CELL ENGINEERING PLATFORM
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)