A food-grade cell dissociation agent via regulatory pre-check framework

Hiroaki Hatano; Misaki Sawada; Misaki Kitsukawa; Azusa Hayakawa; Hiroaki Kondo; Ikko Kawashima

doi:10.1371/journal.pone.0345921

. 2026 Apr 13;21(4):e0345921. doi: 10.1371/journal.pone.0345921

A food-grade cell dissociation agent via regulatory pre-check framework

Hiroaki Hatano ¹, Misaki Sawada ¹, Misaki Kitsukawa ¹, Azusa Hayakawa ¹, Hiroaki Kondo ¹, Ikko Kawashima ^1,^*

Editor: Shengqian Sun²

PMCID: PMC13075713 PMID: 41973685

Abstract

Advancements in cell-based foods require materials that meet the highest food safety standards. Conventional cell dissociation agents, such as animal-derived trypsin, pose significant risks including viral contamination and allergenicity, which hinder their use in food production. To overcome this challenge, we propose a food regulatory pre-check framework, which prioritizes regulatory compliance before functional testing. Following this framework, we developed iDisper, a novel food-grade animal-free cell dissociation reagent composed of approved food additives (papain and trisodium citrate). Specifically, an optimized formulation containing 2 mg/mL papain demonstrated a dissociation efficacy and viability comparable to those of commercial agents in primary avian cells. Furthermore, iDisper demonstrated 8-month stability at −20 °C. This study validated the proposed framework as an effective strategy for developing safe and practical reagents for the cell-based food industry.

Introduction

For commercially viable cell-based products, the entire production process must follow the same rigorous safety standards as conventional foods [1–3]. In the coming decade, compliance with established food safety management systems, such as HACCP and FSSC 22000, will likely be required [4]. These standards apply not only to final products but also to all raw materials including cell-based food. Therefore, comprehensive reassessment of manufacturing processes is essential. Recent reports on cell-based product safety emphasize that strictly controlling and characterizing all production inputs —including the composition of cell manipulation reagents— is a critical challenge for regulatory compliance [5,6]. This necessitates a fundamental shift in development philosophy from the earliest stages of product design; however, such a comprehensive approach has yet to be widely adopted in practice.

Cell dissociation is essential for both the initial cell isolation and subsequent passaging to expand the cell population [7]. However, trypsin, a commonly used cell dissociation agent, is typically derived from porcine sources. Its animal origin poses inherent contamination risks for food applications [8,9]. Alternatives, such as thermoresponsive polymers [10,11] and other commercial reagents, also face regulatory barriers. As exemplified by Japan’s Food Sanitation Act, many countries strictly control the use of substances through a positive list system [12]. Research-stage synthetic polymers are typically excluded from these lists, and agents with undisclosed compositions cannot be used in food manufacturing.

Regulatory scrutiny of animal-derived ingredients is a key hurdle in cell-based food production. Public FDA filings show that manufacturers consistently highlight the risk of viral contamination from porcine trypsin, often requiring additional safety validation [13–17]. Thus, whether from allergens or viruses, the use of animal-derived ingredients is a key consideration in cell-based food production.

Therefore, a dedicated upstream development process is crucial to integrate safe ingredient selection and consistent product definitions. In this study, an “upstream development process” is defined as a framework that extends beyond simply identifying functional ingredients to include: (1) assessing compliance with food regulations, (2) verifying performance, and (3) evaluating the industrial feasibility as the agent for this field. To demonstrate the effectiveness of this framework, its value lies not only in avoiding animal-derived components but also in selecting ingredients with well-characterized, manageable risks within established food safety paradigms.

As a model case, we developed a cell dissociation agent using food-grade ingredients, including papain, with a known allergen profile, including papain. This approach intentionally shifts the challenge of managing the unknown risks of non-food-grade or composition-undisclosed reagents to a known, visible hazard, providing a more transparent path to food safety. The effectiveness of this framework was demonstrated by selecting ingredients in accordance with food regulations and validating the agent’s performance against commercial alternatives. By establishing this foundational perspective, this study not only identifies future industrialization challenges but also paves the way for optimizing the framework through quantitative assessments of supply chain stability, costs, and relative risk.

Materials and methods

iDisper reagent preparation

The iDisper was prepared by mixing food-grade papain (W-40; Amano Enzyme, Aichi, Japan), trisodium citrate (Marugo Corporation, Saitama, Japan), and potassium chloride (Kanto Chemical, Tokyo, Japan) in purified water at final concentrations of 2, 0.37, and 28.72 mg/mL, respectively. The reagents were weighed using a precision electronic balance (FX-5000i; A&D, Tokyo, Japan) and added sequentially to water. To ensure complete dissolution, the mixture was stirred at 550 rpm using a magnetic stirrer for 1 h at room temperature (25°C). The solution was sterilized using a Nalgene™ Rapid-Flow™ Sterile Disposable Filter Unit (0.2 µm pore size; 431433; Thermo Scientific, MA, USA). iDisper was stored at −20 °C until use. Before use, the solution was thawed and heated to 37 °C in a water bath. The physicochemical properties were verified using a pH meter (FP20; Mettler Toledo, Switzerland) and an osmometer (Osmostat OM-6040; ARKRAY, Tokyo, Japan). The pH and osmolality were measured to ensure they fell within the ranges of 5.5–6.0 and 295 ± 5 mOsm/kg, respectively, without requiring any further adjustment. To validate sterility, the filtered solution was plated on Soybean-Casein Digest (SCD) agar and incubated at 35°C. Colony-forming units (CFU) were quantified using an automatic colony counter (Scan500; Interscience, Saint Nom la Bretêche, France). Internal quality control records confirmed 0 CFU across independent manufacturing lots. iDisper was stored at −20 °C until use.

Cell source, cell culture and evaluation method

Primary cells were isolated from the duck liver and chicken muscle. Fertilized duck eggs were purchased from Shiina Hatchery (Japan), and fertilized chicken eggs were obtained from Yamagishi (Wakayama, Japan). Both types of eggs (containing embryo tissue) were incubated in a P-008 incubator (Showa Furanki, Saitama, Japan) maintained at 37 °C and 60% humidity. The incubation periods were set to 14 and 12 days for the duck and chicken eggs, respectively, to ensure appropriate embryonic development. To ensure sterility, the egg surface was sterilized with ethanol, followed by immersion in sodium hypochlorite solution (Kitchen Haiter; Kao, Japan) for 30 min. Embryos were aseptically extracted in a laminar flow hood, and liver tissues were dissected. The tissues were mechanically dissociated in I-MEM without enzymes using a gentleMACS™ Dissociator (Miltenyi Biotec, Germany). The cell suspension was centrifuged at 300 × g for 4 min at room temperature, and the pellet derived from the two embryos was pooled and seeded into a single T175 flask to minimize individual variability. The chicken embryonic fibroblast cell line DF-1 (CRL-12203) was obtained from ATCC.

The cells were maintained and cultured under standard conditions (37°C, 5% CO2) in a basal medium, I-MEM [18], supplemented with 10% Yolk Lipoprotein (YLP), an egg-derived serum substitute [19]. Before cell seeding, the culture vessels were coated with the gelatin-based coating agent iCoater, as described in our previous study [20]. For each passage in the cell-expansion regimen, cells were initially seeded at 25% confluency (5 × 10⁶ cells/T175 flask) based on these counts and cultured until an approximate 4-fold expansion reached approximately 100% confluency (20 × 10⁶ cells/T175 flask), at which point they were detached using iDisper. The detached cells were then recovered by centrifugation at 300 × g for 5 min at room temperature. For specific experiments to determine cell doubling times, cells were cultured and analyzed as they reached approximately 70% confluency.

Cell dissociation efficacy was evaluated using 12-well plates. After removing the culture medium from each well and washing once with PBS (D-PBS(-) without Ca and Mg; 048−29805; FUJIFILM Wako, Tokyo, Japan), 1 mL of either pre-warmed iDisper or a control reagent (TrypLE™ Express; 12605010; Thermo Fisher Scientific, MA, USA) was added. The plates were incubated for 5 min, after which the cells were detached by pipetting. The resulting cell suspension was collected and centrifuged at 300 × g for 5 min at room temperature, and the cell number and viability were measured using an automated cell counter (NucleoCounter® NC-202™; ChemoMetec, Denmark) with Via2-Cassette (941−0024; ChemoMetec, Denmark) containing acridine orange and 4′,6-diamidino-2-phenylindole (DAPI) fluorescent dyes, according to the manufacturer’s instructions. The cell morphology before and after dissociation was observed using a phase-contrast microscope (BZ-X810; Keyence, Tokyo, Japan).

Primary duck-liver-derived cells were seeded in 6-well plates. For the assay, the culture medium was aspirated, and the wells were washed once with 2 mL of I-MEM. Subsequently, iDisper solutions (0.5 mL) containing papain at different concentrations (1, 2, or 4 mg/mL) or the control reagent TrypLE Express were added to each well. Plates were incubated at 37°C, and cell detachment was monitored and imaged at 0, 5, and 10 min using a phase-contrast microscope. After the final point, I-MEM (2.5 mL) was added, and the cells were thoroughly suspended by pipetting. The cell suspension was transferred to a 15 mL tube and centrifuged at 300 × g for 3 min at room temperature. The supernatant was removed, and the cell pellet was resuspended in 1 mL of PBS. The live cell number and particle size distribution were determined using the Cell Counter model R1 (Olympus, Tokyo, Japan) in S1, S2 Figs. For proliferation analysis (S3 Fig), cell confluence (percentage of occupied area) was monitored using the IncuCyte® SX5 Live-Cell Analysis System (Sartorius, Germany). Time-lapse images were captured, and confluence was quantified using the system’s integrated software.

RNA extraction and sequencing

Total RNA was extracted from cell samples of Anas platyrhynchos domestica at various stages of culture using a standard RNA extraction kit, following the manufacturer’s protocol. RNA concentration and purity were assessed using spectrophotometry. After quality assessment, the total RNA samples were sent to Rhelixa (Chiba, Japan) for library preparation and sequencing. Strand-specific libraries were prepared using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and the NEBNext® Ultra™ II Directional RNA Library Prep Kit (Illumina, US). Sequencing was performed using an Illumina NovaSeq 6000 platform (Illumina, US), generating 150 bp paired end reads.

Bioinformatic analysis

Bioinformatic analysis was conducted using Rhelixa. First, the quality of the raw sequence reads was assessed using FastQC (v0.11.7). Adapter sequences and low-quality bases were then trimmed using Trimmomatic (v0.38). The trimmed reads were subsequently aligned to the A. platyrhynchos domesticus reference genome (ZJU1.0, GCF_015476345.1) using HISAT2 (v2.1.0). Gene expression levels were quantified from the mapped reads using featureCounts software (v1.6.3). Raw read counts were normalized and expressed as transcripts per million (TPM) to enable comparisons across samples. The expression profiles of specific genes of interest, such as albumin (ALB) and hepatocyte nuclear factor 4α (HNF4A), were visualized from these TPM values. Differentially expressed genes (DEGs) between sample groups were identified using DESeq2 (v1.24.0).

Mycoplasma detection

To ensure sterility, mycoplasma contamination was assessed in duck liver-derived cells (passages 2 and 5) propagated using the iDisper. Genomic DNA was extracted using the Dneasy® Blood & Tissue Kit (69504; Qiagen, Germany) according to the manufacturer’s instructions. PCR amplification was performed using the e-Myco™ plus Mycoplasma PCR Detection Kit (25237; iNtRON Biotechnology, Korea). The amplification products were visualized using gel electrophoresis to confirm the absence of mycoplasma bands (S4 Fig).

Statistical analysis

All data are expressed as the mean ± standard deviation (SD) from at least three independent biological replicates. Statistical significance was assessed using the Student’s t-test for two-group comparisons or one-way ANOVA with a Bonferroni post-hoc test for multiple comparisons. Statistical analyses were performed using Python with the StatsModel library.

Results and discussion

A food regulatory pre-check framework for upstream development in the cell-based food industry

To commercialize the cell-based food industry, it is essential to develop industrial products that meet strict food safety standards. However, because the industry is still in its infancy, there is generally no concept of a development process, particularly upstream development, that includes the industry’s unique characteristics. Therefore, we proposed a new upstream development framework “Food Regulatory Pre-check” for cell-based food ingredients (Fig 1). The conventional upstream development process typically involves selecting raw ingredients based on their functionality (Step 1) and then immediately preceding to a performance evaluation (Step 3). However, as with trypsin, this approach risks the conclusion that an ingredient performs well but is unsuitable as food.

Fig 1 — The proposed four-step process begins with (1) Concept Planning, where markets are defined. The core of the framework is (2) Ingredient Screening utilizes the “Food Regulation Pre-Check” (yellow box) as a gatekeeper. This step filters infinite candidates down to a finite compliant list based on global regulations and cultural safety (e.g., Kosher status). Viable candidates then undergo (3) a Feasibility Test, where performance is validated against key metrics. Finally, the lead candidate proceeds to (4) Product Profile Definition, which marks the transition to the downstream development process.

A notable feature of our framework is that it prioritizes incorporating “food regulatory compliance assessment” as Step 2. In this step, raw ingredients are rigorously screened for approval as “food” based on criteria set out in Japan’s Food Sanitation Act (Table 1). Consequently, many existing options, such as trypsin (animal-derived), temperature-responsive polymers (not listed), and EDTA (limited use), can be eliminated at this stage. In contrast, trisodium citrate (INS No. 331(iii)) and papain (INS No. 1101(ii)), which are listed as food additives in the International Food Standard Codex Alimentarius, are the most reasonable options from the perspective of regulations, including global imports and exports.

Table 1. Multi-faceted evaluation of candidate cell dissociation technologies based on food safety and manufacturing principles.

Category	Ingredients	Origin/ Overview	Cell Culture Precedent	Codex Number	Singapore	USA	Israel	Japan	Allergen
Plant-Derived Enzymes	Papain #	Papaya-derived	〇	〇 (INS No. 1101(ii))	〇	〇 (21 CFR 184.1585)	〇	〇	×
	Actinidin	Kiwi-derived	×	×	〇	〇	〇	〇	×
	Ficin	Fig-derived	×	〇 (INS No. 1101(iv))	〇	〇 (21 CFR 184.1316)	〇	〇	×
Animal-Derived Enzymes	Trypsin	Porcine pancreas-derived	〇	×	〇	〇 (21 CFR 184.1914	△ (Non Kosher)	〇	×
Animal-Derived Enzymes	Pepsin	Digestive enzymes from pigs, etc.	×	×	〇	〇 (21 CFR 184.1595)	△ (Non Kosher)	〇	×
Chelating Agents	Trisodium citrate #	Organic acid found in citrus fruits	〇	〇 (INS No. 331(iii))	〇	〇 (21 CFR 184.1751)	〇	〇	〇
Chelating Agents	EDTA	Chemically synthesized	〇	〇 (INS No. 385)	〇	〇 (21 CFR 573.360)	△ (Use limit)	△ (Use limit)	〇
Recombinant Enzymes	Recombinant Collagenase	Microbial origin. Degrades collagen. Standard reagent for 3D culture dissociation, etc.	〇	×	×	×	×	×	〇
Others	Thermoresponsive polymer	Synthetic polymer, etc.	×	×	×	×	×	×	〇

Open in a new tab

Legend; 〇: Proven track record, △: Limited track record, × : Significant challenges, #: Materials selected in this study.

The efficiency of this framework is quantitatively supported by the “reduction of search space.” Traditional reagent development often requires the screening of vast, effectively infinite libraries of synthetic compounds. In contrast, this framework utilizes the “Positive List” system of food regulations as a beneficial constraint, immediately reducing the candidate pool from an infinite number to a finite, manageable list at the start of development (Day 0). For example, the number of enzyme preparations with established specifications in the WHO JECFA Database [21] is limited to approximately 100 distinct source organisms. This drastic reduction in the screening scope mathematically validates the high efficiency of this framework compared with conventional blind screening.

This framework also addresses practical business and cultural issues. First, checking regulations early ensures that the product complies with global standards. Second, it ensures “cultural safety” by including religious criteria (e.g., Kosher status in Table 1) from the start. Finally, it aids corporate decision-making. In business, it is often difficult to stop a project once a budget is assigned for testing (Feasibility Test). By requiring a regulatory “Pre-Check,” this framework creates a clear decision point to prevent wasted resources and establishes a formal standard for the industry.

Performance evaluation of iDisper: Dissociation performance, cell versatility, and long-term stability

The iDisper performance was evaluated using an industrially relevant cell population. We used primary duck liver-derived cells, which are used as raw materials for cell-based food. Under standard culture conditions, these cells proliferated as adherent cells and were predominantly mesenchymal cells. To evaluate the performance of the pre-culture step on cell quality, we compared gene expression profiles in cells at an early stage of passage (P0) and late passages (P2 and P6) (Fig 2). As a result, the expression of ALB and HNF4A, which are hepatocyte maturation markers [22], was significantly decreased in P2 and P6 cells compared to that in P0 cells. In addition, the expression levels of COL3A1 and DCN, which are highly expressed in mesenchymal cells [23], were significantly increased. Hepatocytes generally have a low proliferation profile [24]. This suggests that a cell population with a higher ability to adhere to the planar substrate and that is suitable for proliferation may have been selected. This indicated that the characteristics of the cells selected in the standard 2D cell culture were maintained.

Fig 2 — Gene expression in duck liver-derived cells was analyzed at passage 0 (P0), P2, and P6. Expression of hepatocyte maturation markers ALB and HNF4A decreased significantly by P2. Conversely, expression of mesenchymal markers COL3A1 and DCN, associated with proliferation, increased significantly by P2. These changes suggest the selection of a more proliferative cell population during culture expansion. Gene expression values are shown in arbitrary units (a.u.).

Development of iDisper began with trisodium citrate as a gentle baseline chelating agent, considering the delicacy of primary cells [25]. However, when tested on primary duck liver-derived cells, citrate alone failed to achieve complete detachment, as shown by microscopy and low cell counts (Fig 3a-3c). Particle size analysis indicated the presence of large aggregates with citrate alone (S1 Fig). To improve efficacy, we investigated the addition of food-grade protease papain. A comparative evaluation demonstrated that the combination of trisodium citrate and papain significantly improved cell dissociation. A papain concentration of 2 mg/mL yielded a dissociated cell number and a single-cell particle size distribution comparable to the commercial reagent TrypLE™ Express (S2 Fig). In contrast, 1 mg/mL was insufficient for effective dissociation. Although a concentration of 4 mg/mL also induced detachment, it caused a significant delay in subsequent cell proliferation compared to 2 mg/mL (S3 Fig). Although papain alone was effective, the iDisper formulation was finalized by combining papain with trisodium citrate. This design was chosen to mimic the dual mechanism of standard trypsin-EDTA reagents, utilizing proteolysis (2 mg/mL papain) and chelation (citrate) to target distinct cell adhesion factors.

Furthermore, iDisper consistently supported robust expansion through serial passaging, simulating a cell-based food production line (Fig 3d-3e). However, the 4.1% failure rate at passage 0 indicates a limitation of iDisper in overcoming the inherent variability in primary avian cells. This instability at the initial isolation stage acts as a bottleneck, potentially reducing overall production efficiency. Therefore, optimizing the iDisper for robust primary isolation while being effective for expansion remains a critical challenge.

Mycoplasma testing confirmed that the cells passaged with different lots of iDisper (P2 and P5) were negative for contamination (S4 Fig).

To evaluate its broader utility, we tested the iDisper on other avian cell types, including primary chicken muscle cells and DF-1. The iDisper achieved robust cell numbers and high viability across all cell types (Fig 4a). The product showed excellent storage stability, with no performance loss after 8 months at −20 °C (Fig 4b). The detachment performance decreased to approximately 30% after 12 months (S5 Fig). However, a limitation of the current study is that the dissociation efficacy of iDisper was exclusively validated using avian cells. Given that bovine and porcine species are next targets for the cell-based food industry, future studies should validate this versatility across a broad range of industrially relevant species, including mammalian and aquatic cells.

Fig 4 — Performance of freshly prepared iDisper (“Fresh”), iDisper stored for 8 months at −20 °C (“8 month”), and a commercial control (“Reagent”) was compared on duck liver-derived cells **(a)**, chicken muscle-derived cells **(b)**, and the DF-1 cell line **(c)**. Blue and black bars represent total and live cell numbers, respectively. Data are presented as mean ± SD (n = 3). Statistical analyses were performed separately for total and live cell counts using Student’s t-test followed by Bonferroni correction. * p < 0.05, ** p < 0.01 compared to the Reagent group.

We compared iDisper to the animal-free recombinant enzyme TrypLE™ Express to highlight two viable paths for the food industry: the food-grade approach and the recombinant protein approach. Our results demonstrated that iDisper was robust and effective, thus validating the food-grade path for scalable expansion.

Industrial production of food-grade cell culture materials and future challenges

The upstream development process proposed in this study must be integrated with the downstream development process to bring products to market. In a previous study, we established a heat-based sterilization process [20]. However, as iDisper’s core component, papain, is heat-sensitive, an industrial-scale product is likely to be a powder. This is a common formulation for food ingredients such as seasonings or powdered soup bases. Future developments must address key manufacturing parameters for powders, such as controlling the particle size to ensure product uniformity, solubility, and stability [26,27]. Currently, the specific limits of scalability for this powder formulation remain unquantified. Moving beyond laboratory-scale proof-of-concept will necessitate rigorous quantitative evaluations to define the maximum viable batch sizes and the precise moisture thresholds acceptable for long-term storage.

The iDisper is transparently treated as a dispersion with defined allergen characteristics, enabling its management within food safety framework. This directly contrasts with agents with secret compositions, which pose “hidden risks,” make appropriate food safety assessments difficult, and contribute to serious issues such as food allergen recalls in the conventional food industry [28].

By contrast, our framework yields products with visible, manageable risks. The complete composition and our proposed labeling, conceptually based on Japanese food labeling standards, are shown in (Supporting Information; S1 Table). When compared to recent FDA pre-market consultations for cell-cultured meat [13–17]—which rigorously evaluate whether allergenic processing aids remain in the final product—our approach similarly ensures safety by transparently declaring known allergens (e.g., “Contains enzymes derived from papaya”) A logical consequence is that the final cell-based food product may require allergen labeling to ensure consumer safety and choice, which is a standard and established practice in the food industry [29]. The core contribution of this study is to provide transparent starting materials that make allergen risk manageable, shifting the industry’s approach from dealing with unknown variables to overcoming known ones. Transparency is an essential step for downstream producers to validate allergen removal or inactivation processes. For instance, a practical manufacturing process would likely require validated mitigation steps, such as post-dissociation washing steps or terminal heat treatment to inactivate residual enzymes. In terms of temporal stability, the allergen remained structurally robust throughout the process. Our data (Fig 4b) showed stability for 8 months at −20°C, and Vicente et al. [30] reported that papain retains its stability with a half-life of 10 days in aqueous media. This quantitative evidence indicates that the allergen does not degrade naturally during cell dissociation, necessitating active removal strategies.

Conclusions

This study proposed and validated a food regulatory pre-check framework for developing safe, practical, and animal-free materials for the cell-based food industry. The resulting reagent, iDisper, demonstrated dissociation efficacy comparable to that of commercial alternatives and excellent long-term stability. This study shifts the paradigm from managing unknown risks in conventional reagents to addressing the visible and manageable risks of transparently formulated food-grade materials. Future studies should optimize industrial production and validate allergen mitigation strategies.

Supporting information

S1 Fig. Particle size distribution of duck liver-derived cells immediately after detachment.

Green and red bars indicate live and dead cells, respectively. The brightfield images below each graph show the cell suspension used for the measurement. Observationally, the particle size distribution for the iDisper condition (+ Trisodium Citrate + Papain) showed a primary peak between 10–15 µm, similar to the Commercial Reagent. In contrast, the Trisodium Citrate alone and the Negative control conditions showed a main peak closer to 10 µm with a broader distribution, suggesting the presence of cell aggregates. These data are presented as a qualitative reference.

(TIF)

pone.0345921.s001.tif^{(7.5MB, tif)}

S2 Fig. Optimization of papain concentration for effective cell dissociation.

Phase-contrast microscopy images of primary duck liver cells before treatment (Before) and at 10 min after (After) the addition of iDisper containing papain at 1, 2, or 4 mg/mL. The control reagent, TrypLE™ Express, was treated for 5 min. The “After pipetting” column shows the plate surface after cell collection. All cultures were confluent before treatment. The bar chart shows the number of live cells recovered after detachment. Data are presented as mean ± SD (n = 3). Scale bar = 1 mm.

(TIF)

pone.0345921.s002.tif^{(6.7MB, tif)}

S3 Fig. Post-detachment proliferation of cells treated with different papain concentrations.

Growth curves of primary duck liver cells over approximately 7 days following detachment with iDisper containing papain at 1, 2, or 4 mg/mL, or with the control reagent TrypLE™ Express. Cell proliferation was evaluated by measuring cell confluence (%) over time using an IncuCyte® live-cell imaging and analysis system. Data are presented as mean ± SE (n = 3).

(TIF)

pone.0345921.s003.tif^{(7.9MB, tif)}

S4 Fig. Validation of sterility: Mycoplasma PCR test.

Duck liver cells passaged with iDisper were tested using the e-Myco™ plus Kit across three independent lots (Lots A, B, and C). Lanes: Marker: DNA ladder; Positive Control (kit): Positive control showing specific target bands (including 270 bp); Duck liver-derived cell (P2, Lot A): Cells at Passage 2 (n = 3); Duck liver-derived cell (P2, Lot B): Cells at Passage 2 (n = 3); Duck liver-derived cell (P5, Lot C): Cells at Passage 5 (n = 2); Water: Negative control. The presence of internal control bands in all sample lanes confirms successful PCR amplification, while the absence of the specific 270 bp band confirms that all cell cultures were free of mycoplasma contamination.

(TIF)

pone.0345921.s004.tif^{(5.7MB, tif)}

S5 Fig. Evaluation of iDisper stability after long-term storage.

The cell detachment performance of iDisper stored for 12 months at －20°C was compared against that of a commercial reagent. Data are presented as mean ± SD (n = 3). The cell recovery rate after 12 months was below 50% of the control, which did not meet our internal criteria for further statistical analysis.

(TIF)

pone.0345921.s005.tif^{(2.6MB, tif)}

S1 Table. Proposed food label for iDisper designed to declare its ‘manageable risk’.

This label demonstrates our framework for avoiding unknown risks in favor of managing known risks. As established in our previous study [20], classifying products as food and providing transparent information is fundamental for reliable safety management in cellular agriculture.

(DOCX)

pone.0345921.s006.docx^{(16.7KB, docx)}

S1 File. Raw data for Fig2–4’.

(ZIP)

pone.0345921.s007.zip^{(8.2MB, zip)}

S2 File. Raw data for S1-5 Fig.

(ZIP)

pone.0345921.s008.zip^{(89.8MB, zip)}

Acknowledgments

The authors express their sincere gratitude to food regulatory experts and government officials who provided invaluable advice and insights for this study. We also thank all members of the Production Development Department at IntegriCulture Inc. for their helpful discussions

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by the Small Business Innovation Research Program of the Ministry of Agriculture, Forestry and Fisheries of Japan under the “Demonstration of a Production System for Cellular Foods Utilizing CulNet Supernatant” (N020) project. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Post MJ, Levenberg S, Kaplan DL, Genovese N, Fu J, Bryant CJ, et al. Scientific, sustainability and regulatory challenges of cultured meat. Nat Food. 2020;1(7):403–15. doi: 10.1038/s43016-020-0112-z [DOI] [Google Scholar]
2.Stephens N, Di Silvio L, Dunsford I, Ellis M, Glencross A, Sexton A. Bringing Cultured Meat to Market: Technical, Socio-Political, and Regulatory Challenges in Cellular Agriculture. Trends in Food Science & Technology. 2018;78:155–66. doi: 10.1016/j.tifs.2018.04.010 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Navare S, El-Moghazy AY. Food safety considerations in the advancement of cultured meat: Evaluating novel ingredients. Future Foods. 2025;11:100639. doi: 10.1016/j.fufo.2025.100639 [DOI] [Google Scholar]
4.Ovissipour R, Yang X, Saldana YT, Kaplan DL, Nitin N, Shirazi A, et al. Cell-based fish production case study for developing a food safety plan. Heliyon. 2024;10(13):e33509. doi: 10.1016/j.heliyon.2024.e33509 [DOI] [PMC free article] [PubMed] [Google Scholar]
5.World Health Organization WHO, Food and Agriculture Organization of the United Nations FAO. Food safety aspects of cell-based food. 2023. doi: 10.4060/cc4855en [DOI] [Google Scholar]
6.Ong KJ, Johnston J, Datar I, Sewalt V, Holmes D, Shatkin JA. Food safety considerations and research priorities for the cultured meat and seafood industry. Compr Rev Food Sci Food Saf. 2021;20(6):5421–48. doi: 10.1111/1541-4337.12853 [DOI] [PubMed] [Google Scholar]
7.Segeritz CP, Vallier L. Cell culture: growing cells as model systems in vitro. In: Jalali M, Saldanha FYL, Jalali M. Basic science methods for clinical researchers. Boston: Academic Press. 2017. 151–72. doi: 10.1016/B978-0-12-803077-6.00009-6 [DOI] [Google Scholar]
8.Merten O-W. Virus contaminations of cell cultures - A biotechnological view. Cytotechnology. 2002;39(2):91–116. doi: 10.1023/A:1022969101804 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Pinheiro de Oliveira TF, Fonseca AA, Camargos MF, de Oliveira AM, Pinto Cottorello AC, Souza A dos R, et al. Detection of Contaminants in Cell Cultures, Sera and Trypsin. Biologicals. 2013;41(6):407–14. doi: 10.1016/j.biologicals.2013.08.005 [DOI] [PubMed] [Google Scholar]
10.Yamato M, Konno C, Utsumi M, Kikuchi A, Okano T. Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture. Biomaterials. 2002;23(2):561–7. doi: 10.1016/S0142-9612(01)00138-7 [DOI] [PubMed] [Google Scholar]
11.Thummarati P, Laiwattanapaisal W, Nitta R, Fukuda M, Hassametto A, Kino-Oka M. Recent Advances in Cell Sheet Engineering: From Fabrication to Clinical Translation. Bioengineering (Basel). 2023;10(2):211. doi: 10.3390/bioengineering10020211 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Magnuson B, Munro I, Abbot P, Baldwin N, Lopez-Garcia R, Ly K, et al. Review of the regulation and safety assessment of food substances in various countries and jurisdictions. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2013;30(7):1147–220. doi: 10.1080/19440049.2013.795293 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Good Meat, Inc, Polsinelli PC, AR Toxicology, Burdock Group Consultants. Dossier in support of the safety of Good Meat cultured chicken as a human food ingredient. 2022. https://www.fda.gov/media/166346/ [Google Scholar]
14.Wildtype. Pre-Market Safety Consultation Summary Prepared for the US Food and Drug Administration; Cell Culture Consultation 000005; FDA, 2022. https://www.fda.gov/media/186754/download?attachment [Google Scholar]
15.Mission Barns. Mission Barns Cultivated Animal Fat Cells (Pork) Complete Consultation Data Package Public Version. 2022. https://www.fda.gov/media/185700/download?attachment [Google Scholar]
16.UPSIDE Foods. Premarket notice for integral tissue cultured poultry meat; cell culture consultation 000002. 2021. https://www.fda.gov/files/food/published/CellCultureConsultation-000002-Submission-03222023.pdf [Google Scholar]
17.Believer Meats. Safety evaluation of cultured chicken cells produced by Believer Meats. 2024. https://www.fda.gov/media/188066/download?attachment [Google Scholar]
18.Kanayama T, Tanaka K, Shimada H, Ichiyama K, Higuchi S, Kawashima I. Improving the Safety of Cultured Meat Using Basal Medium Prepared Using Food Ingredients. BioRxiv. 2022. doi: 10.1101/2022.03.28.486069 [DOI] [Google Scholar]
19.Murakami H, Okazaki Y, Yamada K, Omura H. Egg yolk lipoprotein, a new supplement for the growth of mammalian cells in serum-free medium : Egg yolk lipoprotein for serum-free growth of cells. Cytotechnology. 1988;1(2):159–69. doi: 10.1007/BF00146817 [DOI] [PubMed] [Google Scholar]
20.Hatano H, Sawada M, Kitsukawa M, Hayakawa A, Kondo H, Kawashima I. A food-grade ready-to-use cell culture coating agent developed via food manufacturing: A dual technical and regulatory approach. ChemRxiv. 2025. doi: 10.26434/chemrxiv-2025-9gfsq [DOI] [Google Scholar]
21.World Health Organization WHO, Food and Agriculture Organization of the United Nations FAO. JECFA Online Database: Evaluations of the Joint FAO/WHO Expert Committee on Food Additives. https://apps.who.int/food-additives-contaminants-jecfa-database/ [Google Scholar]
22.Odom DT, Zizlsperger N, Gordon DB, Bell GW, Rinaldi NJ, Murray HL, et al. Control of pancreas and liver gene expression by HNF transcription factors. Science. 2004;303(5662):1378–81. doi: 10.1126/science.1089769 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Schaefer L, Iozzo RV. Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction. J Biol Chem. 2008;283(31):21305–9. doi: 10.1074/jbc.R800020200 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Ueyama-Toba Y, Tong Y, Yokota J, Murai K, Hikita H, Eguchi H, et al. Development of a hepatic differentiation method in 2D culture from primary human hepatocyte-derived organoids for pharmaceutical research. iScience. 2024;27(9):110778. doi: 10.1016/j.isci.2024.110778 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Nie Y, Walsh P, Clarke DL, Rowley JA, Fellner T. Scalable passaging of adherent human pluripotent stem cells. PLoS One. 2014;9(1):e88012. doi: 10.1371/journal.pone.0088012 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Wünsch I, Finke JH, John E, Juhnke M, Kwade A. The influence of particle size on the application of compression and compaction models for tableting. Int J Pharm. 2021;599:120424. doi: 10.1016/j.ijpharm.2021.120424 [DOI] [PubMed] [Google Scholar]
27.Deng T, Sousa LM, Garg V, Bradley MSA. Segregation of formulated powders in direct compression process and evaluations by small bench-scale testers. Int J Pharm. 2023;647:123544. doi: 10.1016/j.ijpharm.2023.123544 [DOI] [PubMed] [Google Scholar]
28.Yue SR, Shrivastava R, Campbell K, Walker MJ. Food allergen recalls in the United Kingdom: A critical analysis of reported recalls from 2016 to 2021. Food Control. 2023;144:109375. doi: 10.1016/j.foodcont.2022.109375 [DOI] [Google Scholar]
29.FAO, WHO. Risk assessment of food allergens – part 3: Review and establish precautionary labelling in foods of the priority allergens. 2023. doi: 10.4060/cc6081en [DOI] [Google Scholar]
30.Vicente LC, Aires-Barros R, Empis JM. Stability and proteolytic activity of papain in reverse micellar and aqueous media: a kinetic and spectroscopic study. J Chem Technol Biotechnol. 1994;60(3):291–7. doi: 10.1002/jctb.280600310 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0345921.r001

Decision Letter 0

Shengqian Sun

21 Dec 2025

Dear Dr. Kawashima,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 30 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ’Submissions Needing Revision’ folder to locate your manuscript file. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ‘Submissions Needing Revision’ folder to locate your manuscript file.. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ’Submissions Needing Revision’ folder to locate your manuscript file. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ‘Submissions Needing Revision’ folder to locate your manuscript file.

A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled ‘Response to Reviewers’.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled ‘Revised Manuscript with Track Changes’.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled ‘Manuscript’.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols..

We look forward to receiving your revised manuscript.

Kind regards,

Shengqian Sun

Academic Editor

PLOS One

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE’s style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“This research was funded by the Ministry of Agriculture, Forestry and Fisheries of Japan (MAFF) through the Small/Startup Business Innovation Research Program (SBIR), grant number N020.”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Competing Interests section:

“All authors are employees of IntegriCulture Inc. The company developed, and has a commercial interest in, the product described in this manuscript. The authors declare this affiliation as a potential conflict of interest.”

We note that one or more of the authors are employed by a commercial company: name of commercial company.

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors’ salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

4. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor’s input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process.

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

6. We notice that your supplementary table is included in the manuscript file. Please remove them and upload them with the file type ‘Supporting Information’. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list.

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

[Note: HTML markup is below. Please do not edit.]

Reviewers’ comments:

Reviewer’s Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: I Don’t Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: The manuscript introduces a groundbreaking framework for pre-checking food regulations in the development of iDisper, a food-grade cell dissociation reagent crafted from papain and trisodium citrate, aimed at enhancing cell-based food production. The discussion highlights significant safety concerns in cellular agriculture, showcasing encouraging initial findings on the effectiveness of dissociation in avian cells. Nevertheless, considerable methodological, analytical, and interpretive concerns require substantial revisions prior to contemplating publication in PLOS ONE.

Significant Issues

Abstract and Language Issues: Grammatical errors (e.g., "Advancement... require" should be "requires"; "demon-strated" hyphenation artifact) and awkward phrasing diminish the overall clarity. Revise the abstract for clarity and rectify any typographical or formatting mistakes present.

The "Food Regulatory Pre-Check" framework (Fig 1, Table 1) presents an intriguing concept; however, it falls short in terms of thorough validation. Present numerical data that contrasts development timelines and costs with traditional methods, and enhance Table 1 by incorporating global regulatory information beyond Japan, such as the FDA GRAS status for papain. Explain why this represents a "new" framework instead of conventional food additive screening.

The methods presented are lacking in detail: The sourcing of cells from duck liver and chicken muscle does not include necessary ethical approvals, provenance information, or controls for variability. The preparation of iDisper lacks specific details regarding pH levels, mixing conditions, and does not provide validation for sterility beyond the filtration process. It is essential to ensure that statistical methods are supported by the deposition of raw data (ChemRxiv DOI provided, but please verify accessibility). Incorporate comprehensive protocols to ensure reproducibility.

The interpretation of results appears exaggerated: Assertions of "comparable" performance to TrypLE Express (Figs 2-3) overlook significant differences in incubation duration, cell types, and metrics for passaging success. Figure 2d pie charts illustrate significant failure rates (approximately 30-50%) that cannot be accounted for by contamination or reagent-related problems. Examine the constraints of testing exclusively on birds and analyze the duration of papain allergens in a quantitative manner.

Concerns regarding conflict of interest and commercial bias arise from the involvement of all authors from IntegriCulture Inc. (the product developer), particularly in relation to provisional labeling (S1 Table). Offer unbiased validation data or external testing; reduce promotional language (e.g., "Everyone Can Make Cell-Based Food").

Reviewer #2: Hatano and colleagues investigate a novel food-grade animal-free cell dissociation reagent, “iDisper”, to achieve efficient dissociation and viability of primary avian cells (duck liver and chicken muscles), that would be used in downstream cell-based food products e.g. lab grown meat. Commonly used cell dissociation agents (e.g. trypsin) poses contamination risks, such as viral contamination or allergens, for food applications. This manuscript highlights a novel dissociation reagent which includes papain (a protease from unripe papaya) and validating the performance of the new dissociation reagent against commercial alternatives. Furthermore, they propose a new upstream development “Food Regulatory Pre-Check” framework for cell-based food ingredients.

• You state that iDisper could maintain passaging of cells with hepatic stellate identity and stable marker expression, however your results S2 Appendix – S1 indicate a loss of ALB and HNF4A in P2 and P6 cells (both hepatocyte maturation markers). Can you please clarify why you conclude that iDisper maintains cells with hepatic stellate identity i.e. what markers are you looking at to make that conclusion?

• You examined microbial contamination in passaged cells, but did you confirm that cell cultures were mycoplasma free?

• There is no discussion of Sup Fig 3 and 4 in the main manuscript – please include discussion especially since you conclude that papain with trisodium citrate is better than papain alone. You have stated that papain alone could not achieve dissociation to the levels of papain with trisodium citrate (line 133), however you currently have not presented/discussed the results to support that claim (or please move your S2 Appendix discussions into the main body of the manuscript). I see that Sup Fig 3 indicates dissociation of duck liver cells with papain alone at various concentrations, and it appears that papain alone did achieve similar dissociation results compared to the TryplE Express reagent. Hence I’m not convinced that papain + trisodium citrate is required over papain alone.

• Fig 3 – no indication of A, B or C on the figure. Also can you indicate statistical significance on the figures.

• Methods section of the main manuscript could be expanded to include S1 Appendix (do not understand why this methods has been moved to the supplementary section.

• When washing the dissociated cells with PBS, is there calcium and magnesium included in the PBS?

• Details in supplemental methods do not match what is written in main manuscript – for example, in S1 Appendix-S3 Cell detachment assay, you state that the Cell Counter model R1 (Olympus) was used to measure total cell number and viability, but in the main manuscript you state that the NucleoCounter NC-202 was used. Please clarify if both equipment were used.

• Proof-read the manuscript before submission, especially punctuation.

• Table 1 format can be improved.

**********

what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

PLoS One. 2026 Apr 13;21(4):e0345921. doi: 10.1371/journal.pone.0345921.r002

Author response to Decision Letter 1

28 Jan 2026

Dear Editor,

Resubmission of Manuscript ID: PONE-D-25-59887

Title: “A Food-Grade, Animal-Free Cell Dissociation Alternative Product Developed via a Food Regulatory Pre-Check Framework”.

Response to Editor and Reviewers

We would like to express our sincere appreciation of the reviewers for their valuable comments and suggestions. We revised the manuscript based on the comments and suggestions provided below. In this response letter, the reviewers’ comments are presented in italics, the authors’ responses are written in regular font with a five-character indent, and the revised text is in blue.

Editor

We sincerely appreciate the reviewer’s constructive and insightful feedback, which has greatly improved the clarity and quality of our manuscript. We have carefully revised our manuscript in response to these comments. We hope that these revisions adequately address the reviewers’ concerns and that the revised manuscript is now suitable for publication in PLOS One. We look forward to hearing from you.

Journal Requirements

1. Please ensure that your manuscript meets PLOS ONE’s style requirements, including those for file naming

Response 1: We appreciate the guidance. We have carefully reviewed the guidelines and revised the manuscript, including file naming conventions and formatting, to fully comply with PLOS ONE’s style requirements.

2. Please state what role the funders took in the study.

Response 2: We have included the specific role of the funders in the Cover Letter (page 2).

Added text: This study was supported by the Small Business Innovation Research Program of the Ministry of Agriculture, Forestry and Fisheries of Japan under the “Demonstration of a Production System for Cellular Foods Utilizing CulNet Supernatant” (N020) project. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

3.1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study.

Response 3.1: We have amended the Funding Statement to clarify the commercial affiliation and role of the funder.

Added text: The funder provided support in the form of salaries for the authors [HH, MS, MK, AH, HK, and IK], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

3.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Response 3.2: We have updated both the Cover Letter and Competing Interests Statement to explicitly declare commercial affiliation and confirm adherence to journal policies.

Added text: This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Response 4: We acknowledge the policy regarding data availability. We confirm that all data underlying the findings described in this manuscript are fully available without restrictions. The minimal data set underlying the results, including the data in Figures 2 and 3, is provided in the Supporting Information submitted for this revision.

Response 5: The corresponding author (Ikko Kawashima) has a registered ORCID iD [0009-0003-9612-4007]. We ensured that this ID was successfully linked and validated using an Editorial Manager System.

Response 6: We appreciate your guidance on the formatting. We have removed the supplementary table from the main manuscript file and uploaded it as a separate file labeled ‘Supporting Information’ (S1 Table). Additionally, we have ensured that the legends for all the Supporting Information files are correctly listed at the end of the main manuscript, following the References section.

Reviewer: 1

Comment 1: Abstract and Language Issues: Grammatical errors (e.g., "Advancement... require" should be "requires"; "demon-strated" hyphenation artifact) and awkward phrasing diminish the overall clarity. Revise the abstract for clarity and rectify any typographical or formatting mistakes present.

Response 1: We apologize for grammatical errors and awkward phrasing in the original submission. We have thoroughly revised the manuscript, including the abstract, and have had the text proofread by Cactus Communications, Inc. (Tokyo, Japan). Specific corrections, such as "Advancement... requires", and “demonstrated” have been made.

Comment 2-1: The "Food Regulatory Pre-Check" framework (Fig 1, Table 1) presents an intriguing concept; however, it falls short in terms of thorough validation. Present numerical data that contrasts development timelines and costs with traditional methods, and enhance Table 1 by incorporating global regulatory information beyond Japan, such as the FDA GRAS status for papain. Explain why this represents a "new" framework instead of conventional food additive screening.

Response 2-1: We appreciate the reviewer’s suggestion. Although specific financial data and timelines are proprietary and difficult to benchmark owing to the nascency of the industry, we quantitatively validated the framework’s efficiency by demonstrating the "Reduction of Search Space." Unlike traditional screening of effectively infinite chemical libraries, our framework restricts the candidate pool to a finite "Positive List" (e.g., 104 enzyme preparations in the WHO JECFA Database; verified by "Enzyme Preparation" in the JECFA Online Database on January 16, 2026;

https://apps.who.int/food-additives-contaminants-jecfa-database/) from Day 0. This mathematical constraint to have a significantly higher screening efficiency than conventional methods. We have incorporated this rationale into the Discussion section. Additionally, we updated Table 1 to include regulatory information from Singapore (first approval in 2020), the USA (approval in 2023), and Israel (approval in 2024), which currently lead the global cellular agriculture sector.

Revised text (Page 8, Line 168): The efficiency of this framework is quantitatively supported by the "reduction of search space." Traditional reagent development often requires the screening of vast, effectively infinite libraries of synthetic compounds. In contrast, this framework utilizes the "Positive List" system of food regulations as a beneficial constraint, immediately reducing the candidate pool from an infinite number to a finite, manageable list at the start of development (Day 0). For example, the number of enzyme preparations with established specifications in the WHO JECFA Database [19] is limited to approximately 100 distinct source organisms. This drastic reduction in the screening scope mathematically validates the high efficiency of this framework compared with conventional blind screening.

Comment 2-2: Explain why this represents a "new" framework instead of conventional food additive screening.

Response 2-1: We appreciate the reviewer’s comment. The novelty of this framework is that we reversed the screening order. Instead of just looking for "what works," we prioritize "what is allowed" to address three practical realities:

● Global Rules: Conventional methods often identify high-performing ingredients that are banned in some countries. Our method prevents this by checking global regulations first.

● Cultural Safety: Food is a culture. As shown by the inclusion of Kosher status (Table 1), we checked religious and cultural suitability early to avoid ethical conflicts.

● Business Reality: Many companies find it difficult to stop a project once the budget is approved (Step 3). By setting a mandatory "Pre-Check" (Step 2), we create a clear "Stop/Go" point before spending money. Making this common-sense management rule a formal framework is a novel contribution.

Revised text (Page 9, Line 176): This framework also addresses practical business and cultural issues. First, checking regulations early ensures that the product complies with global standards. Second, it ensures "cultural safety" by including religious criteria (e.g., Kosher status in Table 1) from the start. Finally, it aids corporate decision-making. In business, it is often difficult to stop a project once a budget is assigned for testing (Feasibility Test). By requiring a regulatory "Pre-Check," this framework creates a clear decision point to prevent wasted resources and establishes a formal standard for the industry.

Revised text (Page 9, Line 183): Fig 1. "Food Regulatory Pre-Check" upstream development framework. The proposed four-step process begins with (1) Concept Planning, where markets are defined. The core of the framework is (2) Ingredient Screening utilizes the "Food Regulation Pre-Check" (yellow box) as a gatekeeper. This step filters infinite candidates down to a finite compliant list based on global regulations and cultural safety (e.g., Kosher status). Viable candidates then undergo (3) a Feasibility Test, where performance is validated against key metrics. Finally, the lead candidate proceeds to (4) Product Profile Definition, which marks the transition to the downstream development process.

Comment 3: The methods presented are lacking in detail: The sourcing of cells from duck liver and chicken muscle does not include necessary ethical approvals, provenance information, or controls for variability. The preparation of iDisper lacks specific details regarding pH levels, mixing conditions, and does not provide validation for sterility beyond the filtration process. It is essential to ensure that statistical methods are supported by the deposition of raw data (ChemRxiv DOI provided, but please verify accessibility). Incorporate comprehensive protocols to ensure reproducibility.

Response 3: We appreciate the reviewer’s guidance on improving the transparency and reproducibility of our methods. We have addressed each point as follows:

Ethical Information: We added a specific statement regarding ethical compliance to the manuscript.

Revised text (Page 16, Line 295): Compliance with Ethical Standards

In this study, we used duck eggs embryonated before hatching. According to Japanese institutional guidelines and the SCAW Category A classification, these embryos are not defined as experimental animals; therefore, formal approval from the Institutional Animal Care and Use Committee was not required. All procedures were performed in accordance with the principles of animal welfare to minimize potential distress.

Provenance Information of duck liver and chicken muscle cells: We have expanded the "Materials and Methods" section to include detailed preparation conditions and quality control specifications to ensure reproducibility.

Revised text (Page 4, Line 77): Cell Source, Cell Culture and Evaluation Method

Primary cells were isolated from the duck liver and chicken muscle. Fertilized duck eggs were purchased from Shiina Hatchery (Japan), and fertilized chicken eggs were obtained from Yamagishi (Wakayama, Japan). Both types of eggs were incubated in a P-008 incubator (Showa Furanki, Saitama, Japan) maintained at 37 °C and 60% humidity. The incubation periods were set to 14 and 12 days for the duck and chicken eggs, respectively, to ensure appropriate embryonic development. To ensure sterility, the egg surface was sterilized with ethanol, followed by immersion in sodium hypochlorite solution (Kitchen Haiter; Kao, Japan) for 30 min. Embryos were aseptically extracted in a laminar flow hood, and liver tissues were dissected. The tissues were mechanically dissociated in I-MEM without enzymes using a gentleMACS™ Dissociator (Miltenyi Biotec, Germany). The cell suspension was centrifuged, and the pellet derived from the two embryos was pooled and seeded into a single T175 flask to minimize individual variability.

iDisper Preparation and Sterility Validation: We expanded the "Materials and Methods" section to include detailed preparation conditions and quality control specifications to ensure reproducibility.

Revised text (Page 4, Line 62): The iDisper was prepared by mixing food-grade papain (W-40; Amano Enzyme, Aichi, japan), trisodium citrate (Marugo Corporation, Saitama, Japan), and potassium chloride (Kanto Chemical, Tokyo, Japan) in purified water at final concentrations of 2, 0.37, and 28.72 mg/mL, respectively. The reagents were weighed using a precision electronic balance (FX-5000i; A&D, Tokyo, Japan) and added sequentially to water. To ensure complete dissolution, the mixture was stirred at 550 rpm using a magnetic stirrer for 1 h at room temperature (25°C). The solution was sterilized using a Nalgene™ Rapid-Flow™ Sterile Disposable Filter Unit (0.2 µm pore size; 431433; Thermo Scientific, MA, USA). iDisper was stored at −20 °C until use. Before use, the solution was thawed and heated to 37 °C in a water bath. The physicochemical properties were verified using a pH meter (FP20; Mettler Toledo, Switzerland) and an osmometer (Osmostat OM-6040; ARKRAY, Tokyo, Japan). The pH was measured to ensure it fell within the range of 5.5–6.0 without adjustment, and osmolality was adjusted to 295±5 mOsm/kg. To validate sterility, the filtered solution was plated on Soybean-Casein Digest (SCD) agar and incubated at 35°C. Colony-forming units (CFU) were quantified using an automatic colony counter (Scan500; Interscience, Saint Nom la Bretêche, France). Internal quality control records confirmed 0 CFU across independent manufacturing lots. iDisper was stored at −20 °C until use.

Raw Data Accessibility: Regarding the availability of data, we have uploaded the complete raw dataset underlying our statistical analyses as Supporting Information in this resubmission, rather than relying on the external ChemRxiv repository.

Comment 4: The interpretation of results appears exaggerated: Assertions of "comparable" performance to TrypLE Express (Figs 2-3) overlook significant differences in incubation duration, cell types, and metrics for passaging success. Figure 2d pie charts illustrate significant failure rates (approximately 30-50%) that cannot be accounted for by contamination or reagent-related problems. Examine the constraints of testing exclusively on birds and analyze the duration of papain allergens in a quantitative manner.

Response 4: Thank you for your comment. We have revised the manuscript to explicitly attribute the failure rates to raw material variability and provide a quantitative analysis of allergen stability based on data and literature.

Clarification of Figure 2d (Failure Rates): We revised the manuscript to explicitly attribute the failure rates to the current limitations of iDisper during the primary isolation phase. Clarification of Figure 2d (Failure Rates): We clarified that the failure rates shown in Figure 2d, particularly the 4.1% failure rate at passage 0 (P0), highlighted a specific challenge. Although iDisper is highly effective for cell expansion (

Attachment

Submitted filename: Response Letter 20260128.docx

pone.0345921.s010.docx^{(2.2MB, docx)}

PLoS One. doi: 10.1371/journal.pone.0345921.r003

Decision Letter 1

Shengqian Sun

24 Feb 2026

Dear Dr. Kawashima,

Please submit your revised manuscript by Apr 10 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ‘Submissions Needing Revision’ folder to locate your manuscript file.. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ‘Submissions Needing Revision’ folder to locate your manuscript file.. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ‘Submissions Needing Revision’ folder to locate your manuscript file.. When you’re ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the ‘Submissions Needing Revision’ folder to locate your manuscript file.

A letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled ‘Response to Reviewers’.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled ‘Revised Manuscript with Track Changes’.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled ‘Manuscript’.

We look forward to receiving your revised manuscript.

Kind regards,

Shengqian Sun

Academic Editor

PLOS One

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers’ comments:

Reviewer’s Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

Reviewer #1: Abstract and Title: Refine the title for conciseness (e.g., "Food-Grade Cell Dissociation Agent via Regulatory Pre-Check Framework"). Expand abstract to explicitly state key metrics (e.g., 2 mg/mL papain efficacy, 8-month stability).

Introduction: Add 1-2 sentences citing recent cell-based food safety reviews (e.g., refs 3-4) to strengthen context on upstream challenges.

Methods: Specify exact iDisper osmolality adjustment protocol beyond "adjusted to 295±5 mOsm/kg" and include centrifuge RPM for all steps (e.g., 300 g noted inconsistently). Clarify embryo incubation humidity control details.

Results: In Fig 2d, quantify "4.1% failure rate" derivation (e.g., n= total cultures). Move S1 Appendix gene expression (S1 Fig) to main text as it supports phenotypic stability claims.

Discussion: Briefly address scalability for powder formulation (mentioned but not quantified) and potential mammalian cell testing limitations. Compare allergen management to FDA consultations (refs 11-15).

Figures/Tables: Ensure all scale bars are uniform (e.g., Fig 2a: 0.5 mm). Table 1 legend could note "Materials selected" more prominently.

Reviewer #2: (No Response)

**********

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #1: No

Reviewer #2: No

**********

To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation.

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

PLoS One. 2026 Apr 13;21(4):e0345921. doi: 10.1371/journal.pone.0345921.r004

Author response to Decision Letter 2

1 Mar 2026

We would like to express our sincere appreciation to the reviewers for their valuable comments and suggestions. We have carefully addressed all the points raised by the reviewers and have reflected them in the revised manuscript and the accompanying response letter.

Attachment

Submitted filename: Response Letter2.docx

pone.0345921.s011.docx^{(17.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0345921.r005

Decision Letter 2

Shengqian Sun

13 Mar 2026

A food-grade cell dissociation agent via regulatory pre-check framework

PONE-D-25-59887R2

Dear Dr. Kawashima,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information’ link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information’ link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information’ link at the top of the page. For questions related to billing, please contact and clicking the ‘Update My Information’ link at the top of the page. For questions related to billing, please contact billing support....

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Shengqian Sun

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers’ comments:

Reviewer’s Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: (No Response)

**********

Reviewer #1: I recommend that the revised version be accepted for publication in its current form, as it satisfactorily meets the academic and technical requirements expected for inclusion in the journal.

**********

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #1: No

**********

PLoS One. doi: 10.1371/journal.pone.0345921.r006

Acceptance letter

Shengqian Sun

PONE-D-25-59887R2

PLOS One

Dear Dr. Kawashima,

I’m pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps.

Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they’ll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Shengqian Sun

Academic Editor

PLOS One

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Particle size distribution of duck liver-derived cells immediately after detachment.

(TIF)

pone.0345921.s001.tif^{(7.5MB, tif)}

S2 Fig. Optimization of papain concentration for effective cell dissociation.

(TIF)

pone.0345921.s002.tif^{(6.7MB, tif)}

S3 Fig. Post-detachment proliferation of cells treated with different papain concentrations.

(TIF)

pone.0345921.s003.tif^{(7.9MB, tif)}

S4 Fig. Validation of sterility: Mycoplasma PCR test.

(TIF)

pone.0345921.s004.tif^{(5.7MB, tif)}

S5 Fig. Evaluation of iDisper stability after long-term storage.

(TIF)

pone.0345921.s005.tif^{(2.6MB, tif)}

S1 Table. Proposed food label for iDisper designed to declare its ‘manageable risk’.

(DOCX)

pone.0345921.s006.docx^{(16.7KB, docx)}

S1 File. Raw data for Fig2–4’.

(ZIP)

pone.0345921.s007.zip^{(8.2MB, zip)}

S2 File. Raw data for S1-5 Fig.

(ZIP)

pone.0345921.s008.zip^{(89.8MB, zip)}

Attachment

Submitted filename: Response Letter 20260128.docx

pone.0345921.s010.docx^{(2.2MB, docx)}

Attachment

Submitted filename: Response Letter2.docx

pone.0345921.s011.docx^{(17.6KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0345921.ref001] 1.Post MJ, Levenberg S, Kaplan DL, Genovese N, Fu J, Bryant CJ, et al. Scientific, sustainability and regulatory challenges of cultured meat. Nat Food. 2020;1(7):403–15. doi: 10.1038/s43016-020-0112-z [DOI] [Google Scholar]

[pone.0345921.ref002] 2.Stephens N, Di Silvio L, Dunsford I, Ellis M, Glencross A, Sexton A. Bringing Cultured Meat to Market: Technical, Socio-Political, and Regulatory Challenges in Cellular Agriculture. Trends in Food Science & Technology. 2018;78:155–66. doi: 10.1016/j.tifs.2018.04.010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref003] 3.Navare S, El-Moghazy AY. Food safety considerations in the advancement of cultured meat: Evaluating novel ingredients. Future Foods. 2025;11:100639. doi: 10.1016/j.fufo.2025.100639 [DOI] [Google Scholar]

[pone.0345921.ref004] 4.Ovissipour R, Yang X, Saldana YT, Kaplan DL, Nitin N, Shirazi A, et al. Cell-based fish production case study for developing a food safety plan. Heliyon. 2024;10(13):e33509. doi: 10.1016/j.heliyon.2024.e33509 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref005] 5.World Health Organization WHO, Food and Agriculture Organization of the United Nations FAO. Food safety aspects of cell-based food. 2023. doi: 10.4060/cc4855en [DOI] [Google Scholar]

[pone.0345921.ref006] 6.Ong KJ, Johnston J, Datar I, Sewalt V, Holmes D, Shatkin JA. Food safety considerations and research priorities for the cultured meat and seafood industry. Compr Rev Food Sci Food Saf. 2021;20(6):5421–48. doi: 10.1111/1541-4337.12853 [DOI] [PubMed] [Google Scholar]

[pone.0345921.ref007] 7.Segeritz CP, Vallier L. Cell culture: growing cells as model systems in vitro. In: Jalali M, Saldanha FYL, Jalali M. Basic science methods for clinical researchers. Boston: Academic Press. 2017. 151–72. doi: 10.1016/B978-0-12-803077-6.00009-6 [DOI] [Google Scholar]

[pone.0345921.ref008] 8.Merten O-W. Virus contaminations of cell cultures - A biotechnological view. Cytotechnology. 2002;39(2):91–116. doi: 10.1023/A:1022969101804 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref009] 9.Pinheiro de Oliveira TF, Fonseca AA, Camargos MF, de Oliveira AM, Pinto Cottorello AC, Souza A dos R, et al. Detection of Contaminants in Cell Cultures, Sera and Trypsin. Biologicals. 2013;41(6):407–14. doi: 10.1016/j.biologicals.2013.08.005 [DOI] [PubMed] [Google Scholar]

[pone.0345921.ref010] 10.Yamato M, Konno C, Utsumi M, Kikuchi A, Okano T. Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture. Biomaterials. 2002;23(2):561–7. doi: 10.1016/S0142-9612(01)00138-7 [DOI] [PubMed] [Google Scholar]

[pone.0345921.ref011] 11.Thummarati P, Laiwattanapaisal W, Nitta R, Fukuda M, Hassametto A, Kino-Oka M. Recent Advances in Cell Sheet Engineering: From Fabrication to Clinical Translation. Bioengineering (Basel). 2023;10(2):211. doi: 10.3390/bioengineering10020211 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref012] 12.Magnuson B, Munro I, Abbot P, Baldwin N, Lopez-Garcia R, Ly K, et al. Review of the regulation and safety assessment of food substances in various countries and jurisdictions. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2013;30(7):1147–220. doi: 10.1080/19440049.2013.795293 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref013] 13.Good Meat, Inc, Polsinelli PC, AR Toxicology, Burdock Group Consultants. Dossier in support of the safety of Good Meat cultured chicken as a human food ingredient. 2022. https://www.fda.gov/media/166346/ [Google Scholar]

[pone.0345921.ref014] 14.Wildtype. Pre-Market Safety Consultation Summary Prepared for the US Food and Drug Administration; Cell Culture Consultation 000005; FDA, 2022. https://www.fda.gov/media/186754/download?attachment [Google Scholar]

[pone.0345921.ref015] 15.Mission Barns. Mission Barns Cultivated Animal Fat Cells (Pork) Complete Consultation Data Package Public Version. 2022. https://www.fda.gov/media/185700/download?attachment [Google Scholar]

[pone.0345921.ref016] 16.UPSIDE Foods. Premarket notice for integral tissue cultured poultry meat; cell culture consultation 000002. 2021. https://www.fda.gov/files/food/published/CellCultureConsultation-000002-Submission-03222023.pdf [Google Scholar]

[pone.0345921.ref017] 17.Believer Meats. Safety evaluation of cultured chicken cells produced by Believer Meats. 2024. https://www.fda.gov/media/188066/download?attachment [Google Scholar]

[pone.0345921.ref018] 18.Kanayama T, Tanaka K, Shimada H, Ichiyama K, Higuchi S, Kawashima I. Improving the Safety of Cultured Meat Using Basal Medium Prepared Using Food Ingredients. BioRxiv. 2022. doi: 10.1101/2022.03.28.486069 [DOI] [Google Scholar]

[pone.0345921.ref019] 19.Murakami H, Okazaki Y, Yamada K, Omura H. Egg yolk lipoprotein, a new supplement for the growth of mammalian cells in serum-free medium : Egg yolk lipoprotein for serum-free growth of cells. Cytotechnology. 1988;1(2):159–69. doi: 10.1007/BF00146817 [DOI] [PubMed] [Google Scholar]

[pone.0345921.ref020] 20.Hatano H, Sawada M, Kitsukawa M, Hayakawa A, Kondo H, Kawashima I. A food-grade ready-to-use cell culture coating agent developed via food manufacturing: A dual technical and regulatory approach. ChemRxiv. 2025. doi: 10.26434/chemrxiv-2025-9gfsq [DOI] [Google Scholar]

[pone.0345921.ref021] 21.World Health Organization WHO, Food and Agriculture Organization of the United Nations FAO. JECFA Online Database: Evaluations of the Joint FAO/WHO Expert Committee on Food Additives. https://apps.who.int/food-additives-contaminants-jecfa-database/ [Google Scholar]

[pone.0345921.ref022] 22.Odom DT, Zizlsperger N, Gordon DB, Bell GW, Rinaldi NJ, Murray HL, et al. Control of pancreas and liver gene expression by HNF transcription factors. Science. 2004;303(5662):1378–81. doi: 10.1126/science.1089769 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref023] 23.Schaefer L, Iozzo RV. Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction. J Biol Chem. 2008;283(31):21305–9. doi: 10.1074/jbc.R800020200 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref024] 24.Ueyama-Toba Y, Tong Y, Yokota J, Murai K, Hikita H, Eguchi H, et al. Development of a hepatic differentiation method in 2D culture from primary human hepatocyte-derived organoids for pharmaceutical research. iScience. 2024;27(9):110778. doi: 10.1016/j.isci.2024.110778 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref025] 25.Nie Y, Walsh P, Clarke DL, Rowley JA, Fellner T. Scalable passaging of adherent human pluripotent stem cells. PLoS One. 2014;9(1):e88012. doi: 10.1371/journal.pone.0088012 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0345921.ref026] 26.Wünsch I, Finke JH, John E, Juhnke M, Kwade A. The influence of particle size on the application of compression and compaction models for tableting. Int J Pharm. 2021;599:120424. doi: 10.1016/j.ijpharm.2021.120424 [DOI] [PubMed] [Google Scholar]

[pone.0345921.ref027] 27.Deng T, Sousa LM, Garg V, Bradley MSA. Segregation of formulated powders in direct compression process and evaluations by small bench-scale testers. Int J Pharm. 2023;647:123544. doi: 10.1016/j.ijpharm.2023.123544 [DOI] [PubMed] [Google Scholar]

[pone.0345921.ref028] 28.Yue SR, Shrivastava R, Campbell K, Walker MJ. Food allergen recalls in the United Kingdom: A critical analysis of reported recalls from 2016 to 2021. Food Control. 2023;144:109375. doi: 10.1016/j.foodcont.2022.109375 [DOI] [Google Scholar]

[pone.0345921.ref029] 29.FAO, WHO. Risk assessment of food allergens – part 3: Review and establish precautionary labelling in foods of the priority allergens. 2023. doi: 10.4060/cc6081en [DOI] [Google Scholar]

[pone.0345921.ref030] 30.Vicente LC, Aires-Barros R, Empis JM. Stability and proteolytic activity of papain in reverse micellar and aqueous media: a kinetic and spectroscopic study. J Chem Technol Biotechnol. 1994;60(3):291–7. doi: 10.1002/jctb.280600310 [DOI] [PubMed] [Google Scholar]

PERMALINK

A food-grade cell dissociation agent via regulatory pre-check framework

Hiroaki Hatano

Misaki Sawada

Misaki Kitsukawa

Azusa Hayakawa

Hiroaki Kondo

Ikko Kawashima

Roles

Abstract

Introduction

Materials and methods

iDisper reagent preparation

Cell source, cell culture and evaluation method

RNA extraction and sequencing

Bioinformatic analysis

Mycoplasma detection

Statistical analysis

Results and discussion

A food regulatory pre-check framework for upstream development in the cell-based food industry

Fig 1. “Food Regulatory Pre-Check” upstream development framework.

Table 1. Multi-faceted evaluation of candidate cell dissociation technologies based on food safety and manufacturing principles.

Performance evaluation of iDisper: Dissociation performance, cell versatility, and long-term stability

Fig 2. Gene expression changes during serial passage.

Fig 3. Performance of iDisper on primary duck liver-derived cells.

Fig 4. Versatility and stability of iDisper across different cell types.

Industrial production of food-grade cell culture materials and future challenges

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Shengqian Sun

Roles

Author response to Decision Letter 1

Decision Letter 1

Shengqian Sun

Roles

Author response to Decision Letter 2

Decision Letter 2

Shengqian Sun

Roles

Acceptance letter

Shengqian Sun

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases