles in a portion of the density gradient fluid sandwiched between portions of the density gradient fluid that do not include the dispensed volume of the sample particles.

6. The system according to claim 5, wherein: The instructions, when executed by the processing circuitry, further cause the processing circuitry to: dispensing a first portion of the density gradient fluid, the first portion including the dispensing volume of the density modifier but not including the sample particles; dispensing a second portion of the density gradient fluid, the second portion comprising a dispensing volume of the density modifier and the sample particles; and A third portion of the density gradient fluid is dispensed, the third portion including the dispensed volume of the density modifier but not including the sample particles.

7. The system according to claim 6, wherein: The instructions, when executed by the processing circuitry, further cause the processing circuitry to: dispensing the first portion of the density gradient solution by increasing the dispensing volume of the density modifier and decreasing the dispensing volume of deionized water so that the density of the first portion increases along the first portion; dispensing the second portion of the density gradient fluid by increasing the dispensing volume of the density modifier and decreasing the dispensing volume of the sample particles so that the density of the second portion increases along the second portion; and The third portion of the density gradient liquid is dispensed by increasing the dispensing volume of the density adjuster and decreasing the dispensing volume of the deionized water, so that the density of the third portion increases along the third portion.

8. The system according to claim 1, wherein: The instructions, when executed by the processing circuitry, further cause the processing circuitry to: Before dispensing the density gradient liquid, lowering the distal end of the probe toward the bottom of the container; and After dispensing the density gradient, the probe is removed from the container.

9. The system according to claim 1, wherein: The density of the density gradient fluid ranges between 1.0 g / mL and 1.8 g / mL between the first end and the second end.

10. A method for dispensing a density gradient in a container, the method comprising: pumping the sample particles and the density modifier into a mixing chamber in fluid communication with the proximal end of the probe, the mixing chamber mixing the sample particles and the density modifier together; and The density gradient fluid is dispensed into the container through the distal end of the probe, wherein the density of the density gradient fluid varies between a first end and a second end, and at least a portion of the density gradient fluid between the first end and the second end includes a dispensing volume of the sample particles.

11. The method according to claim 10, wherein: The density gradient fluid is a continuous gradient fluid with increasing density between the first end and the second end.

12. The method according to claim 10, wherein: The density gradient solution is a step gradient solution having interfaces of different densities between the first end and the second end.

13. The method according to claim 10, further comprising: The density gradient fluid is dispensed to cover the entire dispensing volume of the density gradient fluid including the sample particles.

14. The method according to claim 10, further comprising: The density gradient fluid is dispensed to include the dispensed volume of the sample particles in a portion of the density gradient fluid sandwiched between portions of the density gradient fluid that do not include the dispensed volume of the sample particles.

15. The method according to claim 14, further comprising: dispensing a first portion of the density gradient fluid, the first portion including the dispensing volume of the density modifier but not including the sample particles; dispensing a second portion of the density gradient fluid, the second portion comprising a dispensing volume of the density modifier and the sample particles; and A third portion of the density gradient fluid is dispensed, the third portion including the dispensed volume of the density modifier but not including the sample particles.

16. The method according to claim 15, further comprising: dispensing the first portion of the density gradient solution by increasing the dispensing volume of the density modifier and decreasing the dispensing volume of deionized water so that the density of the first portion increases along the first portion; dispensing the second portion of the density gradient fluid by increasing the dispensing volume of the density modifier and decreasing the dispensing volume of the sample particles so that the density of the second portion increases along the second portion; and The third portion of the density gradient liquid is dispensed by increasing the dispensing volume of the density adjuster and decreasing the dispensing volume of the deionized water, so that the density of the third portion increases along the third portion.

17. The method according to claim 10, further comprising: before dispensing the density gradient liquid, lowering the distal end of the probe toward the bottom of the container; as well as After dispensing the density gradient, the probe is removed from the container.

18. The method according to claim 10, wherein The density of the density gradient fluid ranges between 1.0 g / mL and 1.8 g / mL between the first end and the second end.

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