3.2 Cell and Cargo
Loading
1. The cells are loaded on top of the 3D NEP Chip (Fig. 1a).
2. A vacuum is applied during cell loading to enhance the contact
of cells to the nanopores (see Note 1).
3. The living cells adhere to the chip surface 4 h after seeding.
4. The solution containing the cargo to be delivered is loaded on
the opposite side within the confines of the PDMS reservoir.
3.3 System Setup
1. All components for the system setup are sterilized with a 70%
alcohol solution and UV-light.
2. The 3D NEP platform is subsequently assembled by compartmentalizing each array of nanochannels with a ~2 mm thick
polydimethylsiloxane (PDMS) stencil on both sides, as well as
interfacing with the proper electrode system required for applying the nanoelectroporating system as shown in Fig. 1b.
3. The cells are electroporated with the optimized setting (see
Note 2).
4. After electroporation, the chip is transferred in a 6-well plate.
5. The cells are then trypsinized to release them from the surface
of chip and adequate amount of cell media is added.
6. The chip surface is gently washed to ensure most of the cells are
released from the surface.
7. The 6-well plate with the cells is left in an incubator (37
C, 5%
CO 2 ) for further processes.
3.4 Adoptive
Immunotherapy
1. CAR plasmid is injected into NK-92 cells with the NEP conditions of a single square wave pulse with 100 V amplitude and
a 20 ms duration (see Note 3).
2. Characterization of CAR plasmid 10 h post transfection in
terms of GFP reporter gene activity is carried out (Fig. 2a).
3. Nikon Ti fluorescence microscope is used for measuring the
fluorescence intensities from cells.
4. Transfection efficiency is determined by enumerating the cells
showing positive GFP fluorescence among all cells.
5. Transfection uniformities are calculated in terms of relative
GFP fluorescence intensities.
3.5 Cell
Reprogramming
1. OSKM factors are pCAG plasmid backbone encoded with
(OCT-4, SOX2, KCL-1, and c-MYC transcription factors,
13 kbp) labeled with YOYO-1 dye.
2. Primary cells (mouse embryonic fibroblasts) are loaded on the
3D NEP platform and cultured for overnight.
3. OSKM factors are prepared in PBS solutions and are loaded in
the bottom chamber of the 3D NEP platform.
3D Nanochannel Electroporation for Macromolecular Nucleotide Delivery
73
Loading
1. The cells are loaded on top of the 3D NEP Chip (Fig. 1a).
2. A vacuum is applied during cell loading to enhance the contact
of cells to the nanopores (see Note 1).
3. The living cells adhere to the chip surface 4 h after seeding.
4. The solution containing the cargo to be delivered is loaded on
the opposite side within the confines of the PDMS reservoir.
3.3 System Setup
1. All components for the system setup are sterilized with a 70%
alcohol solution and UV-light.
2. The 3D NEP platform is subsequently assembled by compartmentalizing each array of nanochannels with a ~2 mm thick
polydimethylsiloxane (PDMS) stencil on both sides, as well as
interfacing with the proper electrode system required for applying the nanoelectroporating system as shown in Fig. 1b.
3. The cells are electroporated with the optimized setting (see
Note 2).
4. After electroporation, the chip is transferred in a 6-well plate.
5. The cells are then trypsinized to release them from the surface
of chip and adequate amount of cell media is added.
6. The chip surface is gently washed to ensure most of the cells are
released from the surface.
7. The 6-well plate with the cells is left in an incubator (37
C, 5%
CO 2 ) for further processes.
3.4 Adoptive
Immunotherapy
1. CAR plasmid is injected into NK-92 cells with the NEP conditions of a single square wave pulse with 100 V amplitude and
a 20 ms duration (see Note 3).
2. Characterization of CAR plasmid 10 h post transfection in
terms of GFP reporter gene activity is carried out (Fig. 2a).
3. Nikon Ti fluorescence microscope is used for measuring the
fluorescence intensities from cells.
4. Transfection efficiency is determined by enumerating the cells
showing positive GFP fluorescence among all cells.
5. Transfection uniformities are calculated in terms of relative
GFP fluorescence intensities.
3.5 Cell
Reprogramming
1. OSKM factors are pCAG plasmid backbone encoded with
(OCT-4, SOX2, KCL-1, and c-MYC transcription factors,
13 kbp) labeled with YOYO-1 dye.
2. Primary cells (mouse embryonic fibroblasts) are loaded on the
3D NEP platform and cultured for overnight.
3. OSKM factors are prepared in PBS solutions and are loaded in
the bottom chamber of the 3D NEP platform.
3D Nanochannel Electroporation for Macromolecular Nucleotide Delivery
73