Multi-pulse transfection methods and cells
Systems and methods are provided for transfecting immune competent cells with RNA at high efficiency and viability.
1 . A method of transfecting mammalian cells with a RNA, comprising:
disposing the mammalian cells with the RNA in a transfection medium, wherein the mammalian cells are NK cells;
applying two, three, or four pulses to the mammalian cells at a field strength of 600-1,400 V/cm and a time constant from about 0.5 msec to about 10 msec to produce transfected mammalian cells.
2 . The method of claim 1 , wherein the transfection medium is an isotonic medium and a growth medium or a high conductance medium.
3 . The method of claim 1 , wherein a time between each pulse is between 1-15 seconds.
4 . The method of claim 3 , wherein the time between each pulse is between 2-10 seconds.
5 . The method of claim 1 , wherein the field strength is applied at a gap width of about 0.1 cm to about 0.4 cm.
6 . The method of claim 1 , wherein the time constant is about 0.5 msec to about 5 msec.
7 . The method of claim 1 , wherein the time constant is about 1 msec to about 3 msec.
8 . The method of claim 1 , wherein the field strength is applied at a voltage of between 150-250 V.
9 . The method of claim 1 , wherein the field strength is 800-1200 V/cm.
10 . The method of claim 1 , wherein the pulses are delivered from a capacitor having a capacitance of between 5-50 μF.
11 . The method of claim 1 , wherein the pulses are delivered from a capacitor having a capacitance of between 10-25 μF.
12 . The method of claim 1 , further comprising a step of culturing the transfected mammalian cells after pulsing.
13 . The method of claim 1 , wherein the transfected mammalian cells have a viability of at least 70%.
14 . The method of claim 1 , wherein the pulses provide a transfection efficiency of at least 80%.
15 . The method of claim 1 , wherein an injected charge is between 1 mC and 30 mC.
16 . A method of transfecting mammalian cells with a RNA, comprising:
disposing the mammalian cells with the RNA in a transfection medium, wherein the mammalian cells are NK cells; and
applying two, three, or four pulses to the mammalian cells at a voltage of 150-250 V, a gap width of about 0.1 cm to about 0.4 cm, and a time constant from about 0.5 msec to about 10 msec to produce transfected mammalian cells, wherein the pulses are delivered from a capacitor having a capacitance of between 5-50 μF.
17 . A method of transfecting mammalian cells with a RNA, comprising:
disposing the mammalian cells with the RNA in a transfection medium, wherein the mammalian cells are NK cells; and
applying two, three, or four pulses to the mammalian cells at a field strength of 600-1,400 V/cm and a time constant from about 0.5 msec to about 10 msec to produce transfected mammalian cells, wherein the pulses are delivered from a capacitor having a capacitance of between 5-50 μF.