Assay Ready Frozen Instant Cells Can Improve Bioassay Performance

introduc on In 2005, a dogma fell sta ng that cryo-preserved mammalian cells have to be pas-saged at least three to four mes before full recovery and can then be applied to a cell based assay. At the annual conference of the Society for Biomolecular Screening (SBS), data had been presented which demonstrat-ed that cryopreserved cells can be used in bioassays instantly a er thawing when they had been frozen properly (personal commu-nica on). Assay ready cells can be used in different assays, e.g. receptor ac vated sec-ond messenger assays (cAMP, IP1, Calcium flux), transporter assays, ion channel patch clamping, kinase, reporter gene and even prolifera on assays. Since this first demonstra on, the use of Frozen Instant Cells commence their trium-phant advance in cell based drug discovery. As high-throughput screening campaigns require large quan es of cells with a con-sistent performance, cell produc on always becomes a bo leneck. With the ability to

prepare assay ready cells in advance in a ho-mogenous and controlled quality, screening campaigns are now much easier to schedule and more reliable [1]. Nowadays, assay ready Frozen Instant Cells are a widely accepted tool not only in drug discovery, but also in toxicology (cytotoxicity- , phospholipidosis) and DMPK (Caco-2 perme-ability) Assays Ready Cells are also a tool for diagnos c monitoring in clinical development or lot release tests for thera-peu c proteins, an bod-ies or vaccines (receptor ac va on and cell prolif-era on, virus infec vity).

material and methods

expansion of assay cells Recombinant cell lines, or unmodified human tumor cell lines, were cul vated in their custom cell cul-ture medium with 10-20%

FBS (PAN Biotech) at 37°C with 5% CO2. Ad-herent cells were detached with Accutase (Sigma Aldrich) and cul vated in CellStacks (Corning) for further expansion (Fig. 1). Sus-pension cells were grown in HyperFlasks (Corning).

cryopreserva on of Frozen Instant Cells Expanded cells were harvested and resus-pended in a cryopreserva on medium op -mized for the par cular cell type. A XSD-Biofill (FuidX) was used to dispense the cells auto-ma cally into 2ml cryovials (Nunc) at densi-

es between 5 to 50 million cells/vial. Finally the cells were cryopreserved in a Cryomed 7452 controlled rate freezer (Thermo Fisher) at a cooling rate of 1°C per minute. Assay ready cells were stored long term in liquid nitrogen.

thawing of Frozen Instant Cells For immediate use, assay ready Frozen In-stant Cells were thawed quickly in a water bath and washed once in recovery buffer. The cells were resuspended in assay medium and kept for 30 minutes at room temperature. Therea er, cells were instantly applied for the par cular assay, e.g. seeded into assay

The reproducibility of a cell based assay depends on the consistency of the cell culture and is o en not easy to obtain. Assay ready cells which have been cryo-preserved at a highly func onal state, can be used like a reagent without prior cul-

va on or passaging. The cells can be prepared in large homogenous batches and get pre-validated to minimize cell culture-dependent variances. Assay ready cells are immediately available, perform reliably, and can provide be er quality of bio-assays in cell based screening, in vitro toxicology and DMPK, as well as in clinical development and quality control.

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Fig.1: Produc on process of assay ready Frozen Instant Cells.

Fig.2: assay ready Frozen Instant Cells of NIH-3T3, MDCK, Caco-2 and THP-1.

NIH-3T3 MDCK

THP-1 Caco2

plates and treated with test compounds.

results quality and reproducibility of assay ready cells Assay ready Frozen Instant Cells were pre-pared from various human and animal cell lines. For quality control, 5% of the batches were thawed and analyzed. All batches dis-played a good homogeneity in cell count (> 90%), a viability above 95%, and a recovery rate of greater than 90%. 24 hours a er pla ng, the assay ready cells did not show elevated levels of debris or dead cells com-pared to a con nuously passaged culture. The morphology of the cells looked unsuspicious (Fig. 2).

For a func onal test, four batches (100 vials) of assay ready Frozen Instant Cells were pre-pared from a recombinant CHO-K1 cell ex-pressing human Ghrelin receptor. Aliquots of each batch (10 million cells/vial) were thawed and seeded into 96-well plates at 50.000 cells per well. Four hours a er seeding, a Ghrelin analogue (L-692-585) was added in serial dilu ons and incubated with the cells for 24 hours. Ghrelin receptor ac va on was meas-ured by IP-One htrf assay (cisbio). All batches of assay ready cells displayed the same re-sponse to the receptor agonist (Fig. 3).

assay ready cells in reporter gene assays A recombinant A549 lung epithelial cell line expressing a SEAP reporter gene upon ac va-

on of the NF B signaling pathway was ex-panded to prepare assay ready. One aliquot of Frozen Instant Cells was thawed and seed-ed into a 96-well plate. Immediately a er pla ng, the cells were incubated with increas-ing concentra ons of the inflammatory cyto-kine TNF , a potent inductor of NF B. SEAP ac vity was quan fied by chemiluminescent substrate (CSPD). For comparison the cells from a con nuously passaged culture were treated the same way. Assay ready cells pro-vided an almost iden cal potency and effica-cy for the TNF as con nuously passaged cells (Fig. 4).

use of assay ready cells in prolifera on assays Assay ready cell were prepared from different human tumor cell line. A er thawing, the cells were directly challenged with cytosta c

5-Fluorouracil (5-FU) for 48 hours. Tumor cells from a con nuously passaged culture were seeded at the same density and treated the same way as the assay ready cells. Growth inhibi on at increasing concentra on of 5FU was detected by a fluorescent dye, which is metabolized by viable cells and gives a sensi ve measure of cell quan ty. Maxi-mum prolifera on obtained a er 48 hours was not significantly impaired for the assay ready frozen tumor cells. The cells showed the same sensi vity to the cytosta c drug as cells from a con nuous culture (Fig. 5)

discussion

The comparison of assay ready cryopreserved cells with cells from a con nuously growing culture revealed no disadvantage of this method. The assay ready cells perform as well as fresh cells in various types of bioas-says, including reporter gene ac va on, sec-ond messenger quan fica on, and even pro-lifera on. Because individual vials of assay ready Frozen Instant Cells are derived from a single validated batch of cells, the reproduci-bility of a cell based assay performed with these cells can be improved. Variances gener-ated by different operators, different media, or serum lots are minimized. Custom Frozen Instant Cell can be stored in liquid nitrogen

for many month without loosing performance to supply bioassays which are a rou nely used over an extended period of

me (e.g. lot release tes ng). The cells can be provided with a GMP compliant documenta-

on.

literature [1] Cawkill D, Eaglestone SS, Evolu on of cell-based reagent

provision. Drug Discovery Today 12/19-20, 820–825; 2007.

Fig.3: ac va on of GHSR by an Ghrelin receptor agonist measured in assay ready Frozen Instant Cells of CHO-GHSR.

Fig.5: growth inhibi on of 5-Fluorouracil (5-FU) tested on assay ready Frozen Instant Cells of different human tumor cell lines.

Fig.4: TNF ac vity measured in assay ready cells (red) and con nuously passaged cells (green) of A549-NF B-SEAP reporter cells.