Patent Publication Number: US-2023132556-A1

Title: Microfluidic chip cell sorting and transfection

Description:
BACKGROUND 
     Transfection is sometimes used to engineer or modify biological cells for immunotherapies such as CAR-T therapy. During transfection, nucleic acids and small proteins are introduced into eukaryotic cells. Electroporation is sometimes used to electrically open pores in the cell membrane for the introduction of the nucleic acids and small proteins. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    is a schematic diagram illustrating portions of an example cell transfection apparatus. 
         FIG.  2    is a flow diagram illustrating an example cell transfection method. 
         FIG.  3    is a schematic diagram illustrating portions of an example cell propagation system comprising the cell transfection apparatus of  FIG.  1   . 
         FIG.  4    is a schematic diagram illustrating portions of an example cell transfection apparatus. 
         FIG.  5    is a schematic diagram illustrating portions of an example cell transfection apparatus. 
         FIG.  6    is a schematic diagram illustrating portions of an example cell transfection apparatus. 
         FIG.  6    is a schematic diagram illustrating portions of an example cell transfection apparatus. 
         FIG.  7    is a schematic diagram illustrating portions of an example cell transfection apparatus. 
         FIG.  8    is a schematic diagram illustrating portions of an example cell transfection apparatus. 
     
    
    
     Throughout the drawings, identical reference numbers designate similar, but not necessarily identical, elements. The FIGS. are not necessarily to scale, and the size of some parts may be exaggerated to more clearly illustrate the example shown. Moreover, the drawings provide examples and/or implementations consistent with the description; however, the description is not limited to the examples and/or implementations provided in the drawings. 
     DETAILED DESCRIPTION OF EXAMPLES 
     Disclosed are example cell transfection apparatus, cell transfection methods and cell propagation systems that provide for the enhanced generation of engineered biological cells. The example apparatus, methods and systems may provide for an automated integrated apparatus or system that reduces contamination, reduces operator error, and reduces labor cost. The example apparatus, method and systems may sort cells prior to transfection to improve homogeneity of the transfected cells which may result in improved testing and therapy outcomes. 
     The example cell transfection apparatus, example cell transfection methods and example cell propagation systems integrate a cell sorter, a cell transfection region and a fluid ejector on a single microfluidic chip. Such integration facilities a compact arrangement that may reduce cost and contamination issues. Such integration may facilitate a streamlined automated process for efficiently and economically generating engineered biological cells for therapeutic and academic purposes. 
     In some implementations, the sorted cells undergo electrotransfection, wherein electrodes in an electroporation region apply an electric field with an amplitude and voltage that induces momentary poration of the cell membrane to allow material to diffuse into the cell. Following the introduction of the material into the cell, the pores are allowed to close. In some implementations, the cells may be diluted and directed across a single cell electroporation region. Each of the cells sequentially passing through the electroporation region experiences same electric field are not perturbed by neighbor cell passing through electroporation region at the same time. This results in a more uniform and reliable transfection of cells. 
     In some implementations, cells are sequentially moved through the single cell electroporation region by pumping devices also integrated into the single microfluidic chip. For example, the pumping devices may comprise inertial pumps. In some implementations, the inertial pumps may comprise thermoresistive electrodes which vaporize liquid to create a bubble which displaces and pumps fluid. 
     In some implementations, cells are sequentially pulled or drawn through the single cell electroporation region by a fluid ejector integrated into the single microfluidic chip. Ejection of fluid by the fluid ejector creates a fluid pressure differential to draw cells, suspended in fluid, across the electroporation region. In some implementations, the fluid ejector may comprise a fluid actuator which displaces fluid through an ejection orifice to dispense the sorted and transfected cells. 
     As noted above, the integration of the cell sorting, cell transfection and fluid ejection into a single microfluidic chip provides a compact and cost-effective solution for generating engineered cells. The compact microfluidic chip may be well adapted for incorporation into a larger, but more compact and cost-effective, cell propagation system. The microfluidic chip may be incorporated into a cell propagation system that also includes a multi-well plate such that different samples of cells may be ejected or dispensed by the fluid ejector into different wells. Propagation of the different cells may be further enhanced through the addition of reagents and liftoff agents supplied by liquid handler. In some implementations, an imager or other sensor may also monitor the propagation of cells in the different wells. Due to compactness of the overall propagation system, it may be more readily housed within an incubator which controls environmental factors such as temperature, humidity and carbon dioxide levels for enhanced cell propagation. 
     The disclosed cell transfection apparatus, cell transfection methods and cell propagation systems may be well suited for use in chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy, a sample of patient&#39;s T cells (part of the patient&#39;s immune system) are collected from blood and then modified or engineered to produce the special CAR constructs on their surface. Such T cells may be engineered to express disease-specific targets which then may be propagated to therapeutic levels before being infused back into the patient from which the cells were taken. 
     With the disclosed cell transfection apparatus, cell transfection methods and cell propagation systems, the T cells collected from the blood of the patient may first be sorted to separate naïve T cells from mature T cells. Through sorting, naïve T cells can be separated from mature T cells for transfection and propagation. The naïve T cells may have a higher therapeutic efficiency than the mature and activated T cells. As a result, transfected cells generated by the cell transfection apparatus, methods and systems may have a higher percentage of naïve T cells. The higher percentage of naïve T cells may lead to a higher drug persistence and enhanced therapeutic results. 
     Disclosed are example cell transfection apparatus that may include a microfluidic chip. The microfluidic chip may include a fluid input port, a cell sorter to sort target cells from non-target cells in fluid received through the input port, a cell transfection region comprising a single cell electroporation region to receive the target cells sorted from the nontarget cells and a fluid ejector to dispense a transfected target cell received from the cell transfection region. 
     Disclosed are example cell transfection methods may include depositing a solution into a port of a microfluidic chip, sorting a target cell from a non-target cell of the solution with a cell sorter on the microfluidic chip, moving the target cell through a single cell electroporation region of the chip, transfecting the electroporated target cell on the chip and dispensing the transfected cell from the chip. 
     Disclosed are example cell propagation system systems that may include a multi-well plate, a microfluidic chip, a liquid handler, a stage and a controller. The microfluidic chip may include a fluid input port to receive a fluid containing target cells, a cell transfection region comprising a single cell electroporation region and a fluid ejector to eject droplets of fluid to pull the solution containing the target cells through the single cell electroporation region. The liquid handler is to exchange media within wells of the well plate. The stages position the multi-well plate relative to the fluid ejector in the liquid handler. The controller may help control signals controlling stage, the fluid ejector in the liquid handler. 
       FIG.  1    is a block diagram schematically illustrating portions of an example cell transfection apparatus  20 . Cell transfection apparatus  20  comprises a microfluidic chip  22  that integrates cell sorting, cell transfection and controlled cell ejection or dispensing into a compact arrangement that may reduce cost and contamination issues. Such integration may facilitate a streamlined automated process for efficiently and economically generating engineered biological cells for therapeutic and academic purposes. 
     Microfluidic chip  22  may comprise a platform or substrate formed by a single layer multiple layers that support or form microfluidic passages, chambers and volumes and that further support electronic elements in the form of transistors, resistors, fluid actuators and their associated electrical conductive wires or traces. The platform or substrate may comprise a silicon-based wafer or other such similar materials used for microfabricated devices (e.g., glass, gallium arsenide, quartz, sapphire, metal, plastics, etc.). In some implementations, microfluidic chip  22  may be formed from a glass reinforced epoxy laminate material such as a glass epoxy laminate such as FR4, wherein microfluidic channels may be formed in the laminate material or may be formed in other structures mounted to the laminate material. 
     As will be appreciated, portions of microfluidic chip  22  may be formed by performing various microfabrication and/or micromachining processes on a substrate to form and/or connect structures and/or components. Microfluidic channels and/or chambers may be formed by performing etching, microfabrication processes (e.g., photolithography), or micromachining processes in a substrate. Accordingly, microfluidic channels and/or chambers may be defined by surfaces fabricated in the substrate of a microfluidic device. In some implementations, microfluidic channels and/or chambers may be formed by an overall package, wherein multiple connected package components combine to form or define the microfluidic channel and/or chamber. 
     In some examples described herein, at least one dimension of a microfluidic channel and/or capillary chamber may be of sufficiently small size (e.g., of nanometer sized scale, micrometer sized scale, millimeter sized scale, etc.) to facilitate pumping of small volumes of fluid (e.g., picoliter scale, nanoliter scale, microliter scale, milliliter scale, etc.). For example, some microfluidic channels may facilitate capillary pumping due to capillary force. In addition, examples may couple at least two microfluidic channels to a microfluidic output channel via a fluid junction. 
     Microfluidic chip  22  comprises fluid input port  30 , cell sorter  34 , cell transfection region  38  and fluid ejector  42 . Fluid input port  30  comprises an opening or passage through which a volume of fluid may be introduced into microfluidic chip  22 . The volume of fluid may contain cells for transfection. The fluid may also contain additional particles and other non-target cells. In some implementations, fluid input port is sized for receiving a pipette or needle by which the fluid may be supplied to microfluidic chip  22 . In some implementations, fluid input port  30  may comprise a fluid coupling for connection to a tube supplying the fluid or connection to an output port of a fluid source. In some implementations, microfluidic chip  22  may be insertable into the fluid source, wherein the fluid input port becomes aligned with the output port of the fluid source. 
     Cell sorter  34  comprises a portion of microfluidic chip  22  that receives the fluid received through fluid input port  30  and that sorts target cells from non-target cells or particles in the fluid. Cell sorter  34  may distinguish between target cells and non-target cells based upon various differences between the target cells and nontarget cells. For example, cell sorter  34  may distinguish between target cells and non-target cells based upon size or using techniques such as negative or positive enrichment, dielectrophoretics or acoustics. As shown by broken lines, target cell  35 , sorted by cell sorter  34 , is pumped or otherwise moved to cell transfection region  38 . 
     Cell transfection region  38  comprises a region of microfluidic chip  22  that carries out transfection with respect to the sorted target cell  35 . Cell transfection region  38  comprises that apply an electric field with an amplitude and voltage that induces momentary poration of the membrane of target cell  35  to allow material to diffuse into the cell  35 . For example, to transfect naïve T cells, transfection region  38  may apply an electric field having 0.01 V/um to 1V/um. In other implementations, the field may have other values depending upon the solution containing the cells. Following the introduction of the material into the cell, the pores are allowed to close. 
     In some implementations, the fluid containing the cell  35  may be diluted and directed across a single cell electroporation region sized such that cells move through the region in single file order, such that a single individual cell  35  undergoes electroporation and electrotransfection at a time. The fluid passage through which cell  35  moves or is contained during electro transfection is sized such that additional neighboring cells are inhibited from entering the same fluid passage and extending alongside, in parallel to, target cell  35  during electroporation and transfection. There are no neighboring cells which may interfere with or perturb the electric field being applied to the target cell  35 . Thus, each of the cells  35  sequentially passing through the cell transfection region  38  and its electroporation region experiences same electric field. This results in a more consistent and uniform transfection of the cells being generated by apparatus  20 . Although such sizes may vary depending upon the cells being engineered, examples of a cross-sectional area of region  38  include, but are not limited to. 5×10 um, 10×10 um,  20 ×20 um, 20×10 um, 50×50 um, etc. In some implementations where larger cells are to be transfected, the cross-sectional dimensions may be greater. 
     In some implementations, cell  35  is sequentially moved through the cell transfection region  38  by pumping devices also integrated into the single microfluidic chip  22 . For example, the pumping devices may comprise inertial pumps. In some implementations, the inertial pumps may comprise thermoresistive electrodes which vaporize liquid to create a bubble which displaces and pumps fluid. 
     Fluid ejector  42  comprises a fluid actuator that dispenses or ejects the transfected target cell  35  through an ejection orifice. In one implementation, fluid ejector  42  may comprise a thermal resistor which, upon receiving electrical current, heats to a temperature above the nucleation temperature of the fluid so as to vaporize a portion of the adjacent fluid to create a bubble which displaces the fluid through the associated ejection orifice. In other implementations, the fluid ejector  42  may comprise other forms of fluid actuators. In other implementations, the fluid ejector  42  may comprise a fluid actuator in the form of a piezo-membrane based actuator, an electrostatic membrane actuator, mechanical/impact driven membrane actuator, a magnetostrictive drive actuator, an electrochemical actuator, and external laser actuators (that form a bubble through boiling with a laser beam), other such microdevices, or any combination thereof. 
     In some implementations, fluid ejector  42  dispenses the transfected target cell  35  into a target location, such as into a well of a multi-well plate. In some implementations, fluid ejector  42  dispenses fluid to pull or draw cell through the single cell transfection region  38 . Ejection of fluid by the fluid ejector  42  creates a fluid pressure differential to draw cells, suspended in fluid, across the transfection region  38 . In some implementations, fluid ejector  42  may be used alone or in combination with other pumps (such as inertial pumps) supported by chip  22  to sequentially move cells sorted by cell sorter  34  through and across cell transfection region  38 . 
       FIG.  2    is a flow diagram of an example cell transfection method  100 . Method  100  facilitates a streamlined automated process for efficiently and economically generating engineered biological cells for therapeutic and academic purposes. Although method  100  is described in the context of being carried out by cell transfection apparatus  20 , it should be appreciated that method  100  may likewise be carried out with any of the following described cell transfection apparatus and cell propagation systems or with similar cell transfection apparatus or cell propagation systems. 
     As indicated by block  104 , a fluid solution is deposited into a port, such as fluid input port  30 , of a microfluidic chip, such as microfluidic chip  22 . The solution may contain target cells  35  which are to be transfected. In some implementations, the fluid solution may be deposited with a pipette or needle. In other implementations, the fluid solution may be deposited via a connection to the fluid input port. 
     As indicated by block  108 , the target cell  35  is sorted from non-target cells of the fluid solution with a cell sorter, such as cell sorter  34 , on the microfluidic chip  22 . The sorting of the target cell or target cells from non-target cells in the fluid solution may be based upon size or using techniques such as negative or positive enrichment, dielectrophoretics or acoustics. 
     As indicated by block  112 , those target cells which have been sorted or separated from other types of cells or other particles are moved through cell transfection region  38  of the chip  22 . In some implementations, the target cells are pumped by pump located on the microfluidic chip  22 . For example, inertial pumps may be formed on microfluidic chip  22  for moving the cells through the cell transfection region. In some implementations, fluid pressures created on the microfluidic chip through the ejection of fluid by fluid ejector may be used to pull or draw cells through and across the cell transfection region. 
     As indicated by block  116 , while the cells are within the cell transfection region or are being moved through the cell transfection region, the cells undergo transfection. In some implementations, the cells undergo electro transfection or electroporation. During electrotransfection or electroporation, electrodes on the microfluidic chip  22  apply an electric field with an amplitude and voltage that induces momentary poration of the membrane of the individual target cell received within the cell transfection region to allow material to diffuse into the cell. Following the introduction of the material into the cell, the pores are allowed to close. As indicated by block  118 , transfected cells are then dispensed from the microfluidic chip  22 . 
       FIG.  3    is a diagram schematically illustrating portions of an example cell propagation system  200 . Cell propagation system  200  facilitates the generation of engineered biological cells as well as the propagation of the engineered biological cells. In addition to microfluidic chip  22  (described above), cell propagation system  200  comprises multi-well plate  250 , stage  252 , media exchange system  254  and controller  260 . 
     Multi-well plate  250  comprises an array of individual wells which are to receive the transfected cells dispensed by fluid ejector  42 . Stage  252  comprises a motorized stage that controllably positions individual wells of multi-well plate  250  opposite to fluid ejector  42  for receiving a transfected cell or multiple transfected cells. Stage  252  may further position the individual wells of multi-well plate  250  opposite to media exchange system  254 . In some implementations where microfluidic chip  22  and media exchange system  254  are themselves movable or positionable relative to multi-well plate  250 , stage  252  may be omitted. 
     Media exchange system  254  exchanges fluid in each of the individual wells of multi-well plates  250  facilitate the propagation of the transfected cells, the growth in number or multiplication of transfected cells. Media exchange system  254  may further remove waste. Media exchange system  254  may comprise liquid handler  262 , reagent sources  264 - 1 - 264 - n  (collectively referred to as reagent sources  264 ) and waste reservoir  266 . Liquid handler  262  pumps or directs the flow of fluid to and from the individual wells of multi-well plate  250 . Liquid handler  262  refreshes media for continuous cell growth. Liquid handler  262  supplies reagents from reagent sources  264  to the transfected cells contained within the wells of multi-well plate  250 . Liquid handler  262  may further withdraw used or exhausted fluid from such wells. In some implementations, liquid handler  262  may comprise an automated pipetting system or peristaltic pump. 
     Controller  260  controls the operation of cell propagation system  200 . Controller  260  comprises a processor  270  that follows instructions contained on a non-transitory computer-readable medium in the form of memory  272 . Such instructions direct the processor  270  to output control signals controlling the pumping or movement of cells from cell sorter  34  through transfection region  38  and the ejection of fluid (with the suspended transfected cells) by fluid ejector  42 . Such control signals may further control the operation of  252  to properly position individual wells of multi-well plate  250  opposite to fluid ejector  42  and to properly position the individual wells of multi-well plate  250  opposite to liquid handler  262 . In some implementations, such control signals may further control the sorting of cells by cell sorter  34  and/or the pumping of fluid from input port  30  to cell sorter  34 . Controller  260  may communicate with the other components of propagation system  200  in a wired or wireless manner. 
     As shown by broken lines, in some implementations, cell propagation system  200  may additionally comprise an imager  280 . Imager  280  may monitor and inspect the cells within multi-well plate  250  during their propagation. Signals from imager  280  may be supplied to controller  260  which, pursuant to the instructions contained in memory  272 , adjusts the exchange of media by media exchange system  254  and/or adjusts the sorting, transfection and dispensing operations carried out on microfluidic chip  22  based upon the signals from imager  280 . Using the information acquired by imager  280 , controller  260  may provide feedback control to enhance the transfection performance, cell viability and cell propagation. In some implementations, imager  280  may be omitted. 
     As further shown by broken lines, in some implementations, cell propagation system  200  may additionally comprise an incubator  290 . Incubator  290  comprises an enclosure containing microfluidic chip  22 , stage  252 , multi-well plate  250  and media exchange system  254 . In some implementations, incubator  290  may additionally contain imager  280 , when provided. In other implementations, imager  280  may image the contents of multi-well plate  253  window or transparent portion of incubator  290 . 
     Incubator  290  facilitates the control of the environment in which the cells are transfected and propagated. For example, incubator  290  may facilitate the control of temperature, humidity and carbon dioxide concentration levels to enhance the propagation of transfected cells. In some implementations, controller  260 , following instructions contained in memory  272 , may output control signals to heaters, humidifiers, vents, fans or carbon dioxide sources connected to the interior of incubator  290  to adjust the temperature, humidity and carbon dioxide levels within incubator  290 . In some implementations, such adjustment may be based upon data obtained from imager  280 . In some implementations, such adjustments may be based upon predetermined timing protocols or schedules. In some implementations, incubator  290  may be omitted. 
     In some implementations, cell propagation system  200  may be used for CAR T cell therapy. As part of the CAR T cell therapy, a sample of patient&#39;s T cells (part of the patient&#39;s immune system) are collected from blood and then modified or engineered to produce the special CARs on their surface. Such T cells may be engineered to express disease-specific targets which then may be propagated to therapeutic levels before being infused back into the patient from which the cells were taken. 
     With cell propagation system  200 , the T cells collected from the blood of the patient are inserted to fluid input port  30  and then sorted by cell sorter  34  to separate naïve engineered T cells from active T cells. Through sorting, naïve T cells can be separated from active T cells. The fluid having the higher concentration of naïve T cells is then transmitted through cell transfection region  38  where the naïve T cells are transfected with CAR plasmids for disease specific targets. Once transfected, cells are dispensed by fluid ejector  42  into designated wells of multi-well plate  250 . Thereafter, the wells containing the transfected cells are repositioned opposite to media exchange system  254  which exchanges cell growth media to enhance the growth or propagation of the transfected cells. The larger number of propagated transfected cells may then be reintroduced into the patient for therapeutic purposes. 
       FIG.  4    schematically illustrates portions of an example cell transfection apparatus  320 . Cell transfection apparatus  320  illustrates one example of how biological cells may be sorted by size for subsequent transfection and dispensing. Similar to cell transfection apparatus  20 , cell transfection apparatus  320  is supported by or integrated into a single microfluidic chip  322 . Microfluidic chip  322  is similar to microfluidic chip  22  except microfluidic chip  322  specifically comprises fluid input port  330 , cell sorter  334 , cell transfection regions  338 - 1 ,  338 - 2  (collectively referred to as transfection regions  338 ) and fluid ejector&#39;s  342 - 1 ,  342 - 2  (collectively referred to as fluid ejectors  342 ). 
     Fluid input port  330  is similar to fluid input port  30  described above. Fluid input port  330  comprises an opening or passage through which a volume of fluid may be introduced into microfluidic chip  322 . The volume of fluid may contain cells for transfection. The fluid may also contain additional particles and other non-target cells. In some implementations, fluid input port  330  is sized for receiving a pipette or needle by which the fluid may be supplied to microfluidic chip  322 . In some implementations, fluid input port  330  may comprise a fluid coupling for connection to a tube supplying the fluid or connection to an output port of a fluid source. In some implementations, microfluidic chip  322  may be insertable into the fluid source, wherein the fluid input port becomes aligned with the output port of the fluid source. 
     Cell sorter  334  sorts cells in the fluid received through input port  330  based upon the size-elasticity and/or shape changing characteristics of such cells. In the example illustrated, cell sorter  334  comprises an array of spaced pillars  350  extending from a floor to roof of fluid passage  352  and forming a filter, wherein pillars  350  are spaced apart from one another by a distance sufficiently large to allow first biological cells  335 - 1  to pass therebetween and sufficiently small to inhibit or block the passage of biological cells  335 - 2 . The spacing between pillars  350  may be dependent upon the expected size of the different biological cells  335  as well as their potentially different elasticities or ability to compress or change shape. In some other implementations, the filter may be formed by horizontal bars extending side to side within passage  352 . In other implementations, cell sorter  334  may comprise a filter or other arrangement of structures that permit the passage of some biological cells while blocking the passage of other biological cells based upon their different sizes. 
     Cell transfection regions  338  branch off of microfluidic passage  352  on opposite sides of the filter form by pillars  350 . Cell transfection regions  338 - 1  and  338 - 2  comprise constricted fluid passages  354 - 1  and  354 - 2  (collectively referred to as passages  354 ), respectively. Passage  354 - 1  is sized to inhibit to biological cells  335 - 1  from moving through passage  354 - 1  in parallel. Rather, passage  354 - 1  is sized social force a sequential ordering of individual cells (single cells) through and across passage  354 - 1 . Similarly, passage  354 - 2  is sized to inhibit to biological cells  335 - 2  from moving through passage  354 - 2  in parallel. Rather, passage  354 - 2  is sized so as to force a sequential ordering of individual cells (single cells) through and across passage  354 - 2 . 
     In some implementations, passages  354 - 1  and  354 - 2  may have different cross-sectional dimensions and/or different cross-sectional shapes from one another based upon the particular cross-sectional dimensions, shape and/or shape changing characteristics of the respective cells  335 - 1  and  335 - 2  that are to be transfected in such regions. In some implementations where cell transfection apparatus  320  is used as part of CAR T therapy, the spacing between pillars  550  is sized so as to allow the passage of naïve T cells and block or inhibit the passage of active T cells. 
     Cell transfection region  338 - 1  further comprises a pair of electrodes  356 - 1  that are electrically connected to a ground  358  and a voltage source  360  such that electric field is created across passage  354 - 1  containing the single cell  335 - 1 . The electric field thus formed has characteristics so as to electrically open pores in the membrane of the individual cell  335 - 1  contained within transfection region  338 - 1  for the introduction of the nucleic acids and small proteins. 
     Similar to cell transfection region  338 -one, cell transfection region  338 - 2  further comprises a pair of electrodes  356 - 2  that are electrically connected to a ground  358  and a voltage source  360 - 2  such that electric field is created across passage  354 - 2  containing the single cell  335 - 2 . The electric field thus formed has characteristics so as to electrically open pores in the membrane of the individual cell  335 - 2  contained within cell transfection region  338 - 2  for the introduction of the nucleic acids and small proteins. In some implementations, the electric fields created across transfection region  338 - 1  and  338 - 2  are different from one another due to the different characteristics of the cells being transfected within such different regions. 
     While within the cell transfection region  338 , the cells  335  undergo electroporation, wherein pores in the membrane are opened. While within passages  354 - 1  and  354 - 2 , cells  335 - 1  and  335 - 2  are transfected with foreign materials such as nucleic acids and small proteins. In implementations where apparatus  320  is used as part of CAR T therapy, the naïve T cells in cell transfection region  338 - 1  may be infused with CAR receptors. 
     Fluid ejectors  342 - 1  and  342 - 2  comprise ejection passages or chambers  364 - 1 ,  364 - 2  (collectively referred to as chambers  364 ), ejection orifices  366 - 1 ,  366 - 2  (collectively referred to as ejection orifices  366 ) and fluid actuator  368 - 1 ,  368 - 2  (collectively referred to as fluid actuator  368 ). Chambers  364 - 1 ,  364 - 2  of fluid ejectors  342  receive transfected cells from their respective transfection region  338 - 1 ,  338 - 2 . Ejection office  366 - 1 ,  366 - 2  extend from their respective chambers  364 - 1 ,  364 - 2 . 
     Fluid actuators  368 - 1 ,  368 - 2  comprise devices that displace fluid within their respective chambers  364 - 1 ,  364 - 2  through their respective ejection orifices  366 - 1 ,  366 - 2 . Such fluid actuator  368  also dispense the transfected cells through the ejection orifices  366 . In one implementation, each of fluid actuators  368  comprises a thermal resistor which, upon receiving electrical current, heats to a temperature above the nucleation temperature of the fluid so as to vaporize a portion of the adjacent fluid to create a bubble which displaces the fluid through the associated ejection orifice. In other implementations, each of fluid actuators  368  may comprise other forms of fluid actuators. In other implementations, the fluid ejector  42  may comprise a fluid actuator in the form of a piezo-membrane based actuator, an electrostatic membrane actuator, mechanical/impact driven membrane actuator, a magnetostrictive drive actuator, an electrochemical actuator, and external laser actuators (that form a bubble through boiling with a laser beam), other such microdevices, or any combination thereof. 
     In the example illustrated, upon receiving a transfected cell  335 - 1 , fluid actuator  368 - 1  may be actuated to dispense eject fluid, containing the transfected cell  335 - 1 , through ejection orifice  366 - 1 , to an underlying target site, well, passage or other receptacle. In some implementations, the dispensing of fluid through ejection orifice  366 - 1  creates a fluid pressure differential which draws fluid and other suspended cells across pillars  350 - 1 . The pressure differential may further draw an individual cell  335 - 1  into transfection region  338 - 1  for transfection. 
     In the example illustrated, upon receiving a transfected cell  335 - 2 , fluid actuator  368 - 2  may be actuated to dispense or eject fluid, containing the transfected cell  335 - 2 , through ejection orifice  366 - 2  to an underlying target site, well, passage or other receptacle. In some implementations, the dispensing of fluid through ejection orifice  366 - 2  creates a fluid pressure differential which draws fluid and other suspended cells across pillars  350 - 2 . The pressure differential may further draw an individual cell  335 - 2  into transfection region  338 - 2  for transfection. In other implementations, additional inertial pumps may be provided in passage  352  or elsewhere for moving cells  335 - 1  through pillars  350  and across transfection region  338 - 1  or for moving cells  335 - 2  through transfection region  338 - 2 . 
     In some implementations, transfection region  338 - 2  and fluid ejector  342 - 2  may be omitted. For example, in some implementations, fluid and cells not passing through the filter formed by pillars  350  may instead be directed to a waste receptacle or port. In some implementations, transfection region  338 - 2  may be omitted, wherein fluid and cells not passing through the filter formed by pillars  350  may be dispensed by fluid ejector  342 - 2  to a waste passage or reservoir. Although microfluidic chip  322  is illustrated as having a single filter, in other implementations, microfluidic chip  322  may comprise multiple different filters which are in series with one another, wherein three or more different biological cells may be separated or sorted from one another, wherein different cells that have been sorted from one another may undergo different transfection procedures or disposal. 
       FIG.  5    schematically illustrates portions of an example cell transfection apparatus  420 .  FIG.  5    illustrates an example of how a microfluidic chip may carry out the sorting of cells based upon affinity prior to transfection and dispensing by the same microfluidic chip. Cell transfection apparatus  420  is similar to cell transfection apparatus  320  described above except that cell transfection apparatus  420  comprises a microfluidic chip  422  comprising cell sorter  434  in place of cell sorter  334  and omitting transfection region  338 - 2  and fluid ejector  342 - 2 . Those remaining components of cell transfection apparatus  420  which correspond to components of cell transfection apparatus  320  are numbered similarly. 
     Cell sorter  434  comprises pillars  450  or other structures which are enriched so as to attract non-target cells  335 - 2  while permitting target cells  335 - 1 , which are less attracted to pillars  450 , to pass by to transfection region  338 - 1 . In some implementations, pillars  450  are enriched with anti-bodies  451  for attracting the nontarget cells  335 - 2  while allowing the target cells  335 - 1  to pass. In implementations where cell transfection apparatus  420  is used as part of a CAR T therapy, the antibodies are chosen so as to attract the active T cells, allowing naïve T cells to pass. Once sorted and separated from cells  335 - 2 , the target cell  335 - 1  may be directed in a single file basis through and across cell transfection region  338 - 1  (described above). Once transfected, the cells  335 - 1  may be controllably and selectively dispensed through ejection orifice  366 - 1  by fluid actuator  368 - 1 . In the example illustrated, the dispensing of fluid by fluid actuator  368 - 1  creates a negative pressure differential which draws or pulls cell  335 - 1  through and across transfection region  338 - 1  in a single file order. In other implementations, inertial pumps may be provided for assisting with the movement of cells  335 - 1  through transfection region  338 - 1  to ejector  342 - 1 . 
       FIG.  6    schematically illustrates portions of an example cell transfection apparatus  520 .  FIG.  6    illustrates an example of how a microfluidic chip may carry out the sorting of cells using dial electrophoretic cell sorting prior to transfection and dispensing by the same microfluidic chip. Cell transfection apparatus  420  is similar to cell transfection apparatus  320  described above except that cell transfection apparatus  420  comprises a microfluidic chip  522  comprising cell sorter  534  in place of cell sorter  334 . Those remaining components of cell transfection apparatus  520  which correspond to components of cell transfection apparatus  320  are numbered similarly. 
     Cell sorter  534  comprises three electrodes forming a separation or sorting region. Electrode  550 - 1  comprises an electrode connected to a ground  358 . Electrode  550 - 2  comprises an electrode connected to a positive voltage source. Electrode  550 - 3  comprise an electrode connected to a negative voltage source. Electrodes  550 - 3  and  550 - 2  cooperate to form a first electric field across fluid passage  361 - 1  which leads to transfection region  338 - 1 . Electrodes  550 - 1  and  550 - 2  cooperate to form a second electric field across fluid passage  361 - 2  which leads to transfection region  338 - 2 . The different electric fields in passages  361 - 1  and  361 - 2  exert different dielectrophoretic forces upon the different cells  335 - 1  and  335 - 2  such that cells  335 - 1  are drawn and directed along passage  361 - 1  while cells  335 - 2  are drawn and directed along passage  361 - 2 . In one implementation, cells  335 - 1  may comprise naïve T cells which are being separated are sorted from active T cells  335 - 2 , the naïve T cells being targeted for transfection in region  338 - 1 . In one example of such an implementation, the electric field applied across passages  361 - 1  and  361 - 2  is in the range of 10V/mm to 200V/mm. In one example, the fields is 80V/mm. In other implementations, the fields may differ and may have other values depending on the flow rate and the separator geometry. When apparatus  520  is used for sorting other types of cells, other electrical fields may be applied across or within passages  361 . 
     As discussed above, once the cells have been transfected, the cells are dispensed by their respective fluid ejector  342 - 1  and  342 - 2 . The dispensing of fluid creates a negative fluid pressure so as to draw cells towards fluid ejectors. In some implementations, inertial pumps may be provided on microfluidic chip  522  to assist in the movement of cells  335 - 1  and  335 - 2  out of the sorting region and through the transfection region  338  towards the fluid ejectors  342 . 
       FIG.  7    is a schematic diagram illustrating portions of an example cell transfection apparatus  620 .  FIG.  7    illustrates an example of how a microfluidic chip may carry out the sorting of cells using acoustics prior to transfection and dispensing by the same microfluidic chip. Cell transfection apparatus  620  is similar to cell transfection apparatus  320  described above except that cell transfection apparatus  620  comprises a microfluidic chip  622  comprising cell sorter  534  in place of cell sorter  334 . Those remaining components of cell transfection apparatus  520  which correspond to components of cell transfection apparatus  320  are numbered similarly. 
     Cell sorter  534  comprises an acoustic cell sorter that separates targeted cells from non-targeted cells or other particles using acoustics. Cell sorter  534  comprises piezo acoustic element  650 , discharge passages  652  and fluid ejectors  654 . Piezo acoustic element  650  surrounds channel or passage  352  and applies a standing surface acoustic wave to passage  352 . Cells in the continuous laminar flow through passage  352  may be separated based upon their volume, density and compressibility. Different acoustic forces result in different displacements of cells, repositioning larger cells closer to the center of passage  352  and smaller cells farther from the center. 
     In the example illustrated, the cells being targeted for transfection, cells  335 - 1 , are larger relative to the non-target cells  335 - 2 . For example, in implementations where cell transfection apparatus  620  is to be used for CAR T therapy, naïve T cells may be different in size than active T cells. Given this relationship between cells  335 - 1  and  335 - 2 , transfection region  338 - 1  and fluid ejector  342 - 1  are generally centered in alignment with the center of passage  352  so as to receive the target cell  335 - 1  flowing along the center of passage  352 . Discharge passages  652  and fluid ejectors  654  are located along the outer periphery of passage  352  to receive the non-target cells  335 - 2 . Fluid ejectors  654  are each similar to fluid ejector  342 - 1  in that each of fluid ejectors  654  comprises an ejection chamber  364 - 1 , an ejection orifice  366 - 1  and a fluid actuator  368 - 1 , each of which has been described above. In some implementations, fluid ejectors  654  may be omitted, where reservoirs for collecting and containing the non-target cell  335 - 2  are provided on microfluidic chip  622 . 
     As should be appreciated, in implementations where the cells being targeted for transfection are smaller than the non-targeted cells, transfection region  33 - 1  and its associated fluid ejector  342 - 1  may be located along the outer internal periphery of passage  352  to receive the smaller target cells, whereas discharge passage  652  and its associated fluid ejector  654  may be generally centered in alignment with the center of passage  352  to receive the non-target cells. In some implementations, piezo acoustic element  650  may continuously surround passage  352 . In other implementations, piezo acoustic element  650  may intermittently extend about passage  352 . 
       FIG.  8    is a schematic diagram illustrating portions of an example cell transfection apparatus  720 .  FIG.  8    illustrates an example of how a microfluidic chip may additionally include inertial pumps to move target cells from input port  330 , across transfection region  338 - 1  to fluid ejector  342 - 1 . Cell transfection apparatus  720  is similar to cell transfection apparatus  420  described above except that cell transfection apparatus  620  comprises a microfluidic chip  722  additionally comprising inertial pump  750 . Those remaining components of cell transfection apparatus  720  which correspond to components of cell transfection apparatus  420  are numbered similarly. 
     Inertial pump  750  is located within passage  352  and sizes to move cells along passage  352  towards transfection region  338 - 1 . In the example illustrated in which non-target cells are sorted from cells targeted for transfection using affinity (such as anti-bodies  451  on pillars  450 ), inertial pump  750  is sized and located such that the displacement forces created by inertial pump  750  are insufficient to detach or separate non-targeted cells from pillars  450 . In the example illustrated, inertial pump  750  comprises fluid actuators  752  supported by microfluidic chip  722 . In some implementations, fluid actuators  752  comprise thermoresistive elements, such as thermal resistive electrodes, formed along the interior surface of passage  352  so as to move fluid in the direction indicated by arrow  753 . 
     Inertial pump  750  may likewise be used in any of the above described cell transfection apparatus  20 ,  320 ,  420 ,  520  and  620 . For example, inertial pump  750  may be located between input port  330  and the particular cell sorter of such apparatus. In some implementations, inertial pump  750  may be located between the cell sorter and the transfection region to pull or draw cells across the cell sorter. In some implementations, the inertial pump  750  may be located so as to also pump or drive cells that have been sorted through and across the transfection region. 
       FIG.  9    is a schematic diagram illustrating portions of an example cell transfection apparatus  820 .  FIG.  9    illustrates an example of how cell propagation may be additionally provided on the same microfluidic chip that carries out cell sorting and cell transfection. Cell transfection apparatus  820  is similar to cell transfection apparatus  720  described above except that cell transfection apparatus  820  comprises a microfluidic chip  822  additionally comprising cell propagation region  860 . The remaining components of cell transfection apparatus  820  which correspond to components of cell transfection apparatus  720  are numbered similarly. 
     Cell propagation region  860  is sandwiched between cell transfection region  338 - 1  and fluid ejector  342 - 1  on microfluidic chip  822 . Cell propagation region  860  comprises cell propagation chamber  862 , reagent supplies  864 - 1 ,  864 - 2  and  864 - 3  (collectively referred to as supplies  864 ), and lift off reagent supply  871 . Cell propagation chamber  862  receives biological cells  351 - 1  that have been transfected by transfection region  338 - 1  and contains such cells during their propagation, multiplication or growth. Cell propagation chamber  862  may contain surfaces to which the propagating cells may adhere. In some implementations, cell propagation chamber  862  may comprise small openings connecting the interior of chamber  862  to atmosphere or to gas sources to assist with gas exchange. 
     Reagent supplies  864  supply reagents such as cell growth media, to chamber  862  to enhance or facilitate the propagation of the transfected cells  351 - 1  within chamber  862 . Each of reagent supplies  864  comprises a reagent source  866  connected to chamber  862  by reagent supply passage  868  that contains an inertial pump  870  to controllably move fluid containing the particular reagent from the reagent source  866  to chamber  862 . Reagent source  866  may comprise a reservoir containing a reagent or may comprise a port for connection to an external reagent source. Examples of reagents include, but are not limited to, hi-glucose Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), Roswell Park Memorial Institute (RPMI) culture medium, fetal bovine serum (FBS), non-essential amino acids, RPMI, trace amounts beta-mercaptoethanol, and assorted antibiotics including penicillin and streptomycin. 
     Lift off supply  871  supplies a lift off reagent to the interior of chamber  862  to assist in the lift off or detachment of the propagated cells  351 - 1  from the propagation surfaces within chamber  862 . Lift off supply  871  comprises lift off reagent source  876  connected to chamber  862  by lift off supply passage  878  which contains an inertial pump  880  to controllably move fluid containing the lift off reagent from the lift off reagent source  876  to chamber  862 . Lift off reagent source  876  may comprise a reservoir containing a lift off reagent or may comprise a port for connection to an external lift off reagent source. 
     As shown by broken lines, in some implementations, microfluidic chip  822  may additionally support a sensor  884 . Sensor  884  extends along chamber  862  to sense the interior of chamber  862 , facilitating a determination of a state of cell propagation within chamber  862 . In some implementations, sensor  884  may comprise an illumination source and a sensor array, such as a liquid crystal display (LCD) sensor array to capture images of the propagating cells  351 - 1  within chamber  862 . In some implementations, sensor  84  may comprise a spectrometer to capture spectrographic information regarding the state of the propagation within chamber  862 . In yet other implementations, sensor  84  may be omitted. 
     Each of the transfection apparatus shown in  FIGS.  4 - 9    may additionally comprise controller  260  (shown in  FIG.  3   ). Controller  260  may be supported by the microfluidic chip that carries out cell sorting, and transfection or may be separate or remote from the microfluidic chip, wherein controller  260  communicates with components of the microfluidic chip in a wired or wireless fashion. Controller  260  may output control signals controlling the fluid ejector(s), the inertial pumps and the supply of power to the electrodes carrying out transfection. Controller  260  may receive signals from sensor  884  or other sensitive determine a state of propagation, wherein controller  260  may control the fluid ejector&#39;s, the inertial pumps in the supply of power to the electrodes carry out transfection additionally based upon information obtained from sensor  884 . Controller  260  may facilitate an automated process for sorting, transfected (potentially propagating) and dispensing biological cells to a target site, such as individual wells of a well plate. Each of such cell transfection apparatus described in  FIGS.  4 - 9    may be utilized as part of cell propagation system  200  (shown and described with respect to  FIG.  3   ), in place of cell transfection apparatus  20 . Each of such cell transfection apparatus  320 ,  420 ,  520 ,  620 ,  720  and  820  may be contained within an incubator  290 , wherein transfected cells are ejected into multi-well plate  250  which is moved by stage  252 . In some implementations, the wells deposited within the multi-well plate  250  may be once again imaged by imager  280  and or further propagated in the multi-well plate  250  through the use of media exchange system  254 . 
     Although the present disclosure has been described with reference to example implementations, workers skilled in the art will recognize that changes may be made in form and detail without departing from the disclosure. For example, although different example implementations may have been described as including features providing various benefits, it is contemplated that the described features may be interchanged with one another or alternatively be combined with one another in the described example implementations or in other alternative implementations. Because the technology of the present disclosure is relatively complex, not all changes in the technology are foreseeable. The present disclosure described with reference to the example implementations and set forth in the following claims is manifestly intended to be as broad as possible. For example, unless specifically otherwise noted, the claims reciting a single particular element also encompass a plurality of such particular elements. The terms “first”, “second”, “third” and so on in the claims merely distinguish different elements and, unless otherwise stated, are not to be specifically associated with a particular order or particular numbering of elements in the disclosure.