Cell engineering platform

Abstract

The current subject matter provides a cell engineering platform of a diagnostic and clinical use scale for vector-free and/or viral delivery of payload/cargo compounds and compositions into non-adherent cells. The platform achieves delivery to a large number of cells quickly in a closed system. Related apparatus, systems, techniques, articles and compositions are also described.

1 . A method comprising:
 filling a chamber of a cell engineering platform with a mixture of cells and a first medium; and   discharging the first medium from the chamber through a filter, leaving the cells deposited on the filter,   wherein the cell engineering platform includes:
 a chamber; 
 a lid disposed at a first end of the chamber; 
 a base disposed at a second end of the chamber; and 
 a filter holder disposed within the chamber. 
   
     
     
         2 . The method of  claim 1 , further comprising spraying a delivery solution that contains a payload to the cells deposited on the filter. 
     
     
         3 . The method of  claim 2 , further comprising applying a stop solution in the chamber. 
     
     
         4 . The method of  claim 1 , further comprising filling the chamber with a second medium to resuspend the cells from the filter. 
     
     
         5 . The method of  claim 4 , wherein the discharged first medium is reused as the second medium. 
     
     
         6 . The method of  claim 4 , further comprising agitating the chamber. 
     
     
         7 . The method of  claim 4 , further comprising extracting the resuspended cells from the chamber. 
     
     
         8 . The method of  claim 4 , wherein the filling the chamber is performed automatically with a pump and a controller. 
     
     
         9 . The method of  claim 1 , further comprising culturing the cells within the chamber. 
     
     
         10 . The method of  claim 1 , wherein the discharging the first medium from the chamber is performed by supplying a positive pressure into the chamber 
     
     
         11 . The method of  claim 1 , wherein the discharging the first medium from the chamber is performed by gravity. 
     
     
         12 . The method of  claim 3 , wherein the applying the stop solution is performed to wash the cells. 
     
     
         13 . The method of  claim 4 , wherein the filling the chamber with the second medium is performed as at least one of a cell wash process, a cell concentration change process, and a cell medium change process. 
     
     
         14 . A system comprising:
 a housing configured to receive a filter plate comprising a well;   a differential pressure applicator configured to apply a differential pressure to the well;   a delivery solution applicator configured to deliver atomized delivery solution to the well;   and   a culture medium applicator configured to deliver a culture medium to the well; wherein the housing comprises:
 a chamber; 
 a lid disposed at a first end of the chamber; and 
 a base disposed at a second end of the chamber, 
 wherein the filter plate is disposed within the chamber. 
   
     
     
         15 . (canceled) 
     
     
         16 . The system of  claim 14 , wherein the differential pressure applicator is connected to the lid of the housing and includes a shower head, which includes a plurality of apertures at an end portion thereof. 
     
     
         17 . The system of  claim 14 , further comprising a stop solution applicator configured to deliver a stop solution to the well, wherein the stop solution applicator delivers the stop solution to the well through a septum provided in the lid of the housing. 
     
     
         18 . The system of  claim 14 , wherein the culture medium applicator delivers the culture medium to the well through a septum provided in the lid of the housing or through a port of the chamber. 
     
     
         19 . The system of  claim 14 , wherein the housing is configured to tilt the filter plate. 
     
     
         20 . The system of  claim 14 , wherein the delivery solution applicator includes a robotic arm and spray head, the robotic arm magnetically coupled to the spray head. 
     
     
         21 . The system of  claim 20 , further comprising a soft elastomeric barrier enclosing the filter plate and spray head, wherein the soft elastomeric barrier separates the robotic arm and the spray head. 
     
     
         22 . The system of  claim 20 , wherein the robotic arm is configured to carry the spray head to a plurality of locations on the filter plate. 
     
     
         23 . The system of  claim 14 , wherein the stop solution applicator and the culture medium applicator are formed integral within the housing as ports, and wherein the housing forms a vessel. 
     
     
         24 . The system of  claim 23 , wherein the vessel includes a waste media outlet for collecting waste media. 
     
     
         25 . The system of  claim 14 , further comprising a soft elastomeric barrier enclosing the filter plate and delivery solution applicator within the housing, the soft elastomeric barrier and the housing forming a bioreactor. 
     
     
         26 . The system of  claim 14 , wherein the housing includes a filter plate base configured to tilt, rotate, and/or vibrate the filter plate. 
     
     
         27 . The system of  claim 14 , wherein the delivery solution applicator includes a spray head, and the spray head is a single-use cartridge. 
     
     
         28 . The system of  claim 14 , wherein the filter plate is sized to hold greater than 1×10 7  T cells. 
     
     
         29 . The system of  claim 14 , wherein the system is configured to automatically:
 provide to the filter plate cells in media;   remove culture media to form a cellular monolayer on top of the filter plate;   apply an atomized delivery solution to the cellular monolayer;   incubate the cells;   and   provide new media to the cellular monolayer.   
     
     
         30 . The system of  claim 29 , wherein the system is configured to automatically tilt, vibrate, and/or rotate the filter plate to re-suspend the cells in the new media. 
     
     
         31 . The system of  claim 29 , wherein the system is configured to repeat the application of the atomized delivery solution, the incubation, and the application of the stop solution. 
     
     
         32 . The system of  claim 14 , wherein the delivery solution applicator includes a nebulizer. 
     
     
         33 . The system of  claim 32 , wherein the delivery solution applicator further includes a mass flow controller or a volumetric flow controller to regulate a gas flow to operate the nebulizer. 
     
     
         34 . The system of  claim 14 , wherein the delivery solution applicator is configured to deliver 10-300 micro liters of the delivery solution per actuation. 
     
     
         35 . The system of  claim 14 , further comprising a temperature control system configured to control a temperature of the delivery solution and/or of the plate comprising the well. 
     
     
         36 . The system of  claim 14 , wherein the delivery solution includes an aqueous solution, the aqueous solution including the payload. 
     
     
         37 .- 40 . (canceled) 
     
     
         41 . The system of  claim 36 , wherein said aqueous solution comprises between 12.5-500 mM KCl. 
     
     
         42 . The system of  claim 36 , wherein said aqueous solution comprises 106 mM KCl. 
     
     
         43 . The system of  claim 14 , further comprising:
 the filter plate, wherein the well is configured to contain a population of non-adherent cells.   
     
     
         44 . The system of  claim 43 , wherein said non-adherent cell comprises a peripheral blood mononuclear cell. 
     
     
         45 . The system of  claim 43 , wherein said non-adherent cell comprises an immune cell. 
     
     
         46 . The system of  claim 43 , wherein said non-adherent cell comprises a T lymphocyte 
     
     
         47 . The system of  claim 36 , wherein said payload comprises a messenger ribonucleic acid (mRNA). 
     
     
         48 . The system of  claim 47 , wherein said mRNA encodes a gene-editing composition. 
     
     
         49 . The system of  claim 48 , wherein said gene editing composition reduces the expression of PD-1. 
     
     
         50 . The system of  claim 47 , wherein said mRNA encodes a chimeric antigen receptor. 
     
     
         51 . The system of  claim 14  for use to deliver a cargo compound or composition to a mammalian cell. 
     
     
         52 . The system of  claim 43 , wherein said population of non-adherent cells comprises a monolayer. 
     
     
         53 . A system comprising:
 a chamber;   a lid disposed at a first end of the chamber;   a base disposed at a second end of the chamber; and   a filter holder disposed within the chamber.   
     
     
         54 . The system of  claim 53 , wherein the filter holder includes a plurality of apertures arranged in a predetermined pattern. 
     
     
         55 . The system of  claim 54 , wherein the filter holder includes a plurality of targets within which the plurality of apertures are arranged to allow cells to be deposited on a filter at areas corresponding to the plurality of apertures. 
     
     
         56 . The system of  claim 53 , further comprising a gasket disposed between the filter holder and the base. 
     
     
         57 . The system of  claim 55 , further comprising a filter holder insert received within the filter holder to accommodate the filter between the filter holder and the filter holder insert. 
     
     
         58 . The system of  claim 57 , wherein the filter holder insert includes a concavely shaped top surface. 
     
     
         59 . The system of  claim 57 , wherein the filter holder insert includes a plurality of openings corresponding to the plurality of targets. 
     
     
         60 . The system of  claim 59 , wherein the filter holder includes three targets. 
     
     
         61 . The system of  claim 59 , wherein the filter holder includes seven targets. 
     
     
         62 . The system of  claim 59 , wherein the filter holder insert includes nineteen targets. 
     
     
         63 . The system of  claim 57 , wherein the plurality of targets are arranged in a pattern selected from a group consisting of a square pattern, a rectangular pattern, a triangular patter, and a linear pattern. 
     
     
         64 . The system of  claim 53 , further comprising a controller configured to operate at least one of a pump, a valve, a heating element, a cooling element, and an agitation device. 
     
     
         65 . The system of  claim 64 , wherein the pump is a peristaltic pump or a positive displacement pump. 
     
     
         66 . The system of  claim 53 , wherein the lid further comprises a pressure port and an aperture. 
     
     
         67 . The system of  claim 53 , wherein the base includes a port for receiving media or for draining the media. 
     
     
         68 . The system of  claim 66 , wherein the lid further comprises a septum. 
     
     
         69 . The system of  claim 68 , wherein the pressure port includes a shower head, which includes a plurality of apertures at an end portion thereof. 
     
     
         70 . The system of  claim 66 , wherein a 0.2 micron filter is connected to the pressure port. 
     
     
         71 . The system of  claim 70 , further comprising a pinch valve that is configured to open and close the pressure port to atmosphere through the 0.2 micron filter. 
     
     
         72 . The system of  claim 53 , wherein the chamber includes a port for extracting processed cells. 
     
     
         73 . The system of  claim 53 , wherein the system is sized to process greater than 1×10 9  T cells. 
     
     
         74 . (canceled)