Highly efficient, large volume flow electroporation.
Electroporation is widely used to transfect and load cells with various molecules. Traditional electroporation using a static mode is typically restricted to volumes less than 1 mL, which limits its use in clinical and industrial bioprocessing applications. Here we report efficient, large volume transfection results by using a scalable-volume electroporation system. Suspended (Jurkat) and adherent cells (10T1/2 and Huh-7) were tested. A large macromolecule, FITC-conjugated dextran (MW=500 kD) was used to measure cell uptake, while a plasmid carrying the gene coding for enhanced green fluorescence protein (eGFP) was used to quantitate the flow electrotransfection efficiency as determined by flow cytometry. The flow electroloading efficiency of FITC-dextran was >90%, while the cell viability was highly maintained (>90%). High flow electrotransfection efficiency (up to 75%) and cell viability (up to 90%) were obtained with processing volumes ranging from 1.5 to 50 mL. No significant difference of electrotransfection efficiency was observed between flow and static electrotransfection. When 50 mL of cell volume was processed and samples collected at different time points during electroporation, the transgene expression and cell viability results were identical. We also demonstrated that DNA plasmid containing EBNA1-OriP elements from Epstein-Barr virus were more efficient in transgene expression than standard plasmid without the elements (at least 500 too 1000-fold increase in expression level). Finally, to examine the feasibility of utilizing flow electrotransfected cells as a gene delivery vehicle, 10T1/2 cells were transfected with a DNA plasmid containing the gene coding for mIL12. mIL12 transfected cells were injected subcutaneously into mice, and produced functional mIL12, as demonstrated by anti-angiogenic activity. This is the first demonstration of efficient, large volume, flow electroporation and the in vivo efficacy of flow electrotransfected cells. This technology may be useful for clinical gene therapy and large-scale bioprocesses.
Li
Lin-Hong
LH
MaxCyte, Inc., 9640 Medical Center Drive, Rockville, MD 20850, USA.
Shivakumar
Rama
R
Feller
Stephanie
S
Allen
Cornell
C
Weiss
Jonathan M
JM
Dzekunov
Sergey
S
Singh
Vin
V
Holaday
John
J
Fratantoni
Joseph
J
Liu
Linda N
LN
D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D018274:Electroporation / Q000379:methods*; D005434:Flow Cytometry; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D009389:Neovascularization, Pathologic; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
D004396:Coloring Agents