Microfluidic chip, system for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates, and system for automated media replenishment

Abstract

A microfluidic chip including a flow junction, which includes a primary inflow channel, a secondary inflow channel converging and meeting the primary inflow channel at an intersection, a primary outflow channel extending from the intersection to form a continuous line with the primary inflow channel, and a secondary outflow channel extending from the intersection, between the secondary inflow channel and the primary outflow channel, so as to form a first angle with the secondary inflow channel and a second angle with the primary outflow channel. A system for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates comprising the microfluidic chip. A system for automated media replenishment comprising the microfluidic chip.

Description

MICROFLUIDIC CHIP, SYSTEM FOR BUFFER EXCHANGE AND HARVESTING OF CELLS FROM MICROCARRIERS AND / OR CELL AGGREGATES, AND SYSTEM FOR AUTOMATED MEDIA REPLENISHMENTCross References to Related Applications

[0001] The present application claims the benefit of the Singapore Patent Application No. 10202403062S filed on 2 October 2024, the entire contents of which are incorporated herein by reference for all purposes.Technical Field

[0002] Various embodiments generally relate to a microfluidic chip. In particular, various embodiments generally relate to a microfluidic chip for buffer / media exchange and / or cell sorting / dissociation. Various embodiments generally also relate to a system for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates, the system including the microfluidic chip. Various embodiments generally further relate to a system for automated media replenishment, the system including the microfluidic chipBackground

[0003] Cell culture systems using microcarriers (MCs) or cell aggregates in bioreactors are widely used for cell expansion in tissue engineering and cell-based therapies. Typically, microcarriers are microbeads (- 100-300 pm) that can be fabricated using either synthetic (e g. dextran, glass, plastic, etc.) or natural materials (e.g. collagen, gelatin, etc.). These microcarriers generally provide large surface area for adherent cells to grow and expand in suspension culture in bioreactors On the other hand, cell aggregates are 3D clusters of cells that form naturally during culture in suspension-based systems. Cell aggregates are generally formed through cell-cell adhesion or intentionally through controlled processes in stem cell differentiation or spheroid formation. Conventional cell recovery from microcarriers or cell aggregates is usually based on manual sample washing and enzyme treatment (e.g. trypsin, collagenase, etc.), followed by centrifugation to quench the enzyme and membrane filtration to remove the microcarriers. The manual operations are time-consuming, prone to inconsistency with user handling, and may introduce the risk of contamination to the cultured cells. Besides, the commonly used sieve-based filtration using cell strainers of mesh size 40pm to 100 gm are non-scalable and not applicable to laboratory-scale productions. Although tangential flow filtration and alternating tangential flow filtration techniques have been developed to scale up the production and alleviate the microcarner deposition on the membrane, they are still facing membrane fouling and cell loss issues. Further, these singleuse products are mostly used for batch harvesting of bioreactor cultures, which will increase the cost of production if adapted in continuous cell manufacturing. In recent years, microfluidic devices have been developed to remove the microcarriers using inertial focusing. However, multiple passes are generally required to achieve high rate of cell recovery, and the prolonged exposure to the shear stress could be another concern that may affect cell properties and functionalities.

[0004] Accordingly, there is a need to provide an improved solution to address some of the problems above.Summary

[0005] According to various embodiments, there is provided a microfluidic chip including a flow junction, which includes a primary inflow channel, a secondary inflow channel converging and meeting the primary inflow channel at an intersection, a primary outflow channel extending from the intersection to form a continuous line with the primary inflow channel, and a secondary outflow channel extending from the intersection, between the secondary inflow channel and the primary outflow channel, so as to form a first angle with the secondary inflow channel and a second angle with the primary outflow channel.

[0006] According to various embodiments, there is provided a system for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates. The system including the microfluidic chip as described herein; a sample source connected to the sample inlet port of the first microfluidic chip; and an incubation module connected to the primary outlet port of the first microfluidic chip.

[0007] According to various embodiments, there is provided a system for automated media replenishment. The system including the microfluidic chip as described herein; a bioreactor connected to the sample inlet port of the microfluidic chip; a pump operable to pump and flow a sample from the bioreactor through the sample inlet port of the microfluidic chip; a fresh medium source connected to the sheath flow inlet port of the microfluidic chip; and a used- medium collection module connected to the secondary outlet port of the microfluidic chip,wherein the primary outlet port of the microfluidic chip is connected back to the bioreactor tank.Brief description of the drawings

[0008] Tn the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the invention. In the following description, various embodiments are described with reference to the following drawings, in which:FIG. 1 A shows a microfluidic chip according to various embodiments;FIG. IB shows a flowjunction of the microfluidic chip of FIG 1 A according to various embodiments;FIG 2 shows results of numerical simulation performed for the flow junction of the microfluidic chip of the various embodiments;FIG. 3 andFTG. 4 show the flow junction of the microfluidic chip of FIG 1 A according to various embodiments;FIG 5 A shows a microfluidic chip according to various embodiments;FIG. 5B shows a perspective view of the microfluidic chip of FIG 5 A according to various embodiments;FIG. 6A shows a microfluidic chip according to various embodiments;FIG. 6B shows a perspective view of the microfluidic chip of FIG 6A according to various embodiments;FIG. 6C shows an example of the flowrate reduction arrangement of the microfluidic chip of FIG 6A according to various embodiments;FIG. 6D shows an example of a series of ridges of a corrugated surface of a mixing channel of the microfluidic chip of FIG. 6A according to various embodiments;FIG. 6E shows another example of the series of ridges of the corrugated surface of the mixing channel of the micro fluidic chip of FIG 6 A according to various embodiments;FIG. 6F shows yet another example of the series of ridges of the corrugated surface of the mixing channel of the microfluidic chip of FIG. 6A according to various embodiments;FIG. 7 shows a system for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates according to various embodiments;FIG. 8A to FIG. 81 illustrate the state of microcarrier and cells as they flow through the system according to various embodiments;FIG. 9A and FIG. 9B show a study to demonstrate the buffer exchange capability of the flowjunction of the first microfluidic chip of the system of FIG. 7A to enhance downstream enzymatic cell dissociation according to various embodiments;FIG. 10A to FIG. 10C show a study to demonstrate the effects of sheath flow rate on buffer exchange and sample concentrating performance of the first microfluidic chip of the system of FIG 7A according to various embodiments;FIG. 11A and FIG. 11B show a study of the flow rates at all outlets of the first microfluidic chip of the system of FIG. 7A at different rate of flow of the sheath flow for optimizing the flow rate and concentration of enzymatic solution (e g trypsin) according to various embodiments;FIG. 12 A to FIG 12C show a study of the mixing performance of the first microfluidic chip of the system of FIG. 7A without and with the disturbance elements in the mixing channel (e g. staggered herringbone structures) of different repeated patterns according to various embodiments;FIG. 13 A to FIG 13E showa study demonstrating cell dissociation from cell aggregates and microcarriers achieved by the first microfluidic chip of the system of FIG 7A according to various embodiments;FIG. 14A to FIG. 14F show a study of the cell sorting / purification performance of the second microfluidic chip of the system of FIG 7A according to various embodiments;FIG. 15A to FIG. 15D show a study on the characterization of the cell purification performance of the second microfluidic chip of the system of FIG. 7A using a mixture of adipose-derived mesenchymal stem cells (ADSCs) and Cytodex-3 microcarriers, which are commonly used in tissue engineering, according to various embodiments;FIG. 16 shows further study and quantification to validate the vortex-assisted sample washing effect, i.e. buffer exchange efficiency, of the flow junction using the second microfluidic chip of the system of FIG. 7A at different rate of flow of the sheath flow according to various embodiments;FIG. 17A and FIG 17B show a study of cell recovery performance of the vortex generated at the flow junction of the microfluidic chip according to various embodiments;FIG. 18A and FIG. 18B show a study to validate the on-chip cell dissociation capability of the microfluidic chip of FIG. 6A according to various embodiments;FIG. 19A to FIG. 19E show a comparison between the manual dissociation, cell viability, cell recovery, cell metabolic activity, and stem cell biomarker expression as compared to the capability of the microfluidic chip of the various embodiments;FIG. 20A to FIG. 20C show the system of FIG. 7 being employed to harvest cells cultured on the surface of Cytodex-3 microcarriers according to various embodiments;FIG. 21 shows a system for automated media replenishment according to various embodiments;FIG. 22 shows a schematic diagram of a flow junction being used to study the effects of channel configuration for vortex generation according to various embodiments; andFIG. 23 A to FIG. 23F show experimental results for various configurations of the flow junction of FIG. 22 according to various embodiments.Detailed description

[0009] Embodiments described below in context of the apparatus are analogously valid for the respective methods, and vice versa. Furthermore, it will be understood that the embodiments described below may be combined, for example, a part of one embodiment may be combined with a part of another embodiment.

[0010] Itshouldbeunderstoodthattheterms“on”, “over”, “top”, “bottom”, “down”, “side”, “back”, “left”, “right”, “front”, “lateral”, “side”, “up”, “down” etc., when used in the following description are used for convenience and to aid understanding of relative positions or directions, and not intended to limit the orientation of any device, or structure or any part of any device or structure. In addition, the singular terms “a”, “an”, and “the” include plural references unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0011] “Cells” refer to biological units capable of growth, proliferation, and metabolic activity, including but not limited to stem cells, progenitor cells, differentiated cells, embryonic stem cells, placenta stem cells and genetically modified cells, whether derived from human or non-human sources. The term encompasses native, cultured, expanded, cryopreserved, genetically modified, or otherwise processed biological cells, including stem cells such as induced pluripotent stem cells (iPSCs), adipose-derived mesenchymal stem cells (ADSCs), or cancer stem cells used in microfluidic platforms for therapeutic, diagnostic, or research applications.

[0012] “Microcarriers” refer to particulate substrates that provide a surface or support for the attachment and growth of adherent cells in suspension-based culture systems. The term encompasses a variety of materials, including but not limited to polymers, glass, hydrogel, or composite materials, which may be coated or functionalized with extracellular matrix proteins,peptides, or other surface chemistries to enhance cell adhesion, proliferation, and viability. Microcarriers may be spherical or irregular in shape, porous or non-porous, and may be biodegradable or non-biodegradable. The term includes commercially available or custom- fabricated micro carriers, whether sterile, pre-treated, or modified, for use in bioreactors, microfluidic systems, or other culture platforms for therapeutic, diagnostic, or research applications.

[0013] “Cell aggregates” refer to clusters of two or more cells that are physically associated through cell-cell adhesion, extracellular matrix deposition, or other biological interactions, and that function as a collective unit in culture or in vivo. The term encompasses naturally occurring or artificially induced aggregates, including spheroids, organoids, embryoid bodies, and microtissues, formed from homogeneous or heterogeneous cell populations Cell aggregates may be generated through spontaneous self-assembly, scaffold-based culture, microcarrier culture, microfluidic manipulation, or other aggregation techniques. They may vary in size, morphology, and cellular composition, and may be used for therapeutic, diagnostic, research, or manufacturing purposes, including tissue engineering, regenerative medicine, and high- throughput screening.

[0014] Various embodiments generally relate to a microfluidic chip. Tn particular, various embodiments generally relate to a microfluidic chip for buffer / media exchange and / or cell sorting / dissociation. According to various embodiments, the microfluidic chip may include a network of channels for manipulating fluids. Tn the various embodiments, buffer / media exchange may include exchanging or replacing a fluid in a fluid flow with another fluid. The fluid may be buffer solution and / or medium (such as culture medium etc ). Further, the buffer / media exchange may occur in a continuous manner instead of by batch. Accordingly, in the various embodiments, the microfluidic chip may be configured to enable buffer / media exchange to occur continuously in an uninterrupted manner, whereby the fluid continues to flow in a steady and constant manner as the fluid is being exchanged or replaced. In the various embodiments, cell sorting may include separating and / or purifying cells from other particles (e g. microcarriers) suspended in the fluid flow. Similarly, cell sorting may occur in a continuous manner instead of by batch. Accordingly, in the various embodiments, the microfluidic chip may be configured to enable cell sorting to occur continuously in an uninterrupted manner, whereby the fluid continues to flow in a steady and constant manner as the cells suspended in the fluid is being separated and / or purified. In various embodiments, cell dissociation may include detaching cells from microcarriers and / or separating cells from each other from cell aggregates so as to result in individual cells being suspended in the fluid flow.According to various embodiments, the microfluidic chip may be configured to enable cell dissociation to occur continuously in an uninterrupted manner, whereby the fluid continues to flow in a steady and constant manner as the cells are detached from the microcarriers and / or separated from cell aggregates.

[0015] Various embodiments generally also relate to a system for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates, the system including the microfluidic chip of the various embodiments. According to various embodiments, the system may be configured to perform a series of automated processes including washing of sample (e g. exchanging of culture medium with buffer solution), and / or enzymatic cell dissociation from microcarriers in the sample with incubation, and / or cell sorting / purifi cation for separating the dissociated cells and the microcarriers suspended in the fluid According to various embodiments, the system may include a first microfluidic chip for washing of sample and adding of enzymatic solution, an incubation module, and a second microfluidic chip for cell sorting / purification. The first microfluidic chip, the incubation module and the second microfluidic chip may be connected in series such that the system may perform the steps of washing of sample, enzymatic cell dissociation, incubation, and cell sorting / purification in sequence and in a continuous manner Tn the various embodiments, with the first microfluidic chip configured for continuous buffer / media exchange, the incubation module configured for inline incubation (i.e. incubation while fluid continue to flow), and the second microfluidic chip configured for continuous cell sorting / purification, the system may achieve sequential and continuous performance of the various steps as the fluid flowthrough the system. In the various embodiments, the fluid flow may serve as a conveyancing mechanism for moving samples through the various apparatus such that the various steps may be performed in sequence continuously. According to various embodiments, for harvesting of cells from cell aggregates, the system may perform the steps of washing of sample, enzymatic cell dissociation, and incubation. Accordingly, the system may include a microfluidic chip for washing of sample and adding of enzymatic solution, and an incubation module to complete the cell dissociation process. Hence, individual separated cells may be collected from the incubation module without requiring further cell sorting / purification.

[0016] Various embodiments generally further relate to a system for automated media replenishment, the system including the microfluidic chip. The system may include a bioreactor containing a medium, whereby a biological process or cell culture is taking place therein. The system may further include a biosensor for measuring one or more chemical or biological property of the sample in the bioreactor. When a measurement of the one or more chemical orbiological property of the sample is outside a predetermined range, a pump may be activated to load the culture into the microfluidic chip. At the same time, fresh medium may be provided to the microfluidic chip. As the culture flows through the microfluidic chip, media exchange may occur continuously in an uninterrupted manner such that the medium in the culture is being exchanged with the fresh medium. Hence, the medium of the culture would be exchanged or replaced with a fresh medium in a continuous manner as the culture flow steadily and constantly through the microfluidic chip. The culture with the fresh medium may then be returned to the bioreactor.

[0017] According to various embodiments, the microfluidic chip may include a flow junction (e.g. at least a four-way flow junction or a multi-way flow junction) configured to generate a vortex-assisted hydrodynamic force capable of enabling buffer / media exchange and / or cell sorting / purification. The flow junction may include a primary inflow channel, a secondary inflow channel, a primary outflow channel and a secondary outflow channel. The primary inflow channel may flow a sample flow (or culture) into and / or through the flow junction, and the secondary inflow channel may flow a sheath flow (e.g. buffer solution / medium) into and / or through the flow junction. Based on the configuration of the primary inflow channel and the secondary inflow channel, the sample flow and the sheath flow may meet at the flowj unction to generate a vortex at the flow junction. The vortex may generate a force to direct (at least a portion of) the fluid of the sample flow into the secondary outflow channel and direct (at least a portion of) the fluid of the sheath flow into the primary outflow channel. At the same time, the cells and particles suspended in the sample flow may be directed from the primary inflow channel into the primary outflow channel, hi light of the effect of the flow junction, the microfluidic chip with the flow junction may be configured for buffer / media exchange and / or cell sorting / purification.

[0018] Accordingly, various embodiments may provide an integrated microfluidic platform (e g. a system) to achieve automated cell harvesting from microcarriers with high throughput and high cell recovery rate. The platform may include a first microfluidic chip with the flow junction, the first microfluidic chip being configured for on-chip cell dissociation after vortex- assisted microcarrier washing (e.g. buffer exchange). The vortex-assisted hydrodynamic force generated in the flow junction may enable washing of microcarriers (e.g. buffer exchange) to enhance enzymatic cell dissociation efficiency (e.g. trypsin efficiency). The on-chip microcarrier washing for the microcarriers enabled by the vortex-assisted first microfluidic chip may minimize the effects of culture media on enzymatic cell dissociation effect, thereby improving the cell dissociation efficiency and saving manpower. Further, the platform mayinclude a second microfluidic chip with the flow junction, the second microfluidic chip being configured for vortex-assisted cell purification (e g. cell sorting). The cell purification may separate dissociated cells from empty carriers in a membrane-free (or filter free) manner, thus allowing continuous processing of high-concentration samples at high through put with high cell recovery and minimal / negligible / no fouling issues. During cell purification, the dissociated cells (which are smaller) may be deflected towards the center of the microvortex while the microcarriers (which are larger) may experience higher inertia and remain in their onginal trajectory at the channel center. Thus, separation of the dissociated cells and the microcarriers may be achieved. Further, by varying the channel outlet hydraulic resistance, efficient sorting of cells into a smaller side outlet associated with the secondary outflow channel while the microcarriers are eluted into an outlet associated with the primary outflow channel as waste may be enabled.

[0019] Tn the various embodiments, the system may recover 90% of the cells, by the actively generated vortex when the samples flowing through the vortex region, within a few milliseconds, thus significantly reducing the duration of exposure to the high shear stress Besides, consistent high cell recoveiy using samples with high cell concentrations (e g. 10 million cells per ml,, a typical concentration in bioreactors) may be achieved by the various embodiments, whereas other conventional technologies may mostly process sample cell concentrations less than 1 million per mL The conventional inertial microfluidic devices focus target cells to a single equilibrium position and, hence, is limited to operate at low cell concentrations (1 million / ml.,) due to steric hindrance On the contrary, various embodiments are configured to deflect high concentrations of cells towards the microvortex centre and, thus, may directly process bioreactor samples of high cell densities (-10 million / mL). In addition, the cell recovery and microcarrier removal performance in the various embodiments may be easily manipulated or varied by adjusting vortex strength via changing the flow rate of the sheath flow; thus enabling the capability of using a single device for various (or different) samples.

[0020] FIG. 1A shows a microfluidic chip 100 according to various embodiments FIG. IB shows a flow junction 110 of the microfluidic chip 100 of FIG. 1A according to various embodiments. According to various embodiments, the flow junction 110 of the microfluidic chip 100 may be configured to generate the vortex-assisted hydrodynamic force. Accordingly, the microfluidic chip 100 may be utilized for vortex-assisted buffer / media exchange and / or vortex-assisted cell sorting / purification. Hence, the microfluidic chip 100 may be part of asystem for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates. The microfluidic chip 100 may also be part of a system for automated media replenishment.

[0021] According to various embodiments, the microfluidic chip 100 may include the flow junction 110. The flow junction 110 may include a primary inflow channel 112 (or a first channel), a secondary inflow channel 114 (or a second channel), a primary outflow channel 116 (or a third channel), and a secondary outflow channel 118 (or a fourth channel). According to various embodiments, the pnmary inflow channel 112 and the secondary inflow channel 114 may converge and meet at an intersection 115. Accordingly, the primary inflow channel 112 and the secondary inflow channel 114 may merge at the intersection 115, and the primary inflow channel 112 and the secondary inflow channel 114 may form an angle therebetween. For example, the angle therebetween may be an angle in a range from 45° to 135°, inclusive; or a range from 50° to 150°, inclusive; or a range from 60° to 120°, inclusive. In some embodiments, for example as shown in FIG. IB, the primary inflow channel 112 and the secondary inflow channel 1 14 may be substantially perpendicular (i.e. the angle therebetween may be 90°) with respect to each other. Accordingly, the primary inflow channel 112 and the secondary inflow channel 114 may meet at right angle with respect to each other. Hence, according to some embodiments, the secondary inflow channel 114 may be converging and meeting the primary inflow channel 112 at the intersection 115 perpendicularly with respect to each other.

[0022] According to various embodiments, the primary outflow channel 1 16 may be extending from the intersection 115 to form a continuous line with the primary inflow channel 112. Accordingly, the primary inflow channel 112 and the primary outflow channel 116 may form a continuous passage for fluid and / or particles to flow from the primary inflow channel 112 through the intersection 115 and into the primary outflow channel 116. For example, the primary outflow channel 116 may form an angle with the primary inflow channel 112, the angle being in a range from 135° to 180°, inclusive; or a range from 140° to 180°, inclusive; or a range from 155° to 180°, inclusive. According to some embodiments, for example as shown in FIG IB, the primary outflow channel 116 and the primary inflow channel 112 may be aligned with each other so as to form the continuous straight line (i.e. the angle therebetween may be 180°). Hence, the primary outflow channel 116 and the primary inflow channel 112 may be in a straight line without forming any obtuse or acute or perpendicular angle (or the primary outflow channel 116 and the primary inflow channel 112 may form a straight angle measuring 180°). Since the secondary inflow channel 114 may be at an angle and / or perpendicular to the primary inflow channel 112, the secondary inflow channel 114 may also be at an angle and / orperpendicular to the pnmary outflow channel 116. Accordingly, when the secondary inflow channel 114 is perpendicular to the primary inflow channel 112, the primary outflow channel 116 and the secondary inflow channel 114 may be at right angle with respect to each other. Hence, in such a configuration, the primary outflow channel 116 may be extending from the intersection 115 perpendicularly with respect to the secondary inflow channel 114.

[0023] According to various embodiments, the secondary outflow channel 1 18 may be extending from the intersection 115 so as to form a first angle with the secondary inflow channel 114 and a second angle with the primary outflow channel 116. Accordingly, the secondary outflow channel 1 18 may be extending between the secondary inflow channel 1 14 and the primary outflow channel 1 16 so as to divide an angle (or a right angle) between the secondary inflow channel 1 14 and the primary outflow channel 1 16 into the first angle and the second angle. Hence, the secondary outflow channel 118 may be extending from the intersection 1 15 in a diverging manner with respect to the primary outflow channel 1 16. For example, the first angle may be in a range from 30° to 105°, inclusive, and the second angle may be in a range from 30° to 90°, inclusive. Hence, in some embodiments, the first angle may be an obtuse angle and the second angle may be an acute angle, or the first angle may be an acute angle and the second angle may be an acute angle, or the first angle may be a right angle and the second angle may be an acute angle, or the first angle may be an acute angle and the second angle may be a right angle. According to some embodiments, for example as shown in FIG IB, the first angle formed between the secondary outflow channel 1 18 and the secondary inflow channel 114 may be an acute angle, and the second angle formed between the secondary outflow channel 118 and the primary outflow channel 116 may be an acute angle.

[0024] According to some embodiments, the flow junction 110 may be a four-way flow junction, for example as shown in FIG. 1A and FIG IB, having the primary inflow channel 112, the secondary inflow channel 114, the primary outflow channel 116, and the secondary outflow channel 118. According to some embodiments, the flow junction 110 may be at least a four-way flow junction. According to some embodiments, the flow junction 110 may be a multi-way flow junction with at least four channels (i.e. the primary inflow channel 112, the secondary inflow channel 114, the primary outflow channel 116, and the secondary outflow channel 118). In the various embodiments, the flow junction 110 may receive two separate fluid inflows, via the primary inflow channel 112 and the secondary inflow channel 114, into the intersection 115, and generate two separate fluid outflows along the primary outflow channel 116 and the secondary outflow channel 118.

[0025] According to some embodiments, for example as shown in FIG. IB, the primary inflow channel 112, the secondary inflow channel 114, the primary outflow channel 116, and the secondary outflow channel 118 may be radiating away from the intersection 115 in a manner whereby the primary inflow channel 112 and the primary outflow channel 116 are extending directly opposite and away from each other, the secondary inflow channel 114 is extending perpendicularly with respect to both the primary inflow channel 1 12 and the primary outflow channel 116, and the secondary outflow channel 118 is extending within a quadrant formed by the primary outflow channel 116 and the secondary inflow channel 114. Accordingly, the secondary outflow channel 1 18 may be angularly spaced, by the second acute angle, from the primary outflow channel 1 16; the secondary inflow channel 1 14 may be angularly spaced, by the first acute angle, from the secondary outflow channel 1 18; and the primary inflow channel 112 may be angularly spaced, by a right angle, from the secondary inflow channel 1 14. Hence, the primary inflow channel 1 12 and the primary outflow channel 1 16 may form a continuous straight channel, with the secondary inflow channel 1 14 being a channel extending perpendicularly from the straight channel so as to for a ‘T’ shape arrangement with the primary inflow channel 1 12 and the primary outflow channel 1 16, and with the secondary outflow channel 118 extending diagonally from the ‘T’ shape arrangement so as to be between the secondary inflow channel 114 and the primary outflow channel 116.

[0026] Referring to FIG. IB, according to various embodiments, the flow junction 110 of the microfluidic chip 100 may generate a vortex 125 within the flow junction 1 10. To generate the vortex 125 within the flow junction 110, a sample flow 122 (i.e. a first fluid flow) may be provided to flow into the flow junction 110 via the primary inflow channel 112 and a sheath flow 124 (i.e. a second fluid flow) may be provided to flow into the flowjunction 110 via the secondary inflow channel 114. As the primary inflow channel 112 and the secondary inflow channel 114 are at an angle and / or perpendicular to each other, the sample flow 122 and the sheath flow 124 may meet at an angle and / or at right angle with respect to each other. When the sample flow 122 and the sheath flow 124 meet at an angle and / or at right angle, the vortex 125 may be generated immediately adjacent to the sheath flow 124 after the confluence of the sample flow 122 and the sheath flow 124. Accordingly, with the secondary outflow channel 118 extending from the intersection 115 and angularly spaced, by the first acute angle, from the secondary inflow channel 114, the vortex 125 may be at an entrance of the secondary outflow channel 118 (i.e. at the starting point of the secondary outflow channel 118). According to various embodiments, the vortex 125 generated at the flow junction 110 may cause or direct (at least a portion of) the fluid 121 of the sample flow 122 (i.e. from the primary inflow channel112) into the secondary outflow channel 118 and (at least a portion of) the fluid 121 of the sample flow 122 (i.e. from the primary inflow channel 112) into the primary outflow channel 116. Similarly, the vortex 125 generated at the flow junction 110 may cause or direct (at least a portion of) the fluid 123 of the sheath flow 124 (i.e. from the secondary inflow channel 114) into the primary outflow channel 116 and (at least a portion of) the fluid 123 of the sheath flow 124 (i.e. from the secondary inflow channel 114) into the secondary outflow channel 118. Accordingly, a first blended flow 126 that may be a blend of the sample flow 122 and the sheath flow 124 (at a first ratio) may flow into the primary outflow channel 116, and a second blended flow 128 that may be a blend of the sample flow 122 and the sheath flow 124 (at a second ratio) may flow into the secondary outflow channel 1 18. According to various embodiments, a magnitude or size of the vortex 125 generated may be varied by varying difference in flow rate between the sample flow 122 and the sheath flow 124. According to various embodiments, due to the change in magnitude or size of the vortex 125 generated at the flow junction 110 cause by the difference in the flow rate between the sample flow 122 and the sheath flow 124, a proportion of the fluid 121 of the sample flow 122 being directed into the secondary outflow channel 118 and the primary outflow channel 116 may be adjusted and / or varied. Similarly, a proportion of the fluid 123 of the sheath flow 124 being directed into the primary inflow channel 116 and the secondary outflow channel 118 may also be adjusted and / or varied. With the proportion of the fluid 121 of the sample flow 122 directed into the secondary outflow channel 118 and the primary outflow channel 1 16 being adjustable as well as the proportion of the fluid 123 of the sheath flow 124 directed into the primary inflow channel 116 and the secondary outflow channel 118 being adjustable, the microfluidic chip 110 having the flow junction 110 may be operable to perform buffer / media exchange and / or cell sorting / purification by selecting the type of flow to be provided via the primary inflow channel 112 and via the secondary inflow channel 114, as well as selecting the difference in flow rate between the flow via the primary inflow channel 112 (i.e. sample flow 122) and via the secondary inflow channel 114 (i.e. sheath flow 124).

[0027] According to some embodiments, as an example, the difference in the flow rate between the sample flow 122 and the sheath flow 124 may be such that the flow rate of the sheath flow 124 may be equal to or greater than 3 times of, or between 2 to 5 times of, or between 3 to 5 times of, or approximately 4 times of, or approximately 4.5 times of the flow rate of the sample flow 122 in order to generate the vortex 125.

[0028] FIG. 2 shows results of numerical simulation performed for the flow junction 110 (e g. a four-way flow junction as shown in FIG. 1 A and FIG. IB) of the microfluidic chip 100of the various embodiments. In the numerical simulation, the sample flow 122 provided along the primary inflow channel 112 was set as 500 pL / min. The sheath flow 124 provided along the secondary inflow channel 114 was varied and its effects on the fluid flow were analyzed by the flow streamlines (see sheath flow streamline and sample flow streamline in FIG. 2). As shown in FIG. 2, the rate of flow of the sheath flow 124 was increased from 1000 μL / min to 2500 pL / min. As the difference in the rate of flow between the sample flow 122 and the sheath flow 124 increases, the magnitude and size of the vortex 125 increases. Further, as also shown, as the rate of flow of the sheath flow 124 was increased from 1000 pL / min to 2000 pL / min, the vortex 125 drove more and more fluid 121 from the sample flow 122 into the secondary outflow channel 1 18 (e g. side channel), while more fluid 123 of the sheath flow 124 entered the primary outflow channel 1 16 (e g main channel). As the rate of flow of the sheath flow 124 further increased to 2500 pL / min, the strength of the vortex 125 became higher, which would reduce the fluid 121 of the sample flow 122 directed into the secondary outflow channel 1 18 and the fluid 123 of the sheath flow 124 directed into the primary outflow channel 1 16 (i.e. reduce the buffer / media exchange performance). Accordingly, the numerical simulation demonstrated that the rate of flow of the sheath flow 124 may be optimized based on the rate of flow of the sample flow 122 to achieve optimum buffer / media exchange performance and / or cell sorting / purifi cation performance.

[0029] Referring back to FIG. 1A and FIG. IB, according to various embodiments, the primary inflow channel 1 12 may be wider than the secondary inflow channel 1 14. Accordingly, a width of the primary inflow channel 112 may be wider than a width of the secondary inflow channel 114. Further, a cross-sectional area of the primary inflow channel 112 may be wider than a cross-sectional area of the secondary inflow channel 114.

[0030] According to some embodiments, the primary inflow channel 112 may be 1.5 to 10 times, or 1.5 to 8 times, or 1.5 to 5 times, larger than a particle in the sample flow 122. For example, a width of the primary inflow channel 112 may be 1.5 to 10 times, or 1.5 to 8 times, or 1.5 to 5 times wider than a width of the particle in the sample flow 122. As another example, a cross-sectional area of the primary inflow channel 112 may be 1.5 to 5 times larger than a cross-sectional area of the particle in the sample flow 122.

[0031] According to various embodiments, the primary outflow channel 116 maybe wider than the primary inflow channel 112. Accordingly, a width of the primary outflow channel 116 may be wider than the width of the primary inflow channel 112. Further, a cross-sectional area of the primary outflow channel 116 may be wider than the cross-sectional area of the primary inflow channel 112.

[0032] According to various embodiments, the primary outflow channel 116 maybe wider than the secondary outflow channel 118. Accordingly, the width of the primary outflow channel 116 may be wider than a width of the secondary outflow channel 118. Further, the cross- sectional area of the primary outflow channel 116 may be wider than a cross-sectional area of the secondary outflow channel 118.

[0033] According to various embodiments, the secondary outflow channel 1 18 maybe wider than the secondary inflow channel 114. Accordingly, the width of the secondary outflow channel 118 may be wider than the width of the secondary inflow channel 114. Further, the cross-sectional area of the secondary outflow channel 1 18 may be wider than the cross- sectional area of the secondary inflow channel 1 14.

[0034] According to various embodiments, the primary outflow channel 1 16 may be tapering in a direction extending away from the intersection 115. Accordingly, the width of the primary outflow channel 1 16 may become narrower or reduced the further it is from the intersection 1 15 (i.e. the width reduces along a length of the primary outflow channel 1 16 as it extends away from the intersection 1 15). Further, the cross-sectional area of the primary outflow channel 1 16 may become narrower or smaller the further it is from the intersection 1 15 (i.e. the size of the cross-sectional area reduces along the length of the primary outflow channel 116 as it extends away from the intersection 115).

[0035] According to various embodiments, the secondary outflow channel 118 may be tapering in a direction extending away from the intersection 1 15. Accordingly, the width of the secondary outflow channel 118 may become narrower or reduced the further it is from the intersection 115 (i.e. the width reduces along a length of the secondary outflow channel 118 as it extends away from the intersection 115). Further, the cross-sectional area of the secondary outflow channel 118 may become narrower or smaller the further it is from the intersection 115 (i.e. the size of the cross-sectional area reduces along the length of the secondary outflow channel 118 as it extends away from the intersection 115).

[0036] According to various embodiments, the microfluidic chip 100 may include a sample inlet port 132 (or a first inlet port) upstream of the primary inflow channel 112 of the flow junction 110. The sample inlet port 132 may serve as an inlet for providing the sample flow 122 into the microfluidic chip 100 for flowing to the primary inflow channel 112 of the flow junction 110. Hence, the sample flow 122 may enter the microfluidic chip 100 via the sample inlet port 132, flow to the primary inflow channel 112 and flow into the flow junction 110 via the primary inflow channel 112. Accordingly, the sample inlet port 132 may be an entry point for the sample flow 122 and the sample inlet port 132 may be in fluid communication with theprimary inflow channel 112 for flowing the sample flow 122 into the flow junction 110. Therefore, in the various embodiments, a bioreactor may be connected to the sample inlet port 132 so as to provide the culture from the bioreactor as the sample flow 122. When the sample flow 122 is the culture from the bioreactor, the sample flow 122 may include culture medium (as the fluid 121 of the sample flow 122) as well as suspended substances in the culture medium. The suspended substances may include, but not limited to, cells and / or microorganism and / or particles and / or microcarriers and / or cell-microcarner complex and / or cell aggregates.

[0037] According to vanous embodiments, the microfluidic chip 100 may include an inertial focusing channel segment 133 between the sample inlet port 132 and the primary inflow channel 1 12 of the flowjunction 1 10. According to various embodiments, the inertial focusing channel segment 133 may be a meandering or serpentine or winding or sinuous channel. Accordingly, as the sample flow 122 flows through the inertial focusing segment 133, the suspended substances in the sample flow 122 may migrate or focus or align or position across streamlines towards a center streamline of the sample flow 122. In the various embodiments, the meandering or serpentine or winding or sinuous channel of the inertial focusing channel segment 133 may cause an inertial force to drive the suspended substances in the sample flow 122 to move or concentrate inwards of the sample flow 122 towards the channel center.

[0038] According to various embodiments, with the inertial focusing channel segment 133 between the sample inlet port 132 and the primary inflow channel 112 of the flowjunction 110, the sample flow 122 may undergo inertial focusing before reaching the primary inflow channel 112 of the flow junction 110. Accordingly, when the sample flow 122 reaches the primary inflow channel 112 of the flow junction 110, the suspended substances of the sample flow 122 may have been arranged along the center of the channel such that the sample flow 122 may flow along the primary inflow channel 112 of the flow junction 110 with the suspended substances along the center of the primary inflow channel 112.

[0039] According to various embodiments, the microfluidic chip 100 may include a sheath flow inlet port 134 (or a second inlet port) upstream of the secondary inflow channel 114 of the flow junction 110. The sheath flow inlet port 134 may serve as an inlet for providing the sheath flow 124 into the microfluidic chip 100 for flowing to the secondary inflow channel 114 of the flow junction 110. Hence, the sheath flow 124 may enter the microfluidic chip 100 via the sheath flow inlet port 134, flow to the secondary inflow channel 114 and flow into the flow junction 110 via the secondary inflow channel 114. Accordingly, the sheath flow inlet port 134 may be an entry point for the sheath flow 124 and the sheath flow inlet port 134 may be in fluid communication with the secondary inflow channel 114 for flowing the sheath flow 124 intothe flow junction 110. Therefore, in the various embodiments, a fluid source (e g. a buffer solution and / or medium solution and / or washing solution etc.) may be connected to the sheath flow inlet port 134 so as to provide the fluid (e g. solution) as the sheath flow 124.

[0040] According to various embodiments, the microfluidic chip 100 may include a flow channel 135 between the sheath flow inlet port 134 and the secondary inflow channel 114 of the flow junction 110. The flow channel 135 may serve to fluidly connects the sheath flow inlet port 134 and the secondary inflow channel 114. Accordingly, the flow channel 135 may directly flow the sheath flow 124 entenng the micro fluidic chip 100 via the sheath flow inlet port 134 to the secondary inflow channel 1 14 such that the secondary inflow channel 114 may flow the sheath flow 124 into the flow junction 1 10. According to various embodiments, the flow channel 135 may include a right-angle bend channel. The sheath flow inlet port 134 may be at the first end of the right-angle bend channel and the secondary inflow channel 114 may be at the second end of the right-angle bend channel.

[0041] According to various embodiments, the microfluidic chip 100 may include a primary outlet port 136 (or a first outlet port) downstream of the primary outflow channel 116 of the flow junction 1 10. The primary outlet port 136 may serve as an outlet for a primary output flow to exit the microfluidic chip 100. According to various embodiments, the first blended flow 126, including a certain portion of the sample flow 122 and a certain portion of the sheath flow 125, may exit the flow junction 110 via the primary outflow channel 116 of the flow junction 110. The first blended flow 126 may then flow through the remaining portion of the micro fluidic chip 100 and subsequently exit the microfluidic chip 100 via the primary outlet port 136. The flow that exits the primary outlet port 136 may be considered as the primary output flow. Accordingly, the primary outlet port 136 may be an exit -point to output the primary output flow from the microfluidic chip 100 and the primary outlet port 136 may be in fluid communication with the primary outflow channel 116 for flowing the first blended flow 126 out of the flow junction 110 so as to subsequently flow through the remaining portion of the microfluidic chip 100 and eventually exit via the primary outlet port 136 of the microfluidic chip 100 as the primary output flow.

[0042] According to various embodiments, the microfluidic chip 100 may include a secondary outlet port 138 (or a second outlet port) downstream of the secondary outflow channel 118 of the flow junction 110. The secondary outlet port 138 may serve as an outlet for a secondary output flow to exit the microfluidic chip 100. According to various embodiments, the second blended flow 128, including a certain portion of the sample flow 122 and a certain portion of the sheath flow 125, may exit the flow junction 110 via the secondary outflowchannel 118 of the flow junction 110. The second blended flow 128 may then flow through the remaining portion of the microfluidic chip 100 and subsequently exit the microfluidic chip 100 via the secondary outlet port 138. The flow that exits the secondary outlet port 138 may be considered as the secondary output flow. Accordingly, the secondary outlet port 138 may be an exit point to output the secondary output flow from the microfluidic chip 100 and the secondary outlet port 138 may be in fluid communication with the secondary outflow channel 118 for flowing the second blended flow 128 out of the flowjunction 110 so as to subsequently flow through the remaining portion of the microfluidic chip 100 and eventually exit via the secondary outlet port 138 of the microfluidic chip 100 as the secondary output flow.

[0043] According to various embodiments, the primary outlet port 136 may be larger than the secondary outlet port 138 Accordingly, a width (or a diameter) of the primary outlet port 136 may be larger than a width (or a diameter) of the secondary outlet port 138. Further, a cross-sectional area of the primary outlet port 136 may be larger than a cross-sectional area of the secondary outlet port 138.

[0044] According to various embodiments, the microfluidic chip 100 may include an inertial focusing channel segment 139 between the secondary outlet port 138 and the secondary outflow channel 1 18 of the flow junction 1 10. According to various embodiments, the inertial focusing channel segment 139 therebetween may be a meandering or serpentine or winding or sinuous channel Accordingly, as the second blended flow 128 flows through the inertial focusing segment 139, any suspended substances in the second blended flow 128 may migrate or focus or align or position across streamlines towards a center streamline of the second blended flow 128. In the various embodiments, the meandering or serpentine or winding or sinuous channel of the inertial focusing channel segment 139 may cause an inertial force to drive the suspended substances in the second blended flow 128 to move or concentrate inwards of the second blended flow 128 towards the channel center.

[0045] According to various embodiments, the microfluidic chip 100 may include an inertial focusing channel segment 137 between the primary outlet port 136 and the primary outflow channel 116 of the flow junction 110. According to various embodiments, the inertial focusing channel segment 137 therebetween may be a meandering or serpentine or winding or sinuous channel Accordingly, as the first blended flow 126 flows through the inertial focusing segment 137, any suspended substances in the first blended flow 126 may migrate or focus or align or position across streamlines towards a center streamline of the first blended flow 126. In the various embodiments, the meandering or serpentine or winding or sinuous channel of the inertial focusing channel segment 137 may cause an inertial force to drive the suspendedsubstances in the first blended flow 126 to move or concentrate inwards of the first blended flow 126 towards the channel center.

[0046] According to various embodiments, in the microfluidic chip 100 as shown in FIG. 1A, the first blended flow 126 from the primary outflow channel 116 of the flow junction 110 may flow through the inertial focusing channel segment 137 and exit the microfluidic chip 100 via the primary outlet port 136 as the primary output flow. According to various embodiments, in the microfluidic chip 100 as shown in FIG. 1A, the second blended flow 128 from the secondary outflow channel 118 of the flow junction 110 may flow through the inertial focusing channel segment 139 and exit the microfluidic chip 100 via the secondary outlet port 138 as the secondary output flow.

[0047] According to various embodiments, the rate of flow of the sheath flow 124 provided to the microfluidic chip 100, via the sheath flow inlet port 134, may be optimized relative to the rate of flow of the sample flow 122 provided to the microfluidic chip 100, via the sample inlet port 132 such that the sheath flow 124 (from the secondary inflow channel 114) and the sample flow 122 (from the primary inflow channel 112) may meet at the flow junction 1 10 to generate the vortex 125 for directing most of the fluid 123 of the sheath flow 124 into the primary outflow channel 1 16 and most of the fluid 121 of the sample flow 122 into the secondary outflow channel 118. Accordingly, the first blended flow 126 exiting the flow junction 110 via the primary outflow channel 116 may include a greater proportion of the fluid 123 of the sheath flow 124 than the fluid 121 of the sample flow 122, while the second blended flow 128 exiting the flow junction 110 via the secondary outflow channel 118 may include a greater proportion of the fluid 121 of the sample flow 122 than the fluid 123 of the sheath flow 124. In this manner, buffer / media exchange may occur continuously in an uninterrupted manner as the sample flow 122 and the sheath flow 124 are continuously flow through the microfluidic chip 100.

[0048] FIG. 3 shows the flow junction 110 (e g. a four-way flow junction) of the microfluidic chip 100 ofFIG. 1A according to various embodiments. As shown in FIG. 3, when the sample flow 122 provided to the microfluidic chip 100 contain suspended substances 321a (e g. microcarriers with cells attached thereon, or cell-microcarrier complex, or cell aggregates), in the fluid 121 of the sample flow 122, the rate of flow of the sheath flow 124 provided to the microfluidic chip 100, via the sheath flow inlet port 134, may be optimized relative to the rate of flow of the sample flow 122 provided to the microfluidic chip 100, via the sample inlet port 132 such that the sheath flow 124 (from the secondary inflow channel 114) and the sample flow 122 (from the primary inflow channel 112) may meet at the flowjunction 110 to generate the vortex 125 for buffer / media exchange so as to result in sample washing (i.e. to exchange the fluid that carries or suspend the suspended substances 321a). During sample washing, most of the fluid 121 of the sample flow 122 may be directed into the secondary outflow channel 118 while the suspended substance 321a may maintain its trajectory, along with the remaining fluid 121 of the sample flow 122, into the primary outflow channel 1 16. At the same time, most of the fluid 123 of the sheath flow 124 may also be directed into the primary outflow channel 116 while some of the fluid 123 of the sheath flow 124 may be directed into the secondary outflow channel 118. In this manner, the fluid 121 of the sample flow 122 that originally carries or suspends the suspended substances 321 a in the primary inflow channel 1 12 may be mostly replaced by the fluid 123 of the sheath flow 124 when the suspended substances 321 a reach the primary outflow channel 1 16 of the flow junction 1 10. Accordingly, the first blended flow 126 exiting the flow junction 110 via the primary outflow channel 1 16 may include the suspended substance 321 a together with a greater proportion of the fluid 123 of the sheath flow 124 than the fluid 121 of the sample flow 122, while the second blended flow 128 exiting the flow junction 1 10 via the secondary outflow channel 1 18 may include a greater proportion of the fluid 121 of the sample flow 122 than the fluid 123 of the sheath flow 124. In this manner, washing of the sample (via buffer / media exchange) may occur continuously in an uninterrupted manner as the sample flow 122 and the sheath flow 124 are continuously flow through the microfluidic chip 100.

[0049] FIG. 4 shows the flow junction 1 10 (e.g. a four-way flow junction) of the microfluidic chip 100 ofFIG. 1A according to various embodiments As shown in FIG. 4, when the sample flow 122 provided to the microfluidic chip 100 contain two or more suspended substances, such as first suspended substances 321b (e.g. cells) and second suspended substances 321c (e.g. microcarriers), in the fluid 121 of the sample flow 122, the rate of flow of the sheath flow 124 provided to the microfluidic chip 100, via the sheath flow inlet port 134, may be optimized relative to the rate of flow of the sample flow 122 provided to the microfluidic chip 100, via the sample inlet port 132 such that the sheath flow 124 (from the secondary inflow channel 114) and the sample flow 122 (from the primary inflow channel 112) may meet at the flow junction 110 to generate the vortex 125 for separating / sorting the first suspended substances 321b and second suspended substances 321c (or purifying the first suspended substances 321b or the second suspended substances 321c). In separating / sorting the first suspended substances 321b and the second suspended substances 321c, the first suspended substance 321b may be directed, along with most of the fluid 121 of the sample flow 122, into the secondary outflow channel 118 while the second suspended substance 321c maymaintain its trajectory, along with the remaining fluid 121 of the sample flow 122, into the primary outflow channel 116. At the same time, most of the fluid 123 of the sheath flow 124 may also be directed into the primary outflow channel 116 while some of the fluid 123 of the sheath flow 124 may be directed into the secondary outflow channel 118. In the various embodiments, the first suspended substances 321b and second suspended substances 321c may differ in sizes and / or mass and / or weight. Thus, an inertia of the first suspended substances 321b and an inertia of the second suspended substances 321c when passing through the flow junction 110 may differ. For example, the first suspended substances 321b may have a smaller size and / or mass and / or weight as compared to the second suspended substances 321 c. Thus, when the vortex 125 direct most of the fluid 121 of the sample flow 122 into the secondary outflow channel 1 18, the first suspended substances 321b with lower inertia may follow most of the fluid 121 of the sample flow 122 into the secondary outflow channel 118. On the other hand, the second suspended substances 321 c with higher inertia may resist the deflection of the fluid 121 of the sample flow 122 and resist to change its state of motion so as to stay in motion to continue its trajectory so as to enter into the primary outflow channel 1 16. Accordingly, the first blended flow 126 exiting the flow junction 1 10 via the primary outflow channel 1 16 may include the second suspended substance 321 c together with a greater proportion of the fluid 123 of the sheath flow 124 than the fluid 121 of the sample flow 122, while the second blended flow 128 exiting the flow junction 110 via the secondary outflow channel 118 may include the first suspended substance 321b together with a greater proportion of the fluid 121 of the sample flow 122 than the fluid 123 of the sheath flow 124. In this manner, sorting / purifi cation may occur continuously in an uninterrupted manner as the sample flow 122 and the sheath flow 124 are continuously flowthrough the microfluidic chip 100.

[0050] FIG. 5 A shows a microfluidic chip 500 according to various embodiments. FIG. 5B shows a perspective view of the microfluidic chip 500 according to various embodiments. According to various embodiments, the microfluidic chip 500 of FIG 5A and FIG 5B contains all the features of the microfluidic chip 100 of FIG. 1A and FIG IB. Accordingly, all features, changes, modifications and variations that are applicable to the microfluidic chip 100 of FIG. 1A and FIG. IB are also applicable to the microfluidic chip 500 of FIG. 5A and FIG 5B. Therefore, elements which are the same as those described earlier are assigned the same reference numerals, and repetition of their explanations is omitted for brevity. The following descriptions focusing on the additional features / elements / limitations of the microfluidic chip 500 of FIG 5 A and FIG. 5B.

[0051] According to various embodiments, the microfluidic chip 500 may, similar to the microfluidic chip 100 of FIG. 1A and FIG. IB, include the flow junction 110 having the primary inflow channel 112, the secondary inflow channel 114, the primary outflow channel 116 and the secondary outflow channel 118. Further, the microfluidic chip 500, may, similar to the microfluidic chip 100 of FIG. 1A and FIG. IB, include the sample inlet port 132, the sheath flow inlet port 134, the primary outlet port 136 and the secondary outlet port 138. Furthermore, the microfluidic chip 500, may, similar to the microfluidic chip 100 of FIG. 1A and FIG. IB, include the inertial focusing channel segment 133 between the sample inlet port 132 and the primary inflow channel 122, the inertial focusing channel segment 137 between the primary outflow channel 116 and the primary outlet port 136, and the inertial focusing channel segment 139 between the secondary outflow channel 118 and the secondary outlet port 138.

[0052] Referring to FIG 5A and FIG. 5B, according to various embodiments, the microfluidic chip 500 may further include a mixing channel 542 between the primary inlet port 132 and the primary inflow channel 122 of the flow junction 1 10. Further, the mixing channel 542 may be upstream of the inertial focusing channel segment 133. Accordingly, the mixing channel 542 may be between the primary inlet port 132 and the inertial focusing channel segment 133. Thus, a flow line from the primary inlet port 132 to the primary inflow channel 122 of the flow junction 110 may be in the order from the primary inlet port 132, followed by the mixing channel 542, followed by the inertial focusing channel segment 133, and to the primary inflow channel 122 of the flow junction 110. Hence, the sample flow 122 may flow into the microfluidic chip 500 via the primary inlet port 132, through the mixing channel 542, and through the inertial focusing channel segment 133 before reaching the primary inflow channel 122 of the flow junction 110.

[0053] According to various embodiments, when the sample flow 122 provided to the microfluidic chip 500 contain two or more suspended substances, such as the first suspended substances 321b (e.g. cells) and the second suspended substances 321c (e.g. microcarriers), in the fluid 121 of the sample flow 122, the mixing channel 542 may disperse or distribute the two or more suspended substances in the sample flow 122 such that the sample flow 122 may be prepared into an even or uniform suspension before flowing through the remaining part of the microfluidic chip 500.

[0054] According to various embodiments, the mixing channel 542 may include one or more disturbance elements 544 to disturb or interfere or disrupt or interrupt the flow so as to promote and / or cause mixing of the flow. According to various embodiments, the one or moredisturbance elements 544 may include corrugations or undulations along the mixing channel 542 to disturb or interfere or disrupt or interrupt the flow.

[0055] According to various embodiments, the mixing channel 542 may include a corrugated surface 546. The corrugated surface 546 may be a stretch of a surface of the mixing channel 542 with a series of raised area so as to form a rippled or wavy surface. Accordingly, the corrugated surface 546 may create a series of constriction along the mixing channel 542 to disturb or interfere or disrupt or interrupt the flow so as to promote and / or cause mixing of the flow. According to various embodiments, the corrugated surface 546 may be a base surface and / or a side surface and / or a top surface of the mixing channel 542. Referring to FIG. 5 A and FIG 5B, the corrugated surface 546 of the mixing channel 542 may be the base surface of the mixing channel 542.

[0056] According to various embodiments, the corrugated surface 546 of the mixing channel 542 may be formed by a series of ridges 548. The series of ridges 548 of the corrugated surface 546 may be a series of protrusions or ribs or raised elements protruding from the surface of the mixing channel 542. Accordingly, the series of ridges 548 may form the series of constriction along the mixing channel 542. In the various embodiments, segments of the mixing channel 542 between adjacent ridges 548 may maintain a constant width and / or cross-sectional area with that of channel segments of the microfluidic chip 500 immediately before and after the mixing channel 542.

[0057] According to some embodiments, the corrugated surface 546 may be a stretch of a surface of the mixing channel 542 with a series of alternating raised area and recessed area so as to form the rippled or wavy surface, ha such embodiments, the corrugated surface 546 of the mixing channel 542 may be formed by a series of alternating ridges 548 and furrows. The ridges 548 may be protrusions or ribs or raised elements protruding from the surface of the mixing channel 542 while the furrows may be indentation or depression recessed into the surface of the mixing channel 542. Accordingly, the ridges 548 may form constrictions while the furrows may expand the channel.

[0058] Referring back to FIG. 5A and FIG. 5B, according to various embodiments, each ridge 548 of the series of ridges 548 may be of an arrowhead shape. The arrowhead shape may include, but not limited to, an inverted V-shape, a chevron shape, or an angle bracket arrow. According to some embodiments, the series of ridges 548 may be uniformly spaced apart. According to some embodiments, the series of ridges 548 may be spaced apart in a non-uniform manner. According to some embodiments, the arrowhead shape may be symmetrical. According to some embodiments, the arrowhead shape may be asymmetrical. As shown inFIG. 5A, in an example embodiment, the series of ridges 548 may be uniformly spaced apart with each ridge 548 being an asymmetrical arrowhead shape, and at least two adjacent ridges 548a, 548b may be different, whereby a first ridge 548a may have an arrowhead tip offset towards one side of the arrowhead shape while a second ridge 548b may have an arrowhead tip offset towards another side of the arrowhead shape.

[0059] According to various embodiments, the mixing channel 542 may be extending in a serpentine or meandering or winding or sinuous manner. Accordingly, mixing channel 542 may be compact while providing sufficient distance for the sample flow 122 to undergo adequate mixing.

[0060] FIG. 6 A shows a microfluidic chip 600 according to various embodiments. FIG. 6B shows a perspective view of the microfluidic chip 600 according to various embodiments. According to various embodiments, the microfluidic chip 600 of FIG 6A and FIG 6B contains all the features of the microfluidic chip 100 of FIG. 1 A and FIG. IB. Accordingly, all features, changes, modifications and variations that are applicable to the microfluidic chip 100 of FIG. 1 A and FIG. IB are also applicable to the microfluidic chip 600 of FIG. 6A and FIG 6B. Therefore, elements which are the same as those described earlier are assigned the same reference numerals, and repetition of their explanations is omitted for brevity. The following descriptions focusing on the additional features / elements / limitations of the microfluidic chip 600 of FIG. 6A and FIG. 6B

[0061] According to various embodiments, the microfluidic chip 600 may, similar to the microfluidic chip 100 of FIG. 1A and FIG IB, include the flow junction 110 having the primary inflow channel 112, the secondary inflow channel 114, the primary outflow channel 116 and the secondary outflow channel 118. Further, the microfluidic chip 600, may, similar to the microfluidic chip 100 of FIG 1A and FIG. IB, include the sample inlet port 132, the sheath flow inlet port 134, the primary outlet port 136 and the secondary outlet port 138. Furthermore, the microfluidic chip 600, may, similar to the microfluidic chip 100 of FIG. 1A and FIG. IB, include the inertial focusing channel segment 133 between the sample inlet port 132 and the primary inflow channel 122, the inertial focusing channel segment 137 between the primary outflow channel 116 and the primary outlet port 136, and the inertial focusing channel segment 139 between the secondary outflow channel 118 and the secondary outlet port 138.

[0062] Referring to FIG 6A and FIG. 6B, according to various embodiments, the microfluidic chip 600 may further include a flowrate reduction arrangement 650. The flowrate reduction arrangement 650 may be configured to retard or slow down or decelerate a flow. Inthe various embodiments, the flowrate reduction arrangement 650 may be between the primary outflow channel 116 of the flow j unction 110 and the primary outlet port 136 of the microfluidic chip 600. Accordingly, the flowrate reduction arrangement 650 may retard or slow down or decelerate the flow (e.g. the first blended flow 126) from the primary outflow channel 116 of the flowjunction 110.

[0063] According to various embodiments, the inertial focusing channel segment 137 may be between the primary outflow channel 116 of the flow junction 110 and the flowrate reduction arrangement 650. Accordingly, the first blended flow 126 from the primary outflow channel 116 of the flow junction 110 may undergo focusing to align any suspended substances in the first blended flow 126 towards the center streamline of the first blended flow 126 before going through the flowrate reduction arrangement 650. Hence, the suspended substances of the first blended flow 126 may be at the center of the flow as the first blended flow 126 undergoes rate of flow retardation through the flowrate reduction arrangement 650.

[0064] According to various embodiments, the flowrate reduction arrangement 650 may direct a fluid flow with reduced flowrate into a flow channel 662 The flow channel 662 may serve as a conveyance channel or a transport channel for flowing the fluid flow with reduced flowrate to another portion or part of the microfluidic chip 600. Accordingly, since the first blended flow 126 is flowed through the flowrate reduction arrangement 650 to reduce the rate of flow, the first blended flow 126 with the reduced flowrate may be directed into the flow channel 662 after passing through the flowrate reduction arrangement 650.

[0065] FIG. 6C shows an example of the flowrate reduction arrangement 650 of the microfluidic chip 600 according to various embodiments. Referring to FIG 6A to FIG. 6C, according to various embodiments, the flowrate reduction arrangement 650 may include a branching junction 651. According to various embodiments, the branching junction 651 may include a main channel 652 branching into two or more branch channels 654, 656, 658. The branching junction 651 may be a flow network or a flow node for splitting the main channel 652 into the two or more branch channels 654, 656, 658. According to various embodiments, the branching junction 651 may be configured such that, when the flow from the main channel 652 is split or distributed into the two or more branch channels 654, 656, 658, a rate of flow in each branch channel 654, 656, 658 may be slower than a rate of flow in the main channel 652. In this manner, the flowrate reduction arrangement 650 may achieve reduction in the rate of flow via the branching junction 651.

[0066] According to various embodiments, the branching junction 651 of the flowrate reduction arrangement 650 may include the main channel 652 branching into a first side branchchannel 654, a middle branch channel 656, and a second side branch channel 658, i.e. the main channel 652 may be split into three branch channels 654, 656, 658. According to various embodiments, the middle branch channel 656 may form a continuous straight line with the main channel 652. Accordingly, the middle branch channel 656 and the main channel 652 may be aligned with each other so as to form the continuous straight line. Further, according to various embodiments, the middle branch channel 656 and the main channel 652 may be coaxial such that respective channel axes may coincide with each other. According to various embodiments, the first side branch channel 654 and the second side branch channel 658 may be on two opposite sides of the middle branch channel 656. Accordingly, the first side branch channel 654 may be alongside the middle branch channel 656 on a first side of the middle branch channel 656 and the second side branch channel 658 may be alongside the middle branch channel 656 on a second side of the middle branch channel 656, whereby the first side of the middle branch channel 656 and the second side of the middle branch channel 656 may be the two opposite sides of the middle branch channel 656.

[0067] According to various embodiments, the branching junction 651 of the flow rate reduction arrangement 650 may be connected to the inertial focusing channel segment 137 with the main channel 652 connected to the inertial focusing channel segment 137 for receiving the first blended flow 126 from the inertial focusing channel segment 137. Accordingly, with the branching junction 651 of the flowrate reduction arrangement 650 having the main channel 652 branching into the first side branch channel 654, the middle branch channel 656, and the second side branch channel 658 as described earlier, the first blended flow 126 from the inertial focusing channel segment 137 may flow into the flowrate reduction arrangement 650 with the first blended flow 126 having the suspended substances aligned or concentrated at the center thereof. Further, since the middle branch channel 656 and the main channel 652 are aligned with each other to form the continuous straight line, (at least a portion of) the fluid of the first blended flow 126 together with suspended substances aligned or concentrated at the center thereof may flow into the middle branch channel 656. The remaining fluid of the first blended flow 126 may flow into the first side branch channel 654 and the second side branch channel 658. As the rate of flow in each of the first side branch channel 654, the middle branch channel 656, and the second side branch channel 658 may be slower than the rate of flow in the main channel 652, a resultant flow in the middle branch channel 656 with (at least a portion of) the fluid of the first blended flow 126 together with suspended substances aligned or concentrated at the center thereof may have a reduced rate of flow as compared to the original rate of flowin the main channel 652. Thus, the resultant flow in the middle branch channel 656 may be the first blended flow 126 with a reduced rate of flow.

[0068] According to various embodiments, the middle branch channel 656 of the branching junction 651 of the flow rate reduction arrangement 650 may form a continuous passage with the flow channel 662. Accordingly, a flow may exit the flow rate reduction arrangement 650 via the middle branch channel 656 of the branching junction 651 into the flow channel 662 so as to subsequently flow into the other portions of the microfluidic chip 600. Since the resultant flow in the middle branch channel 656 may be the first blended flow 126 with the reduced rate of flow, the first blended flow 126 with the reduced rate of flow may flow from the middle branch channel 656 of the branching junction 651 into the flow channel 662. Therefore, in the various embodiments, the first blended flow 126 may flow from into the flow rate reduction arrangement 650 via the main channel 652 of the branching junction 651 and may exit the flow rate reduction arrangement 650 (as the first blended flow 126 with the reduced rate of flow) via the middle branch channel 656 of the branching junction 651 .

[0069] According to some embodiments, each of the first side branch channel 654 and the second side branch channel 658 may be wider than the middle branch channel 656. Accordingly, a width of each of the first side branch channel 654 and the second side branch channel 658 may be wider than a width of the middle branch channel 656. Further, a cross- sectional area of each of the first side branch channel 654 and the second side branch channel 658 may be wider than a cross-sectional area of the middle branch channel 656.

[0070] According to some embodiments, the middle branch channel 656 and the main channel 652 may have an equal width. Accordingly, a width of the middle branch channel 656 may be equal to a width of the main channel 652. Further, a cross-sectional area of the middle branch channel 656 may be equal to a cross-sectional area of the main channel 652.

[0071] According to various embodiments, the microfluidic chip 600 may include a first branch outlet 653 downstream of the first side branch channel 654 and a second branch outlet 657 downstream of the second side branch channel 658. Accordingly some of the fluid of the first blended flow 126 flowing into the first side branch channel 654 and the second side branch channel 658 may be discharged from the microfluidic chip 600 via the first branch outlet 653 and the second branch outlet 657.

[0072] According to various embodiments, the microfluidic chip 600 may include a three- way junction 660 downstream of the flow rate reduction arrangement 650. Accordingly, the three-way junction 660 may be between the primary outlet port 136 and the flow rate reduction arrangement 650. Hence, the flow rate reduction arrangement 650 may be between the three-way junction 660 and the inertial focusing channel segment 137. Thus, a flow line from the flow junction 110 to the primary outlet port 136 may be in the order from the primary outflow channel 116 of the flow junction 110, followed by the inertial focusing channel segment 137, followed by the flow rate reduction arrangement 650, followed by the three-way junction 660, and to the primary outlet port 136. Therefore, the three-way junction 660 may also be between the flow junction 1 10 and the primary outlet port 136. In the various embodiments, the first blended flow 126 may exit the flow junction 110 via the primary outflow channel 116 of the flow junction 110, flow through the inertial focusing channel segment 137, flow through the flow rate reduction arrangement 650, and flow through the three-way junction 660 before reaching the primary outlet port 136.

[0073] According to various embodiments, the three-way junction 660 may include a solution inflow channel 664 that may merge with the flow channel 662 into a merged channel 666. Accordingly, at the three-way junction 660, the flow channel 662 and the solution inflow channel 664 may converge or join or unit together so as to provide a joint exit via the merged channel 666. Tn the various embodiments, a solution-flow may be provided via the solution inflow channel 664 to merge with the first blended flow 126 (having the reduced rate of flow) from the flow rate reduction arrangement 650. Thus, the solution inflow channel 664 of the three-way junction 660 may serve to introduce the solution-flow to combine with the first blended flow 126 (having the reduced rate of flow) from the flow rate reduction arrangement 650. Hence, the three-way junction 660 may provide a means to add or introduce an additional fluid (as the solution-flow) into the microfluidic chip 600 for adding to the first blended flow 126 (having the reduced rate of flow) from the flow rate reduction arrangement 650.

[0074] According to various embodiments, the microfluidic chip 600 may include a solution inlet port 668 (or a third inlet port) upstream of the solution inflow channel 664 of the three- way junction 660. The solution inlet port 668 may serve as an inlet for providing the solutionflow into the microfluidic chip 600 for flowing to the solution inflow channel 664 of the three- way flow junction 660. Hence, the solution-flow may enter the microfluidic chip 600 via the solution inflow channel 664, flow to the solution inflow channel 664 and into the three-way flow junction 660 via the solution inflow channel 664. Accordingly, the solution inlet port 668 may be an entry point for the solution-flow and the solution inlet port 668 may be in fluid communication with the solution inflow channel 664 for flowing the solution-flow into the three-way flow junction 660. Therefore, in the various embodiments, a fluid source (or solution source) may be connected to the solution inlet port 668 so as to provide the fluid from the fluid source as the solution-flow.

[0075] According to various embodiments, the microfluidic chip 600 may further include a mixing channel 672 downstream of the three-way junction 660. Accordingly, the mixing channel 672 may be between the three-way junction 660 and the pnmary outlet port 136. Hence, the three-way junction 660 may be between the flow rate reduction arrangement 650 and the mixing channel 672. Thus, a flow line from the flow junction 110 to the primary outlet port 136 may be in the order from the primary outflow channel 116 of the flow junction 110, followed by the inertial focusing channel segment 137, followed by the flow rate reduction arrangement 650, followed by the three-way junction 660, followed by the mixing channel 672, and to the primary outlet port 136. Therefore, the mixing channel 672 may also be between the flowjunction 110 and the primary outlet port 136. Tn the various embodiments, the first blended flow 126 may exit the flow junction 1 10 via the primary outflow channel 1 16 of the flow junction 110, flow through the inertial focusing channel segment 137, flow through the flow rate reduction arrangement 650, flow through the three-way junction 660 and flow through the mixing channel 672 before reaching the primary outlet port 136.

[0076] According to various embodiments, with the solution-flow introduced into the microfluidic chip 500 via the three-way junction 660, the solution-flow and the first blended flow 126 (having the reduced rate of flow) from the flow rate reduction arrangement 650 may be combined and flow from the three-way junction 660 via the merged channel 666 into the mixing channel 672. According to various embodiments, the mixing channel 672 may promote the mixing of the solution-flow and the first blended flow 126 (having the reduced rate of flow) so as to produce an even or uniform fluid serving as the primary output flow for flowing out of the primary outlet port 136.

[0077] According to various embodiments, the mixing channel 672 may be extending in a serpentine or meandering or winding or sinuous manner. Accordingly, mixing channel 672 may be compact while providing sufficient distance for the solution-flow and the first blended flow 126 to undergo adequate mixing.

[0078] According to various embodiments, the mixing channel 672 may include one or more disturbance elements 674 to disturb or interfere or disrupt or interrupt the flow so as to promote and / or cause mixing of the flow. According to various embodiments, the one or more disturbance elements 674 may include corrugations or undulations along the mixing channel 672 to disturb or interfere or disrupt or interrupt the flow.

[0079] According to various embodiments, the mixing channel 672 may include a corrugated surface 676. The corrugated surface 676 may be a stretch of a surface of the mixing channel 672 with a series of raised area so as to form a rippled or wavy surface. Accordingly,the corrugated surface 676 may create a series of constriction along the mixing channel 672 to disturb or interfere or disrupt or intermpt the flow so as to promote and / or cause mixing of the flow. According to various embodiments, the corrugated surface 676 may be a base surface and / or a side surface and / or a top surface of the mixing channel 672. Referring to FIG. 6A, the corrugated surface 676 of the mixing channel 672 may be the base surface of the mixing channel 672.

[0080] According to various embodiments, the corrugated surface 676 of the mixing channel 672 may be formed by a series of ridges 678. The series of ridges 678 of the corrugated surface 676 may be a series of protrusions or ribs or raised elements protruding from the surface of the mixing channel 672. Accordingly, the series of ridges 678 may form the series of constriction along the mixing channel 672. In the various embodiments, segments of the mixing channel 672 between adjacent ridges 678 may maintain a constant width and / or cross-sectional area with that of the merged channel 666 of the three-way junction 660 immediately before the mixing channel 672.

[0081] According to some embodiments, the corrugated surface 676 may be a stretch of a surface of the mixing channel 672 with a series of alternating raised area and recessed area so as to form the rippled or wavy surface. Tn such embodiments, the corrugated surface 676 of the mixing channel 672 may be fonned by a series of alternating ridges 678 and furrows. The ridges 678 may be protrusions or ribs or raised elements protruding from the surface of the mixing channel 672 while the furrows may be indentation or depression recessed into the surface of the mixing channel 672. Accordingly, the ridges 678 may fonn constrictions while the furrows may expand the channel

[0082] Referring back to FIG. 6A, according to various embodiments, each ridge 678 of the series of ridges 678 may be of an arrowhead shape. The arrowhead shape may include, but not limited to, an inverted V-shape, a chevron shape, or an angle bracket arrow. According to some embodiments, the series of ridges 678 may be uniformly spaced apart. According to some embodiments, the series of ridges 678 may be spaced apart in a non-uniform manner. According to some embodiments, the arrowhead shape may be symmetrical. According to some embodiments, the arrowhead shape may be asymmetrical.

[0083] According to various embodiments, with the ridges 678 of the series of ridges 678 being in the form of the arrowhead shape, the corrugated surface 676 of the mixing channel 672 may be of a herringbone pattern. Accordingly, the ridges 678 of the series of ridges 678 in the arrowhead shape may be arranged into the herringbone pattern to form the corrugated surface 676 of the mixing channel 672.

[0084] As shown in FIG. 6A, in an example embodiment, the series of ridges 678 may be uniformly spaced apart with each ridge 678 being an asymmetrical arrowhead shape, and at least two adjacent ridges 678a, 678b may be different, whereby a first ridge 678a may have an arrowhead tip offset towards one side of the arrowhead shape while a second ridge 678b may have an arrowhead tip offset towards another side of the arrowhead shape.

[0085] FIG. 6D shows an example of the series of ridges 678 of the corrugated surface 676 of the mixing channel 672 according to various embodiments. As shown, the series of ridges 678 may include a first set of ridges 678a and a second set of ndges 678b arranged in an alternating manner. Each ridge 678 of the first set of ridges 678a and the second set of ridges 678b may be of an asymmetrical arrowhead shape. Further, each set of the first set of ridges 678a and the second set of ridges 678b may include three ridges 678. The ridges 678 within the first set of ridges 678a may be the same with each other, and the ridges 678 within the second set of ridges 678b may be the same with each other. However, the ridges 678 of the first set of ridges 678a may be different from the ridges 678 of the second set of ridges 678b. For example, each ridge 678 of the first set of ridges 678a may have an arrowhead tip offset towards one side of the arrowhead shape while each ridge 678 of the second set of ridges 678b may have an arrowhead tip offset towards another side of the arrowhead shape.

[0086] FIG. 6E shows another example of the series of ridges 678 of the corrugated surface 676 of the mixing channel 672 according to various embodiments. The example as shown in FIG 6E differs from the example as shown in FIG. 6D in that each set of the first set of ridges 678a and the second set of ridges 678b may include five ridges 678.

[0087] FIG. 6F shows yet another example of the series of ridges 678 of the corrugated surface 676 of the mixing channel 672 according to various embodiments. The example as shown in FIG. 6F differs from the examples as shown in FIG. 6D and FIG. 6E in that each set of the first set of ridges 678a and the second set of ridges 678b may include seven ridges 678.

[0088] FIG. 7 shows a system 701 for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates according to various embodiments. According to various embodiments, the system 701 may be an integrated platform for automated cell recovery from microcarriers and / or cell aggregates in suspension cell cultures. In the various embodiments, the system 701 may provide an integrated microfluidic on-chip cell dissociator and / or cell purificator. The vortex-assisted microcarriers washing, the enzymatic (e.g. trypsin) cell dissociation of cell-microcarrier complex and / or cell aggregates, and the vortex-assisted separation of dissociated cells from microcarriers provided in the system 701 of the various embodiments maybe label-free, membrane-free, and scalable for higher throughput. Thesystem 701 may eliminate the manual steps and operations of the conventional method. The system 701 may also benefit both small and large-scale bioreactor productions by enabling automated and continuous cell harvesting with higher cell recovery and throughput.

[0089] According to various embodiments, the system 701 may include the microfluidic chip 600 of FIG. 6A and FIG. 6B as a first microfluidic chip 782 and / or the microfluidic chip 500 of FIG. 5 A and FIG. 5B as a second microfluidic chip 784. The first microfluidic chip 782, being the microfluidic chip 600 of FIG. 6A and FIG. 6B may serve as a microfluidic cell dissociator. The second microfluidic chip 784 being the microfluidic chip 500 of FIG. 5 A and FIG 5B may serve as a microfluidic vortex-assisted cell purificator. In the various embodiments, the first microfluidic chip 782, serving as the microfluidic cell dissociator, may have the flow junction 1 10 to serve as a vortex-assisted microcarrier washing module for buffer exchange to minimize the effect of components in culture media (e.g. FBS) on subsequent enzyme performance, the inertia focusing channel 137 serving as a sample concentrator to concentrate the sample, the flow reduction arrangement 650 to reduce the flow rate so as to save enzyme consumption in cell dissociation process, and the mixing channel 672 serving as a herringbone-assisted micromixing module for efficient mixing of microcarrier samples and the enzyme (e g. trypsin) added via the three-way junction 660. The first microfluidic chip 782 (being the microfluidic chip 600 of FIG. 6A and FIG. 6B) may include one inlet (e g. the sample inlet port 132) for loading of cell-mi crocarri er complex samples and / or cell aggregates samples, one inlet (e g the sheath flow inlet port 134) for sheath flow to induce the vortex for buffer exchange, one inlet (e.g. the solution inlet port 688) for enzyme solution (e.g. trypsin), three waste outlets (e.g. the secondary outlet port 138, the first branch outlet 653, and the second branch outlet 657), and one sample outlet (e.g. primary outlet port 136) that may be connected to an incubation module 786 (e g. a long segment of tubing for in-line incubation to allow trypsin to take effect). The disturbance elements 544 of the mixing channel 542 of the first microfluidic chip 782 (e.g. the herringbone structure) may induce chaotic flow to accelerate the mixing of microcarriers and trypsin. The second microfluidic chip 784 (being the microfluidic chip 500 of FIG 5 A and FIG. 5B) may include two inlets (e.g. the sample inlet port 132 and the sheath flow inlet port 134) and two outlets (e.g. the primary outlet port 136 and the secondary outlet port 138) The sample inlet port 132 may be for loading the mixture of cells and microcarriers from the incubation module 786, and the other inlet (e g. the sheath flow inlet port 134) may provide the sheath flow 124 to induce the vortex that actively extracts the cells from the sample flow 122 (i.e. the mixture cells and microcarrier from the incubation module 786). The two outlets e.g. the primary outlet port 136 and the secondary outlet port138) may be configured for the collection of microcarriers (e g. via the primary outlet port 136) and purified cells (e g. via the secondary outlet port 138), respectively. The width of the microcarrier outlet (e.g. the primary outlet port 136) may be twice the width of the cell outlet (e.g. the secondary outlet port 138) to ensure microcarriers flow into the mam outlet (e.g. the primary outlet port 136) and reduce the cell dilution by the sheath flow 124 while maintaining the cell recovery performance.

[0090] According to some embodiments, both the first microfluidic chip 782 (being the microfluidic chip 600 of FIG. 6A and FIG. 6B serving as the microfluidic cell dissociator) and / or the second microfluidic chip 784 (being the microfluidic chip 500 of FIG. 5A and FIG. 5B serving as the cell purificator) may be fabricated using polydimethylsiloxane (PDMS) via standard soft lithography techniques. Accordingly, the first microfluidic chip 782 and / or the second microfluidic chip 784 may be fabricated with low cost and good biocompatibility.

[0091] According to various embodiments, the first microfluidic chip 782 and / or the second microfluidic chip 784 may be primed with Bovine Serum Albumin (BSA) solution to prevent particle adhesion on the channel walls. In an example, a 10% BSA solution may be used.

[0092] According to various embodiments, a sample source 788 may be connected to the sample inlet port 132 of the first microfluidic chip 782, a buffer solution source 787 may be connected to the sheath flow inlet port 134 of the first microfluidic chip 782, and an enzymatic solution source 789 may be connected to the solution inlet port 688 of the first microfluidic chip 782. Accordingly, the sample (e.g. the microcarrier samples) from the sample source 788 (e g. bioreactor), the buffer solution (e g. Phosphate-buffered saline, PBS) from the buffer solution source 787, and the enzymatic solution (e.g. trypsin) from the enzymatic solution source 789 may be loaded into the three inlets separately.

[0093] According to various embodiments, the sample from the sample source 788 may enter the first microfluidic chip 782 via the sample inlet port 132 to form the sample flow 122. The cell-microcarrier complex and / or the cell aggregates in the sample flow 122 may first align to the channel centre due to the inertial focusing effect as the sample flow 122 flows through the inertia focusing channel segment 133 (e g. the serpentine channel). Subsequently, when the sheath flow 124 is applied through the sheath flow inlet port 134 using a pump 792 (e.g. a syringe pump) at a higher flow rate (e.g. greater than 3 times of that of the sample flow 122), the vortex 125 may be generated at the intersection 115 of the flow junction 110 to perform buffer exchange. After the vortex-assisted buffer exchange, the cell-microcarrier complex and / or the cell aggregates in the first blended flow 126 exiting via the primary outflow channel 116 of the flow junction 110 may be aligned to the channel centre again by the inertial focusingchannel segment 137 (e.g. a serpentine channel), before reaching the flowrate reduction arrangement 650 (i.e. a flow rate reduction region). After that, the cell-microcarrier complex and / or cell aggregates in the resultant flow from the flowrate reduction arrangement 650 (e g. the first blended flow 126 with the reduced flowrate) may be mixed with the enzymatic solution (e.g. trypsin), which may be added at the three-way junction 660, as they flow through the mixing channel 672 with the disturbance elements 674 (e.g. the herringbone structures).

[0094] According to various embodiments, the primary outlet port 136 of the first microfluidic chip 782 (e g. the microfluidic chip 600 of FIG. 6A and FIG. 6B serving as the cell dissociation device) may be connected to the incubation module 786. According to some embodiments, the incubation module 786 may include a long tubing 785 maintained at a predetermined temperate (e.g. at a temperature of 37 °C; or at a temperature within a range from 36 °C to 38 °C, inclusive; or at a temperature within a range from 35 °C to 40 °C; or at a temperature within a range from 30 °C to 45 °C). According to some embodiments, the long tubing 785 may have a length sufficient to ensure a fluid flowing through the long tubing 785 may require at least a predetermined amount of time such that the fluid may be incubated in the incubation module 786 for at least the predetermined amount of time. In other words, the fluid may take at least the predetermined amount of time to flow through the long tubing 785. According to some embodiments, the predetermined amount of time may be set to ensure complete cell dissociation. In an example, the predetermined amount of time may be around 10 minutes or 15 minutes or 20 minutes.

[0095] According to some embodiments, when the sample flow 122 contain only cell aggregates, the incubated flow exiting the incubation module 786 may contain the dissociated cells (i.e. individual separated cells). Accordingly, a cell collection module may be connected to the incubation module 786 for collecting the dissociated cells. Hence, the cell collection module may be downstream of the incubation module 786 to collect the dissociated cells.

[0096] According to some embodiments, when the sample flow 122 contain cellmicrocarrier complex and / or a combination of cell-microcarrier complex and cell aggregates, the incubated flow with the dissociated cells and the microcarriers exiting the incubation module 786 may be flowed into the second microfluidic chip 784 (e g. the microfluidic chip 500 of FIG. 5 A and FIG. 5B serving as the microfluidic cell purificator) for cell recovery and microcarrier removal. According to various embodiments, the incubation module 786 may be connected to the sample inlet port 132 of the second microfluidic chip 784. Accordingly, the incubated flow with the dissociated cells and the microcarriers may flow into the second microfluidic chip 784 via the sample inlet port 132 to serve as the sample flow 122 of thesecond microfluidic chip 784. In the various embodiments, the microcarriers and the cells in the sample flow 122 of the second microfluidic chip 784 may be initially randomly distributed in the channel. After flowing through the inertial focusing channel segment 133 (e.g. a serpentine channel), the microcarriers (e g. -175 pm) may be aligned to the centre of the channel due to the inertial focusing effect, while the cells (e g. -18 pm) may remain randomly dispersed in the channel due to the small size and lower inertial forces Similar to the first microfluidic chip 782, the sheath flow 124 may be applied, via the sheath flow inlet port 134, to induce the vortex 125 at the intersection 115 of the flow junction 110. When the aligned microcarriers and the randomly distributed cells flowthrough this vortex region, the vortex 125 may focus the smaller cells / particles into a band which is deflected towards the vortex centre due to the centrifugal effect. The larger microcarriers experiencing higher inertia may be directed into the primary outflow channel 116 of the flow junction 110 so as to be subsequently eluted via the main outlet (e.g. the primary outlet port 136). In contrast, the vortex-induced deflected cells may be sorted into the secondary outflow channel 1 18 of the flow junction 110 so as to be recoverable from the side outlet (e g. the secondary outlet port 138) to achieve separation. According to various embodiments, the rate of flow of the sheath flow 124 may be adjusted to maximize the cell recovery from secondary outlet port 138 and ensure complete microcarrier removal from the primary outlet port 136.

[0097] FIG. 8A to FIG. 81 illustrate the state of the microcarrier 725 and cells 727 as they flow through the system 701 according to various embodiments According to various embodiments, when the sample from the sample source 788 enters the first microfluidic chip 782 via the sample inlet port 132 to form the sample flow 122 of the first microfluidic chip 782, the sample flow 122 of the first microfluidic chip 782 may include the microcarriers 725 with the cells 727 attached thereon, e.g. cell-microcarrier complex (see FIG. 8A). The microcarriers 725 with the cells 727 attached thereon (or thecell-microcarrier complex) may serve as the suspended substances 321a in the fluid 121 of the sample flow 122. Further, the fluid 121 of the sample flow 122 may be a culture medium. According to various embodiments, after the buffer exchange at the flow junction 110, the microcarriers 725 with the cells 727 attached thereon (or the cell-microcarrier complex) may continue to serve as the suspended substances 321a in the fluid of the first blended flow 126 exiting via the primary outflow channel 116 of the flow junction 110. However, the fluid (e.g. culture medium) of the first blended flow 126 may be the buffer solution 729a as a result of the buffer exchange (see FIG. 8B). According to various embodiments, at the three-way junction 660, the microcarriers 725 with the cells 727 attached thereon (or the cell-microcarrier complex) may continue to serve asthe suspended substances 321a in the fluid of the first blended flow 126 with the reduced flow rate. However, the fluid may include the buffer solution 729a and the enzymatic solution 729b in an unmixed state (see FIG. 8C). According to various embodiments, after passing through the mixing channel 672, the buffer solution 729a and the enzymatic solution 729b (e.g. trypsin) may be mixed into a mixture 729c carrying the microcarriers 725 with the cells 727 attached thereon (see FIG. 8D).

[0098] According to various embodiments, the mixture 729c containing the microcarriers 725 with the cells 727 attached thereon (or the cell-microcamer complex) may then flow through the incubation module 786. Tn the incubation module 786, the microcarriers 725 with the cells 727 attached thereon (or the cell-microcarrier complex) may undergo enzymatic incubation (e g. trypsin incubation) to cause dissociation of the cells 727 from the microcarriers 725. After going through the incubation module 786, cells dissociation may be achieved such that the cells 727 may be detached from the microcarriers 725. Accordingly, the cells 727 may become the first suspended substances 321b in the mixture 729c and the microcarriers 725 may become the second suspended substances 321 c in the mixture 729c (see FIG. 8E).

[0099] According to various embodiments, the mixture 729c with the cells 727 (i.e. the first suspended substances 321b) and the microcarriers 725 (i.e. the second suspended substances 321c) separately suspended in the mixture 729c may be provided to the second microfluidic chip 784 via the sample inlet port 132 to form the sample flow 122 of the second microfluidic chip 784. Accordingly, the sample flow 122 of the second microfluidic chip 784 may include the detached cells 727 serving as the first suspended substances 321b, the detached microcarriers 725 serving as the second suspended substances 321c, and the mixture 729c serving as the fluid 121 of the sample flow 122 of the second microfluidic chip 784. According to various embodiments, the sample flow 122 of the second microfluidic chip 784 may first flow through the mixing channel 542 after entering the second microfluidic chip 784 via the sample inlet port 132. As the sample flow 122 of the second microfluidic chip 784 flows through the mixing channel 542, the cells 727 (i.e. the first suspended substances 321b) and the microcarriers 725 (i.e. the second suspended substances 321c) may be dispersed or distributed or mixed such that the sample flow 122 of the second microfluidic chip 784 may become an even or uniform suspension (e g. see FIG. 8F). According to various embodiments, the sample flow 122 of the second microfluidic chip 784 may then flow through the inertial focusing channel segment 133 of the second microfluidic chip 784. As the sample flow 122 of the second microfluidic chip 784 pass through the inertial focusing channel segment 133, the microcarriers 725 (i.e. the second suspended substances 321c) may be focused into the centerof the sample flow 122 while the cells 727 (i.e. the first suspended substances 321b) remain randomly distributed throughout the sample flow 122 (e.g. see FIG. 8G). According to various embodiments, the sample flow 122 of the second microfluidic chip 784 may subsequently flow through the flow junction 110. At the flow junction 110 of the second microfluidic chip 784, the sheath flow 124 of the second microfluidic chip 784 may generate the vortex 125 at the flowjunction 1 10 of the second microfluidic chip 784 to cause cells sorting / purification. Tn the various embodiments, the vortex 125 at the flow junction 110 of the second microfluidic chip 784 may deflect the fluid 121 of the sample flow 122 into the secondary outflow channel 118 of the flow junction 1 10 while carrying the cells 727 (i.e. the first suspended substances 321b) along with it while the microcarriers 725 (i.e. the second suspended substances 321 c) may carry on with the trajectory into the primary outflow channel 1 16 of the flow junction 1 10 along with the fluid 123 of the sheath flow 124. In this manner, the cells 727 (i.e. the first suspended substances 321b) and the microcarriers 725 (i.e. the second suspended substances 321 c) may be separated such that the cells 727 (i .e. the first suspended substances 321b) may flow via the secondary outflow channel 1 18 towards the secondary outlet port 138 of the second microfluidic chip 784 and the microcarriers 725 (i.e. the second suspended substances 321 c) may flow via the primary outflow channel 1 16 towards the primary outlet port 1 6 of the second microfluidic chip 784. According to various embodiments, the microcarriers 725 (i.e. the second suspended substances 321c) may be collected at the primary outlet port 136 (see FIG 8H) and the cells 727 (i.e. the first suspended substances 321b) may be collected at the secondary outlet port 138 (see FIG. 81)[000100] Accordingly, the system 701 of the various embodiments may automate buffer exchange and microfluidic cell harvesting from microcarrier / 3D cell aggregate cultures, whereby the automated workflow may include on-chip vortex-assisted sample washing, concentrating, enzymatic cell dissociation, incubation, and / or vortex-assisted cell purification from microcarriers.[000101] The following relates to studies and experiments relating to the system 701 according to various embodiments.[000102] FIG. 9A and FIG. 9B show a study to demonstrate the buffer exchange capability of the flow junction 110 (e.g. a four-way flow junction) of the first microfluidic chip 782 of the system 701 to enhance downstream enzymatic cell dissociation according to various embodiments In the experiments, the sample flow 122 of the first microfluidic chip 782 of the system 701 (e g. the microcarrier sample in culture media) was loaded into the sample inlet port 132 at 500 pL / min, and the sheath flow 124 of the first microfluidic chip 782 (e g. thePBS) was loaded into the sheath flow inlet port 134 at 1900 pL / min. As shown in FIG. 9A, the fluid 123 of the sheath flow 124 of the first microfluidic chip 782 (e.g. the PBS buffer) together with the cell-laden microcarriers may enter the primary outflow channel 116 of the flow junction 110 (e.g. the main channel), while the fluid 121 of the sample flow 122 (e.g. the culture media) may enter the secondary outflow channel 118 of the flow junction 110 (e g. the side channel). According to the various embodiments, the fluid 121 of the sample flow 122 flowing into the secondary outflow channel 118 of the flow junction 110 may also contain dead cells. As shown in FIG. 9B, after the automatic sample washing, a shorter time may be required for the cells to detach from the microcarriers, which may help or contribute to shortening the time required for incubation and may further simplify the system 701 of the various embodiments. [000103] FIG. 10 A to FIG. 10C show a study to demonstrate the effects of sheath flow rate on buffer exchange and sample concentrating performance of the first microfluidic chip 782 of the system 701 according to various embodiments. Tn the experiments, the fluid flow at the vortex region of the flow junction 110 was demonstrated by fluorescein isothiocyanate (FTTC) dye, and the trajectory of microcarriers was captured viahigh -speed camera. As shown in FIG.10A, when the sample flow 122 with fluorescent FTTC and the sheath flow 124 (e.g. PBS) were loaded into the sample inlet port 132 (e.g. entering from the left in the photograph) and sheath flow inlet port 134 (e.g. entering from the bottom in the photograph) respectively, the sample flow 122 with fluorescent FITC (which is appearing bright in the images) was shown to be partially removed from entering the primary outflow channel 1 16 (e g the main channel) at a low sheath flow rate (1500 pL / min). As the rate of flow of the sheath flow 124 was increased to 2500 pL / min, a higher proportion of the sample flow 122 with fluorescent FITC was shown to have entered the secondary outflow channel 118 (e.g. the side channel), and the primary outflow channel 116 (e g. the main channel) was shown to be mostly covered by the sheath flow 124 (e.g. PBS, which is appearing dark in the images). Accordingly, the experiment illustrated good buffer exchange performance. Further, as shown in FIG. 10B, in spite of this vortex-deflected flow, the microcarriers from the sample flow 122 may still maintain the aligned flow and enter the primary outflow channel 116 (e g. the main channel) due to their large sizes. Thus, the experiment results further illustrated good buffer exchange performance. After buffer exchange, the first blended flow 126 with microcarriers (from the primary outflow channel 116) flowed through the inertial focusing channel segment 137 (e g. the serpentine focusing channel) and reached the flowrate reduction arrangement 650 serving as a sample concentrating compartment. As shown in FIG. 10C, the stacked high-speed images showed that the microcarriers may pass through the flowrate reduction arrangement 650, i.e. may enterthe middle branch channel 656 (e.g. the centre channel), successfully under all the tested flow rates, except for a few microcarrier aggregates that may possibly be deflected and exited via the first side branch channel 654 and / or the second side branch channel 658 (e g. the side channels).[000104] FIG. 11A and FIG. 11B show a study of the flow rates at all outlets of the microfluidic chip 782 of the system 701 (i.e. the primary outlet port 136 [labelled as Chipl -01 in the figure], the secondary outlet port 138 [labelled as Chipl -04 in the figure], the first branch outlet 653 [labelled as Chipl-O2 in the figure], and the second branch outlet 657 [labelled as Chipl -03]) at different rate of flow of the sheath flow 124 for optimizing the flow rate and concentration of enzymatic solution (e g. trypsin) according to various embodiments. Tn the experiments conducted for the study, the rate of flow of the sample flow 122 was 500 pL / min, and the solution inlet port 688 (e g. trypsin inlet) was blocked in these experiments as it would not affect the fluid flow in the first microfluidic chip 782 before the mixing channel 672 (e g. herringbone mixing compartment). As shown in FIG 1 1 A, the flow rates at all outlets (i.e. the primary outlet port 136, the secondary outlet port 138, the first branch outlet 653, and the second branch outlet 657) increases as the rate of flow of the sheath flow 124 increases. As shown in FIG. 1 IB, the ratio of the flow at the different outlets (i.e. the primary outlet port 136 [labelled as Chip 1-01 in the figure], the secondary outlet port 138 [labelled as Chip 1-04 in the figure], the first branch outlet 653 [labelled as Chipl -02 in the figure) remained consistent even when the sheath flow rate increases. It is noticeable that the flow rate of the primary outlet port 136 (e.g. sample outlet) was decreased to less than 300 pL / min, which may help to save enzymatic solution (e.g. trypsin) consumption.[000105] FIG. 12A to FIG 12C show a study of the mixing performance of the first microfluidic chip 782 of the system 701 without and with the disturbance elements 674 in the mixing channel 672 (e g. staggered herringbone structures) of different repeated patterns, hi the experiments for the study, the three-way junction 660 and the mixing channel 672 was simulated with a device having two inlets and one outlet with a mixing channel 672. Four types of mixing channels 672 were studied. The first type of mixing channel 672 (labelled as plain in the figures) is a plain channel without any disturbance elements 674. The second type of mixing channel 672 (labelled as SH3 in the figures) is the variant as shown in FIG. 6D wherein each set of the first set of ridges 678a and the second set of ridges 678b includes three ridges 678. The third type of mixing channel 672 (labelled as SH5 in the figures) is the variant as shown in FIG. 6E wherein each set of the first set of ridges 678a and the second set of ridges 678b includes five ridges 678. The fourth type of mixing channel 672 (labelled as SH7 in thefigures) is the variant as shown in FIG. 6F wherein each set of the first set of ridges 678a and the second set of ridges 678b may include seven ridges 678. In the various experiments, FITC dye and PBS buffer were loaded into the two inlets, and the mixing was demonstrated by the fluorescent coverage across the mixing channel 672 at different positions of the mixing channel 672. As shown in FIG. 12B, the fluorescent images showed that FITC covered only half of the channel at the inlet region in all the variants, regardless of whether the mixing channel 672 is plain or has the disturbance elements 674 (e.g. herringbone features), due to laminar flow. As shown, the second type of mixing channel 672 (SH3) with 3-by-3 repeated ridges (e g. staggered herringbone features) may achieve 100% FITC coverage after the shortest distance of flow, suggesting a strong chaotic mixing effect in the mixing channel 672, whereas the first type of mixing channel 672 that is plain and without any disturbance elements 674 (e.g. without herringbone features) failed to achieve complete mixing. This may be due to limited diffusion effects under laminar flow From the study, the optimized first microfluidic chip 672 may be provided with the second type of mixing channel 672 (SH3) having 3-by-3 repeated ridges (e g. 3-by-3 staggered herringbone structures).[000106] FIG. 13 A to FIG. 13E show a study demonstrating cell dissociation achieved by the system 701 according to various embodiments. In the experiments conducted, enzymatic solution (e.g. trypsin (10x) solution) and samples (cell-microcarrier complex and cell aggregates) were directly provided into the mixing channel 672 (e.g. a herringbone device) at flow rates of 100 pl / min and 500 pl / min, respectively, and the incubation module 786 (e.g. inline incubation in a long tubing) was connected to the outlet of the mixing channel 672 FIG. 13B and FIG. 13C show the results after 10 minutes of incubation for the sample containing cell aggregates. As shown, complete cell dissociation from cell aggregates with high cell viability (strong Calcein AM signal) was achieved after 10 minutes incubation in the tubing. FIG 13D and FIG. 13E show the results after 10 minutes of incubation for the sample containing cell-microcarrier complex. Similarly, FIG. 13D shows that all the cells were detached from microcarriers after enzymatic incubation (e.g. trypsin incubation). FIG. 13E also shows that Calcein AM staining also verified the high viability of the detached cells. Thus, the experiments have illustrated the feasibility of the various embodiments to facilitate automated on-chip cell dissociation from both cell-microcarrier complex and cell aggregates.[000107] FIG. 14A to FIG. 14F show a study of the cell sorting / purification performance of the second microfluidic chip 674 of the system 701 according to various embodiments. In the experiments conducted, a mixture of 10 pm polystyrene beads and Cytodex-3 microcarriers was loaded into the sample inlet port 132 of the second microfluidic chip 674 at 500 pL / min.As shown in FIG. 14 A, in the absence of sheath flow 124 (i.e. sheath flow 124 is at 0 gL / min), the 10 pm polystyrene beads were randomly distributed in the flow junction 110 and exited both the primary outflow channel 116 and the secondary outflow channel 118. As shown in FIG. 14B, the larger microcarriers were aligned at the centre of the primary inflow channel 112 by the inertial focusing effect and exited via the primary outflow channel 116 regardless of the rate of flow of the sheath flow 124. As the rate of flow of the sheath flow 124 was increased from 0 to 1800 gL / min, the vortex 125 (e.g. microvortex) was formed and became stronger, thus focusing the 10 pm polystyrene beads into a band towards the vortex centre at the side and sorting the 10 gm polystyrene beads into the secondary outflow channel 128. However, when the rate of flow of the sheath flow 124 was increased to 2500 gL / min, more 10 gm polystyrene beads start to exit via the primary outflow channel 116. This may be due to the turbulence caused by the stronger vortex. In contrast, microcarrier separation was unaffected by rate of flow of the sheath flow 124 tested. To further investigate the fluid flow and distribution at the vortex region, FITC solution and 2 gm polystyrene beads were used to visualize the fluid flow. FIG. 14C demonstrates that the non-fluorescent PBS from the sample inlet port 132 (500 gL / min) mostly flowed into primary outflow channel 116, while the fluorescent FITC from the sheath flow inlet port 134 (1900 gL / min) entered both the primary outflow channel 116 and the secondary outflow channel 118. Similarly, an inverse flow pattern was observed when the PBS and FITC flow were swapped and pumped via the sheath flow inlet port 134 and sample inlet port 132, respectively (see FIG. 14D). The streamlines of the 2 pm polystyrene beads roughly indicated microvortex region, which also clearly demonstrates that the majority of the 2 gm polystyrene beads from the sample inlet port 132 exited via the secondary outflow channel 118 (see FIG. 14E), while those from the sheath flow inlet port 134 were entering both the primary outflow channel 116 and the secondary outflow channel 118 (see FIG. 14F).[000108] FIG. 15 A to FIG. 15D show a study on the characterization of the cell purification performance of the second microfluidic chip 674 of the system 701 using a mixture of adipose- derived mesenchymal stem cells (ADSCs) and Cytodex-3 microcarriers, which are commonly used in tissue engineering, according to various embodiments. Consistent with the beads characterization as shown in FIG. 14A and FIG. 14B, FIG 15A shows that the ADSCs were uniformly distributed in the channel and exited both the primary outflow channel 116 (labelled as Chip2-01 in the figure) and the secondary outflow channel 118 (labelled as Chip2-O2 in the figure), and the aligned microcarriers exited via the primary outflow channel in the absence of sheath flow 124 (i.e. sheath flow 124 at 0 gL / min). As also shown, when the sheath flow 124is at 1900 pL / min, the vortex 125 caused by the sheath flow 124 (i.e. sheath flow 124 at 1900 pL / min ) drove the cells into the secondary outflow channel 118 to achieve good separation between the cells and microcarriers. FIG. 15B demonstrates that cell recovery via the secondary outlet port 138 was significantly increased as the sheath flow 124 increased from 0 to 1800 pL / min. At higher rate of flow of the sheath flow 124 (> 2200 pL / min), the cell recovery via the secondary outlet port 138 may decrease due to the turbulence of the strong vortex. FIG. 15C also show that high microcarner (MC) removal via the primary outlet port 136 was also achieved when rate of flow of the sheath flow 124 is between 1900 pL / min and 2200 pL / min. Therefore, rate of flow of the sheath flow 124 at 1900 pL / min was selected for subsequent experiments to study the effects of sample concentration on cell recovery performance. In the subsequent experiments, ADSC suspensions of concentrations ranging from 0.2 million / mL to 10 million / mL were prepared and loaded into the sample inlet port 132 at 500 pL / min. FIG. 15D shows that consistent cell recovery for all tested cell concentrations was observed when a 1900 pL / min sheath was applied. Thus, the experiments show that sample concentration has negligible effect on cell recovery. Further, it was shown that the system 701 (as well as the second microfluidic chip 784) may be capable to process the raw samples in industrial-scale production with cell density as high as 10 million / mL.[000109] FIG. 16 shows further study and quantification to validate the vortex-assisted sample washing effect, i.e. buffer exchange efficiency, of the flow junction 110 using the second microfluidic chip 784 of the system 701 at different rate of flow of the sheath flow 124 according to various embodiments. A fluorescent solution (FITC) and non-fluorescent PBS were loaded into the sample inlet port 132 (500 pL / min) and the sheath flow inlet port 134 of the second microfluidic chip 784, respectively. Solutions from the primary outlet port 136 and the secondary outlet port 138 were collected separately, and fluorescent intensity was analysed using a plate reader. The FITC residue in the solution from the primary outlet port 136 (labelled as 01 in FIG. 16) decreased from 35.6% to as low as 3.1% as the rate of flow of the sheath flow 124 increased from 1000 pL / min to 2200 pL / min. The FITC residue in the solution from the primary outlet port 136 (labelled as 01 in FIG. 16) then slightly increased to 3.5% at 2500 pL / min, which is consistent with the fluorescent images shown in the previously experiments shown by FIG. 10A. Therefore, as little as 3.1% culture media residue may be achieved after buffer exchange (washing) in the vortex region at a rate of flow of 2200 pL / min for the sheath flow 124. This may ensure efficient enzymatic solution (e.g. trypsin) activity downstream, for example in the first microfluidic chip 782 of the system 701.[000110] FIG. 17A and FIG. 17B show a study of cell recovery performance of the vortex 125 generated at the flow junction 110 of the microfluidic chip 100, 500, 600 according to various embodiments. In the experiments conducted, FITC (provided to the sample inlet port 132) and PBS (provided to the sheath flow inlet port 134) were loaded into the microfluidic chip 100, 500, 600. The amount of FITC recovered from the primary outlet port 136 (labelled as 01 in FIG. 17A) and the secondary outlet port 138 (labelled as 02 in FIG 17A and FIG. 17B) was evaluated based on the fluorescent intensity and the volume of the samples collected from each outlet. FIG. 17A shows that 88% of the FITC was recovered from the secondary outlet port 138 (labelled as 02 in FIG. 17A) when the rate of flow of the sheath flow 124 is at 2000 pL / min and 2200 pL / min, indicating that the highest cell recovery may be achieved when the rate of flow of the sheath flow 124 is within this range. In subsequent experiments, as different cell types may vary in size, the effect of particle size on recovery rate was also studied by loading polystyrene beads of different sizes into the sample inlet port 132 and with the rate of flow of the sheath flow 124 (via the sheath flow inlet port 134) at 2200 pL / min. Tn the various embodiments, the polystyrene bead sizes ranged from 1 pm to 15 pm, covering the typical size range of commonly used cells in the lab. FIG. 17B shows that polystyrene beads of all sizes within this range may be recovered with a recovery rate higher than 80%, demonstrating the versatility of the microfluidic chip 100, 500, 600 of the various embodiments to process different cell types.[000111] FIG. 18A and FIG. 18B show a study to validate the on-chip cell dissociation capability of the microfluidic chip 600 of the various embodiments — which integrates vortex- assisted sample washing, concentration, enzymatic solution (e.g. trypsin) mixing, and in-line incubation. In the experiments, dissociation of 3D cell aggregates was first explored. FIG. 18A demonstrates the flow trajectory of ADSC aggregates provided via sample inlet port 132 and the rate of flow of the sheath flow 124 provide via the sheath flow inlet port was at 500 pL / min and 2000 pL / min, respectively. As shown, in the flow junction 110 (e g. the buffer exchange region), culture media and cell debris were removed, and the aggregates were rinsed with clean PBS. Subsequently, in the flow rate reduction arrangement 650 (e g. the concentrator region), the aggregates were focused into the middle branch channel 656 (e g. the centre channel) while excess buffer was removed through the first side branch channel 654 and the second side branch channel 658 (e.g. two side branch channels). Further, by adjusting the rate of flow of the enzymatic solution (e.g. trypsin) and the configuration of the incubation module 786 (e g. incubation tubing) connected to the primary outlet port 136, the incubation duration was tuned to determine the optimal time for complete dissociation of the aggregates. As shown in FIG.18B, Hoechst (DAPI) staining of samples collected from primary outlet port 136 showed that a 10-minute incubation was sufficient to achieve complete dissociation. Further, Calcein AM staining indicated that the majority of cells remained viable after on-chip dissociation. Together, the microfluidic chip 600 of the various embodiments may enable direct single-cell collection from the primary outlet port 136 without requiring any manual sample preparation steps (e g., centrifugation or washing to remove serum from the culture medium), and a 10- minute incubation was found to be optimal for on-chip dissociation of cell aggregates.[000112] FIG. 19A to FIG. 19E show a comparison between the manual dissociation, cell viability, cell recovery, cell metabolic activity, and stem cell biomarker expression as compared to the capability of the microfluidic chip 100, 500, 600 of the various embodiments. Tn the experiments, cell viability was evaluated based on fluorescent images of live / dead-stained cells, showing that both on-chip and manual dissociation achieved -97% viability of the harvested cells (see FIG 19A and FIG. 19B). Cell recovery — defined as the number of single cells harvested from 1 mL of aggregate sample — showed no significant difference between the on-chip and manual methods (see FIG. 19C). Similarly, comparable levels of cell metabolic activity were observed after on-chip and manual dissociation, as measured by a commercial MTT assay kit (ab211091 , Abeam) (see FIG. 19D). Furthermore, on-chip dissociation, like manual dissociation, may not affect the expression of key stem cell markers, including CD73 and CD90 (see FIG. 19E). Together, these results demonstrate that on-chip dissociation of ADSC aggregates may achieve similar cell yield (recovery) and quality (viability, metabolic activity, and stem cell marker expression) as manual dissociation. Thus, various embodiments may replace labour-intensive manual processing with the microfluidic chip 100, 500, 600, thereby saving manpower and minimizing the risk of contamination.[000113] FIG. 20A to FIG. 20C show the system 701 of the various embodiments being employed to harvest cells cultured on the surface of Cytodex-3 microcarriers. Similar to cell aggregates, the experiments demonstrated that the first microfluidic chip 782 of the system 701 (labelled as Chip 1 in the figure) may also dissociate cells cultured on the surface of Cytodex- 3 microcarriers. Effective sample washing via buffer exchange and sample concentration was also achieved when the rate of flow of the sample flow 122 is at 500 pL / min and rate of flow of the sheath flow rate 124 is at 2000 pL / min (see FIG. 20 A). Cells remained well-spread on the microcarrier surface after passing through the vortex region of the flow junction 110, suggesting minimal impact of the vortex 125 on the microcarriers. FIG. 20B shows that cells may be completely detached from the microcarriers after passing through the first microfluidic chip 782, where the sample was washed, concentrated, and mixed with enzymatic solution (e g.trypsin), followed by in-line incubation. The resulting mixture of cells and microcarriers was then introduced into the second microfluidic chip 784 of the system 701 (labelled as Chip 2 in the figure), where microcarriers were removed and single cells were recovered following vortex-assisted separation at the flow junction 110 when the rate of flow of the sheath flow 124 is at 2200 pL / min (see FIG. 20C).[000114] Through the various studies and experiments conducted, it has been shown that the first microfluidic chip 782 of the system 701 — featuring sample washing, concentration, and trypsin mixing — followed by the in-line incubation was capable of successfully dissociating cells from both cell aggregates and cell -microcarrier complex, achieving high cell yield and quality Downstream membrane-free cell purification from microcarriers was further accomplished using the second microfluidic chip 784 of the system 701 with vortex-assisted separation. The entire process using the system 701 of the various embodiments may be closed- loop and may not require human intervention. As such, the system 701 of the various embodiments may be integrated with a bioreactor for automated cell harvesting, offering potential benefits including reduced labour and cost, minimized contamination risk, and improved production efficiency in cell manufacturing.[000115] Referring back to FIG. 7, according to various embodiments, the system 701 for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates may include the first microfluidic chip 782 based on the microfluidic chip 600 of FIG. 6A and FIG. 6B. The system 701 may also include the sample source 788 connected to the sample inlet port 132 of the first microfluidic chip 782. Further, the incubation module 786 may be downstream of the first microfluidic chip 782.[000116][000117] According to various embodiments, the sample source 788 may include, but not limited, to a bioreactor, a cell culture platform, or a sample storage. According to various embodiments, the sample source 788 may contain a fluid medium with cell-microcarrier complex (i.e. microcarriers with cells attached thereon) and / or cell aggregates suspended therein serving as the sample.[000118] When the sample contains only cell aggregates, the incubation module 786 of the system 701 may be connected to the primary outlet port 136 of the first microfluidic chip 782 and a cell collection module may be connected to the outlet of the incubation module for collecting the dissociated cells.[000119] When the sample contains the cell-microcarrier complex or a combination of cellmicrocarrier complex and cell aggregates, the system 701 may also include the secondmicrofluidic chip 784 based on the microfluidic chip 500 of FIG. 5A and FIG. 5B. The incubation module 786 may be connected between the primary outlet port of the first microfluidic chip 782 and the sample inlet port 132 of the second microfluidic chip 784. Furthermore, a microcarrier collection module 781 may be connected to the primary outlet port 136 of the second microfluidic chip 784 for collecting the dissociated microcarriers and a cell collection module 783 may be connected to the secondary outlet port 138 of the second microfluidic chip 782 for collecting the dissociated cells.[000120] According to various embodiments, the incubation module 786 may include a channel 786a (or a tube) and a temperature regulating arrangement 786b associated with the channel 786a (or the tube) According to some embodiments, the channel 786a (or the tube) may be a helical channel (or a helical tube). An example of the helical channel (or the helical tube) may be a coiled tubing. According to some embodiments, when the sample contains only cell aggregates, a first end of the channel 786a (or the tube) may be connected to the primary outlet port 136 of the first microfluidic chip 782 and the second end of the channel 786a (or the tube) may be connected to the cell collection module. According to some embodiments, when the sample contains the cell-microcarrier complex or a combination of cell -microcarrier complex and cell aggregates, a first end of the channel 786a (or the tube) may be connected to the primary outlet port 136 of the first microfluidic chip 782 and the second end of the channel 786a (or the tube) may be connected to the sample inlet port 132 of the second microfluidic chip 784. Accordingly, in these embodiments, the channel 786a of the incubation module 786 may fluidly interconnect the primary outlet port 136 of the first microfluidic chip 782 and the sample inlet port 132 of the second microfluidic chip 784. According to various embodiments, the temperature regulating arrangement 786b may be configured to maintain the channel 786a at an incubation temperature at approximately 37°C; or within a range from 36°C to 38°C, inclusive; or within a range from 35°C to 40°C, inclusive; or within a range from 30°C to 45°C, inclusive. According to some embodiments, the temperature regulating arrangement 786b may include, but not limited to, a series of heating elements coupled to the channel 786a (or the tube), a temperature regulated enclosure surrounding the channel 786a (or the tube), or a water bath in which the channel 786a (or the tube) may be submerged According to various embodiments, the incubation module 786 may further include a temperature control unit 786c operable to control the temperature regulating arrangement 786b for regulating a temperature of the channel 786a (or the tube). According to some embodiments, the temperature control unit 786c may include a processor or a microcontroller or any processor unit capable to control the temperature regulating arrangement 786b.[000121] According to various embodiments, the system 701 may include a pump 791 associated with the sample source 788. The pump 791 may be operable to generate a flow from the sample source 788 to the sample inlet port 132 of the first microfluidic chip 782. Accordingly, the pump 791 may be activated to pump the sample from the sample source 788 into the first microfluidic chip 782 via the sample inlet port 132 thereof. According to some embodiments, the pump 791 may include a peristaltic pump.[000122] According to various embodiments, the system 701 may include the buffer solution source 787 associated with the first microfluidic chip 782. The buffer solution source 787 may be connected to the sheath flow inlet port 134 of the first microfluidic chip 782. According to some embodiments, the buffer solution source 787 may include, but not limited to, a buffer solution tank, a buffer solution container, or a buffer solution vessel.[000123] According to various embodiments, the system 701 may include the pump 792 associated with the buffer solution source 787. The pump 792 may be operable to generate a flow from the buffer solution source 787 to the sheath flow inlet port 134 of the first microfluidic chip 782. Accordingly, the pump 792 may be activated to pump the buffer solution from the buffer solution source 787 into the first microfluidic chip 792 via the sheath flow inlet port 134 thereof According to some embodiments, the pump 792 may include a syringe pump or a peristaltic pump.[000124] According to various embodiments, the pump 791 for pumping the sample into the first microfluidic chip 782 and the pump 791 for pumping the buffer solution into the first micro fluidic chip 782 may be operated in a manner such that a rate of flow of the buffer solution being pump through the sheath flow inlet port 134 of the first microfluidic chip 782 may be equal to or greater than 3 times of, or between 2 to 5 times of, or between 3 to 5 times of, or approximately 4 times of, or approximately 4.5 times of a rate of flow of the sample being pump through the sample inlet port 132 of the first microfluidic chip 784.[000125] According to various embodiments, the system 701 may include a further buffer solution source 787a associated with the second microfluidic chip 784. The further buffer solution source 787a may be connected to the sheath flow inlet port 134 of the second microfluidic chip 784. According to some embodiments, the further buffer solution source 787a may include, but not limited to, a buffer solution tank, a buffer solution container, or a buffer solution vessel.[000126] According to various embodiments, the system 701 may include the pump 792a associated with the further buffer solution source 787a. The pump 792a may be operable to generate a flow from the buffer solution source 787a to the sheath flow inlet port 134 of thesecond microfluidic chip 784. Accordingly, the pump 792a may be activated to pump the buffer solution from the further buffer solution source 787a into the second microfluidic chip 784 via the sheath flow inlet port 134 thereof. According to some embodiments, the pump 792a may include a syringe pump or a peristaltic pump.[000127] According to various embodiments, the pump 792a for pumping the buffer solution into the second microfluidic chip 784 may be operated in a manner such that a rate of flow of the buffer solution being pump through the sheath flow inlet port 134 of the first microfluidic chip 782 may be equal to or greater than 3 times of, or between 2 to 5 times of, or between 3 to 5 times of, or approximately 4 times of, or approximately 4.5 times of a rate of flow of the incubated sample flowing through the sample inlet port 132 of the second microfluidic chip 784 from the incubation module 786.[000128] According to various embodiments, the system 701 may include a medium collection module 796 associated with the first microfluidic chip 782. The medium collection module 796 may be connected to the secondary outlet port 138 of the first microfluidic chip 782. Accordingly, the fluid medium of the sample that is being directed into the secondary outflow channel 1 18 of the flow junction 1 10 of the first microfluidic chip 782 may be discharged into the medium collection module 796. According to some embodiments, the medium collection module 796 may include, but not limited to, a medium collection tank or a medium collection container or a medium collection vessel.[000129] According to various embodiments, the system 701 may include a waste collection arrangement 795 associated with the first microfluidic chip 782. The waste collection arrangement 795 may be connected to the first branch outlet 652 and the second branch outlet 657 of the flowrate reduction arrangement 650 of the first microfluidic chip 782. Accordingly, the fluid that is being directed into the first side branch channel 654 and the second side branch channel 658 of the branch junction 651 of the flowrate reduction arrangement 650 may be discharged into the waste collection arrangement 795. According to some embodiments, the waste collection arrangement 795 may include, but not limited to, a waste collection tank or a waste collection container or a waste collection vessel. Further, the waste collection arrangement 795 may include one waste collection tank or waste collection container or waste collection vessel associated with each of the first branch outlet 652 and the second branch outlet 657.[000130] According to various embodiments, the flowrate reduction arrangement 650 of the first microfluidic chip 782 of the system 701 may be configured to reduce a rate of flow by 6 to 9 times, or 5 to 10 times. Accordingly, a rate of flow at the middle branch channel 656 ofthe flowrate reduction arrangement 650 may be 6 to 9 times, or 5 to 10 times, slower than a rate of flow at the main channel 652 of the flowrate reduction arrangement 650.[000131] According to various embodiments, the system 701 may include the enzymatic solution source 789 associated with the first microfluidic chip 782. The enzymatic solution source 789 may be connected to the solution inlet port 668 of the first microfluidic chip 782. According to some embodiments, the enzymatic solution source 789 may include, but not limited to, an enzymatic solution tank, an enzymatic solution container, or an enzymatic solution vessel. According to some embodiments, the enzymatic solution may include, but not limited to, trypsin.[000132] According to various embodiments, the system 701 may include the pump 794 associated with the enzymatic solution source 789. The pump 794 may be operable to generate a flow from the enzymatic solution source 789 to the solution inlet port 668 of the first microfluidic chip 782. Accordingly, the pump 794 may be activated to pump the enzymatic solution from the enzymatic solution source 789 into the first microfluidic chip 782 via the solution inlet port 668 thereof According to some embodiments, the pump 794 may include a syringe pump or a peristaltic pump.[000133] According to various embodiments, the microcarrier collection module 781 may include, but not limited to, a microcarrier collection tank or a microcarrier collection container or a microcarrier collection vessel. According to various embodiments, the cell collection module 783 may include, but not limited to, a cell collection tank or a cell collection container or a cell collection vessel.[000134] Various embodiments have provided a system 701 (e g. a label-free microfluidic system) for buffer exchange and cell harvesting from microcarrier and / or cell aggregates in continuous cell manufacturing. The system 701 may include the first microfluidic chip 782 for on-chip microcarrier washing and cell dissociation. The first microfluidic chip 782 may include 3 inlets (one for cell-laden microcarrier sample [i.e. the sample inlet port 132], one for sheath flow [i.e. the sheath flow inlet port 134], and one for trypsin [i.e. the solution inlet port 668) and 4 outlets (three waste outlets [i.e. the secondary outlet port 138, the first branch outlet 653 and the second branch outlet 657], and one target containing a mixture of trypsin and microcarrier samples for processing by the second microfluidic chip 784 [i.e. the primary outlet port 136]). The first microfluidic chip 782 may include the flow junction 110 for vortex- assisted microcarrier washing, the flowrate reduction arrangement 650 for microcarrier concentrating (with flow rate reduction), and the mixing channel 672 for herringbone-induced mixing of microcarriers and trypsin. The system 701 may further include the incubation module786 including a long tubing for incubation, the long tubing positioned between the first micro fluidic chip and the second microfluidic chip (placed in 37 °C environment), wherein the first end of the tubing is connected to the primary outlet port 136 of the first microfluidic chip 782. The incubation module 786 may enable the cell dissociation to complete. The system 701 may further include the second microfluidic chip 784 for vortex-assisted cell purification and removal of micro carriers. The second microfluidic chip 784 may include two inlets (one sample inlet for loading mixture of cells and microcarriers [i.e. the sample inlet port 132], and one inlet for sheath flow [i.e. the sheath flow inlet port 138]) and two outlets (for the collection of purified cells and microcarriers, respectively [i.e. the secondary outlet port 138 and the primary outlet port 136 respectively). The second end of the tubing of the incubation module 786 may be connected to the sample inlet 132 of the second microfluidic chip 784. The second microfluidic chip 784 may enable cell sorting / purification.[000135] Advantageously, the various embodiments have included an on-chip microcarrier sample washing (to replace culture media and resuspend microcarriers in sheath buffer (e g. PBS)) by vortex-assisted buffer exchange prior to trypsin treatment which is more efficient to detach cells. This may eliminate the need to use manual centrifugation and pipetting (which is the gold standard in conventional methods). Further, the various embodiments may also achieve higher cell recovery rate (-90%) in the second microfluidic chip 784 due to vortex- assisted deflection of smaller cells into the target outlet. This membrane-free approach may greatly minimize cell loss. The various embodiments may also allow simple optimization for different cell types by adjusting the sheath flow rate in the same microfluidic chip. The vortex- assisted media exchange and cell sorting functions may also be achieved by the sheath-induced vortex, and the vortex strength may be optimized by adjusting sheath flow rate. In addition, the additional sheath flow in the various embodiments automatically dilutes and quenches the trypsin effect to prevent cells from being over-digested to maintain cell viability and functionalities.[000136] FIG. 21 shows a system 2101 for automated media replenishment according to various embodiments The system 2101 may include the microfluidic chip 100 of FIG 1A. Further, the system 2101 may include a bioreactor 2187 connected to the sample inlet port 132 of the micro fluidic chip 100. The bioreactor 2187 may be supporting an ongoing cell culture / cultivation process. Accordingly, the cell culture / cultivation process may be in progress in the bioreactor 2187.[000137] According to various embodiments, the system 2101 may include a pump 2191. The pump 2191 may be operable to pump and flow a sample from the bioreactor 2187 throughthe sample inlet port 132 of the microfluidic chip 100 so as to flow the sample into the microfluidic chip 100. According to some embodiments, the pump 2191 may include a syringe pump or a peristaltic pump.[000138] According to various embodiments, the system 2101 may include a fresh medium source 2187. The fresh medium source 2187 may be connected to the sheath flow inlet port 134 of the microfluidic chip 100. Accordingly, the fresh medium source 2187 may provide a source of fresh medium for flowing into the microfluidic chip 100 via the sheath flow inlet port 134.[000139] According to various embodiments, with the fresh medium flowing into the microfluidic chip 100 via the sheath flow inlet port 134 and the sample flowing into the microfluidic chip 100 via the sample inlet port 132, the fresh medium may flow into the flow junction 110 of the microfluidic chip 100 via the secondary inflow channel 114 while the sample may flow into the flow junction 110 of the microfluidic chip 100. Accordingly, the fresh medium and the sample may meet at the flow junction 110 and generate the vortex 125. The vortex 125 may then deflect the used-medium in the sample into the secondary outflow channel 118 while the fresh medium may enter the primary outflow channel 116. At the same time, the cell aggregate or microcarriers with cells from the sample may continue to flow into the primary outflow channel 116. In this manner, the medium in the sample may be replaced by fresh medium due to the medium exchange at the flow junction 110.[000140] According to various embodiments, the system 2101 may include a used-medium collection module 2196. The used-medium collection module 2196 may be connected to the secondary outlet port 138 of the microfluidic chip 100. Accordingly, used-medium may be discharged from the microfluidic chip 100 into the used-medium collection module 2196 via the secondary outlet port 138.[000141] According to various embodiments, the primary outlet port 136 of the microfluidic chip 100 may be connected back to the bioreactor 2187. Accordingly, the microfluidic chip 100 may return the sample with fresh medium back into the bioreactor 2187 for continuing the cell culture / cultivation.[000142] According to various embodiments, the system 2101 may further include one or more biosensors 2198. The one or more biosensors 2198 may be configured to sense or detect or measure a chemical or biological property of the sample. According to some embodiments, the one or more biosensors 2198 may be disposed in the bioreactor 2187 for sensing or detecting or measuring a chemical or biological property of the sample in the bioreactor 2187.For example, the chemical or biological property of the sample may include, but not limited to, pH level, glucose level, gas composition, nutrients level, oxygen level, or carbon dioxide level. [000143] According to various embodiments, the pump 2191 may be activated to pump the sample into the microfluidic chip 100 when the biological property measured by the one or more biosensors 2198 is outside a predetermined range. Accordingly, the one or more biosensors 2198 may monitor the condition of the cell culture / cultivation in the bioreactor 2187. Upon detection that the condition for the cell culture / cultivation is sub-optimal, the pump 2191 may be activated to replace the medium of the sample with fresh medium such that the sample in the cell culture / cultivation may be maintained at an optimal condition for cell cul ture / cul ti vati on .[000144] According to various embodiments, the system 2101 may include aprocessor 2199. The processor 2199 may be coupled to the one or more biosensors 2198 and further coupled to the pump 2191. Accordingly, the processor 2199 may receive a measurement of the chemical or biological property of the sample from the one or more biosensors 2198. Further, the processor 2199 may control the operation of the pump 2191. According to various embodiment, the processor 2199 may be configured to activate the pump 2191 to flow the sample from the bioreactor 2187 to the microfluidic chip 100 based on the measurement of the chemical or biological property, by the one or more biosensors 2198, being outside the predetermined range. Hence, the processor 2199 may monitor the measurements from the one or more biosensors 2198 and control the pump 2191 to flow the sample from the bioreactor 2187 to the microfluidic chip 100 for medium exchange when the measurement is outside the predetermined range. The predetermined range may be a range of the chemical or biological property that provides the optimal condition for cell culture / cultivation.[000145] The following relates to studies and experiments relating to the flow junction 110 according to various embodiments. FIG. 22 shows a schematic diagram of a flow junction 2210 being used to study the effects of channel configuration for vortex generation. The flow junction 2210 may include the primary inflow channel 112, the secondary inflow channel 114 and an outflow channel 117. The outflow channel 117 may be an inversed-funnel shape. Hence, the outflow channel 117 may be expanding outwards or may be widening. In the various studies and experiments, the angle a between the primary inflow channel 112 (e.g. for sample inflow) and the secondary inflow channel 114 (e.g. for sheath inflow) was varied; the angle b between the secondary inflow channel 114 and the outflow channel 117 was varied; and the angle c [being the widening angle] of the outflow channel 117 was varied. The angle c of the outflow channel 117 of the flow junction 2210 may be transformed into the angle between the primaryoutflow channel 116 and the secondary outflow channel 118 of the flow junction 110 of the various embodiments. In the various studies and experiments, fluorescent beads of 2pm were loaded into the sample flow (i.e. via the primary inflow channel 112) at 500 pm / min, and non- fluorescent Phosphate-Buffered Saline (PBS) was used as sheath flow (i.e. via the secondary inflow channel 114) at 1000 pm / min and 2500 pm / min.[000146] FIG. 23 A shows the experimental results when the angle a was 60°, angle b was 105°, and angle c was 30°. The top fluorescent image shows the experimental results when the sheath flow was at 1000 pm / min and the bottom fluorescent image shows the experimental results when the sheath flow was at 2500 pm / min. FIG. 23B shows the experimental results when the angle a was 60°, angle b was 90°, and angle c was 60°. The top fluorescent image shows the experimental results when the sheath flow was at 1000 pm / min and the bottom fluorescent image shows the experimental results when the sheath flow was at 2500 pm / min. FIG. 23C shows the experimental results when the angle a was 90°, angle b was 75°, and angle c was 30°. The top fluorescent image shows the experimental results when the sheath flow was at 1000 pm / min and the bottom fluorescent image shows the experimental results when the sheath flow was at 2500 pm / min FIG. 23D shows the experimental results when the angle a was 90°, angle b was 60°, and angle c was 60°. The top fluorescent image shows the experimental results when the sheath flow was at 1000 pm / min and the bottom fluorescent image shows the experimental results when the sheath flow was at 2500 pm / min. FIG. 23E shows the experimental results when the angle a was 120°, angle b was 45°, and angle c was 30°. The top fluorescent image shows the experimental results when the sheath flow was at 1000 pm / min and the bottom fluorescent image shows the experimental results when the sheath flow was at 2500 pm / min. FIG. 23F shows the experimental results when the angle a was 120°, angle b was 30°, and angle c was 60°. The top fluorescent image shows the experimental results when the sheath flow was at 1000 pm / min and the bottom fluorescent image shows the experimental results when the sheath flow was at 2500 pm / min. The fluorescent images from FIG. 23A to FIG. 23F demonstrated that micro-vortex may be generated in the flow junction 2210 for all configurations when the sheath flow rate is at 2500 pm / min. However, for sheath flow rate at 1000 pm / min, micro-vortex was observed only in FIG. 23D (when the angle a was 90°, angle b was 60°, and angle c was 60°) and FIG. 23F (when the angle a was 120°, angle b was 30°, and angle c was 60°). Accordingly, the experimental results suggest that having a larger angle a and a larger angle may contribute to the micro-vortex generation. Further, the experimental results also shown that micro-vortex may be successfully generated to achieve media / buffer exchange and particle sorting (for cell sorting / purification) in all the testedchannel configuration (i.e. for angle a being within 60° to 120°, inclusive, and angle c being within 30° to 60°, inclusive) as long as the sheath flow rate was optimized. Accordingly, these results indicated the potential of micro-vortex generation in a wide range of channel configurations for the flow junction 110 of the various embodiments. In particular, variations in the angle between the primary inflow channel 112 and the secondary inflow channel 114 of the flow junction 110 as well as variations in the angle between the primary outflow channel 116 and the secondary outflow channel 118 of the flow junction 110 may be possible to achieve media / buffer exchange and particle sorting (for cell sorting / purification).[000147] Various embodiments have provided a microfluidic chip, suitable for use as a label- free microfluidic cell dissociator. The microfluidic chip integrating a vortex-assisted sample washing module, a herringbone micromixer, and an in-line incubation module for on-chip cell dissociation from microcarriers and cell aggregates. Various embodiments have also provided a microfluidic chip, suitable for use as a label-free microfluidic vortex-assisted cell purificator, for gentle cell harvesting and removal of microcarriers at high cell concentrations Various embodiments have also provided the generation of a vortex (or microvortex) using a perpendicular sheath flow of higher flow rate to deflect the sample flow stream for buffer exchange and washing of cell-laden microcarriers by replacing sample culture media with the sheath buffer from the sheath inlet. Various embodiments have also provided the generation of a vortex (or microvortex) using a perpendicular sheath flow of higher flow rate to focus smaller cells into a band towards the vortex centre for efficient cell sorting. Various embodiments have also provided a system with an integrated microfluidic workflow to achieve automated and continuous sample processing in a closed setup. The system of the various embodiments may also provide tuneable particle size-based separation based on the control of sheath fluid flow rates.[000148] While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes, modification, variation in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

Claims

Claims1. A microfluidic chip comprising: a flowjunction comprising a primary inflow channel, a secondary inflow channel converging and meeting the primary inflow channel at an intersection with respect to each other, a primary outflow channel extending from the intersection to form a continuous line with the primary inflow channel, and a secondary outflow channel extending from the intersection, between the secondary inflow channel and the primary outflow channel, so as to form a first angle with the secondary inflow channel and a second angle with the primary outflow channel2. The microfluidic chip according to claim 1 , wherein the primary inflow channel is wider than the secondary inflow channel3. The microfluidic chip according to claim 1 or 2, wherein the primary outflow channel is wider than the primary inflow channel4. The microfluidic chip according to any one of claims 1 to 3, wherein the primary outflow channel is wider than the secondary outflow channel5. The microfluidic chip according to any one of claims 1 to 4, wherein the primary outflow channel is tapering in a direction extending away from the intersection.

6. The microfluidic chip according to any one of claims 1 to 5, wherein the secondary outflow channel is tapering in a direction extending away from the intersection.

7. The microfluidic chip according to any one of claims 1 to 6, wherein the first angle is an acute angle and the second angle is an acute angle.

8. The microfluidic chip according to any one of claims 1 to 7, further comprising a sample inlet port upstream of the primary inflow channel of the flowjunction.

9. The microfluidic chip according to claim 8, further comprising an inertial focusing channel segment between the sample inlet port and the flow junction.

10. The microfluidic chip according to claim 8 or 9, further comprising a primary outlet port downstream of the primary outflow channel of the flowjunction, a secondary outlet port downstream of the secondary outflow channel of the flow junction, and a sheath flow inlet port upstream of the secondary inflow channel of the flow junction.11 . The microfluidic chip according to claim 10, further comprising a flowrate reduction arrangement between the primary outflow channel of the flow junction and the primary outlet port, wherein the flowrate reduction arrangement directs a fluid flow with reduced flowrate into a flow channel.

12. The microfluidic chip according to claim 1 1 , wherein the flow rate reduction arrangement comprises a branching junction, wherein a main channel branches into two or more branching channels13. The microfluidic chip according to claim 12, wherein, at the branching junction, the main channel branches into a first side branch channel, a middle branch channel, and a second side branch channel, wherein the middle branch channel forms a continuous straight line with the main channel, wherein the first side branch channel and the second side branch channel are on two opposite sides of the middle branch channel, wherein the middle branch channel forms a continuous passage with the flow channel.

14. The microfluidic chip according to any one of claims 11 to 13, further comprising a further inertial focusing channel segment between the primary outflow channel of the fourway flow junction and the flow rate reduction arrangement.

15. The microfluidic chip according to any one of claims 11 to 14, further comprising a three-way junction downstream of the flow rate reduction arrangement, wherein the three-way junction comprises a solution inflow channel that merges with the flow channel into a merged channel, wherein a solution inlet port is upstream of the solution inflow channel.

16. The microfluidic chip according to claim 15, further comprising a mixing channel downstream of the three-way junction, wherein the mixing channel comprises a corrugated surface.

17. The microfluidic chip according to claim 16, wherein the corrugated surface is formed by a series of ridges.

18. The microfluidic chip according to claim 17, wherein each ridge is of an arrowhead shape such that the corrugated surface is of a herringbone pattern.

19. The microfluidic chip according to claim 18, wherein the arrowhead shape is an asymmetric arrowhead shape.

20. The microfluidic chip according to claim 19, wherein the series of ridges comprises a first set of ridges with a first asymmetric arrowhead shape and a second set of ridges with a second asymmetric arrowhead shape, wherein the first asymmetric arrowhead shape and the second asymmetric arrowhead shape are different from each other.

21. The micro fluidic chip according to any one of claims 15 to 20, wherein the mixing channel extends in a serpentine manner22. The micro fluidic chip according to any one of claims 1 to 21, wherein the secondary inflow channel of the flow junction is perpendicular to the primary inflow channel.

23. The micro fluidic chip according to any one of claims 1 to 22, wherein the primary outflow channel is aligned to the primary inflow channel to form a continuous straight line.

24. A system for buffer exchange and harvesting of cells from microcarriers and / or cell aggregates, the system comprising the microfluidic chip according to any one of claims 15 to 21;a sample source connected to the sample mlet port of the microfluidic chip; and an incubation module connected to the primary outlet port of the microfluidic chip.

25. The system according to claim 24, further comprising a cell collection module downstream of the incubation module, wherein the incubation module comprises a channel or a tube, a temperature regulating arrangement associated with the channel or the tube, wherein a first end of the channel or the tube is connected to the primary outlet port of the microfluidic chip, and a second end of the channel or the tube is connected to the cell collection module for cells collection, wherein the temperature regulating arrangement is configured to maintain the channel or the tube at an incubation temperature within a range from 30°C to 45°C, inclusive.

26. The system according to claim 24, further comprising a further microfluidic chip based on the microfluidic chip according to claim 10, the further microfluidic chip connected downstream of the incubation module; a microcarrier collection module connected to the primary outlet port of the further microfluidic chip; and a cell collection module connected to the secondary outlet port of the further microfluidic chip27. The system according to claim 26, wherein the incubation module comprises a channel or a tube, a temperature regulating arrangement associated with the channel or the tube, wherein a first end of the channel or the tube is connected to the primary outlet port of the microfluidic chip, and a second end of the channel or the tube is connected to the sample inlet port of the further microfluidic chip, wherein the temperature regulating arrangement is configured to maintain the channel or tube at an incubation temperature within a range from 30°C to 45°C, inclusive.

28. The system according to any one of claims 24 to 27, wherein the incubation module comprises a temperature control unit operable to control the temperature regulating arrangement for regulating a temperature of the channel.

29. The system according to any one of claims 24 to 28, further comprising a pump operable to generate a flow from the sample source to the sample inlet port of the microfluidic chip.

30. The system according to any one of claims 24 to 29, further comprising a buffer solution source connected to the sheath flow inlet port of the microfluidic chip.

31. The system according to claim 30 insofar as to be dependent on claim 26, further comprising a further buffer solution source coupled to the sheath inlet port of the further microfluidic chip32. The system according to any one of claims 24 to 31 , further comprising a medium collection module connected to the secondary outlet port of the microfluidic chip.

33. The system according to any one of claims 24 to 32, further comprising an enzymatic solution source connected to the solution inlet port of the microfluidic chip.

34. A system for automated media replenishment, the system comprising the microfluidic chip according to claim 10; a bioreactor connected to the sample inlet port of the microfluidic chip; a pump operable to pump and flow a sample from the bioreactor through the sample inlet port of the microfluidic chip; a fresh medium source connected to the sheath flow inlet port of the microfluidic chip; and a used-medium collection module connected to the secondary outlet port of the microfluidic chip, wherein the primary outlet port of the microfluidic chip is connected back to the bioreactor tank.

35. The system according to claim 34, further comprising a biosensor disposed in the bioreactor tank to sense a chemical or biological property of the sample,36. The system according to claim 35, further comprising a processor coupled to the biosensor to receive a measurement of the chemical or biological property of the sample and further coupled to the pump to control an operation of the pump, wherein the processor is configured to activate the pump to flow the sample from the bioreactor to the microfluidic chip based on the measurement of the chemical or biological property, by the biosensor, being outside a predetermined range.

Images