Transfection optimization for primary human CD8+ cells

Abstract

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by >95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.

Viable and efficient electroporation-based genetic manipulation of unstimulated human T cells

“…Since unstimulated cells are, on average, smaller than the activated cells, we wanted to test whether a higher voltage setting would improve the efficiency as others have noted on cell size and electroporation efficiency (Shirley et al, 2014;Gehl, 2003) . We then performed electroporation at 2200V as was suggested by Jay Levy's group for plasmid electroporation of unstimulated CD8+ T cells using the Neon electroporation machine (Liu et al, 2011) . By electroporating unstimulated cells at the 2200V setting, we achieved a relatively higher efficiency even within the naive subpopulation (overall efficiency: 54.3% versus 0.3% at 2200V versus 1600V, respectively).…”

“…Therefore, their optimal electroporation settings might be different given that smaller cells require a higher voltage (Shirley et al, 2014;Gehl, 2003) . Jay Levy's group electroporated unstimulated CD8+ T cells with plasmids and achieved 59.6% electro-transfection efficiencies with a viability of 34.6% at 2200 V 20 ms 1 pulse setting using the same electroporation device (Neon, Thermo Fisher) (Liu et al, 2011) . When we tried the same settings for electroporating unstimulated cells with our GFP plasmid, we achieved an average of %54.3 electro-transfection efficiency across 3 donors (Figure 3a, orange bars).…”

“…An earlier study using phytohemagglutinin (PHA)-activated human T lymphocytes resulted in very low transgene expression (15%) (Van Tendeloo et al, 2000) . In 2011, a broad optimization study using the Neon electroporation machine showed 59.6% efficiency and 34.6% viability in unstimulated CD8+ T cells (Liu et al, 2011) . Later, in 2013, another group showed that CD3/CD28-activated T cells were vulnerable to plasmid electroporation by nucleofection and because of this, plasmid electroporation in activated cells was not achieved (Chicaybam et al, 2013) .…”

Cancer diagnosis and immunotherapy in the age of CRISPR

“…One issue with plasmid‐based delivery of CRISPR components is the prolonged presence of SpCas9 nuclease, which may lead to increased off‐target activity, as well as the reported ability of primary T‐cells to recognize cytosolic DNA and prompt antiviral or apoptotic responses . High levels of cellular toxicity have been associated with DNA plasmid delivery to primary human T‐cells, and are probably a major contributor to the low editing efficiency observed following plasmid nucleofection. This led to the investigation of non‐DNA based systems for transient exposure of T‐cells to SpCas9, similar to what had been previously demonstrated as effective using TALEN and megaTAL nucleases.…”

“…One issue with plasmid-based delivery of CRISPR components is the prolonged presence of SpCas9 nuclease, which may lead to increased off-target activity, as well as the reported ability of primary T-cells to recognize cytosolic DNA and prompt antiviral or apoptotic responses. 62 High levels of cellular toxicity have been associated with DNA plasmid delivery to primary human T-cells, 63 Substantially greater efficiency was obtained when purified SpCas9 ribonucleoparticles (RNPs-SpCas9 complexed with a specific gRNA 7,64,65 ) were delivered to primary T cells. 66,67 Using a gRNA targeting the CXCR4 HIV coreceptor, a frequency of gene inactivation greater than 50% was observed.…”