# Optimization of secure workstation for manipulation of 35S-labelled molecules: a case of in vitro cell metabolic labelling

Mathilde Audry, Mathieu Coureuil, Alexandre Hinzpeter, Patricia Frot, Olivier Saltiel, Meriem Kadri, Marie-Lène Gaab, Anne-Sophie Armand, Nathalie Servel

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Mathilde Audry, Mathieu Coureuil, Alexandre Hinzpeter, Patricia Frot, Olivier Saltiel, et al. Optimization of secure workstation for manipulation of 35S-labelled molecules: a case of in vitro cell metabolic labelling. Radioprotection, 2023, Radioprotection, 58 (3), pp.213 - 220. 10.1051/radio-pro/2023015. hal-05114086

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# Optimization of secure workstation for manipulation of ³⁵S-labelled molecules: a case of *in vitro* cell metabolic labelling

M. Audry, M. Coureuil, A. Hinzpeter, P. Frot, O. Saltiel, M. Kadri, M.-L. Gaab, A.-S. Armand and N. Servel* ID

Institut Necker Enfants Malades, Radioactivity core facility, INSERM U1151, CNRS UMR 8253, Université Paris Cité, Faculté de Santé, 156 rue de Vaugirard, 75015 Paris, France.

Received: 10 January 2023 / Accepted: 12 April 2023

**Abstract** – Pulse-chase and cell metabolic experiments involving ³⁵S-labelled molecules can be detrimental to your health. Suppliers and literature suggest the use of activated charcoal to limit contamination without any precise specification. The aim of this study is to fill in this gap. Here, we perform the usual procedures, which consist in working with the available equipment according to recommendations of radioprotection. We carried out radiotoxicological exams as well as dosimetric follow-ups of the workers. As this procedure leads to internal contamination of the operators and of surface contamination at the workplace, we tested a more secure workstation with collective protection equipment that we validated as limiting contaminations for workers. This study proposes a method to use ³⁵S-labelled molecules in a more secure way, which could be beneficial for the scientific community.

**Keywords:** pulse-chase / Sulphur 35 / internal contamination evaluation / metabolic labelling

## 1 Introduction

Procedures, such as pulse-chase and cell metabolite labelling experiments based on ³⁵S-labelled molecules, could be sources of contamination to the operators. Cells are incubated in the presence of ³⁵S-labelled molecules at 37 °C for several hours, producing ³⁵S-labelled volatile species (Simonnet and Simonnet, 1990; Raisonnier, 2004; Delacroix et al., 2006; Harvard Campus Services, 2012). These volatile species accumulate in the incubator, which poses a threat to the users when they have to open the incubator and manipulate cells. In our facility, cells were incubated with or without ³⁵S-Methionine in conjunction with labelled cysteine. Both of these could be metabolized into volatile species such as SO₂ (Fig. 1), which are difficult to estimate the amount of. As a consequence, we observed internal and surface contaminations. In this study, we designed a workflow of how to rationally optimize the safety of the workstation. As suppliers did not take responsibility to validate biological safety cabinet with adequate filters as safe from radiological contamination, we designed a set-up to better trap volatile ³⁵S species that we deemed free of contamination risk.

## 2 Materials and methods

### 2.1 Materials

Radioactive sources were constituted with either a mix of ³⁵S-labelled methionine and cysteine (Perkin Elmer SAS, France) or ³⁵S-labelled cysteine (Hartmann Analytic, Deutschland). The first radioactive solution presented an activity of 74 MBq, or 2 mCi, whereas the latter one an activity of 37 MBq or 1 mCi. Activated carbon filter masks FFP2 (VWR International, France) were available in the facility, as well as urine collection bottles and RPL dosimeters. Biological sample inactivation was performed with Surfanios Premium (Dominique Dutscher, France) and radioactive surface decontamination with TFD4 detergent.

Two incubators were used in this study, a large one #1 (CO₂ Galaxy S+, RS Biotech, UK) and a small one #2 (CO₂ MCO-5AC 49 L, Panasonic). High safe scintillation liquid Optiphase 3 (Perkin Elmer, France) and scintillation counter Hidex 300SL (Lablogic S.A., France) were used to measure radioactivity.

The collective protection equipments available in the facility include glove box G-Safe (Jacomex, France) with activated charcoal filter and, after optimization, a Class II biological safety cabinet HERAsafe KSP12 (Thermo Electron Corporation) with 12 activated charcoal filters and a fume cupboard with activated charcoal filters.

<footnote>*Corresponding author: nathalie.servel@inserm.fr</footnote>
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Sulphur amino-acid catabolism produces light and extremely volatile compounds such as SO₂. From Raisonnier (2004).</figcaption></figure>

## 2.2 Pulse-chase experiments before optimization

Epithelial cells (HeLa) were cultured in the presence of ³⁵S-labelled methionine and cysteine to measure protein half-life as described (Lukacs et al., 1994). The radioactive solution was collected and introduced into the cell culture medium inside a glove box, even if 74 MBq were allowed to be manipulated on the bench (Delacroix et al., 2006). The radioactive medium was added to the cells on the bench behind a Plexiglas screen. Then, culture plates were placed in the incubator monitoring area (Fig. 2). During this procedure, the operator was using individual protection equipment, as FFP2 masks to avoid contamination from ³⁵S-vapours and wearing a single pair of gloves. These masks were equipped with an activated carbon filter to protect the handlers against inhalation of vapours or aerosols outside the glove box. The activated carbon filter had a specific FFP2 classification with EN 149-2001 certification, ensuring 92% 0.6 µm particle filtration. A babyline (ionization chamber) was installed near the handling areas to detect possible dose rates in the air and gloves contamination has been verified by Geiger counter. The experiment was repeated three times by the same operator. Laboratory's temperature was measured between 30 °C and 40 °C.

We performed individual dosimetric monitoring by RPL dosimeter for external contamination reporting. Radiotoxicological examinations were performed to report internal contamination by inhalation. Radiotoxicological monitoring of the operator consisted of urine collection 24 h following the last experiment (3 times 74 MBq manipulated). Surface wipe tests were performed and counted for 10 minutes after the second and the third experiments as described in Figure 3.

## 2.3 Metabolic labelling after optimization

The scientific aim of the study was to evaluate if a bacterial strain (Neisseria meningitidis) was able to be supplied with a Sulphur species from human Cerebral

Microvascular Endothelial Cells (hCMEC/D3), a fully differentiated brain endothelial cell line derived from human brain capillaries (Weksler et al., 2005). hCMEC/D3 were incubated overnight in the presence of 5 MBq ³⁵S-labelled cysteine, onto Cultrex rat collagen type I-coated dishes (R&D) in Endothelial Cell Basal Medium-2 (Lonza) supplemented with 5% of fetal calf serum, 1.4 µM hydrocortisone (Lonza), 5 µg/mL ascorbic acid (Lonza), 1 ng/mL b-FGF (Lonza), at 37 °C in 5% CO₂. Cells were then infected with 10⁷ bacteria to assess the release of Sulphur-containing species by cells and the potent acquisition by bacteria. Neisseria meningitidis strain 2C4.3 was used in this study and cultures as described previously (Nassif et al., 1993). Four hours later, endothelial cells were washed and supernatants were processed for further experiments (Fig. 4).

Urine of two workers was collected within the first hour and 24 h following the experiment. Nose blowing tests were carried out by the two workers 6 h after the beginning. For this particular test, the user blew his nose in a handkerchief, put this tissue in a vial with scintillation liquid. Surface wipe tests were performed in the incubator and carried out at the end of the first experiment as well as one month later.

## 2.4 Radioactivity handling

Biological samples containing supernatants and solids were inactivated with surfanios and stored in a dedicated basket for radioactive waste.

## 2.5 Internal contamination monitoring

### 2.5.1 Urinary radiotoxicological analysis

Urinary radiotoxicological analysis was performed by the Institut de Radioprotection et de Sûreté Nucléaire (Vésinet, France). Urines were collected by the operator from the first urines after the experiment to 24 h.
## PULSE-CHASE EXPERIMENT BAD PRACTICE

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Pulse-chase experiment procedure before optimization: 1) radioactive mix was carried out in the glove box; 2) radioactive medium was added to the cells into dishes, on the bench behind a screen and with activated carbon FFP2 mask; 3) the pulse is performed at 37°C in the CO₂ incubator from 30 min to 24 h; 4) media were collected after 30 min to 6 h of pulse and cells were washed for chase on the bench with mask; 5) 24 h after the pulse, media were collected and cells washed for chase without mask and; 6) cells were collected to extract proteins for SDS PAGE analysis in the same room. (original illustration by Solveig Colomb).</figcaption></figure>

### 2.5.2 Nose blowing test

The operator blew his nose in a small piece of tissue that fitted into the vial for liquid scintillation counting and added 3 mL of Optiphase 3 scintillation liquid (Perkin Elmer). A negative control was scheduled with a handkerchief in scintillation liquid without any nose blowing. The counting was performed for 10 minutes per point.

## 2.6 External contamination monitoring

### 2.6.1 Surface wipe test

Surface contamination was assessed by wiping 100 cm² with a Whatman glass microfiber filter (grade GF/A, diameter

42.5 mm, VWR, France) soaked in an ethanol/water mixture (50/50). The filter was then introduced into a 6 mL vial and 3 mL of Optiphase 3 scintillation liquid was added before counting for 10 minutes per point. A negative control was performed with a glass fiber filter soaked in the same mixture without any wiping.

## 3 Results

Pulse chase or metabolic labelling experiments using ³⁵S-labelled molecules could be sources of contamination, as incubation of cells at 37°C for several hours increases the generation of ³⁵S-labelled volatile species (Fig. 1). For this
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Laboratory #1 map before optimization. Surface wipe tests were carried out at time point #1 (the week following the first day of experiment), on bench 1 (6 Bq/cm²), bench 2 (4 Bq/cm²), bench 3 (0.6 Bq/cm²), bench 4 (18 Bq/cm²) and on floor 5 (n.s.) and at time point #2 (two months after the last experiment) on fridge's door 6 (0.44 Bq/cm²) and on floor 5 (300 Bq/cm²).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 4.** ³⁵S-labelled cysteine incorporated by endothelial cells (metabolic labelling). hCMEC/D3 were incubated overnight in the presence of 5 MBq ³⁵S-labelled cysteine, onto Cultrex rat collagen type I-coated dishes, at 37 °C in 5% CO₂. Cells were then infected with 10⁷ bacteria to assess the release of Sulphur-containing species by cells and the potent acquisition by bacteria. Four hours later, endothelial cells were washed and supernatants were processed for further experiments. ³⁵S-activity was assessed in all fractions: bacteria in suspension and in medium, in endothelial cells: bacteria inside the cells and in the cytoplasm.</figcaption></figure>

reason, we measured internal and external contaminations for experiments.

## 3.1 Internal contamination

Sulphur radiokinetic curves show that 80% of ³⁵S are excreted within 6 h following cell treatment. We considered that urine should be collected from the start of the experiment (Soufre-35-Sheet ED4307-INRS, 2009). Radiotoxicological monitoring of the operator consisted of urine collection 24 h following the pulse-chase experiment. Contamination with ³⁵S was found on the sample collected the same day of the experiment.

Sulphur is a low-energy beta emitter (max energy = 168 keV).

Calculation of the committed dose (E) as shown below.

This is given from the formula indicated in the INRS sheet of Sulphur 35:

$$E(\text{Sv}) = I(\text{Bq}) \times \text{DPUI}(\text{Sv}/\text{Bq}),$$

where E is the committed effective dose in Sievert (Sv), I is the activity incorporated during contamination in Becquerel (Bq), and DPUI is the dose per unit of incorporation, i.e., the committed effective dose for 1 Bq incorporated by inhalation.

$$I = \frac{Am}{F(t)},$$

where Am is the activity measured on day t after contamination, F(t) is the fraction excreted at day t for 1 Bq incorporated.
The values of F(t) are given in Soufre-35-Sheet ED4307-INRS (2009).

The values of DPUI are shown in JORF n°0262 du 13/11/ 2003 (2003).

- A = 74 Bq/24 h (activity measured in urine during 24 h after exposure);

- F(t) at day 1 = 2.9 × 10⁻¹ (in Bq per Bq incorporated);

- DPUI = 1.1 × 10⁻³ µSv (inhalation is chosen as the route of exposure and the 5 µm particle form is chosen);

- Embodied activity (inhaled): I = 74/2.9 × 10⁻¹ = 255 Bq.

Calculation of the committed dose, taking the corresponding DPUI, as follows:

$$E(\mu\text{Sv}) = 255 \times 1.1 \times 10^{-3} = 280 \times 10^{-4} \mu\text{Sv} \\ = 0.28\mu\text{Sv}.$$

The committed effective dose was equal to 0.28 µSv, demonstrating the internal contamination of the operator.

## 3.2 External contamination

To assess external contamination, we performed surface wipe tests after the second and third pulse-chase experiments at the indicated points (Fig. 3). We obtained 6 Bq/cm² on bench #1; 4 Bq/cm² on bench #2; 0.6 Bq/cm² on bench #3 and 18 Bq/cm² on bench #4. These measurements clearly showed external contamination during these experiments, despite the absence of floor contamination. This protocol implies that the cells were in contact with CO₂ injected into the incubator during overnight incubation. Consequently, the plates were not tightly closed and radioactive vapours could escape and spread into the incubator and room. As a precaution, we installed a Babyline ionization chamber near the handling areas to detect possible dose rates. However, no emission was detected, possibly due to the very low energy levels emitted by Sulphur. Contaminated surfaces were cleaned with TFD4 cleaner.

Two months after the last Sulphur-35 experiment stopped due to internal contamination, we performed routine surface wipe test measurements. We obtained two positive results: 300 Bq/cm² on point #5 and 0.44 Bq/cm² on point #6. #5 was situated on the floor, while #6 corresponded to the door of the fridge; both areas were negative during the previous surface wipe test measures. These data suggest that this late contamination could correspond to evaporating ³⁵S species falling down from the air, weeks after the experiments, were initially performed, maybe enhanced by hot temperatures in our laboratory at that time (35–40 °C) due to air conditioning problems.

## 3.3 Evaluation of a new set-up to avoid contamination during pulse-chase experiments using ³⁵S-labelled molecules

To avoid contamination while manipulating cells incubated with ³⁵S-labelled molecules, we adapted a previous setup that we tested for internal and external contamination. Briefly,

radioactive sources were manipulated into a biological safety cabinet KSP12 until cell washings included (Fig. 5). Incubation of cells at 37 °C for metabolic labelling experiments was performed in the 50 L-CO₂ incubator, which was placed under a fume cupboard. In both cases, radioactive vapours enriched in ³⁵S sulfur were trapped by activated charcoal filters: One at the top of the fume hood and twelve at the bottom of the KSP12 biological safety cabinet. Metabolic labelling experiments were performed in this optimized workstation.

Using nose blowing and urine radiotoxicological examination, we tested internal contamination of two operators using this optimized setup for their experiments. We were not able to detect any activity above background neither in nose blowing tests nor in urine of both workers, suggesting no contamination. External contamination was assessed using surface wipe tests that were performed as described in Figure 6. We measured 7.8 Bq/cm² in the cell incubator and 0.3 Bq/cm² in the KSP12 biological safety cabinet. The radioactive vapours likely dissipated from the incubator in the fume cupboard and not into the room.

These experiments were later repeated 5 times incubating each about 5 MBq to cultured cells without any internal contamination detected for the operators. One month later, surface wipe tests revealed 52 Bq/cm² in the cell incubator and 0.3 Bq/cm² in the fume cupboard. All the samples taken from the floor, benches, shelves or freezer door, gave values under background, indicating that ³⁵S-vapours were confined into the incubator and the fume cupboard with the optimized setup.

Even though only 5 MBq were involved per experiment in the second method, internal contamination would have been detected by nose blowing and surface wipe tests.

## 4 Discussion

Cell incubation with ³⁵S-labelled molecules is often linked to internal contamination by inhalation due to highly volatile ³⁵S species. We evaluated internal contamination of operators to 0.28 µSv, without any confinement provisions apart from opening the radioactive vial into the glove box. Compared to the annual dose limits for the public at 1 mSv and the recommended dose for Category B workers at 6 mSv, this effective dose is very low. ³⁵S is highly soluble and is homogeneously distributed in the organism after incorporation. Organic Sulphur is retained for a period of 140 days (Soufre-35-Fiche 4307-INRS, 2009). Therefore, limiting any internal contamination would benefit workers. Here, personal protective equipment was insufficient to completely protect the operator; therefore, collective protective equipment should be used to avoid further contamination. Accordingly, we adapted the setup to limit internal and external radioactive contaminations during pulse-chase or cell metabolic experiments involving ³⁵S-labelled molecules.

According to the literature and University radioprotection officer (Meisenhelder and Semba, 2006; Meisenhelder and Bursik, 2007; Harvard Campus Services, 2012), radioactive vapour could be trapped using: i) activated charcoal in the incubator (without air circulating, the efficacy might be very low). So, we have chosen to open the vial in the glove box or in the biological safety cabinet with carbon filters; ii) an incubator with activated charcoal filter and ventilation, a system which is not supplied; iii) an incubator in the fume cupboard, which is
PULSE-CHASE EXPERIMENT GOOD PRACTICE

<figure><img src="image_6.png" /></figure>

Urine collection from the beginning of the pulse to 24h.

<caption>**Fig. 5.** Schema of good practice: 1) to mix radioactive medium in the KSP12 class II biological safety cabinet: 5 MBq incorporated of <sup>35</sup>S Cys; 2) to add radioactive medium on cell culture in the cabinet; 3) to incorporate <sup>35</sup>S Cys *in vivo* in cultured cells, at 37 °C, 5% CO<sub>2</sub>, 30 min to 24 h (pulse); 4) to collect radioactive medium to wash (chase) from 30 min to 24 h. This step is performed in the KSP12 class II biological safety cabinet; 5) to collect cells, to count in scintillation liquid radioactive fraction. Urine collection from beginning of the pulse to 24 h. Nose blowing test at the 6 h point. Surface wipe tests at the end of the experiment and one month later. Laboratory temperature should be around 25 °C.</caption>

suitable for vapour trapping; iv) biological safety cabinet with
activated charcoal filters. Among the recommendations,
Meisenhelder and Semba (2006) propose to place cell dishes
into a Plexiglas box. This suggestion is not applicable in our
case since the cells need air exchange that would be limited by
a Plexiglas box.

Apart from the glove box for pipetting the radioactive
source, we chose a small CO₂ incubator (50 L CO₂ incubator,
Panasonic), which we introduced into a fume cupboard for the
cell incubation step at 37°C. Air extraction efficiency was
checked before the pulse-chase experiment. For washing steps,
cell culture dishes were manipulated in a microbiological
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Map of the optimized setup. Surface wipe tests were carried out (i) the week following the first-test experiment (5 MBq manipulated), the only positive measures were obtained in the incubator 2 (7.8 Bq/cm²) and on the KSP12 class II biological safety cabinet bench (0.3 Bq/cm²). Benches, shelves and floor were negative; (ii) one month after manipulating 28 MBq, two points were positive: incubator 2 (52 Bq/cm²) and fume cupboard (0.3 Bq/cm²). Floor, KSP12, benches, shelves or fridge's door were under the background.</figcaption></figure>

security station with activated charcoal filters (KSP12 biological safety cabinet, Thermo Electron SAS). These experiments were performed in parallel for independent scientific projects. They were not for radioprotection purposes *per se*, but were carried out on two cell models (HeLa cells or endothelial hCMEC/D3 cells) with two different methods, i.e., pulse chase and metabolic labelling. Both cell types had incorporated ³⁵S-labelled cysteine after 4 to 16 h incubation at 37°C. In both protocols, 90% of the activity was found in cell media (source of ³⁵S evaporation).

These results demonstrate that this adapted procedure limits internal contamination.

## 5 Conclusion

In this work, we designed a secure workstation to manipulate ³⁵S-labelled molecules in cell cultures, with validated and convenient procedures. In these conditions, surface wipe tests, nose blowing tests and urinary radiotoxicological assays should be negative, as these procedures prevent from internal contaminations of operators. This study is a response to Bourguignon (2021) who insisted on radiological protection research coming from French academic institutions.

## Conflict of interest

The authors declare that they have no conflict of interest.

## Funding

This work was supported by INSERM.

## Ethical approval

Ethical approval was not required.

## Informed consent

This article does not contain any studies involving human subjects.

## Authors contributions

M. Audry, M. Coureuil, A. Hinzpeter and N. Servel performed experiments. O. Saltiel, M. Kadri, P. Frot and N. Servel analyzed data. N. Servel designed the study. M.-L. Gaab and N. Servel contributed to the methodology. M. Coureuil, A.-S. Armand and N. Servel wrote the manuscript.

## Acknowledgements

We would like to thank Solveig Colomb from LISAA (L'Institut Supérieur des Arts Appliqués, Paris) who illustrated bad and good practices and Nathaniel Fathe Henneman for editing this manuscript.

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**Cite this article as:** Audry M, Coureuil M, Hinzpeter A, Frot P, Saltiel O, Kadri M, Gaab M-L, Armand A-S, Servel N. 2023. Optimization of secure workstation for manipulation of ³⁵S-labelled molecules: a case of *in vitro* cell metabolic labelling. *Radioprotection* 58(3): 213–220