Sampling device and system

Abstract

Provided herein are devices, systems, and methods of using the devices and systems that enable manual and automated sampling and preparation of biological samples for evaluation. Any number of samples can be obtained, including nano / micro / microfluidic quantities. The samples include cells and / or other biological particles that are suspended or grown on a culture medium such as microcarriers, and can be obtained from one or more vessels (e.g., single well plates, vials, flasks, or bioreactors). The instruments to which the samples are transferred can include any analytical instrument, such as optical or laser force cytometry instruments.

Description

Technical Field

[0001] Embodiments of this disclosure relate to apparatus, systems, and methods for implementing any number of manual and automated sampling operations, including nano / micro / microfluidic sampling. Samples are obtained from one or more vessels, prepared for evaluation, and transferred to a separate instrument for analysis. Vessels may include containers ranging from single-well or vials to flasks, bioreactors, or other containers. Samples may include cells and / or other biological particles that can be suspended or grown on a culture medium (e.g., microcarriers). The instrument to which the sample is transferred may include any analytical instrument, such as an optical or laser-force cytology instrument. Background Technology

[0002] In biopharmaceutical analysis, the composition of biological samples can be complex. The diversity of biological matrices presents significant challenges to sample preparation for the analysis of target substances in these samples. In addition to analytes, samples often contain a large number of interfering substances, including endogenous substances, metabolites, and contaminants. An ideal sample pretreatment technique should remove interfering substances to the greatest extent possible, be applicable to a wide range of samples, and be compatible with a wide range of analytical instruments. Most samples must be properly treated for separation, purification, enrichment, and chemical modification to meet the requirements of analytical instruments such as laser force cytology (LFC) analyzers, high-performance liquid chromatography (HPLC), mass spectrometry (MS), etc. Current methods are complex, labor-intensive, error-prone, and in some cases, even harmful to the environment or technicians. Current methods also have many other drawbacks, including the need for large quantities of reagents, high testing costs, and increased complexity due to low recoveries and below-standard precision. Furthermore, these methods are not conducive to online processing and automation.

[0003] Therefore, there is a need for efficient equipment, systems, and methods for obtaining biological samples and processing and preparing such samples for use in analytical instruments. Preferably, such equipment, systems, and methods should be easy to implement, low-cost, efficient, reliable, and compatible with instruments (e.g., laser-force cytology instruments). Summary of the Invention

[0004] Systems, methods, and apparatus for preparing samples for analysis include obtaining a sample from a container, processing the sample, and transporting the sample to an analytical instrument. The system includes a sample extraction device, one or more control valves, one or more dilution devices, one or more mixing devices, and an analytical instrument interface is provided herein. In embodiments, the analytical instrument includes… machine.

[0005] In some embodiments, the invention further includes a microcarrier separation device capable of separating biological particles from microcarriers, including using enzymatic, chemical, thermal, or mechanical methods. Additional features of the invention include processing samples by separating, purifying, enriching, chemically modifying, and purifying the biological particles from other sample components. Attached Figure Description

[0006] Figure 1 A schematic diagram is provided illustrating the overall setup of the novel sampling system of the present invention: a sample containment container (e.g., a bioreactor 100), a sample extraction tube (e.g., a sterile immersion tube 102), a control valve (104A), a dilution device (110), an instrument interface (115) (i.e., a microfluidic or microfluidic chip or fluid manifold), and an analytical instrument (113). A container (112) for waste collection is also shown. Figure 1 This includes a reservoir / vessel (106B) that can be used for various purposes, such as, for example, storing solutions for processing (i.e., dilution or rinsing). In some embodiments, the analytical instrument (113) is... Machine (LumaCyte, LLC., Virginia, USA).

[0007] Figure 2 A schematic diagram is provided illustrating the overall setup of the novel sampling system of the present invention, further including a microcarrier separation device (119). A vessel (117) optionally contains a microcarrier enzyme / chemical solution.

[0008] Figures 3A-3C A schematic diagram is provided, which illustrates the overall setup of the novel sampling system including the present invention in a segmented flow. Figure 3A The overall configuration of the segmented stream is shown, with particular emphasis on the sampling steps; Figure 3B It provides configuration for incorporating the purification process. Figure 3C A configuration is provided that allows the incorporation of cleaning fluid or solution into the system from a separate container (106B).

[0009] Figure 4 A multiplexing system including a sampling system is provided, in which samples are obtained from two bioreactors (100(i) and 100(ii)).

[0010] Figure 5 Embodiments of a novel sampling system of the present invention are provided, the novel sampling system including one or more features that enable feedback monitoring of process control using a control system (e.g., a monitoring system).

[0011] Figure 6Embodiments of the novel sampling system of the present invention are provided, the novel sampling system including a built-in dual bioreactor system demonstrating continuous production.

[0012] Figure 7 An embodiment of the novel sampling system of the present invention is provided, which includes a combined dilution device and an instrument interface.

[0013] Figure 8 Embodiments of the novel sampling system of the present invention are provided, which includes a combined dilution device and instrument interface, thereby allowing non-segmented continuous flow and rapid sample preparation. Figure 8 A configuration with four integrated chambers is specifically provided.

[0014] Figure 9 An embodiment of the novel sampling system of the present invention is provided, which includes a separate dilution device and an instrument interface.

[0015] Figure 10 A microfluidic T-type mixing and dilution device with microfluidic channels is provided.

[0016] Figure 11 A microfluidic T-type mixing and dilution device with microfluidic channels is provided, wherein the buffer channels are offset from each other at the mixing crossover point.

[0017] Figure 12 A schematic diagram of an embodiment of a dilution device is provided, wherein the dilution device is a microfluidic multiplexer chip.

[0018] Figures 13A-13G An embodiment of the instrument interface and the sequence of steps for using the instrument interface are provided. Figure 13A A representative schematic diagram is provided, showing the sampling manifold being distributed into the orifice plate. Figure 13A The diagram illustrates a diluted sample traveling from a dilution device through a series of three valves, with a dispensing manifold (sampling manifold dispensing into the orifice plate) located between the three valves. Figure 13B An embodiment is shown in which the fluid flow from the dilution device includes cells to be introduced into the analytical instrument. Figure 13C This illustrates how the orifice plate moves to the injection position, and how the motion platform incorporates a mechanism for vertical movement. Figure 13D A top view is shown illustrating how multiple manifolds can be positioned so that multiple samples from the manifolds can be filled in series (one sample at a time from one manifold) or in parallel (multiple samples simultaneously from multiple manifolds into one or more well plates). Figure 13E An embodiment is shown in which the injection tubing is small enough to pass through both the dispensing manifold and the injection manifold, thereby forming a direct flow path from the orifice plate to the analytical instrument. Figure 13FAn embodiment demonstrating the sequence of introducing microcarrier-isolated cells into an analytical instrument using a combined dispensing manifold and injection manifold is shown. Figure 13G It shows in Representative data collected from a mixture of isolated cells and microcarriers using a laser-force cytology instrument.

[0019] Figure 14 provides a schematic diagram of an embodiment of a device for removing microcarriers from cells.

[0020] Figures 15A-15C A schematic diagram of an embodiment of a device for removing microcarriers from cells is provided: Figure 15A Embodiments of a combined microcarrier removal device are provided, wherein the input of the device includes microcarriers having attached cells or other biological products from a bioreactor or other source, the microcarriers enter a removal chamber, in which a substance for separation and / or anti-adhesion is introduced via a separate input, and then the microcarriers travel through a reaction zone, in which the biological products are separated from the microcarriers. Figure 15B The sedimentation of microcarriers and cells was shown. Figure 15C Examples illustrating the separation of multiple cell types based on differences in sedimentation velocity are provided.

[0021] Figure 16 A schematic diagram of an embodiment of a device for removing microcarriers from cells with a vertical design is provided.

[0022] Figure 17 A schematic diagram of an embodiment of a device for removing microcarriers from cells is provided.

[0023] Figure 18 A schematic diagram of an embodiment of a device for removing microcarriers from cells is provided, the device including guiding the microcarriers downward using a laser.

[0024] Figure 19 A schematic diagram of an embodiment of a device for removing microcarriers from cells is provided. In some embodiments, such as Figure 19 The general diagram illustrates the separation and isolation mechanism for isolating cells (or biological particles) from microcarriers. Density gradients can be modified and customized to separate fluids (or density and phase).

[0025] Figure 20 A schematic diagram of an embodiment of a device for removing microcarriers from cells is provided. In some embodiments, the device is designed and tailored such that the density and phase difference between the two layers, as shown in the figure, creates a high-density aqueous “plug” or sac, into which cells can fall but microcarriers cannot.

[0026] Figure 21 A schematic diagram of an embodiment of a device for removing microcarriers from cells is provided.

[0027] Figure 22 A schematic diagram is provided illustrating an embodiment of a non-helical concentrating method that can be used to separate free cells (306) from microcarriers (304). Detailed Implementation

[0028] The invention has been described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in practice without departing from the scope or spirit of the invention. Those skilled in the art will recognize that these features can be used alone or in any combination, depending on the requirements and specifications of a given application or design. Those skilled in the art will recognize that the systems and devices of embodiments of the invention can be used with any method of the invention, and any system and device of the invention can be used to perform any method of the invention. Embodiments including various features may also consist of or substantially consist of these various features. Other embodiments of the invention will be apparent to those skilled in the art in light of the specification and practice of the invention. The description of the invention provided is merely exemplary in nature, and therefore, variations without departing from the essence of the invention are intended to be within the scope of the invention.

[0029] Before explaining at least one embodiment of the present invention in detail, it should be understood that the invention is not limited in its application to the details of the construction and arrangement of the components set forth in the following description or illustrated in the accompanying drawings. The invention can have other embodiments or be practiced or implemented in various ways. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood or used by one of ordinary skill in the art to which this technique and method are covered.

[0031] The texts and references mentioned herein are incorporated in their entirety, including PCT / US2017 / 068373 (filed December 23, 2017 and published June 27, 2019 as WO2019 / 125502A1), PCT / US2019 / 023130 (filed March 20, 2019 and published September 26, 2019 as WO2019 / 183199A1), PCT / US2019 / 026335 (filed April 8, 2019 and published October 10, 2019 as WO2019 / 195836A1), U.S. Provisional Patent Application Serial No. 62 / 897,437 filed September 9, 2019, and U.S. Provisional Patent Application Serial No. 63 / 049,499 filed July 8, 2020.

[0032] The novel inventions described herein include devices, systems, and methods for using these devices and systems, which enable the manual and automated acquisition and preparation of biological samples for evaluation by analytical machines, such as laser force analyzers. Samples can be acquired from any vessel (including but not limited to bioreactors), and in any quantity, including nano / micro / microfluidic quantities.

[0033] The novel devices and systems described herein include one or more microfluidic sampling devices in which samples are withdrawn from one or more bioreactors or (one or more) other containers and subsequently introduced into an analytical instrument. As used herein, the terms “bioreactor” and “container” are used interchangeably and can be understood to include any vessel for storing or handling cells (including cultured cells), such as flasks, bottles, test tubes, slides, bags, microtiter plates, microtiter dishes, multiwell plates, culture dishes, permeable carriers, etc. The system may optionally include various features and capabilities for enabling sample preparation for analysis. In embodiments, the system provides the ability to dilute cells from the bioreactor to a desired concentration with a specific buffer or fluid. The system may also optionally include the ability to isolate adherent cells grown on microcarriers or other suitable culture media and subsequently separate suspended cells from the microcarriers before introduction into the analytical instrument.

[0034] The devices and systems envisioned herein include features that support processes that enable samples to flow from one location to another. All fluids in all embodiments discussed herein can be moved from one location to another by means of pressure or vacuum-driven flow, via peristaltic pumps, syringe pumps, diaphragm pumps, etc., whereby the direction of flow is determined by the forces, valve configurations, and piping involved.

[0035] Figure 1 One embodiment of the system is provided. An extraction device (e.g., a sterile infusion tube (102)) for acquiring the sample is placed in a bioreactor (100) so that the system is close to the sample. During sampling, fluid from the bioreactor is drawn upward into the infusion tube (102) and then through a valve (104A) before flowing into a dilution device (110). A solution (106B) for dilution or reagent addition (if desired) flows through a valve (116) and into the dilution device (110) to dilute the cells to a desired target concentration. The flow rate of (106B) can be adjusted and customized according to methods known to those skilled in the art to achieve a range of target concentrations. The sample then proceeds to an instrument interface (115), which is designed to present the sample in a manner that allows for a nimble and robust introduction of the analytical instrument (114). Figure 1As indicated between (110) and (115), the system will have the ability to move reagents, buffer solutions, and cleaning solutions in multiple directions. It is important to note that the dilution device (110) and instrument interface (115) can be as follows: Figure 1 The illustrated individual device, or the two devices that can be coupled together on a single combined device performing both functions. For example... Figure 1 As shown, waste (112) can be discharged from the dilution device (110) or, as needed, from the instrument interface (115).

[0036] Figure 2 An optional addition of equipment is shown when adherent cells are grown on microcarriers within a bioreactor. In this embodiment, the cell-containing sample attached to the microcarriers is removed from the bioreactor (100) and first introduced into a microcarrier separation device (109), which segregates the cells from the microcarriers and separates the suspended cells and microcarriers into different fluid streams. Microcarrier separation devices (which also do not perform the segregation step) are known to those skilled in the art and, as used herein, include all such devices that separate cells from other devices and enable the separation of cells so that the cells can be used for analytical purposes. Examples include HARVESTAINE. TM The system (Thermo Fisher Scientific, Massachusetts, USA) and other filter-based systems are designed for the large-scale separation of cells from microcarriers after dissociation. After processing, the dissociated cells continue into a dilution device (110), while the microcarriers exit the device and enter waste (119). Cells can be separated from the microcarriers via several methods, including enzymatic, chemical, thermal, or mechanical methods. Chemical and enzymatic methods may require the addition of a solution (117) such as trypsin, ethylenediaminetetraacetic acid (EDTA), or other suitable enzymes or chemicals to the microcarrier separation device (109). Once the cells have been dissociated and separated from the microcarriers, the remainder of the system functions as described above, with the sample flowing through the dilution device (110) before being introduced into the analytical instrument (114), and then through the instrument interface (115). It is important to note that the microcarrier separation device (109), dilution device (110), waste collection device (112), and instrument interface (115) can be as follows: Figure 2 The individual devices shown can be coupled together to form a combined device that performs all three functions, or two devices that perform all three functions when used together.

[0037] Figure 1 and Figure 2The embodiments presented allow for continuous sampling. This means the system continuously removes samples from the bioreactor (100) to prevent backflow and contamination. The removed volume is low enough not to adversely affect the overall process and can be adjusted as needed depending on cell concentration, process design, and sampling regime. Valve (104A) is either an active control valve capable of closing as needed, or a passive one-way check valve that only allows flow out of the bioreactor to further prevent backflow and contamination. Although in Figure 1 and Figure 2 The embodiment shown depicts only one valve, but it is conceivable that multiple valves of one or more types, including multi-port / multi-way valves, can be used at any point throughout the fluid system as needed. The valves can be manually controlled or automatically controlled via electronic or computerized mechanisms.

[0038] Figure 3A , Figure 3B and Figure 3C A segmented (rather than continuous) sampling regime is provided. In the sampling step, the sample from the bioreactor is aspirated by the aforementioned force and proceeds through a series of valves (104A and 104B). The first of the valves connects a purification fluid (108) to the sample line, thereby allowing post-cleaning sample analysis. Figure 3A In this process, the valve is open to allow the sample to pass through, but closed to the purge fluid to ensure that no purge fluid mixes with the sample. The sample then travels through valve 104B, where it comes into contact with a solution (106A) for dilution. To minimize the amount of sample removed from the bioreactor and to prevent sample settling in the pipeline, the dilution solution in 106A is intended to rapidly drive the sample to the dilution device (110). Here, additional solutions (one or more types) (106B) for dilution can be introduced to rapidly dilute the sample to the appropriate concentration before it is introduced into the analytical instrument (114) and before it enters the instrument interface (115). After sample preparation, waste (112) exits the dilution device. Although Figures 3A-3C Not shown, but a segmented sampling regime can also be used with a microcarrier system that also includes a microcarrier separation device (109), such as Figure 2 As shown.

[0039] Figure 3BThe configuration of the post-analysis purification step is shown. A first three-way valve (104A) is open for the purification fluid but closed for the sample. A second three-way valve (104B) is closed for the solution (106A) but open to allow purification to pass through the tubing into the dilution device (110). Valve (116) is also closed to prevent dilution of the purification fluid in the dilution device. The purification fluid continues through the instrument interface (115) and into the injection port of the analytical instrument (114). In this system, the entire tubing between each sample is cleaned, ensuring sample sterility and preventing contamination. Once the entire tubing has been cleaned, waste (112) exits the dilution device (110) or the instrument interface (115).

[0040] Figure 3C It shows Figure 3B The subsequent cleaning steps: To allow the purification fluid (108) to exit the line and prepare the line for the next sample, dilution fluid is used to clean the line. Valve 104A remains closed to the sample and to the purification fluid. Valve 104B is opened, allowing the solution from 106A to be introduced into the line and into the dilution device (110). Valve 116 is also opened, allowing the solution from 106B to enter the dilution device, thoroughly cleaning the dilution device for the next sample. Buffer fluid (106B) is then injected through the instrument interface (115) and into the analytical instrument (114) to clean the line before it exits into waste (112).

[0041] It should be noted that the above-described embodiments, designed for sampling from multiple bioreactors, can also be used. Figure 1 , Figure 2 , Figure 3A , Figure 3B and Figure 3C The concepts described herein are used for implementation. An implementation is, for example... Figure 4 As shown, samples are obtained from two separate bioreactors (100(i) and 100(ii)) and introduced into the analytical instrument (114). In the illustrated embodiment, samples from each individual reactor flow to separate, distinct dilution devices (110(i) and 110(ii)) before being combined into a multiplexed instrument interface (125). The multiplexed instrument interface (125) accepts streams from multiple inputs that are sequentially guided through the outputs and into the analytical instrument (114). The samples remain distinct to allow for accurate sampling from each individual bioreactor (110). The specific design for achieving this is described subsequently. Appropriate physical separation or cleaning steps are employed to maintain sample distinctness and sample separation. Although Figure 4 Two reactors are shown, but multiple reactors can be connected.

[0042] Additional features of the invention include the previously described embodiments and, as well as... Figure 5The procedure in the continuous feedback monitoring system is illustrated. In one embodiment, the condition of the bioreactor (100) is maintained or adjusted by introducing changes in input 1 (118) and input 2 (120), although any number of inputs can be used in alternative embodiments. During operation, an analytical instrument (114) monitors the condition of the bioreactor to adjust the conditions within the bioreactor, which may involve adjusting the input streams from input 1 (118) and / or input 2 (120). In one embodiment, measurements from the analytical instrument are then sent to a control system (122), which directs changes to the bioreactor (100) as needed. The control system includes monitoring components, such as a computer capable of manually or automatically adjusting changes to the bioreactor or changes within the bioreactor based on data collected from one or more devices. These changes may include, but are not limited to, introducing reagents or other solutions to introduce nutrients, adjusting pH, modifying cell culture conditions or changing cell states, and changes in gas concentration or injection rate, temperature, or mixing speed. This aspect allows for real-time monitoring and adjustment of the production pipeline, thereby allowing the bioreactor to be operated in a more precise manner to increase production time, efficiency, quality, or any combination thereof. A variety of biological products can be introduced, and a variety of bioreactors can be used in this setup. The setup can also be operated in batch, staggered feeding, or continuous operation modes.

[0043] In an additional embodiment, the device and system also include a built-in dual bioreactor system. One embodiment is, for example... Figure 6 As shown, a first bioreactor (124) serves as the input to a second bioreactor (92), and an analytical instrument (114) is connected to the second bioreactor (92). During operation, the analytical instrument (114) monitors the conditions and any changes occurring within the bioreactor (92). The measurements and information can then be sent to a computer (122), which communicates with the bioreactors to adjust their interactions with each other. The analytical instrument can be paired with multiple bioreactors, each connected to a source bioreactor (124), or each connected to a discrete source bioreactor itself. The sampling, purification, and cleaning procedures are similar to the steps described in detail above. The setup can also be operated in batch, batch feed, or continuous operation modes.

[0044] Figure 7An embodiment of the combined dilution device (110) and instrument interface (115) is presented. It is an integral chamber (128) with three ports at the base of the chamber. The dilution port (144) allows fluid from (106B) to pass through a two-way valve (116) to fill the chamber for rapid dilution. The waste port (148) allows fluid in the chamber to exit the chamber to discharge waste (112). The injection port (146) allows the sample from the bioreactor (102), plus (if needed) a fast-moving buffer fluid (106A), to meet at valve 104B and enter the chamber. During the sampling step, the waste valve (126) is closed to prevent fluid from leaving the chamber. The sample and dilution solution enter and fill the chamber from their respective ports until the appropriate concentration is reached. Once the correct concentration is reached, the fluid enters the analytical instrument (114), where the sample is analyzed for the current state of the bioreactor. After sample analysis, the waste discharge valve (126) is opened, allowing the remaining fluid to be discharged into the waste. The waste discharge valve and the valve from the buffer solution are then closed again. Figures 3A-3C (106A and 106B). Purification fluid (108) is drawn into the integrated chamber to fill it, and then into the analyzer to clean and sterilize the tubing in preparation for the next run. Remaining purification fluid in the chamber is discharged to waste (112) by opening valve (126). Next, valve (126) closes again, the valve to the buffer fluid is opened, and the integrated chamber is filled with buffer to wash away the purification fluid. The buffer flow then travels to the analyzer for a period of time before exiting the integrated chamber via waste (112). The integrated chamber (128) can be shaped in various ways, as long as it can contain an appropriate volume of fluid and the diluted sample can exit the chamber and enter the analyzer (114).

[0045] In configurations using multiple bioreactors, there is a corresponding number of integral chambers (128), all of which can be housed in the same material or separated from each other as needed. Each integral chamber is... Figure 3A The connection to each bioreactor is repeated a corresponding number of times so that all bioreactors, valves, dilution devices, and tubing reach a single analytical instrument. Each bioreactor has its own set of fluids (106A, 106B, 108) to ensure sterility and efficiency of fluid movement. In a single chamber, each buffer port (144) is connected to its own buffer (106B), each injection port (146) is connected via its own three-way valve (104B) to its own bioreactor dip tube (102) and rapid-moving buffer (106A), and each waste port (148) leads to common waste (112). This configuration allows for non-segmented continuous flow and rapid sample preparation, preventing waiting times. Figure 8An embodiment using this configuration of four integrated chambers is provided. In the illustrated diagram, an integrated chamber (128-2) is shown during the sampling step, where a sample from a specific chamber is entering the analytical instrument (114) for measurement. The integrated chamber (128-1) represents the sample that runs before the sample in (128-2). As described above and Figure 3B As shown, the integrated chamber (128-1) is undergoing a purification and cleaning procedure. The integrated chamber (128-3) is being prepared for the analysis of the next sample. The waste valve (126-3) is closed, and buffer solutions (106A-3 and 106B-3) are introduced into the integrated chamber along with the sample (102B-3) from bioreactor 3. Appropriate dilution occurs before sample run so that the analytical instrument can immediately begin measurements from (128-3) once the sample from (128-2) is completed. The integrated chamber (128-n) represents all other integrated chambers in the system. Waste (112-n) and the valves (106A-n) and (106B-n) for buffering are opened, allowing constant flow through the integrated chamber. This constant flow procedure can be performed for 1 to n bioreactors using the integrated chamber (128-n) and eliminates interruptions in fluid flow, maintaining an acceptable environment for sample passage. The constant flow of the buffer solution also makes the use of a purge fluid optional after each sample from a particular bioreactor, thereby increasing the sampling rate and reducing sample contact with harsh environments. The purge fluid (108) can be used to clean up the system at the end of bioreactor operation. Samples are transferred from each sampling chamber to the analytical instrument via connection mechanisms (e.g., tubing). In some embodiments, connection is facilitated by the use of multi-port valves.

[0046] In systems where a continuously flowing buffer is selected and no purge fluid is used until the end, samples from the bioreactor (100) can also be continuously aspirated into its specific integrated chamber (128). Continuous sample aspiration while keeping valves (104A) and (104B) open ensures that between measurements in a particular bioreactor, none of the samples aspirated from the dip tube (102) will fall back into the bioreactor, thus contaminating the entire reactor. Using the fluid forces listed above, the flow rates of samples and buffer entering integrated chambers not currently being sampled by the analytical instrument can be minimized, thus avoiding wasted resources. All fluid flow rates can be variable to allow for precise and accurate real-time adjustments to ensure sampling integrity.

[0047] It is important to note that Figure 7 and Figure 8The embodiments described herein can also be used as a dilution device (110) instead of the combined dilution device (100) and instrument interface (115). When used as a dilution device, the sample leaves the dilution device (110) and enters a separate instrument interface (115) before being introduced into the analytical instrument (114). This interface can be used to perform other treatments on the sample, including separating the sample using droplets or plugs, or performing other treatments as needed to properly interact with the analytical instrument. Figure 9 An example illustrating this is shown.

[0048] Another embodiment of the dilution device (110) is as follows: Figure 10 As shown. Figure 10 This is a schematic diagram of a microfluidic T-type mixing and dilution apparatus with microfluidic channels of any micro-size and shape required for proper dilution to the appropriate sample concentration. In this diagram, the sample from the dip tube (102) enters the microfluidic T-type mixer through the sample introduction channel (134). Buffer solutions (106A or 106B) for rapid sample movement and dilution, if needed, enter through buffer channels (136) on either side of the apparatus and collide (approximately) perpendicular to the sample and in a straight line with each other at the mixing crossover point (160). The collision of a large volume of buffer solution and a small volume of sample at right angles disrupts the laminar flow path, mixes, and dilutes the sample to the desired concentration. The sample then flows directly into the analyzer (114) or into the instrument interface (115) before entering the analyzer (114). Purifying fluid (108) is listed as an optional source of one of the buffer channels (136) to demonstrate how purifying fluid can enter the microfluidic T-type mixer for cleaning during the aforementioned purification process.

[0049] For multiple bioreactors, multiple microfluidic T-mixers can be used in parallel. Figure 10 Alternatively, a microfluidic T-type mixer may have multiple sample introduction channels (134) that intersect at multiple buffer channels (136) at multiple mixing crossover points (160).

[0050] Figure 11 Another embodiment of the dilution device (110) is shown. This microfluidic offset T-type mixer is... Figure 10 Similar to the configuration in this invention, but the buffer channels are offset from each other at the mixing crossover point (162) by a distance sufficient to subject the sample to rapid impact that disrupts the laminar flow lines. This configuration ensures potentially more efficient mixing and dilution of the sample before it enters the analytical instrument (114). As with other configurations of this invention, Figure 11 The channels shown can be modified in size, shape, and placement depending on the nature of the sample being evaluated and other considerations such as flow, analytical parameters, and the characteristics being evaluated.

[0051] For multiple bioreactors, multiple microfluidic offset T-mixers (10) can be used in parallel, or the microfluidic offset T-mixers can have multiple sample introduction channels (134) that intersect at multiple offset buffer channels (136) at multiple offset mixing intersections (162).

[0052] Figure 12 Another embodiment of the dilution device (110) is shown, wherein the dilution device is a microfluidic multiplexer chip. In this embodiment, samples from multiple bioreactors enter the multiplexer chip via multiple sample inlet channels (134), which converge to a mixing and dilution position (142). The source of the sample inlet channel is indicated by a valve (104B), as the sample, buffer solution (106A), and purge fluid (108) all arrive at the chip via valve (104B). For a bioreactor that is not currently being sampled, the corresponding chip inlet valve (140) is closed to prevent backflow of another sample and contamination of other lines. A sample from one bioreactor is pushed onto the chip by buffer solution 106A and proceeds to the mixing and dilution position (142), where buffer solution from (106B) rapidly enters through a port or buffer channel to dilute and mix the sample. The diluted sample then exits the multiplexer chip and is either directly introduced into the analytical instrument (114) for measurement, or first introduced into the instrument interface (115) before being introduced into the analytical instrument (114). After sample analysis, the above-described purification and cleaning procedures, the multiplexer chip is cleaned and prepared for the next sample by allowing the correct fluid to pass through the inlet channel (134).

[0053] An implementation of the instrument interface (115) is as follows: Figures 13A-13D As shown, Figures 13A-13D The sequence of steps for using the instrument interface (115) is shown. Figure 13A In one embodiment shown, a diluted sample from the dilution device (110) travels through three valves (202A, 204, and 202B), with a distribution manifold (206) located between the three valves. When not sampling, the fluid flow from the dilution device (110) flows to waste (112) during a constant flow process. The distribution manifold has a dispensing needle or tube (208), which typically closes its flow via a three-way valve (204). The fluid flow from (110) can primarily be dilution fluid to maintain a constant flow of fluid through the device or to flush away previous samples. During sampling, the fluid flow from (110) will include cells to be introduced into the analytical instrument (114). Figure 13BOne embodiment illustrating this is shown; a valve (204) opens to the dispensing needle (208), while a valve (202B) for discharging waste closes, forcing the diluted sample from the dilution device (110) to be dispensed into the orifice plate (212). In one embodiment, the dispensing manifold (206) may include or be attached to its own pumping system, such as a pressure-driven flow or syringe, peristaltic pump, or diaphragm pump. The orifice plate (212) may be any number of orifices (e.g., 384, 192, 96, 48, 24, 12, or 6) and may be custom or standard geometry. The orifice plate (212) will be placed on and held by a motion platform (218) that allows for three-dimensional axes of motion. Once a specified volume of diluted sample is dispensed from the dilution manifold (206) into a single orifice, the orifice plate is moved along a path (222) via the motion platform to the injection manifold (214) located at or below the analyzer (114), such that the injection needle (216) is immersed in the particular sample. Figure 13C The diagram illustrates how the orifice plate moves to the injection position, with the motion platform integrated into a mechanism (220) for vertical movement. The sample is then fed into the analytical instrument (114) for measurement. After the sample has been filled into the orifice plate, valve (204) closes the dispensing needle, and valve (202B) opens to purge waste and restore the constant flow process. Subsequent samples can be dispensed to any location within the orifice plate in a similar manner. The motion platform (218) allows any orifice in the plate to be addressed by both the dispensing manifold (206) and the injection manifold (214).

[0054] In a configuration with multiple bioreactors (e.g., where the number of bioreactors is N), there is a corresponding number of distribution manifolds (206) (e.g., where the number of distribution manifolds is M), where M is the same or different number from N, depending on the configuration and settings. Figure 13D A top view is shown, illustrating how multiple manifolds can be positioned so that multiple samples from multiple manifolds can be filled in series (one sample at a time from one manifold) or in parallel (multiple samples simultaneously from multiple manifolds into one or more well plates). Figure 13D A top view illustration of the movement path (222) of the orifice plate (212) between the dispensing manifold and the injection manifold (214) via the motion platform (218) is also shown.

[0055] Figures 13A-13D The embodiments shown may also include the use of multiple well plates. This embodiment also includes the movement of any components, such as the various manifolds, well plates, and injection and dispensing needles required for the appropriate functionality and form of a continuous microfluidic sampling device.

[0056] Figures 13A-13DThe illustrated embodiment depicts two separate locations for the dispensing manifold (206) and the injection manifold (214). However, alternative embodiments combine them into a single connection location within (or outside) the analyzer, such as... Figure 13E As shown. In this case, the sample will be aspirated from the bioreactor and fed into the well plate via the dispensing manifold. The injection path or conduit (380) will travel from the analytical instrument through both the dispensing manifold and the injection manifold. Figure 13E As shown, one embodiment exists in which the injection tubing is small enough to pass through both the dispensing manifold (206) and the injection manifold (214), thus forming a direct flow path from the orifice plate to the analyzer. In other words, fluid from the dispensing manifold side (via valves 202A and 202B) does not come into contact with the fluid in the injection tubing. This is crucial because it maintains two discrete flow paths and allows reagents to be dispensed or removed from the orifice without interrupting the injection flow path of the sample into the analyzer. A hermetically sealed connector or port (390) can be attached as needed to create a sealed or separated flow. In addition to the injection tubing (380) traveling through the top and bottom of the manifold in the illustrated embodiment, Figure 13E The embodiments also depict two ports for reagent or sample entry (shown on the left and right sides). However, additional embodiments may exist with only one port in addition to the injection tubing, or with two or more ports in addition to the injection tubing, or the position of the injection or additional ports may be adjusted.

[0057] Figure 13F The sequence of introducing microcarrier-isolated cells into the analytical instrument using a combined dispensing manifold (206) and injection manifold (214) is described, in conjunction with... Figure 13E The illustrated embodiment is similar. In the first step, microcarriers with attached cells (312) are dispensed from the bioreactor (100) or other external source into the well plates (212) via a dispensing manifold. Subsequently, the cell-containing microcarriers are allowed to settle before the supernatant solution is removed. Then, different solutions are added through the dispensing manifold and into the well plates. Any number of aspiration and dispensing cycles can be run as needed, such as Figure 13F As indicated by the bidirectional arrows. After these cycles are completed, reagents designed for cell isolation are added to the wells and incubated for a period of time. During this incubation, mixing may occur as needed. At the end of the incubation period, the cells (306) will be isolated from the now uncoated / naked microcarriers (304). Subsequently, the combined solutions can be mixed. After a brief settling period, the microcarriers will be at the bottom of the wells, but the cells will be thoroughly mixed due to the significant difference in settling rates between the two substances. This will allow the injection needle (216) to preferentially sample the cells rather than the microcarriers. Although Figure 13F The valve is shown, but other designs or structures (e.g.) Figure 13EThe manifold described herein can be used to preferentially guide and / or isolate various flow streams moving from the sampling container to the orifice plate and from the orifice plate to the analytical instrument.

[0058] Figure 13G It shows Representative data were collected from a mixture of isolated cells and microcarriers using a laser-force cytology instrument. Vero cells were grown on microcarriers in serum-free or serum-containing media. At harvest, cells were manually isolated from the microcarriers, and the mixture of cells and microcarriers was then loaded into 96-well plates for use with... Perform the analysis. Figure 13G i.- Figure 13G iv. Several are shown The overall average of the measurements (including speed, size, and centrifugation rate, as well as the average acquisition time to reach a target cell count of 300) across multiple wells under both media conditions is presented. Figure 13G v. shows a scatter plot of representative dimensions versus velocity for two media conditions. Each symbol represents data from a single cell.

[0059] As part of the bioreactor sample processing procedure, the present invention also includes a device (109) for removing microcarriers from cells, and a schematic diagram of such an embodiment is shown in Figure 14. This device can be used in conjunction with any embodiment of the sampling system described herein, or as a standalone device to assist the user in performing bioproduct analysis or purification of samples in a bioreactor. Two embodiments of the device are schematically shown (Figure 14(A) and Figure 14(B)). In both embodiments, the input to the device consists of cells attached to the microcarriers; however, in the first device, the separation of cells from the microcarriers occurs in a separate sub-device (220), where the physical separation of the separated cells and empty microcarriers becomes a physically separated flow. Therefore, the output of the separation device (220) will be a flow of free cells dispersed with microcarriers. The output of the separation sub-device (220) will then be the input (230) of the separation sub-device, and the output of the separation sub-device (230) will be cells in one channel or tube and microcarriers in another channel or tube. In a second embodiment of the device, separation and isolation occur within the same device (240), the output of which is cells in one channel or tube and microcarriers in another. In any microcarrier removal device embodiment, the mode of cell separation from the microcarriers can be chemical, biochemical (e.g., using enzymes such as trypsin or similar proteases), mechanical, thermal, optical, electrical, or any combination thereof. Furthermore, the separation force that physically separates the separated cells from the microcarriers can be optical, gravitational, electrical, magnetic, fluid, mechanical, or any combination thereof. In one embodiment used for sampling, the cell output stream is ultimately introduced into an analytical instrument (114). However, alternative embodiments may direct the cell stream or microcarrier stream to any number of analytical, purification, or collection devices / instruments. Furthermore, multiple cell types may be included in co-culture on the same microcarriers or cultured on different microcarriers. In this case, additional elements may be introduced to separate the separated cells based on size, density, dielectric potential, optical, magnetic, or other means.

[0060] A specific implementation of the combined microcarrier removal device is as follows: Figure 15AAs shown. The input to the device includes microcarriers (312) with attached cells or other biological products from a bioreactor (100) or other source. The microcarriers (312) enter the removal chamber via a large introduction channel (314), where optionally trypsin or any substance for separation and / or anti-adhesion is introduced via a separate input (302). The microcarriers then travel through a reaction zone (308), in which the biological products are separated from the microcarriers. The reaction zone (308) may be relatively longer or shorter than shown in Figure 15, and the curves are intended to illustrate this. The length and size of the reaction zone (308) can be related to time, channel length, inertial focusing, or any combination thereof. Although shown horizontally in Figure 15, the reaction zone can also be vertical (parallel to the direction of gravity), as shown in Figure 15. Figure 16 As shown, or at an angle relative to gravity, to facilitate efficient cell separation. At the end of the reaction zone (308), most (if not all) of the cells / bioproducts (306) have been separated from the microcarriers (304) and flow together in the same channel. As they enter the separation chamber (320), the microcarriers (304) separate from the cells (306) due to their larger size. The size of the chamber (320) allows the microcarriers to fall into the waste discharge channel (119) due to their higher settling velocity, while the free cells move to the dilution device (110) or another separate device.

[0061] Figure 15B Another embodiment of the device is shown, which includes an additional horizontal channel (307) containing few or trace amounts of cells, as the cells have also settled and moved to the dilution device (110) or to another separate device at a lower cell entry position.

[0062] Figure 15C Another embodiment is shown, in which an additional cell type (305), which is different in characteristics from (306), is grown together with (306) on a microcarrier. Although Figure 15C Cells growing together on the same microcarrier are shown, but they could also grow on completely different microcarriers, and the device would function in a similar manner. Cell types (305) could also be subpopulations of (306) that possess different properties in some desired way, such as increased yield of the target product or enhanced developmental capacity in the bioreactor. In this embodiment, one or more additional cell channels (309) are included to separate multiple cell types and populations based on differences in sedimentation velocities.

[0063] Figure 16An additional embodiment including a vertical reaction zone (308) is shown. In this embodiment, the separation chamber (320) remains horizontal and is configured such that the microcarriers (304) fall into a flow stream separate from the cells (306), which subsequently allows them to be separated into different outlet channels, in this embodiment, the different outlet channels being microcarrier waste (119) and a dilution device (110).

[0064] Figure 17 Another embodiment of the device is shown, in which the reaction zone (308) is vertical, but as the separated cells (306) and microcarriers (304) enter the separation zone (320), an applied force (325) is used to preferentially push the microcarriers (306) into the separated fluid flow and into the waste (119), while the free cells move to the dilution device (110) or another separate device. This separation occurs as the cells (306) and microcarriers (304) pass through the force interaction zone (330) and experience different forces. The illustrated embodiment shows an optical force that creates the force interaction zone (330) based on the shape of a beam. However, the force can also be electrical, magnetic, fluid, mechanical, or any combination thereof. As shown, the force (325) acts horizontally, opposite to the fluid flow as the cells (306) and microcarriers (304) enter the separation zone (320). However, forces can also act on these species before or during their transition from vertical direct current to horizontal flow, so the force (325) will act orthogonally or at an angle.

[0065] Figure 18 Another embodiment illustrating this is in which a force (325) acts at an angle to but in the flow direction, pushing the microcarrier (304) downward into the lower fluid flow, then out through the bottom of the chamber and into the waste (119). In this embodiment, the angle can be varied as needed to maximize separation efficiency.

[0066] Another implementation of the microcarrier separation device is as follows: Figure 19As shown. After the reaction zone (308) (which, as previously described, can be horizontal or vertical), free cells (306) and microcarriers (304) enter the separation zone (320), in which a higher-density fluid (350) is introduced from a lower channel that meets and combines with the upper channel carrying the cells (306) and microcarriers (304). The density of the fluid (350) will be higher than that of the fluid in which the cells (306) and microcarriers (304) travel, and due to the density difference between the two, the higher-density species (which, as shown, is a cell, but could actually be a cell or a microcarrier) descends below the density gradient (360), while the lower-density species remain in the upper portion of the channel. After a certain length of channel that allows separation to occur, the higher-density species exits through the lower channel, while the lower-density species exits through the upper channel. As shown, the lower-density microcarriers travel through to 119, while the higher-density cells move to the dilution device (110) or another separate device. Although shown as different interfaces, the density gradient can also be continuous rather than discrete, depending on the fluid composition and the geometry and operating conditions within the device.

[0067] Figure 20 It shows functions similar to Figure 19 Another embodiment of the device is shown. However, the higher-density fluid (350) is broken down into discrete plugs or droplets (365) by traveling through a higher-density liquid (e.g., an oil or aqueous two-phase system (ATPS)) of the separation phase. When the upper and lower channels of the device meet, the higher-density fluid remains in the discrete segment due to the difference in both density and phase, and cells (306) are able to fall into the fluid plugs or droplets (365), but cells (306) are prevented from entering (355) due to the phase difference. The size of the plugs or droplets (365) can also be adjusted to be small enough to prevent microcarriers (304) from falling in due to size exclusion.

[0068] Figure 21Another embodiment of the apparatus for removing microcarriers from cells is shown. Following the reaction zone (308) (which, as previously described, can be horizontal or vertical), free cells (306) and microcarriers (304) flow into the channel and encounter a selective barrier (370) that excludes only cells based on size. This barrier can be a mesh or membrane with pores of appropriate size to allow cells (306) to pass through, or it can be a series of pillars or other physical barriers spaced apart in such a way that only cells (306) are allowed to pass through. Microcarriers (304), due to their much larger size, cannot move through the barrier but instead slide down the barrier by sedimentation and exit the apparatus through the waste channel (119). Cells (306) move through the barrier (370) to a dilution device (110) or another separate device. The flow through the apparatus can be continuous or pulsating to remove any microcarriers (304) adhering to the barrier (370).

[0069] Figure 22 Another embodiment capable of separating free cells (306) from microcarriers (304) is shown. Following the reaction zone (308) (which, as previously described, can be horizontal or vertical), the free cells (306) and microcarriers (304) flow into a channel or series of channels designed to separate the cells based on inertial forces generated by the curvature and / or shape of the channel's cross-section. As the channel rotates, the microcarriers (304) move into different fluid layers and are thus able to be separated into individual channels capable of flowing to waste (119). The cells (306) remain in separate layers and move to a dilution device (110) or another separate device. The inertial force can also be combined with a separate force (e.g., an optical force (using a laser (320) with a beam overlapping the channel (325)), mechanical force, magnetic force, or electrical force) positioned in a manner that enhances the efficiency of the inertial separation and allows it to operate under a wider range of flow conditions.

[0070] This article provides a novel system and method for preparing biological samples for analysis, comprising the following steps: obtaining a sample from a container, processing the sample, and transporting the sample to an analytical instrument. The system includes tools for extracting the sample from the container, one or more control valves, one or more reagent addition, dilution, or concentration mechanisms, one or more mixing mechanisms, and an analytical instrument interface.

[0071] Novel systems and methods can be used to evaluate biological samples, including but not limited to cells, cell fragments, cell components, viruses, bacteria, microorganisms, pathogens, macromolecules, carbohydrates, genetic material, nucleic acids, DNA, RNA, transcription factors, amino acids, peptides, proteins, lipids, enzymes, metabolites, antibodies, or receptors. As envisioned herein, containers for removing biological samples are known to those skilled in the art and include bioreactors, flasks, bottles, test tubes, slides, bags, microtiter plates, microtiter dishes, multiwell plates, culture dishes, permeable carriers, etc. In various embodiments, the analytical instrument may include laser power analyzers, sequencing instruments, PCR analyzers, high-performance gas or liquid chromatography (HPLC) or mass spectrometry (MS) machines; in certain specific embodiments, the analytical instrument includes… The machine (LumaCyte, LLC., Virginia, USA). Additional features of the system include a sample extraction device comprising a sterile dip tube and / or a microcarrier separation device. As known to those skilled in the art, microcarriers are carrier matrices that allow the growth of adherent cells / bioparticles in a bioreactor; the system described herein is capable of separating adherent cells / bioparticles from microcarriers using methods including fluidic, enzymatic, biological, chemical, electrical, magnetic, thermal, optical, mechanical, or gravitational methods. In some embodiments, microcarriers are separated from bioparticles by various methods, including but not limited to: gravity in horizontal channels, using fluids of different densities due to density differences between cells, using active force in vertical channels with different outlet locations, using active force in horizontal channels with different outlet locations, using meshes based on size, angled meshes, columns, or other structures where the flow is continuous or pulsating, and / or using inertial fluid forces or inertia combined with others (e.g., optical or electrical) based on size to improve separation efficiency.

[0072] The system and method envisioned in this paper allow for the processing of biological particles from other sample components, thereby enabling purification, cleansing, enrichment, and chemical modification.

[0073] As envisioned herein, biological samples are transported from containers to analytical instruments using pressure or vacuum-driven flow via peristaltic pumps, syringe pumps, or diaphragm pumps, with the flow direction determined by mechanisms known to those skilled in the art, including, for example, valve configurations and / or piping.

[0074] Sampling can be continuous or segmented, and in some embodiments, the system may include a multiplexing system consisting of sampling systems, wherein samples are obtained from more than one bioreactor.

[0075] In some embodiments, dilution, mixing, and interaction occur within the same device.

[0076] In some embodiments, the systems and methods envisioned herein include one or more features that enable feedback monitoring of process control using a control system.

[0077] Additional features of some embodiments include microfluidic T-type mixing and dilution devices with microfluidic channels; such embodiments may also include configurations of basic T-type, offset T-type, parallel T-type, T-type with multiple discrete inputs, and T-type with multiplexed inputs combined into one.

[0078] In some embodiments, the system and method also include a sampling manifold chip / cup and an interface to an autosampler (and variants).

[0079] As used herein, the term bioparticle includes, but is not limited to, cells, cell fragments, cell components, viruses, bacteria, microorganisms, pathogens, macromolecules, sugars, genetic material, nucleic acids, DNA, RNA, transcription factors, amino acids, peptides, proteins, lipids, enzymes, metabolites, antibodies, receptors, analytes of interest, etc. Biological samples include specimens derived from living organisms, such as, but not limited to, blood, urine, tissues, organs, saliva, DNA / RNA, hair, nail clippings, or any other cells or fluids, whether for research purposes or as residual specimens collected during diagnostic, therapeutic, or surgical procedures.

[0080] As used herein, the term “microfluidic channel” includes openings, orifices, gaps, conduits, pathways, chambers, or recesses in a device, wherein the size of the microfluidic channel is sufficient to allow the passage or analysis of one or more biological particles.

[0081] As used herein, reagents and solutions suitable for use in this invention include any substances required for the analysis and processing of biological samples. Examples of such reagents and solutions include, but are not limited to, enzymes, fluorophores, oligonucleotides, primers, barcodes, buffers, deoxyribonucleotide triphosphates, detergents, lysis agents, reducing agents, chelating agents, oxidizing agents, nanoparticles, antibodies, enzymes, temperature-sensitive enzymes, pH-sensitive enzymes, photosensitizers, reverse transcriptases, proteases, ligases, polymerases, restriction enzymes, transposases, nucleases, protease inhibitors, and nuclease inhibitors.

[0082] As will be appreciated, the channels and / or connecting segments described herein can be coupled to any of a variety of different fluid sources or receiving components (including reservoirs, pipes, manifolds, or other fluid components of systems). Furthermore, the channel structures can have different geometries: for example, a microfluidic channel structure can have more than one channel connection point, and a microfluidic channel structure can have 2, 3, 4, or 5 (or more) channel segments. Additionally, fluid can be guided to flow along one or more channels or reservoirs via one or more fluid flow units. Fluid flow units can include compressors (e.g., providing positive pressure), pumps (e.g., supplying negative pressure), actuators, etc., to control the flow of fluid. Fluid can also be controlled or otherwise managed via applied pressure differential, centrifugal force, electric pumping, vacuum, capillary or gravity flow, etc.

Claims

1. A system for preparing biological samples for analysis, the system comprising: The biological sample, wherein the biological sample comprises one or more biological particles; A sample container configured to at least store or prepare the biological sample; A microcarrier separation device in fluid communication with the sample container, the microcarrier separation device being configured to separate at least one of the one or more biological particles from the microcarrier, the microcarrier separation device being further configured to separate the biological particles and the microcarrier into different channels, wherein the separation device utilizes density difference, size exclusion, fluid force, enzymatic reaction, biological process, chemical process, electric force, magnetic field, thermal disturbance, optical force, mechanical force, gravity, or a combination thereof to separate the biological particles and the microcarrier; One or more dilution devices in fluid communication with the microcarrier separation device, the one or more dilution devices being configured to receive the at least one biological particle, receive a solution comprising a diluent, a reagent, or both, dilute the at least one biological particle to a desired concentration, or discharge waste; and An interface in fluid communication with an analytical instrument, wherein the interface is configured to introduce the at least one biological particle at the desired concentration into the analytical instrument, wherein the analytical instrument includes a label-free laser-force cytology analyzer.

2. The system according to claim 1, wherein the one or more bioparticles comprise: Cells, cell fragments, cell components, viruses, bacteria, microorganisms, pathogens, macromolecules, sugars, genetic material, nucleic acids, DNA, RNA, transcription factors, amino acids, peptides, proteins, lipids, enzymes, metabolites, antibodies, or receptors.

3. The system according to claim 1, wherein the container comprises one or more bioreactors, flasks, bottles, test tubes, glass slides, bags, microtiter plates, microtiter dishes, multi-well plates, culture dishes, permeable carriers, or combinations thereof.

4. A method for preparing a biological sample for analysis, the method comprising: Provide the system as described in claim 1; The biological sample is obtained from the sample-containing container configured to: store or prepare the biological sample, wherein the biological sample contains biological particles; Processing the biological sample, wherein processing the biological sample includes separating at least one biological particle from other sample components, purifying the biological sample, enriching the biological sample, and chemically modifying the biological sample; and The biological sample is transported to the analytical instrument.

5. The method according to claim 4, further comprising purifying the sample pipeline through which the biological sample flows after the analytical instrument analyzes the biological sample.

6. The method of claim 4, further comprising using pressure or vacuum to drive the flow, via a peristaltic pump, syringe pump or diaphragm pump, to transport the biological sample from the sample container to the analytical instrument.

7. The method of claim 6, wherein the flow direction is further determined by valve configuration and piping.

8. The system of claim 1, wherein the sampling of the analytical instrument is continuous or wherein the sampling is segmented.

9. The system of claim 1, wherein the system is a multiplexing system including a sampling system, wherein samples are obtained from one or more bioreactors.

10. The method of claim 4, further comprising using the same apparatus for dilution, mixing, and interaction with the analytical instrument.

11. The system of claim 1, further comprising a process control system in communication with at least one of the sample container, separation device, one or more dilution devices, interface, analytical instrument, one or more control valves or combinations thereof.

12. The system of claim 1, wherein the one or more dilution devices, interfaces or both comprise a microfluidic T-type mixing and dilution device having microfluidic channels.

13. The system of claim 12, comprising a basic T-type, an offset T-type, a parallel T-type, a T-type having multiple discrete inputs, and a T-type having multiplexed inputs combined into one.

14. The system of claim 1, further comprising a sampling manifold chip / cup and an interface to an autosampler.

15. The system of claim 1, wherein the microcarriers are separated from the biological particles by gravity in a horizontal channel.

16. The system of claim 1, wherein fluids of different densities are used to separate the microcarriers from the biological particles due to density differences between cells.

17. The system of claim 1, wherein the microcarrier is separated from the bioparticles by an active force in vertical channels with different outlet positions.

18. The system of claim 1, wherein the microcarrier is separated from the bioparticles by an active force in a horizontal channel with different outlet positions.

19. The system of claim 1, wherein microcarriers are separated from the biological particles based on size using a mesh, angled mesh, column or other structure, wherein the flow is continuous or pulsating.

20. The system of claim 1, wherein inertial fluid force, optical force, electricity, or a combination thereof are used to separate the microcarrier from the biological particles to improve separation efficiency.

21. The system of claim 1, wherein the analytical instrument is configured to measure the velocity, size, centrifugation rate, or a combination thereof of the at least one biological particle in the biological sample.

22. The system of claim 1, further comprising a sample extraction device including a sterile immersion tube for extracting the biological sample from the sample containing container, wherein the sample extraction device is configured to obtain a plurality of biological samples from a plurality of containers.

23. The system of claim 1, wherein the microcarrier is separated from the biological particles based on size using the following method: i) Inertial hydrodynamics; and ii) Optical force, electric force, or optical force and electric force.

24. The system of claim 1, wherein the at least one biological particle is a cell.

25. The method of claim 4, wherein the at least one biological particle is separated by the microcarrier separation device in fluid communication with the sample containing container.

26. The method of claim 4, wherein processing the biological sample further comprises diluting the biological sample using the one or more dilution devices in fluid communication with the microcarrier separation device.

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