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MaxCyte Platform > Technology > Cell Cryopreservation

MaxCyte electroporation can transfect billions of cells in a single 30 minute large scale run. Transfected cells can be used immediately following electroporation in a wide range of high throughput assays, protein production or clinical cell therapy applications. If more suitable to scheduling, transfected cells can also be aliquoted and cryopreserved for future use. MaxCyte has developed several cryopreservation protocols that enable cell archiving while maximizing cell viability and target expression upon thawing for a variety of cell types.

Cells can be cryopreserved without a loss in transgene expression or viability

The ability to cryopreserve cells following electroporation greatly increases the utility of transiently transfected cells. Cells transfected via MaxCyte electroporation can be cryopreserved and stored post transfection with minimal effects on cell viability, or transgene expression.

To examine the effects of cell cryopreservation cells were transfected with a GFP expression plasmid via MaxCyte electroporation, divided into two groups, and analyzed immediately post electroporation or following cryopreservation. GFP expression was concentration-dependent in both cell populations and only slightly decreased following cryopreservation (Figure 1). Results from the control cell populations (non-electroporated cells and cells electroporated, but without DNA) show no evidence of decreases in viability illustrating that electroporation itself does not have a negative impact on cell viability.

Cryopreservation Effects on Transfection Efficiency and Cell Viability Cryopreservation Effects on Transfection Efficiency and Cell Viability
Figure 1. Cryopreservation Effects on Transfection Efficiency and Cell Viability. HEK 293T cells were transfected in OC-400 (small scale) processing assemblies with pGFP and divided into two groups: 1) plated immediately for FACS analysis, or 2) cryopreserved and analyzed post thaw. GFP expression was analyzed by FACS.

Cryopreservation does not affect transgene expression

Experiments were conducted to examine the effects of cryopreservation on expression of a transgene, as well as cell performance in downstream assays. Cells were transfected using the MaxCyte STX with an NFκB reporter plasmid, split in two populations and processed either with or without cryopreservation. Cells from both populations were treated with TNFα and luciferase was measured as a read-out of NFκB activation. Assay sensitivity was dependent on the concentration of the NFκB reporter plasmid used during transfection (Figure 2). Cryopreserved cells showed comparable concentration-dependent responses to TNFα as freshly transfected cells, indicating that cryopreservation did not affect transgene expression.

Cryopreservation Effects on NFκB Activation Assay Cryopreservation Effects on NFκB Activation Assay
Figure 2. Cryopreservation Effects on NFκB Activation Assay. HEK 293T cells were transfected in OC-400 (small scale) processing assemblies with an NFκB reporter plasmid and divided into two groups: 1) plated immediately for use in assay, or 2) cryopreserved and assayed post thaw. For the NFκB assays, cells were seeded in 96 well plates (50k cells/well) and treated with TNFα 4 hours post plating. Cells were incubated overnight and luciferase activity measured.

Optimization of cryopreservation protocols

MaxCyte scientists have examined the effects of culturing cells for various lengths of time prior to cryopreservation on transfection efficiency and levels of cell viability. A variety of cell types including HEK, CHO and Jurkat cells, have been tested and while specific cell types may have slightly increased viability and transfection efficiency using specific conditions, overall cell health and transgene expression are not significantly affected. This provides an additional level of flexibility in cell handling post transfection and scheduling of downstream cell applications.

The studies in the figures below evaluate the affects of cell incubation post electroporation on transgene expression and general cell health. U2OS and SH-SY5Y cells were transfected using small scale electroporation with a GFP expression plasmid, split it 3 populations and incubated for various lengths of time prior to cryopreserving the cells. The U2OS study showed that the fresh versus frozen cell populations had very minor differences. Additionally, the results showed that both populations of cryopreserved cells proliferated and exhibitied strong GFP expression post thawing.

U2OS cell cryopreservation
Figure 3 U2OS cell cryopreservation. U2OS cells were electroporated with 200μg/mL of pGFP and divided into 3 groups. 1/3 of the cells were analyzed immediately after a 20 minute recovery period. 1/3 of the cells were cryopreserved following the 20 minute recovery period. The remaining 1/3 was plated and incubated overnight prior to cryopreservation. Cells were frozen in a cryopreservation medium containing 90% serum/10%DMSO.

Similar experiments with SH-SY5Y cells showed that all three populations of cryopreserved cells proliferated and exhibited strong GFP expression post thawing. Cell handling conditions prior to cryopreservation did have an impact on the viability of thawed cells. Cell incubated for an hour or overnight post transfection had increased viability compared to immediate freezing. Incubating in medium for 1 hour often provides a good compromise between immediate cryopreservation and overnight culture prior to freezing if time is constrained.
SH-SY5Y cell cryopreservation
Figure 4 SH-SY5Y cell cryopreservation. SH-SY5Y cells were electroporated with 100μg/mL of pGFP and divided into 3 groups. 1/3 of the cells were frozen immediately after a 20 minute recovery period, 1/3 of the cells were frozen following a 1 hour incubation in complete medium. The remaining 1/3 was plated and incubated overnight, harvested via trypsinization and frozen. All three cell populations were frozen in a cryopreservation medium containing 90% serum/10%DMSO.

Cryopreservation does not significantly impact performance in downstream receptor assays

Receptors, including GPCRs, ion channels, and nuclear receptors are common targets of HTS and HCS campaigns. Flexibility in assay scheduling, data quality and reproducibility, and assay scalability are important criteria when deciding on technologies to be used for screening and profiling assays. MaxCyte electroporation fulfills these needs by reproducibly transfecting a large number of cells which can be immediately used or cryopreserved. The data in this section directly assess the effects of cryopreservation on the performance of transfected cells in common downstream receptor assays using side by side comparisons. These results highlight the universal ability to cryopreserve electroporated cells without sacrificing assay performance.

GPCR Assay
Cells were transfected in two separate runs using small scale electroporation or a single run using large scale electroporation. Following transfection half of each cell population was cryopreserved prior to ligand stimulation and cAMP measurement. Similar ligand responses were seen for both fresh and frozen cells. Additionally, both small scale electroporation experiments produced similar results demonstrating the reproducibility of the MaxCyte system. The size of the electroporation run did not have an effect on the results illustrating the seamless scalability of MaxCyte electroporation.

Cell Cryopreservation
Figure 2. cAMP GPCR Assay: Fresh vs. Frozen Cell Assay Performance. HEK 293F suspension cells were transfected with a GPCR expression plasmid (100µg/mL) using small scale MaxCyte electroporation. Half of the transfected cells were cryopreserved 18 hours post transfection. GPCR activity was assayed by cAMP ELISA at 18 hrs post electroporation (fresh cells) or at 1 hour post thawing (frozen cells).
Ion Channel Assay
To assess the effects of cell cryopreservation and archiving on the performance of transfected cells in downstream ion channel assays, CHO cells were transfected with a Kv1.5 expression plasmid in bulk using MaxCyte flow electroporation and cryopreserved 48 hours post transfection. Transfected cells were thawed approximately one or five months after cryopreservation and assayed using the IonWorks Quattro. Cells cryopreserved for either 1 or 5 months demonstrated no significant differences in seal resistance, expression levels or current amplitudes. Cryopreserved cells also exhibited seal resistances, expression levels and current amplitudes comparable to those of freshly transfected cells (data not shown).
Table 1. Large Scale Transfection and Cryopreservation of CHO Cells for Ion Channel Assays
Table 1. Large Scale Transfection and Cryopreservation of CHO Cells for Ion Channel Assays. 8x108 CHO K1 cells were transfected with 150 µg/mL of Kv1.5 α-subunit plasmid DNA via flow electroporation. Transfected cells were cultured @ 37°C for 24 hours, transferred to a 28°C incubator for an additional 24 hours, then suspended in freezing medium and cryopreserved in liquid nitrogen using standard methodology. Thawed cells were assayed in single hole (SH) and population patch clamp (PPC) modes on the IonWorks Quattro. Data courtesy of BioFocus.
Nuclear Receptor Assay
The data in Figure 3 summarize the results from a luciferase reporter gene assay that measured nuclear receptor activity in freshly transfected versus cryopreserved cells. Calculated IC50 values for a reference nuclear receptor inhibitor were comparable for both cell populations. These data demonstrate that downstream assay performance is not impacted by cell cryopreservation.
Figure 3. Nuclear Receptor Assay in Cryopreserved Jurkat Cells
Figure 3. Nuclear Receptor Assay in Cryopreserved Jurkat Cells. Jurkat cells were co-transfected via small scale electroporation with a reporter plasmid in which luciferase was expressed from a minimal promoter containing multiple GAL4 UAS sequences and with an activator plasmid encoding a constitutively expressed fusion protein in which a GAL4 DNA binding domain was linked to a nuclear receptor ligand binding domain. Cells transfected with 200 µg/mL of the plasmid mixture were plated in 384-well plates immediately after electroporation (fresh cells) or cryopreserved after electroporation and plated after thawing (frozen cells). Cells were treated with varying concentrations of inhibitor immediately after plating, and luciferase activity measured following a 5 hour incubation.