An anhydrobiotic cell line expressing odorant receptors shows odorant responses after dry storage

Fuse, Hiroto; Cornette, Richard; Miyata, Yugo; Tokumoto, Shoko; Shimura, Sachiko; Araneda, Ricardo C.; Abshire, Pamela; Kikawada, Takahiro; Anderson, Roy; Haider, Redwan; Smela, Elisabeth

doi:10.1038/s41598-025-19627-x

Download PDF

Article
Open access
Published: 10 October 2025

An anhydrobiotic cell line expressing odorant receptors shows odorant responses after dry storage

Hiroto Fuse^1,2,
Richard Cornette¹,
Yugo Miyata³,
Shoko Tokumoto^1,4,
Sachiko Shimura¹,
Ricardo C. Araneda⁵,
Pamela Abshire⁶,
Takahiro Kikawada^1,2,
Roy Anderson⁷,
Redwan Haider⁷ &
…
Elisabeth Smela⁷

Scientific Reports volume 15, Article number: 35533 (2025) Cite this article

1774 Accesses
2 Altmetric
Metrics details

Subjects

Abstract

Odorant receptor-expressing cells have been shown to recognize various odors, which has brought them to the interest of the growing field of cell-based olfactory sensors. However, cell cultures are difficult to use outside a laboratory because of their continuous need for controlled conditions. In this study, the odorant receptor DmOr47a, the co-receptor DmOrco, and the calcium-sensing fluorescent protein GCaMP6f were stably expressed in a Pv11 cell line (Pv11-00443-Or47a), which is desiccatable. This cell line not only retained desiccation tolerance, but also showed dose-dependent fluorescence responses to the DmOr47a ligand pentyl acetate that were recovered 12 h after rehydration. Even more importantly, Pv11-00443-Or47a showed a response to the agonist of DmOrco just 1 h after rehydration, even upon inhibition of protein synthesis. This result demonstrates for the first time that a transmembrane protein can be dry-stored in an orthologous cell culture system. This work also constitutes an initial step towards the development of improved desiccatable sensing cells for use in portable devices.

Molecular recognition of an odorant by the murine trace amine-associated receptor TAAR7f

Article Open access 30 August 2024

Conserved pattern-based classification of human odorant receptor multigene family

Article Open access 08 November 2024

Engineered odorant receptors illuminate the basis of odour discrimination

Article 30 October 2024

Introduction

Olfactory systems across species identify odors with high selectivity and sensitivity. Attention has thus turned to the possibility of utilizing olfactory receptors (ORs) in odor identification devices. Olfactory receptors are membrane proteins, which can be divided into mainly two types: the G protein-coupled receptor (GPCR) type found in vertebrates and the ligand-gated ion channel (LIC) type found in insects. In the case of insects, the receptors consist of a hetero-tetramer comprising an odorant-specific OR and an olfactory receptor co-receptor (Orco). This complex itself functions as a ligand-gated cation channel^1,2.

Like other membrane proteins, ORs have limited stability in isolation³, therefore it is beneficial to express them in cells. It is also beneficial to maintain stable cell lines, rather than to repeatedly transiently transfect batches of cells with receptors, and to have methods of creating cell lines that could be applied with other receptors. Since no stable cell lines derived from olfactory sensory neurons currently exist, there is interest in expressing ORs in heterologous cell lines. In insects, cell expression of only two membrane proteins, an OR and Orco, yields responses to odorants. This system is therefore easier to use than the more complex GPCR-based olfactory system. In fact, previous research has shown that it is possible to express insect ORs in various cell lines and evaluate their responsiveness to odorants^4,5,6.

However, a challenge to cell-based sensing is continuously keeping the cells alive in an aqueous culture medium and within a narrow temperature range, which limits device storability and portability. Although different cell shipping methods have been developed, cell viability at room temperature usually lasts from a few days to a week⁷. Cell-based sensors have thus been largely confined to the infrastructure of the laboratory.

One solution to this problem is to use a cell line that can survive long periods outside laboratory settings. The Pv11 cells, derived from the non-biting midge Polypedilum vanderplanki, have such capabilities⁸. During the larval stage, P. vanderplanki can withstand almost complete desiccation through an ametabolic state called anhydrobiosis^9,10. Like P. vanderplanki larvae, Pv11 cells also have the ability to tolerate desiccation and can be stored in the dry state at room temperature for over a year¹¹. This desiccation tolerance is induced by immersing the cells in a highly concentrated trehalose solution for 48 h¹¹. Furthermore, an exogenous gene expression system has been established for Pv11 cells¹², and Pv11 cells expressing luciferase as a foreign protein were successfully stored in a dry state at room temperature for over a year, while retaining the enzyme activity¹³. However, there has as yet been no proof that membrane proteins such as ORs can be preserved as functional in desiccated Pv11 cells. To our knowledge no previous report has shown the dry preservation of functional membrane proteins in a heterologous cell culture expression system. Thus, this work sought to verify whether functional ORs could be dry-preserved on the surface of Pv11 cells.

Here, we show that Pv11 cells can be engineered to express an OR, that they respond to their target odorant with a fluorescence signal, and retain their desiccation tolerance. This could potentially be a critical breakthrough for cell-based olfactory sensing, although significant further work is needed to develop the cells and technology.

We selected an insect OR derived from the fly Drosophila melanogaster, a species phylogenetically closely related to P. vanderplanki and that has well-characterized ligands. The fluorescent calcium indicator GCaMP6f was employed to evaluate olfactory receptor function, as has been done in previous studies^5,6. Upon ligand binding and opening of the channel-receptor complex, the influx of Ca²⁺ triggers a fluorescence signal that is dependent on the intracellular Ca²⁺ concentration. GCaMP6f, DmOrco and DmOr47a were knocked in at the genomic region downstream of the Pv.00443 gene, which is under the control of the strongest promoter in Pv11 cell line¹⁴and a stable expression cell line was established. This stable cell line was dried and stored at room temperature. After rehydration the ligand response of the cells was evaluated.

Results

Establishment of a Pv11 cell line stably expressing DmOr47a

Preliminary screening was done with transient expression of GCaMP6f, DmOrco and DmOr47a.This showed that the proteins were functional in Pv11 cells (Fig. S1) and that DmOr47a protein localized to the cell membrane surface (Fig. S2). To generate a cell line stably expressing DmOr47a, the three genes were knocked-in to the Pv11 genome using the CRISPR/Cas9 system combined with the precise integration of target chromosome (CRIS-PITCh) method, as described previously¹⁵. The genes were integrated downstream of the Pv.00443 gene, whose expression is regulated by the strong 121 promoter, allowing the maximal expression level in Pv11 cells¹⁴. A guide RNA was designed to target the 5’-flanking site of the Pv.00443 stop codon, and two donor vectors were constructed to produce polycistronic expression of three genes: the first Pv.00443, GCaMP6f, and DmOrco, and the second Pv.00443, zeocin resistance gene (Zeo^R), and DmOr47a (Fig. 1A). The Cas9 gRNA-expression vectors were co-transfected into Pv11 cells together with the donor vectors.

After zeocin selection and single cell sorting for GCaMP6f fluorescence, a cell line expressing GCaMP6f, DmOrco, DmOr47a, and Zeo^R in a biallelic conformation was obtained (Fig. 1A). Genomic PCR of this monoclonal cell line confirmed the presence of a 3562 bp band corresponding to “P2A-GCaMP6f-P2A-Orco” and a 2281 bp band corresponding to “P2A-Zeo^R-P2A-Or47a”, whereas in wild-type Pv11 cells the primers generated only a 619 bp band corresponding to the 3’-region of Pv.00443 gene (Fig. 1B, Fig. S3). The DmOr47a-expressing monoclonal cell line, named Pv11-00443-Or47a, was thus knocked-in as expected.

We also confirmed that genome editing did not affect the desiccation tolerance of Pv11-00443-Or47a. One hour after rehydration, dried Pv11-00443-Or47a showed a viability of 26.9%, which was comparable to the viability of wild-type Pv11 cells (Fig. 1C).

We next investigated the ligand response of Pv11-00443-Or47a. For reference, exposure to 50 µM ionomycin produced a maximal fluorescence response (average ∆F/F₀ = 0.84). The addition of the DmOrco agonist VUAA1 at 500 µM induced an average response of ∆F/F₀ = 0.53, confirming the sufficient expression of functional DmOrco (Fig. 1D). Furthermore, the ligand of Or47a, pentyl acetate (PA), at a concentration of 1 mM elicited an average fluorescent response ∆F/F₀ = 0.19, which was significantly higher than the response to the control solvent DMSO 0.5%, ∆F/F₀ = 0.05. Fluorescence imaging showed that most of the cells responded to 1 mM PA (Fig. 1D). These results show the successful engineering a Pv11-00443-Or47a sensor cell line that recognizes, with a fluorescence response, the odorant PA and the agonist VUAA1 before dry storage while retaining its desiccation tolerance.

Evaluation of the dose-response to PA in the Pv11-00443-Or47a cell line

Pv11-00443-Or47a was exposed to PA at concentrations between 0 and 1 mM, and the response was dose-dependent (Fig. 2A). Exposure to 1 µM PA did not induce a significant fluorescent response, compared to the control solvent 0.5% DMSO (Fig. 2A, B). In contrast, 10 µM PA induced a significantly higher response than control, and the amplitude of the response was then stable up to 1 mM (Fig. 2B). The dose-response curve exhibited a baseline at ∆F/F₀ = 0.04 and a maximal plateau response of ∆F/F₀ = 0.24 (Fig. 2B). The calculated EC₅₀ for PA was 3.4 µM (95% confidence interval (CI) 1.9 µM – 6.0 µM, n = 6–13).

Dried Pv11-00443-Or47a cells fully recover their function 24 h after rehydration

To investigate whether Pv11-00443-Or47a would retain its sensor function through desiccation and dry storage, the cells were desiccated and stored for 14 days in the dry state prior to rehydration. Response to PA was observed 24 h after rehydration. As in Pv11-00443-Or47a prior to desiccation, the cells showed a dose-dependent response to PA (Fig. 2C). Although some cells responded to PA at 1 µM (Fig. 2C), on average the response was not significant (∆F/F₀ = 0.05), compared to the control solvent 0.5% DMSO (Fig. 2D). However, concentrations of 5 µM, 10 µM, 100 µM, and 1 mM elicited significant increases in fluorescence (∆F/F₀ = 0.16, ∆F/F₀ = 0.13, ∆F/F₀ = 0.27 and ∆F/F₀ = 0.21, respectively), compared to the solvent control (Fig. 2D). The baseline of the dose-response curve was ∆F/F₀ = 0.04 and the maximal plateau was at ∆F/F₀ = 0.24 (Fig. 2D). The calculated EC₅₀ for PA in rehydrated cells was 8.1 µM (95% CI 4.6 µM – 14.2 µM, n = 7–16), which is slightly higher than the EC₅₀ prior to desiccation (compare Fig. 2B), but within the same magnitude order.

DmOrco and GCaMP6f partially retain their function after dry storage

The response of Pv11-00443-Or47a cells was first investigated just 1 h after rehydration. Rehydrated cells had a significant response to 50 µM ionomycin (Fig. 3A), showing that GCaMP6f was functional, although the fluorescence amplitude was 5-fold lower than the response observed for cells prior to desiccation (Figs. 1D and 3A). Similarly, rehydrated Pv11-00443-Or47a had a significant response to 500 µM VUAA1, showing that DmOrco retained function, although the fluorescence response was 3.3-fold weaker than prior to desiccation (Figs. 1D and 3A). In contrast, the cells did not show a significant response to 1 mM PA, suggesting that DmOr47a function was lost during dehydration and rehydration (Fig. 3A).

To verify whether the observed responses of rehydrated Pv11-00443-Or47a were due to dry-preserved DmOrco and GCaMP6f proteins or due to de-novo synthesized proteins, desiccated cells were rehydrated in culture medium containing the translation inhibitor cycloheximide (CHX). One hour after rehydration Pv11-00443-Or47a again showed significant responses to 50 µM ionomycin and 500 µM VUAA1, with fluorescence changes not significantly different from cells rehydrated without CHX, ∆F/F₀ = 0.17 and ∆F/F₀ = 0.14, respectively (Fig. 3A, B). Cells rehydrated with CHX once more showed no significant response to 1 mM PA (Fig. 3B).

Response to PA after rehydration depends on de novo protein synthesis

After 24 h post-rehydration, the cells showed significant responses not only to 500 µM VUAA1 and 50 µM ionomycin, but also to 1 mM PA, compared to the control DMSO, with average fluorescence change responses of ∆F/F₀ = 0.81, ∆F/F₀ = 1.03, and ∆F/F₀ = 0.21, respectively (Fig. 3C). The ligand responses were similar to, or even stronger than, those from cells prior to desiccation (Fig. 1D). Specifically, responses to 1 mM PA 24 h after rehydration and prior to desiccation were not significantly different (Fig. 1D). However, the responses to 500 µM VUAA1 and 50 µM ionomycin were significantly higher 24 h after rehydration (Fig. 3C) than before desiccation (Fig. 1D), with p-values of 0.000002 and 0.002211, respectively. These results show that not only DmOr47a, but also GCaMP6f and DmOrco fully recovered their function 24 h after rehydration.

Pv11-00443-Or47a rehydrated in the presence of the translation inhibitor CHX were also tested 24 h after rehydration. The results showed significant responses only to 50 µM ionomycin (∆F/F₀ = 0.16) and 500µM VUAA1 (∆F/F₀ = 0.12), but not to 1 mM PA (∆F/F₀ = 0.10) (Fig. 3D). This, in combination with the smaller amplitude of fluorescence response to ionomycin and VUAA1 reported above, suggests that the complete recovery of the PA response 24 h after rehydration (Figs. 2 and 3C) depends on de novo synthesis of GCaMP6f, DmOrco, and DmOr47a proteins.

In conclusion, Pv11-00443-Or47a cells in the dry state can at least partly preserve the function of intracellular GCaMP6f and membrane-bound DmOrco. In contrast, DmOr47a function was either not preserved or was too weak for detection just after rehydration, requiring de novo protein synthesis for full recovery of its ligand response.

Ligand responses and cell growth after rehydration

As presented above, the response of Pv11-00443-Or47a to PA was recovered 24 h after rehydration. To determine the time course of the response recovery to PA, cells were monitored during the 48 h after rehydration. As described previously, there was no significant response to 1 mM PA just 1 h after rehydration, nor was there a significant difference 6 h after rehydration (Fig. 4A). In contrast, the cells showed a significant response between 12 and 48 h after rehydration (Fig. 4A).

It is worth noting that the response to PA observed here was lower in amplitude (average ∆F/F₀ around 0.11) than in previous experiments (∆F/F₀ = 0.24) at the same 24 h mark (Figs. 2D and 3C). This difference is probably due to a lower cell viability after rehydration observed in this experiment (13% of live cells) compared to other experiments (Fig. 1C). In addition, the ∆F/F₀ of the control 0.5% DMSO was fluctuating between samples (Fig. 4A) and a variable proportion of the cells responded positively to the control 0.5% DMSO, which constitutes a problem to be solved in future works. Nevertheless, this variation of the response to 0.5% DMSO did not affect the significance of the responses to PA observed here (Fig. 4A).

The average raw fluorescence of individual cells was also obtained for both the baseline and at the highest response to 1 mM PA starting before desiccation and continuing for 48 h after rehydration (Fig. S4). The GCaMP6f fluorescence was lower by about 20% 1–6 h after rehydration, compared to cells before desiccation. Starting twelve hours after rehydration, the fluorescence recovered (Fig. S4). This suggests that not only DmOr47a, but also GCaMP6f require de novo protein synthesis.

One hour after rehydration, the concentration of live Pv11-00443-Or47a cells was 3.8 × 10⁵ cells/mL and remained stable within 48 h following rehydration (Fig. 4B). Significant cell growth was observed starting 72 h after rehydration and reached a plateau at 1.8 × 10⁶ cells/mL after 144 h (without changing culture medium) (Fig. 4B). Taken together, these data indicate that Pv11-00443-Or47a can significantly detect PA 12–48 h after rehydration, but after 72 h, this response is likely to be influenced by cell proliferation.

Discussion

In the present work, we successfully generated a Pv11 cell line stably expressing the functional insect OR, DmOr47a. The detection of its ligand, PA, occurred in a dose-dependent manner, with an estimated range of 5–100 µM. Importantly, Pv11-00443-Or47a cells retain desiccation tolerance and storability in the dry form at room temperature. We showed that (1) in the Pv11 cell system, DmOr47a showed an EC₅₀ of 1.9–6.0 µM for PA, consistent with values reported in other expression systems, (2) DmOr47a function was lost during dry-storage, but it recovered upon de novo protein synthesis 12–48 h after rehydration, and (3) the co-receptor protein DmOrco was partly preserved in the dry state, independently from de novo protein synthesis. Each of these points is discussed below, along with thoughts on dry storage and on limitations of this work.

Pv11-00443-Or47a cell line shows ligand responses within the range observed in other cultured cell expression systems

Pv11-00443-Or47a stably expresses GCaMP6f, DmOrco and DmOr47a. The three proteins were functional, with the latter two responding to their respective ligands (Fig. 1D). DmOr47a responded to PA in a dose-dependent manner with a calculated EC₅₀ of 3.4 µM (Fig. 2B), comparable to or better than the response reported in other cell lines. For example, DmOr47a expressed in HEK293 showed a similar EC₅₀ of 2.2 µM⁶. From another report in HEK293 cells, the apparent EC₅₀ for PA was 50 µM¹⁶. In Xenopus oocytes, a dose-dependent response of DmOr47a to PA was observed from 50 µM to 300 µM¹ (suggesting a higher EC₅₀). A high apparent EC₅₀ was also reported for PA in transgenic D. melanogaster expressing GCaMP6m and DmOr47a¹⁷. After rehydration, Pv11-00443-Or47a showed a somewhat higher calculated EC₅₀ of 8.1 µM (Fig. 2D), similar to the EC₅₀ of 14 µM reported for the co-expression of DmOr47a with DmOrco¹⁸. As shown by the values listed above, the responses of DmOr47a vary between expression systems. Even under highly standardized conditions, EC₅₀ values derived from receptor-ligand interactions often show 2–4-fold variability. These differences can be attributed to factors such as the amount of Or47a expressed at the surface of the cells, the proportion of co-receptor Orco (supplementary Fig. S2) or its origin¹⁸, the expression level and sensitivity of GCaMP protein, and the cell membrane environment.

After rehydration, DmOr47a function is restored upon de novo protein synthesis

One hour after rehydration, no significant response to PA was detected (Fig. 3A). However, the response to PA recovered to a level similar to that observed prior to desiccation 24 h after rehydration (Figs. 1D and 3C), but this recovery was not observed in the presence of a protein synthesis inhibitor (Fig. 3D). These results suggest that DmOr47a is insufficiently protected in dry cells to observe significant ligand response just after rehydration and that DmOr47a function is recovered upon de novo protein synthesis. Newly synthesized DmOr47a (and probably DmOrco and GCaMP6f) allowed recovery at a significant level starting 12 h after rehydration (Fig. 4A). Beyond 48 h after rehydration, cell growth resumed (Fig. 4B) but the resulting effect on the stability of PA detection remains to be investigated.

Concerning the stability of exogenous proteins, our previous work reported that GFP expression was stable through multiple passages for 1 year¹⁹, but the stability of exogenous proteins expressed in the cytosol following rehydration was only assessed for luciferase and suggested no significant change of activity 1 h after rehydration¹³. Here, our data suggest that cytosolic GCaMP6f was not fully preserved during the first 1–6 h post-rehydration (Fig. S4). Nevertheless, the remaining GCaMP6f was sufficient to reveal significant response of DmOrco to 500 µM VUAA1 even 1 h after rehydration (Fig. 3). Since the function of DmOr47a is directly dependent on the presence of functional co-receptor DmOrco, it is reasonable to conclude that de novo synthesis of both proteins is required to recover response to PA after 12 h of rehydration. The low fluorescence levels shortly after rehydration further suggest that de novo synthesis of GCaMP6f is also needed.

Co-receptor DmOrco was partially preserved in dry Pv11-00443-Or47a cells

One important finding here is that DmOrco protein retained at least partial function at least partially during dry storage and immediately after rehydration (Fig. 3). Our previous work showed that the enzyme luciferase can be preserved in the cytosol of desiccated Pv11 cells, showing enzymatic activity immediately after rehydration¹³. Using the same translation inhibitor experiment, we showed here that at least a fraction of DmOrco protein was preserved in the dry state and remained functional at the Pv11 cell surface immediately after rehydration. To our knowledge, this is the first report of dry preservation of a membrane protein in a cell culture orthologous expression system. (For further discussion, see supplementary material Sect. 2.1.). From previous reports, we expect that trehalose²⁰ and chaperone proteins such as Late Embryogenesis Abundant (LEA) proteins^21,22 played a protective role. The reason why DmOrco, but not DmOr47a, was preserved during dry storage is unclear, but we hypothesize that with only partial protection the expression of DmOr47a prior to desiccation was too low to allow detectable ligand response just after rehydration.

Potential of dry storage at room temperature for shipping and long-term preservation

Concerning the stability of dry-stored Pv11-00443-Or47a cells, most experiments in the present study were performed after a 14-days desiccation and dry storage period. Previous studies have shown that Pv11 cells are almost completely dried 36 h after transfer into a desiccator¹¹. Thus, the 14-day dry storage period ensured complete desiccation of the cells. Under these conditions, Pv11-00443-Or47a cells showed 27% viability one hour after rehydration, confirming that desiccation tolerance was not altered in this monoclonal cell line compared to the original Pv11 cells.

In previous studies, longer dry storage periods yielded lower viability. For example, dry storage of Pv11 cells for 9 months reduced cell viability to only 7% ¹¹. Similarly, luciferase-expressing Pv11 cells stored for more than one year showed less than 3% viability after rehydration; however, surviving cells still showed luciferase activity¹³.

In the present study, Pv11-00443-Or47a cells were desiccated in NARO, Japan and shipped to the University of Maryland, where the cells were preserved at room temperature for over 3 years (from July 2019 to September 2022) prior to successful rehydration. This demonstrates that OR-expressing Pv11 cells can be handled and preserved at room temperature, resulting in significantly lowering shipping cost, while remaining functional after rehydration and subsequent culture (Fig. S5). The cell line also retained some sensitivity to PA and VUAA1 through multiple desiccation-rehydration cycles and culture over a period of 4 years (from September 2020 through October 2024) (Fig. S6). Furthermore, individual cells responses to successive exposures to 1 mM PA in a perfusion assay showed that, even after multiple dry storage and culture passages, Pv11-00443-Or47a responses were not limited to a single exposure (Fig. S7); the cells are not easily damaged after a robust calcium response.

Limitations of this work and future perspectives

Although we showed that Pv11-00443-Or47a cells can be stored in the dry state, shipped at room temperature and stored for long periods while recovering their function after rehydration and subsequent passages, this work has limitations, and several issues must be addressed before practical application of these cells in odor sensors. First, the fluorescence signals from GCaMP6f are faint and require long exposure times (1–3 s) for detection. This limitation affects the stability and reproducibility of quantitative measurements. Furthermore, the viability rate of the cells after rehydration (i.e. the number of cells responding to the ligand) and their physiological state (i.e. the amount of OR in the cell membranes) are subject to variation and influence the stability of the responses to PA with a noticeable shift in EC₅₀ compared to the responses prior to desiccation (Fig. 2A and B).

To solve these problems, improvement of the expression system could include an increase in GCaMP6f, DmOrco and DmOr47a protein levels, which would allow brighter and more stable fluorescent responses to PA. Higher DmOr47a protein levels prior to desiccation would also be expected to lead to a significant response to PA immediately after rehydration.

DmOr47a was expressed in this work in a polycistronic system, producing a large protein including the Pv.00443 gene product, zeocin resistance protein, and DmOr47a (Fig. 1A). Subsequent cleavage of the P2A peptide generated three independent proteins. However, polycistronic expression systems with P2A peptide can show lower expression levels, especially for the protein in the third position²³. Recent work on Pv11 cells allowed efficient protein expression from genes directly under the control of the strong 121 promoter and inserted in newly identified genomic safe harbors^14,19. We anticipate that this alternative approach will enhance DmOr47a expression and allow response to its ligand immediately after rehydration at values close to those observed before desiccation.

The effect of cell viability on the responses to PA could potentially be overcome by normalizing the responses to an internal fluorescent marker or to the maximal response to VUAA1. Individual cell recordings could also mitigate response variability after rehydration.

Finally, although depending partly on cell viability and cell density, the response to the control 0.5% DMSO was variable between samples (e.g. Figure 4A, or Fig. 2D vs. Fig. 3C, D). It is unknown why a variable proportion of the cells in different experiments responded positively to DMSO, but possible reasons include culture conditions or cell physiology after rehydration. Since this variability of the control could affect the detection of faint odorant responses, it may be better to avoid the use of DMSO as a solvent in future work.

Conclusions

To summarize, DmOr47a, DmOrco, and GCaMP6f were successfully expressed in the desiccation-tolerant Pv11 cell line and showed a dose-dependent response to the DmOr47a ligand PA prior to desiccation and also following dry storage and 24 h after rehydration. Our data also provide the first evidence that a transmembrane protein, DmOrco, can be functionally preserved in the dry state in Pv11 cells. Future optimization of our expression system in Pv11 cells should enable stronger and more stable ligand responses and also increase the panel of ORs that can be functionally expressed in Pv11 cells.

This work represents a first step toward the development of cell-based odorant sensors storable at room temperature and practically usable outside of the laboratory. The use of Pv11 cells has several advantages for cell-based sensing. Since Pv11 cells are insect cells, they can be maintained in culture at room temperature without carbon dioxide gas. Secondly, Pv11 cells are non-adherent, floating in the medium, so the culture can be rapidly scaled up. Our long-term aim is to integrate dry-stored sensor Pv11 cells into miniaturized detector devices^24,25.

Methods

Cell culture

Pv11 cells were cultured as described previously^11,15. Briefly, Pv11 cells were cultured in IPL-41 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2.6 g/L tryptose phosphate broth (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 10% (v/v) fetal bovine serum, and 0.05% (v/v) of an antibiotic and antimycotic mixture (AA mix; penicillin, amphotericin B, and streptomycin; Millipore Sigma, Burlington, MA, USA). Cells were passaged at a concentration of 3 × 10⁵ cells/mL weekly and incubated at 25 °C.

Expression vectors for gene knock-in

Total RNA was extracted from dissected heads of Drosophila melanogaster (Canton-S) flies with ReliaPrep™ RNA Tissue Miniprep System (Promega, Madison, WI). RNA was then reverse transcribed to cDNA with PrimeScript II 1st strand cDNA Synthesis Kit (Takara, Kusatsu, Japan) and this cDNA was used as a template to clone DmOrco and DmOr47a coding regions. The GCaMP6f coding sequence was cloned from the plasmid vector pGP-CMV-GCaMP6f (Addgene #40755). The expression vectors constructed for transient expression (Supplementary methods 2.1.), namely pPv121-Orco (Supplementary data 8), pPv121-Or47a (Supplementary data 11), pPv121-GCaMP6f (Supplementary data 12), and an expression vector including Zeo^R, pCR4-Zeocin-P2A (Supplementary data 13), were used as templates and amplified by PCR with primers so that the encoding nucleotide sequence for the P2A peptide (GSGATNFSLLKQAGDVEENPGP) was added to the 5’-end of the sequences encoding each gene. PCR products were used as inserts and pCR4-Pv.00443#1µH-P2A-BbsI¹⁵ was used as a vector, which contains the guide RNA (gRNA) target and microhomology, P2A, and BbsI sequences. The vector was treated with the restriction enzyme BbsI (New England Biolabs) and PCR product inserts corresponding to P2A-GCaMP6f and P2A-Orco were assembled using NEBuilder HiFi Assembly kit (New England Biolabs), generating the donor vector pCR4-Pv.00443#1µH-P2A-GCaMP6f-P2A-Orco (Supplementary data 15). Alternatively, pCR4-Pv.00443#1µH-P2A-MCS (supplementary data 14) was used as a backbone vector containing the guide RNA (gRNA) target and microhomology, P2A, and multiple cloning site sequences. pCR4-Pv.00443#1µH-P2A-MCS and the PCR products corresponding to Zeocin-P2A and Or47a were treated with the restriction enzymes BamHI, HindIII and XhoI before performing a ligation that generated the donor vector pCR4-Pv.00443#1µH-P2A-Zeocin^R-P2A-Or47a (supplementary data 16). The primer oligonucleotides used to build these expression vectors are listed in table S2.

Knock-in into Pv11 genome and monoclonal cell selection

Integration of the target sequences into the Pv11 genome downstream to Pv.00443 gene was performed using the CRIS-PITCh genome editing method²⁶ as described previously¹⁵. In detail, a mixture of 0.3 µg of each donor vector obtained above, 5 µg of gRNA expression vector pPvU6b-DmtRNA-Pv.00443#1¹⁵, and 5 µg of Cas9 expression vector pPv121-SpCas9¹⁵ was transfected into Pv11 cells, following the electroporation protocol described previously¹² (for details, see Supplementary methods 3.2.). Transfected cells were cultured in 2 mL of supplemented IPL-41 medium for 5 days before performing zeocin selection treatment by inseminating 10⁵ cells/mL in supplemented IPL-41 medium containing zeocin at a final concentration of 400 µg/mL and incubating the cells for 1 week under zeocin selection. Then, half the medium was replaced by fresh supplemented IPL-41 medium containing zeocin (400 µg/mL), and the cells were again selected over a second week. After these 2 weeks of zeocin selection, cells were transferred into fresh supplemented IPL-41 medium without zeocin and cultured for one more week to let the selected cells grow. After this week of recovery, the cells were subjected to single cell sorting in order to obtain a monoclonal cell line. Single cell sorting was performed with a MoFlo Astrios cell-sorter (Beckman Coulter, Brea, CA) equipped with 355 and 488 nm lasers, as described previously¹⁵. One thousand wild-type Pv11 cells were seeded as feeder cells in each well of a 96-well plate prior to sorting. The cells were stained with DAPI (Dojindo, Kumamoto, Japan) and then DAPI and GCaMP6f were excited with 355 nm and 488 nm lasers, respectively. DAPI- and GCaMP6f-positive cells were selected for single cell sorting, and the obtained single cells were grown for 2 weeks with feeder cells. Then, zeocin selection was performed as above to eliminate the feeder cells. The obtained monoclonal cell line was named Pv11-00443-Or47a. The Pv11-00443-Or47a cell line was passaged and cultured at 25 °C for 8 days in a 25 cm² flask (BD Falcon, 353018) prior to a ligand binding assay.

Genomic PCR

Genomic DNA was extracted from the obtained monoclonal cells with FavorPrep Blood Genomic DNA Extraction Mini Kit (Favorgen, Taiwan). To confirm precise knock-in, the knock-in target region of the Pv.00443 gene was amplified by PCR using KOD One PCR Master Mix-Blue (Toyobo, Osaka, Japan) with the following primer set: 5′-GCCAAAGCGAGCCAATTCAA-3′ and 5′-GGGTGTTATTGCTACTTTAATGCGT-3′. The presence of the knock-in band was verified by electrophoresis of the PCR product. After gel purification of the PCR products, sequencing was carried out with BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Tokyo, Japan) and precise integration of the target genes into Pv11 genome was verified.

Glass coating for immobilizing cells

Experiments were performed in 96-well plates (EZview culture plate LB glass bottom; AGC Techno Glass, Shizuoka, Japan). The coating protocol was described previously²⁷. In detail, Cellmatrix type I-C (3 mg/mL; Nitta Gelatin Inc., Osaka, Japan) was diluted 10-fold in HCl (1 mM; pH 3), spread on the glass bottom of each well of the 96-well plate, and incubated for 60 min at room temperature for collagen coating. The wells were then washed twice with milliQ water and left to dry. Subsequently, BAM powder (Sunbright OE-040-CS; NOF corporation, Tokyo, Japan) was dissolved in dimethyl sulfoxide (DMSO) (Fujifilm Wako, Osaka, Japan) to obtain a 10 mM solution. This concentrated BAM solution was diluted with phosphate buffered saline (PBS(-): Fujifilm Wako) to a concentration of 100 µM in 1% DMSO. The BAM solution was added to each well of the collagen-coated 96-well plate and incubated for 60 min at 37 °C. The 96-well plate was then washed once with PBS(-) and twice with MilliQ water before drying.

Ligand binding assay

OR-expressing cells were diluted into modified artificial cerebrospinal fluid buffer (aCSF: 125 mM NaCl; 2.5 mM KCl; 1.25 mM Na₂HPO₄; 10 mM HEPES; 4.5 mM CaCl₂; pH 7.4) at a concentration of 1 × 10⁷ cells/mL and applied to each well of the BAM-coated 96-well plate as described above for 60 min at room temperature to achieve cell attachment. (Note: the aCSF contained no trehalose). Once cells adhered to the bottom of the wells, aCSF buffer with any remaining floating cells was removed and replaced by fresh aCSF buffer for the ligand binding assay.

Ionomycin (> 95% purity; Fujifilm Wako, Osaka, Japan) was used as an ionophore to artificially increase intracellular calcium concentration and evaluate the GCaMP6f fluorescence response. VUAA1 (> 98% purity; Sigma-Aldrich, Tokyo, Japan) was used as the DmOrco co-receptor agonist. The ligand PA (> 97% purity; Fujifilm Wako) was used to induce the responses of DmOr47a. These chemicals were first dissolved in dimethyl sulfoxide (DMSO; Fujifilm Wako) and then diluted in modified aCSF buffer at the following final concentrations: 1% DMSO, 100 µM ionomycin, 1mM VUAA1, and 2 mM PA. To evaluate the Or47a ligand dose-response, the PA was diluted to final concentrations of 2 µM, 20 µM, 200 µM and 2 mM in modified aCSF buffer, 1% DMSO. Fifty µL of these ligand solutions were added to the 50 µL aCSF buffer covering the attached cells and mixed gently so that the cells were exposed to a final ligand concentration diluted by one half.

Image acquisition was performed with an Axio Observer 7 fluorescence inverted microscope (Carl Zeiss, Oberkochen, Germany) equipped with a 10x objective (EC PlnN 10x/0.3 PhI DICI, Carl Zeiss) and a high-resolution camera (Axiocam 506 mono, Carl Zeiss), using Zen 3.2 (Zen pro) software. GCaMP6f fluorescence excitation was obtained with an LED at 475 nm through 38 HE Green filter set (EX BP 470/40, BS FT 495, EM BP 525/50). Nine frames, one taken every 10 s, were acquired at an exposure of 400 ms for a region of interest (ROI) of 2048 × 2048 pixels with resolution binning of 2 × 2. The first 3 frames corresponded to the baseline and the remaining 6 frames were acquired after addition of the ligand. These time lapse pictures were analyzed with Fiji (ImageJ v.2.1.0) software²⁸measuring the fluorescence intensities of the whole ROI or alternatively of individual cells for Fig. 2A and B and S7. The time series of fluorescence intensity change (∆F/F₀) was calculated as follows: ∆F/F₀ = (F_max-F₀)/F₀, where F₀ is the fluorescence intensity of the frame preceding ligand addition and F_max is the maximal fluorescence intensity measured during the 6 frames following ligand addition.

Evaluation of cell viability after desiccation, dry storage, and rehydration

Pv11 cells and Pv11-00443-Or47a cells were desiccated following the protocol described previously^11,19. Cells were first treated with a trehalose mixture (9 volumes of 600 mM trehalose for 1 volume of supplemented IPL-41 culture medium) at a concentration of 2 × 10⁷ cells/mL and incubated for 48 h at 25 °C. After trehalose treatment, cells were centrifuged at 300 g for 5 min and recovered into fresh trehalose mixture at a concentration of 1 × 10⁸ cells/mL. A drop of 40 µL of these cells in fresh trehalose mixture was deposited at the center of a tissue culture dish (BD Falcon, 353001). Twenty of these dishes, covered with their lids but unsealed, were transferred to a plastic desiccator (250 × 250 × 250 mm, AsOne, UD-1) containing 1 kg of silica gel (Toyota Kako Co., Ltd., Toyota Silica Gel) to reach a relative humidity < 10% at 25 °C. Cells were almost completely desiccated after 36 h¹¹. The cells were kept in the desiccator for 14 days.

Cells were rehydrated by adding 1 mL of supplemented IPL-41 medium to the tissue culture dish with dried cells and incubating at 25 °C. The trehalose concentration was thus reduced to 24 mM, which does not affect Pv11 growth. One hour after rehydration, cells were double-stained with Hoechst 33,342 (Hoechst; Dojindo, Kumamoto, Japan) at a final concentration of 2 µg/mL and with propidium iodide (PI; Dojindo, Kumamoto, Japan) at a final concentration of 0.75 µg/mL in supplemented ILP-41 culture medium.

Image acquisition was performed with an all-in-one fluorescence microscope (BZ-X710, Keyence). The excitation/emission wavelengths were 544 nm / 605 nm and 405 nm / 460 nm for PI and Hoechst, respectively. The number of dead cells (PI-positive cells) was subtracted from the total number of cells (Hoechst-positive cells) to obtain the number of live cells. Cell viability was expressed as a percentage of live cells, relative to the total number. For the ligand binding assay of rehydrated cells, the cells taken between 1 and 48 h after rehydration were collected by centrifugation at 300 g for 5 min and recovered into aCSF buffer. Cell attachment and ligand binding assays were performed as described above.

Assessment of protein dry preservation with a translation inhibitor

To examine if the ligand response observed after rehydration was due to dry-preserved proteins, i.e. proteins produced before desiccation that were preserved during the drying and rehydration process, or to de novo synthesized proteins, we used cycloheximide (CHX, Sigma-Aldrich, St Louis, MO), a translation inhibitor for eukaryotic cells that interferes with elongation in protein synthesis. Dried Pv11-00443-Or47a cells were rehydrated with 1 mL of complete ILP-41 medium containing CHX at 0.35 mM, an optimal concentration determined previously¹³. Cells were collected 1–24 h after rehydration and subjected to the ligand binding assay as described above, except that cells were recovered into aCSF buffer containing 0.35 mM CHX for cell attachment.

Statistics

GraphPad Prism 8 software (GraphPad, San Diego, CA) was used for statistical analyses. Differences between ligand samples and solvent control (DMSO 0.5%) were examined for statistical significance by one-way ANOVA corrected with post-hoc Dunnett test. In paired experiments (Fig. 4A), statistical differences were calculated with Student’s t test. Dose-response fit curves were obtained by nonlinear regression with the equation log(agonist) vs. response (three parameters) to determine bottom and top responses and EC₅₀.

Data availability

The DNA sequences generated during the current study are available in the NCBI repository with accession numbers PV368676, PV368677, PV368678, PV368679, PV368680, PV368681, PV368682, PV368683, and PV368684.

References

Sato, K. et al. Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452, 1002–1006. https://doi.org/10.1038/nature06850 (2008).
Article ADS CAS PubMed Google Scholar
Butterwick, J. A. et al. Cryo-EM structure of the insect olfactory receptor Orco. Nature 560, 447–452. https://doi.org/10.1038/s41586-018-0420-8 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Qin, C. et al. Artificial olfactory biohybrid system: an evolving sense of smell. Adv. Sci. (Weinh). 10, e2204726. https://doi.org/10.1002/advs.202204726 (2023).
Article CAS PubMed Google Scholar
del Mármol, J., Yedlin, M. A. & Ruta, V. The structural basis of odorant recognition in insect olfactory receptors. Nature 597, 126–131. https://doi.org/10.1038/s41586-021-03794-8 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Termtanasombat, M. et al. Cell-Based odorant sensor array for odor discrimination based on insect odorant receptors. J. Chem. Ecol. 42, 716–724. https://doi.org/10.1007/s10886-016-0726-7 (2016).
Article CAS PubMed Google Scholar
Zboray, K. et al. High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers. Sci. Rep. 13, 21757. https://doi.org/10.1038/s41598-023-47455-4 (2023).
Article ADS CAS PubMed PubMed Central Google Scholar
Heydarzadeh, S., Kia, S. K., Boroomand, S. & Hedayati, M. Recent developments in cell shipping methods. Biotechnol. Bioeng. 119, 2985–3006. https://doi.org/10.1002/bit.28197 (2022).
Article CAS PubMed Google Scholar
Nakahara, Y. et al. Cells from an anhydrobiotic chironomid survive almost complete desiccation. Cryobiology 60, 138–146. https://doi.org/10.1016/j.cryobiol.2009.10.004 (2010).
Article PubMed Google Scholar
Cornette, R. & Kikawada, T. The induction of anhydrobiosis in the sleeping chironomid: current status of our knowledge. IUBMB Life. 63, 419–429. https://doi.org/10.1002/iub.463 (2011).
Article CAS PubMed Google Scholar
Hinton, H. E. A fly larva that tolerates dehydration and temperatures of -270° to + 102°C. Nature 188, 336–337 (1960).
Article ADS Google Scholar
Watanabe, K., Imanishi, S., Akiduki, G., Cornette, R. & Okuda, T. Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration. Cryobiology 73, 93–98 (2016).
Article PubMed Google Scholar
Sogame, Y. et al. Establishment of gene transfer and gene silencing methods in a desiccation-tolerant cell line, Pv11. Extremophiles 21, 65–72. https://doi.org/10.1007/s00792-016-0880-4 (2017).
Article CAS PubMed Google Scholar
Kikuta, S. et al. Towards water-free biobanks: long-term dry-preservation at room temperature of desiccation-sensitive enzyme luciferase in air-dried insect cells. Sci. Rep. 7, 6540. https://doi.org/10.1038/s41598-017-06945-y (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Miyata, Y. et al. Identification of a novel strong promoter from the anhydrobiotic midge, with conserved function in various insect cell lines. Sci. Rep. 9 https://doi.org/10.1038/s41598-019-43441-x (2019).
Miyata, Y. et al. Cas9-mediated genome editing reveals a significant contribution of calcium signaling pathways to anhydrobiosis in Pv11 cells. Sci. Rep. 11, 19698 https://doi.org/10.1038/s41598-021-98905-w (2021).
Article ADS CAS Google Scholar
Miazzi, F. et al. Optimization of insect odorant receptor trafficking and functional expression via transient transfection in HEK293 cells. Chem. Senses. 44, 673–682. https://doi.org/10.1093/chemse/bjz062 (2019).
Article CAS PubMed PubMed Central Google Scholar
Lüdke, A., Kumaraswamy, A. & Galizia, C. G. Olfactory receptor responses to pure odorants in. Eur. J. Neurosci. 61 https://doi.org/10.1111/ejn.70036 (2025).
Takaku, T., Tonooka, Y., Takahashi, Y. & Kitamoto, S. Enhanced sensitivity of chimeric insect olfactory co-receptors for detecting odorant molecules. Biochem. Biophys. Res. Commun. 726 https://doi.org/10.1016/j.bbrc.150273 (2024).
Miyata, Y. et al. Identification of genomic safe harbors in the anhydrobiotic cell line, Pv11. Genes-Basel 13, 406. https://doi.org/10.3390/genes13030406 (2022).
Article CAS PubMed PubMed Central Google Scholar
Sakurai, M. et al. Vitrification is essential for anhydrobiosis in an African chironomid, Polypedilum vanderplanki. Proc. Natl. Acad. Sci. U S A. 105, 5093–5098 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Kikawada, T. et al. Dehydration-induced expression of LEA proteins in an anhydrobiotic chironomid. Biochem. Biophys. Res. Commun. 348, 56–61 (2006).
Article ADS CAS PubMed Google Scholar
Hatanaka, R. et al. An abundant LEA protein in the anhydrobiotic midge, PvLEA4, acts as a molecular shield by limiting growth of aggregating protein particles. Insect Biochem. Mol. Biol. 43, 1055–1067 (2013).
Article CAS PubMed Google Scholar
Liu, Z. Q. et al. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci. Rep. 7 https://doi.org/10.1038/s41598-017-02460-2 (2017).
Hirata, Y., Morimoto, Y., Nam, E. & Takeuchi, S. Portable biohybrid odorant sensors using cell-laden collagen micropillars. Lab. Chip. 19, 1971–1976. https://doi.org/10.1039/c9lc00131j (2019).
Article CAS PubMed Google Scholar
Choi, K. et al. In 2022 IEEE International Symposium on Circuits and Systems (ISCAS). 2092–2096 (IEEE).
Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K. T. & Yamamoto, T. MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the pitch systems. Nat. Protoc. 11, 118–133. https://doi.org/10.1038/nprot.2015.140 (2016).
Article CAS PubMed Google Scholar
Fuse, H., Kikawada, T. & Cornette, R. Effective methods for immobilization of non-adherent Pv11 cells while maintaining their desiccation tolerance. Cytotechnology 75, 491–503. https://doi.org/10.1007/s10616-023-00592-0 (2023).
Article CAS PubMed PubMed Central Google Scholar
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods. 9, 676–682. https://doi.org/10.1038/Nmeth.2019 (2012).
Article CAS PubMed Google Scholar

Download references

Acknowledgements

This work was mainly supported by NARO and, for work at UMD by the National Science Foundation under the following grants for UMD: NSF-EAGER: CBET 1842315, CBET BIOSENS 2316199, and EFRI ELiS 2318027. We thank Tomoe Shiratori and Yoko Saito for their help in the maintenance and preparation of cell cultures. We are also grateful to Masami Shimoda for providing the fruit flies that allowed cloning of OR genes and Ruilong Hu in the Araneda Lab for conducting preliminary experiments in pharmacology.

Author information

Authors and Affiliations

Division of Biomaterial Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), 1-2 Owashi, Tsukuba, Ibaraki, 305-0851, Japan
Hiroto Fuse, Richard Cornette, Shoko Tokumoto, Sachiko Shimura & Takahiro Kikawada
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwa, Chiba, 277-8562, Japan
Hiroto Fuse & Takahiro Kikawada
Department of Medical Chemistry, Medical Research Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, 113-8510, Japan
Yugo Miyata
Intractable Disease Research Center, Graduate School of Medicine, Juntendo University, Tokyo, 113-8421, Japan
Shoko Tokumoto
Department of Biology, College of Computer, Mathematical, and Natural Sciences, University of Maryland, Bioscience Research Bldg. R-1114, College Park, MD, 20742, USA
Ricardo C. Araneda
Department of Electrical and Computer Engineering, A. James Clark School of Engineering, University of Maryland, 2112 Glenn L. Martin Hall, College Park, MD, 20742, USA
Pamela Abshire
Department of Mechanical Engineering, A. James Clark School of Engineering, University of Maryland, 2112 Glenn L. Martin Hall, College Park, MD, 20742, USA
Roy Anderson, Redwan Haider & Elisabeth Smela

Authors

Hiroto Fuse
View author publications
Search author on:PubMed Google Scholar
Richard Cornette
View author publications
Search author on:PubMed Google Scholar
Yugo Miyata
View author publications
Search author on:PubMed Google Scholar
Shoko Tokumoto
View author publications
Search author on:PubMed Google Scholar
Sachiko Shimura
View author publications
Search author on:PubMed Google Scholar
Ricardo C. Araneda
View author publications
Search author on:PubMed Google Scholar
Pamela Abshire
View author publications
Search author on:PubMed Google Scholar
Takahiro Kikawada
View author publications
Search author on:PubMed Google Scholar
Roy Anderson
View author publications
Search author on:PubMed Google Scholar
Redwan Haider
View author publications
Search author on:PubMed Google Scholar
Elisabeth Smela
View author publications
Search author on:PubMed Google Scholar

Contributions

The authors confirm contribution to the paper as follows: study conception and design: RC, TK, HF, YM, ST; data collection: HF, RC, SS, RA, RH, ES; analysis and interpretation of results: HF, RC, RA, RCA, ES; draft manuscript preparation: HF, RC, ES, PA, RCA. Author. All authors reviewed the results and approved the final version of the manuscript.

Corresponding author

Correspondence to Richard Cornette.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Fuse, H., Cornette, R., Miyata, Y. et al. An anhydrobiotic cell line expressing odorant receptors shows odorant responses after dry storage. Sci Rep 15, 35533 (2025). https://doi.org/10.1038/s41598-025-19627-x

Download citation

Received: 14 March 2025
Accepted: 09 September 2025
Published: 10 October 2025
Version of record: 10 October 2025
DOI: https://doi.org/10.1038/s41598-025-19627-x