Methods and systems for mechanoporation-based high-throughput payload delivery into biological cells

Described herein are methods and systems for mechanoporation-based high-throughput payload delivery into biological cells. For example, one system can process at least 1 billion cells per minute or at least 25 billion cells per minute, which is substantially greater than conventional methods. A cell processing apparatus comprises a processing assembly formed by stacking multiple processing components. Each processing component comprises channels, which may be used for filtration, mechanoporation, and/or separation of cells in the cell media. This functionality depends on the configuration of each channel. For example, each channel comprises one or more ridges such that each ridge forms a processing gap with an adjacent one of the processing components. The ridges may extend to the side walls or form a bypass gap with the wall. The processing gaps can be specially configured to compress cells as the cells pass through these gaps thereby initiating the mechanoporation process.

BACKGROUND

Intracellular delivery has many valuable applications, such as gene transfection, editing, cell labeling, and cell interrogation. However, conventional delivery methods (e.g., microinjection, electroporation, chemical poration, and sonoporation) have demonstrated low delivery efficiencies and cell viability, especially for large molecules (e.g., molecules with sizes of at least 2000 kDa) and large particles (e.g., particles with sizes of at least 50 nanometers). Furthermore, many conventional delivery methods are not able to process cells at high rates. For example, cells often require individual handling, which significantly slows down processing speeds. What is needed are new methods and systems for high-throughput payload delivery into biological cells.

SUMMARY

Described herein are methods and systems for mechanoporation-based high-throughput payload delivery into biological cells. For example, one system can process at least 1 billion cells per minute or at least 25 billion cells per minute, which is substantially greater than conventional methods. A cell processing apparatus comprises a processing assembly formed by stacking multiple processing components. Each processing component comprises channels, which may be used for filtration, mechanoporation, and/or separation of cells in the cell media. This functionality depends on the configuration of each channel. For example, each channel comprises one or more ridges such that each ridge forms a processing gap with an adjacent one of the processing components. The ridges may extend to the side walls or form a bypass gap with the wall. The processing gaps can be specially configured to compress cells as the cells pass through these gaps thereby initiating the mechanoporation process.

DETAILED DESCRIPTION

In the following description, numerous specific details are outlined to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well-known process operations have not been described in detail to avoid obscuring the present invention. While the invention will be described in conjunction with the specific examples, it will be understood that it is not intended to limit the invention to the examples.

INTRODUCTION

Microfluidic techniques provide new opportunities for processing and manipulation of biological cells, such as the delivery of payload into cells for gene engineering and other applications. For purposes of this disclosure, a “microfluidic” technique is defined as a process of passing fluid through a channel, which has the smallest dimension of less than 1 millimeter. For example, an apparatus may include one or more constrictions forming a gap that is less than 1 millimeter.

A specific example of a microfluidic technique is mechanoporation, which involves mechanical actions on cells to deliver a payload into the cells. For example, cells are passed through narrow gaps (which may be also referred to as constrictions). The gaps are formed by ridges, e.g., extending from one wall toward another wall. The ridges (and as a result the gaps) can have specific geometry (e.g., sharp ridge). Furthermore, cells can be flown at certain processing conditions (e.g., high linear flow rates). Various combinations of these structural and processing conditions can cause rapid cell compression resulting in some cell volume losses. When the cells pass each constriction and are allowed to recover, the cells return to the original volume by absorbing surrounding media, which comprises a payload. As such, a combination of the volume loss followed by the volume gain results in a very efficient payload transfer into the cells. This payload transfer can be referred to as a convective delivery to differentiate from, e.g., diffusion-based delivery during which the payload is driven through the cell membranes by the concentration gradient. However, the diffusion-based delivery takes a long time and is limited to small-sized payloads. Mechanoporation provides substantially faster transfers and is less impacted by the payload size.

The cell compression is achieved in flow channels, each comprising one or more ridges. As noted above, each ridge forms a gap, with the gap size (G) being smaller than the cell diameter (D), at least for cells in a relaxed state/non-compressed state. A combination of the gap size, ridge geometry, and the linear flow rate causes cell compression and volume loss. The mechanoporation with volume change should be distinguished from other microfluidic techniques, which are not based on volume changes and one of which is briefly mentioned above. For example, other microfluidic techniques (e.g., membrane shearing) involve changing the porosity of cell membranes, thereby enabling diffusion-based payload delivery. More specifically, membrane shearing does not involve rapid compression and volume changes. Instead, the cells are passed through tapered funnels that form full circumferential contact with the cell membranes. This full circumferential contact ensures that a large portion of the cell membranes experiences shearing, resulting in the membrane poration with pores sufficiently large for payload diffusion in the cell interior.

One aspect of microfluidic techniques is the small size of flow channels, resulting in flow conditions represented by low Reynolds numbers (e.g., less than 1). As such, fluid viscosity is a dominant factor in these flow conditions. Furthermore, the overall throughput of a single channel is limited. For example, a volumetric flow rate of a single channel having a width of 1 millimeter can be less than 1 ml/min. This flow rate corresponds to the processing speed of about 107cells per hour. At the same time, industrial applications require much larger processing speeds, such as 109or 1010cells per hour, which has significantly limited the adoption of microfluidic techniques in the past.

Furthermore, processing speeds should be maintained at or close to set values despite all possible process and material variations. For example, cell media often contains unwanted particles, abnormal cells, and other such components, which are not able to pass through microchannels, especially through gaps formed by ridges within the channels. Such media components can accumulate in some channels and even block the flow through the channels, thereby reducing the throughput capacity of the overall device.

Different approaches are available to increase the processing speed of a channel. For example, cell concentration in provided cell media, which flows through the channel, can be increased. However, higher cell concentrations (e.g., greater than 10 million cells/mL) can lead to undesirable cell-to-cell collisions. These cell-to-cell collisions can damage the cells thereby reducing cell recovery and viability. Furthermore, these collisions may cause channel clogging, resulting in reduced processed speed.

Another approach is increasing the linear flow speed by increasing the pressure differential across the channel. For example, a linear speed of 1 m/s may require a pressure differential as high as 106Pa in some channels. Such high-pressure differentials require complex and expensive equipment. Furthermore, high flow speed can be damaging to cells.

Another approach for increasing the processing speed involves using multiple parallel channels. This approach produces a proportional increase in the volumetric flow rate without requiring an increase in linear flow speeds/pressure differentials, cell concentrations, and other like methods. However, this multi-channel approach requires special considerations for maintaining the same or similar conditions in all channels, operating in parallel. Maintaining this processing condition uniformity can be challenging. For example, all channels need to have similar linear flow rates of the media, cell concentrations in the media, payload concentrations in the media, and the like. Furthermore, these conditions have to be maintained across multiple processing runs/different batches.

Described herein are methods and systems for mechanoporation-based high-throughput payload delivery into populations of biological cells, which address various challenges described above. Specifically, a cell processing apparatus includes a processing assembly, which can be replaceable and/or disposable in some examples. At least individual components of the processing assembly can be easily replaced, e.g., after processing, which in some instances can cause partial clogging of channels. For example, a pressure differential (for a given volumetric flow) is measured across the processing assembly and the entire processing assembly or some components of the assembly are replaced when this pressure differential reaches or exceeds a certain threshold. Furthermore, components of the processing assembly can be replaced to form different configurations of the processing assembly, e.g., different mechanoporation characteristics. Finally, individual components are easy to manufacture, e.g., using injection molding.

Cell Processing Apparatus Examples

FIG.1AandFIG.1Bare schematic representations of cell processing apparatus100(in an assembled form and exploded view), in accordance with some examples. Cell processing apparatus100comprises processing assembly110, inlet component102, and stopper104. Processing assembly110is disposed between inlet component102and stopper104. More specifically, each inlet component102and stopper104is sealed against processing assembly110. Collectively, processing assembly110, inlet component102, and stopper104define and enclose cavity107of cell processing apparatus100. Cavity107can be also referred to as an inlet opening. In some examples, cell processing apparatus100also comprises distribution component106, positioned inside cavity107.

Referring toFIG.1AandFIG.1B, inlet component102comprises inlet103providing fluidic access to cavity107. During the operation of cell processing apparatus100, cell media containing a population of cells is delivered through inlet103into cavity107. Distribution component106then uniformly distributes the cell media to different portions of cavity107and toward processing assembly110.

Processing assembly110comprises multiple channels139, which allow the cell media to pass through processing assembly110while being subjected to the mechanoporation in each channel. In some examples, processing assembly110comprises at least about 100 channels, at least about 500 channels, or even at least about 1000 channels. Referring to the example inFIG.1AandFIG.1B, each channel139extends radially from primary axis101of cell processing apparatus100through processing assembly110. However, other examples (e.g., when channels139extend parallel to each other) are also within scope.

FIG.2Ais another schematic representation of cell processing apparatus100, from a different angle that may be referred to as a bottom perspective view. In this example, stopper104does not include any outlets. Instead, an outlet may be provided in an enclosure (not shown inFIGS.1A,1B, and2A). Various examples of inlets and outlets are further described below.

FIG.2Bis an expanded view of processing assembly110inFIG.2A, showing processing components119, forming processing assembly110. For example, processing components119are stacked together along primary axis101of cell processing apparatus100(in the Z-direction). In this example, primary axis101may be also referred to as a center axis. Specifically, processing components119are shaped as rings with primary axis101extending through the centers of these rings.

More specifically,FIG.2Billustrates processing component111, second processing component112, and third processing component113, stacked together along the Z-axis. Processing components119may include any number of processing components (e.g., one, two, three, four, five, or more). This number depends on the desired processing speed/processing throughput of cell processing apparatus100as further described below. It should be noted that a larger number of processing components119also allows using cell processing apparatus100for a longer duration. Specifically, the channels of these processing components119can get clogged over time. A larger number of processing components119corresponds to a larger number of channels, which would take longer to clog (e.g., cell processing apparatus100at a reduced capacity can continue to operate as the channels continue to clog). However, increasing the number of processing components119can present various challenges for the uniform distribution of the cell media to each channel. As noted above, each channel needs to process cells in substantially the same manner, e.g., at the same flow rate, pressure, media concentration, and the like.

Referring toFIG.2C, processing component111comprises channels139. Each processing component111may include any number of channels. Similar to the number of processing components, this channel number depends on the desired processing speed/throughput of cell processing apparatus100. Therefore, the overall processing throughput of cell processing apparatus100depends on the throughput of each channel, the number of channels in each processing component, and the number of processing components in the apparatus (as shown by the following formula):
Apparatus Throughput=Channel Throughput×Channels per Component×No of Components

In some examples, each of processing components119has the same design, e.g., to ensure processing consistency and interoperability. For example, processing components119may be supplied as consumables and assembled into processing component111before the use of cell processing apparatus100.

In some examples, each processing component119is fabricated individually, e.g., using injection molding and/or thermal embossment. An injection molding tool can be formed by CNC machining and/or nickel plating (e.g., the tool portions used to form ridges140). Unlike other components of processing components119, ridges140have many small features that require significant precision.

Before stacking these processing components119, each processing component119comprises an open channel with ridges140positioned on the bottom of the channel and extending toward the opening. Once this processing component119is stacked with another processing component119, the channel is closed and ridges140facing his other processing component119that form a gap with each of these ridges140. In some examples, each processing component119comprises all channel walls as, e.g., is schematically shown inFIG.2F. For example, each of processing components119can be formed from two portions that are bonded together to form enclosed channels130. These processing components119can be stacked together to form cell processing apparatus100. In some examples, individually formed processing components119, each comprising multiple enclosed channels can be used to improve the uniformity of channel dimensions. The material used for channels139may be a thermal plastic, such as cyclic olefin copolymers (COC), cyclic olefin polymers (COP), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), and polystyrene (PS). Furthermore, non-thermoplastic materials like glass, silicon, metals, and the like, may be used.

In some examples, two adjacent processing components119are bonded together using different methods, such as thermal bonding, adhesive bonding, solvent bonding, ultrasound bonding, laser welding, pressure-sensitive adhesive, and ultraviolet (UV) glue. Furthermore, in some examples, a seal is formed between two adjacent processing components119to prevent cell media from flowing between these components. The seal may be formed in addition or instead of bonding the components. In some examples, the seal is formed by bonding. In some examples, the seal is formed by a gasket. One example of the seal is shown inFIGS.2D and2E. Specifically, processing component111comprises sealing channel125while second processing component112comprises sealing protrusion126. When processing component111is stacked with second processing component112, sealing protrusion126is inserted into sealing channel125forming a seal. It should be noted that each of sealing protrusion126and sealing channel125(as well as the seal formed by these components) is circumferentially closed.FIG.2Billustrates an example where two adjacent ones of processing components119directly interface with each other, without any intermediate components.

Examples of Mechanoporation Features Positioned in Each Channel

Referring toFIG.2C, each channel130comprises one or more ridges140. Each one of these ridges140is configured to compress cells as the cells pass through a gap formed by this ridge. In some examples, ridges140are configured to simultaneously compress multiple cells distributed along the ridge length, such as from 1 cell to 100 cells, or more than 100 cells. In other words, multiple cells can be compressed by the same since the length of the ridge is much greater than the diameter of the ridge. As such, the number of ridges140in each channel130depends on the number of compressions needed during each pass through cell processing apparatus100. In some examples, the number of ridges140in each channel130is 1 to 50 or, more specifically, 2 to 20 such as 5 to 15. In some examples, the number of ridges in each channel130is the same for all channels of cell processing apparatus100. In some examples, the overall length of each channel130between about 0.05 millimeters to 100 millimeters or, more specifically, between about 1 millimeter and 10 millimeters. Shorter channel lengths are beneficial for reducing the pressure required to produce desired flow speed. In some examples, ridges140are distributed uniformly along the channel length. Alternatively, ridges140can be clustered closer to the channel outlet, e.g., to enable cell focusing prior to the cell interaction with ridges140. In some examples, ridges140are clustered closer to the channel inlet, e.g., to allow longer time for cells to remain in the fluid with higher speed after interactions with ridges140. In some examples, the channel inlet and/or the channel outlet are located at the front and back edges of processing components119. In some examples, the channel inlet and/or outlet can be arranged through channel wall132of processing component119, e.g., to reduce the amount of dust entering each channel130due to the fabrication process.

Furthermore referring toFIG.2C, each channel130is positioned between two dividers120. In some examples, the width of divider120is from 1 micrometer to 100 micrometers, or from 0.1 mm to 0.5 mm, or from 0.5 mm to 10 mm. Furthermore, each channel130is defined by first divider wall121, second divider wall122, and channel wall132. Ridges140protrude from channel wall132and may extend between and first divider wall121and second divider wall122. However, the height of ridges140is smaller than the height of first divider wall121and second divider wall122. In some examples, the height of first divider wall121is the same as the height of second divider wall122. In some examples, the height of the divider120is from 1 micrometer to 50 micrometers, or from 20 micrometers to 500 micrometers, or from 0.1 millimeters to 10 millimeters, or more than 10 millimeters. The channel width in the Y direction defines the number of cells that are processed by compressive ridges140simultaneously. Wider channels allow for a larger amount of cells to be compressed in parallel. Wider channels, however, are more likely to deform (e.g., under the internal pressure within the channels) affecting the uniformity of the gap formed by ridges140. Narrow channels, on the other hand, are more prone to clogging. In some examples, the channel width is from about 10 micrometers to about 1 millimeter, or more specifically from about 0.1 millimeters to 0.5 millimeters, or from about 0.4 millimeters to 0.8 millimeters. In some examples, the channel width is from about 1 millimeter to about 10 millimeters, or wider than 5 millimeters.

FIG.2Dillustrates a side cross-sectional view of processing component111and second processing component112, prior to stacking these components. Processing component111comprises first surface123, formed by divider120, and facing second processing component112. Second processing component112comprises second surface124, facing away from divider120of this component and facing first processing component111.

FIG.2Eillustrates a side cross-sectional view of processing component111and second processing component112, after stacking these components/after forming processing assembly110. At this stage, first surface123of processing component111contacts second surface124of second processing component112, thereby isolating adjacent channels130. In some examples, first surface123is bonded and/or sealed to second surface124. The height of these channels (in the Z-direction) is defined by the height of the divider walls (e.g., first divider wall121shown inFIG.2D). Because ridges140are shorter than the divider walls, ridges140of processing components111form gaps141with second surface124of second processing component112. These gaps141are specifically configured to compress cells as these cells pass through each of gaps141and cause the volumetric change in each cell. Additional gap features will now be described with reference toFIG.2F.

Referring toFIG.2F, each gap141is identified with a corresponding height, labeled as “H”. The gap height is selected such that cells are compressed to pass through gap141. In other words, the gap height is smaller than the cell size (H<D). It should be noted thatFIG.2Fillustrates an example in which the cross-sectional profile of ridge140is rectangular. However, other shapes of the profile are also within the scope, e.g., cylindrical, trapezoidal, or triangular. In some embodiments, the plurality of compressive surfaces may be orthogonal.

Another characterization, at least of rectangular ridges140, is the length of ridge surface142in the X direction (identified as “L”), which may be also referred to as the ridge thickness. In some examples, the ridge surface length is between about 1 micrometer and 100 microns or, more specifically between about 20 micrometers and 50 micrometers. This length, together with the linear flow rate, defines the period during which a cell is compressed by the ridge. In some examples, ridge surface142is parallel to second surface124. In other words, gap141is defined by two parallel surfaces. These parallel compressive surfaces allow for a uniform compression for the entire cell. Additionally, the compression surfaces can be converging and/or diverging. It should be noted that in addition to the cell compression, ridges140also produce hydrodynamic mixing within the cell media

Referring toFIG.2F, gap141is selected based on the cell size, compression needed, and other characteristics of mechanoporation. In some examples, the gap height (H) is between 1 micrometer and 20 micrometers, or between 10 micrometers and 100 micrometers or, more specifically, between 3 micrometers and 8 micrometers. Furthermore, the gap height (H) may be also defined relative to the cell size (D), which is defined as the average largest cross-sectional dimension of cells. More specifically, the ratio of the gap height to the cell size (H/D) defines cell compression. In some examples, this H/D ratio is between 25% and 75% or, more specifically, between 30% and 60%. Internal channel height (IH) defines the flow speed between ridges and the time that the cell spends between consecutive compressions. In some examples, the IH is between about 2 micrometers and 100 micrometers or more specifically between 5 micrometers and 10 micrometers or between 10 micrometers and 15 micrometers. In some examples, IH is between about 10 micrometers and 1 mm or more specifically between 50 micrometers and 100 micrometers.

The gap height (H) may be the same for all ridges140in the same channel. Alternatively and with reference toFIG.2F, the gap height (H) may be different for different ridges140. For example, the gap height decreases along the direction of the flow thereby subjecting the cells to higher compression as the cells flow through cell processing apparatus100. In some examples, when larger cells are compressed in larger gaps, these cells may retain a flattened (pancake-like) shape and can then pass through smaller gaps, again without being removed from the flow. This feature may be referred to as a staged-compression. Furthermore, the smaller gaps may start processing smaller cells, if present (e.g., in a diverse population). Thus, using gap size varying along the channel can improve convective intracellular delivery to heterogeneous cell populations. Furthermore, such channels with varying compression gaps can be used to improve cell sorting by reducing the influence of cell size heterogeneity.

Intracellular delivery is controlled by cell compression rate, which is a rate of volume loss by cells in order to pass through the gap formed by a ridge. The cell compression rate can be determined by the flow rate, ridge geometry, a ratio of the gap height to the cell size, ridge width, ridge angle, and compressive surface coating. Furthermore, it has been found that the volume loss (Vloss) increases with the increase in the cell compression rate. Various processing and device characteristics may be specifically selected to achieve the desired cell compression rate. In some examples, ridges140are oriented at an angle (a) between 0 degrees to 90 degrees relative to a central flow axis109in each channel as, e.g., is schematically shown inFIGS.2G-2J. More specifically, the ridge angle (a) is between 10 degrees to 30 degrees, or between 30 degrees and 90 degrees, more specifically about 45 degrees. In some examples, ridges140form a chevron as, e.g., is shown inFIG.2G. Alternatively, ridges140are straight, e.g., as shown inFIG.2H. In some examples, ridges140are curved as, e.g., is shown inFIGS.21and2J. Furthermore, in some examples, ridges140can extend both sidewalls, e.g., as shown inFIG.2Gor, form a sidewall gap with at least one of the sidewall, e.g., as shown inFIG.2H-2J. This sidewall gap may be referred to as a gutter and may receive uncompressible cells (e.g., pushed along ridges140into the sidewall gap) thereby reducing the risk of channel clogging.

It should be noted that the cross-sectional shape of ridges140(shown inFIG.2F) defines the cell compression profile. In some examples, the shape is rectangular (shown inFIG.2F), or trapezoidal, or triangular. In some examples, ridge surface142forming compression gap141is substantially flat (e.g., parallel to the second surface124), or tilted forming a gap varying along the X-axis. In some examples, ridge surface142is nearly cylindrical.

Referring toFIG.2F, spaces within the channel between adjacent ridges140and after the last ridge and outlet may be referred to as recovery spaces145. The length of recovery spaces145(in the X direction) between two adjacent ridges may be referred to as ridge spacing (S). The ridge spacing depends on the flow rate, cell characteristics, levels of the previous compression, and such. In some examples, the ridge spacing is between 1 micrometer and 100 micrometers or between 50 micrometers and 10000 microns such as between 200 micrometers and 500 micrometers. The volume of each recovery space145and the flow rate determines the average recovery time, i.e., the time that the cells spend in recovery space145before being subjected to another compression. It has been found that volume gain (Vgain) is increased when the recovery time is increased. The recovery time can be increased by increasing the length of recovery spaces145.

Processing Assembly Enclosure Examples

As described above, processing assembly110comprises multiple processing components119stacked along primary axis101. Each of processing components119comprises multiple channels139extending in the direction perpendicular to primary axis101and configured for flowing cells. Processing assembly110can be enclosed in various enclosure examples, which isolate processing assembly110from the environment, provide support to processing assembly110, and ensure uniform flow of the cell media into each channel139.

FIG.3Ais a schematic exploded view of an example of cell processing apparatus100comprising inlet component310(comprising inlet103) and outlet component320(comprising outlet105), sealed to each other, and enclosing processing assembly110comprising multiple processing components119.FIG.3Bis a schematic cross-sectional view of cell processing apparatus100inFIG.3A. Specifically, inlet component310and outlet component320define interior cavity330, which houses processing assembly110. The shape of interior cavity330is specifically defined to ensure that the cell media (generally flowing through cell processing apparatus100along the Z-axis) is evenly distributed to each channel139of processing assembly110. It should be noted that multiple channels139are positioned on each processing component119and offset with respect to each other along the X-axis. Furthermore, multiple processing components119are stacked together along the Y-axis, forming processing assembly110. As such, the shape of interior cavity330provides uniform cell media distribution along the X-axis as well as along the Y-axis while the cell media is delivered from inlet103to each channel139, e.g., to ensure the same flow through each channel139. Similarly, the shape of interior cavity330provides the uniform cell media collection along the X-axis as well as along the Y-axis while the cell media exits from each channel139and is directed to outlet105(e.g., to ensure uniform resistance to the overall flow). It should be noted that the cell media generally flows along the Z-axis. Additional features of interior cavity330and channels139are shown inFIGS.3C-3F, illustrating inlet component310. It should be noted that inlet component310and outlet component320can have a symmetrical design as, e.g., is shown inFIG.3B.

FIG.3Cis a schematic top view of inlet component310of cell processing apparatus100inFIG.3A. Inlet component310is shown to support processing assembly110. For example, processing assembly110can be inserted into inlet component310(or outlet component320) during the assembly of cell processing apparatus100before attaching and sealing inlet component310and outlet component320relative to each other.FIG.3Dis an expanded top view of a portion of inlet component310inFIG.3C, illustrating channels139and ridges140in processing component119. While only the top processing component119is visible inFIGS.3C and3D, any number of processing components119can be stacked along the Y-axis.FIGS.3E and3Fare schematic cross-sectional views of inlet component310inFIG.3C, illustrating a portion of interior cavity330and processing components119positioned within interior cavity330, in accordance with some examples.

FIG.4Ais a schematic perspective view of yet another example of cell processing apparatus100formed by processing components119stacked between inlet plate430and outlet plate440.FIG.4Bis a schematic exploded view of cell processing apparatus100inFIG.4A. In this example, processing assembly110is formed by eight processing components119. However, any number of processing components119is within the scope.FIG.4Cis a top schematic view of processing component119, used in the cell processing apparatus inFIG.4A, illustrating inlet opening410and outlet opening420.FIG.4Calso illustrates multiple channels139extending between inlet opening410and outlet opening420. In this example, each inlet opening410and outlet opening420has a triangular boundary within a plane perpendicular to primary axis101(and parallel to the X-Z plane inFIG.4C). In some examples, a triangular boundary shape prevents the formation of stagnant zones in the flow.

During the operation of cell processing apparatus100, the cell media enters cell processing apparatus100through inlet plate430and is directed to inlet opening410of processing component119, adjacent to inlet plate430. It should be noted that inlet openings410of all processing components119can coincide and form a continuous tunnel through cell processing apparatus100between inlet plate430and outlet plate440. Similarly, outlet openings420of all processing components119can coincide and form a continuous tunnel through cell processing apparatus100between inlet plate430and outlet plate440. In some examples, the cross-section of inlet opening410is the same, and a mirror image of the cross-section of outlet opening420as, e.g., is shown inFIG.4C.

Returning to the operation examples, once the cell media is within inlet opening410of processing component119, adjacent to inlet plate430, a portion of the cell media flows through channels139of this processing component119into outlet opening420. The remaining cell media is directed to inlet openings410of other processing components119. Eventually, all cell media goes through channels139of processing components119and into the tunnel formed by outlet openings420of processing components119and then removed from cell processing apparatus100.

In some examples, cell processing apparatus100is assembled by stacking several substantially identical cell processing components119. While the tunnels, formed by inlet openings410and outlet openings420of processing components119, provide uniform distribution and collection of the cell media, these tunnels also require a significant volume of the cell media to fill the tunnels. At least some (or all) of this cell media cannot be recovered from the tunnels. As such, the tunnels represent “dead volume” within cell processing apparatus100. This “dead volume” can be reduced by special protrusions extending into these tunnels. Specifically, when these external protrusions (e.g., provided as a part of inlet plate430and outlet plate440) allow processing components119to be the same and be replaced when needed (e.g., when channels139of processing components119become blocked). These protrusions features will now be described with reference toFIGS.4D-4G.

Specifically,FIG.4Dis a schematic perspective view cell processing apparatus100with processing components119stacked between inlet plate430and outlet plate440.FIG.4Eis a schematic cross-sectional view of cell processing apparatus100inFIG.4D.FIG.4Fis an exploded cross-sectional view of cell processing apparatus100inFIG.4D, providing additional representation of some components and features. Inlet plate430comprises inlet protrusion432extending into outlet opening420of each of processing components119and occupying a different volume in outlet openings of different ones processing components119. Specifically, inlet protrusion432is tapered and blocks the most volume in outlet opening420of the closest (adjacent) processing component119and the least volume in outlet opening420of the furthest processing component119. As such, the unblocked volume is the largest in the closest (adjacent) processing component119since outlet opening420carries the least amount of cell media (out of all processing components119), i.e., the only the cell media that have flown through this closest (adjacent) processing component119. Outlet opening420of the next processing component119carries the cell media that have flown through this next processing component119also received from the closest (adjacent) processing component119. Finally, outlet opening420of the furthest processing component119carries all cell media going through all processing components119, thereby needing the most unblocked volume. In a similar manner and with reference toFIG.4G, outlet plate440comprises outlet protrusion442extending into inlet opening410of each of processing components119and occupying a different volume in outlet opening420of a different one processing components119. The same principle of different volumetric flow rates through each inlet opening410applies on the inlet side. This matching of different volumetric flowrates (at different positioned within the inlet and outlet tunnels) and different cross-sectional areas of these tunnels (provided by different blocked volumes by inlet protrusion432and outlet protrusion442) produce more uniform linear flow rates within cell processing apparatus100as will now be described with reference toFIG.4H

Cell media is supplied into each one of channels139using various distribution pathways. These pathways are specifically designed such that the linear flow rate through each channel139is substantially the same. This flowrate uniformity ensures that all cells are processed in a similar manner, e.g., subjected to the same compression rate, for the same period, and allowed the same relaxation time. These pathways are provided by various components of cell processing apparatus100such as distribution component106, processing assembly110, and/or other components.

FIG.4Hillustrates a cross-sectional side of a portion of cell processing apparatus100, in accordance with some examples. Specifically,FIG.4Hillustrates distribution component106, outer wall150, and processing assembly110, positioned between distribution component106and outer wall150. The space between distribution component106and processing assembly110is used to supply the cell media to channels139within processing assembly110. The space between outer wall150and processing assembly110is used to remove the cell media that passed through channels139.FIG.4Halso illustrates the cell media is being delivered to the space between distribution component106and processing assembly110at the top of cell processing apparatus100, adjacent to inlet component102. The cell media is being removed from the space between outer wall150and processing assembly110at the bottom of cell processing apparatus100, adjacent to stopper104.

The cross-section inFIG.4Hillustrates six channels139stacked vertically between inlet component102and stopper104. As the cell media enters the space between distribution component106and processing assembly110the cell media is directed into channels139. For clarity, a channel formed by processing component111and second processing component112may be referred to as a first channel, while a channel formed between second processing component112and third processing component113may be referred to as a second channel. As the cell media enters the first channel, the volumetric flow rate of cell media traveling into space between distribution component106and processing assembly110past the first channel is less. Additional reduction on the volumetric flow rate appears after each new channel as each channel allows some of the cell media to flow through the channel. If the cross-section of the space between distribution component106and processing assembly110remains constant, then the linear flow rate of the cell media will drop proportionally to the volumetric flow rate. The width reduction (W1>W2>W3) shown inFIG.4Hallows maintaining the linear flow rate of the cell media substantially constant within the space between distribution component106and processing assembly110. Similar but the inverse process occurs in the space between outer wall150and processing assembly110. The volumetric flow rate in that space increases from top to bottom as additional cell media is received from each new channel. As such, the width of this space is increased from top to bottom (W1<W′2<W′3). Overall, the cross-sectional area of supply channels varies between channel layers such that the linear flow velocity is uniform throughout all parts of cell processing apparatus100. This can prevent the formation of stagnation zones in the flow.

In some examples, one or more of the processing components comprise distribution and/or collecting pathways. These pathways should be distinguished from processing channels, which comprise compressive ridges.FIG.5Aillustrates an example of distribution pathway160, arranged into a tree-like structure. Each distribution pathway channels branches to two identical sub-pathways, at each branching level, to provide equal flow conditions for each branch (e.g., using specific diameters in each branch of these pathways). These sub-pathways may be also referred to as branching pathways. This branching structure ensures that each channel139has the same linear flow rate. Furthermore, when the cross-section of all channels139is the same, the volumetric flow rate is also the same. WhileFIG.5Aillustrates a two-way split, one having ordinary skill in the art would understand that a split includes any number of sub-pathways (two, three, four, and so on). Furthermore, whileFIG.5Aillustrates two levels of splits, one having ordinary skill in the art would understand that a split includes any number of these split levels (two, three, four, and so on).

Overall, the configuration is shown inFIG.5Aand other like configurations can be used to ensure that all processing channels are supplied by identical amounts of media, reagents, and cells which are required for maintaining product quality and consistency. To maintain similar flow velocity in the distribution and collection channels, the total channel cross-section area (i.e. the sum of channel cross-section areas at each level of branching) can be kept constant at each branching level (distributing and collecting), such that Atotal=N*A, where A=H*W and N it the number of channels. The channel dimensions can be set such that W/H did not exceed 20 due to fabrication constraints, such as wall sagging when channels are excessively wide. The number of parallel microchannels can be limited by the fabrication process, the footprint of the microchannel layout with distributing and collecting channel networks, air bubble formation in microchannels preventing or altering the fluid flow, and the cross-sectional dimensions of the supply channels. Similarly branching channel structures can be used at the outlet to collect the media and processed cells. Such arrangement of the outlet section can be used to ensure similar flow resistance over each processing channel139.

FIG.5Billustrates another example of distribution pathway160provided in processing components or, more specifically, in second processing component112, stacked between processing component111and third processing component113. In this example, distribution pathway160is in fluidic communication with two channels, channel130formed by processing component111and second processing component112, and also channel130formed by second processing component112and third processing component113. As such, distribution pathway160can be provided in every other processing component.FIG.5Cillustrates another component where distribution pathway160is provided in each processing component.

Distribution pathway160is shown inFIGS.5B and5Ccan be used for more efficient use of reagents (e.g., reduce the volume of expensive reagents needed). In these examples, reagents can be supplied directly into the processing channels130using distribution pathway160without premixing with media-containing cells, at least outside premixing of processing assembly110. This approach can be used also when reagents are unstable and degrade in cell media. In some examples, the flow rate of reagents is lower than the flow rate of cell media by at least about 10, 100, or even 1000 times. In these examples, distribution pathways160can have much smaller cross-sectional areas in comparison to processing channels130. Furthermore, the pressure in distribution pathways160is matched or exceeds the pressure in processing channels130to prevent the backflow of cell media into distribution pathways160.

Arrangements of processing channels130in processing components119may depend on the shape of processing components119and overall processing assembly110. For example,FIG.2Aillustrates round processing components119. In this example, processing channels130extend radially, e.g., from primary axis101of cell processing apparatus100. When processing components are rectangular, processing channels130may extend parallel to each other. The use of circular layers has the advantage of a simpler stack design where leaks can be prevented using ring-shaped rubber gaskets placed between individual layers. In contrast, using rectangular-shaped layers can require the use of gaskets to prevent fluid leads at the corners to the assembly that are more prone to leaking.

In some examples, processing components119, which comprise ridges140are alternated with reagent delivery components, which do not have ridges. In these examples, processing channels130may be formed entirely by processing components119or by stacking processing components119with reagent delivery components.

Channel design can include structural elements enhancing the mixing of the reagents with media such as pillars, ridges, channel constrictions. Actuated mixing elements can be included such as magnetic beads, magnetic filaments, and acoustically driven filaments. The mixing ensures uniformity of the media.

In some examples, the apparatus can include a plurality of cell processing apparatuses100arranged in parallel. The use of multiple parallel devices simultaneously can be limited by the requirement for the overall flow rate in the apparatus. In some examples, the flow is supplied sequentially to different devices. The flow is directed to the next device when significant clogging is detected reducing the flow rate. In some examples, the flow switching from one device to another is controlled by monitoring the flow rate within the supply channel. In some examples, a plurality of cell processing apparatuses100are connected in series to provide multistep mechanoporation. In some examples, the serial connection of multiple cell processing apparatuses100is implemented to improve delivery efficiency or to deliver different payloads sequentially into the cell population.

Filter and Separation Integration Examples

In some examples, cell processing apparatus100is configured to perform additional functions, besides mechanoporation. Furthermore, one of these additional functions may be performed in the same processing assembly where mechanoporation is performed. For example, processing components may be configured to perform filtering and cell separation.

FIG.6Ais a schematic illustration of cell processing apparatus100comprising processing assembly110, which in turn comprises primary filter510, mechanoporation520, and separator530. Each of primary filter510, mechanoporator520, and separator530is formed by one or more processing components. However, the configurations of these processing components is different for each of primary filter510, mechanoporator520, and separator530as further described below with reference toFIGS.6B and6C.FIG.6Aalso illustrates pre-filter502and mixer504as additional components of cell processing apparatus100. These components may be separate from processing assembly110.

Cells are initially delivered into pre-filter502. The purpose of the pre-filter is to remove (from the media) any synthetic and biological particles that are significantly greater than the average cell size and can lead to clogging of the processing microchannels. Some examples of the pre-filters include, but are not limited to, arrays of posts with separation comparable to the average cell size, and cross-channel ridges forming a gap that is similar to the gap in the processing channel.

Pre-filtered cells are then delivered from pre-filter502into mixer504. In some examples, media (e.g., a liquid base) and/or payload are also delivered into mixer504. Mixer504combined the cells with the media and payload, forming a cell media. The cell media is then delivered into processing assembly110. More specifically, the cell media is first delivered into primary filter510where cells are filtered based on their compressibility. In some examples, cells are mixed with payload using a mixer504after primary filter510. The cells, which passed primary filter510, are then delivered to mechanoporator520. Mechanoporator's functions are described above. Processed cells are then flown from mechanoporator520to separator530. Each of these components will now be described in more detail.

Similar to mechanoporator520, primary filter510is formed from processing components, which are stacked together along primary axis101of cell processing apparatus100. The processing components of primary filter510may be stacked together with the processing components of mechanoporator520. However, the processing components of primary filter510are different from the processing components of mechanoporator520as further described below with reference toFIGS.6B and6C. Primary filter510is configured to capture abnormal cells and other particles that due to their size or mechanical properties cannot pass through the gaps of mechanoporator520and can be stuck in the processing channels of mechanoporator520, leading to clogging of mechanoporator520.

In some examples, the linear flow velocity of the cell media through primary filter510is less than the linear flow velocity through mechanoporator520(e.g., at least about 2 times less, at least about 5 times less, or even about 10 times less). The lower flow velocity through primary filter510is used to prevent damage of cells as cells pass near captured particles and uncompressible cells in primary filter510. In some examples, the difference in the linear flow velocity is achieved by using a larger number of processing components (and corresponding channels) in primary filter510than in mechanoporator520as, e.g., is schematically shown inFIG.6B.FIG.6Billustrates cell processing apparatus100comprising processing assembly110formed by primary filter510and mechanoporator520. The number of processing components in primary filter510is greater than in mechanoporator520, e.g., at least about 2 times greater, at least about 5 times greater, or even about 10 times greater. Assuming that each processing component in primary filter510and mechanoporator520has the same number of channels and these channels have the same average cross-section, then the ratio of the linear flowrate through each channel in primary filter510and the linear flowrate through each channel in mechanoporator520is inverse-proportional to the ration of the number of processing components in primary filter510to the number of processing components in mechanoporator520. As such, the number of processing components in primary filter510, mechanoporator520, and other sub-assemblies of processing assembly110can be used to control linear flow rates through each of these sub-assemblies. It should be noted that the volumetric flow rate through each of these sub-assemblies is the same. Overall, primary filter510, mechanoporator520, and other sub-assemblies can be integrated into the same processing assembly110. Alternatively, these sub-assemblies can be standalone components.

Referring toFIG.6A, after passing through mechanoporator520, the cell media can be supplied to separator530that separates processed cells and from the rest of the cell media (e.g., media and remaining payload). These media and remaining payload can be recycled (e.g., supplied back to mixer504) where these components are combined with new cells, additional payload, and/or additional media (e.g., to achieve the desired composition of the cell media supplied into processing assembly). The amount of new reagent can be defined based on the separation efficiency of separator530.

Referring toFIG.6C, in some examples, separator530comprises diagonal ridges140that concentrate cells along one side of the channel, e.g., first divider wall121. The spacing between the ends of ridges140and first divider wall121may be referred to as sidewall gap143(or “gutter”). As the cells flow through the channel and encounter ridges140, ridges140direct the cells toward sidewall gap143while allowing the rest of the media (including the remaining payload) to flow through the separation gap between ridges140and another wall of the channel. The separation gap is similar to gap141described above with reference toFIG.2Fand used to compress cells. Separator530also comprises first outlet146, aligned with sidewall gap143, and used to remove processes cells from separator530. Furthermore, separator530comprises second outlet147for removing the rest of the media (separated from the processed cells). As noted above, this media can be returned back to mixer504.

In some examples, cell processing apparatus100comprises sensors for process control, such as pressure, temperature, and oxygen sensors. For example, pressure sensors are positioned in an inlet (before the processing channel) and in an outlet (after processing channels) to determine the pressure drop across the processing channels or, more specifically, across primary filter510and/or across mechanoporator520. The monitoring pressure differential can be used to determine clogging. In some examples, chemical sensors are used to control cell conditions. Flow sensors can be used to control flow rates. The signals from the sensors can be used to control the operations of various devices (e.g., pumps, cell media supply, and the like). For example, pressure and flow data can be used to control the functionality of the filtration element. When the filtration element traps a significant amount of particles and abnormal cells, the reduced flow rate or increased pressure can be used to interrupt the processing and replace or flush the filtration element.

FIG.7Ais a schematic representation of another example of processing system190comprising cell processing apparatus100. Cell processing apparatus100comprises inlet103and outlet105. Inlet103is used for connection to cell media source191, such as a sterile bag. Outlet105is used for connection to cell media receiver192, such as a sterile bag. While not specifically identified inFIG.7A, processing system190can also include various valves, connectors, and the like.

Referring toFIGS.7B and7C, cell processing apparatus100comprises cell media collector710comprising collector cavity712and collector port720. During the operation of processing system190, collector cavity712is first filled with the cell media (e.g., from cell media source191). For example, a reduced pressure (e.g., between 1 Pa and 1 kPa) can be created in collector cavity712, using a device (e.g., vacuum pump) fluidically coupled to collector port720. This reduced pressure causes the cell media to fill collector port720. It should be noted that during this cavity-filling operation, the flow through outlet105is blocked (e.g., using an outlet valve). Once collector cavity712is filled, the flow through inlet103is blocked (e.g., using an inlet valve), while the flow-through outlet105is enabled. Collector cavity712can be pressurized (e.g., between 105Pa and 106), using the same or a different device fluidically coupled to collector port720. This pressure causes the cell media to flow through processing components119or, more specifically, through channels139in processing components119.

Referring toFIG.7C, outlet105is fluidically coupled to each of the multiple channels139. Collector cavity712is fluidically coupled with the collector port720, inlet103, and each of multiple channels139. Collector port720can be used for connection to a gas flow source. In this example, inlet103is positioned closer to processing assembly110than to collector port720. This approach exerts less mechanical stress on the cell media (gentler filling) compared to the top filling. Furthermore, bottom filling generates less foaming. In some examples, a top-filled device is equipped with a liquid guide feature to prevent the cell media (liquid) from falling down/splashing in a collector cavity.

FIGS.7D and7Eas well asFIGS.8A and8Bare schematic views of additional examples of cell processing apparatus100, in which inlet103is positioned closer to collector port720than to processing assembly110. It should be noted that during filling, some air bubbles can be introduced to cell processing apparatus100. The top filling approach minimizes bubble formation by releasing air from the top. If the liquid is filled from the bottom and there is some air inside the liquid initially, the air has to pass through the liquid to escape, which may cause bubbles and confuse the liquid level sensors. The top filling approach helps with preventing bubble formation, e.g., allowing air to escape from the top and avoid bubble trapping.

Referring toFIG.7E, in some examples, cell media collector710further comprises one or more level sensors714for measuring one or more levels of cell media within the collector cavity712. Some examples of level sensors714include, but are not limited to, capacitance sensors, ultrasonic sensors, and magnetic sensors. When magnetic sensors are used, a floater with a magnet can be positioned in the cavity. The floater changes the position with the level of the cell media.

Operating Method Examples

FIG.9is a process flowchart corresponding to mechanoporation method900of processing cells using cell processing apparatus100, in accordance with some examples. Various examples of cell processing apparatus100are described above.

Mechanoporation method900comprises (block910) flowing the cell media comprising cells through inlet103of cell processing apparatus100. In some examples, the cell media is agitated in cell media source191to prevent cell gravitational sedimentation. Such agitation can be achieved by a mechanical or magnetic agitator causing temporal or continuous media motion in the cell media source191.

Mechanoporation method900also comprises (block920) distributing the cell media within cell processing apparatus100among multiple channels139in each of processing components119stacked along primary axis101of cell processing apparatus100.

Mechanoporation method900further comprises (block930) flowing the cell media through multiple channels139. Each channel139comprises one or more ridges140. Each ridge140forms gap141with an adjacent one of processing components119such that gap141is smaller than the diameter of the cells in the cell media. Flowing the cell media through multiple channels139causes the compression of cells while cells pass through gap141as, e.g., described above with reference toFIG.2F.

Furthermore, flowing the cell media through multiple channels139is performed while a portion of cell media experiences the same pressure upon entry into each of multiple channels139. This same pressurization feature is achieved by an air pump or another pressure source.

CONCLUSION

Although the foregoing concepts have been described in some detail for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. It should be noted that there are many alternative ways of implementing the processes, systems, and apparatuses. Accordingly, the present examples are to be considered illustrative and not restrictive.