instant use of assay ready thp‐1 cells to test for skin sensitization (h‐clat) ·...

contact: Dr. Oliver Wehmeier, acCELLerate GmbH, [email protected]

Instant Use of Assay Ready THP‐1 Cells to Test for Skin Sensitization (h‐CLAT)Vladimir Mazurov, Julie Walbech, Alexander Loa, Oliver WehmeieracCELLerate GmbH, Hamburg, Germany

introductionRecently, a cell based in vitro model has have been approved by the OECD (Test N° 442E) to assess the skin sensitizing hazard of chemicals. One of the tests applied is the human Cell Line Activation Test (h‐CLAT) which

uses monocytic THP‐1 cells as a surrogate for dendritic cells. These cells express CD86 and CD54 upon activation through sensitizing molecules at subtoxic concentrations. However, it can be tedious to set up a reliable

and reproducible h‐CLAT because the performance of cultivated THP‐1 can vary a lot depending on culture conditions and individual handling. In addition, it is known known that THP‐1 cells are very sensitive to

cryopreservation and usually need a couple of days to overcome a crisis of reduced viability when resuscitated from a frozen stock. Here we demonstrate an optimized protocol to freeze and recover THP‐1 for instant

use in cell based assays. Assay ready THP‐1 cells display a steadily high viability. The cells can be applied in h‐CLAT skin sensitization testing immediately after thawing. No prior cultivation or passaging of the cells is

required. By preparing a prequalified batch of assay ready THP‐1 cells the culture dependent variations in assay performance can be eliminated. We show that assay ready THP‐1 cells are equally susceptible to skin

sensitizers like cells from a continuously passaged culture.

discussionA major obstacle of the h‐CLAT assay is the reproducible cultivation of the THP‐1 cells. The concentration rage at which skin sensitization can be measured without significantly reducing viability, is very tight. A healthy

and highly viable culture of THP‐1 cells is therefore of the essence. THP‐1 are known to recover badly from suboptimal cultivation or cryopreservation. It usually takes at least a week of intensive care until the cells regain

an acceptable viability. Because the overall fitness of the cells has a significant impact on their sensitivity to sensitizers, reproducibility of the h‐CLAT very much depends on the cell culture quality and is therefore difficult

to control. We here demonstrated an optimized freezing process to prepare assay ready THP1 cells. These cells recover with high viability and functionality and can be thawed, plated and used directly in h‐CLAT skin

sensitization assay. Assay ready THP‐1 cells demonstrate very good vial‐to‐vial and batch‐to‐batch reproducibility in skin sensitization h‐CLAT assay. Based on the present results, applying of assay ready THP‐1 cells in h‐

CLAT is suggested for reducing variable results and saving the time for h‐CLAT assay preparation.

preparation of assay ready frozen THP‐1 cellsTHP‐1, obtained from CLS Cell Line Services (Heidelberg), were cultivated according to the supplier’s

recommended culture conditions and kept at a cell density below 1 million cells/ml. For optimal

cryopreservation the cells were harvested in the logarithmic growth phase by low g‐force centrifugation

(80 x g) and were resuspended in a serum free freezing‐medium (NFM‐G1) containing 5% DMSO for

cryoprotection. The cells were automatically dispensed into cryovials at 10 million cells/vial using a XSD‐

Biofill decapping and filling device (FuidX) and were frozen in a Cryomed 7452 controlled rate freezer

(Thermo Fisher) at a cooling rate of 1°C per minute. Cryopreserved cells were stored in vapor phase of

liquid nitrogen (Fig.1).

Fig. 1.: Production of assay ready frozen cells

large scale cell production

automatic dispensing into cryo vials

controlled ratecryopreservation

long term storage in liquid nitrogen

aliquots of assay ready cells

For recovery the assay ready THP‐1 cells have been quickly thawed at 37°C, immediately diluted in an

RPMI based recovery buffer and centrifuged at 80x g. The cell pellet was resuspended in assay medium

(RPMI 1640, 20% FBS, 2mM L‐Gln, 1mM NaPy 10mM Hepes, 50µM 2‐MEtOH) and seeded in a 6‐well

plate at 4E5 cell/ml. Viability of the cells was determined over the course of 2 days by Propidium Iodide

(PI) staining of damaged cells (Fig. 2).

Expression of CD54 and CD86 was detected by fluorescently (FITC) labelled antibodies (α‐CD54 (RR1/1);

LifeTechnologies & α‐CD86 (2331); BD Biosciences) and analyzed by Flow Cytometry (Cytomix FC500)

(Fig. 6).

The treatment of THP‐1 cells with skin sensitizers resulted in a significant expression of CD54 and CD86

(red histograms) compared to untreated control cells (grey histograms) indicating an activation of the

cells triggered by the OECD proficiency chemicals. Lactic acid treatment did not result in a shift of CD54

and CD86 expression indicating no activation of monocytes. Viability of cells was measured with

propidium iodide staining. Assay ready THP‐1 cells showed comparable response sensitizing and non‐to

sensitizing substances as continuously passaged THP‐1 cells demonstrated by similar activation levels of

CD54 and CD86.

0%

20%

40%

60%

80%

100%

0 h 24 h 48 h

viab

le cells [%]

hours after thawing

continously passaged standard freezing assay ready

Fig.3.: Morphology of assay ready THP‐1 cells.

Fig.2.: Long term viability of assay ready THP‐1 cells

Reference Chemical in vitro Classification

2,4‐Dinitrochlorbenzene Extreme Sensitizer

4‐Phenylenediamine Strong Sensitizer

Nickel Sulfate Moderate Sensitizer

2‐Mercaptobenzothiazole Moderate Sensitizer

Lactic Acid Non‐Sensitizer

Tab. 1.: OECD recommended chemicals for demonstrating proficiency with the h‐CLAT

human Cell Line Activation Test (h‐CLAT)To determine the skin sensitizing potential of chemicals the h‐CLAT is modeling one key element of the

complex process, the activation of dendritic cells expressed by an increase of CD86 and CD54 at the cell

surface. Cells from a high viability maintenance culture as well as assay ready THP‐1 cells, recovered as

described above, were seeded into a 24‐well plate at 1 million cells/well. After seeding the cells were

immediately challenged for 24 hours with four reference sensitizers (Tab 1). Fig. 6.: Expression of CD54 & CD86 in THP‐1 cells after activation by skin sensitizers

Directly after thawing (0h) the assay ready THP‐1 cells as well as cells which have been classically frozen

in culture medium, with 10% DMSO both display a high viability above 95% comparable to cells from a

continuously passaged maintenance culture (light grey). 24 hours later, cells from the standard process

display a significant decrease in viability (dark grey) which fully recovers only after 10 days of culture (not

shown). However, cells which have been frozen according to the optimized assay ready protocol (red)

does not go through a crises and display a constantly high viability after 24 and even 48 hours. The cells

have a round and evenly shaped morphology after thawing (Fig. 3).

Untreted cells (control) Sensitizer treated cells

Control: MFI 18,2%, Viability 98,8%Lactic Acid: MFI 6,4%, Viability 94,5%


Control: MFI 18,2%, Viability 98,8%NiSO4: MFI 65,6%, Viability 90,7%

Control: MFI 0,9%, Viability 95,8%DNCB: MFI 16,2%, Viability 94,3%

Control: MFI 3,9%, Viability 99,7%2‐Mercapt.: MFI 89,9%, Viability 99,5%




Control: MFI 2,4%, Viability 97,8%4‐Pnenyl.: MFI 1,3%, Viability 97,8%

Assay Read

y Cells

ContiniouslyPassagedCells

Control: MFI 0,2%, Viability 98,8%4‐Pnenyl.: MFI 0,7%, Viability 87,8%

Lactic acid (2 mg/ml) DNCB (3 µg/ml)NiSO4 (100 µg/ml) 2‐Mercaptobenzothiazole (100 µg/ml) 4‐Phenylenediamine (20 µg/ml)


CD54

Lactic acid (2 mg/ml) DNCB (3 mg/ml)NiSO4 (100 µg/ml) 2‐Mercaptobenzothiazole (100 mg/ml) 4‐Phenylenediamine (20 µg/ml)

Control: MFI 4,5%, Viability 98,8%Lactic Acid: MFI 5,1%, Viabiltiy 94,8%



Control: MFI 3,3%, Viability 97,4%NiSO4: MFI 26,4%, Viabiltiy 77,7%


Control: MFI 8,3%, Viability 99,5%2‐Mercapt: MFI 22,7%, Viability 98,3%

Control: MFI 2,7%, Viability 99,7%4‐Pneyl: MFI 14,8%, Viability 94,8%



Control: MFI 3,3%, Viability 97,4%Phenyl: MFI 29,9%, Viability 85,1%

Assay Read

y Cells

ContinuouslyPassegedCells


CD86

instant use of assay ready thp‐1 cells to test for skin sensitization (h‐clat) ·...

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