The present disclosure provides a cell growth, buffer exchange, and/or cell concentration device that may be used as a stand-alone device or as a module configured to be used in an automated multi-module cell processing environment.
In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Genome editing with engineered nucleases is a method in which changes to nucleic acids are made in the genome of a living organism. Certain nucleases create site-specific double-strand breaks at target regions in the genome, which can be repaired by nonhomologous end-joining or homologous recombination resulting in targeted edits. Nucleases can be used to introduce one or more edits into multiple cells simultaneously, allowing for the production of libraries of cells with one or more edits in the cellular genome. These methods, however, generally have not been compatible with automation due to low transformation and editing efficiencies and challenges with cell growth and selection. In addition to genome editing, other multi-step cell processes would benefit from automation, including genome engineering, hybridoma production, and induction of protein synthesis.
In order to obtain an adequate number of cells for transformation or transfection, cells typically are grown to a specific optical density in milliliter or liter volumes in medium appropriate for the growth of the cells of interest; however, for effective transformation or transfection, it is desirable to decrease the volume of the cells as well as render the cells competent via buffer or medium exchange. Thus, one sub-component or module that is essential to cell processing systems for the processes listed above is a module or component that can grow, perform buffer exchange, and/or concentrate cells and render them competent so that they may be transformed or transfected with the nucleic acids needed for engineering or editing the cell's genome.
There is thus a need for automated stand-alone cell growth, buffer exchange, and/or concentration devices as well as cell growth and/or concentration modules that may be one module in a multi-module cell processing instruments where the cell growth and/or concentration modules are capable of growing, concentrating and rendering competent cells in an efficient and automated fashion. The present invention addresses this need.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
The present disclosure provides a cell growth and/or concentration device that not only grows and concentrates cells, but also in some aspects renders the cells being concentrated competent via medium/buffer exchange. The cell growth and/or concentration device may be used as a stand-alone device or as one module in a multi-module cell processing instrument. Also described are automated multi-module cell processing instruments including the cell growth and/or concentration devices or modules and methods of using the cell growth and/or concentration devices or modules. The cell growth and/or concentration device described herein operates using tangential flow filtration (TFF), also known as crossflow filtration), in which the majority of the feed flows tangentially over the surface of the filter. Tangential flow filtration reduces cake formation compared to dead-end filtration, in which the feed flows into the filter. Secondary flows relative to the main feed are also exploited to generate shear forces that prevent filter cake formation and membrane fouling thus maximizing particle recovery. The terms “cell growth, buffer exchange and/or concentration device”, “cell growth, buffer exchange, and/or concentration module”, “cell growth and/or concentration device”, “cell growth and/or concentration module”, “TFF device”, and “TFF module” are equivalent.
Thus, there is provided a tangential flow filtration (TFF) device comprising 1) a tangential flow assembly comprising: a retentate member comprising an upper surface and a lower surface with a retentate channel structure defining a flow channel disposed on the lower surface of the retentate member and first and second retentate ports wherein the first retentate port is disposed at a first end of the channel structure and the second retentate port is disposed at a second end of the channel structure, and wherein the first and second retentate ports traverse the first member from the lower surface to the upper surface; a permeate member comprising an upper surface and a lower surface with a permeate channel structure defining a flow channel disposed on the upper surface of the permeate member and at least one permeate port, wherein the at least one permeate port is disposed at a first end of the permeate channel structure, wherein the at least one permeate port traverses the permeate member from the lower surface to the upper surface, and wherein the channel structures of the retentate and permeate members mate to form a single flow channel; and a membrane disposed between the retentate and permeate members thereby bifurcating the single flow channel into upper and lower portions; 2) a reservoir assembly comprising a first retentate reservoir fluidically coupled to the first retentate port, a second retentate reservoir fluidically coupled to the second retentate port and a reservoir top disposed over the first and second retentate reservoirs; 3) a pneumatic assembly configured to apply pressure to move liquid through the single flow channel via negative and positive pressure applied to the first and second retentate reservoirs, to monitor pressure in the retentate reservoirs, and to monitor flow in the flow channel; 4) an interface between the pneumatic assembly and the reservoir top; and 5) means to couple the retentate member, the membrane, the permeate member, and the reservoir assembly.
In some aspects of this embodiment of a TFF, wherein the single flow channel has a serpentine configuration and in some aspects, the channel structure has an undulating geometry. In some aspects, the length of the single flow channel is from 100 mm to 500 mm, or from 150 mm to 400 mm, or from 200 mm to 350 mm.
In some embodiments, the reservoir assembly further comprises a first permeate reservoir fluidically coupled to the at least one permeate port. In some aspects, there is a second permeate port disposed at a second end of the permeate channel structure and the second permeate port also is fluidically coupled to the first permeate reservoir.
In some aspects, the reservoir assembly further comprises a buffer reservoir fluidically coupled to at least one of the first and second retentate reservoirs.
In some aspects, the cross section of the flow channel is rectangular or trapezoidal, and in some aspects, the cross section of the flow channel is 300 μm to 700 μm wide and 300 μm to 700 μm high. In yet other aspects, the cross section of the flow channel is generally circular, and the cross section of the flow channel is 300 μm to 700 μm in radius.
In some aspects, the reservoir assembly further comprises a gasket disposed on the reservoir top of the reservoir assembly and the gasket comprises a pneumatic port and a fluid transfer port for each of the first and second retentate reservoirs. In some aspects of this embodiment, the flow channel has a channel structure with a serpentine configuration that crisscrosses the retentate and permeate members, and in some aspects, the channel structure has other curved geometries. In yet other aspects, the TFF device has a serpentine configuration and an undulating geometry. In some aspects, the footprint length of the channel structure is from 10 mm to 1000 mm, from 60 mm to 200 mm, or from 80 mm to 100 mm. In some aspects, the entire footprint width of the channel structure is from 10 mm to 120 mm, from 40 mm to 70 mm, or from 50 mm to 60 mm.
In some aspects, the cross section of the flow channel is rectangular. In some aspects, the cross section of the flow channel is 5 μm to 1000 μm wide and 5 μm to 1000 μm high, 300 μm to 700 μm wide and 300 μm to 700 μm high, or 400 μm to 600 μm wide and 400 μm to 600 μm high. In other aspects, the cross section of the flow channel is circular, elliptical, trapezoidal, or oblong, and is 100 μm to 1000 μm in hydraulic radius, 300 μm to 700 μm in hydraulic radius, or 400 μm to 600 μm in hydraulic radius.
In some aspects, the means to couple or secure the retentate member, permeate member and membrane together is use of a pressure sensitive adhesive. In other aspects, the retentate member, permeate member and membrane are coupled or secured together by fasteners such as screws or clamps. In other aspects, the retentate member, permeate member and membrane are coupled or secured together by solvent bonding. In other aspects, the retentate member, permeate member and membrane are coupled or secured together by ultrasonic welding. In yet other aspects, the retentate member, permeate member and membrane are coupled or secured together by mated fittings.
Again, in some aspects, the channel structure has a serpentine configuration with local curved geometries that crisscrosses the retentate and permeate members; and in some aspects, the TFF device further comprises retentate reservoirs coupled to the retentate ports.
Also provided is an automated multi-module cell processing instrument comprising the tangential flow filtration device, and further comprising a transformation module and an automated liquid handling device configured to move liquids from the TFF device to the transformation module. In some aspects the automated multi-module cell processing system further comprises a reagent cartridge, and in some aspects, the reagent cartridge further comprises the transformation module. In some aspects, the transformation module is a flow-through electroporation device. In some aspects, there is also included in the automated multi-module cell processing instrument an isolation and editing module. In some aspects, the isolation and editing module is a solid wall isolation and editing module. In some aspects of the automated multi-module cell processing instrument, there is a growth module separate from the tangential flow filtration device.
Other embodiments provide method for growing a cell sample, comprising the steps of: providing one of the tangential flow filtration (TFF) devices described herein; providing a cell sample; placing the cell sample into the first retentate reservoir; passing the cell sample through the retentate channel structure for a length of the channel structure until the cell sample is transported into and retained within the second retentate reservoir; collecting filtrate through the permeate port; passing the cell sample from the second reservoir through the retentate channel structure for the length of the retentate channel structure until the cell sample is transported into and retained within the first reservoir; collecting filtrate through the permeate port; monitoring growth of the cell sample in the retentate reservoirs; repeating the passing, collecting, passing, collecting and monitoring steps until the cell sample has reached a desired stage of growth; and collecting the cell sample.
In some aspects, there is further provided the step of bubbling an appropriate gas through the cell culture while the cell culture is in one or both of the first and second reservoirs. In some aspects, growth of the cell sample is measured by optical density. In some aspects, medium is added to the cell sample in the first and/or second retentate reservoir to refresh the medium to enhance cell growth.
Also provided is a method for concentrating a cell sample, comprising the steps of providing tangential flow filtration (TFF) device; providing a cell sample in a first medium; placing the cell sample into the first retentate reservoir; passing the cell sample from the first retentate reservoir through the retentate channel structure for a length of the channel structure until the cell sample is transported into and retained within the second retentate reservoir; collecting filtrate through the permeate port; passing the cell sample from the second retentate reservoir through the retentate channel structure for the length of the channel structure until the cell sample is transported into and retained within the first retentate reservoir; collecting filtrate through the permeate port; and repeating the passing and collecting steps until the cell sample is concentrated to a desired volume.
In some aspects, this method further comprises the steps of adding a second medium to the cells in the first and/or second reservoirs where the second medium is different from the first medium, and repeating the passing and collecting steps until the cell sample is suspended in the second medium.
These aspects and other features and advantages of the invention are described below in more detail.
The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.
All of the functionalities described in connection with one embodiment are intended to be applicable to the additional embodiments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.
The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and genetic engineering technology, which are within the skill of those who practice in the art. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green and Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014); Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017); Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, eds., John Wiley & Sons 1998); Mammalian Chromosome Engineering—Methods and Protocols (G. Hadlaczky, ed., Humana Press 2011); Essential Stem Cell Methods, (Lanza and Klimanskaya, eds., Academic Press 2011); Neumann, et al., Electroporation and Electrofusion in Cell Biology, Plenum Press, New York, 1989; and Chang, et al., Guide to Electroporation and Electrofusion, Academic Press, California (1992), all of which are herein incorporated in their entirety by reference for all purposes. CRISPR-specific techniques can be found in, e.g., Genome Editing and Engineering from TALENs and CRISPRs to Molecular Surgery, Appasani and Church (2018); and CRISPR: Methods and Protocols, Lindgren and Charpentier (2015); both of which are herein incorporated in their entirety by reference for all purposes.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “the instrument” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.
The present disclosure relates to a cell growth, concentration and medium exchange device/module for growing and concentrating cells and, in some embodiments, rendering cells competent. The cell growth and/or concentration device/module (e.g., tangential flow filtration module or TFF module) can be used as a stand-alone device or as one module as part of an automated multi-module cell processing instrument. The automated multi-module cell processing instrument can be used to process many different types of cells in a controlled, contained, and reproducible manner, including bacterial cells, yeast cells, mammalian cells, other non-mammalian eukaryotic cells, plant cells, fungi, and the like. The cell processes that may be performed include genome engineering, cell transformation, cell culture and/or selection, genome editing and recursive editing, protein production and production of hybridomas.
The present disclosure provides a cell growth, buffer exchange, and/or concentration device (module) that not only grows and concentrates cells, but also in some aspects renders the cells being concentrated competent via medium/buffer exchange. The tangential flow filtration device or TFF device may be used as a stand-alone device or, in some embodiments, as one module in a multi-module cell processing instrument. Also described are automated multi-module cell processing instruments and systems including the TFF devices or modules and methods of using the TFF devices or modules. The TFF cell growth and/or concentration device described herein operates using tangential flow filtration (TFF), also known as crossflow filtration, in which the majority of the feed flows tangentially over or across the surface of the filter thereby reducing cake (retentate) formation as compared to dead-end filtration, in which the feed flows into the filter. Secondary flows relative to the main feed are also exploited to generate shear forces that prevent filter cake formation and membrane fouling thus maximizing particle recovery, as described below.
The TFF device described herein was designed to take into account two primary design considerations. First, the geometry of the TFF device leads to filtration of a cell culture over a large surface area so as to minimize processing time. Second, the design of the TFF device is configured to minimize filter fouling.
The length 110 and width 112 of the channel structure 116 may vary depending on the volume of the cell culture to be grown and the optical density of the cell culture to be concentrated. The length 110 of the channel structure 116 typically is from 1 mm to 300 mm, or from 50 mm to 250 mm, or from 60 mm to 200 mm, or from 70 mm to 150 mm, or from 80 mm to 100 mm. The width of the channel structure 116 typically is from 1 mm to 120 mm, or from 20 mm to 100 mm, or from 30 mm to 80 mm, or from 40 mm to 70 mm, or from 50 mm to 60 mm. The cross-section configuration of the flow channel 102 may be round, elliptical, oval, square, rectangular, trapezoidal, or irregular. If square, rectangular, or another shape with generally straight sides, the cross section may be from about 10 μm to 1000 μm wide, or from 200 μm to 800 μm wide, or from 300 μm to 700 μm wide, or from 400 μm to 600 μm wide; and from about 10 μm to 1000 μm high, or from 200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400 μm to 600 μm high. If the cross section of the flow channel 102 is generally round, oval or elliptical, the radius of the channel may be from about 50 μm to 1000 μm in hydraulic radius, or from 5 μm to 800 μm in hydraulic radius, or from 200 μm to 700 μm in hydraulic radius, or from 300 μm to 600 μm wide in hydraulic radius, or from about 200 to 500 μm in hydraulic radius. Moreover, the volume of the channel in the retentate 122 and permeate 120 members may be different depending on the depth of the channel in each member.
When looking at the top view of the TFF device/module of
The overall work flow for cell growth comprises loading a cell culture to be grown into a first retentate reservoir (not shown), optionally bubbling air or an appropriate gas through the cell culture, passing or flowing the cell culture through the first retentate port then tangentially through the TFF channel structure while collecting medium or buffer through one or both of the permeate ports 106, collecting the cell culture through a second retentate port 104 into a second retentate reservoir (not shown), optionally adding additional or different medium to the cell culture and optionally bubbling air or gas through the cell culture, then repeating the process, all while measuring, e.g., the optical density of the cell culture in the retentate reservoirs continuously or at desired intervals. Measurements of optical densities (OD) at programmed time intervals are accomplished using a 600 nm Light Emitting Diode (LED) that has been columnated through an optic into the retentate reservoir(s) containing the growing cells. The light continues through a collection optic to the detection system which consists of a (digital) gain-controlled silicone photodiode. Generally, optical density is shown as the absolute value of the logarithm with base 10 of the power transmission factors of an optical attenuator: OD=−log 10 (Power out/Power in). Since OD is the measure of optical attenuation—that is, the sum of absorption, scattering, and reflection—the TFF device OD measurement records the overall power transmission, so as the cells grow and become denser in population, the OD (the loss of signal) increases. The OD system is pre-calibrated against OD standards with these values stored in an on-board memory accessible by the measurement program.
In the channel structure, the membrane bifurcating the flow channels retains the cells on one side of the membrane (the retentate side) and allows unwanted medium or buffer to flow across the membrane into a filtrate or permeate side (e.g., permeate member 120) of the device. Bubbling air or other appropriate gas through the cell culture both aerates and mixes the culture to enhance cell growth. During the process, medium that is removed during the flow through the channel structure is removed through the permeate/filtrate ports 106. Alternatively, cells can be grown in one reservoir with bubbling or agitation without passing the cells through the TFF channel from one reservoir to the other.
The overall work flow for cell concentration using the TFF device/module involves flowing a cell culture or cell sample tangentially through the channel structure. As with the cell growth process, the membrane bifurcating the flow channels retains the cells on one side of the membrane and allows unwanted medium or buffer to flow across the membrane into a permeate/filtrate side (e.g., permeate member 120) of the device. In this process, a fixed volume of cells in medium or buffer is driven through the device until the cell sample is collected into one of the retentate ports 104, and the medium/buffer that has passed through the membrane is collected through one or both of the permeate/filtrate ports 106. All types of prokaryotic and eukaryotic cells-both adherent and non-adherent cells—can be grown in the TFF device. Adherent cells may be grown on beads or other cell scaffolds suspended in medium that flow through the TFF device.
The medium or buffer used to suspend the cells in the cell concentration device/module may be any suitable medium or buffer for the type of cells being transformed or transfected, such as SOC, MEM, DMEM, IMDM, RPMI, Hanks', PBS and Ringer's solution, where the media may be provided in a reagent cartridge as part of a kit. For culture of adherent cells, cells may be disposed on beads, microcarriers, or other type of scaffold suspended in medium. Most normal mammalian tissue-derived cells-except those derived from the hematopoietic system—are anchorage dependent and need a surface or cell culture support for normal proliferation. In the rotating growth vial described herein, microcarrier technology is leveraged. Microcarriers of particular use typically have a diameter of 100-300 μm and have a density slightly greater than that of the culture medium (thus facilitating an easy separation of cells and medium for, e.g., medium exchange) yet the density must also be sufficiently low to allow complete suspension of the carriers at a minimum stirring rate in order to avoid hydrodynamic damage to the cells. Many different types of microcarriers are available, and different microcarriers are optimized for different types of cells. There are positively charged carriers, such as Cytodex 1 (dextran-based, GE Healthcare), DE-52 (cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based, Sigma-Aldrich Labware), and HLX 11-170 (polystyrene-based); collagen- or ECM—(extracellular matrix) coated carriers, such as Cytodex 3 (dextran-based, GE Healthcare) or HyQ-sphere Pro-F 102-4 (polystyrene-based, Thermo Scientific); non-charged carriers, like HyQ-sphere P 102-4 (Thermo Scientific); or macroporous carriers based on gelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GE Healthcare).
In both the cell growth and concentration processes, passing the cell sample through the TFF device and collecting the cells in one of the retentate ports 104 while collecting the medium in one of the permeate/filtrate ports 106 is considered “one pass” of the cell sample. The transfer between retentate reservoirs “flips” the culture. The retentate and permeate ports collecting the cells and medium, respectively, for a given pass reside on the same end of TFF device/module 100 with fluidic connections arranged so that there are two distinct flow layers for the retentate and permeate/filtrate sides, but if the retentate port 104 resides on the retentate member of device/module 100 (that is, the cells are driven through the channel above the membrane and the filtrate (medium) passes to the portion of the channel below the membrane), the permeate/filtrate port 106 will reside on the permeate member of device/module 100 and vice versa (that is, if the cell sample is driven through the channel below the membrane, the filtrate (medium) passes to the portion of the channel above the membrane). Due to the high pressures used to transfer the cell culture and fluids through the flow channel of the TFF device, the effect of gravity is negligible. The TFF device or module can be seen more clearly in
At the conclusion of a “pass” in either of the growth and concentration processes, the cell sample is collected by passing through the retentate port 104 and into the retentate reservoir (not shown). To initiate another “pass”, the cell sample is passed again through the TFF device, this time in a flow direction that is reversed from the first pass. The cell sample is collected by passing through the retentate port 104 and into retentate reservoir (not shown) on the opposite end of the device/module from the retentate port 104 that was used to collect cells during the first pass. Likewise, the medium/buffer that passes through the membrane on the second pass is collected through the permeate port 106 on the opposite end of the device/module from the permeate port 106 that was used to collect the filtrate during the first pass, or through both ports. Alternatively, there may be a single permeate reservoir configured to collect the permeate fluid as is the case with the TFF embodiment depicted in
Note that there is one retentate port and one permeate port on each “end” (e.g., the narrow edges) of the TFF device/module. The retentate and permeate ports on the left side of the device/module will collect cells (flow path at 160) and medium (flow path at 170), respectively, for the same pass. Likewise, the retentate and permeate ports on the right side of the device/module will collect cells (flow path at 160) and medium (flow path at 170), respectively, for the same pass. In this embodiment, the retentate is collected from ports 104 on the top surface of the TFF device, and filtrate is collected from ports 106 on the bottom surface of the device. The cells are maintained in the TFF flow channel above the membrane 124, while the filtrate (medium) flows through membrane 124 and then through ports 106; thus, the top/retentate ports and bottom/filtrate ports configuration is practical. It should be recognized, however, that other configurations of retentate and permeate ports may be implemented such as positioning both the retentate and permeate ports on the side (as opposed to the top and bottom surfaces) of the TFF device.
In
Again, there is one retentate port and one permeate/filtrate port on each “end” (e.g., the narrow edges) of this embodiment of a TFF device/module. The retentate and permeate/filtrate ports on the left side of the device/module will collect cells (flow path at 160) and medium (flow path at 170), respectively, for the same pass. Likewise, the retentate and permeate/filtrate ports on the right side of the device/module will collect cells (flow path at 160) and medium (flow path at 170), respectively, for the same pass. In this embodiment, the retentate is collected from ports 104 on the top surface of the TFF device, and filtrate is collected from ports 106 on the bottom surface of the device. The cells are maintained in the TFF flow channel above the membrane 124, while the filtrate (medium) flows through membrane 124 and then through ports 106. In
Medium exchange (during cell growth) or buffer exchange (during cell concentration or rendering the cells competent) is performed on the TFF device/module by adding fresh medium to growing cells (that is, refreshing medium to replace depleted nutrients) or by adding a desired buffer to the cells concentrated to a desired volume; for example, after the cells have been concentrated at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more. A desired exchange medium or exchange buffer is added to the cells either by addition to the retentate reservoir (e.g., to cells in retentate reservoir 130) or medium or buffer may be added through the membrane from the permeate/filtrate side and the process of passing the cells through the TFF device 100 is repeated until the cells have been grown to a desired optical density or concentrated to a desired volume in the exchange medium or buffer. This process can be repeated any number of desired times so as to achieve a desired level of exchange of the buffer and a desired volume of cells. As described in the Example 2, in the context of cell concentration, the exchange buffer may comprise, e.g., glycerol or sorbitol thereby rendering the cells competent for transformation in addition to decreasing the overall volume of the cell sample.
The TFF device 100 may be fabricated from any robust material in which channels and channel branches may be milled including stainless steel, silicon, glass, aluminum, or plastics including cyclic-olefin copolymer (COC), cyclo-olefin polymer (COP), polystyrene, polyvinyl chloride, polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA), polysulfone, and polyurethane, and co-polymers of these and other polymers. If the TFF device/module is disposable, preferably it is made of plastic. In some embodiments, the material used to fabricate the TFF device/module is thermally-conductive so that the cell culture may be heated or cooled to a desired temperature. In certain embodiments, the TFF device is formed by precision mechanical machining, laser machining, electro discharge machining (for metal devices); wet or dry etching (for silicon devices); dry or wet etching, powder or sandblasting, photostructuring (for glass devices); or thermoforming, injection molding, hot embossing, or laser machining (for plastic devices) using the materials mentioned above that are amenable to this mass production techniques.
The retentate reservoirs are fluidically coupled to the upper portion of the flow channel (e.g., the portion of the flow channel disposed in the retentate member), and the buffer or medium reservoir is fluidically coupled to the retentate reservoirs. Also seen in this assembled view of TFF device 1100 is membrane 1124, permeate member 1120 which, as described previously, comprises on its top surface the lower portion of the tangential flow channel (not shown), where the channel structures of the retentate member 1122 and permeate member 1120 (neither shown in this view) mate to form a single flow channel. Beneath and adjacent to permeate member 1120 is a gasket 1140, which is interposed between permeate member 1120 and a filtrate (or permeate) reservoir 1142. The permeate/filtrate reservoir 1142 is in fluid connection with the lower portion of the flow channel (e.g., the portion of the flow channel disposed in the permeate member) as a receptacle for the filtrate or permeate that is removed from the cell culture. In operation, top 1144, combined reservoir and retentate member structure 1150, membrane 1124, permeate member 1120, gasket 1140, and permeate/filtrate reservoir 1142 are coupled and secured together to be fluid- and air-tight. The assembled TFF device 1100 typically is from 4 to 25 cm in height, or from 5 to 20 cm in height, or from 7 to 15 cm in height; from 5 to 30 cm in length, or from 8 to 25 cm in length, or from 10 to 20 cm in length; and is from 3 to 15 cm in depth, or from 5 to 10 cm in depth. An exemplary TFF device is 11 cm in height, 12 cm in length, and 8 cm in depth. The retentate reservoirs, buffer or medium reservoir, and tangential flow channel-forming structures may be configured to be cooled to 4° C. for cell maintenance, and 30° C. for cell growth. The dimensions for the serpentine channel recited above, as well as the specifications and materials for the filter and the TFF device apply to the embodiment of the device shown in
Also seen in this exploded view of TFF device 1100 is permeate member 1120 which, as described previously comprises on its top surface the lower portion of the tangential flow channel 1102 (seen on the top surface of permeate member 1120), where the upper and lower portions of the channel structures of the retentate member 1122 and permeate member 1120, respectively, when coupled mate to form a single flow channel (the membrane that is interposed between the retentate member 1122 and permeate member 1120 in operation is not shown). Beneath permeate member 1120 is gasket 1140, which in operation is interposed between permeate member 1120 and a filtrate (or permeate) reservoir 1142. In operation, top 1144, combined reservoir and retentate member structure 1150, membrane (not shown), permeate member 1120, gasket 1140, and permeate/filtrate reservoir 1142 are coupled and secured together to be fluid- and air-tight. In
For growing E. coli cells, the TFF is chilled to 4° C. prior to loading the cell sample into RR1, and the cells are passed through the TFF flow channel with aeration (bubbling) in the retentate reservoirs. Once the proper OD is reached, the E. coli cells are concentrated and buffer exchange is performed to render the cells competent with, e.g., glycerol-containing buffer. For growing yeast cells, the TFF is heated to 30° C. for growth in the TFF device with aeration. Once the desired OD is reached, the yeast cells are conditioned with aeration and then are concentrated and resuspended in buffer, such as buffer containing lithium acetate and DTT (dithiothreitol) (or DTT/TCEP (tris(2-carboxyethyl)phosphine)) to render the yeast cells competent. In either example, the cells are loaded into the TFF device, electroporation buffer is loaded into the buffer reservoir. During concentration, electroporation buffer is added to the retentate reservoirs from the buffer reservoirs and the cells are both concentrated and rendered electrocompetent. During a “pass”, air pressure and flow rate are monitored. When fluid has been “pushed” into a reservoir, the flow rate spikes because fluid is no longer being pushed in the system and air begins flowing through the retentate channel, thus signaling the end of a pass. The process of transferring fluid from one reservoir to the other reservoir is a “pass”, and one to many passes may be performed to arrive at the proper buffer exchange and/or concentration desired (e.g., a concentration “round”). In some embodiments, fluid on the permeate side of the channel may be pulled across the membrane to assist in dislodging cells from the membrane on the retentate side of the membrane. After dislodging the cells, buffer may be added to one of the reservoirs and pressure applied to “sweep” the cells into the opposite reservoir.
In one embodiment, the TFF device or module constantly measures cell culture growth, and in some aspects, cell culture growth is measured via optical density (OD) of the cell culture in one or both of the retentate reservoirs and/or in the flow channel of the TFF device. Optical density may be measured continuously (kinetic monitoring) or at specific time intervals; e.g., every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or so on minutes. Alternatively, OD can be measured at specific time intervals early in the cell growth cycle, and continuously after the OD of the cell culture reaches a set point OD. The TFF module is controlled by a processor, which can be programmed to measure OD constantly or at intervals as defined by a user. A script on, e.g., the reagent cartridge(s) may also specify the frequency for reading OD, as well as the target OD and target time. Additionally, a user manually can set a target time at which the user desires the cell culture hit a target OD. To accomplish reaching the target OD at the target time, the processor measures the OD of the growing cells, calculates the cell growth rate in real time, and predicts the time the target OD will be reached. The processor then automatically adjusts the temperature of the TFF module (and the cell culture) as needed. Lower temperatures slow growth, and higher temperatures increase growth. In addition, the processor may be programmed to inform a user of the progress of cell growth, buffer exchange, and/or cell concentration by altering the user via, e.g., cell phone or other personal digital device. Aside from OD, other properties of the cell culture can be measured, such as impedance of the culture, measurement of metabolic by-products or measurement of other cellular characteristics that correlate with the rate of growth of the cell culture.
In
As in the tangential flow channel 102 configuration seen in
As described above, the overall work flow for cell growth comprises loading a cell culture to be grown into a first retentate reservoir 1252, optionally bubbling air or an appropriate gas through the cell culture, passing or flowing the cell culture through the first retentate port 1228 then tangentially through the TFF channel structure while collecting medium or buffer through one (or both, depending on the embodiment) of the permeate/filtrate ports 1226, collecting the cell culture through a second retentate port 1228 into a second retentate reservoir 1252, optionally adding additional or different medium to the cell culture and optionally bubbling air or gas through the cell culture, then repeating the process, all while measuring, e.g., the optical density of the cell culture in the retentate reservoirs continuously or at desired intervals. Measurements of optical densities (OD) at programmed time intervals are accomplished using a 600 nm Light Emitting Diode (LED) that has been columnated through an optic into the retentate reservoir(s) containing the growing cells. In the channel structure, the membrane bifurcating the flow channels retains the cells on one side of the membrane (the retentate side) and allows unwanted medium or buffer to flow across the membrane into a filtrate or permeate side of the device. Bubbling air or other appropriate gas through the cell culture both aerates and mixes the culture to enhance cell growth. During the process, medium that is removed during the flow through the channel structure is removed through the permeate/filtrate port(s) 1226 and is collected in the permeate reservoir 1254. Alternatively, cells can be grown in one reservoir with bubbling or agitation without passing the cells through the TFF channel from one reservoir to the other.
The overall work flow for cell concentration using the TFF device/module involves flowing a cell culture or cell sample tangentially through the channel structure. As with the cell growth process, the membrane bifurcating the flow channels retains the cells on one side of the membrane (retentate) and allows unwanted medium or buffer (permeate) to flow across the membrane into a permeate side (e.g., permeate member 1220) of the device. In this process, a fixed volume of cells in medium or buffer is driven through the device until the cell sample is collected into one of the retentate reservoirs 1252, and the medium/buffer that has passed through the membrane is collected through one (or both, depending on the embodiment) of the permeate/filtrate port(s) 1226 into permeate reservoir 1254.
Membrane or filter 1224 is seen at center in
Additionally seen in
Permeate/filtrate member 1220 is seen in the middle of
The bottom figure of
Like in other embodiments described herein, the TFF device or module depicted in
In a summary of steps for concentrating E. coli cells, cells that have been grown to a desired OD are transferred from, e.g., a cell growth device such as seen in
In the present system, the flow meter that is coupled directly to the retentate reservoir to which the cell culture is being transferred is monitored to determine when the cells have been thoroughly transferred to that reservoir. That is, flow meter FM2 is read to ascertain whether the cell culture has been completely transferred to RR2, rather than FM1 being monitored to ascertain whether the cell culture has been entirely transferred from RR1. This practice is different from how such monitoring is accomplished typically. The reason behind this practice is that at times the volume of the cell culture is quite small and a good deal of the culture may have evacuated a retentate reservoir, but reside primarily within the TFF flow channel. By monitoring the flow meter coupled to the retentate reservoir to which the cell culture is being transferred (again, monitoring FM1 when RR1 is the retentate reservoir to which the cell culture is being transferred, and monitoring FM2 when RR2 is the retentate reservoir to which the cell culture is being transferred), one can detect when the entirety of the cell culture has been transferred from the transferring reservoir, through the flow channel and into the receiving reservoir.
In some implementations, the reagent cartridges 210 are disposable kits comprising reagents and cells for use in the automated multi-module cell processing/editing instrument 200. For example, a user may open and position each of the reagent cartridges 210 comprising various desired inserts and reagents within the chassis of the automated multi-module cell editing instrument 200 prior to activating cell processing. Further, each of the reagent cartridges 210 may be inserted into receptacles in the chassis having different temperature zones appropriate for the reagents contained therein.
Also illustrated in
Inserts or components of the reagent cartridges 210, in some implementations, are marked with machine-readable indicia (not shown), such as bar codes, for recognition by the robotic handling system 258. For example, the robotic liquid handling system 258 may scan one or more inserts within each of the reagent cartridges 210 to confirm contents. In other implementations, machine-readable indicia may be marked upon each reagent cartridge 210, and a processing system (not shown, but see element 237 of
Inside the chassis 290, in some implementations, will be most or all of the components described in relation to
In one embodiment, the reagent reservoirs or reservoirs 304 of reagent cartridge 300 are configured to hold various size tubes, including, e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorf or microcentrifuge tubes. In yet another embodiment, all reservoirs may be configured to hold the same size tube, e.g., 5 ml tubes, and reservoir inserts may be used to accommodate smaller tubes in the reagent reservoir. In yet another embodiment-particularly in an embodiment where the reagent cartridge is disposable—the reagent reservoirs hold reagents without inserted tubes. In this disposable embodiment, the reagent cartridge may be part of a kit, where the reagent cartridge is pre-filled with reagents and the receptacles or reservoirs sealed with, e.g., foil, heat seal acrylic or the like and presented to a consumer where the reagent cartridge can then be used in an automated multi-module cell processing instrument. As one of ordinary skill in the art will appreciate given the present disclosure, the reagents contained in the reagent cartridge will vary depending on work flow; that is, the reagents will vary depending on the processes to which the cells are subjected in the automated multi-module cell processing instrument, e.g., protein production, cell transformation and culture, cell editing, etc.
Reagents such as cell samples, enzymes, buffers, nucleic acid vectors, expression cassettes, proteins or peptides, reaction components (such as, e.g., MgCl2, dNTPs, nucleic acid assembly reagents, gap repair reagents, and the like), wash solutions, ethanol, and magnetic beads for nucleic acid purification and isolation, etc. may be positioned in the reagent cartridge at a known position. In some embodiments of cartridge 300, the cartridge comprises a script (not shown) readable by a processor (not shown) for dispensing the reagents. Also, the cartridge 300 as one component in an automated multi-module cell processing instrument may comprise a script specifying two, three, four, five, ten or more processes to be performed by the automated multi-module cell processing instrument. In certain embodiments, the reagent cartridge is disposable and is pre-packaged with reagents tailored to performing specific cell processing protocols, e.g., genome editing or protein production. Because the reagent cartridge contents vary while components/modules of the automated multi-module cell processing instrument or system may not, the script associated with a particular reagent cartridge matches the reagents used and cell processes performed. Thus, e.g., reagent cartridges may be pre-packaged with reagents for genome editing and a script that specifies the process steps for performing genome editing in an automated multi-module cell processing instrument, or, e.g., reagents for protein expression and a script that specifies the process steps for performing protein expression in an automated multi-module cell processing instrument.
For example, the reagent cartridge may comprise a script to pipette competent cells from a reservoir, transfer the cells to a transformation module, pipette a nucleic acid solution comprising a vector with expression cassette from another reservoir in the reagent cartridge, transfer the nucleic acid solution to the transformation module, initiate the transformation process for a specified time, then move the transformed cells to yet another reservoir in the reagent cassette or to another module such as a cell growth module in the automated multi-module cell processing instrument. In another example, the reagent cartridge may comprise a script to transfer a nucleic acid solution comprising a vector from a reservoir in the reagent cassette, nucleic acid solution comprising editing oligonucleotide cassettes in a reservoir in the reagent cassette, and a nucleic acid assembly mix from another reservoir to the nucleic acid assembly/desalting module, if present. The script may also specify process steps performed by other modules in the automated multi-module cell processing instrument. For example, the script may specify that the nucleic acid assembly/desalting reservoir be heated to 50° C. for 30 min to generate an assembled product; and desalting and resuspension of the assembled product via magnetic bead-based nucleic acid purification involving a series of pipette transfers and mixing of magnetic beads, ethanol wash, and buffer.
As described in relation to
Additional details of the FTEP devices are illustrated in
In the FTEP devices of the disclosure, the toxicity level of the transformation results in greater than 30% viable cells after electroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells following transformation, depending on the cell type and the nucleic acids being introduced into the cells.
The housing of the FTEP device can be made from many materials depending on whether the FTEP device is to be reused, autoclaved, or is disposable, including stainless steel, silicon, glass, resin, polyvinyl chloride, polyethylene, polyamide, polystyrene, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers of these and other polymers. Similarly, the walls of the channels in the device can be made of any suitable material including silicone, resin, glass, glass fiber, polyvinyl chloride, polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers of these and other polymers. Preferred materials include crystal styrene, cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), which allow the device to be formed entirely by injection molding in one piece with the exception of the electrodes and, e.g., a bottom sealing film if present.
The FTEP devices described herein (or portions of the FTEP devices) can be created or fabricated via various techniques, e.g., as entire devices or by creation of structural layers that are fused or otherwise coupled. For example, for metal FTEP devices, fabrication may include precision mechanical machining or laser machining; for silicon FTEP devices, fabrication may include dry or wet etching; for glass FTEP devices, fabrication may include dry or wet etching, powderblasting, sandblasting, or photostructuring; and for plastic FTEP devices fabrication may include thermoforming, injection molding, hot embossing, or laser machining. The components of the FTEP devices may be manufactured separately and then assembled, or certain components of the FTEP devices (or even the entire FTEP device except for the electrodes) may be manufactured (e.g., using 3D printing) or molded (e.g., using injection molding) as a single entity, with other components added after molding. For example, housing and channels may be manufactured or molded as a single entity, with the electrodes later added to form the FTEP unit. Alternatively, the FTEP device may also be formed in two or more parallel layers, e.g., a layer with the horizontal channel and filter, a layer with the vertical channels, and a layer with the inlet and outlet ports, which are manufactured and/or molded individually and assembled following manufacture.
In specific aspects, the FTEP device can be manufactured using a circuit board as a base, with the electrodes, filter and/or the flow channel formed in the desired configuration on the circuit board, and the remaining housing of the device containing, e.g., the one or more inlet and outlet channels and/or the flow channel formed as a separate layer that is then sealed onto the circuit board. The sealing of the top of the housing onto the circuit board provides the desired configuration of the different elements of the FTEP devices of the disclosure. Also, two to many FTEP devices may be manufactured on a single substrate, then separated from one another thereafter or used in parallel. In certain embodiments, the FTEP devices are reusable and, in some embodiments, the FTEP devices are disposable. In additional embodiments, the FTEP devices may be autoclavable.
The electrodes 408 can be formed from any suitable metal, such as copper, stainless steel, titanium, aluminum, brass, silver, rhodium, gold or platinum, or graphite. One preferred electrode material is alloy 303 (UNS330300) austenitic stainless steel. An applied electric field can destroy electrodes made from of metals like aluminum. If a multiple-use (i.e., non-disposable) flow-through FTEP device is desired—as opposed to a disposable, one-use flow-through FTEP device—the electrode plates can be coated with metals resistant to electrochemical corrosion. Conductive coatings like noble metals, e.g., gold, can be used to protect the electrode plates.
As mentioned, the FTEP devices may comprise push-pull pneumatic means to allow multi-pass electroporation procedures; that is, cells to electroporated may be “pulled” from the inlet toward the outlet for one pass of electroporation, then be “pushed” from the outlet end of the flow-through FTEP device toward the inlet end to pass between the electrodes again for another pass of electroporation. This process may be repeated one to many times.
Depending on the type of cells to be electroporated (e.g., bacterial, yeast, mammalian) and the configuration of the electrodes, the distance between the electrodes in the flow channel can vary widely. For example, where the flow channel decreases in width, the flow channel may narrow to between 10 μm and 5 mm, or between 25 μm and 3 mm, or between 50 μm and 2 mm, or between 75 μm and 1 mm. The distance between the electrodes in the flow channel may be between 1 mm and 10 mm, or between 2 mm and 8 mm, or between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall size of the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to 12 cm in length, or 4.5 cm to 10 cm in length. The overall width of the FTEP device may be from 0.5 cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cm to 2.5 cm, or from 1 cm to 1.5 cm.
The region of the flow channel that is narrowed is wide enough so that at least two cells can fit in the narrowed portion side-by-side. For example, a typical bacterial cell is 1 μm in diameter; thus, the narrowed portion of the flow channel of the FTEP device used to transform such bacterial cells will be at least 2 μm wide. In another example, if a mammalian cell is approximately 50 μm in diameter, the narrowed portion of the flow channel of the FTEP device used to transform such mammalian cells will be at least 100 μm wide. That is, the narrowed portion of the FTEP device will not physically contort or “squeeze” the cells being transformed.
In embodiments of the FTEP device where reservoirs are used to introduce cells and exogenous material into the FTEP device, the reservoirs range in volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to 5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute, or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute. The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi, or from 3-5 psi.
To avoid different field intensities between the electrodes, the electrodes should be arranged in parallel. Furthermore, the surface of the electrodes should be as smooth as possible without pin holes or peaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. In another embodiment of the invention, the flow-through electroporation device comprises at least one additional electrode which applies a ground potential to the FTEP device.
The drive engagement mechanism 412 engages with a motor (not shown) to rotate the vial. In some embodiments, the motor drives the drive engagement mechanism 412 such that the rotating growth vial 400 is rotated in one direction only, and in other embodiments, the rotating growth vial 400 is rotated in a first direction for a first amount of time or periodicity, rotated in a second direction (i.e., the opposite direction) for a second amount of time or periodicity, and this process may be repeated so that the rotating growth vial 400 (and the cell culture contents) are subjected to an oscillating motion. Further, the choice of whether the culture is subjected to oscillation and the periodicity therefor may be selected by the user. The first amount of time and the second amount of time may be the same or may be different. The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1, 2, 3, 4 or more minutes. In another embodiment, in an early stage of cell growth the rotating growth vial 400 may be oscillated at a first periodicity (e.g., every 60 seconds), and then a later stage of cell growth the rotating growth vial 400 may be oscillated at a second periodicity (e.g., every one second) different from the first periodicity.
The rotating growth vial 400 may be reusable or, preferably, the rotating growth vial is consumable. In some embodiments, the rotating growth vial is consumable and is presented to the user pre-filled with growth medium, where the vial is hermetically sealed at the open end 404 with a foil seal. A medium-filled rotating growth vial packaged in such a manner may be part of a kit for use with a stand-alone cell growth device or with a cell growth module that is part of an automated multi-module cell processing system. To introduce cells into the vial, a user need only pipette up a desired volume of cells and use the pipette tip to punch through the foil seal of the vial. Open end 404 may optionally include an extended lip 402 to overlap and engage with the cell growth device. In automated systems, the rotating growth vial 400 may be tagged with a barcode or other identifying means that can be read by a scanner or camera (not shown) that is part of the automated system.
The volume of the rotating growth vial 400 and the volume of the cell culture (including growth medium) may vary greatly, but the volume of the rotating growth vial 400 must be large enough to generate a specified total number of cells. In practice, the volume of the rotating growth vial 400 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50 mL, or from 12-35 mL. Likewise, the volume of the cell culture (cells+growth media) should be appropriate to allow proper aeration and mixing in the rotating growth vial 400. Proper aeration promotes uniform cellular respiration within the growth media. Thus, the volume of the cell culture should be approximately 5-85% of the volume of the growth vial or from 20-60% of the volume of the growth vial. For example, for a 30 mL growth vial, the volume of the cell culture would be from about 1.5 mL to about 26 mL, or from 6 mL to about 18 mL.
The rotating growth vial 400 preferably is fabricated from a bio-compatible optically transparent material- or at least the portion of the vial comprising the light path(s) is transparent. Additionally, material from which the rotating growth vial is fabricated should be able to be cooled to about 4° C. or lower and heated to about 55° C. or higher to accommodate both temperature-based cell assays and long-term storage at low temperatures. Further, the material that is used to fabricate the vial must be able to withstand temperatures up to 55° C. without deformation while spinning. Suitable materials include cyclic olefin copolymer (COC), glass, polyvinyl chloride, polyethylene, polyamide, polypropylene, polycarbonate, poly(methyl methacrylate (PMMA), polysulfone, polyurethane, and co-polymers of these and other polymers. Preferred materials include polypropylene, polycarbonate, or polystyrene. In some embodiments, the rotating growth vial is inexpensively fabricated by, e.g., injection molding or extrusion.
The motor 438 engages with drive mechanism 412 and is used to rotate the rotating growth vial 400. In some embodiments, motor 438 is a brushless DC type drive motor with built-in drive controls that can be set to hold a constant revolution per minute (RPM) between 0 and about 3000 RPM. Alternatively, other motor types such as a stepper, servo, brushed DC, and the like can be used. Optionally, the motor 438 may also have direction control to allow reversing of the rotational direction, and a tachometer to sense and report actual RPM. The motor is controlled by a processor (not shown) according to, e.g., standard protocols programmed into the processor and/or user input, and the motor may be configured to vary RPM to cause axial precession of the cell culture thereby enhancing mixing, e.g., to prevent cell aggregation, increase aeration, and optimize cellular respiration.
Main housing 436, end housings 452 and lower housing 432 of the cell growth device 430 may be fabricated from any suitable, robust material including aluminum, stainless steel, and other thermally conductive materials, including plastics. These structures or portions thereof can be created through various techniques, e.g., metal fabrication, injection molding, creation of structural layers that are fused, etc. Whereas the rotating growth vial 400 is envisioned in some embodiments to be reusable, but preferably is consumable, the other components of the cell growth device 430 are preferably reusable and function as a stand-alone benchtop device or as a module in a multi-module cell processing system.
The processor (not shown) of the cell growth device 430 may be programmed with information to be used as a “blank” or control for the growing cell culture. A “blank” or control is a vessel containing cell growth medium only, which yields 100% transmittance and 0 OD, while the cell sample will deflect light rays and will have a lower percent transmittance and higher OD. As the cells grow in the media and become denser, transmittance will decrease and OD will increase. The processor (not shown) of the cell growth device 430 may be programmed to use wavelength values for blanks commensurate with the growth media typically used in cell culture (whether, e.g., mammalian cells, bacterial cells, animal cells, yeast cells, etc.). Alternatively, a second spectrophotometer and vessel may be included in the cell growth device 430, where the second spectrophotometer is used to read a blank at designated intervals.
In use, cells are inoculated (cells can be pipetted, e.g., from an automated liquid handling system or by a user) into pre-filled growth media of a rotating growth vial 400 by piercing though the foil seal or film. The programmed software of the cell growth device 430 sets the control temperature for growth, typically 30° C., then slowly starts the rotation of the rotating growth vial 400. The cell/growth media mixture slowly moves vertically up the wall due to centrifugal force allowing the rotating growth vial 400 to expose a large surface area of the mixture to a normal oxygen environment. The growth monitoring system takes either continuous readings of the OD or OD measurements at pre-set or pre-programmed time intervals. These measurements are stored in internal memory and if requested the software plots the measurements versus time to display a growth curve. If enhanced mixing is required, e.g., to optimize growth conditions, the speed of the vial rotation can be varied to cause an axial precession of the liquid, and/or a complete directional change can be performed at programmed intervals. The growth monitoring can be programmed to automatically terminate the growth stage at a pre-determined OD, and then quickly cool the mixture to a lower temperature to inhibit further growth.
One application for the cell growth device 430 is to constantly measure the optical density of a growing cell culture. One advantage of the described cell growth device is that optical density can be measured continuously (kinetic monitoring) or at specific time intervals; e.g., every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. While the cell growth device 430 has been described in the context of measuring the optical density (OD) of a growing cell culture, it should, however, be understood by a skilled artisan given the teachings of the present specification that other cell growth parameters can be measured in addition to or instead of cell culture OD. As with optional measure of cell growth in relation to the solid wall device or module described supra, spectroscopy using visible, UV, or near infrared (NIR) light allows monitoring the concentration of nutrients and/or wastes in the cell culture and other spectroscopic measurements may be made; that is, other spectral properties can be measured via, e.g., dielectric impedance spectroscopy, visible fluorescence, fluorescence polarization, or luminescence. Additionally, the cell growth device 430 may include additional sensors for measuring, e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.
Another module useful in multi-module cell processing is a solid wall isolation, incubation, and normalization (SWIIN) module.
The SWIIN module 550 in
In this
In this embodiment of a SWIIN module, the perforated member includes through-holes to accommodate ultrasonic tabs disposed on the permeate member. Thus, in this embodiment the perforated member is fabricated from 316 stainless steel, and the perforations form the walls of microwells while a filter or membrane is used to form the bottom of the microwells. Typically, the perforations (microwells) are approximately 150 μm-200 μm in diameter, and the perforated member is approximately 125 μm deep, resulting in microwells having a volume of approximately 2.5 nl, with a total of approximately 200,000 microwells. The distance between the microwells is approximately 279 μm center-to-center. Though here the microwells have a volume of approximately 2.5 nl, the volume of the microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl, and even more preferably from 2 to 4 nl. As for the filter or membrane, like the filter described previously, filters appropriate for use are solvent resistant, contamination free during filtration, and are able to retain the types and sizes of cells of interest. For example, in order to retain small cell types such as bacterial cells, pore sizes can be as low as 0.10 μm, however for other cell types (e.g., such as for mammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm or more. Indeed, the pore sizes useful in the cell concentration device/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm, 0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. The filters may be fabricated from any suitable material including cellulose mixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, or glass fiber.
The cross-section configuration of the mated serpentine channel may be round, elliptical, oval, square, rectangular, trapezoidal, or irregular. If square, rectangular, or another shape with generally straight sides, the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to 12 mm wide, or from 5 mm to 10 mm wide. If the cross section of the mated serpentine channel is generally round, oval or elliptical, the radius of the channel may be from about 3 mm to 20 mm in hydraulic radius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mm in hydraulic radius.
As in previous embodiments, disposed between serpentine channels 560a and 560b is perforated member 501 (adjacent retentate member 504) and filter 503 (adjacent permeate member 508), where filter 503 is swaged with perforated member 501. Serpentine channels 560a and 560b can have approximately the same volume or a different volume. For example, each “side” or portion 560a, 560b of the serpentine channel may have a volume of, e.g., 2 mL, or serpentine channel 560a of permeate member 508 may have a volume of 2 mL, and the serpentine channel 560b of retentate member 504 may have a volume of, e.g., 3 mL. The volume of fluid in the serpentine channel may range from about 2 mL to about 80 mL, or about 4 mL to 60 mL, or from 5 mL to 40 mL, or from 6 mL to 20 mL (note these volumes apply to a SWIIN module comprising a, e.g., 50-500K perforation member). The volume of the reservoirs may range from 5 mL to 50 mL, or from 7 mL to 40 mL, or from 8 mL to 30 mL or from 10 mL to 20 mL, and the volumes of all reservoirs may be the same or the volumes of the reservoirs may differ (e.g., the volume of the permeate reservoirs is greater than that of the retentate reservoirs).
The serpentine channel portions 560a and 560b of the permeate member 508 and retentate member 504, respectively, are approximately 200 mm long, 130 mm wide, and 4 mm thick, though in other embodiments, the retentate and permeate members can be from 75 mm to 400 mm in length, or from 100 mm to 300 mm in length, or from 150 mm to 250 mm in length; from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from 100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from 4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness. Embodiments the retentate (and permeate) members may be fabricated from PMMA (poly(methyl methacrylate) or other materials may be used, including polycarbonate, cyclic olefin co-polymer (COC), glass, polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone, polyurethane, and co-polymers of these and other polymers. Preferably at least the retentate member is fabricated from a transparent material so that the cells can be visualized (see, e.g.,
Because the retentate member preferably is transparent, colony growth in the SWIIN module can be monitored by automated devices such as those sold by JoVE (ScanLag™ system, Cambridge, Mass.) (also see Levin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growth for, e.g., mammalian cells may be monitored by, e.g., the growth monitor sold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One, 11(2):e0148469 (2016)). Further, automated colony pickers may be employed, such as those sold by, e.g., TECAN (Pickolo™ system, Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.); Molecular Devices (QPix 400™ system, San Jose, Calif.); and Singer Instruments (PIXL™ system, Somerset, UK).
Due to the heating and cooling of the SWIIN module, condensation may accumulate on the retentate member which may interfere with accurate visualization of the growing cell colonies. Condensation of the SWIIN module 550 may be controlled by, e.g., moving heated air over the top of (e.g., retentate member) of the SWIIN module 550, or by applying a transparent heated lid over at least the serpentine channel portion 560b of the retentate member 504. See, e.g.,
In SWIIN module 550 cells and medium—at a dilution appropriate for Poisson or substantial Poisson distribution of the cells in the microwells of the perforated member—are flowed into serpentine channel 560b from ports in retentate member 504, and the cells settle in the microwells while the medium passes through the filter into serpentine channel 560a in permeate member 508. The cells are retained in the microwells of perforated member 501 as the cells cannot travel through filter 503. Appropriate medium may be introduced into permeate member 508 through permeate ports 511. The medium flows upward through filter 503 to nourish the cells in the microwells (perforations) of perforated member 501. Additionally, buffer exchange can be effected by cycling medium through the retentate and permeate members. In operation, the cells are deposited into the microwells, are grown for an initial, e.g., 2-100 doublings, editing is induced by, e.g., raising the temperature of the SWIIN to 42° C. to induce a temperature inducible promoter or by removing growth medium from the permeate member and replacing the growth medium with a medium comprising a chemical component that induces an inducible promoter.
Once editing has taken place, the temperature of the SWIIN may be decreased, or the inducing medium may be removed and replaced with fresh medium lacking the chemical component thereby de-activating the inducible promoter. The cells then continue to grow in the SWIIN module 550 until the growth of the cell colonies in the microwells is normalized. For the normalization protocol, once the colonies are normalized, the colonies are flushed from the microwells by applying fluid or air pressure (or both) to the permeate member serpentine channel 560a and thus to filter 503 and pooled. Alternatively, if cherry picking is desired, the growth of the cell colonies in the microwells is monitored, and slow-growing colonies are directly selected; or, fast-growing colonies are eliminated.
Imaging of cell colonies growing in the wells of the SWIIN is desired in most implementations for, e.g., monitoring both cell growth and device performance and imaging is necessary for cherry-picking implementations. Real-time monitoring of cell growth in the SWIIN requires backlighting, retentate plate (top plate) condensation management and a system-level approach to temperature control, air flow, and thermal management. In some implementations, imaging employs a camera or CCD device with sufficient resolution to be able to image individual wells. For example, in some configurations a camera with a 9-pixel pitch is used (that is, there are 9 pixels center-to-center for each well). Processing the images may, in some implementations, utilize reading the images in grayscale, rating each pixel from low to high, where wells with no cells will be brightest (due to full or nearly-full light transmission from the backlight) and wells with cells will be dim (due to cells blocking light transmission from the backlight). After processing the images, thresholding is performed to determine which pixels will be called “bright” or “dim”, spot finding is performed to find bright pixels and arrange them into blocks, and then the spots are arranged on a hexagonal grid of pixels that correspond to the spots. Once arranged, the measure of intensity of each well is extracted, by, e.g., looking at one or more pixels in the middle of the spot, looking at several to many pixels at random or pre-set positions, or averaging X number of pixels in the spot. In addition, background intensity may be subtracted. Thresholding is again used to call each well positive (e.g., containing cells) or negative (e.g., no cells in the well). The imaging information may be used in several ways, including taking images at time points for monitoring cell growth. Monitoring cell growth can be used to, e.g., remove the “muffin tops” of fast-growing cells followed by removal of all cells or removal of cells in “rounds” as described above, or recover cells from specific wells (e.g., slow-growing cell colonies); alternatively, wells containing fast-growing cells can be identified and areas of UV light covering the fast-growing cell colonies can be projected (or rastered with shutters) onto the SWIIN to irradiate or inhibit growth of those cells. Imaging may also be used to assure proper fluid flow in the serpentine channel 560.
While cells are being processed for electroporation, pre-assembled vector backbones+expression/editing cassettes (e.g., editing vectors, including libraries or editing vectors) are provided 611 and are transferred to the flow-through electroporation device.
After electroporation 613, the transformed cells optionally are transferred 614 to a growth vial 220 to, e.g., recover from the transformation process and be submitted to selection and editing. Once the transformed cells have recovered, been selected (e.g., by an antibiotic or other reagent added from the reagent cartridge) and/or genome editing has taken place, the transformed cells may be removed from the instrument and used in further research 618, or transferred 615 into the TFF module 222 for buffer or medium exchange and/or to be concentrated and rendered competent for another round of transformation. The competent cells may then be collected in an empty vessel 206 in the wash cartridge. All or some of steps 601-605 and 611-615 may be repeated for recursive rounds of genome editing 617.
As described above, the reagent cartridges are used as components in an automated multi-module processing instrument. A general exemplary embodiment of a multi-module cell processing diagram is shown in
In some embodiments, after recovery the cells are transferred to a storage module 712 to be stored at, e.g., 4° C. or frozen. The cells can then be retrieved from a retrieval module 714 and, e.g., used for protein expression or other studies performed off-line. The automated multi-module cell processing instrument is controlled by a processor 750 configured to operate the instrument based on user input and/or one or more scripts, which may be associated with the reagent cartridge or other module. The processor 750 may control the timing, duration, temperature, and other operations (including, e.g., dispensing reagents) of the various modules of the instrument 700 as specified by one or more scripts. In addition to or as an alternative to the one or more scripts, the processor may be programmed with standard protocol parameters from which a user may select; alternatively, a user may select one or all parameters manually. The script may specify, e.g., the wavelength at which OD is read in the TFF module, the target OD to which the cells are grown, the target time at which the cells will reach the target OD, and/or the volume to which the cells should be concentrated. The processor may update the user (e.g., via an application to a smart phone or other device) as to the progress of the cells in the cell growth module, electroporation device, filtration module, recovery module, etc. in the automated multi-module cell processing instrument.
A second embodiment of an automated multi-module cell processing instrument is shown in
In addition to the reservoir for storing the cells, the reagent cartridge may include a reservoir for storing editing cassettes 816 and a reservoir for storing a vector backbone 818. Both the editing cassettes and the vector backbone are transferred from the reagent cartridge to a nucleic acid assembly module 820, where the editing cassettes are inserted into the vector backbone. The assembled nucleic acids may be transferred into an optional purification module 822 for desalting and/or other purification procedures needed to prepare the assembled nucleic acids for transformation. Once the processes carried out by the assembly/purification module 822 are complete, the assembled nucleic acids are transferred to a transformation module 808, which already contains the cell culture grown to a target OD, rendered competent and concentrated. In the transformation module 808, the nucleic acids are introduced into the cells. Following transformation, the cells are transferred into a combined recovery and editing module 812. As described above, in some embodiments the automated multi-module cell processing instrument 800 is a system that performs gene editing such as an RNA-direct nuclease editing system. For examples of multi-module cell editing instruments, see U.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat. No. 10,329,559, issued 25 Jun. 2019; and U.S. Pat. No. 10,323,242, issued 18 Jun. 2019; and U.S. Ser. No. 16/412,175, filed 14 May 2019; Ser. No. 16/412,195, filed 14 May 2019; and Ser. No. 16/423,289, filed 28 May 2019, all of which are herein incorporated by reference in their entirety. In the recovery and editing module 810, the cells are allowed to recover post-transformation, and the cells express the nuclease and editing oligonucleotides to effect editing in desired genes in the cells.
Following editing, the cells are transferred to a storage module 814, where the cells can be stored at, e.g., 4° C. until the cells are retrieved for further study. The multi-module cell processing instrument is controlled by a processor 850 configured to operate the instrument based on user input, as directed by one or more scripts, or as a combination of user input or a script. The processor 850 may control the timing, duration, temperature, and operations of the various modules of the instrument 800 and the dispensing of reagents from the reagent cartridge. The processor may be programmed with standard protocol parameters from which a user may select, a user may specify one or more parameters manually or one or more scripts associated with the reagent cartridge may specify one or more operations and/or reaction parameters. In addition, the processor may notify the user (e.g., via an application to a smart phone or other device) that the cells have reached a target OD, been rendered competent and concentrated, and/or update the user as to the progress of the cells in the various modules in the multi-module instrument.
As described above, in one embodiment the automated multi-module cell processing instrument 800 is a nucleic acid-guided nuclease editing system. Multiple nuclease-based systems exist for providing edits into a cell and each can be used in either single editing systems as could be performed in the automated instrument 700 of
A third embodiment of a multi-module cell processing instrument is shown in
After selection, the cells may be transferred to an editing module 928 where providing conditions for the cells to edit, e.g., if editing is driven by an inducible promoter. After editing, the cells are transferred back to a TFF module 904 where the edited cells are allowed to grow, and then buffer or medium exchange is performed once again and the cells are rendered competent once again in preparation for transfer to the transformation module 908. Note that in the case of a SWIIN, for example, selection, editing and growth all take place in the same module.
In transformation module 908, the cells are transformed by a second set of editing cassettes (or other type of cassette) and the cycle is repeated until the cells have been transformed and edited by a desired number of, e.g., editing cassettes. As discussed above in relation to
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Other equivalent methods, steps and compositions are intended to be included in the scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.
Both bacterial (E. coli) and yeast (S. cerevisae) cells were cultures in the TFF device. E. coli cells were grown on LB medium with 25 μg/mL chloramphenicol, and S. cerevisae were grown in YDP medium with 100 μg/mL carbenicol. For both E. coli and S. cerevisae, starter culture was grown overnight, and a 1/100 dilution of the starter cultures were grown in 30 mL of the appropriate medium in the device. The initial culture was loaded into one of the retentate reservoirs. Bubbling of the culture at 20 psi was performed while the cell cultures resided in the reservoirs. The cultures were then transferred through the flow channel at 30 psi to the other retentate reservoir where bubbling (aeration) of the cultures at 20 psi effected mixing. The transfer and bubbling processes were repeated several times. Fresh medium was added to the cell cultures as old medium was removed from the cultures during the transfer of the cell culture through the flow channel. The results of culturing E. coli in the TFF device vs. a traditional shaker culture is shown in
The TFF module as described above in relation to
First, a 20 ml culture of E. coli in LB grown to OD 0.5-0.62 was passed through the TFF device in one direction, then passed through the TFF device in the opposite direction. At this point the cells were concentrated to a volume of approximately 5 ml. Next, 50 ml of 10% glycerol was added to the concentrated cells, and the cells were passed through the TFF device in one direction, in the opposite direction, and back in the first direction for a total of three passes. Again the cells were concentrated to a volume of approximately 5 ml. Again, 50 ml of 10% glycerol was added to the 5 ml of cells and the cells were passed through the TFF device for three passes. This process was repeated; that is, again 50 ml 10% glycerol was added to cells concentrated to 5 ml, and the cells were passed three times through the TFF device. At the end of the third pass of the three 50 ml 10% glycerol washes, the cells were again concentrated to approximately 5 ml of 10% glycerol. The cells were then passed in alternating directions through the TFF device three more times, wherein the cells were concentrated into a volume of approximately 400 μl.
The same process was repeated with yeast cell cultures. A yeast culture was initially concentrated to approximately 5 ml using two passes through the TFF device in opposite directions. The cells were washed with 50 ml of IM sorbitol three times, with three passes through the TFF device after each wash. After the third pass of the cells following the last wash with IM sorbitol, the cells were passed through the TFF device two times, wherein the yeast cell culture was concentrated to approximately 525 μl.
Singleplex automated genomic editing using MAD7 nuclease was successfully performed with an automated multi-module instrument such as that shown in
An ampR plasmid backbone and a lacZ_F172* editing cassette were assembled via Gibson Assembly® into an “editing vector” in an isothermal nucleic acid assembly module included in the automated instrument. lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicates that the edit happens at the 172nd residue in the lacZ amino acid sequence. Following assembly, the product was de-salted in the isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The assembled editing vector and recombineering-ready, electrocompetent E. Coli cells were transferred into a transformation module for electroporation. The cells and nucleic acids were combined and allowed to mix for 1 minute, and electroporation was performed for 30 seconds. The parameters for the poring pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1; polarity, +. The parameters for the transfer pulses were: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−. Following electroporation, the cells were transferred to a recovery module (another growth module) and allowed to recover in SOC medium containing chloramphenicol. Carbenicillin was added to the medium after 1 hour, and the cells were allowed to recover for another 2 hours. After recovery, the cells were held at 4° C. until recovered by the user.
After the automated process and recovery, an aliquot of cells was plated on MacConkey agar base supplemented with lactose (as the sugar substrate), chloramphenicol and carbenicillin and grown until colonies appeared. White colonies represented functionally edited cells, purple colonies represented un-edited cells. All liquid transfers were performed by the automated liquid handling device of the automated multi-module cell processing instrument.
The result of the automated processing was that approximately 1.0E0.3 total cells were transformed (comparable to conventional benchtop results), and the editing efficiency was 83.5%. The lacZ_172 edit in the white colonies was confirmed by sequencing of the edited region of the genome of the cells. Further, steps of the automated cell processing were observed remotely by webcam and text messages were sent to update the status of the automated processing procedure.
Recursive editing was successfully achieved using the automated multi-module cell processing system. An ampR plasmid backbone and a lacZ_V10* editing cassette were assembled via Gibson Assembly® into an “editing vector” in an isothermal nucleic acid assembly module included in the automated system. Similar to the lacZ_F172 edit, the lacZ_V10 edit functionally knocks out the lacZ gene. “lacZ_V10” indicates that the edit happens at amino acid position 10 in the lacZ amino acid sequence. Following assembly, the product was de-salted in the isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The first assembled editing vector and the recombineering-ready electrocompetent E. coli cells were transferred into a transformation module for electroporation. The cells and nucleic acids were combined and allowed to mix for 1 minute, and electroporation was performed for 30 seconds. The parameters for the poring pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1; polarity, +. The parameters for the transfer pulses were: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−. Following electroporation, the cells were transferred to a recovery module (another growth module) allowed to recover in SOC medium containing chloramphenicol. Carbenicillin was added to the medium after 1 hour, and the cells were grown for another 2 hours. The cells were then transferred to a centrifuge module and a media exchange was then performed. Cells were resuspended in TB containing chloramphenicol and carbenicillin where the cells were grown to OD600 of 2.7, then concentrated and rendered electrocompetent.
During cell growth, a second editing vector was prepared in the isothermal nucleic acid assembly module. The second editing vector comprised a kanamycin resistance gene, and the editing cassette comprised a galK Y145* edit. If successful, the galK Y145* edit confers on the cells the ability to uptake and metabolize galactose. The edit generated by the galK Y154* cassette introduces a stop codon at the 154th amino acid residue, changing the tyrosine amino acid to a stop codon. This edit makes the galK gene product non-functional and inhibits the cells from being able to metabolize galactose. Following assembly, the second editing vector product was de-salted in the isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The assembled second editing vector and the electrocompetent E. Coli cells (that were transformed with and selected for the first editing vector) were transferred into a transformation module for electroporation, using the same parameters as detailed above. Following electroporation, the cells were transferred to a recovery module (another growth module), allowed to recover in SOC medium containing carbenicillin. After recovery, the cells were held at 4° C. until retrieved, after which an aliquot of cells were plated on LB agar supplemented with chloramphenicol, and kanamycin. To quantify both lacZ and galK edits, replica patch plates were generated on two media types: 1) MacConkey agar base supplemented with lactose (as the sugar substrate), chloramphenicol, and kanamycin, and 2) MacConkey agar base supplemented with galactose (as the sugar substrate), chloramphenicol, and kanamycin. All liquid transfers were performed by the automated liquid handling device of the automated multi-module cell processing system.
In this recursive editing experiment, 41% of the colonies screened had both the lacZ and galK edits, the results of which were comparable to the double editing efficiencies obtained using a “benchtop” or manual approach.
While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, 16.
This application is a continuation of U.S. patent application Ser. No. 16/516,701, filed 5 Sep. 2019, which claims priority to U.S. patent application Ser. Nos. 62/728,365, filed 7 Sep. 2018; 62/857,599, filed 5 Jun. 2019; and 62/867,415, filed 27 Jun. 2019.
Number | Date | Country | |
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62728365 | Sep 2018 | US | |
62857599 | Jun 2019 | US | |
62867415 | Jun 2019 | US |
Number | Date | Country | |
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Parent | 16561701 | Sep 2019 | US |
Child | 16798302 | US |