Enhancement of Hanwoo bovine muscle satellite cell proliferation and differentiation using edible chitosan-starch scaffolds

Bosung Kim; Yeonsu Jo; Doyeon Kim; Xiangzi Li; Sungkwon Park

doi:10.25259/JKSUS_472_2025

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Research Article

2026

:38;

4722025

doi:

10.25259/JKSUS_472_2025

Enhancement of Hanwoo bovine muscle satellite cell proliferation and differentiation using edible chitosan-starch scaffolds

Bosung Kim^a, Yeonsu Jo^a,, Doyeon Kim^a, Xiangzi Li^b, Sungkwon Park^a,*

aDepartment of Food Science and Biotechnology, Sejong University, 209 NEUNGDONG RO YULGOK GWAN 511, Seoul, 05006, Gyungki, Republic of Korea

bDepartment of Engineering Research Center of North-East Cold Region Beef Cattle Science & Technology Innovation, Yanbian University, Yanbian, Yenji, 12345, China

* Corresponding author: E-mail address: sungkwonpark@sejong.ac.kr (S Park)

Received: 2025-03-05, Accepted: 2025-10-20, Epub ahead of print: 2025-12-27, Published: 2026-01-16

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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Abstract

The present study evaluated an edible tapioca starch-based scaffold for in vitro 3D culture of Hanwoo bovine muscle satellite cells (BMSCs). We fabricated biomimetic scaffolds comprising chitosan, tapioca starch, and gelatin, and examined the effect of starch concentration (1, 2, 3, and 4%) on scaffold properties and BMSCs behavior. Structural analysis indicated that as the starch concentration increased, the scaffold surfaces remained smooth, while internal porosity and water absorption also increased. These findings were confirmed via scanning electron microscopy (SEM), as no statistical differences in mechanical properties were observed across the different starch concentrations. Cell attachment, proliferation, and viability were measured after 1, 3, 5, and 7 days of culture. The results indicated that high starch concentrations led to significantly enhanced cell attachment, increased proliferation rates, and improved viability, likely due to improved medium absorption and nutrient and oxygen supplies (P<0.0001). Additionally, myotube-like structures were observed in cells cultured with the scaffold in a spinner flask, indicating the potential for differentiation. Given the growing importance of cell-based food production, the edibility and capability of the Chitosan/Tapioca scaffold to support 3D cell culture make it a strong candidate for such applications. Collectively, these findings support the in vitro potential of the edible chitosan-tapioca scaffold for 3D BMSCs culture and may inform advances in cell-based food research.

Keywords

Cell-based food

Chitosan

Gelatin

Hanwoo muscle satellite cell

Starch

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1. Introduction

With the global rise in population, the concomitant increase in beef consumption is projected to continue until 2050 (Kim et al., 2022; Liu et al., 2023; Odegard and van der Voet, 2014; Parlasca and Qaim, 2022; Turk, 2016). However, this trend presents challenges, including environmental issues, animal welfare concerns, and land usage associated with livestock production (Kim et al., 2024; Ramachandraiah and Hong, 2020). To address these issues, various countries and research laboratories are developing alternative protein sources, including cell-based foods (Kim et al., 2023; Park et al., 2023). The development of suitable scaffolds is essential for 3D cultures. The extracellular matrix (ECM) provides a physical environment for cell attachment and interactions in vivo, and scaffolds mimic ECM functions in vitro (Bomkamp et al., 2022). Scaffolds for cell-based food production should support cell attachment and growth using edible, food-grade materials (Benny et al., 2022; Nurul Alam et al., 2024; Rao et al., 2023), and mechanical stability is important for in vitro handling and culture. Although much prior work has focused on protein-based scaffolds, optimizing edible polysaccharide-protein composites and selecting food-safe crosslinking strategies remains important for in vitro 3D culture (Seah et al., 2022).

Starch, a polysaccharide composed of glucose units linked by glycosidic bonds, is commonly used in the food and other industries, owing to its processable physical properties, nontoxicity, biocompatibility, and affordability (Benalaya et al., 2024; Díaz-Montes, 2022). The properties of starch are influenced by the amylose-to-amylopectin ratio, which depends on the plant source (Alcázar-Alay and Meireles, 2015; Leloup et al., 1991). An increased amylopectin content enhances viscosity and water absorption, which are crucial for the mechanical properties of the scaffold (Roslan et al., 2016). Starch gelatinization involves amylose dissolution and amylopectin swelling, which are processes that enhance moisture absorption and are widely used in medical tissue engineering and biodegradable films (Sundaram et al., 2008; Xie et al., 2013; Zou et al., 2012).

Chitosan, composed of D-glucosamine and N-acetyl-D-glucosamine linked by β-(1-4) glycosidic bonds, is a biocompatible polymer with high viscosity and nontoxicity (Wang and Zhuang, 2022). Its polycationic nature in acidic solutions and structural similarity to glycosaminoglycans endow it with antibacterial properties against fungi, bacteria, and viruses (Kassem et al., 2019). Chitosan has various applications in food packaging, drug encapsulation, and water pollutant removal. It is also suitable for cell-based food scaffolds owing to its biocompatibility and cost-effectiveness (Fan et al., 2012; Xu et al., 2005).

Gelatin is a component of connective tissues, such as the skin, muscle, and bones, in mammals, including humans, and is derived from the breakdown of collagen proteins (Mikhailov, 2023). It can be produced not only from meat, such as beef and pork, but also from fish (Davidenko et al., 2016). Gelatin contains an arginine–glycine–aspartate (RGD) sequence, which is a cell adhesion motif that has been widely used as a scaffold in tissue engineering owing to its excellent cell affinity (Kim et al., 2017; Wang et al., 2013). Genipin, a nontoxic crosslinker from Gardenia jasminoides (Ellis fruit), enhances scaffold hardness by reacting with the lysine and hydroxylysine residues of gelatin (Kirchmajer et al., 2013). Thus, genipin cross-linking of chitosan/starch and gelatin improves the mechanical properties of cell-based foods (Roy and Rhim, 2022; Wang et al., 2020). Although gelatin-based scaffolds are commonly used in cell-based food production, reports on the use of tapioca starch in scaffolds are scarce.

In the present study, we aimed to develop a biomimetic scaffold consisting of chitosan, tapioca starch, and gelatin to support the growth of Hanwoo muscle satellite cells. We investigated the cell attachment efficiency, proliferation rate, and activity based on the starch concentration in chitosan. This study evaluates the in vitro 3D culture performance of an edible chitosan-tapioca scaffold and highlights starch as a viable edible component for myogenic culture. Although confined to in-vitro data, the study provides insights that could facilitate future cell-based food scaffold development and highlights starch as a promising edible component.

2. Materials and Methods

2.1 Muscle satellite cell isolation and cell culture

Bovine muscle satellite cells (BMSCs) were isolated from the rump muscles of three Hanwoo cattle (32-month-old) obtained from Farmstory Hannaeng Bio & Food Co., Ltd. (Chungbuk, South Korea). Tissues were immediately immersed in Dulbecco’s phosphate-buffered saline (98% DPBS; Welgene, Gyeongsan, Korea) containing 2% antibiotic-antimycotic (AA; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and stored at 4°C. Muscle tissues were rinsed with 70% ethanol, and residual fat impurities were removed using sterile tweezers and scissors. Subsequently, 30 g of muscle tissue was added to 50 mL conical tubes containing a pronase solution (Sigma P5147; 0.8 mg/mL of DPBS). The tubes were placed in a water bath at 37°C for 40 min and shaken every 10 min. The tubes were then centrifuged at 1200 × g for 15 min at 4°C, and the supernatant was discarded. The pellet was resuspended in DPBS and centrifuged at 1200 × g for 15 min at 4°C. The pellet was vortexed with 40 mL DMEM-F12 (Gibco) and centrifuged at 200 × g for 5 min at 4°C. The supernatant was filtered through a 100 µm cell strainer (Corning Inc., Corning, NY, USA) into a new 50 mL tube. DMEM was then added to the tube, and the sample was centrifuged at 1200 × g for 15 min at 4°C to harvest a cell pellet. The harvested cell pellets were stored in cryovials by allotting 1 g of muscle chunks per vial with DMEM-F12 containing AA, fetal bovine serum (FBS; Gibco), and dimethyl sulfoxide.

The growth medium (GM) consisted of DMEM-F12/GlutaMax (Gibco) containing 1% AA and 10% FBS. The cells were subcultured with 0.05% trypsin-EDTA (Gibco) until reaching approximately 70-80% confluency. Cells were cultured in a 37°C CO₂ incubator, and the culture medium was replaced every other day.

2.2 Preparation of chitosan/tapioca scaffolds

The chitosan-to-tapioca ratio used in this experiment has been presented in Table 1. The chitosan solution was prepared by stirring chitosan powder (deacetylation degree of 75%; Sigma-Aldrich, St. Louis, MO, USA) in sterilized 1% acetic acid with distilled water (DW) at 300 × g. The stirring speed was reduced to 80 × g for 30 min when the solution thickened. The tapioca solution was prepared by dispersing the tapioca starch (Heungyildang, Seoul, Korea) in sterilized DW at 73°C and 300 × g for 30 min until it became gelatinized. The chitosan and tapioca solutions (w/w 1:1) were then mixed at 80 × g for 30 min. The mixed chitosan/tapioca solution was centrifuged at 3000 × g for 10 min at 25°C. The chitosan/tapioca mixture was added dropwise to the cross-linking solution, which included 5% sodium tripolyphosphate (STPP; Sigma-Aldrich, St. Louis, MO, USA) and sodium trimetaphosphate (STMP; w/w 1:99; degree of deacetylation 75%; Sigma-Aldrich, St. Louis, MO, USA) solution using a 1 mL syringe. The mixture in the form of spheres was then stirred at room temperature at 350 × g for 24 h. The prepared chitosan/tapioca scaffold and the 4% gelatin solution with DW (v/v 1:3; Sigma -Aldrich, St. Louis, MO, USA) were placed in a centrifuge tube and stirred in a water bath at 50°C for 1 h. The coated chitosan/tapioca scaffolds were washed three times with DW, placed in a centrifuge tube with a 0.1% genipin solution (scaffold: genipin solution, v/v 1:3; Challenge Bioproducts, Touliu, Taiwan), and then cross-linked at 60°C and 100 × g in a water bath for 3 h. The scaffolds were washed three times with sterilized DW after cross-linking. The cross-linked chitosan/tapioca scaffolds were freeze-dried for one day.

Table 1. Different formulations of chitosan/tapioca composite scaffolds.

	Chitosan/Tapioca ^* (CS/TA)	Tapioca (w/v)	Chitosan (w/v)
CS/TA-1^*	5:5	1%	5%
CS/TA-2		2%
CS/TA-3		3%
CS/TA-4		4%

* Chitosan (CS), Tapioca (TA), Chitosan/Tapioca (CS/TA)

2.3 Scaffold structure analysis

The structures of the scaffolds were investigated using scanning electron microscopy (SEM; Hitachi Hightech, Tokyo, Japan). The surfaces and interiors of the freeze-dried scaffolds were observed under magnifications of 30, 100, and 200X. The obtained images were analyzed, and the pore sizes were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

2.4 Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy was performed using the Attenuated Total Reflectance (ATR) method with a Nicolet NEXUS 470 FT-IR spectrometer (Thermo Fisher Scientific) to confirm the chemical composition and cross-linking reaction of the scaffolds. The spectrum data were obtained in the wavenumber range of 600 cm^-1 to 4000 cm^-1. Each spectrum was the average of multiple scans.

2.5 Water absorption

Water absorption was evaluated by soaking the scaffolds in PBS at room temperature for 0, 1, 2, 4, and 8 h. The immersed scaffolds absorbed excess PBS with a filter paper, and were weighed. The water absorption was calculated using the following formula:

(1)

W % = (W_{wet} - W_{dry}) / W_{dry} \times 100 %

2.6 Textural properties

The physical properties of the chitosan/tapioca scaffolds were assessed using a TA.XTplusC texture analyzer (TA; Stable Micro Systems, Surrey, UK). The scaffolds were hydrated in Dulbecco’s Phosphate Buffered Saline (DPBS) for 3 h and cut into 15 mm × 15 mm × 15 mm pieces. The samples were compressed twice to 70% of their original height. The test speed was 1.0 mm/s, and the trigger force was 0.1 N. Data were analyzed using NexygenPlus™ software (AMETEK Lloyd Instrument Ltd., Hampshire, UK). Hardness (kgf), adhesiveness (kgf·mm), springiness, chewiness (kgf), cohesiveness, and resilience were analyzed. The tests were performed in triplicate.

2.7 Cell adhesion and proliferation

Seventeen scaffolds were seeded per well in 24-well plates. BMSCs were seeded at a density of 2 × 10⁴ cells/mL per well on the scaffolds, and the medium was added after 3 h. Scaffolds with attached cells were cultured at 37°C in a CO₂ incubator, and the medium was changed every 48 h. The cells attached to the scaffolds were analyzed on days 1, 3, 5, and 7.

Cell counting was performed by staining the nuclei with Hoechst 33342 (Cat # H3570; Invitrogen, Carlsbad, CA, USA) and counting the number of cells attached to the scaffolds using ImageJ software. Measurements were performed on five scaffolds, and the average was calculated.

2.8 3-(4,5-Dimethylthiazol -2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

Three scaffolds were placed per well in a 96-well plate, and the cells were seeded at a density of 1×10⁴ cells/mL per well. The MTS assay was performed on days 1, 3, 5, and 7. The scaffolds were transferred to a new 96-well plate, and 100 µL of GM was added. Then, 10 µL of MTS solution (CellTiter 96 Aqueous MTS Reagent Powder; Promega, Madison, WI, USA) were added to each well, and the plate was incubated for 4 h. The MTS solution in which the scaffolds were immersed was transferred to a new plate, and the absorbance was measured at 490 nm using a microplate spectrophotometer (Multiskan Sky; Thermo Fisher Scientific).

2.9 Cell staining

Staining was performed on days 1, 3, 5, and 7 to observe the number and morphology of cells attached to the scaffolds. Nuclei were stained with Hoechst 33342 (1:1500), while the cytoplasm was stained with rhodamine 6G (Cat#R4127; Sigma-Aldrich). Cells adhered to the scaffolds were fixed in 4% paraformaldehyde for 10 min. Hoechst 33342 was added to the scaffolds, which were incubated at 37°C for 5 min. The staining solution was then discarded, and the cells were incubated for an additional 15 min. The scaffolds were then washed with DPBS and observed under an inverted light microscope (Nikon, Tokyo, Japan). Staining was performed in the dark.

2.10 Statistical analysis

All experiments were conducted in triplicate, and the results are presented as the mean ± standard deviation. Data were analyzed using one-way or two-way ANOVA in Prism v9.4.0 (GraphPad, San Diego, CA, USA) with Tukey’s post hoc test for multiple comparisons. Results were considered statistically significant at p < 0.05 (*p < 0.05, **p < 0.01, ***p <0.001, and ****p < 0.0001).

3. Results

3.1 Morphological and structural analyses

The morphologies of the scaffolds (CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4) with different tapioca starch concentrations were similar in size, ranging from 1 to 2 mm (Fig. 1). The scaffold surface is critical as it serves as the site for direct cell attachment. Additionally, the porous structure facilitates the delivery of nutrients and oxygen necessary for cell growth in 3D scaffolds. Therefore, SEM was used to examine the surface and internal structures. The surfaces of all the scaffolds exhibited a non-porous structure, with slight wrinkles appearing at lower starch concentrations. After cutting the scaffolds in half, internal porous structures with porosity levels of 19.1, 29.7, 33.4, and 44.2% were observed. The porosity of the scaffolds increased with increasing starch concentration. Therefore, higher starch concentrations may improve the supply of nutrients and oxygen to cells that adhered to the scaffolds.

Morphology of chitosan/tapioca (CS/TA) scaffolds. (Scale bar = 500 µm). CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 = 5% CS and 1%, 2%, 3%, and 4% TA, respectively.

3.2 Fourier transform infrared spectroscopy

To understand the interactions and cross-linking reactions among the components of all scaffolds (CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4), a comparative analysis using FTIR spectra was conducted. The infrared spectra of chitosan, tapioca starch, gelatin, genipin, and chitosan-starch scaffolds have been presented in Fig. 2(a). A strong characteristic O–H stretching peak was observed at 3351 cm⁻¹ for all groups. In the tapioca starch spectrum, the peak at 2929 cm⁻¹ corresponded to CH vibrations, whereas the peaks in the chitosan spectrum at 1553 cm⁻¹ and 1665 cm⁻¹ corresponded to NH bending and CO stretching, respectively. The chitosan peak at 1655 cm⁻¹ (CO) shifted to 1632 cm⁻¹ in the starch/tapioca scaffold, while the peak at 1553 cm⁻¹ (NH) shifted to 1531 cm⁻¹, indicating interactions between the amino group of chitosan and the hydroxyl groups of starch and cross-linking agents (Fig. 2b). Furthermore, the peak at 1267 cm⁻¹ corresponded to PO vibrations in the chitosan/starch scaffold spectrum, and the peak at 1025 cm⁻¹ corresponded to phosphorus-oxygen-carbon (P-O-C) vibrations, indicating that starch and chitosan were cross-linked with sodium trimetaphosphate and sodium trimetaphosphate.

FTIR spectroscopy spectra of chitosan/tapioca (CS/TA). (a) FTIR was used to measure the CS/TA scaffold and the components of the scaffold. (b) FTIR spectra of CS/TA-4. CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 = 5% CS and 1%, 2%, 3%, and 4% TA, respectively.

3.3 Water absorption and degradation

The water absorption characteristics of CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 were analyzed after hydration in DPBS for 1, 2, 3, 4, and 8 h (Fig. 3). All the scaffolds exhibited the highest water absorption at 1 h, with minimal changes observed beyond that point, regardless of the starch concentration. As the starch concentration increased, more water was absorbed, with CS/TA-4 exhibiting substantially higher water absorption than the other scaffolds. Chitosan is a hydrophilic polymer due to the presence of amino and hydroxyl groups (Narayanan et al., 2018). Additionally, tapioca starch has a high hydroxyl group content, which facilitates strong interactions with water molecules (Judawisastra et al., 2017). Therefore, as the starch concentration increased, more hydrogen bonds were formed between chitosan and tapioca starch, leading to increased water absorption. Once the porous network became saturated and osmotic balance was reached in DPBS, further water uptake was limited by charge screening and network constraints. Extending hydration beyond 1 h resulted in minimal additional mass gain.

Water absorption of chitosan/tapioca (CS/TA) scaffolds. The results are expressed as means ± standard deviation (n = 3). The asterisks indicate significant differences (**p <0.01). CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 = 5% CS and 1%, 2%, 3%, and 4% TA, respectively.

3.4 Textural property analysis

The TA of the scaffolds (CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4) was evaluated using a TA instrument. As depicted in Table 2, although hardness and springiness did not exhibit statistically significant differences among the scaffolds, numerical variations were observed. Cohesiveness, chewiness, and resilience showed decreasing trends with increasing starch concentration; however, these trends were not statistically significant.

Table 2. Textural properties of scaffolds.

Treatment	Texture profile
Treatment	Hardness (kgf)	Adhesiveness (kgf.mm)	Springiness	Chewiness (kgf)	Cohesiveness	Resilience
CS/TA-1	5.88±0.25^a	0.04±0.04^b	0.88±0.04^a	3.7±0.20^ab	0.72±0.05^a	0.36±0.05^a
CS/TA-2	6.78±0.05^a	0.08±0.01^ab	0.86±0.05^a	4.01±0.13^a	0.67±0.02^ab	0.30±0.03^ab
CS/TA-3	5.95±0.79^a	0.11±0.00^a	0.64±0.10^a	2.35±0.38^b	0.62±0.08^ab	0.26±0.03^b
CS/TA-4	6.19±0.82^a	0.11±0.05^a	0.68±0.17^a	2.44±0.50^b	0.58±0.08^b	0.24±0.02^b

Textural profile analysis of each scaffold: Chitosan (CS), Tapioca (TA), Chitosan/Tapioca (CS/TA); results are expressed as mean ± standard deviation (n = 3)

a, b Values with different letters within the same column are significantly different (p <0.05)

CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 = 5% CS and 1%, 2%, 3%, and 4% TA, respectively

3.5 Cell proliferation and viability

BMSCs were seeded onto the scaffolds (CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4) and cultured for 7 d to confirm cell attachment and proliferation (Table 3). As presented in Fig. 4, on day 1, no statistically significant differences in cell numbers were observed among the scaffolds, although CS/TA-4 exhibited numerically higher cell growth than the other scaffolds. Additionally, all scaffolds exhibited steady cell proliferation after day 3, indicating that they provided a stable structure and adequate adhesion capacity. Although CS/TA-3 had fewer cells than CS/TA-2 up to day 5, it exhibited a higher cell attachment on day 7 as the concentration of tapioca starch increased. The number of cells attached to CS/TA-4 was higher than that attached to the other scaffolds on days 1, 3, 5, and 7 (p < 0.05).

Table 3. Staining images of BMSCs in CS/TA scaffolds with different starch concentrations on day 7.

		CS/TA-1	CS/TA-2	CS/TA-3	CS/TA-4
40X	Hoechst 33342
	Rhodamine 6G
	Merge
100X	Hoechst 33342
	Rhodamine 6G
	Merge

Chitosan (CS), Tapioca (TA), Chitosan/Tapioca (CS/TA); images of adherent cells were observed at 40x and 100x; scale bar: 1000µm (40X), 200µm (100X); CS/TA-1, CS/TA-2, CS/TA-3, CS/TA-4 = 5% CS and 1%, 2%, 3%, 4% TA, respectively.

Cell number of BMSCs after proliferation on the chitosan/tapioca (CS/TA) scaffold. The results are expressed as mean ± standard deviation (n = 5). The asterisks indicate significant differences (*p <0.05, **p <0.01, and ****p <0.0001). CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 = 5% CS and 1%, 2%, 3%, and 4% TA, respectively.

Cell viability was assessed using the MTS assay, which revealed that cell viability consistently increased on all scaffolds, with CS/TA-4 exhibiting substantially higher viability than that of CS/TA-1 on day 7 (Fig. 5). These findings indicate that the scaffolds efficiently supplied the oxygen and nutrients necessary for cell growth. Additionally, an increase in the tapioca starch concentration led to higher cell attachment and proliferation rates.

Cell viability of BMSCs after proliferation on the chitosan/tapioca (CS/TA) scaffold. The results are expressed as mean ± standard deviation (n = 3). The asterisks indicate significant differences (*p <0.05). CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 = 5% CS and 1%, 2%, 3%, and 4% TA, respectively.

3.6 Muscle satellite cell differentiation on scaffold

CS/TA-4, which exhibited the highest cell attachment and proliferation rate, was cultured in a differentiation medium (DM) for 5 d to assess cell differentiation ability. The cytoplasm was stained with rhodamine 6G, and the nuclei were stained with Hoechst 33342 to observe the morphology of the differentiated cells at 40X, 100X, and 200X magnifications. A comparative analysis between 2D and 3D cultures revealed myotube-like structures, suggesting myogenic progression; quantitative differentiation metrics such as the fusion index and myotube diameter, and length were not assessed. Apparent cell fusion was observed, consistent with early differentiation, and cell attachment appeared higher in 3D culture than in 2D culture, as shown in Fig. 6. Notably, the scaffold maintained its structure after culturing in a spinner flask for 12 d, with no observed cell detachment. This result indicates that the scaffold might maintain its attachment ability and structural stability for an extended period, even under certain physical stresses.

Myogenic differentiation of BMSCs on chitosan/tapioca scaffold, CS/TA-4. Images of adherent cells were observed at 40X, 100X, and 200X. Scale bar: 1000 µm (40X), 400 µm (100X), 200 µm (200X). CS/TA-1, CS/TA-2, CS/TA-3, and CS/TA-4 = 5% CS and 1%, 2%, 3%, and 4% TA, respectively.

4. Discussion

In this study, we developed a scaffold using a chitosan/tapioca starch material, and the attachment and growth of BMSCs seeded on the scaffold were analyzed based on the starch concentration. According to Xiang et al., plant-based proteins generally have a lower molecular weight, higher hydrophilicity, and better support for cell attachment than animal-based proteins (Xiang et al., 2022). This suggests that the plant-based components in the chitosan/tapioca starch used in our experiments were effective for cell attachment. Increasing the starch concentration in the current study did not result in substantial changes in the scaffold size. SEM analysis revealed that the scaffold surfaces were smooth and non-porous, whereas the internal porosity increased with higher starch concentrations. Additionally, water absorption was enhanced at higher starch concentrations, with CS/TA-4 exhibiting substantially higher water absorption than the other starch concentrations. Tapioca starch, in particular, possesses the highest water absorption capacity among starches (Othman et al., 2019). Lin et al. also confirmed that water absorption increases with increasing starch concentration (Lin et al., 2021). The stronger O-H peak in CS/TA-4 at 3351 cm^-1 in the FTIR analysis in the present study suggests a higher number of hydrogen bonds with water molecules, which is consistent with the greater water absorption observed for CS/TA-4; this interpretation is correlative and does not establish a mechanism. Similarly, Lin et al. reported an O–H peak at 3435 cm^-1 (Lin et al., 2021). The similarity in these findings suggests that the starch structure within the composite scaffold was preserved after cross-linking.

In the present study, textural analysis revealed that CS/TA-2 and CS/TA-4 exhibited higher hardness than CS/TA-1 and CS/TA-3, but these differences were not statistically significant. Chewiness and cohesiveness likewise showed no significant change with starch concentration. The stiffness of the tissue matrix affects stem cell differentiation and promotes cell attachment and alignment; however, we detected no statistically significant differences in mechanical properties across the starch concentrations tested. Accordingly, the present dataset does not support attributing the observed cell responses to stiffness. Similar findings were reported by Hussain, who observed that as starch concentration increased, there was a corresponding increase in hardness, cohesiveness, springiness, adhesiveness, and chewiness (Hussain, 2015).

Proper sterilization of materials is essential for successful cell attachment to scaffolds (Dai et al., 2016). Without adequate sterilization, there is a risk of contamination and potential failure (Serrano-Aroca et al., 2022). Various sterilization methods for scaffold fabrication include radiation, heat, chemical, and ultraviolet light treatments (Horakova et al., 2020; Łopianiak and Butruk-Raszeja, 2020; Suamte et al., 2023). Among these, heat and ultraviolet treatments are particularly cost-effective in the production of cell-based foods (Tchonkouang et al., 2023). These methods are extensively used in the food industry and are recognized as safe for ensuring food safety. In our experiments, sterilization was performed using standard procedures as described in Methods; we note its general importance here, although it was not a variable tested in this study.

In the present study, the cell attachment and proliferation rates increased with higher starch concentrations, with CS/TA-4 exhibiting substantially higher values than those of the other scaffolds. Furthermore, cell viability consistently increased in all scaffolds over 4 d, indicating efficient oxygen and nutrient supply to the attached cells. Enhanced water absorption supports nutrient transport to the attached cells, thereby affecting cell attachment and proliferation. Thus, the high porosity and water absorption properties of CS/TA-4 likely contributed to improved cell attachment and growth. This finding is consistent with earlier studies reporting that high starch concentrations promote cell attachment in scaffolds made of tapioca starch and alginate (Lee et al., 2024; Lin et al., 2021). Alternative contributors, such as differences in gelatin content or crosslinking efficiency, may also influence cell outcomes and are noted as potential explanations. Overall, increasing the starch concentration in the scaffold composition was associated with increased absorption, porosity, and higher cell attachment and growth in vitro.

5. Conclusions

In this study, we developed a biomimetic scaffold using chitosan, tapioca starch, and gelatin to support the growth of Hanwoo muscle satellite cells. Scaffold porosity and water absorption increased with higher starch concentrations, leading to improved cell attachment, proliferation, and viability over several days of culture. Moreover, the observed myotube formation indicates the potential of the scaffold to support cell differentiation. Cell-based food production is emerging as an important industry, and the scaffold for producing cell-based foods should be edible and capable of supporting cell culture. The CS/TA scaffold developed in our study can provide a suitable matrix for 3D cell culture, highlighting the potential of starch as a scaffold material. Hence, the CS/TA scaffold has potential for application in cell-based food production, contributing to the growth of the cell-based food industry.

CRediT authorship contribution statement

Bosung Kim: Conceptualization, formal analysis, writing-review and editing. Yeonsu Jo: Formal analysis, methodology, writing-orginal draft. Doyeon Kim: Formal analysis, investigation. Sungkwon Park: Conceptualization, writing - original draft, writing - review & editing.

Declaration of competing interest

The authors declare that they have no competing financial interests or personal relationships that could have influenced the work presented in this paper.

Declaration of Generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of Artificial Intelligence (AI)-Assisted Technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Funding

This study was conducted with the support of the Technology Innovation Program [20012411, Alchemist Project] funded by the Ministry of Trade, Industry, and Energy (MOTIE).

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[6] Davidenko N., Schuster C.F., Bax D.V., Farndale R.W., Hamaia S., Best S.M., Cameron R.E.. Evaluation of cell binding to collagen and gelatin: A study of the effect of 2D and 3D architecture and surface chemistry. J Mater Sci Mater Med. 2016;27:148. https://doi.org/10.1007/s10856-016-5763-9
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[7] Díaz-Montes E.. Polysaccharides: Sources, characteristics, properties, and their application in biodegradable films. Polysaccharides. 2022;3:480-501. https://doi.org/10.3390/polysaccharides3030029
[Google Scholar]

[8] Fan W., Yan W., Xu Z., Ni H.. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Surf B Biointerfaces. 2012;90:21-27. https://doi.org/10.1016/j.colsurfb.2011.09.042
[Google Scholar]

[9] Horakova J., Klicova M., Erben J., Klapstova A., Novotny V., Behalek L., Chvojka J.. Impact of various sterilization and disinfection techniques on electrospun poly-ε-caprolactone. ACS Omega. 2020;5:8885-8892. https://doi.org/10.1021/acsomega.0c00503
[Google Scholar]

[10] Hussain S.. Native rice starch and linseed gum blends: Effect on the pasting, thermal and rheological properties. Czech J Food Sci. 2015;33:556-563. https://doi.org/10.17221/243/2015-cjfs
[Google Scholar]

[11] Jiang G., Ameer K., Kim H., Lee E. J., Ramachandraiah K., Hong G. P.. Strategies for sustainable substitution of livestock meat. Foods. 2020;9:1227. https://doi.org/10.3390/foods9091227
[Google Scholar]

[12] Judawisastra H., Sitohang R.D.R., Marta L., Mardiyati. Water absorption and its effect on the tensile properties of tapioca starch/polyvinyl alcohol bioplastics. IOP Conf Ser: Mater Sci Eng. 2017;223:012066. https://doi.org/10.1088/1757-899x/223/1/012066
[Google Scholar]

[13] Kassem A., Ayoub G.M., Malaeb L.. Antibacterial activity of chitosan nano-composites and carbon nanotubes: A review. Sci Total Environ. 2019;668:566-576. https://doi.org/10.1016/j.scitotenv.2019.02.446
[Google Scholar]

[14] Kim A.Y., Kim Y., Lee S.H., Yoon Y., Kim W.H., Kweon O.K.. Effect of gelatin on osteogenic cell sheet formation using canine adipose-derived mesenchymal stem cells. Cell Transplant. 2017;26:115-123. https://doi.org/10.3727/096368916X693338
[Google Scholar]

[15] Kim B., Ko D., Choi S.H., Park S.. Bovine muscle satellite cells in calves and cattle: A comparative study of cellular and genetic characteristics for cultivated meat production. Curr Res Food Sci. 2023;7:100545. https://doi.org/10.1016/j.crfs.2023.100545
[Google Scholar]

[16] Kim B., Min Y., Jeong Y., Ramani S., Lim H., Jo Y., Kim W., Choi Y., Park S.. Comparison of growth performance and related gene expression of\n muscle and fat from landrace, Yorkshire, and duroc and Woori black\n pigs. J Anim Sci Technol. 2023;65:160-174. https://doi.org/10.5187/jast.2022.e93
[Google Scholar]

[17] Kirchmajer D.M., Watson C.A., Ranson M.. Gelapin, a degradable genipin cross-linked gelatin hydrogel. RSC advances. 2013;3:1073-1081. https://doi.org/10.22175/mmb.17645.
[Google Scholar]

[18] Kirchmajer D.M., Watson C.A., Ranson M., Panhuis M.. Gelapin, a degradable genipin cross-linked gelatin hydrogel. RSC Adv. 2013;3:1073-1081. https://doi.org/10.1039/c2ra22859a
[Google Scholar]

[19] Lee J.Y., Kamel J., Yadav C.J., Yadav U., Afrin S., Son Y.M., Won S.Y., Han S.S., Park K.M.. Production of plant-based, film-type scaffolds using alginate and corn starch for the culture of bovine myoblasts. Foods. 2024;13:1358. https://doi.org/10.3390/foods13091358
[Google Scholar]

[20] Leloup V.M., Colonna P., Buleon A.. Influence of amylose-amylopectin ratio on gel properties. J Cereal Sci. 1991;13:1-13. https://doi.org/10.1016/s0733-5210(09)80023-4
[Google Scholar]

[21] Lin C.W., Wu P.T., Liu K.T., Fan Y.J., Yu J.. An environmental friendly tapioca starch-alginate cultured scaffold as biomimetic muscle tissue. Polymers (Basel). 2021;13:2882. https://doi.org/10.3390/polym13172882
[Google Scholar]

[22] Liu J., Chriki S., Kombolo M., Santinello M., Pflanzer S.B., Hocquette É, Ellies-Oury M.P., Hocquette J.F.. Consumer perception of the challenges facing livestock production and meat consumption. Meat Sci. 2023;200:109144. https://doi.org/10.1016/j.meatsci.2023.109144
[Google Scholar]

[23] Łopianiak I., Butruk-Raszeja B.A.. Evaluation of sterilization/disinfection methods of fibrous polyurethane scaffolds designed for tissue engineering applications. Int J Mol Sci. 2020;21:8092. https://doi.org/10.3390/ijms21218092
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[24] Mikhailov O.V.. Gelatin as it is: History and modernity. Int J Mol Sci. 2023;24:3583. https://doi.org/10.3390/ijms24043583
[Google Scholar]

[25] Narayanan A., Kartik R., Sangeetha E., Dhamodharan R.. Super water absorbing polymeric gel from chitosan, citric acid and urea: Synthesis and mechanism of water absorption. Carbohydr Polym. 2018;191:152-160. https://doi.org/10.1016/j.carbpol.2018.03.028
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[26] Nurul Alam A.M.M., Kim C.J., Kim S.H., Kumari S., Lee E.Y., Hwang Y.H., Joo S.T.. Scaffolding fundamentals and recent advances in sustainable scaffolding techniques for cultured meat development. Food Res Int. 2024;189:114549. https://doi.org/10.1016/j.foodres.2024.114549
[Google Scholar]

[27] Odegard I.Y.R., van der Voet E.. The future of food — Scenarios and the effect on natural resource use in agriculture in 2050. Ecological Economics. 2014;97:51-59. https://doi.org/10.1016/j.ecolecon.2013.10.005
[Google Scholar]

[28] Othman S.H., Kechik N.R.A., Shapi’i R.A., Talib R.A., Tawakkal I.S.M.A.. Water sorption and mechanical properties of starch/chitosan nanoparticle films. J Nanomater. 2019;2019:1-12. https://doi.org/10.1155/2019/3843949
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[29] Park S., Lee H., Jung S., Choi B., Lee M., Jung S.Y., Lee S.T., Lee S., Hong J.. Cost-effective culture medium for cell-based future foods. ACS Sustainable Chem Eng. 2023;11:13868-13876. https://doi.org/10.1021/acssuschemeng.3c02972
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[30] Parlasca M.C., Qaim M.. Meat Consumption and sustainability. Annu Rev Resour Econ. 2022;14:17-41. https://doi.org/10.1146/annurev-resource-111820-032340
[Google Scholar]

[31] Rao K.M., Choi S.M., Han S.S.. A review on directional muscle cell growth in scaffolding biomaterials with aligned porous structures for cultivated meat production. Food Res Int. 2023;168:112755. https://doi.org/10.1016/j.foodres.2023.112755
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[32] Roslan, M.R., Nasir, N.F.M., Cheng, E.M., Amin, N.A.M., 2016. Tissue engineering scaffold based on starch: A review. 2016 International Conference on Electrical, Electronics, and Optimization Techniques (ICEEOT) Chennai, India, 1857-1860. https://doi.org/10.1109/iceeot.2016.7755010

[33] Roy S., Rhim J.W.. Genipin-crosslinked gelatin/chitosan-based functional films incorporated with rosemary essential oil and quercetin. Materials (Basel). 2022;15:3769. https://doi.org/10.3390/ma15113769
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[34] Seah J.S.H., Singh S., Tan L.P., Choudhury D.. Scaffolds for the manufacture of cultured meat. Crit Rev Biotechnol. 2022;42:311-323. https://doi.org/10.1080/07388551.2021.1931803
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[35] Serrano-Aroca Á, Cano-Vicent A., Sabater I, Serra R., El-Tanani M., Aljabali A., Tambuwala M.M., Mishra Y.K.. Scaffolds in the microbial resistant era: Fabrication, materials, properties and tissue engineering applications. Mater Today Bio. 2022;16:100412. https://doi.org/10.1016/j.mtbio.2022.100412
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[36] Suamte L., Tirkey A., Barman J., Jayasekhar Babu P.. Various manufacturing methods and ideal properties of scaffolds for tissue engineering applications. Smart Mater Manuf. 2023;1:100011. https://doi.org/10.1016/j.smmf.2022.100011
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[37] Sundaram J., Durance T.D., Wang R.. Porous scaffold of gelatin-starch with nanohydroxyapatite composite processed via novel microwave vacuum drying. Acta Biomater. 2008;4:932-942. https://doi.org/10.1016/j.actbio.2008.01.019
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[38] Tchonkouang R.D., Lima A.R., Quintino A.C., Cristofoli N.L., Vieira M.C.. UV-C light: A promising preservation technology for vegetable-based nonsolid food. Prod Foods. 2023;12:3227. https://doi.org/10.3390/foods12173227
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[39] Turk J.. Meeting projected food demands by 2050: Understanding and enhancing the role of grazing ruminants. J Anim Sci. 2016;94:53-62. https://doi.org/10.2527/jas.2016-0547
[Google Scholar]

[40] Wang F., Li Y., Shen Y., Wang A., Wang S., Xie T.. The functions and applications of RGD in tumor therapy and tissue engineering. Int J Mol Sci. 2013;14:13447-13462. https://doi.org/10.3390/ijms140713447
[Google Scholar]

[41] Wang J., Zhuang S.. Chitosan-based materials: Preparation, modification and application. J Cleaner Prod. 2022;355:131825. https://doi.org/10.1016/j.jclepro.2022.131825
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[42] Wang Z., Liu H., Luo W., Cai T., Li Z., Liu Y., Gao W., Wan Q., Wang X., Wang J., Wang Y., Yang X.. Regeneration of skeletal system with genipin crosslinked biomaterials. J Tissue Eng. 2020;11:2041731420974861. https://doi.org/10.1177/2041731420974861
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[43] Xiang N., Yao Y., Yuen J.S.K., Stout A.J., Fennelly C., Sylvia R., Schnitzler A., Wong S., Kaplan D.L.. Edible films for cultivated meat production. Biomaterials. 2022;287:121659. https://doi.org/10.1016/j.biomaterials.2022.121659
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[44] Xie F., Pollet E., Halley P.J., Avérous L.. Starch-based nano-biocomposites. Prog Polym Sci. 2013;38:1590-1628. https://doi.org/10.1016/j.progpolymsci.2013.05.002
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[45] Xu Y.X., Kim K.M., Hanna M.A., Nag D.. Chitosan–starch composite film: Preparation and characterization. Ind Crops Prod. 2005;21:185-192. https://doi.org/10.1016/j.indcrop.2004.03.002
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[46] Zou W., Yu L., Liu X., Chen L., Zhang X., Qiao D., Zhang R.. Effects of amylose/amylopectin ratio on starch-based superabsorbent polymers. Carbohydr Polym. 2012;87:1583-1588. https://doi.org/10.1016/j.carbpol.2011.09.060
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Enhancement of Hanwoo bovine muscle satellite cell proliferation and differentiation using edible chitosan-starch scaffolds

Abstract

Keywords

Cell-based food

Chitosan

Gelatin

Hanwoo muscle satellite cell

Starch

1. Introduction

2. Materials and Methods

2.1 Muscle satellite cell isolation and cell culture

2.2 Preparation of chitosan/tapioca scaffolds

2.3 Scaffold structure analysis

2.4 Fourier transform infrared spectroscopy

2.5 Water absorption

2.6 Textural properties

2.7 Cell adhesion and proliferation

2.8 3-(4,5-Dimethylthiazol -2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

2.9 Cell staining

2.10 Statistical analysis

3. Results

3.1 Morphological and structural analyses

3.2 Fourier transform infrared spectroscopy

3.3 Water absorption and degradation

3.4 Textural property analysis

3.5 Cell proliferation and viability

3.6 Muscle satellite cell differentiation on scaffold

4. Discussion

5. Conclusions

CRediT authorship contribution statement

Declaration of competing interest

Declaration of Generative AI and AI-assisted technologies in the writing process

Funding

References

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