Assay Ready Frozen Instant Cells Can Improve Bioassay Performance

introduction

In 2005 a dogma felt which says cryo-

preserved mammalian cells have to be pas-

saged at least three to four times before they

have fully recovered and can be applied in a

cell based assay. At the annual conference of

the Society for Biomolecular Screening (SBS)

data had been presented which demonstrate

that cryopreserved cells can be used in bioas-

says instantly after thawing when they had

been frozen properly (personal communica-

tion). Assay ready cells can be used in differ-

ent assays, e.g. receptor activated second

messenger assays (cAMP, IP1, Calcium flux),

transporter assays, ion channel patch clamp-

ing, kinase, reporter gene and even prolifera-

tion assays.

Since this first demonstration the use of Fro-

zen Instant Cells commence their triumphant

advance in cell based drug discovery. As high-

throughput screening campaigns require

large quantities of cells with a consistent

performance, cell production is always a

bottleneck. With the ability to prepare assay

ready cells in advance in a homogenous and

controlled quality, screening campaigns be-

came much easier to schedule and more reli-

able [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 permea-

bility) as well as for diagnostic monitoring in

clinical development or

lot release tests for thera-

peutic proteins, antibod-

ies or vaccines (receptor

activation, cell prolifera-

tion, virus infectivity).

material and methods

expansion of assay cells

Recombinant cell lines or

unmodified human tumor

cell lines were cultivated

in their custom cell cul-

ture medium with 10-20%

FBS (PAN Biotech) at 37°C

with 5% CO2. Adherent cells were detached

with Accutase (Sigma Aldrich) and cultivated

in CellStacks (Corning) for further expansion

(Fig. 1). Suspension cells were grown in Hy-

perFlasks (Corning).

cryopreservation of Frozen Instant Cells

Expanded cells were harvested and resus-

pended in a cryopreservation medium opti-

mized for the particular cell type. A XSD-Biofill

(FuidX) was used to dispense the cells auto-

matically into 2ml cryovials (Nunc) at densi-

ties 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 Instant

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. There-

after, cells were instantly applied for the par-

ticular assay, e.g. seeded into assay plates

and treated with test compounds.

The reproducibility of a cell based assay depends on the consistency of the cell

culture and is often not easy to obtain. Assay ready cells which have been cryo-

preserved at a highly functional state can be used like a reagent without prior culti-

vation or passaging. The cells can be prepared in large homogenous batches and

got pre-validated to minimize cell culture dependent variances. Assay ready cells

are immediately available, perform reliably, and can provide a better quality of

bioassays in cell based screening, in vitro toxicology and DMPK, as well as in clinical

development and quality control.

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Fig.1: Production 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

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 after

plating the assay ready cells did not show

elevated levels of debris or dead cells com-

pared to a continuously passaged culture. The

morphology of the cells looked unsuspicious

(Fig. 2).

For a functional 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 after seeding, a Ghrelin

analogue (L-692-585) was added in serial

dilutions and incubated with the cells for 24

hours. Ghrelin receptor activation 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 activa-

tion of the NFB signaling pathway were

expanded to prepare assay ready. One ali-

quot of Frozen Instant Cells were thawed and

seeded into a 96-well plate. Immediately

after plating the cells were incubated with

increasing concentrations of the inflammato-

ry cytokine TNF, a potent inductor of NFB.

SEAP activity was quantified by chemilumi-

nescent substrate (CSPD). For comparison the

cells from a continuously passaged culture

were treated the same way. Assay ready cells

provided an almost identical potency and

efficacy for the TNF as continuously pas-

saged cells (Fig. 4).

use of assay ready cells in proliferation assays

Assay ready cell were prepared from different

human tumor cell line. After thawing, the

cells were directly challenged with cytostatic

5-Fluorouracil (5-FU) for 48 hours. Tumor

cells from a continuously passaged culture

were seeded at the same density and treated

the same way as the assay ready cells.

Growth inhibition at increasing concentration

of 5FU were detected by a fluorescent dye

which is metabolized by viable cells and gives

a sensitive measure of cell quantity. Maxi-

mum proliferation obtained after 48 hours

was not significantly impaired for the assay

ready frozen tumor cells. The cells showed

the same sensitivity to the cytostatic drug as

cells from a continuous culture (Fig. 5)

discussion

The comparison of assay ready cryopreserved

cells with cells from a continuously growing

culture revealed no disadvantage of this

method. The assay ready cells perform as

good as fresh cells in various types of bioas-

says including reporter gene activation, sec-

ond messenger quantification and even pro-

liferation. Because individual vials of assay

ready Frozen Instant Cells derive from a sin-

gle validated batch of cells, the reproducibil-

ity of a cell based assay performed with these

cells can be improved. Variances generated

by different operators or 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 routinely

used over an extended period of time (e.g.

lot release testing). The

cells can be provided with

a GMP compliant docu-

mentation.

literature

[1] Cawkill D, Eaglestone SS,

Evolution of cell-based reagent

provision. Drug Discovery Today

12/19-20, 820–825; 2007.

Fig.3: activation of GHSR by an Ghrelin receptor agonist

measured in assay ready Frozen Instant Cells of CHO-GHSR.

Fig.5: growth inhibition of 5-Fluorouracil (5-FU) tested on assay ready Frozen

Instant Cells of different human tumor cell lines.

Fig.4: TNF activity measured in assay ready cells (red) and

continuously passaged cells (green) of A549-NFB-SEAP

reporter cells.